CN106673866A - Bacillus subtilis microbial fertilizer and preparation method thereof - Google Patents
Bacillus subtilis microbial fertilizer and preparation method thereof Download PDFInfo
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- CN106673866A CN106673866A CN201710097177.1A CN201710097177A CN106673866A CN 106673866 A CN106673866 A CN 106673866A CN 201710097177 A CN201710097177 A CN 201710097177A CN 106673866 A CN106673866 A CN 106673866A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Life Sciences & Earth Sciences (AREA)
- Pest Control & Pesticides (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Molecular Biology (AREA)
- Fertilizers (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for preparing bacillus subtilis microbial fertilizer. The method comprises the following steps: S1, preparing a first liquid culture medium; S2, preparing a second liquid culture medium; S3, culturing a strain to obtain a first nutrient solution; S4, continuously culturing to obtain a second nutrient solution; and S5, fermenting a solid culture medium to obtain the bacillus subtilis microbial fertilizer. The preparation method can be used for preparing the bacillus subtilis microbial fertilizer with excellent technical effect by the unique combination and synergizing of multiple technical characteristics, can meet the wide requirement in the technical field of agriculture, and has excellent application prospect and industrial production potential.
Description
Technical field
The present invention relates to a kind of microbial manure and preparation method thereof, more specifically, is related to a kind of bacillus subtilises
Microbial manure and preparation method thereof, belongs to microorganism culturing and microbial manure applied technical field.
Background technology
Bacillus subtilises (Bacillus subtillis) belong to bacillus and further belong to gram sun
Property bacterium, in individual cells form, without pod membrane, usually (0.7-0.8) × (2-3) microns.
Bacillus subtilises are a kind of probiotic bacterias, and it has various biological functions, in can be applicable to multiple technical fields, example
Such as：It has that reproduction speed is fast, produces raw digestive enzyme in a large amount of, improves feed nutrition quality, improving fertilizer efficiency, can be used as microorganism
Complicated Organic substance, promotion in pesticide, degraded feedstuff absorbs, strengthens animal immunizing power and premunition etc., so as in animal husbandry, raise
All have in the multiple fields such as material industry, pesticide, fertilizer, sterilization, oil exploitation, gardens industry, protein and peptide synthesis and widely should
With, and its application and new purposes are also always in research and expansion.
Wherein, application of the bacillus subtilises in biological organic fertilizer field is increasingly subject to the concern of people, because micro- life
Thing fertilizer have not available for chemical fertilizer it is various a little, will not for example cause soil compaction, noresidue, safety non-toxic, right
Water body and environmental contaminants etc., have therefore suffered from the very big concern of people, have caused research and development enthusiasm, and achieve it is many into
Really, for example：
The Water Soluble Compound fertilizer and preparation method thereof that CN102329166A discloses a kind of activating soil, can be no-tillage, by
Following percentage by weight raw material is made：Carbamide 15%, potassium dihydrogen phosphate 50%, humic acid 25%, bacillus subtilis microbial agent
10%；Preparation process is：1) carbamide is crushed；2) bacillus subtilis microbial agent is cultivated；3) by urea powder, humic acid, di(2-ethylhexyl)phosphate
Hydrogen potassium is added into successively continuous reaction kettle；4) bacillus subtilis microbial agent is added to carry out secondary mixing；5) packaging and storage.It takes
Obtained following beneficial effect：Not only had quick-acting again with long-lasting, can directly provide nutrition for crop can also activate soil
By fixed nutrition in earth；Can activating soil can also suppress soil pathogenic bacteria；Yield can be increased can also improving quality；Can promote
Entering root system development can also improve soil water-reataining fertilizer-keeping ability；Effectively can also reduce chemical fertilizer consumption by culture fertility；This product
Water solubility reaches more than 98%, can apply or use fertilization gun soil injection with water punching, simple and easy to do, saves work(and saves money.
CN102391948A discloses a kind of straw fermentation complex microbial inoculant, and it is by following materials by weight percentage preparation
Into：Bacillus subtilises 30%, trichoderma aureoviride 10%, saccharomyces cerevisiae 12%, aspergillus oryzae 20% expands penicillium sp 18%, green wood
Mould 10%.The microbial inoculum of the present invention can accelerate the decomposition rate of organic matter, and agricultural crop straw can be completely decomposed also once all left and right
Field, straw decomposition blackening, reaches and becomes thoroughly decomposed；Nutrient release in straw, the growth for later stage crop is accelerated to provide more simultaneously
Nutrient, maintain and increase soil fertility, improve the soil organism, improve soil physico-chemical property, second stubble crop output increased 5%
More than, can be widely applied to Oryza sativa L., Semen Tritici aestivi, seeding corn and other crops straw-returning.
CN102417413A discloses a kind of composite microbiological fertilizer, and it is combined into by bacillus subtilises with other strains
Complex micro organism fungicide, complex micro organism fungicide is composited again with turf or differentiation coal, inorganic additive.With existing micro- life
Thing fertilizer is compared, and the composite microbiological fertilizer is in addition to the effect with existing microbial manure, wherein the humic acidss for containing point
Solution bacterium can degrade turf and differentiation coal in humic acidss, so as to increased the fertilizer efficiency of microbial manure so as to promote plant growth,
The advantages of improving crop yield and improved soil.
CN102515891A discloses a kind of microbial manure, the microbial fermentation solution comprising following ratio：Photosynthetic bacteria,
Lactic acid bacteria, actinomycetes, trichodermaharzianum bacterium, bacillus subtilis fermentation liquor volume ratio are 1: 0.8~1: 1.2~1.4：0.6~
0.8:1~1.2.The fertilizer can improve the utilization ratio of fertilizer, and also can play that preventing and treating plant is withered, disease work
With.
CN102515954A discloses a kind of verticillium wilt bio-organic fertilizer special, by biocontrol water bacillus subtilises
(Bacillus subtilis) NCD-2, colloid bacillus cereus (Bacillus mucilaginosus Krassilnikov) are huge
Bacterium anthracoides (Bacillus megaterium) with adsorbing medium absorption after by 6-4: 3-2: 3-2 mass ratio is mixed into
Composite bacteria agent capable, then mix with hydrolysis residue.Additionally provide the production method of the biological organic fertilizer.The verticillium wilt special bio
Fertilizer can significantly improve the fertilizer efficiency of bio-feritlizer, have obvious prevention effect to the disease of crop, greatly reduce sick seedling rate,
Simultaneously crop yield can be improved than more comprehensively providing the nutrient needed for plant growth.
