CN102701831A - Microbial fertilizer and preparation method thereof - Google Patents
Microbial fertilizer and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a microbial fertilizer and a preparation method thereof. The fertilizer after being applied to soil can improve the soil fertility, reduce the usage amount of chemical fertilizers and increase the crop yield. The microbial fertilizer is prepared by mixing azotobacter chroococcum strain X11, a fermentation broth of bacillus cereus strain B81, turfy soil and diatomaceous earth. The preparation method comprises the following steps: (1) activating bacteria; (2) preparing a seed liquid and fermenting in a fermentation tank; (3) preparing a culture medium; (4) inoculating and culturing the bacteria; (5) fermenting in another fermentation tank; (6) disinfecting the turfy soil and the diatomaceous earth; (7) mixing the bacteria liquid with the adsorbents to obtain a living bacteria dry powder preparation of the microbial fertilizer. The microbial fertilizer can be used in agriculture, flower cultivation, maize cultivation, vegetable cultivation, potato cultivation and wheat cultivation, has positive agricultural applicability, and is simple, low in cost and easy to implement.
Description
Technical field
The present invention relates to belong to biotechnology, production in light industry technical field, relate in particular to a kind of microbial fertilizer and preparation method thereof.
Background technology
The usage quantity of chemical fertilizer and agricultural chemicals increases year by year in the agriculture prodn, causes the problems such as degeneration, the deterioration of the ecological environment of soil, and the Sustainable development of the safety of agricultural-food and agricultural is constituted a threat to and challenges.Microbial fertilizer is to make farm crop obtain the products similar of specific fertilizer effect by the microbial life activity; Be that effective mushroom and sorbing material are mixed the composite bio-fertilizer of processing; The use of microbial fertilizer can effectively reduce the use of chemical fertilizer and agricultural chemicals, can promote farm crop growth, increase output and improve crops quality.
Summary of the invention
Goal of the invention of the present invention will provide a kind of preparation microbial fertilizer and preparation method thereof exactly, in this incorporation of fertilizerin the soil after, can increase soil fertility, reduce chemical fertilizer use, improve the output of farm crop.
The present invention obtains blown-ball Azotobacter X11 bacterial strain and bacillus cereus B81 bacterial strain by separating in the soil of area, Qingdao.The present invention adopts Ah Xu shellfish solid medium dull and stereotyped :/1000 milliliters of glucose 10 grams ,/1000 milliliters of potassium hydrogenphosphate 0.2 grams ,/1000 milliliters of sal epsom 0.2 grams; / 1000 milliliters of sodium-chlor 0.2 grams; / 1000 milliliters of calcium sulfate 0.2 grams ,/1000 milliliters of lime carbonate 2 grams ,/1000 milliliters of agar 15-20 grams; PH7.0-7.2 sterilized 30 minutes for 112 ℃-115 ℃; With the even spreading of rich soil small-particle on this Ah Xu shellfish culture medium flat plate; Cultivated 48-72 hour down at 28 ℃ again; When treating to grow around the ped muddiness, translucent gluey bacterium colony, picking colony is done line separation, picking list bacterium colony on Ah Xu shellfish culture medium flat plate; The purifying of on Ah Xu shellfish flat board, ruling again is up to obtaining purebred blown-ball Azotobacter X11 bacterial strain.
This bright employing nutrient agar medium solid medium flat board :/1000 milliliters of Carnis Bovis seu Bubali cream 3 grams ,/1000 milliliters of peptone 10 grams ,/1000 milliliters of sodium-chlor 5 grams ,/1000 milliliters of agar 15-20 grams, pH7.4-7.6 sterilized 20-30 minute for 121 ℃; Rich soil is done 10 times of serial dilutions with saline water; Each extent of dilution is got the 0.1-0.2 milliliter and is coated on this nutrient agar medium solid medium flat board; Cultivated 36-48 hour down at 37 ℃ again; Picking list bacterium colony, the purifying of on nutrient agar plate, ruling again is up to obtaining purebred bacillus cereus B81 bacterial strain.
