CN113480366A - Microbial liquid bacterial fertilizer - Google Patents

Microbial liquid bacterial fertilizer Download PDF

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Publication number
CN113480366A
CN113480366A CN202110960591.7A CN202110960591A CN113480366A CN 113480366 A CN113480366 A CN 113480366A CN 202110960591 A CN202110960591 A CN 202110960591A CN 113480366 A CN113480366 A CN 113480366A
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fermentation
liquid
culture
bacillus subtilis
culture medium
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姜红军
李建全
王姗姗
陈相辉
邱彦国
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Shandong De Ming Xing Biotechnology Co ltd
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Shandong De Ming Xing Biotechnology Co ltd
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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
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • 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
    • C05G3/80—Soil conditioners
    • 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
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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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Abstract

The invention discloses a microbial liquid bacterial fertilizer. The invention comprises bacillus subtilis fermentation liquor, EM (effective microorganisms) fermentation liquor, lactobacillus plantarum fermentation liquor, yeast fermentation liquor, polyglutamic acid, water and seaweed crystals; the volume ratio of the bacillus subtilis fermentation liquor to the EM (effective microorganisms) fermentation liquor to the lactobacillus plantarum fermentation liquor to the yeast fermentation liquor to the polyglutamic acid to the water to the seaweed crystals is (10-15): (25-28): (15-17): 10: 5: 800: 5. the microbial fertilizer can effectively promote the growth of apple plants, reduce the application amount of the fertilizer, reduce the production cost, play the aims of reducing weight and improving efficiency, and play a positive role in the sustainable development of modern agriculture.

Description

Microbial liquid bacterial fertilizer
Technical Field
The invention relates to the technical field of agricultural fertilizers, in particular to a microbial liquid bacterial fertilizer.
Background
The apple is a fruit rich in minerals and vitamins, is deeply loved by people and is one of the most frequently eaten fruits by people. The apple producing area is mainly distributed in Liaoning, Hebei, Shanxi, Shandong, Shaanxi, Gansu, Sichuan, Yunnan and Tibet areas in China. At present, in the planting process of apples, people generally fertilize according to the fertilizer-requiring period of fruit trees and the nutritional status of the fruit trees. For example, organic fertilizer and phosphorus and potassium fertilizers are applied to the fruit trees after the apples are harvested and before the apple trees fall leaves; applying a quick-acting nitrogen fertilizer to the fruit trees in the sprouting period of the fruit trees; applying quick-acting nitrogen fertilizer and boron fertilizer to the fruit trees after the fruit trees fall flowers; in the flower bud differentiation period of fruit trees, the fertilizer to be applied is mainly compounded by phosphorus and potassium; after picking the apples, urea is applied. The fertilization process is tedious, consumes a large amount of manpower and material resources, and chemical fertilizer also brings adverse effects to the development of agriculture towards the environmental protection and the economic direction, so more and more researchers put research and development hot spots on the microbial fertilizer.
The microbial fertilizer is a novel fertilizer in modern agricultural development for ensuring crops to obtain sufficient nutrient substances through the life activities of active microorganisms, has various nutrient elements and a large number of active microorganisms, can improve the environment in soil and improve the soil fertilizer supply and disease resistance, thereby achieving the purpose of improving the quality of agricultural products and being very beneficial to the development of agriculture towards the environmental protection and the economic direction.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a microbial liquid bacterial fertilizer.
The technical scheme of the invention is as follows:
a microbial liquid bacterial fertilizer comprises Bacillus subtilis fermentation broth, EM (effective microorganisms) fermentation broth, Lactobacillus plantarum fermentation broth, yeast fermentation broth, polyglutamic acid, water and seaweed crystals; the volume ratio of the bacillus subtilis fermentation liquor to the EM (effective microorganisms) fermentation liquor to the lactobacillus plantarum fermentation liquor to the yeast fermentation liquor to the polyglutamic acid to the water to the seaweed crystals is (10-15): (25-28): (15-17): 10: 5: 800: 5.
preferably, the bacillus subtilis fermentation liquor is a bacterial liquor with the bacillus subtilis content of more than 500 × 108CFU/mL, which is obtained by culturing and fermenting bacillus subtilis which is obtained by breeding in corn root soil according to a conventional dilution plate separation method and is used as a strain; the EM bacteria fermentation liquor, the lactobacillus plantarum fermentation liquor and the yeast fermentation liquor used in the invention are obtained by culturing according to the method of the known technology.
