CN106434481B - The isolation and purification method of Facultative Halophiles and isolated saline-alkali tolerant bacterial strain and its application - Google Patents
The isolation and purification method of Facultative Halophiles and isolated saline-alkali tolerant bacterial strain and its application Download PDFInfo
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- 238000000746 purification Methods 0.000 title abstract description 17
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- 244000005700 microbiome Species 0.000 claims abstract description 38
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- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 16
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
Abstract
The present invention relates to microorganism fields, isolation and purification method and isolated saline-alkali tolerant bacterial strain and its application in particular to a kind of Facultative Halophiles.Bacillus amyloliquefaciens selected by the present invention (Bacillus amyloliquefaciens) are that the microorganism with significant salt resistance alkali effect screened is extracted in the alkaline land soil of the Hetao area, the ability with outstanding improvement Hetao area soil vitality.This screening technique provided by the invention, the microorganism for providing high-salt tolerance energy has been screened from for examination soil sample using the training method of concentration and separation, with with strong points, the ideal effect of the selection result, to be provided safeguard for period short, quick alkaline land soil reparation.
Description
Technical field
The present invention relates to microorganism fields, isolation and purification method in particular to a kind of Facultative Halophiles and isolated
Saline-alkali tolerant bacterial strain and its application.
Background technique
Salinization of soil problem is one of the environmental problem of current global most serious, including population expansion etc. various aspects because
Element constantly makes the mankind develop and use large area soil and produces new Secondary Saline, accelerates the process of the salinization of soil.
Saline-alkali soil is raw to plant because containing excessive salinity, noxious material, basicity is excessive and undesirable soil physical property
It is long to generate inhibiting effect, cause local area ecological to deteriorate.Under the premise of promotion ecological benefits are attached most importance to now, biological modification measure is
It has become a hot topic of research, and the improving effect in salt-soda soil should not be overlooked in microorganism.How much microorganism is soil fertility or salt-soda soil
One index, salt-soda soil micro organism quantity is few, is to be not easy existence procreation under alkaline condition due to microorganism beneficial bacterium group.It is main
It wants just not converting organic matter without microorganism the reason is that be unfavorable for microbe survival under saline-alkali environment, thus raw not at plant
The bioprotein and amino acid needed.Salt-soda soil is not improved only according to bio-fertilizer, it is necessary to implement " improvement salt-soda soil system
Improvement project " could improve salt-soda soil to improve the matched multiple products in salt-soda soil and corresponding technology, activate organic matter, increase
Add micro organism quantity.The more, the more, soil productivity is better for profitable strain microorganism for organic matter in soil.
In soil, water, in air and animal and plant body, different types of microorganism overwhelming majority is to mix to live in one
Rise, when it is desirable that obtaining a certain microorganism, just must from the microbe groups mixed sub-argument it, to be contained only
A kind of this pure culture of microorganism, this method for obtaining pure culture are known as the isolation and purification of microorganism.In order to obtain certain
The pure culture of microorganism, usually according to the microorganism to nutrition, the requirement of pH value, the conditions such as oxygen is different, and it is suitable to supply it
Suitable condition of culture, or certain inhibitor is added and causes only to be conducive to the environment that this bacterium grows and other bacterium is inhibited to grow, to wash in a pan
Some other unwanted microorganism is eliminated, then with dilution spread flat band method or dilutes a mixing flat board method or plate streaking partition method etc.
Microorganism is isolated and purified, until obtaining pure bacterial strain.
If want increase salt-soda soil in microorganism quantity, the isolation and purification to salt-durable microbe in its soil be it is capable it
An effective important means.The salt-soda soil in Hetao Plain, Inner Mongolia area is based on sulfate, chloride, mostly moderate and again
Salt-soda soil is spent, Soil Microorganism flora number of species are relatively fewer, it is desirable to isolate purpose strain and be not easily accomplished, especially
It is resistant to the bacterial strain of high concentration sodium chloride.There are no the effective skills for being directed to saline and alkaline geomicrobes isolation and purification in the prior art
Art.