Task of the present invention is accomplished by following technical scheme:
A kind of microbial fertilizer, this microbial fertilizer are by blown-ball Azotobacter X11 bacterial strain and bacillus cereus B81 bacterial strain, add what sorbent material turfy soil and zeyssatite were prepared from, and the viable count of two kinds of bacteriums in microbial fertilizer is >=2 hundred million viable bacteria/grams;
A kind of making method of microbial fertilizer may further comprise the steps successively:
1) activation of bacterial classification calibrating:
The freeze-drying lactobacillus of aseptic unlatching blown-ball Azotobacter X11 bacterial strain; Be inoculated in Ah Xu the shellfish liquid tube substratum; Cultivated 18-72 hour at 20 ℃-45 ℃, streak inoculation is dull and stereotyped in Ah Xu shellfish solid medium then, cultivates 18-72 hour at 20 ℃-45 ℃; Choose colonies typical, carry out the calibrating of morphology and characteristic; The freeze-drying lactobacillus of aseptic unlatching bacillus cereus B81 bacterial strain; Be inoculated in the nutrient broth liquid tube substratum; Cultivated 18-48 hour at 20 ℃-45 ℃, streak inoculation is dull and stereotyped in the nutrient agar medium solid medium then, cultivates 18-48 hour at 20 ℃-45 ℃; Choose colonies typical, carry out the calibrating of morphology and characteristic;
2) preparation of seed liquor:
Pure growth with the blown-ball Azotobacter X11 bacterial classification of assay approval is inoculated in Ah Xu the shellfish liquid nutrient medium, 20 ℃-45 ℃ shake-flask culture 18-72 hour, carry out pure bacterium and bacterium property flag check; Pure growth with the bacillus cereus B81 bacterial strain bacterial classification of assay approval is inoculated in the nutrient broth liquid nutrient medium, 20 ℃-45 ℃ shake-flask culture 18-48 hour, carry out pure bacterium and bacterium property flag check;
3) preparing culture medium:
Ah Xu shellfish substratum: glucose, potassium hydrogenphosphate, sal epsom, sodium-chlor, calcium sulfate, lime carbonate are added in the zero(ppm) water, and above-mentioned each component is prepared by the component of the contained weight (g) of 1000 milliliters solution in zero(ppm) water :/1000 milliliters of glucose 5-15 grams ,/1000 milliliters of potassium hydrogenphosphate 0.1-0.4 grams; / 1000 milliliters of sal epsom 0.1-0.4 grams; / 1000 milliliters of sodium-chlor 0.1-0.4 grams ,/1000 milliliters of calcium sulfate 0.1-0.4 grams ,/1000 milliliters of lime carbonate 1-8 grams; After the said mixture heating for dissolving; Adjust pH 7.0-7.4 sterilized 20-30 minute down for 112 ℃-115 ℃, promptly processed the cultivation blown-ball Azotobacter and used liquid nutrient medium.
Nutrient broth medium: Carnis Bovis seu Bubali cream, peptone, sodium-chlor are added in the zero(ppm) water; Above-mentioned each component is prepared by the component of the contained weight (g) of 1000 milliliters solution in zero(ppm) water :/1000 milliliters of Carnis Bovis seu Bubali cream 2-8 grams ,/1000 milliliters of peptone 8-12 grams ,/1000 milliliters of sodium-chlor 3-7 grams; After the said mixture heating for dissolving; Adjust pH 7.2-7.6 sterilized 20-30 minute down for 121 ℃, promptly processed the cultivation bacillus cereus and used liquid nutrient medium.