CN102757275A discloses a kind of bio-feritlizer for aquaculture pond and preparation method thereof, the bio-fertilizer
Material is made up of sodium humate, carbamide, calcium superphosphate, bacillus subtilises and clinoptilolite powder, the humic acidss based on its parts by weight
100~200 parts of sodium, 100~200 parts of carbamide, 100~200 parts of calcium superphosphate, tiltedly 100~200 parts of bacillus subtilises, a boiling
200~600 parts of stone powder, is mixed with by each component and is formed；The bio-feritlizer by reasonable formula can quick-acting rich water, adjust
Water quality, improves substrate, and the bio-feritlizer can also be tied in addition to supply aquatile directly utilizes with the harmful substance in sediment
Close, release toxicity, while improving the metabolism of algae, increase water body dissolved oxygen；Its preparation method is simple, is adapted to large-scale industrial
The needs of production.
CN102757277A discloses a kind of rice biological fertilizer and preparation method thereof and bacillus subtilises.The preparation
Method is added to deposit number in solid material for the bacillus subtilises of CGMCC No.6190 ferments to bacillus subtilis bacterium number
At least 1.2 × 1010Individual/g, and bacillus subtilis spore rate is at least up to 60%, miscellaneous bacteria rate less than 20%, then will fermentation
Good solid material is mixed with humic acid, agricultural rare-earth, the fowl and animal excrement after fermenting that becomes thoroughly decomposed, silicon-calcium-magnesium fertilizer and amino acid fertilizer
It is even to obtain final product rice biological fertilizer.The invention using the organic matter of the fermentation of becoming thoroughly decomposed such as bacillus subtilis and chicken manure of mutation, humic acidss,
The material mixings such as silicon-calcium-magnesium fertilizer, agricultural rare-earth, amino acid fertilizer, form compound Oryza sativa L. microbial manure, it is possible to increase Oryza sativa L.
Tiller number, in advance rice plant of tillering stage, increase rice root weight, deepen Oryza sativa L. leaf color, improve water temperature, promote Oryza sativa L. under cold stress more
Good growth.
CN105541423A discloses a kind of microbial manure containing bacillus subtilises, is related to agricultural technology field,
The formula of the microbial manure is made up of organic fertilizer and microbial bacteria, by being used as micro- life using organic fertilizer and microbial bacteria
The constituent of thing fertilizer, significantly improves the growing environment of crop, the nutrient for needed for it provide not only growth, and
Its ability for adapting to environment is also improved, promotes crop that good growth situation is still presented in colder environment.
CN105254424A discloses a kind of microbial manure prepared by bacillus subtilises and preparation method thereof, and this is withered
Careless bacillus cereuss preserving number obtains culture, gained culture and grass for the microorganism fungus kind of CGMCC No.5091 through culture
Powdered carbon is mixed to get microorganism fungus kind agent, and gained microorganism fungus kind agent is mixed with common fertilizer forms again, wherein described micro-
The cultural method of biological inoculum culture is as follows：1) during 1 volume bacteria suspension to be accessed No. 1 culture medium of 20 times of volumes, 30-38 DEG C
Aerobic culture 8 hours；2) by step 1) culture fluid that obtains is added in No. 2 culture medium of about 20 times of volumes, 30-38 DEG C of aerobic
Culture 8 hours；3) by step 2) culture fluid that obtains is added in No. 3 culture medium of about 20 times of volumes, and 35-38 DEG C of culture 8 is little
When, obtain final product can production culture.
CN104774112A discloses a kind of preparation method of organic composite fertilizer, including the step for mixing following component
Suddenly, Bacillus globigii spores powder 0.3%；Aspergillus oryzae spore powder 0.4%；Organic fertilizer 30%；Humic acid 50%；Balance of load
Body；Bacillus globigii spores powder is identical with the preparation method of aspergillus oryzae spore powder, through slant culture and seed culture, obtains
Spore powder.The organic fertilizer is obtained organic matter and other effective ingredient by biodegradation is then passed through, and does not result in soil
Scleroma, it is to avoid cause soil pollution.
CN104671848A discloses a kind of composite microbial manure and its production method, the composite microbial manure,
It is made up of aspergillus niger culture, bacillus subtilis microbial agent, experiment shows：Grain yield 8-30% can be improved using this product, is carried
High yield of vegetables 10-40%, can also improve vegetable, the quality of grain and local flavor.The amount of fertilizer described in every mu of land use is 50-
100 grams, be current one of less composite microbial manure of usage amount both at home and abroad.
CN104311225A discloses a kind of efficient sterilizing fertilizer, and it is made up of following weight portion：Plant ash 8-15 parts, it is multiple
Conjunction microbial bacterial agent 3-7 parts, nitrate 4-6 parts, zinc sulphate heptahydrate 8-14 parts, organic acid 5-9 parts, potassium dihydrogen phosphate 4-11 parts,
Tea seed episperm carbon granule 3-5 parts, calcium superphosphate 5-9 parts, ammonium hydrogencarbonate 3-8 parts, aminoacid 2-5 parts, potassium sulfate 1-4 parts, hay spore
Bacillus microbial inoculum 5-7 parts, antibacterial 6-10 parts.The fertilizer has certain bactericidal action, while with the addition of microbial inoculum, improves soil
Earth, it is therefore prevented that soil it is hardened.
CN104291951A discloses a kind of fertilizer for improving soil acidity or alkalinity, and it is made up of following weight portion：Hay bud
Spore bacillus 2-7 parts, kieselguhr 3-6 parts, dalcium biphosphate 6-10 parts, sodium glutamate 1-5 parts, peat soil 3-8 parts, animal MAOFEN 2-
5 parts, ammonium molybdate tetrahydrate 3-9 part, zinc sulphate heptahydrate 5-9 parts, poly-aspartate 5-8 parts, potato class straw 8-14 parts, straw ashes 1-
5 parts, analysis for soybean powder 2-8 part, ammonium chloride 3-6 parts, Radix Aconiti Brachypodi Radix Aconiti Szechenyiani 5-11 parts, limestone 1-6 parts.The fertilizer can improve soil
Acid-base value, improve soil fertility, improve the growing environment of crop.
