CN115044509B - Preparation method of growth-promoting microbial agent - Google Patents

Preparation method of growth-promoting microbial agent Download PDF

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CN115044509B
CN115044509B CN202210717657.4A CN202210717657A CN115044509B CN 115044509 B CN115044509 B CN 115044509B CN 202210717657 A CN202210717657 A CN 202210717657A CN 115044509 B CN115044509 B CN 115044509B
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microbial agent
growth
streptomyces
bacillus
fermentation
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张海涛
张磊
徐长青
徐东升
于文慧
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SHANDONG SANFANG CHEMICAL INDUSTRY GROUP CO LTD
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Abstract

The invention discloses a preparation method of a growth-promoting microbial agent, and belongs to the technical field of organic fertilizer and microbial fertilizer manufacturing. The invention screens and compounds three microorganism strains, firstly cultures streptomyces corchondri, then adds Erwinia amylovora and Bacillus paratlicheniformis to form the core component of the functional microbial agent after mixed culture. The microbial agent can effectively promote the absorption and utilization of nutrients in soil by crops, promote the growth of the crops, has small application amount and stable and durable fertilizer efficiency, is suitable for being applied to various crops, can greatly improve the agricultural planting efficiency and the crop quality, realizes the recycling of waste biological straws, and has wide social and economic benefits.