4) fermentation culture:
The bacteria suspension of the blown-ball Azotobacter X11 of above-mentioned assay approval is inoculated the nutrient solution that Ah Xu shellfish substratum is housed by the 5-15% inoculum size in fermentor tank, cultivate, culture temperature is 20-37 ℃, and incubation time is 48-96 hour;
The bacteria suspension of the bacillus cereus B81 of above-mentioned assay approval is inoculated the nutrient solution that nutrient broth medium is housed by the 5-15% inoculum size in fermentor tank, cultivate, culture temperature is 25-45 ℃, and incubation time is 48-72 hour;
5) the nutrient solution cell concentration detects:
Aseptic technique is got and is cultivated Ah Xu the shellfish substratum that blown-ball Azotobacter X11 is arranged, and in microscopically counting thalline quantity, requires thalline quantity to reach 20,000,000,000/milliliter with blood cell counting plate;
Aseptic technique is got and is cultivated the nutrient broth medium that bacillus cereus B81 is arranged, and in microscopically counting thalline quantity, requires thalline quantity to reach 2,000,000,000/milliliter with blood cell counting plate; The ratio that the inspection gemma generates, and the spore rate of will seeking survival reaches more than 90%;
6) collect thalline:
With the overdue culture of above-mentioned cultivation, after pure bacterium experimental verification did not have living contaminants, the centrifugal supernatant that goes obtained spissated wet thallus;
7) carrier sterilization:
Turfy soil and zeyssatite were sterilized 100 ℃-160 ℃ heating in 1-4 hour.
8) preparation microbial fertilizer bacterium powder:
With the turfy soil and the zeyssatite of sterilization, ratio is 10%-90%: 90%-10%, mixes stirring with above-mentioned spissated wet thallus, and is dry under 40 ℃-70 ℃, processes dry powder, is microbial fertilizer;
Microbial fertilizer according to the invention and preparation method thereof, said sorbent material are turfy soil and zeyssatite, and the sorbent material proportion is 95%-99% (weight fraction), and all the other are thalline and substratum, and the viable count of said preparation is >=2 hundred million viable bacteria/grams.
The beneficial effect of microbial fertilizer of the present invention and preparation method thereof is: this product can be applicable to agricultural; Be applied to flower culture; Be applied to maize culture; Be applied to vegetable growing; Be applied to the potato cultivation; Be applied to wheat cultivation.This product has positive agriculture practicality, and simple, cost is low, be prone to implement.
Embodiment
Be described further in the face of microbial fertilizer of the present invention and preparation method thereof down.
Microbial fertilizer of the present invention, this microbial fertilizer are by blown-ball Azotobacter X11 bacterial strain and bacillus cereus B81 bacterial strain, add what sorbent material turfy soil and zeyssatite were prepared from, and the viable count of two kinds of bacteriums in microbial fertilizer is >=2 hundred million viable bacteria/grams;
Said sorbent material is turfy soil and zeyssatite, and the sorbent material proportion is 95%-99% (weight fraction), and all the other are thalline and substratum, and the viable count of said preparation is >=2 hundred million viable bacteria/grams.
Described preparation method is following:
1) activation of bacterial classification calibrating:
The freeze-drying lactobacillus of aseptic unlatching blown-ball Azotobacter X11 bacterial strain; Be inoculated in Ah Xu the shellfish liquid tube substratum; Cultivated 18-72 hour at 20 ℃-45 ℃, streak inoculation is dull and stereotyped in Ah Xu shellfish solid medium then, cultivates 18-72 hour at 20 ℃-45 ℃; Choose colonies typical, carry out the calibrating of morphology and characteristic; The freeze-drying lactobacillus of aseptic unlatching bacillus cereus B81 bacterial strain; Be inoculated in the nutrient broth liquid tube substratum; Cultivated 18-48 hour at 20 ℃-45 ℃, streak inoculation is dull and stereotyped in the nutrient agar medium solid medium then, cultivates 18-48 hour at 20 ℃-45 ℃; Choose colonies typical, carry out the calibrating of morphology and characteristic;
2) preparation of seed liquor:
Pure growth with the blown-ball Azotobacter X11 bacterial classification of assay approval is inoculated in Ah Xu the shellfish liquid nutrient medium, 20 ℃-45 ℃ shake-flask culture 18-72 hour, carry out pure bacterium and bacterium property flag check; Pure growth with the bacillus cereus B81 bacterial strain bacterial classification of assay approval is inoculated in the nutrient broth liquid nutrient medium, 20 ℃-45 ℃ shake-flask culture 18-48 hour, carry out pure bacterium and bacterium property flag check;
3) preparing culture medium:
Ah Xu shellfish substratum: glucose, potassium hydrogenphosphate, sal epsom, sodium-chlor, calcium sulfate, lime carbonate are added in the zero(ppm) water, and above-mentioned each component is prepared by the component of the contained weight (g) of 1000 milliliters solution in zero(ppm) water :/1000 milliliters of glucose 5-15 grams ,/1000 milliliters of potassium hydrogenphosphate 0.1-0.4 grams; / 1000 milliliters of sal epsom 0.1-0.4 grams; / 1000 milliliters of sodium-chlor 0.1-0.4 grams ,/1000 milliliters of calcium sulfate 0.1-0.4 grams ,/1000 milliliters of lime carbonate 1-8 grams; After the said mixture heating for dissolving; Adjust pH 7.0-7.4 sterilized 20-30 minute down for 112 ℃-115 ℃, promptly processed the cultivation blown-ball Azotobacter and used liquid nutrient medium.