In view of this, the present invention is specifically proposed.
Summary of the invention
Based in the prior art, relatively for saline-alkali tolerant micro organism quantity in the salt-soda soil in Hetao Plain, Inner Mongolia area
Few problem, the first object of the present invention is that providing one plant is capable of the microorganism of high salt tolerance stress, and then salt is effectively performed
Alkali land soil.
The second object of the present invention is to provide the isolation and purification method of Facultative Halophiles described in one kind, above-mentioned high salt tolerance stress
Microorganism i.e. to pass through this method isolated.
The third object of the present invention is to provide the isolation and purification method of Facultative Halophiles described in one kind in identification salt-soda soil soil
There are salt tolerant bacillus and the application of salinization and alkalization is judged whether in sample.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
One plant of salt tolerant bacteria strain, the bacterial strain are bacillus amyloliquefaciens (Bacillus amyloliquefaciens),
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC13060；When preservation
Between are as follows: on 09 29th, 2016.
The strain source is in Hetao Plain, Inner Mongolia salt-soda soil.
By identification, which belongs to Bacteriophyta bacillus suborder bacillus subtilis Pseudomonas bacterium,
With extensive energetic supersession mechanism, energy photosynthesis, aerobic growth under illumination condition；It can be up to 120g/L's in salinity
It remains to preferably grow in culture medium, there is the application value of improvement alkaline land soil well.Since the bacterial strain is by Inner Mongol Furukawa
Set Plain is separated, thus its alkaline land soil for being particularly suitable for improvement Hetao Plain, Inner Mongolia.
Liquid culture be it is light yellow, there is a small amount of flocky precipitate in bottom of bottle and bottle wall, and 2 are formed on solid medium
Dotted bacterium colony, the edge of~3mm is irregular, rough, and centre is prominent.
Salt tolerant bacteria strain as described above is in the application being used as in soil conditioner.
Preferably, the soil conditioner is mainly made of following component:
Mineral material, activator A, microbial bacterial agent, for providing the activator B of nutrition for the microbial bacterial agent；
The mineral material includes attapulgite, sepiolite, zeolite；
The main component of the activator A is organic acid；
The bacterium bacterial strain contained in the microbial bacterial agent is bacillus amyloliquefaciens provided by the invention.
A kind of isolation and purification method of Facultative Halophiles, comprising the following steps:
1) it, will be inoculated in after diluting in nutrient broth enriched medium for examination salt-soda soil soil sample, the training of aerobic constant temperature shaking table
Mixed bacteria liquid is obtained after supporting；
2) mixed bacteria liquid, is inoculated into the flat lining out culture of nutrient broth agar, therefrom picking meets gemma bar
The bacterium colony of bacterium form；
3), step 2) is repeated until obtaining the single bacterium colony of form；
4) the single bacterium colony of the form, is subjected to Zengjing Granule；
5), the bacterium that step 4) culture obtains is inoculated on slant medium and cultivate simultaneously cryo-conservation；
6) the isolated microorganism of step 5), is subjected to screening training in the resistance screening culture medium containing sodium chloride
It supports, obtains Facultative Halophiles.
The type main bacteria of Soil Microorganism, actinomyces, photosynthetic bacteria, fungi etc..It is main from the quantity of strain
If bacterium is dominant, and is reported according to documents and materials, most of salt-enduring strain is the bacillus in bacterium, therefore this hair
It is bright that the fundamental type of enriched medium is set to nutrient broth medium.
In addition, can clearly learn in an embodiment of the present invention, using screening technique provided by the invention, specific
(Hetao Plain, Inner Mongolia salt-soda soil) is screened in soil, can obtain the bacillus of a kind of saline-alkali tolerant, and only it is each
From salt resistance ability difference.