CN103833427A discloses a kind of water-soluble bio-feritlizer, and its active component is bacillus subtilises
BacillussubtilisLiu-3 thalline, preparation process is：1) it is grown on solid medium with connecing collarium and take
The ring of BacillussubtilisLiu-3 bacterium 1, in accessing the fluid medium of 5mL, 35-37 DEG C, 300-400r/min, shaking table training
Foster 20-24h；2) by the first order seed culture fluid 5mL for obtaining, in accessing 1000mL fluid mediums, 35-37 DEG C, 300-400r/
Min, shaking table culture 20-24h；3) by the secondary seed culture fluid 1000mL for obtaining, in accessing 30L fluid mediums, 35-37
DEG C, 200r/min, fermentation culture 70-72h, fermentation liquid is that every mL contains more than 200,000,000 bacterium.The fertilizer can be used for land for growing field crops work
Thing, fruit tree and cultivation of various vegetable melon and fruits etc., can promote plant growing and improve the yield of crop, with considerable economy
Benefit and social benefit.
The method that CN103664259A discloses a kind of utilization duck excrement just fermenting and producing bio-organic fertilizer, including following step
Suddenly：Step one, the bacillus subtilises for taking equivalent, lactic acid bacteria, yeast etc., spread cultivation and prepare leaven；Step 2, to duck excrement just
Leaven is accessed in raw material, fermentation reactor system pile in kiln formula solid fermentation tunnel, one time fermentation after 15-35 days are loaded after adding water
Become thoroughly decomposed and complete；Step 3, the duck excrement that one time fermentation is become thoroughly decomposed just are banked up naturally, and to room temperature, then ferment in second time is completed；Step 4, general
The duck excrement of ferment in second time just adopts the straight fire drying of revolving drum drier.Using the method for present invention offer to duck manure fermentation,
Using the synergism of the various microorganisms, make duck excrement just be converted to the bio-organic fertilizer utilized beneficial to plant growing, apply
After entering soil, soil organic matter content is high, and quality is relatively preferable, comprehensive nutrition can be provided for crops, so as to improve agriculture
Crop yield, economic benefit is higher.
CN102765971A discloses a kind of amino acid fertilizer and preparation method thereof, by weight amino acid fertilizer by
Following components is constituted, enzymatic microorganism 1-2 part；Fowl and animal excrement 20-30 parts；Herba Artemisiae Annuae slag 50-70 parts；Wherein enzymatic microorganism is by bacillus subtilis
Bacterium (Bacillus subtilis.) RB and commercially available enzymatic microorganism are constituted, and bacillus subtilises RB is preserved in China General Microbiological bacterium
Preservation administrative center is planted, preserving number is CGMCC NO.6268；The effect that bacillus subtilises RB processes Herba Artemisiae Annuae slag is high, made by
Nutrient enriches in amino acid fertilizer, and rich in aminoacid, realizing turns waste into wealth, the aminoacid that can provide high-quality for agriculture and forestry production
Fertilizer.
CN102515891A discloses a kind of microbial manure, the microbial fermentation solution comprising following ratio：Photosynthetic bacteria,
Lactic acid bacteria, actinomycetes, trichodermaharzianum bacterium, bacillus subtilis fermentation liquor volume ratio are 1:0.8-1:1.2-1.4:0.6-0.8:1-
1.2.The fertilizer can improve the utilization ratio of fertilizer, and also can play a part of to prevent and treat that plant is withered, disease.
As described above, the multiple-microorganism fertilizer comprising bacillus subtilises is disclosed in prior art, but for new
Microorganism Bacillus subtilis fertilizer and preparation method thereof, still suffer from the necessary and demand for continuing to study, this is currently micro- life
Study hotspot and emphasis in thing fertilizer field, the power that even more present invention is accomplished is located and basis is leaned on.
The content of the invention
In order to seek microorganism Bacillus subtilis fertilizer and preparation method thereof, present inventor has performed substantial amounts of deeply grind
Study carefully, after creative work has been paid, so as to complete the present invention.
Specifically, the present invention relates to following aspects.
One side, the present invention relates to a kind of preparation method of microorganism Bacillus subtilis fertilizer, methods described bag
Include following steps：
S1：Prepare first liquid culture medium；
S2：Prepare second liquid culture medium；
S3：Spawn culture obtains the first culture fluid；
S4：Continue culture and obtain the second culture fluid；
S5：Solid-substrate fermentation process, obtains the microorganism Bacillus subtilis fertilizer.
In the preparation method of the microorganism Bacillus subtilis fertilizer of the present invention, step S1 is specific as follows：
S1-1：Following base is weighed respectively：Trace element water-soluble liquid 40-50ml, Vitamin E 1-2g, threonine
300-400mg, L-PROLINE 200-300mg, glucose 3-4.5g, sucrose 2-4g, agar 5-6g, peptone 6-8g, yeast leaching
Cream 1.5-3g, 10-12g Semen sojae atricolor powder and Carnis Bovis seu Bubali cream 3-5g；
S1-2：Above-mentioned base is added in deionized water, is sufficiently stirred for, and deionized water is settled to 1000ml,
Obtain the first liquid culture medium.
Wherein, the trace element water-soluble liquid is by 4g zinc nitrates, 3.5g zinc sulfate, 3g magnesium nitrates, 1g cobaltous chlorides, 2g nitre
Sour copper, 2.5g calcium chloride, 4g manganese chlorides, 8g ferrous chlorides and 3g iron chloride are completely dissolved in 1000ml distilled water and obtain
's；Then 40-50ml therein is measured, the 40-50ml trace element water-soluble liquid as used in above-mentioned steps S1-1.
In the preparation method of the microorganism Bacillus subtilis fertilizer of the present invention, step S2 is specific as follows：
S2-1：With step S1-1；
Namely another name takes following base：Trace element water-soluble liquid 40-50ml, vitamin e1-2g, threonine 300-
400mg, L-PROLINE 200-300mg, glucose 3-4.5g, sucrose 2-4g, agar 5-6g, peptone 6-8g, yeast extract
1.5-3g, 10-12g Semen sojae atricolor powder and Carnis Bovis seu Bubali cream 3-5g；
S2-2：The base and 25-40ml inorganic matters mixed liquors are added in deionized water and are sufficiently stirred for, and is spent
It is 6.4-7.2 that ionized water is settled to 1000ml and adjusts pH value, that is, obtain the second liquid culture medium.
Wherein, the inorganic matters mixed liquor is by 5g Na2SO4、2g NaCl、12g K2HPO4、4g Na3PO4、7g
NH4Cl, 2.5g boric acid, 3g KNO3It is added in 1000ml distilled water with 5g KI and is sufficiently stirred for obtained from dissolving；Then measure
25-40ml inorganic matters mixed liquors used in 25-40ml therein, as above-mentioned steps S2-2.
Wherein, pH value is adjusted to into 6.4-7.2 in step S2-2, for example, can is 6.4,6.6,6.8,7 or 7.2, most preferably
For 6.8.
The pH value adjusts to those skilled in the art very conventional technique means, and here is no longer retouched in detail
State.
