CN106883998A - A kind of preparation method of microorganism multifunctional fertilizer is constituted with fermentation medium - Google Patents
A kind of preparation method of microorganism multifunctional fertilizer is constituted with fermentation medium Download PDFInfo
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- CN106883998A CN106883998A CN201710179484.4A CN201710179484A CN106883998A CN 106883998 A CN106883998 A CN 106883998A CN 201710179484 A CN201710179484 A CN 201710179484A CN 106883998 A CN106883998 A CN 106883998A
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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
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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
- 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
- C05G5/00—Fertilisers characterised by their form
- C05G5/20—Liquid fertilisers
- C05G5/23—Solutions
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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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Abstract
Preparation method the invention discloses a kind of microorganism multifunctional fertilizer is constituted with fermentation medium, belongs to microbial manure preparing technical field, more particularly to a kind of preparation method of microorganism fertilizer.The preparation method of microorganism multifunctional fertilizer includes following operating procedure：（1）Culture medium is prepared；（2）Actication of culture；（3）It is prepared by liquid seeds；（4）Liquid state fermentation；（5）It is prepared by seed dressing fertilizer, foliar fertilizer；（6）It is prepared by ground leaven.Fermentation medium is constituted：Glucose 15000g, peptone 10000g, yeast extract 5000g, KH2PO41300g, K2HPO41000g, MgSO4·7H2O 500g, MnSO4·H2O 100g, FeSO4·7H2O 100g, CaC12·2H2O 1500g, add water to 1000L, pH 7.0 7.5.The preparation method is simple to operate, low cost, obtained microorganism fertilizer can controlling plant diseases, can promote plant growth again, reduce the consumption of chemical fertilizer and agricultural chemicals, can effectively solve the problems such as pollution by pesticides, hardening soil.
Description
Technical field
The present invention relates to microbial manure preparing technical field, and in particular to a kind of preparation method of microorganism multifunctional fertilizer
Constituted with fermentation medium.
Background technology
China human mortality is more, and per capita area of cultivated farmland is few, improves crop yield significant.Soil application is a kind of normal
Yield Increase Way.At present, the fertilizer for agriculturally using includes microbial manure and chemical fertilizer.Chronic administration chemical fertilizer meeting
Cause soil hardening；And " green ", the health idea of " environmental protection " are currently based on, people are to pollution-free, safety, the green food of health
The demand of product increasingly increases so that microbial manure enjoys favor.In addition, applying for chemical fertilizer can be reduced using microbial manure
With, improve soil, improve quality of agricultural product；And microbial manure have it is long-acting, nontoxic, pollution-free, economize on resources, low cost
The features such as, the shortcoming and defect of chemical fertilizer can be made up.
Ground leaven is the important channel of plant absorption nutritional ingredient, and foliar fertilizer is the important benefit of plant absorption nutritional ingredient
Fill.With the aggravation that various pest and disease damages in growing process influence, if energy antagonism is planted again using that can promote plant growth
The microbial manure of thing disease, is beneficial to the increasing both production and income of plant, crop, improves household economy interests.Therefore, it can utilize
The strain excellent of research and development, is fermented, the tunning of acquisition, is prepared into multi-functional micro- lifes such as ground leaven, foliar fertilizer, seed dressing fertilizer
Thing fertilizer.
The content of the invention
Bacillus subtilis is applied the present invention relates to one kind（Bacillus subtilis）BS501a prepares microorganism fertilizer
The culture medium prescription of method and fermenting and producing microorganism fertilizer, obtained microorganism multifunctional fertilizer can controlling plant diseases, and energy
Promote plant growth, solve problems of the prior art.
The present invention is achieved through the following technical solutions：
A kind of preparation method of microorganism multifunctional fertilizer, including following operating procedure：
（1）Culture medium is prepared：Strain activation and culture base, seed culture medium and fermentation medium, high-temperature sterilization standby are prepared respectively
With；
The strain activation and culture base is：Peptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1L, pH7.0-7.5；
The seed culture medium is：Peptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1L, pH7.0-7.5；
The fermentation medium is：Glucose 15000g, peptone 10000g, yeast extract 5000g, KH2PO41300g,
K2HPO41000g, MgSO4·7H2O 500g, MnSO4·H2O 100g, FeSO4·7H2O 100g, CaC12·2H2O
1500g, adds water to 1000L, pH 7.0-7.5；
（2）Actication of culture：Picking slant preservationBacillus subtilisBS501a is inoculated in step（1）Obtained strain
In activation medium, in 35-37 DEG C, 180-220r/min shaking table shaken cultivation 16-24h must activate bacillus subtilis bacterium solution；
（3）It is prepared by liquid seeds：By 5%-10%（Percentage by volume）Inoculum concentration by step（2）The bacillus subtilis of activation
Bacterium solution is inoculated in step（1）In obtained seed culture medium, in 35-37 DEG C, lead to filtrated air culture 16-24h, obtain liquid strain
Son；
（4）Liquid state fermentation：By 5%-10%（Percentage by volume）Inoculum concentration by step（3）Obtained liquid seeds are inoculated in
Step（1）In obtained fermentation medium, in 30 DEG C, lead to filtrated air culture 48h, through count plate, viable count up to 1 ×
109 - 9×109CFU/mL, obtains final product thalline culture living；
（5）It is prepared by foliar fertilizer and seed dressing fertilizer：By step（4）Obtained thalline culture living stands, bacterial sediment.Remove thalline,
Obtain final product microorganism foliage fertilizer and seed dressing fertilizer；
（6）The preparation of ground leaven：By step（4）The thalline culture aeration-drying living of acquisition, that is, obtain ground leaven.