Preferably, the isolation and purification method of Facultative Halophiles as described above, in step 6), the specific mistake of the screening and culturing
Journey are as follows:
Micro-biological samples are cultivated 1~9 time in the resistance screening culture medium containing sodium chloride, when incubation times are greater than 1
When secondary, in every adjacent resistance screening culture medium used of culture twice, the sodium chloride concentration once cultivated afterwards is not less than previous
It is secondary, and the microorganism fungus kind that the strain source once cultivated afterwards survives in preceding primary culture.
The strain for various concentration sodium chloride sensitivity can be screened with the method, that is, the system can be filtered out quickly
Different degrees of salt-enduring strain.
It is further preferred that in step 6), in the resistance screening culture medium of adjacent concentration gradient, rear primary culture institute
It is previous 1.5~5 times with the sodium chloride concentration in culture medium.
The NaCl concentration gradient of specific resistance screening culture medium may be configured as 10g/L, 30g/L, 50g/L, 70g/L,
100g/L, 150g/L, 200g/L, etc..
According to practice of the inventor in soil improvement, it is considered that the bacterial strain salt tolerant degree screened then has in 100g/L
There is preferable application value.
Preferably, the isolation and purification method of Facultative Halophiles as described above, in step 6), the resistance containing sodium chloride
Screening and culturing medium includes following component:
Peptone 8g/L~12g/L, beef extract 2g/L~4g/L, sodium chloride 5g/L~200g/L, yeast powder 2g/L~4g/
L, sodium citrate 2g/L~4g/L；Solvent is water.
Resistance screening culture medium can be solid medium (adding the agar of 10g/L~20g/L again), can also be trained with liquid
Support base.
Preferably, the isolation and purification method of Facultative Halophiles as described above, in step 2), the nutrient broth agar plate
In culture medium include following component:
Peptone 8g/L~12g/L, beef extract 2g/L~4g/L, sodium chloride 4g/L~6g/L, agar 10g/L~20g/L,
Nystatin 40mg/L~60mg/L；Solvent is water.
Nystatin has conjugated polyene macrolide structure, can inhibit the activity of fungi and skin moss bacterium, to bacterium without suppression
Production is used.
Preferably, the isolation and purification method of Facultative Halophiles as described above, in step 1), the nutrient broth Zengjing Granule
Base includes following component:
Peptone 8g/L~12g/L, beef extract 2g/L~4g/L, sodium chloride 4g/L~6g/L；Solvent is water；
In step 4), culture medium used in the Zengjing Granule includes following component:
Peptone 8g/L~12g/L, beef extract 2g/L~4g/L, sodium chloride 4g/L~6g/L, yeast powder 2g/L~4g/L,
Sodium citrate 2g/L~4g/L；Solvent is water；
In step 5), the slant medium includes following component:
Peptone 8g/L~12g/L, beef extract 2g/L~4g/L, sodium chloride 4g/L~6g/L, agar 10g/L~20g/L；
Solvent is water.
Preferably, the isolation and purification method of Facultative Halophiles as described above, in step 4), the culture of the Zengjing Granule is whole
Point by the gemma rate of culture bacterium reach 90% or more and then terminate culture.
Preferably, the isolation and purification method of Facultative Halophiles as described above, which is characterized in that step 5) specifically includes:
Strain after step 4) Zengjing Granule is lined on slant medium, 28 DEG C~32 DEG C are cultivated 2~3 days, are terminated
Culture is placed on 4 DEG C of refrigerators and saves.
There are salt tolerant bacillus whether identifying in the soil sample of salt-soda soil for the isolation and purification method of Facultative Halophiles as described above
And judge the application of salinization and alkalization.
If there are objective microbes in sample, corresponding micro- life can be obtained according to screening technique provided by the invention
Object；If objective microbe is not present in sample, the process of screening calibrating can also be equally repeated, to determine whether with target
The ability of microorganism and the microorganism salt tolerant illustrates the salinization and alkalization in pedotheque source place if salt resistance ability is stronger
It is higher, and habitat is severe, is not suitable for crop growth, this equally can achieve the purpose for reducing cost.Therefore, affiliated technology neck
The technical staff in domain can repeat technical solution provided by the invention, and be able to solve application technical problem to be solved, reach
To the effect of the technical solution.