The inventors discovered that, when pH value is 6.8, best technique effect can be obtained, this should be withered under the pH value
Careless bacillus cereuss have optimum growth and breed environment.
And, the inventors discovered that, it is extra inorganic in step S2-2 when on the basis of the S1-1 of step S1, continuously adding
During thing mixed liquor, in can remarkably promoting and improving the culture fluid of bacillus subtilises second (the second culture fluid i.e. in step S4)
Viable bacteria content, this should be when adding inorganic matters mixed liquor, can further improve and promote on the basis of the first culture medium
The growth of bacillus subtilises and breeding (and then also improving final fertilizer performance) are entered, inventor is intended to carry out follow-up
Research.
In the preparation method of the microorganism Bacillus subtilis fertilizer of the present invention, step S3 is specific as follows：
Bacillus subtilis strain is carried out into slant tube culture, bacillus subtilises bacterium colony is obtained, then using inoculation
Ring connects 1 ring and is inoculated in first liquid culture medium described in 120ml, the vibration training under temperature T1 and rotating speed are for 140-160 rev/min
Foster 30-34 hours, so as to obtain first culture fluid.
Wherein, the slant tube culture is the routine techniquess in this area, and here is no longer described in detail.
Wherein, those skilled in the art may be selected any of bacillus subtilis strain, for example, can use and protect
The numbering for ensconcing Chinese agriculture Microbiological Culture Collection administrative center is ACCC10619 or the bacillus subtilis of ACCC03120 etc.
Bacteria strain, or be deposited in China Microbial Culture Preservation Commission's common micro-organisms center numbering be CGMCC No.0954,
The bacillus subtilis strain of CGMCC No.1019, CGMCC No.2099 etc., or can be bought with commercial channel acquisition it is many
Bacillus subtilis strain is planted, here is no longer described in detail.
Wherein, temperature T1 is 35-37 DEG C, for example, can be 35 DEG C, 36 DEG C or 37 DEG C.
In the preparation method of the microorganism Bacillus subtilis fertilizer of the present invention, step S4 is specific as follows：
According to 1.5-1.9:100 volume ratio, first culture fluid is inoculated in the second liquid culture medium, and
Temperature T2 is cooled to, is then stirred with 160-170 rev/min of speed and is divulged information (be passed through air), cultivate 18-22 hours,
So as to obtain second culture fluid, the cultural method is completed.
Wherein, temperature T2 is 28-32 DEG C, for example, can be 28 DEG C, 29 DEG C, 30 DEG C, 31 DEG C or 32 DEG C, and preferably T1-T2 is (i.e.
The difference of temperature T1 and temperature T2) it is 5-7 DEG C, most preferably 6 DEG C, i.e., most preferably T1-T2=6 DEG C.
Inventor has found, total viable count and spore that the difference of temperature T1 and T2 can be in the culture fluid of appreciable impact second
Rate, this should be that suitable temperature reduces that the numerous of strain can be further enhancing under the synergism of second liquid culture medium
Grow and cultivate one's ability, so as to achieve superior technique effect, also improve the performance of final microbial manure.
Wherein, the ventilating ratio of ventilation is 1:0.7-0.8, for example, can be 1:0.7、1:0.75 or 1:0.8.
In the preparation method of the microorganism Bacillus subtilis fertilizer of the present invention, step S5 is specific as follows：
S5-1：Weigh respectively 3 weight portion cottonseed meal, 1 weight portion Testa Tritici, 2 weight portion peanut cakes, 2.5 weight portion dry silkworm excrements, 10
The corn stalk powder of weight portion drying, 0.1 weight portion brown sugar, 0.6 weight portion analysis for soybean powder, 2 weight portion fishbone powder, 4 weight portions are done
Dry bagasse, 0.008 part by weight of cellulose enzyme, 1.8 weight portion Dry chick faeces, 0.1 amount part ammonium chloride, 1 weight portion molasses and
0.08 weight potassium fulvate, mix homogeneously, then 130-140 DEG C of high-temperature vapor fully sterilize, obtain base material；
S5-2：Drench into deionized water in the base material, and fully turn, until the biodiversity percentage composition of base material is
40-46%, obtains the solid medium；
S5-3：Under aseptic conditions, second culture fluid is inoculated in the solid medium, and at 36-40 DEG C
Under carry out solid fermentation culture 50-60 hours, it is always 43 ± 2% that period keeps biodiversity percentage composition, after the completion of fermentation
Obtain fermented product；The fermented product is carried out into hot air drying at 70-80 DEG C, until biodiversity percentage composition≤8%, cooling
To room temperature and 80 mesh sieves were crushed, that is, obtained the microorganism Bacillus subtilis fertilizer.
Wherein, in step S5-3, second culture fluid is 1 with the mass ratio of the solid medium:18-
24, for example can be 1:18、1:20、1:22 or 1:24.
Second aspect, the present invention relates to the microorganism Bacillus subtilis fertilizer according to obtained in above-mentioned preparation method
Material.
The preparation method of the present invention passes through unique multiple technical characteristics synergism each other and mutually promotes, from
And good technique effect is achieved, can provide with high activity, highdensity bacillus subtilis bacteria culture fluid, and then obtain
Microorganism Bacillus subtilis fertilizer with excellent properties, it is latent so as to industrially having a good application prospect and producing
Power.
3rd aspect, the present invention relates to purposes of the microorganism Bacillus subtilis fertilizer in agriculture field.
Find that the microorganism Bacillus subtilis fertilizer has superperformance through research, so as to can in agriculture field,
For example in the plantation and/or cultivation of crops, veterinary antibiotics, flowers or nursery stock etc. have a good application prospect and potentiality.
As described above, the invention provides a kind of microorganism Bacillus subtilis fertilizer and preparation method thereof, the preparation
Method is by being mutually combined between unique multiple technical characteristics and cooperates with, so as to obtain with excellent technique effect most
Whole microorganism Bacillus subtilis fertilizer, disclosure satisfy that the widespread demand of agricultural technology field, have a good application prospect and
Industrial production potential.
Specific embodiment
Below by specific embodiment, the present invention is described in detail, but the purposes of these exemplary embodiments and
Purpose is only used for enumerating the present invention, not constitutes any type of any restriction to the real protection scope of the present invention, more non-to incite somebody to action
Protection scope of the present invention is confined to this.
Preparation example 1：The preparation of trace element water-soluble liquid
By 4g zinc nitrates, 3.5g zinc sulfate, 3g magnesium nitrates, 1g cobaltous chlorides, 2g copper nitrates, 2.5g calcium chloride, 4g manganese chlorides,
8g ferrous chlorides and 3g iron chloride are completely dissolved in 1000ml distilled water, so as to obtain trace element water-soluble liquid.