The application method of mentioned microorganism multifunctional fertilizer：When seed dressing is used, seed dressing fertilizer 100 times of dilutions of water, seed dressing is used；
When foliage spray is used, foliar fertilizer 100 times of dilutions of water are sprayed in crops seedling stage foliage-spray, fruit phase；Ground leaven is in cultivated
Ground is sowed when planting.
The beneficial effects of the invention are as follows：
1st, bacillus subtilis of the present invention has promotion plant growth and antagonistic phytopathogen function concurrently, and gained is prepared through its fermentation
Microorganism multifunctional fertilizer have concurrently promote plant growth, preventing and treating plant leaf surface disease and fruit disease double action, can reduce
Chemical fertilizer and pesticide dosage, improve soil, improve farm environment quality, improve the quality of agricultural product.Bacillus subtilis of the present invention
Can successfully colonize in plant rhizosphere, body surface or internal, secretion antibacterial material suppresses growth of pathogenic bacteria, with biocontrol effect, to many
Planting plant, crop disease fungal pathogenses has very strong inhibitory action；
2nd, in preparation method of the present invention, fermentation medium can lift the quantity of bacillus subtilis in tunning；By micro-
Biological metabolism, improves the secretory volume of antagonism metabolite, while viable bacteria body promoting growth of plants is given full play to, can guarantee that and is planting
Effective biological bactericide effect is played in the whole growth cycle of thing or crop, strengthens plant or crop disease-resistant ability.Culture
Nitrogen, phosphorus, potassium, and various mineral matters are rich in base, soil fertility can be strengthened, reduce soil applying fertilizer amount.
Specific embodiment
The optimization of the orthogonal experiment method of embodiment 1 obtains fermentation medium
To improveB. subtilisThe antagonistic ability of BS501a tunnings, using single factor experiment and optimization of orthogonal test
Fermentation medium is constituted and fermentation condition, is obtained optimal medium composition and is：Glucose 1.5%, peptone 1%, yeast extract 0.5%,
KH2PO4 0.13%, K2HPO4 0.1%, MgSO4·7H2O 0.05%, MnSO4·H2O 0.01%, FeSO4·7H2O 0.01%
, CaC12·2H2O 0.15%, pH 7.0；Most suitable 30 DEG C of fermentation temperature, most suitable fermentation time 48h.
The bacillus subtilis BS501a zymotic fluid antimicrobial spectrums of embodiment 2
The antimicrobial spectrum of bacillus subtilis BS501a is surveyed using dual test.Because the plant pathogenic fungi speed of growth is different, it is
The accurate antagonistic activity for judging bacillus subtilis BS501a, using bacillus subtilis BS501a and plant pathogenic fungi by stages
Or the method for same period culture.Plant pathogenic fungi to growing speed, it is first withered in diagonal inoculation on fresh PDA flat board
Careless bacillus BS501a, after 26 DEG C of culture 24h, test fungal bacteria cake is moved into flat board center, then continues to cultivate 48h, is surveyed
Amount antagonism bandwidth.The plant pathogenic fungi medium to the speed of growth, using in fresh PDA flat board center inoculation test fungal
Bacteria cake, while being inoculated with bacillus subtilis BS501a bacterial strains in diagonal around bacteria cake, 26 DEG C of culture 72h measure antagonism bandwidth
Degree.To growing slow plant pathogenic fungi, using in fresh PDA flat board center inoculation test fungal bacteria cake, 26 DEG C of trainings
After supporting 24h, bacillus subtilis BS501a is inoculated with diagonal on flat board, continues to cultivate 72h, measure antagonism bandwidth.Knot
Fruit is as shown in table 1.