Preferably, the preparation method of the bacillus subtilis fermentation liquor comprises the following steps:
1) activated culture of strains
Culturing the bacillus subtilis strain for 12-24h by a planar solid culture medium at 34-36 ℃ to obtain a bacillus subtilis unicellular colony; culturing the bacillus subtilis unicellular colony for 12-24h at 34-36 ℃ by a slant solid culture medium to obtain an activated bacillus subtilis strain;
2) obtaining of fermentation Strain
Inoculating the activated bacillus subtilis strain into a liquid culture medium, and performing shake culture at 34-36 ℃ and 200rpm for 48h to obtain a seed bacterial liquid; then transferring the seed bacterial liquid to a seed rejuvenation culture medium, and performing shake culture at 34-36 ℃ and 200rpm for 48 h; adding glycerol with the volume of 20% of the bacterial liquid into the bacterial liquid obtained by the enlarged culture, and storing at-80 ℃ to obtain a zymocyte;
3) obtaining of zymophyte liquid
Inoculating the fermentation strain into a fermentation culture medium subjected to sterilization treatment according to the inoculation amount of 6-8% by volume for fermentation culture; wherein, the fermentation culture is divided into three stages of early fermentation stage and late fermentation stage; the fermentation culture is carried out for 0-12h as the early stage of fermentation, and the initial stage of fermentation is carried out under the process conditions of the culture temperature of 34-36 ℃, the rotating speed of 170-240rpm, the ventilation quantity of 1:0.7vvm and the dissolved oxygen saturation of more than or equal to 10 percent; the fermentation culture time is 12-24h, which is the later stage of fermentation, and the later stage of fermentation is carried out under the process conditions of the culture temperature of 36-38 ℃, the stirring speed of 450-.
Preferably, in step 1), the formulations of the planar solid medium and the slant solid medium are the same, and both formulations are: 5g of peptone, 4g of NaCl, 1.5g of beef extract, 10g of agar and 1L of deionized water; and the plane solid culture medium and the inclined plane solid culture medium are sterilized and then used for subsequent bacterial culture.
Preferably, in the step 2), the formula of the liquid culture medium is 4g of glucose, 9g of soybean meal, 5g of agar, 10g of NaCl, 10g of peptone, 5g of yeast powder and 1L of deionized water; the liquid culture medium is sterilized and then used for culturing the activated bacillus subtilis strain.
Preferably, in the step 2), the rejuvenation medium is prepared from 4g of glucose, 4g of peptone, 4g of yeast powder and KH2PO4 0.1g、ZnSO4 2g、NaCl 4g、MnCl2 4g、Mg(NO3)2 3g、FeCl26g and 1L of deionized water; the rejuvenation culture medium is used for fermentation culture of fermentation strains subsequently after sterilization.
Preferably, in step 3), the formula of the fermentation medium is: 36kg of bean cake powder, 3.6kg of glucose, 3.3kg of beef extract, 33kg of corn flour and CaCl2 3.0kg、KH2PO4 1.5kg、MgSO4·7H2O 1.5kg、NH4Cl 5.5kg、NaCl 1.5kg、MnSO4 150g、KNO32.3kg, 720g of neutral protease (enzyme activity of 50000U/g) and 330g of moderate temperature alpha-amylase (enzyme activity of 5000U/g).
Preferably, the sterilized fermentation medium is prepared by the following method: adding 500L of deionized water into the fermentation tank, and adding bean cake powder, glucose, beef extract, corn flour and CaCl into the fermentation tank according to a certain proportion2、KH2PO4、MgSO4·7H2O、NH4Cl、NaCl、MnSO4And KNO3Then heating to 40-44 ℃, adding protease, and carrying out enzymolysis for 1.5h at a stirring speed of 100-150 rpm; then heating to 65-70 DEG CAdding medium-temperature alpha-amylase, and carrying out enzymolysis for 1.5h at a stirring speed of 100-150 rpm; after the enzymolysis is finished, the fermentation medium is sterilized for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.11MPa, and then the sterilized fermentation medium can be prepared.
A preparation method of a microbial liquid bacterial fertilizer comprises the steps of taking bacillus subtilis fermentation liquor, EM (effective microorganisms) fermentation liquor, lactobacillus plantarum fermentation liquor, yeast fermentation liquor, polyglutamic acid, water and seaweed crystals according to a specified ratio, and stirring and uniformly mixing the raw materials at the temperature of 34-36 ℃ for 20min to obtain the microbial liquid bacterial fertilizer.