Preparation example 2：The preparation of inorganic matters mixed liquor
By 5g Na2SO4、2g NaCl、12g K2HPO4、4g Na3PO4、7g NH4Cl, 2.5g boric acid, 3g KNO3And 5g
KI is added to stirring and dissolving in 1000ml distilled water, so as to obtain inorganic matters mixed liquor.
Preparation example 3：The preparation (i.e. step S5-1 and S5-2) of solid medium
S5-1：Weigh respectively 3 weight portion cottonseed meal, 1 weight portion Testa Tritici, 2 weight portion peanut cakes, 2.5 weight portion dry silkworm excrements, 10
The corn stalk powder of weight portion drying, 0.1 weight portion brown sugar, 0.6 weight portion analysis for soybean powder, 2 weight portion fishbone powder, 4 weight portions are done
Dry bagasse, 0.008 part by weight of cellulose enzyme, 1.8 weight portion Dry chick faeces, 0.1 amount part ammonium chloride, 1 weight portion molasses and
0.08 weight potassium fulvate, mix homogeneously, then 130-140 DEG C of high-temperature vapor fully sterilize, obtain base material；
S5-2：Drench into deionized water in the base material, and fully turn, until the biodiversity percentage composition of base material is
43%, obtain the solid medium.
Unless otherwise stated, following all of trace element water-soluble liquid, inorganic matters mixed liquor and solid medium are equal
Corresponding above-mentioned preparation example 1-3 is obtained.
Embodiment 1
S1：Prepare first liquid culture medium
S1-1：Following base is weighed respectively：Trace element water-soluble liquid 45ml, Vitamin E 1.5g, threonine
350mg, L-PROLINE 250mg, glucose 3.75g, sucrose 3g, agar 5.5g, peptone 7g, yeast extract 2.25g, 11g are big
Semen Glycines powder and Carnis Bovis seu Bubali cream 4g；
S1-2：Above-mentioned base is added in deionized water, is sufficiently stirred for, and deionized water is settled to 1000ml,
Obtain the first liquid culture medium；
S2：Prepare second liquid culture medium
S2-1：With step S1-1；
S2-2：The base and 32.5ml inorganic matters mixed liquors are added in deionized water and are sufficiently stirred for, and is spent
It is 6.8 that ionized water is settled to 1000ml and adjusts pH value, obtains the second liquid culture medium；
S3：Spawn culture obtains the first culture fluid
By bacillus subtilis strain, (numbering from Chinese agriculture Microbiological Culture Collection administrative center is
The bacillus subtilis strain of ACCC10619) slant tube culture is carried out, bacillus subtilises bacterium colony is obtained, then using connecing
Kind ring connects 1 ring and is inoculated in first liquid culture medium described in 120ml, under temperature T1 (being 36 DEG C) and rotating speed are for 150 revs/min
Shaken cultivation 32 hours, so as to obtain first culture fluid；
S4：Continue culture and obtain the second culture fluid
According to 1.7:100 volume ratio, first culture fluid is inoculated in the second liquid culture medium, and is lowered the temperature
To temperature T2 (being 30 DEG C), then being stirred with 160 revs/min of speed and divulged information, (ventilating ratio is 1:0.75), cultivate 20 hours,
So as to obtain second culture fluid (being named as P1)；
S5：Solid medium culture, obtains microorganism Bacillus subtilis fertilizer
S5-1 to S5-2：See above-mentioned preparation example 3；
S5-3：Under aseptic conditions, second culture fluid is inoculated in the solid medium (the second culture fluid with
The mass ratio of solid medium is 1:21) solid fermentation culture 55 hours, and at 38 DEG C is carried out, period keeps biodiversity hundred
Divide content to be always 43 ± 2%, fermented product is obtained after the completion of fermentation；The fermented product is carried out into hot air drying at 75 DEG C, directly
To biodiversity percentage composition≤8%, it is cooled to room temperature and crushed 80 mesh sieves, that is, obtains the microorganism Bacillus subtilis
Fertilizer, is named as F1.
Embodiment 2
S1：Prepare first liquid culture medium
S1-1：Following base is weighed respectively：Trace element water-soluble liquid 40ml, Vitamin E 2g, threonine 300mg,
L-PROLINE 300mg, glucose 3g, sucrose 4g, agar 5g, peptone 6g, yeast extract 3g, 10g Semen sojae atricolor powder and Carnis Bovis seu Bubali cream 5g；
S1-2：Above-mentioned base is added in deionized water, is sufficiently stirred for, and deionized water is settled to 1000ml,
Obtain the first liquid culture medium；
S2：Prepare second liquid culture medium
S2-1：With step S1-1；
S2-2：The base and 25ml inorganic matters mixed liquors are added in deionized water and are sufficiently stirred for, and spend from
It is 6.8 that sub- water is settled to 1000ml and adjusts pH value, obtains the second liquid culture medium；
S3：Spawn culture obtains the first culture fluid
By bacillus subtilis strain, (numbering from Chinese agriculture Microbiological Culture Collection administrative center is
The bacillus subtilis strain of ACCC10619) slant tube culture is carried out, bacillus subtilises bacterium colony is obtained, then using connecing
Kind ring connects 1 ring and is inoculated in first liquid culture medium described in 120ml, under temperature T1 (being 35 DEG C) and rotating speed are for 140 revs/min
Shaken cultivation 34 hours, so as to obtain first culture fluid；
S4：Continue culture and obtain the second culture fluid
According to 1.5:100 volume ratio, first culture fluid is inoculated in the second liquid culture medium, and is lowered the temperature
To temperature T2 (being 29 DEG C), then being stirred with 170 revs/min of speed and divulged information, (ventilating ratio is 1:0.7), cultivate 22 hours,
So as to obtain second culture fluid (being named as P2)；
S5：Solid medium culture, obtains microorganism Bacillus subtilis fertilizer
S5-1 to S5-2：See above-mentioned preparation example 3；
S5-3：Under aseptic conditions, second culture fluid is inoculated in the solid medium (the second culture fluid with
The mass ratio of solid medium is 1:18) solid fermentation culture 60 hours, and at 36 DEG C is carried out, period keeps biodiversity hundred
Divide content to be always 43 ± 2%, fermented product is obtained after the completion of fermentation；The fermented product is carried out into hot air drying at 70 DEG C, directly
To biodiversity percentage composition≤8%, it is cooled to room temperature and crushed 80 mesh sieves, that is, obtains the microorganism Bacillus subtilis
Fertilizer, is named as F2.