Nutrient broth medium: Carnis Bovis seu Bubali cream, peptone, sodium-chlor are added in the zero(ppm) water; Above-mentioned each component is prepared by the component of the contained weight (g) of 1000 milliliters solution in zero(ppm) water :/1000 milliliters of Carnis Bovis seu Bubali cream 2-8 grams ,/1000 milliliters of peptone 8-12 grams ,/1000 milliliters of sodium-chlor 3-7 grams; After the said mixture heating for dissolving; Adjust pH 7.2-7.6 sterilized 20-30 minute down for 121 ℃, promptly processed the cultivation bacillus cereus and used liquid nutrient medium.
4) fermentation culture:
The bacteria suspension of the blown-ball Azotobacter X11 of above-mentioned assay approval is inoculated the nutrient solution that Ah Xu shellfish substratum is housed by the 5-15% inoculum size in fermentor tank, cultivate, culture temperature is 20-37 ℃, and incubation time is 48-96 hour;
The bacteria suspension of the bacillus cereus B81 of above-mentioned assay approval is inoculated the nutrient solution that nutrient broth medium is housed by the 5-15% inoculum size in fermentor tank, cultivate, culture temperature is 25-45 ℃, and incubation time is 48-72 hour;
5) the nutrient solution cell concentration detects:
Aseptic technique is got and is cultivated Ah Xu the shellfish substratum that blown-ball Azotobacter X11 is arranged, and in microscopically counting thalline quantity, requires thalline quantity to reach 20,000,000,000/milliliter with blood cell counting plate;
Aseptic technique is got and is cultivated the nutrient broth medium that bacillus cereus B81 is arranged, and in microscopically counting thalline quantity, requires thalline quantity to reach 2,000,000,000/milliliter with blood cell counting plate; The ratio that the inspection gemma generates, and the spore rate of will seeking survival reaches more than 90%;
6) collect thalline:
With the overdue culture of above-mentioned cultivation, after pure bacterium experimental verification did not have living contaminants, the centrifugal supernatant that goes obtained spissated wet thallus;
7) carrier sterilization:
Turfy soil and zeyssatite were sterilized 100 ℃-160 ℃ heating in 1-4 hour.
8) preparation microbial fertilizer bacterium powder:
(ratio is 10%-90%: 90%-10%) mix stirring with above-mentioned spissated wet thallus, 40 ℃-70 ℃ times dryings, process dry powder, be microbial fertilizer with turfy soil and the zeyssatite of sterilization;
Strain separating:
Vinelandii separate: the present invention adopts Ah Xu shellfish solid medium dull and stereotyped :/1000 milliliters of glucose 10 grams ,/1000 milliliters of potassium hydrogenphosphate 0.2 grams ,/1000 milliliters of sal epsom 0.2 grams; / 1000 milliliters of sodium-chlor 0.2 grams; / 1000 milliliters of calcium sulfate 0.2 grams ,/1000 milliliters of lime carbonate 2 grams ,/1000 milliliters of agar 15-20 grams; PH7.0-7.2 sterilized 30 minutes for 112 ℃-115 ℃; With the even spreading of rich soil small-particle on this Ah Xu shellfish culture medium flat plate; Cultivated 48-72 hour down at 28 ℃ again; When treating to grow around the ped muddiness, translucent gluey bacterium colony, picking colony is done line separation, picking list bacterium colony on Ah Xu shellfish culture medium flat plate; The purifying of on Ah Xu shellfish flat board, ruling again is up to obtaining purebred blown-ball Azotobacter X11 bacterial strain.