Compared with the prior art, the invention has the following beneficial effects:
1) the fertilizer contains beneficial microorganisms, can effectively improve the physical and chemical properties of soil, promotes the growth of apple plants, reduces the application amount of chemical fertilizers, reduces the production cost, achieves the purpose of increasing the yield and income of crops, and plays a positive role in the sustainable development of modern agriculture; moreover, the microbial fertilizer is suitable for various crops, has wide application range, and breaks through the characteristics of specificity and limitation of the traditional fertilizer;
2) the invention adopts the formula that the spore bacteria content is more than or equal to 500 multiplied by 108The high spore content of the CFU/mL bacillus subtilis fermentation liquid is realized according to the special fermentation process design in the application, specifically, after the bacillus subtilis strain is subjected to slant activation and seed rejuvenation, the high-activity fermentation seed is obtained, and then the fermentation formula and the fermentation process are specially designed for fermentation, so that the viable bacteria content and the spore bacteria amount of the fermentation liquid are effectively improved.
Detailed Description
The first embodiment is as follows:
a microbial liquid bacterial fertilizer comprises the following components: 200ml of bacillus subtilis fermentation liquid, 500ml of EM (effective microorganisms) fermentation liquid, 320ml of lactobacillus plantarum fermentation liquid, 200ml of yeast fermentation liquid, 100ml of polyglutamic acid, 1600ml of water and 100ml of seaweed crystals. The microbial liquid bacterial manure is prepared by the following steps: and putting the bacillus subtilis fermentation liquor, the EM fermentation liquor, the lactobacillus plantarum fermentation liquor, the yeast fermentation liquor, the polyglutamic acid, the water and the seaweed crystal into a tank body, and stirring for 20min at the temperature of 34-36 ℃ to uniformly mix to obtain the microbial liquid bacterial fertilizer.
The EM bacteria fermentation broth, the lactobacillus plantarum fermentation broth and the yeast fermentation broth used in the first embodiment are obtained by culturing according to the methods of the known technology, wherein the viable bacteria content of the EM bacteria fermentation broth is greater than 100 × 108 CFU/mL, the viable bacteria content of the lactobacillus plantarum fermentation liquor is more than 80 multiplied by 108 CFU/mL, the viable bacteria content of the yeast fermentation liquid is more than 300 CFU/mL; the bacillus subtilis fermentation broth used in the first embodiment is obtained by fermentation culture according to a new self-designed fermentation culture path.
In this embodiment, the bacillus subtilis fermentation liquid is a bacterial liquid obtained by culturing and fermenting bacillus subtilis, which is obtained by breeding bacillus subtilis in corn root soil according to a conventional dilution plate separation method, as a bacterial strain, and the bacillus subtilis fermentation liquid has a bacillus content of more than 500 × 108CFU/mL, specifically, the bacillus subtilis fermentation liquid is prepared by the following method:
1) activated culture of strains
Culturing the bacillus subtilis strain for 12 hours at 34 ℃ by using a planar solid culture medium to obtain a bacillus subtilis unicellular colony; culturing the bacillus subtilis unicellular colony for 24 hours at 34 ℃ by a slant solid culture medium to obtain an activated bacillus subtilis strain;
2) obtaining of fermentation Strain
Inoculating the activated bacillus subtilis strain into a liquid culture medium, and performing shake culture at 34 ℃ and 200rpm for 48 hours to obtain a seed bacterium liquid; then transferring the seed bacterial liquid to a seed rejuvenation culture medium, and performing shake cultivation for 48 hours at 34 ℃ and 200 rpm; adding glycerol with the volume of 20% of the bacterial liquid into the bacterial liquid obtained by the enlarged culture, and storing at-80 ℃ to obtain a zymocyte;
3) obtaining of zymophyte liquid
Inoculating the fermentation strain into a fermentation culture medium subjected to sterilization treatment according to the inoculation amount of 6 percent of the volume percentage for fermentation culture; wherein, the fermentation culture is divided into three stages of early fermentation stage and late fermentation stage; the fermentation culture is carried out for 0-12h at the early stage of fermentation under the process conditions of the culture temperature of 34 ℃, the rotation speed of 170rpm, the ventilation volume of 1:0.7vvm and the dissolved oxygen saturation of more than or equal to 10%; the fermentation culture time is 12-24h, and the fermentation culture time is at the culture temperature of 36 deg.C, the stirring speed of 450rpm, the ventilation rate of 1:1.2vvm, and the dissolved oxygen saturation rate of more than 10%.