Compared with prior art, the invention has the benefit that
1), the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) selected by the present invention are in the river bend
The microorganism with significant salt resistance alkali effect screened is extracted in area's alkaline land soil, and there is the outstanding improvement Hetao area
The ability of soil vitality.
2), this screening technique provided by the invention is screened from for examination soil sample using the training method of concentration and separation
Provide high-salt tolerance can microorganism, there is with strong points, the ideal effect of the selection result, to for the period it is short, take effect
Fast alkaline land soil reparation provides safeguard.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) provided by the present application are preserved in the micro- life of China
Object culture presevation administration committee common micro-organisms center, preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology, the academy of sciences, state；The preservation time are as follows: on 09 29th, 2016, deposit number CGMCC NO.13060.Through preservation
Center was detected as survival strains on 09 29th, 2016.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
A kind of microbe to screen method is present embodiments provided, this method comprises the following steps:
It 1), will be for being inoculated in nutrient broth enriched medium after examination salt-soda soil soil sample 1:1000 dilution, in 30 DEG C of conditions
Under, aerobic constant temperature culture shaking table culture is carried out, revolving speed 200rpm cultivates 3 days, obtains mixed bacteria liquid；
The nutrient broth enriched medium includes following component: peptone 8g/L, beef extract 4g/L, sodium chloride 4g/L；
Solvent is water；
2) mixed bacteria liquid, is inoculated into the flat lining out culture of nutrient broth agar, therefrom picking meets gemma bar
The bacterium colony of bacterium form；
The form of bacillus single colonie is generally white, and periphery is irregular, and centre is prominent, Gram-positive, microscope
Observation can be appreciated that transparent gemma occurs.
Culture medium in the nutrient broth agar plate includes following component: peptone 12g/L, beef extract 2g/L, chlorine
Change sodium 6g/L, agar 10g/L~20g/L, nystatin 60mg/L；Solvent is water.
3), step 2) is repeated until obtaining the single bacterium colony of form；30 DEG C of constant temperature incubations are cultivated 2~4 days every time；
4) the single bacterium colony of the form, is subjected to Zengjing Granule；30 DEG C of constant temperature incubations, the gemma rate for cultivating bacterium reach 90%
Culture is then terminated above；
Culture medium used in the Zengjing Granule includes following component: peptone 8g/L, beef extract 4g/L, sodium chloride 6g/
L, yeast powder 2g/L, sodium citrate 2g/L；Solvent is water；
5), the strain after step 4) Zengjing Granule is lined on slant medium, 28 DEG C~32 DEG C are cultivated 2~3 days, knot
Beam culture is placed on 4 DEG C of refrigerators and saves；
The slant medium includes following component: peptone 12g/L, beef extract 4g/L, sodium chloride 4g/L, agar 10g/
L；Solvent is water.
6) the isolated microorganism of step 5), is subjected to screening training in the resistance screening culture medium containing sodium chloride
It supports:
The resistance screening culture medium containing sodium chloride includes following component: peptone 8g/L, beef extract 2g/L, chlorination
Sodium Xg/L, yeast powder 2g/L, sodium citrate 4g/L；Solvent is water；
Sodium chloride X is set as different concentration gradients in above-mentioned resistance screening culture medium, specifically, specific concentration gradient
It is designed as 5g/L, 10g/L, 30g/L, 50g/L, 70g/L, 100g/L, step-sizing salt-enduring strain.It is chosen under 10% salt ion
Well-grown bacterial strain.
Activated single strain bacterium solution 0.1ml in enriched medium is drawn, the salt culture medium of the above various concentration is coated on
On, 30 degree are cultivated 3 days, are chosen at the good bacterium colony of 100g/L salinity growth, after activating repeatedly, 4 degree are stored in nutrient meat
It is spare on soup agar slant.