Embodiment 3
S1：Prepare first liquid culture medium
S1-1：Following base is weighed respectively：Trace element water-soluble liquid 50ml, Vitamin E 1g, threonine 400mg,
L-PROLINE 200mg, glucose 4.5g, sucrose 2g, agar 6g, peptone 8g, yeast extract 1.5g, 12g Semen sojae atricolor powder and beef
Cream 3g；
S1-2：Above-mentioned base is added in deionized water, is sufficiently stirred for, and deionized water is settled to 1000ml,
Obtain the first liquid culture medium；
S2：Prepare second liquid culture medium
S2-1：With step S1-1；
S2-2：The base and 40ml inorganic matters mixed liquors are added in deionized water and are sufficiently stirred for, and spend from
It is 6.8 that sub- water is settled to 1000ml and adjusts pH value, obtains the second liquid culture medium；
S3：Spawn culture obtains the first culture fluid
By bacillus subtilis strain, (numbering from Chinese agriculture Microbiological Culture Collection administrative center is
The bacillus subtilis strain of ACCC10619) slant tube culture is carried out, bacillus subtilises bacterium colony is obtained, then using connecing
Kind ring connects 1 ring and is inoculated in first liquid culture medium described in 120ml, under temperature T1 (being 37 DEG C) and rotating speed are for 160 revs/min
Shaken cultivation 30 hours, so as to obtain first culture fluid；
S4：Continue culture and obtain the second culture fluid
According to 1.9:100 volume ratio, first culture fluid is inoculated in the second liquid culture medium, and is lowered the temperature
To temperature T2 (being 31 DEG C), then being stirred with 160 revs/min of speed and divulged information, (ventilating ratio is 1:0.8), cultivate 18 hours,
So as to obtain second culture fluid (being named as P3)；
S5：Solid medium culture, obtains microorganism Bacillus subtilis fertilizer
S5-1 to S5-2：See above-mentioned preparation example 3；
S5-3：Under aseptic conditions, second culture fluid is inoculated in the solid medium (the second culture fluid with
The mass ratio of solid medium is 1:24) solid fermentation culture 50 hours, and at 40 DEG C is carried out, period keeps biodiversity hundred
Divide content to be always 43 ± 2%, fermented product is obtained after the completion of fermentation；The fermented product is carried out into hot air drying at 80 DEG C, directly
To biodiversity percentage composition≤8%, it is cooled to room temperature and crushed 80 mesh sieves, that is, obtains the microorganism Bacillus subtilis
Fertilizer, is named as F3.
The Performance of the second culture fluid
According to the traditional test methods in this area, the culture fluid of difference second obtained to embodiment 1-3 is being prepared
Afterwards, colony counting investigation is carried out at once, viable count and spore rate in per milliliter of (ml) fermented product have been investigated respectively, as a result see below
Table 1.
Table 1
As can be seen here, the hay bud with high viable count and high spore rate can be obtained in the step of preparation method of the present invention S4
The culture fluid of spore bacillus second, so as to prepare for the most excellent microbial manure of its subsequent step S5 good basis is provided.
Below to affecting the multiple of step S4 the second culture fluid performance and final microorganism Bacillus subtilis fertilizer performance
Technical characteristic is investigated.
Comparative example 1-6：The investigation of culture medium
Comparative example 1-3：Except the second liquid culture medium in S4 respectively by embodiment 1-3 replaces with first liquid culture medium
Outward (i.e. whole process cultivated using first liquid culture medium), other operations are constant, embodiment 1-3 so as to repetitive operation,
Comparative example 1-3 is respectively obtained, the culture fluid of gained second is sequentially named as into DP1, DP2 and DP3, and by the final hay bud of gained
Spore bacillus microorganisms fertilizer is sequentially named as DF1, DF2 and DF3.
Comparative example 4-6：Except the first liquid culture medium in S3 respectively by embodiment 1-3 replaces with second liquid culture medium
Outward (i.e. whole process cultivated using second liquid culture medium), other operations are constant, embodiment 1-3 so as to repetitive operation,
Comparative example 4-6 is respectively obtained, the culture fluid of gained second is sequentially named as into DP4, DP5 and DP6, and by the final hay bud of gained
Spore bacillus microorganisms fertilizer is sequentially named as DF4, DF5 and DF6.
Colony counting investigation is carried out to DP1-DP6 according to above-mentioned identical method, per milliliter (ml) fermentation has been investigated respectively
(unit is for viable count in thing：×109Cfu/ml) and spore rate, as a result see the table below 2.
Wherein, contrasted for convenience, the data of P1-P3 are together listed.
Table 2
Wherein, the data of P1-P3 carry out in that same order respectively correspondence, such as spore rate (%),
" 97.6,97.1,97.4 " mean P1 for 97.6, P2 be 97.1 and P3 be 97.4.The numbered corresponding relation of institute so (
It is also, with same implication, no longer to list one by one when the time comes in following all forms), this is no longer going to repeat them.
As can be seen here, in different step, the species of culture medium selects to be produced not final result with compositional selecting
Predictable impact；The viable count of DP4-DP6 will be significantly better than DP1-DP3, but spore rate is very nearly the same.This is further proved
Only successively using the unique first liquid culture medium and second liquid culture medium of the present invention, could obtain best technology and imitate
Really.
Comparative example 7-10：The investigation of pH in step S2-2
The pH value (other operations are constant) in step S2-2 shown in following table is respectively adopted, so as to enforcement is repeated respectively
Example 1-3, obtain comparative example 7-10 (by the final microorganism Bacillus subtilis fertilizer of gained be sequentially named as DF7, DF7, DF8,
DF9 and DF10), use the work of pH value, correspondence embodiment, the name of the second culture fluid of final gained and same procedure test
(unit is bacterium number：×109) and spore rate data see the table below 3 cfu/ml.Wherein, contrasted for convenience, by the number of P1-P3
According to together listing.
Table 3
Wherein, " -- " represents that the corresponding relation of embodiment 1-3 is respectively itself.
As can be seen here, pH value in step S2-2 adjusts extremely important, when for 6.8 when can obtain best technology and imitate
Really, the value is deviateed bigger, then viable count and spore rate reduce more obvious (see DP7-DP10), it is non-that this proves that the determination of pH value has
Apparent property, and effect is unexpected.