In the invention, in step 1), the formula of the planar solid culture medium and the formula of the inclined solid culture medium are the same, and both the formulas are as follows: 5g of peptone, 4g of NaCl, 1.5g of beef extract, 10g of agar and 1L of deionized water; the planar solid culture medium and the inclined plane solid culture medium are sterilized at the high temperature of 120 ℃ for 30min and then used for subsequent bacterial culture; in the step 2), the formula of the liquid culture medium comprises 4g of glucose, 9g of soybean meal, 5g of agar, 10g of NaCl, 10g of peptone, 5g of yeast powder and 1L of deionized water; the liquid culture medium is used for culturing the activated bacillus subtilis strain after being sterilized; in the step 2), the rejuvenation medium comprises 4g of glucose, 4g of peptone, 4g of yeast powder and KH2PO4 0.1g、ZnSO4 2g、NaCl 4g、MnCl2 4g、Mg(NO3)2 3g、FeCl26g and 1L of deionized water; the rejuvenation culture medium is sterilized at the high temperature of 120 ℃ for 30min and then used for carrying out fermentation culture on the fermentation strain subsequently;
in the invention, the formula of the fermentation medium for sterilization treatment used in the step 3) is as follows: 36kg of bean cake powder, 3.6kg of glucose, 3.3kg of beef extract, 33kg of corn flour and CaCl2 3.0kg、KH2PO4 1.5kg、MgSO4·7H2O 1.5kg、NH4Cl 5.5kg、NaCl 1.5kg、MnSO4 150g、KNO32.3kg, 720g of neutral protease (enzyme activity 50000U/g) and 330g of moderate temperature alpha-amylase (enzyme activity 5000U/g); and the sterilized fermentation medium used in step 3) is prepared by the following method: adding 500L deionized water into 1000L fermentation tank, adding bean cake powder, glucose, beef extract, corn flour, and CaCl into the fermentation tank2、KH2PO4、MgSO4·7H2O、NH4Cl、NaCl、MnSO4And KNO3Then heating to 40 ℃, adding protease, and carrying out enzymolysis for 1.5h at the stirring speed of 100 rpm; then heating to 65 ℃, adding medium-temperature alpha-amylase, and carrying out enzymolysis for 1.5h at the stirring speed of 100 rpm; after the enzymolysis is finished, the fermentation medium is sterilized for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.11MPa, and then the sterilized fermentation medium can be prepared.
Example two:
a microbial liquid bacterial fertilizer comprises the following components: 240ml of bacillus subtilis fermentation liquid, 560ml of EM (effective microorganisms) fermentation liquid, 340ml of lactobacillus plantarum fermentation liquid, 200ml of yeast fermentation liquid, 100ml of polyglutamic acid, 1600ml of water and 100ml of seaweed crystals. The microbial liquid bacterial manure is prepared by the following steps: and putting the bacillus subtilis fermentation liquor, the EM fermentation liquor, the lactobacillus plantarum fermentation liquor, the yeast fermentation liquor, the polyglutamic acid, the water and the seaweed crystal into a tank body, and stirring for 20min at the temperature of 34-36 ℃ to uniformly mix to obtain the microbial liquid bacterial fertilizer.
The EM bacteria fermentation broth, the lactobacillus plantarum fermentation broth and the yeast fermentation broth used in the second embodiment are obtained by culturing according to the methods of the known technologies, wherein the viable bacteria content of the EM bacteria fermentation broth is greater than 100 × 108 CFU/mL, the viable bacteria content of the lactobacillus plantarum fermentation liquor is more than 80 multiplied by 108 CFU/mL, the viable bacteria content of the yeast fermentation liquid is more than 300 CFU/mL; the bacillus subtilis fermentation broth used in the second example is obtained by fermentation culture according to a new fermentation culture path designed by itself.