Embodiment 2
A kind of microbe to screen method is present embodiments provided, this method comprises the following steps:
It 1), will be for being inoculated in nutrient broth enriched medium after examination salt-soda soil soil sample 1:1000 dilution, in 30 DEG C of conditions
Under, aerobic constant temperature culture shaking table culture is carried out, revolving speed 200rpm cultivates 3 days, obtains mixed bacteria liquid；
The nutrient broth enriched medium includes following component: peptone 12g/L, beef extract 2g/L, sodium chloride 6g/L；
Solvent is water；
2) mixed bacteria liquid, is inoculated into the flat lining out culture of nutrient broth agar, therefrom picking meets gemma bar
The bacterium colony of bacterium form；
The form of bacillus single colonie is generally white, and periphery is irregular, and centre is prominent, Gram-positive, microscope
Observation can be appreciated that transparent gemma occurs.
Culture medium in the nutrient broth agar plate includes following component: peptone 8g/L, beef extract 4g/L, chlorination
Sodium 4g/L, agar 20g/L, nystatin 40mg/L；Solvent is water.
3), step 2) is repeated until obtaining the single bacterium colony of form；30 DEG C of constant temperature incubations are cultivated 2~4 days every time；
4) the single bacterium colony of the form, is subjected to Zengjing Granule；30 DEG C of constant temperature incubations, the gemma rate for cultivating bacterium reach 90%
Culture is then terminated above；
Culture medium used in the Zengjing Granule includes following component: peptone 12g/L, beef extract 2g/L, sodium chloride 4g/
L, yeast powder 4g/L, sodium citrate 4g/L；Solvent is water；
5), the strain after step 4) Zengjing Granule is lined on slant medium, 28 DEG C~32 DEG C are cultivated 2~3 days, knot
Beam culture is placed on 4 DEG C of refrigerators and saves；
The slant medium includes following component: peptone 8g/L, beef extract 2g/L, sodium chloride 6g/L, agar 20g/
L；Solvent is water.
6) the isolated microorganism of step 5), is subjected to screening training in the resistance screening culture medium containing sodium chloride
It supports:
The resistance screening culture medium containing sodium chloride includes following component: peptone 12g/L, beef extract 4g/L, chlorine
Change sodium Xg/L, yeast powder 4g/L, sodium citrate 2g/L；Solvent is water；
Sodium chloride X is set as different concentration gradients in above-mentioned resistance screening culture medium, specifically, specific concentration gradient
It is designed as 5g/L, 10g/L, 15g/L, 30g/L, 50g/L, 70g/L, 100g/L, 150g/L, step-sizing salt-enduring strain.It chooses
Well-grown bacterial strain under 10% salt ion.
Activated single strain bacterium solution 0.1ml in enriched medium is drawn, the salt culture medium of the above various concentration is coated on
On, 30 degree are cultivated 3 days, are chosen at the good bacterium colony of 100g/L salinity growth, after activating repeatedly, 4 degree are stored in nutrient meat
It is spare on soup agar slant.
Embodiment 3
A kind of microbe to screen method is present embodiments provided, this method comprises the following steps:
It 1), will be for being inoculated in nutrient broth enriched medium after examination salt-soda soil soil sample 1:1000 dilution, in 30 DEG C of conditions
Under, aerobic constant temperature culture shaking table culture is carried out, revolving speed 200rpm cultivates 3 days, obtains mixed bacteria liquid；
The nutrient broth enriched medium includes following component: peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L；
Solvent is water；
2) mixed bacteria liquid, is inoculated into the flat lining out culture of nutrient broth agar, therefrom picking meets gemma bar
The bacterium colony of bacterium form；
The form of bacillus single colonie is generally white, and periphery is irregular, and centre is prominent, Gram-positive, microscope
Observation can be appreciated that transparent gemma occurs.
Culture medium in the nutrient broth agar plate includes following component: peptone 10g/L, beef extract 3g/L, chlorine
Change sodium 5g/L, agar 15g/L, nystatin 50mg/L；Solvent is water.