Genus bacillus separates: this bright employing nutrient agar medium solid medium flat board :/1000 milliliters of Carnis Bovis seu Bubali cream 3 grams, and/1000 milliliters of peptone 10 grams ,/1000 milliliters of sodium-chlor 5 grams ,/1000 milliliters of agar 15-20 grams, pH7.4-7.6 sterilized 20-30 minute for 121 ℃; Rich soil is done 10 times of serial dilutions with saline water; Each extent of dilution is got the 0.1-0.2 milliliter and is coated on this nutrient agar medium solid medium flat board; Cultivated 36-48 hour down at 37 ℃ again; Picking list bacterium colony, the purifying of on nutrient agar plate, ruling again is up to obtaining purebred bacillus cereus B81 bacterial strain.
Bacterial strain screening:
1) screening of nitrogen-fixing bacteria:
Take by weighing 50 gram soil, place 500 milliliters of triangular flasks of the bacterium of going out in advance, add 1 gram N.F,USP MANNITOL; Behind the mixing, add water to 50% of water retaining capacity, the amount of pressing 5%-20% then inserts vinelandii; Tampon places 28 ℃ to cultivate 10-15 days beyond the Great Wall, accurately takes by weighing 1 gram soil after cultivation finishes; With the total nitrogen content in the nitrogen determination measured soil, contrast does not add bacterium, chooses the strong bacterial strain of nitrogen fixing capacity.
2) screening of phosphate-solubilizing bacteria:
Dull and stereotyped primary dcreening operation: the phosphorus decomposing screening culture medium is dull and stereotyped :/1000 milliliters of N.F,USP MANNITOL 10 grams; / 1000 milliliters of calcium phosphate 0.2 grams ,/1000 milliliters of sal epsom 0.2 grams ,/1000 milliliters of sodium-chlor 0.2 grams; / 1000 milliliters of calcium sulfate 0.2 grams; / 1000 milliliters of lime carbonate 0.1-0.2 grams ,/1000 milliliters of agar 15-20 grams, pH7.0-7.2; On phosphorus decomposing screening culture medium flat board, place 28 ℃ to cultivate 3-10 days microbionation (dibbling), observe periphery of bacterial colonies transparent circle production, choose and produce the big bacterial strain of phosphorus decomposing circle.
The phosphorus decomposing ability detects: the phosphorus decomposing ability detects substratum :/1000 milliliters of N.F,USP MANNITOL 10 grams; / 1000 milliliters of secondary calcium phosphate 0.3 grams ,/1000 milliliters of calcium phosphate 0.2 grams ,/1000 milliliters of sal epsom 0.2 grams; / 1000 milliliters of sodium-chlor 0.2 grams; / 1000 milliliters of calcium sulfate 0.2 grams ,/1000 milliliters of lime carbonate 0.1-0.2 grams, pH7.0-7.2; 100 milliliters of aforesaid liquid substratum are packed in 500 milliliters the triangular flask, after the sterilization, inoculate bacterial strain to be sieved,, cultivated 7 days, measure the content of the capacitive phosphorus in the fermented liquid, get the high bacterial strain of titanium pigment in the substratum with molybdenum antimony resistance colorimetric method at 28 ℃ of 130rpm.
Fungus characteristic:
The characteristic of blown-ball Azotobacter:
Described blown-ball Azotobacter single bacterium colony on Ah Xu shellfish culture medium flat plate be garden shape, translucent, oyster white, surface wettability, smooth, center projections, thick, the edge is more neat; Produce the chocolate pigment in late stage of culture (after 2-7 days); Make bacterium colony be chocolate; Mostly be circle or oval at the microscopically cell, size is 2.4-4.2 * 1.8-3.2 micron, Dan Sheng, twin (usually link to each other in twos and be figure of eight arrangement) or irregular group heap shape; Gram-negative, peritrichous is arranged, pod membrane is arranged, form packing, the starch hydrolysis is positive, through being accredited as blown-ball Azotobacter.