In the second embodiment, the bacillus subtilis fermentation liquid is obtained by breeding bacillus subtilis in corn root soil according to a conventional dilution plate separation method, and the bacillus content of the fermentation liquid obtained by culture and fermentation is more than 500 × 108The CFU/mL bacterial liquid, specifically the Bacillus subtilis fermentation liquid is prepared by the following method:
1) activated culture of strains
Culturing the bacillus subtilis strain for 12 hours at 36 ℃ by using a planar solid culture medium to obtain a bacillus subtilis unicellular colony; culturing the bacillus subtilis unicellular colony for 12h at 36 ℃ by using a slant solid culture medium to obtain an activated bacillus subtilis strain;
2) obtaining of fermentation Strain
Inoculating the activated bacillus subtilis strain into a liquid culture medium, and performing shake culture at 36 ℃ and 200rpm for 48 hours to obtain a seed bacterium liquid; then transferring the seed bacterial liquid to a seed rejuvenation culture medium, and performing shake cultivation for 48 hours at 36 ℃ and 200 rpm; adding glycerol with the volume of 20% of the bacterial liquid into the bacterial liquid obtained by the enlarged culture, and storing at-80 ℃ to obtain a zymocyte;
3) obtaining of zymophyte liquid
Inoculating the fermentation strain into a fermentation culture medium subjected to sterilization treatment according to the inoculation amount of 7 percent of the volume percentage for fermentation culture; wherein, the fermentation culture is divided into three stages of early fermentation stage and late fermentation stage; the fermentation culture is carried out for 0-12h at the early stage of fermentation under the process conditions of the culture temperature of 36 ℃, the rotation speed of 240rpm, the ventilation volume of 1:0.7vvm and the dissolved oxygen saturation of more than or equal to 10%; the fermentation culture time is 12-24h, and the fermentation culture time is at the culture temperature of 37 deg.C, stirring speed of 500rpm, ventilation rate of 1:1.2vvm, and dissolved oxygen saturation rate of more than 10%.
In the invention, in step 1), the formula of the planar solid culture medium and the formula of the inclined solid culture medium are the same, and both the formulas are as follows: 5g of peptone, 4g of NaCl, 1.5g of beef extract, 10g of agar and 1L of deionized water; the planar solid culture medium and the inclined plane solid culture medium are sterilized at the high temperature of 120 ℃ for 30min and then used for subsequent bacterial culture; in the step 2), the formula of the liquid culture medium comprises 4g of glucose, 9g of soybean meal, 5g of agar, 10g of NaCl, 10g of peptone, 5g of yeast powder and 1L of deionized water; the liquid culture medium is used for culturing the activated bacillus subtilis strain after being sterilized; in the step 2), the rejuvenation medium comprises 4g of glucose, 4g of peptone, 4g of yeast powder and KH2PO4 0.1g、ZnSO4 2g、NaCl 4g、MnCl2 4g、Mg(NO3)2 3g、FeCl26g and 1L of deionized water; the rejuvenation culture medium is sterilized at 120 ℃ for 30minThen the fermentation culture is carried out on the fermentation strain subsequently;
in the invention, the formula of the fermentation medium for sterilization treatment used in the step 3) is as follows: 36kg of bean cake powder, 3.6kg of glucose, 3.3kg of beef extract, 33kg of corn flour and CaCl2 3.0kg、KH2PO4 1.5kg、MgSO4·7H2O 1.5kg、NH4Cl 5.5kg、NaCl 1.5kg、MnSO4 150g、KNO32.3kg, 720g of neutral protease (enzyme activity 50000U/g) and 330g of moderate temperature alpha-amylase (enzyme activity 5000U/g); and the sterilized fermentation medium used in step 3) is prepared by the following method: adding 500L deionized water into 1000L fermentation tank, adding bean cake powder, glucose, beef extract, corn flour, and CaCl into the fermentation tank2、KH2PO4、MgSO4·7H2O、NH4Cl、NaCl、MnSO4And KNO3Then heating to 44 ℃, adding protease, and carrying out enzymolysis for 1.5h at the stirring speed of 130 rpm; then heating to 67 ℃, adding medium-temperature alpha-amylase, and carrying out enzymolysis for 1.5h at the stirring speed of 130 rpm; after the enzymolysis is finished, the fermentation medium is sterilized for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.11MPa, and then the sterilized fermentation medium can be prepared.
Example three:
a microbial liquid bacterial fertilizer comprises the following components: 300ml of bacillus subtilis fermentation liquid, 540ml of EM (effective microorganisms) fermentation liquid, 300ml of lactobacillus plantarum fermentation liquid, 200ml of yeast fermentation liquid, 100ml of polyglutamic acid, 1600ml of water and 100ml of seaweed crystals. The microbial liquid bacterial manure is prepared by the following steps: and putting the bacillus subtilis fermentation liquor, the EM fermentation liquor, the lactobacillus plantarum fermentation liquor, the yeast fermentation liquor, the polyglutamic acid, the water and the seaweed crystal into a tank body, and stirring for 20min at the temperature of 34-36 ℃ to uniformly mix to obtain the microbial liquid bacterial fertilizer.