Comparative example 11-16：The investigation of T1-T2 differences in step S3
The difference (other operations are constant) of concrete T1, T2 and T1-T2 in step S3 shown in following table is respectively adopted, from
And embodiment 1-3 is repeated respectively, comparative example 11-16 is obtained, T1, T2 and T1-T2 used in it, correspondence embodiment and most
The eventually name of the second culture fluid of gained see the table below 4 (unit of wherein T1, T2 and T1-T2 be DEG C).
Table 4
(unit is to test viable count by the same way：×109Cfu/ml) and spore rate, data see the table below 5.Wherein,
Contrasted for convenience, the data of P1-P3 are together listed.
Table 5
As can be seen here, the difference of T1 and T2 equally has the impact of highly significant for final result in step S3, is on duty
Be worth for 6 DEG C when, can obtain best technique effect, and when for 5 DEG C or 7 DEG C when, then effect reduces obvious, and for same
Deviation value (1 DEG C), the viable count of DP11-DP13 will be significantly better than DP14-DP16, but spore rate is inferior to DP14-DP16, nothing
Method realizes optimum while viable count and spore rate, and this also demonstrates when only difference is 6 DEG C and could obtain best balance
Can, reach both optimal effectiveness.
As can be seen here, only with best effects can just be obtained using step S1-S4 in preparation method of the present invention
Two culture fluid, so as to lay a good foundation for the preparation of bacillus subtilises fertilizer in subsequent step S5, and ensure that it has most
Good technique effect (many data for seeing below are proved).
The Performance of final fertilizer
A, the investigation of colony counting
According to the traditional test methods in this area, the different bacillus subtilises obtained to each embodiment and comparative example
Fertilizer carries out colony counting investigation after preparing, at once, and viable count in every gram of fermented product has been investigated respectively, and (unit is：
Hundred million/gram) and spore rate, as a result see the table below 6.
Wherein, the numerical value of multiple fertilizer of identical group takes its average.
Table 6
As can be seen here：1st, the F1-F3 that the inventive method is obtained has very high viable count and spore rate；2 and ought make always
During with first liquid culture medium or second liquid culture medium, viable count and spore rate are significantly reduced, and are especially used always
Reduce becoming apparent from (see DF4-DF6) during second liquid culture medium；3rd, pH value in step S2-2 adjusts extremely important, when for 6.8
When can obtain best technique effect, it is bigger to deviate the value, then viable count and spore rate reduce more obvious (see DF7-DF10),
This proves that the determination of pH value has unobviousness, and effect is unexpected；4th, in step S3 the difference of T1 and T2 for most
Termination fruit equally has the impact of highly significant, when difference is 6 DEG C, can obtain best technique effect, and when for 5 DEG C or
When 7 DEG C, then effect reduces obvious, and for same deviation value (1 DEG C), the viable count of DF11-DF13 will be significantly better than
DF14-DF16, but spore rate is inferior to DF14-DF16, and this proves only have difference to obtain best balance when being 6 DEG C
Energy.
The investigation of B, heat-resisting quantity
Each embodiment and comparative example 40 minutes after different bacillus subtilises fertilizer are obtained, respectively in different temperature
Constant temperature is kept for 20 minutes under degree (70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C), then measures its viable count again, and with initial viable count
Calculated, so as to obtain high temperature after survival rate, as a result see the table below 7.
Wherein, multiple fertilizer of identical group take its average.
Table 7
As can be seen here：1st, the F1-F3 that the inventive method is obtained has excellent high temperature resistant property, and this is prevented certainly in storage
Body ferments and reduces active significant；2 and the fertilizer resistance to elevated temperatures of all comparative examples, especially higher than 80 DEG C
When there is significantly reduction, more particularly DF7-DF8 and DF11-DF16 reduces becoming apparent from, bacterium colony resistance to elevated temperatures now
Significantly reduce, this should be result in its flora and send out because the temperature gap in pH value change and step S3 in step S2 changes
Caused by educating existing defects.
The investigation of C, storage stability
By each embodiment and comparative example after different bacillus subtilises fertilizer are obtained, keep in dark place under room temperature certain
Time, and viable count when investigating different time respectively and calculated, so as to obtain during different storages with initial viable count
Between after survival rate, as a result see the table below 8.
Wherein, multiple fertilizer of identical group take its average.
Table 8
As can be seen here：1st, the F1-F3 that the inventive method is obtained has excellent storage stability, longer such that it is able to store
Time；2 and the storage stability of comparative example fertilizer then has significantly reduction, especially DF4-DF8 and DF11-DF13 is reduced most
For notable, this prove in cross-reference, step S2 of step the first and second fluid medium pH value select and step S3 in
Temperature gap changes can unpredictably affect final performance.
From the data in above-mentioned A-C, the microbial manure that the inventive method is obtained has excellent multiple performance,
Fertilizer efficiency, high temperature resistant and storage stability aspect can obtain best effect, so as to can be applicable to crops, veterinary antibiotics, flowers
Or nursery stock etc. plantation and/or cultivate, have a good application prospect and potentiality in agriculture field, be specifically shown in as follows.
Plantation culture Performance
I, corn planting volume increase are investigated
According to per 100 square metres as one piece of sample plot (corn variety be good beautiful 99), be divided into matched group, chemical group,
Microbial manure group, it is specific as follows：
Matched group：Any fertilizer is not used.
Chemical group：Apply normal chemical fertilizer, i.e. N, P, K fertile, 5kg ammonium chloride, 5kg potassium sulfate and 3kg phosphorus can be used
Sour ammonium is applied fertilizer.
Microbial manure group：Respectively using the microbial manure of the 5kg embodiment of the present invention or comparative example, (each sample plot is only
Using a kind of microbial manure), and N, P, K of the same ratio of the amount of additional chemical group 1/4 are fertile.
According to normal field management and after results, per mu yield yield is converted to, as a result see the table below 9.
Table 9
As can be seen here, microbial manure F1-F3 of the invention has effect of increasing production the most excellent, increases relative to chemical group
Produce>10%, although other contrast fertilizer will be significantly below F1-F3, but still chemical group is significantly better than, also achieve good
Effect of increasing production.
II, Cymbidium ensifolium (L.) Sw. cultivation are investigated
Collect fertile soil (organic matter 2.9%, nitrate nitrogen 187.2mg/kg, available phosphoruss 100.3mg/kg, effective potassium
87.5mg/kg, pH value is that matched group simply uses the base soil, and microbial manure group is then used respectively 6.2) as base soil is cultivated
Mass ratio 10:1 base soil and a kind of mixture (every plant of Cymbidium ensifolium (L.) Sw. only uses microbial manure) of each microbial manure, enter
The same cultivation mode of row and daily management.