The bacillus subtilis BS501a antimicrobial spectrums of table 1
| Test fungal | Antagonism bandwidth/mm |
| Pyricularia oryzae | （7.4±0.23）a |
| Cucumber sickle-like bacteria | （6.98±0.21）a |
| Standing grain life is rotten mould | （3.06±0.15）b |
| Withered germ of water-melon | （5.25±0.34）c |
| Capsicum wilt bacterium | （4.73±0.30）c |
| Corn Fusorium moniliforme Sheldon | （3.50±0.21）d |
| Corn Green Wilt Disease | （4.25±0.16）cd |
| Cochliobolus sativus | （8.58±0.47）a |
| Southern corn leaf blight | （7.18±0.32）b |
| Fusarium graminearum | （5.38±0.36）c |
| Valsa mali | （5.00±0.20）c |
| Botryosphaeria berengeriana f. sp | （4.72±0.38）c |
| Botrytis cinerea pers | （8.03±0.71）ab |
| Rhizoctonia cerealis | （9.10±0.12）a |
| Tomato early blight bacterium | （8.65±0.43）ab |
| Cotton-wilt fusarium | （7.10±0.21）bc |
| Verticillium dahliae | （7.25±0.09）b |
Note：English alphabet is identical to represent that difference is not notable, differs expression significant difference.
With antagonism bandwidth 4.25mm as critical point, antagonism bandwidth is strong inhibitory action, antagonism bandwidth more than 4.25mm
Degree is that inhibitory action is weak less than 4.25mm.Dual test result shows Bacillus subtillis BS501a zymotic fluids to wheat sharp eyespot
Bacterium, Cochliobolus sativus, fusarium graminearum, tomato early blight bacterium, botrytis cinerea pers, cucumber sickle-like bacteria, cotton wilt
Bacterium, verticillium dahliae, capsicum wilt bacterium, Rhizoctonia solani Kuhn, southern corn leaf blight, withered germ of water-melon, apple decay
Germ, Botryosphaeria berengeriana f. sp inhibitory action it is strong；To corn Ralstonia solanacearum, corn Fusarium moniliforme, the rotten mould inhibitory action of standing grain life
It is weaker.
The Bacillus subtillis BS501a zymotic fluids biological safety of embodiment 3 is determined
According to pharmacopeia, the biological safety of Bacillus subtillis BS501a zymotic fluids is detected using acute toxicity testing.By withered grass
Bacillus BS501a zymotic fluid freeze-dryings, are made freeze-dried powder.Freeze-dried powder physiological saline is diluted to 5 × 10 successively-2g/
ML, 5 × 10-3G/mL, 5 × 10-4G/mL, 5 × 10-5G/mL, 5 × 10-6The various concentrations such as g/mL.By 48 3 week old kunming mices
The nursing observation of 7d is carried out, mouse free choice feeding, drinking-water in the observation period.After observation period terminates, mouse is randomly divided into 6 groups, often
Group 8.Fasting before mouse administration, but can't help water.After overnight, weigh.5 groups of mouse are taken, according to body weight, every its mouse oral gavage
0.2ml Bacillus subtillis BS501a zymotic fluid freeze-dried powder solution, freezes 5 groups of mouse Bacillus subtillis BS501a zymotic fluids
The dosage of dry powder is respectively 500mg/kg, 50 mg/kg, 5 mg/kg, 0.5 mg/kg, 0.05 mg/kg.6th group of mouse is set to
Blank control group, oral gavage 0.2ml physiological saline.2h feeds, Continuous Observation 7d are spaced after administration.Observed in feeding process small
Mouse outward appearance, spiritual expression, feeding situation, changes of weight.Dissected after 7d, observe internal organ pathological change.
According to State Bureau of Technical Supervision GB15670-1995 acute oral toxicity grade scales, Bacillus subtillis BS501a
The LD50 values of zymotic fluid freeze-dried powder>500 mg/kg, it was demonstrated that belong to low toxicity level, meet green bio agricultural chemicals requirement.
The Bacillus subtillis potted plant prevention effect of BS501a zymotic fluids of embodiment 5
By paddy rice cultivate 3 weeks or so, whne most of paddy rice it is long to three leaves wholeheartedly when, using leaf from watering can to paddy rice and stem sprinkling rice
Seasonal febrile diseases bacterium spore suspension 5mL（The Tween 80 for plus 0.5%）, after sprinkling 24h, in foliage spray bacillus subtilis BS501a fermentations
Clear liquid（The Tween 80 for plus 0.5%）Paddy rice is prevented and treated, once a day, each 5mL.Control is processed with deionized water.28 DEG C of bars
Under part, 100% illumination 14h, dark 10h alternate cultures 6d（The timing since magnaporthe grisea spore suspension is sprayed）, investigation paddy rice dye
State of an illness condition.Investigation grade scale is in units of blade（IRRI）：
0 grade is disease-free
1 grade is that leaf spot lesion is less than 5, and length is less than 1cm
3 grades is leaf spot lesion 6-10, and part scab length is more than 1cm
5 grades is leaf spot lesion 11-25, is partly linked to be piece, accounts for area 10%-25%
7 grades are leaf spot lesion more than 26, are partly linked to be piece, account for area 26%-50%
9 grades are linked to be piece for leaf spot lesion, account for blade area more than 50% or all withered.