The EM bacteria fermentation broth, the lactobacillus plantarum fermentation broth and the yeast fermentation broth used in the third embodiment are obtained by culturing according to the methods of the known technology, wherein the viable bacteria content of the EM bacteria fermentation broth is greater than 100 × 108 CFU/mL, viable bacteria content of Lactobacillus plantarum fermentation brothGreater than 80X 108 CFU/mL, the viable bacteria content of the yeast fermentation liquid is more than 300 CFU/mL; the bacillus subtilis fermentation broth used in the third example is obtained by fermentation culture according to a new self-designed fermentation culture path.
In the third embodiment, the bacillus subtilis fermentation liquid is obtained by breeding bacillus subtilis as a strain in corn root soil according to a conventional dilution plate separation method, and the bacillus content of the fermentation liquid obtained by culture and fermentation is more than 500 × 108The CFU/mL bacterial liquid, specifically the Bacillus subtilis fermentation liquid is prepared by the following method:
1) activated culture of strains
Culturing the bacillus subtilis strain for 24 hours at 35 ℃ by using a planar solid culture medium to obtain a bacillus subtilis unicellular colony; culturing the bacillus subtilis unicellular colony for 24 hours at 35 ℃ by using a slant solid culture medium to obtain an activated bacillus subtilis strain;
2) obtaining of fermentation Strain
Inoculating the activated bacillus subtilis strain into a liquid culture medium, and performing shake culture at 35 ℃ and 200rpm for 48 hours to obtain a seed bacterium liquid; then transferring the seed bacterial liquid to a seed rejuvenation culture medium, and performing shake cultivation for 48 hours at 35 ℃ and 200 rpm; adding glycerol with the volume of 20% of the bacterial liquid into the bacterial liquid obtained by the enlarged culture, and storing at-80 ℃ to obtain a zymocyte;
3) obtaining of zymophyte liquid
Inoculating the fermentation strain into a fermentation culture medium subjected to sterilization treatment according to the inoculation amount of 8 percent of volume percentage for fermentation culture; wherein, the fermentation culture is divided into three stages of early fermentation stage and late fermentation stage; the fermentation culture is carried out for 0-12h at the early stage of fermentation under the process conditions of the culture temperature of 35 ℃, the rotation speed of 200rpm, the ventilation volume of 1:0.7vvm and the dissolved oxygen saturation of more than or equal to 10%; the fermentation culture time is 12-24h, and the fermentation culture time is at the later stage of fermentation under the process conditions of culture temperature of 35 deg.C, stirring speed of 490rpm, ventilation amount of 1:1.2vvm, and dissolved oxygen saturation of more than or equal to 10%.
In the present invention, in step 1), the planar solid culture is performedThe formula of the medium and the formula of the slant solid medium are the same, and both the formulas are as follows: 5g of peptone, 4g of NaCl, 1.5g of beef extract, 10g of agar and 1L of deionized water; the planar solid culture medium and the inclined plane solid culture medium are sterilized at the high temperature of 120 ℃ for 30min and then used for subsequent bacterial culture; in the step 2), the formula of the liquid culture medium comprises 4g of glucose, 9g of soybean meal, 5g of agar, 10g of NaCl, 10g of peptone, 5g of yeast powder and 1L of deionized water; the liquid culture medium is used for culturing the activated bacillus subtilis strain after being sterilized; in the step 2), the rejuvenation medium comprises 4g of glucose, 4g of peptone, 4g of yeast powder and KH2PO4 0.1g、ZnSO4 2g、NaCl 4g、MnCl2 4g、Mg(NO3)2 3g、FeCl26g and 1L of deionized water; the rejuvenation culture medium is sterilized at the high temperature of 120 ℃ for 30min and then used for carrying out fermentation culture on the fermentation strain subsequently;
in the invention, the formula of the fermentation medium for sterilization treatment used in the step 3) is as follows: 36kg of bean cake powder, 3.6kg of glucose, 3.3kg of beef extract, 33kg of corn flour and CaCl2 3.0kg、KH2PO4 1.5kg、MgSO4·7H2O 1.5kg、NH4Cl 5.5kg、NaCl 1.5kg、MnSO4 150g、KNO32.3kg, 720g of neutral protease (enzyme activity 50000U/g) and 330g of moderate temperature alpha-amylase (enzyme activity 5000U/g); and the sterilized fermentation medium used in step 3) is prepared by the following method: adding 500L deionized water into 1000L fermentation tank, adding bean cake powder, glucose, beef extract, corn flour, and CaCl into the fermentation tank2、KH2PO4、MgSO4·7H2O、NH4Cl、NaCl、MnSO4And KNO3Then heating to 42 ℃, adding protease, and carrying out enzymolysis for 1.5h at the stirring speed of 150 rpm; then heating to 68 ℃, adding medium-temperature alpha-amylase, and carrying out enzymolysis for 1.5h at the stirring speed of 150 rpm; after the enzymolysis is finished, the fermentation medium is sterilized for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.11MPa, and then the sterilized fermentation medium can be prepared.