3), step 2) is repeated until obtaining the single bacterium colony of form；30 DEG C of constant temperature incubations are cultivated 2~4 days every time；
4) the single bacterium colony of the form, is subjected to Zengjing Granule；30 DEG C of constant temperature incubations, the gemma rate for cultivating bacterium reach 90%
Culture is then terminated above；
Culture medium used in the Zengjing Granule includes following component: peptone 10g/L, beef extract 3g/L, sodium chloride 5g/
L, yeast powder 3g/L, sodium citrate 3g/L；Solvent is water；
5), the strain after step 4) Zengjing Granule is lined on slant medium, 28 DEG C~32 DEG C are cultivated 2~3 days, knot
Beam culture is placed on 4 DEG C of refrigerators and saves；
The slant medium includes following component: peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, agar 15g/
L；Solvent is water.
6) the isolated microorganism of step 5), is subjected to screening training in the resistance screening culture medium containing sodium chloride
It supports:
The resistance screening culture medium containing sodium chloride includes following component: peptone 10g/L, beef extract 3g/L, chlorine
Change sodium Xg/L, yeast powder 3g/L, sodium citrate 3g/L；Solvent is water；
Sodium chloride X is set as different concentration gradients in above-mentioned resistance screening culture medium, specifically, specific concentration gradient
It is designed as 5g/L, 10g/L, 30g/L, 50g/L, 70g/L, 100g/L, 120g/L, 200g/L, step-sizing salt-enduring strain.It chooses
Well-grown bacterial strain under 10% salt ion.
Activated single strain bacterium solution 0.1ml in enriched medium is drawn, the salt culture medium of the above various concentration is coated on
On, 30 degree are cultivated 3 days, are chosen at the good bacterium colony of 100g/L salinity growth, after activating repeatedly, 4 degree are stored in nutrient meat
It is spare on soup agar slant.
Experimental example 4
According to the culture of Examples 1 to 3 using the salinity for being resistant to 100g/L as standard, 5 plants of salt tolerant gemma bars are filtered out altogether
Bacterium.It is respectively labeled as b1, b2, b3, b4, b5.Wherein, b2 and b3 comes from embodiment 1, and b4 comes from embodiment 2, b1 and b5 from real
Apply example 3.
Salt tolerant degree verification result
The salt tolerant degree of 15 bacillus of table is verified
Note: "+" expression represents strain to be tested energy normal growth on saliferous plate, and "-" indicates that strain to be tested is flat in saliferous
It cannot be grown on plate.
Different "+" indicates the power of salt resistance ability.
4. conclusion
The 5 plants of salt tolerant bacillus screened can grow under 100g/L sodium chloride concentration, wherein only b1 and b5
It can be grown under 120g/L concentration, and b1 growth fraction b5 is good；Above 5 plants of bacterium cannot grow in concentration 200g/L concentration；With
The most suitable growth salinity of upper 5 plants of bacterium b2/b3 is 1%~5%, b1, and the most suitable growth salinity of b5 is 1%, 5%~10%
Between without apparent growth differences.In general, b1 is the strongest bacterial strain of salt resistance ability of this test choosing weight, by physiology
Biochemical identification is accredited as bacillus licheniformis.
Application examples 1
According to the culture of Examples 1 to 3, microorganism in the resistance screening culture medium containing various concentration sodium chloride is counted
Survival rate, be shown in Table 2.
In the fluid nutrient medium for the 100ml that the statistical method of survival rate is inoculated into different salt ionic concentrations for slant strains,
30 DEG C, 200rpm constant temperature incubation 48h takes a bacterium solution in every 4 hours, by detection bacterium solution absorbance and plate count, determines bacterium
The survival number of strain, is compared with 0 hour initial bacterium amount, obtains survival rate；
By in the culture medium of the strain inoculated of survival to next salt ionic concentration after 48h, aforesaid operations are repeated.
The survival rate of 25 bacillus of table is verified
As can be known from the above table in embodiment 3 Facultative Halophiles content highest, thus its salinization and alkalization highest for corresponding to soil sample is (main
See the salt resistance ability of 10g/L).