After sprinkling magnaporthe grisea spore suspension 2-3d, just it can be seen that Pyricularia oryzae infects vestige on rice leaf.Not
The rice leaf for spraying bacillus subtilis BS501a fermentation supernatants is infected significantly by Pyricularia oryzae, has bright on most of blade
Aobvious rice blast scab, and number is more, area is big, and also part scab has diffused to whole blade, and the blade caught an illness is obvious
Jaundice, as burning.The paddy rice of bacillus subtilis BS501a fermentation supernatants is sprayed, most of blade keeps bud green
Color, small part is infected, and leaf spot lesion number is few, area is small.Specific disease index and prevent and treat efficiency and be shown in Table 2.
Table 2 bacillus subtilis BS501a fermentation supernatants prevent and treat efficiency
Note：
Growth promoting function of the bacillus subtilis BS501a zymotic fluids of embodiment 6 to paddy rice
Healthy seed rice is immersed in 100 times of bacillus subtilis fermentation liquors of dilution after 24h, is sowed in plastics yarn disk, table
Face uniform fold spun yarn, simulates natural lighting in multiband incubator, cultivates nursery, optionally adds clear water along yarn disk edge.
Each treatment 100.Control Seed soaking.When paddy rice it is long to two leaves wholeheartedly when survey the fresh weight of germination percentage and rice shoot.As a result table
Bright, compared with the control, rice shoot fresh weight improves 15.93%, but fermentation after bacillus subtilis BS501a zymotic fluid immersion treatments
Liquid does not influence on the germination percentage of rice paddy seed.
Claims (2)
1. a kind of preparation method of microorganism multifunctional fertilizer is constituted with fermentation medium, it is characterized by：Including following operating procedure：
（1）Culture medium is prepared：Strain activation and culture base, seed culture medium and fermentation medium, high-temperature sterilization standby are prepared respectively
With；
The strain activation and culture base is：Peptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1L, pH 7.0-7.5；
The seed culture medium is：Peptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1L, pH 7.0-7.5；
The fermentation medium is：Glucose 15000g, peptone 10000g, yeast extract 5000g, KH2PO41300g, K2HPO4
1000g, MgSO4·7H2O 500g, MnSO4·H2O 100g, FeSO4·7H2O 100g, CaC12·2H2O 1500g, add water
To 1000L, pH 7.0-7.5；
（2）Actication of culture：The bacillus subtilis of picking slant preservation（Bacillus subtilis）BS501a is inoculated in step
（1）In obtained strain activation and culture base, in 35-37 DEG C, 180-220r/min shaking table shaken cultivation 16-24h must activate withered grass
Bacillus bacterium solution；
（3）It is prepared by liquid seeds：By 5%-10%（Percentage by volume）Inoculum concentration by step（2）The bacillus subtilis of activation
Bacterium solution is inoculated in step（1）In obtained seed culture medium, in 35-37 DEG C, blowing air culture 16-24h obtains liquid seeds；
（4）Liquid state fermentation：By 5%-10%（Percentage by volume）Inoculum concentration by step（3）Obtained liquid seeds are inoculated in step
Suddenly（1）In obtained fermentation medium, in 30 DEG C, lead to filtrated air culture 48h, through count plate, viable count up to 3 ×
109-9×109CFU/mL, obtains final product thalline culture living；
（5）It is prepared by foliar fertilizer and seed dressing fertilizer：To step（4）Obtained thalline culture living stands, and bacterial sediment removes thalline,
Obtain final product microorganism foliage fertilizer and seed dressing fertilizer；
（6）The preparation of ground leaven：By step（4）The thalline culture aeration-drying living of acquisition, that is, obtain ground leaven.
2. fermentation medium according to claim 1 is constituted, it is characterized in that：Glucose 15000g, peptone 10000g, ferment
Female cream 5000g, KH2PO41300g, K2HPO41000g, MgSO4·7H2O 500g, MnSO4·H2O 100g, FeSO4·7H2O
100g, CaC12·2H2O 1500g, add water to 1000L, pH 7.0-7.5.
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Cited By (1)
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Effective date of registration: 20211210 Address after: 047600 Huang Ya Dong Zhen Shui Yi Cun, Licheng County, Changzhi City, Shanxi Province Patentee after: Licheng Xinwang organic fertilizer Co.,Ltd. Address before: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Patentee before: Liu Jiaojiao Effective date of registration: 20211210 Address after: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Patentee after: Liu Jiaojiao Address before: 450001 science and Technology Department of Henan University of technology, No.100 Lianhua street, Zhengzhou high tech Industrial Development Zone, Henan Province Patentee before: He'nan University of Technology |