And (3) application effect comparison:
the microbial liquid bacterial manure prepared in the first, second and third examples is used for testing an apple orchard in an enchance city, the testing time is 03-10 months 2020, the number of tested fruit trees in each microbial liquid bacterial manure is 10, and the tested apple varieties are as follows: gold is commander. In addition, in order to compare the effect of the microbial liquid bacterial manure and the chemical fertilizer on the fruit tree setting rate of the fruit tree in the first to third embodiments, the invention particularly provides a comparative example, namely, the fruit tree is subjected to chemical fertilizer application according to the conventional manner.
The manner of applying the microbial liquid bacterial manure prepared in the first, second and third embodiments to fruit trees is as follows: and (3) carrying out flushing application on the microbial liquid bacterial manure according to the use amount of 2L/mu in the flowering period, the fruit setting period, the fruit growing period and the fruit expanding period of the apples.
The fruit setting number and the yield per mu of the three microbial liquid bacterial manure fertilization and the chemical fertilizer fertilization are shown in the following table:
Figure 75176DEST_PATH_IMAGE002
as can be seen from the table, the microbial liquid bacterial manure fertilization in the first to third examples has greatly improved fruit setting number and yield per mu compared with chemical fertilizers, high fruit setting rate and obvious yield increasing effect.

Claims (9)

1. A microbial liquid bacterial fertilizer is characterized in that: comprises bacillus subtilis fermentation liquor, EM (effective microorganisms) fermentation liquor, lactobacillus plantarum fermentation liquor, yeast fermentation liquor, polyglutamic acid, water and seaweed crystals; the volume ratio of the bacillus subtilis fermentation liquor to the EM (effective microorganisms) fermentation liquor to the lactobacillus plantarum fermentation liquor to the yeast fermentation liquor to the polyglutamic acid to the water to the seaweed crystals is (10-15): (25-28): (15-17): 10: 5: 800: 5.
2. the microbial liquid bacterial fertilizer of claim 1, wherein: what is needed isThe bacillus subtilis fermentation liquor is prepared by breeding bacillus subtilis in corn root soil according to a conventional dilution plate separation method, and the bacillus content of the fermentation liquor obtained by culture and fermentation is more than 500 multiplied by 108CFU/mL of bacterial liquid.
3. The microbial liquid bacterial manure of claim 2, wherein: the preparation method of the bacillus subtilis fermentation liquor comprises the following steps:
1) activated culture of strains
Culturing the bacillus subtilis strain for 12-24h by a planar solid culture medium at 34-36 ℃ to obtain a bacillus subtilis unicellular colony; culturing the bacillus subtilis unicellular colony for 12-24h at 34-36 ℃ by a slant solid culture medium to obtain an activated bacillus subtilis strain;
2) obtaining of fermentation Strain
Inoculating the activated bacillus subtilis strain into a liquid culture medium, and performing shake culture at 34-36 ℃ and 200rpm for 48h to obtain a seed bacterial liquid; then transferring the seed bacterial liquid to a seed rejuvenation culture medium, and performing shake culture at 34-36 ℃ and 200rpm for 48 h; adding glycerol with the volume of 20% of the bacterial liquid into the bacterial liquid obtained by the enlarged culture, and storing at-80 ℃ to obtain a zymocyte;
3) obtaining of zymophyte liquid
Inoculating the fermentation strain into a fermentation culture medium subjected to sterilization treatment according to the inoculation amount of 6-8% by volume for fermentation culture; wherein, the fermentation culture is divided into three stages of early fermentation stage and late fermentation stage; the fermentation culture is carried out for 0-12h as the early stage of fermentation, and the initial stage of fermentation is carried out under the process conditions of the culture temperature of 34-36 ℃, the rotating speed of 170-240rpm, the ventilation quantity of 1:0.7vvm and the dissolved oxygen saturation of more than or equal to 10 percent; the fermentation culture time is 12-24h, which is the later stage of fermentation, and the later stage of fermentation is carried out under the process conditions of the culture temperature of 36-38 ℃, the stirring speed of 450-.