Per 2 days or 3 days record its growth traits after the cultivation of Cymbidium ensifolium (L.) Sw. seedling, investigate respectively after 30 days, 60 days and 140 days
Growthform (index such as such as leaf color and thickness, whole height, healthy and strong degree), as a result see the table below 10.
Table 10
Wherein, with the growthform of matched group benchmark as a comparison, comprehensive morphological is defined as into "+", then by microorganism
Fertilizer group is contrasted with it, and according to the degree for improving " ++ " or " +++ " is defined as, i.e. the excellent degree of growthform is：+
++>++>+。
As can be seen here, there is F1-F3 of the invention optimal form to improve, enable to that Cymbidium ensifolium (L.) Sw. is more healthy and strong, blade more
Plump, color is more blackish green, and other contrast microbial manure prolongations then over time, for the improvement of form is less than F1-
F3, especially DF4-DF8, at the 60th day F1-F3 has been worse than.
In sum, the invention provides a kind of microorganism Bacillus subtilis fertilizer and preparation method thereof, the preparation
Method is by being mutually combined between unique multiple technical characteristics and cooperates with, so as to obtain with excellent technique effect most
Whole microorganism Bacillus subtilis fertilizer, disclosure satisfy that the widespread demand of agricultural technology field, have a good application prospect and
Industrial production potential.
It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to limit the protection model of the present invention
Enclose.Additionally, it will also be appreciated that after the technology contents for having read the present invention, those skilled in the art can make each to the present invention
Plant and change, change and/or modification, all these equivalent form of value equally falls within the guarantor that the application appended claims are limited
Within the scope of shield.
Claims (10)
1. a kind of preparation method of microorganism Bacillus subtilis fertilizer, methods described comprises the steps：
S1：Prepare first liquid culture medium；
S2：Prepare second liquid culture medium；
S3：Spawn culture obtains the first culture fluid；
S4：Continue culture and obtain the second culture fluid；
S5：Solid-substrate fermentation process, obtains the microorganism Bacillus subtilis fertilizer.
2. preparation method as claimed in claim 1, it is characterised in that：Step S1 is specific as follows：
S1-1：Following base is weighed respectively：Trace element water-soluble liquid 40-50ml, Vitamin E 1-2g, threonine 300-
400mg, L-PROLINE 200-300mg, glucose 3-4.5g, sucrose 2-4g, agar 5-6g, peptone 6-8g, yeast extract
1.5-3g, 10-12g Semen sojae atricolor powder and Carnis Bovis seu Bubali cream 3-5g；
S1-2：Above-mentioned base is added in deionized water, is sufficiently stirred for, and deionized water is settled to 1000ml, obtains final product
To the first liquid culture medium.
3. preparation method as claimed in claim 2, it is characterised in that：Wherein, the trace element water-soluble liquid is by 4g nitric acid
Zinc, 3.5g zinc sulfate, 3g magnesium nitrates, 1g cobaltous chlorides, 2g copper nitrates, 2.5g calcium chloride, 4g manganese chlorides, 8g ferrous chlorides and 3g chlorine
Change ferrum to be completely dissolved in obtained from 1000ml distilled water.
4. the preparation method as described in any one of claim 1-3, it is characterised in that：Step S2 is specific as follows：
S2-1：With step S1-1；
S2-2：The base and 25-40ml inorganic matters mixed liquors are added in deionized water and are sufficiently stirred for, and uses deionization
Water is settled to 1000ml and adjusts pH value for 6.4-7.2, and most preferably 6.8, that is, obtain the second liquid culture medium.
5. preparation method as claimed in claim 4, it is characterised in that：Wherein, the inorganic matters mixed liquor is by 5g Na2SO4、
2g NaCl、12g K2HPO4、4g Na3PO4、7g NH4Cl, 2.5g boric acid, 3g KNO31000ml distilled water is added to 5g KI
In be sufficiently stirred for dissolving obtained from.
6. the preparation method as described in any one of claim 1-5, it is characterised in that：Step S3 is specific as follows：
Bacillus subtilis strain is carried out into slant tube culture, bacillus subtilises bacterium colony is obtained, then 1 is connect using inoculating loop
Ring is inoculated in first liquid culture medium described in 120ml, in temperature T1 and rotating speed be 140-160 rev/min of lower shaken cultivation 30-
34 hours, so as to obtain first culture fluid；
Wherein, temperature T1 is 35-37 DEG C.
7. the preparation method as described in any one of claim 1-6, it is characterised in that：Step S4 is specific as follows：
According to 1.5-1.9:100 volume ratio, first culture fluid is inoculated in the second liquid culture medium, and is lowered the temperature
To temperature T2, then stirred with 160-170 rev/min of speed and divulged information (be passed through air), cultivate 18-22 hours, so as to
Obtain second culture fluid；
Wherein, temperature T2 is 28-32 DEG C, and preferably T1-T2 is 5-7 DEG C, most preferably 6 DEG C, i.e., most preferably T1-T2=6 DEG C.
8. the preparation method as described in any one of claim 1-7, it is characterised in that：Step S5 is specific as follows：
S5-1：3 weight portion cottonseed meal, 1 weight portion Testa Tritici, 2 weight portion peanut cakes, 2.5 weight portion dry silkworm excrements, 10 weight are weighed respectively
The dry corn stalk powder of part, 0.1 weight portion brown sugar, 0.6 weight portion analysis for soybean powder, 2 weight portion fishbone powder, 4 weight portions are dried
Bagasse, 0.008 part by weight of cellulose enzyme, 1.8 weight portion Dry chick faeces, 0.1 amount part ammonium chloride, 1 weight portion molasses and 0.08
Weight potassium fulvate, mix homogeneously, then 130-140 DEG C of high-temperature vapor fully sterilize, obtain base material；
S5-2：Drench into deionized water in the base material, and fully turn, until the biodiversity percentage composition of base material is 40-
46%, obtain the solid medium；
S5-3：Under aseptic conditions, second culture fluid is inoculated in the solid medium, and is entered at 36-40 DEG C
Row solid fermentation culture 50-60 hours, it is always 43 ± 2% that period keeps biodiversity percentage composition, is obtained after the completion of fermentation
Fermented product；The fermented product is carried out into hot air drying at 70-80 DEG C, until biodiversity percentage composition≤8%, is cooled to room
Temperature simultaneously crushed 80 mesh sieves, that is, obtain the microorganism Bacillus subtilis fertilizer.
9. the microorganism Bacillus subtilis fertilizer that the preparation method according to any one of claim 1-9 is prepared.
10. microorganism Bacillus subtilis fertilizer purposes in agriculture field described in claim 9.
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