The characteristic of bacillus cereus:
Under irregular, opaque, white, the wax shape of bacillus cereus single bacterium colony on the nutrient agar flat board, edge irregular; Examine under a microscope shaft-like, single arrangement that thalline is, the catenation of accidental one-tenth, Gram-positive; Size is 0.5-0.8 * 1.4-2.5 micron, peritrichous, central spore ovalize; The starch hydrolysis is positive, and protease-producing is positive, through being accredited as bacillus cereus.
The blown-ball Azotobacter that the present invention uses has stronger nitrogen fixing capacity, can make nitrogen content >=10 mg/ml in the substratum; Bacillus cereus has stronger phosphorus decomposing ability, and in fermenting experiment, comparing increase titanium pigment amount with contrast is 52.3-95.4%.
The composition of Ah Xu the shellfish substratum that described fermentor tank is equipped with is following: glucose 5 grams, 10 grams, 15 grams; Potassium hydrogenphosphate 0.1 gram, 0.2 gram, 0.3 gram, 0.4 gram; Sal epsom 0.1 gram, 0.2 gram, 0.3 gram, 0.4 gram; Sodium-chlor 0.1 gram, 0.2 gram, 0.3 gram, 0.4 gram; Calcium sulfate 0.1 gram, 0.2 gram, 0.3 gram, 0.4 gram; Lime carbonate 1 gram, 3 grams, 5 grams, 8 grams; All the other add to 1000 milliliters for water.
Concrete fermentation condition of Ah Xu shellfish substratum of the present invention and result see table one.
Table one, the concrete fermentation condition of Ah Xu shellfish substratum and result:
The composition of the nutrient broth medium that described fermentor tank is equipped with is following: Carnis Bovis seu Bubali cream 2 grams, 4 grams, 5 grams, 8 grams; Peptone 8 grams, 10 grams, 12 grams; Sodium-chlor 3 grams, 5 grams, 7 grams; All the other add to 1000 milliliters for water.
Concrete fermentation condition of nutrient broth medium of the present invention and result see table two.
Table two, the concrete fermentation condition of nutrient broth medium and result:
Best zymotechnique instance of the present invention: the top condition of using Ah Xu shellfish substratum fermentative prodn blown-ball Azotobacter is: the initial pH7.2 of substratum; 30 ℃ of culture temperature; Fermentor tank rotating speed 160rpm; Air flow 4l/min, under this top condition, can ferment produces the viable bacteria greater than 2,000,000,000/milliliter.The top condition of applied nutrition broth culture fermentative prodn bacillus cereus is: the initial pH7.2 of substratum, and 30 ℃ of culture temperature, fermentor tank rotating speed 160rpm, air flow 4l/min, under this top condition, can ferment produces the viable bacteria greater than 2,000,000,000/milliliter
The microbial fertilizer application example of preparation of the present invention:
(1) be applied to maize culture: have the big field of corn to impose this microbial fertilizer in kind, apply 8 kilograms/mu-23 kilograms/mu respectively, cooperate to impose the diammonium phosphate chemical fertilizer, control group only imposes the diammonium phosphate chemical fertilizer of convention amount; From using the result to see, the corn of using microbial fertilizer increases than control group corn grain number per spike, and single-strain grain weight has also increased; The corn of using microbial fertilizer increases 10.5%-25.6% than control group corn yield.
(2) be applied to tomato cultivation: have the tomato field to impose this microbial fertilizer in kind, apply with 8 kilograms/mu amounts, cooperate to impose the diammonium phosphate chemical fertilizer, the control group tomato only imposes the diammonium phosphate chemical fertilizer; See that from application effect the economic characters performances such as individual plant fruit number, single plant yield, single fruit weight that impose the tomato of microbial fertilizer all are better than the control group that only imposes the diammonium phosphate chemical fertilizer, the tomato of using microbial fertilizer increases by 10.6% than control group tomato yield.
(3) use the potato cultivation: have the potato field to impose this microbial fertilizer in kind, apply with 18 kilograms/mu-30 kilograms/mu amount respectively, cooperate to impose the diammonium phosphate chemical fertilizer, the control group potato only imposes the diammonium phosphate chemical fertilizer; Individual plant commodity potato number, the individual plant commodity potato weight of using the potato of microbial fertilizer all are higher than control group, and the potato of using microbial fertilizer increases by 15.6% than control group potato output.