Application examples 2
Bacterium is made in the bacillus amyloliquefaciens screened in embodiment 4 (Bacillus amyloliquefaciens)
Agent.
1), actication of culture
The Bacillus amyloliquefaciens strain of cryo-conservation is inoculated in LB liquid medium, shake culture 20h at 30 DEG C
~for 24 hours, obtained fermentation liquid is as inoculation liquid；
2), the preparation of seed liquor
The inoculation liquid for accessing step 1) preparation into LB liquid medium according to the ratio of percent by volume 2%~5%,
Shake culture 12h~16h, obtains seed liquor at 30 DEG C.
3), fermented and cultured
Seed liquor obtained by step 2) and fermentation medium are contained into fermentation according to the access of 1%~2% ratio of percent by volume
It ferments in the fermentor of culture medium, obtains the liquid bacterial agent of bacillus amyloliquefaciens.
Wherein: the constituent and its mass percent of the fermentation medium be glucose 0.5%, corn flour 2%,
Soybean cake powder 0.5%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.15%, calcium carbonate 0.15%, manganese sulfate 0.0001%, Yu Zhe
For water；
The ferment tank condition is that cultivation temperature is 28 DEG C~32 DEG C, and ventilatory capacity is 1:(0.5~1.6), stirring speed
Degree is 200rpm~400rpm, and incubation time is for 24 hours~36h；
Microbial inoculum obtained above is liquid bacterial agent, and suitable matrix is added after centrifugation and is mixed up to solid powder.It is described micro-
The gemma number of bacillus amyloliquefaciens is 1 × 10 in bacteria agent9~10CFU/ml, usually 2 × 109CFU/ml or so.
A kind of soil conditioner and preparation method thereof, which includes following component:
Mineral material, activator A, activator B, mentioned microorganism microbial inoculum；
The mineral material includes the attapulgite, sepiolite, zeolite of 100~200 mesh；The attapulgite, Hai Pao
Stone, zeolite mass ratio be 1:0.5:1；
The composition of the activator A is the mixture of citric acid and amino acetonitrile bisulfate that mass ratio is 1:0.02；
The activator B includes yeast leaching liquor, peptone and amino acid；The yeast leaching liquor, peptone and amino
The mass ratio of acid is 1:2:1；
The soil conditioner preparation method the following steps are included:
1), mineral material and activator A are mixed by the mass ratio of 1:0.02,180 DEG C are reacted 3h in a kettle, are obtained
To the first reactant；
2) microbial bacterial agent and activator B are mixed by the mass ratio of 1:1.5,12h is cultivated in the fermenter in 30 DEG C and obtains
To the second reactant；
3), first reactant is mixed with second reactant by the mass ratio of 1:0.03, is granulated to get described
Soil conditioner.
Soil conditioner provided by the invention is verified to the improved effect of Hetao area saline-alkali soil.
Test material and method
Test site: test is carried out in May, 2015 in October, 2015 in Inner Mongol Bayannur.
Test material:
N P and K ternary compound fertilizer, soil conditioner (attapulgite, sepiolite, zeolite, salt resistance alkali microbial inoculum, activator)；
Sunflower seeds (kind 590).
Experimental design:
One mus of testing ground area, four processing are set altogether, are respectively:
Processing one: conventional to apply nitrogen, phosphorus and potassium fertilizer (NPK group)；
Processing two: NPK+ application attapulgite, sepiolite, zeolite, three match the soil conditioning provided with the application example
The proportion (BN1 group) of agent；
The soil conditioner (BN2 group) that processing three: NPK+ is prepared by the application example method；
Test procedure:
Test sets four processing altogether, 0.07 hectare is averagely divided into four cells, each cell is applied by experimental design
Fertilizer, nitrogen, phosphorus and potassium fertilizer ternary compound fertilizer are disposably applied as base manure, and 60~80 kilograms per acre.Two, three, four difference of processing is in seedling stage
It carries out conditioner with flower bud phase to impose, seedling stage imposes 30 kilograms per acre, and flower bud phase imposes 20 kilograms (foliage-spray can be used) per acre.