4. The method for preparing a liquid microbial fertilizer as claimed in claim 3, wherein the method comprises the following steps: in the step 1), the formula of the planar solid culture medium and the formula of the inclined solid culture medium are the same, and both the formulas are as follows: 5g of peptone, 4g of NaCl, 1.5g of beef extract, 10g of agar and 1L of deionized water; and the plane solid culture medium and the inclined plane solid culture medium are sterilized and then used for subsequent bacterial culture.
5. The method for preparing a liquid microbial fertilizer as claimed in claim 3, wherein the method comprises the following steps: in the step 2), the formula of the liquid culture medium comprises 4g of glucose, 9g of soybean meal, 5g of agar, 10g of NaCl, 10g of peptone, 5g of yeast powder and 1L of deionized water; the liquid culture medium is sterilized and then used for culturing the activated bacillus subtilis strain.
6. The method for preparing a liquid microbial fertilizer as claimed in claim 3, wherein the method comprises the following steps: in the step 2), the rejuvenation medium comprises 4g of glucose, 4g of peptone, 4g of yeast powder and KH2PO4 0.1g、ZnSO4 2g、NaCl 4g、MnCl2 4g、Mg(NO3)2 3g、FeCl2 6g and 1L of deionized water; the rejuvenation culture medium is used for fermentation culture of fermentation strains subsequently after sterilization.
7. The method for preparing a liquid microbial fertilizer as claimed in claim 3, wherein the method comprises the following steps: in the step 3), the formula of the fermentation medium is as follows: 36kg of bean cake powder, 3.6kg of glucose, 3.3kg of beef extract, 33kg of corn flour and CaCl2 3.0kg、KH2PO4 1.5kg、MgSO4·7H2O 1.5kg、NH4Cl 5.5kg、NaCl 1.5kg、MnSO4 150g、KNO3 2.3kg, 720g of neutral protease (enzyme activity of 50000U/g) and 330g of moderate temperature alpha-amylase (enzyme activity of 5000U/g).
8. The method for preparing a liquid microbial fertilizer as claimed in claim 3, wherein the method comprises the following steps: the sterilized fermentation medium is prepared byThe preparation method comprises the following steps: adding 500L of deionized water into the fermentation tank, and adding bean cake powder, glucose, beef extract, corn flour and CaCl into the fermentation tank according to a certain proportion2、KH2PO4、MgSO4·7H2O、NH4Cl、NaCl、MnSO4And KNO3Then heating to 40-44 ℃, adding protease, and carrying out enzymolysis for 1.5h at a stirring speed of 100-150 rpm; then heating to 65-70 ℃, adding medium-temperature alpha-amylase, and carrying out enzymolysis for 1.5h at a stirring speed of 100-150 rpm; after the enzymolysis is finished, the fermentation medium is sterilized for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.11MPa, and then the sterilized fermentation medium can be prepared.
9. A method for preparing a microbial liquid bacterial fertilizer according to claim 1, which is characterized by comprising the following steps: taking bacillus subtilis fermentation broth, EM (effective microorganisms) fermentation broth, lactobacillus plantarum fermentation broth, yeast fermentation broth, polyglutamic acid, water and seaweed crystals according to a specified ratio, and uniformly mixing the raw materials at the temperature of 34-36 ℃ for 20min to obtain the microbial liquid bacterial fertilizer.
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CN101795685A (en) * 2007-06-08 2010-08-04 卑尔根技术股份有限公司 hydroxyproline compositions and uses thereof
CN107141028A (en) * 2017-05-31 2017-09-08 烟台海能生物肥料有限公司 A kind of preparation method of alga fertilizer
CN110054516A (en) * 2019-05-08 2019-07-26 内蒙古佰惠生新农业科技股份有限公司 A kind of beet liquid fertilizer specially prepared using yeast concentrate
CN110964676A (en) * 2020-01-04 2020-04-07 山东得和明兴生物科技有限公司 Method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101795685A (en) * 2007-06-08 2010-08-04 卑尔根技术股份有限公司 hydroxyproline compositions and uses thereof
CN107141028A (en) * 2017-05-31 2017-09-08 烟台海能生物肥料有限公司 A kind of preparation method of alga fertilizer
CN110054516A (en) * 2019-05-08 2019-07-26 内蒙古佰惠生新农业科技股份有限公司 A kind of beet liquid fertilizer specially prepared using yeast concentrate
CN110964676A (en) * 2020-01-04 2020-04-07 山东得和明兴生物科技有限公司 Method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus

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