(4) be applied to wheat cultivation: have the big field of wheat to impose this microbial fertilizer in kind, apply with 15 kilograms/mu amounts, cooperate to impose the diammonium phosphate chemical fertilizer, the control group wheat only imposes the diammonium phosphate chemical fertilizer; See that from application effect the wheat of using microbial fertilizer increases by 5.3% than control group wheat yield.
(5) be applied to the Plantula Brassicae chinensis cultivation: have the big field of Plantula Brassicae chinensis to impose this microbial fertilizer in kind, apply with 7.5 kilograms/mu amounts, cooperate to impose the diammonium phosphate chemical fertilizer, the control group Plantula Brassicae chinensis only imposes the diammonium phosphate chemical fertilizer; See from application effect, use the blade of the Plantula Brassicae chinensis of this microbial fertilizer to increase than control group, the Plantula Brassicae chinensis of using microbial fertilizer increases by 6.7% than control group yield of pakchoi.
Above embodiment describes preferred implementation of the present invention; Be not that scope of the present invention is limited; Design under the prerequisite of spirit not breaking away from the present invention; Various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (3)
1. microbial fertilizer, it is characterized in that: this microbial fertilizer is by blown-ball Azotobacter X11 bacterial strain and bacillus cereus strain B81 fermented liquid, mixes with turfy soil and zeyssatite;
The viable count of said blown-ball Azotobacter in microbial fertilizer is >=2 hundred million viable bacteria/grams, and the viable count of bacillus cereus in microbial fertilizer is >=2 hundred million viable bacteria/grams.
2. the making method of a microbial fertilizer is characterized in that, may further comprise the steps successively:
1) activation of bacterial classification calibrating:
The freeze-drying lactobacillus of aseptic unlatching blown-ball Azotobacter X11 bacterial strain; Be inoculated in Ah Xu the shellfish liquid tube substratum; Cultivated 18-72 hour at 20 ℃-45 ℃, streak inoculation is dull and stereotyped in Ah Xu shellfish solid medium then, cultivates 18-72 hour at 20 ℃-45 ℃; Choose colonies typical, carry out the calibrating of morphology and characteristic; The freeze-drying lactobacillus of aseptic unlatching bacillus cereus B81 bacterial strain; Be inoculated in the nutrient broth liquid tube substratum; Cultivated 18-48 hour at 20 ℃-45 ℃, streak inoculation is dull and stereotyped in the nutrient agar medium solid medium then, cultivates 18-48 hour at 20 ℃-45 ℃; Choose colonies typical, carry out the calibrating of morphology and characteristic;
2) preparation of seed liquor:
Pure growth with the blown-ball Azotobacter X11 bacterial classification of assay approval is inoculated in Ah Xu the shellfish liquid nutrient medium, 20 ℃-45 ℃ shake-flask culture 18-72 hour, carry out pure bacterium and bacterium property flag check; Pure growth with the bacillus cereus B81 bacterial strain bacterial classification of assay approval is inoculated in the nutrient broth liquid nutrient medium, 20 ℃-45 ℃ shake-flask culture 18-48 hour, carry out pure bacterium and bacterium property flag check;
3) preparing culture medium:
Ah Xu shellfish substratum: glucose, potassium hydrogenphosphate, sal epsom, sodium-chlor, calcium sulfate, lime carbonate are added in the zero(ppm) water, and above-mentioned each component is prepared by the component of the contained weight (g) of 1000 milliliters solution in zero(ppm) water :/1000 milliliters of glucose 5-15 grams ,/1000 milliliters of potassium hydrogenphosphate 0.1-0.4 grams; / 1000 milliliters of sal epsom 0.1-0.4 grams; / 1000 milliliters of sodium-chlor 0.1-0.4 grams ,/1000 milliliters of calcium sulfate 0.1-0.4 grams ,/1000 milliliters of lime carbonate 1-8 grams; After the said mixture heating for dissolving; Adjust pH 7.0-7.4 sterilized 20-30 minute down for 112 ℃-115 ℃, promptly processed the cultivation blown-ball Azotobacter and used liquid nutrient medium.