When test harvest, every cell singles are singly received, and are carried out sunflower 100-grain weight and yield detection, are carried out after every cell harvest
P in soil H, organic matter, conductivity, exchangeable sodium, basicity data monitoring.
Three, test result and analysis
Influence of the conditioner to sunflower yield:
The test data respectively handled by comparative analysis, the soil conditioner (BN2 group) that each group and the application example are provided
It compares, as the result is shown: crop 100-grain weight ratio NPK, BN1 processing of BN2 processing have been respectively increased 21.15%, 17.96%；
Sunflower yield ratio NPK, BN1, BN2 processing of BN2 processing have been respectively increased 22.93%, 18.36%.
Influence of the conditioner to soil physical and chemical property:
After sunflower harvest, soil five indices are detected, detection data is shown: the P in soil H ratio of BN2 processing
NPK, BN1 processing reduce 15.94%, 11.44% respectively；Soil total salt ratio NPK, BN1 processing of BN2 processing reduce respectively
27.6%, 23.0%；The soil exchangeable sodium of BN2 processing has dropped 57.6%, 33.6% compared with NPK, BN1 processing respectively；
NPK, BN1, BN2, three processing alkalization of soils degree be 25.46% respectively, 20.32%, the soil sun of 8.87%, BN2 processing
Ion exchange capacity has increased separately 32.5%, 25.3% compared with NPK, BN1 processing.
Influence of 3 conditioner of table to soil physical and chemical property
Four, conclusion (of pressure testing)
Soil conditioner product is imposed on the basis of conventional fertilizer application can make sunflower increase production 15.85%, wherein concave-convex
Stick soil, sepiolite, zeolite, salt resistance alkali microbial inoculum and the effect of activator synergy are best, it can be seen that this agriculture Hetao area soil
Earth conditioner can significantly improve alkaline land soil, and culture fertility increases production extra earning.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (2)
1. one plant of salt tolerant bacteria strain, which is characterized in that the bacterial strain is bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens), it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number
Are as follows: CGMCC13060；The preservation time are as follows: on 09 29th, 2016.
2. salt tolerant bacteria strain described in claim 1 is in the application being used as in soil conditioner.
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| CN106947725B (en) * | 2017-05-24 | 2020-05-22 | 北京本农科技发展有限公司 | Microbial agent and application thereof in saline-alkali soil improvement |
| CN107502579B (en) * | 2017-09-28 | 2020-03-24 | 山东省城市供排水水质监测中心 | Screening and separating method for chlorine-resistant bacteria and Gordonia terranei strain obtained by screening and separating method |
| CN107879852A (en) * | 2017-11-29 | 2018-04-06 | 大工（青岛）新能源材料技术研究院有限公司 | A kind of edaphon modifying agent and preparation method thereof |
| CN108728103A (en) * | 2018-04-17 | 2018-11-02 | 仁维国际股份有限公司 | The extracting method of geobiont element |
| CN109135754A (en) * | 2018-09-04 | 2019-01-04 | 孙爱友 | A kind of microorganism in situ cultural method for agricultural soil |
| CN109370598A (en) * | 2018-09-06 | 2019-02-22 | 中海新世纪量子生物科技（深圳）有限责任公司 | Administer leavening, composite bacteria agent, the Preparation method and use in salt-soda soil |
| CN109136153B (en) * | 2018-09-29 | 2021-08-03 | 中国科学院成都生物研究所 | Salt-tolerant bacillus with plant growth promoting effect |
| CN110684683A (en) * | 2019-09-10 | 2020-01-14 | 北方民族大学 | Bacillus amyloliquefaciens, microbial inoculum and application |
| CN112243814A (en) * | 2020-10-16 | 2021-01-22 | 北方民族大学 | Method for screening microbial strains for improving saline-alkali resistance of rice through field test |
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