Nutrient broth medium: Carnis Bovis seu Bubali cream, peptone, sodium-chlor are added in the zero(ppm) water; Above-mentioned each component is prepared by the component of the contained weight (g) of 1000 milliliters solution in zero(ppm) water :/1000 milliliters of Carnis Bovis seu Bubali cream 2-8 grams ,/1000 milliliters of peptone 8-12 grams ,/1000 milliliters of sodium-chlor 3-7 grams; After the said mixture heating for dissolving; Adjust pH 7.2-7.6 sterilized 20-30 minute down for 121 ℃, promptly processed the cultivation bacillus cereus and used liquid nutrient medium.
4) fermentation culture:
The bacteria suspension of the blown-ball Azotobacter X11 of above-mentioned assay approval is inoculated the nutrient solution that Ah Xu shellfish substratum is housed by the 5-15% inoculum size in fermentor tank, cultivate, culture temperature is 20-37 ℃, and incubation time is 48-96 hour;
The bacteria suspension of the bacillus cereus B81 of above-mentioned assay approval is inoculated the nutrient solution that nutrient broth medium is housed by the 5-15% inoculum size in fermentor tank, cultivate, culture temperature is 25-45 ℃, and incubation time is 48-72 hour;
5) the nutrient solution cell concentration detects:
Aseptic technique is got and is cultivated Ah Xu the shellfish substratum that blown-ball Azotobacter X11 is arranged, and in microscopically counting thalline quantity, requires thalline quantity to reach 20,000,000,000/milliliter with blood cell counting plate;
Aseptic technique is got and is cultivated the nutrient broth medium that bacillus cereus B81 is arranged, and in microscopically counting thalline quantity, requires thalline quantity to reach 2,000,000,000/milliliter with blood cell counting plate; The ratio that the inspection gemma generates, and the spore rate of will seeking survival reaches more than 90%;
6) collect thalline:
With the overdue culture of above-mentioned cultivation, after pure bacterium experimental verification did not have living contaminants, the centrifugal supernatant that goes obtained spissated wet thallus;
7) carrier sterilization:
Turfy soil and zeyssatite were sterilized 100 ℃-160 ℃ heating in 1-4 hour.
8) preparation microbial fertilizer bacterium powder:
With the turfy soil and the zeyssatite of sterilization, ratio is 10%-90%: 90%-10%, mixes stirring with above-mentioned spissated wet thallus, and is dry under 40 ℃-70 ℃, processes dry powder, is microbial fertilizer;
3. according to said microbial fertilizer of claim 1 and preparation method thereof; It is characterized in that: said sorbent material is turfy soil and zeyssatite; The sorbent material proportion is 95%-99% (weight fraction), and all the other are thalline and substratum, and the viable count of said preparation is >=2 hundred million viable bacteria/grams.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103130531A (en) * | 2013-04-01 | 2013-06-05 | 黑龙江旺土肥业有限公司 | Process to prepare liquid microbe organic fertilizer by means of animal blood enzymolysis and fermentation synchronous method |
| CN103130531B (en) * | 2013-04-01 | 2014-12-10 | 黑龙江旺土肥业有限公司 | Process to prepare liquid microbe organic fertilizer by means of animal blood enzymolysis and fermentation synchronous method |
| CN103553817A (en) * | 2013-11-08 | 2014-02-05 | 苏州仁成生物科技有限公司 | Efficient microbial fertilizer as well as preparation method thereof |
| CN103613449A (en) * | 2013-11-08 | 2014-03-05 | 苏州仁成生物科技有限公司 | Fertilizer with high slow release fertilizer efficiency produced by secondary fermentation and preparation method thereof |
| CN103613449B (en) * | 2013-11-08 | 2016-04-27 | 苏州仁成生物科技有限公司 | A kind of second order fermentation produces high slowly-releasing fertilizer efficiency fertilizer and preparation method thereof |
| CN112899188A (en) * | 2021-01-29 | 2021-06-04 | 西南大学 | Microbial agent for promoting crop root development and preparation and application thereof |
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