Description

Preparation method of growth-promoting microbial agent
Technical Field
The invention belongs to the technical field of organic fertilizer and microbial fertilizer manufacturing, and particularly relates to a preparation method of a growth-promoting microbial agent.
Background
The rapid development of the fertilizer industry greatly promotes the development of agriculture in China, and the grain yield is steadily improved year by year, thereby playing an important role in guaranteeing the national grain safety. However, the large amount of chemical fertilizer is put into the agricultural product, so that the quality of the agricultural product is reduced, the soil is hardened, the environment is polluted, and the sustainable agricultural development road of green, organic, ecological and environmental protection in China is seriously affected. The microbial fertilizer has the characteristics of improving soil structure, improving crop quality, improving plant disease and pest resistance and the like, is a requirement for developing green and ecological agriculture, and has wide application prospect.
The development of the microbial fertilizer industry rapidly develops, and the method is gradually changed from small workshops and soil to large-scale, standardized and industrialized methods. Registration of microbial fertilizers, identification requirements of agricultural microbial products, production technical specifications, effectiveness evaluation, market supervision, biosafety guidelines and other relevant standards are gradually perfected. The microbial fertilizer is also developed into a compound bacterial fertilizer from a bacterial fertilizer with single function, and base fertilizers, organic-inorganic compound bacterial fertilizers, genetic engineering bacterial fertilizers, biological organic fertilizers and the like are sequentially produced. At present, microbial fertilizers in China can be divided into 3 types of powder, granules, liquid and the like according to dosage forms, wherein the powder type has longer shelf life, but is easy to agglomerate when wetted; the quality guarantee period of the granular bacterial fertilizer is longer, but the processing mode can greatly reduce or even kill the microbial activity; the liquid bacterial fertilizer has the most effective viable bacteria, but is not suitable for transportation and preservation. The common microbial dosage forms in China are powder. The microbial fertilizer usage amount in China is rapidly increased, but a larger gap exists between the microbial fertilizer usage amount and the chemical fertilizer usage amount, which is related to the insufficient importance of the microbial fertilizer in China. The microbial fertilizer has higher market price and uneven product quality, the used strain has bad character and activity is easy to be influenced by environment, and farmers lack knowledge on the microbial fertilizer, and the microbial fertilizer is not scientific in operation in the application process, which easily causes bad effect, thereby influencing the popularization of the microbial fertilizer in production.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a preparation method of a biological microbial agent, which is prepared by mixing microbial agent microorganisms and a carrier and granulating, wherein the biological activity of the obtained microbial agent is high, the microbial agent is stable and durable, the growth-promoting effect can be continuously and effectively exerted, and meanwhile, the microbial agent has good inhibition effect on part of pathogenic bacteria.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the preparation method of the growth-promoting microbial agent comprises the following steps:
(1) Activating the strain: respectively activating Erwinia amylovora, streptomyces corchomicus and bacillus paralicheniformis by using a culture medium, and culturing for 12-18 hours at 28-30 ℃ to obtain an activated strain;
(2) Preparing seed liquid: respectively inoculating the activated strains into culture bottles filled with liquid culture medium, shake culturing at 28-32deg.C and rotation speed of 160-200r/min to logarithmic phase, and taking out as seed solution;
(3) And (3) performing expansion culture: inoculating streptomyces corchondri seed liquid into a fermentation culture medium according to the volume ratio of 5-8%, wherein the fermentation temperature is 28-30 ℃, the rotation speed is 200-300r/min, and the fermentation time is 12h; simultaneously inoculating the Erwinia amylovora and the Bacillus paratungensis seed solution into a fermentation medium, and performing mixed culture at 28-30deg.C at 200-300r/min for 24-36 hr; the fermentation is completed to obtain mixed bacterial liquid, and the mixed bacterial powder is obtained by spray drying;
(4) Preparing a biochar carrier: crushing and drying the waste biological straws, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring the mixture into a tube furnace in a nitrogen atmosphere, heating to 600 ℃ at the heating rate of 5 ℃/min, and keeping for 1-2h for carbonization and activation; then, soaking the mixture in 5mol/L hydrochloric acid solution for 1-2h, and repeatedly washing the mixture with deionized water until the pH value is unchanged; finally, placing the obtained powder into a vacuum drying oven at 105 ℃ for drying for 24 hours to obtain biomass charcoal;
(5) Uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, granulating by a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min, fully and uniformly mixing the materials, uniformly spraying 5% polydienol aqueous solution and a proper amount of distilled water, granulating the materials, and drying after granulating to obtain the growth-promoting microbial agent.
Further, the collection number of the erwinia amylovora in the step (1) is CGMCC 1.7276, which is purchased from China general microbiological culture collection center (CGMCC), north Qinghai West Lu No. 1, no. 3 in the Korean region of Beijing, and the collection time is 2008, 3 months and 1 day; the preservation number of the streptomyces corchondri is CGMCC 4.7392, and the streptomyces corchondri is purchased from China general microbiological culture collection center (CGMCC), the North western road No. 1 and No. 3 of the Korean region of Beijing, and the preservation time is 2016, 11, 5 and 5; the preservation number of the bacillus vice is CGMCC 1.15832, and the bacillus vice is purchased from China general microbiological culture collection center (CGMCC), and is 1 # 3 in North west way of the Korean area of Beijing, and the preservation time is 2016, 9, 20 days
Further, the liquid culture medium in the step (2) comprises 2-6% of corn flour, 2-6% of beef extract, 0.01-0.05% of magnesium sulfate, 0.01-0.03% of calcium chloride and the balance of water.
Further, the fermentation medium in the step (3) comprises the following components: glucose 1%, starch 1.5%, bean cake powder 1.5%, yeast powder 1.8%, peptone 0.2%, calcium carbonate 0.3%, magnesium sulfate 0.03%, and water in balance.
Further, the added volume ratio of the Erwinia amylovora seed liquid, the auxiliary bacillus licheniformis seed liquid and the streptomyces cannabinus seed liquid in the step (3) is 1:1:1.
Further, the waste biological straw in the step (4) is wheat or corn straw.
Furthermore, the growth-promoting microbial agent contains effective viable count more than or equal to 30 hundred million/g, and the application method is as follows: based on the conventional fertilization, 3-5 kg/mu of microbial agent is applied during soil preparation.
Advantageous effects
Aiming at the defects of low activity, single function, unstable action and the like of microbial agent fertilizer in the prior art, the invention screens and compounds three microbial strains, firstly cultures streptomyces corchorusii, and then adds Erwinia amylovora and Bacillus paratyphenius for mixed culture to form the core component of the functional microbial agent. The streptomyces corchondri can produce organic acidic substances such as succinic acid, lactic acid and the like, and can carry out acidolysis on insoluble phosphorus in soil to release phosphate ions; simultaneously, hydroxyl and carboxyl in the organic acid are combined with metal ions in the mineral substance to destroy the crystal structure and lead the mineral substance to decompose, so that potassium element in the indissolvable mineral substance in the soil is released for plant growth and development. The Erwinia amylovora can decompose phosphate components in soil through phosphatase, nuclease and the like generated in the metabolic activity process of the Erwinia amylovora, so that the utilization efficiency of the plant to phosphorus elements is improved; the bacillus paratungensis can produce active substances such as indoleacetic acid, cytokinin and the like in the metabolic process, promote plant germination and stem and leaf growth, promote lateral bud generation and plant flowering and fruiting, and improve fruiting rate. The three bacteria cooperate with each other and cooperate to promote the growth of crops.
In order to improve the bioactivity and stability of active bacteria, the invention prepares biomass charcoal by using waste biological straws, pre-activates the waste biological straws by using sodium carbonate, improves the internal porosity of the biomass charcoal, can excite the activity of bacteria, and adsorbs and protects the microorganism bacteria to continuously and effectively play roles, on one hand, the recycling of waste biological resources is realized, and on the other hand, the waste biological straws are used as carriers of microbial agents, so that microorganisms are effectively protected, slow release of active substances is realized, and the fertilizer efficiency is effectively prolonged.
In conclusion, the microbial agent can effectively promote the absorption and utilization of nutrients in soil by crops, promote the growth of the crops, has small application amount and stable and durable fertilizer efficiency, is suitable for being applied to various crops, can greatly improve the agricultural planting efficiency and the crop quality, and simultaneously realizes the recycling of waste biological straws, thereby having wide social and economic benefits.
Drawings
FIG. 1 is an SEM image of the internal structure of the microbial inoculum obtained in example 3 and comparative example 1 of the invention.
Detailed Description
The technical scheme of the present invention is further described below with reference to specific examples, but is not limited thereto.
Example 1
The preparation method of the growth-promoting microbial agent comprises the following steps:
(1) Activating the strain: respectively activating Erwinia amylovora, streptomyces corchomicus and bacillus paralicheniformis by using a culture medium, and culturing for 12 hours at 28-30 ℃ to obtain an activated strain;
(2) Preparing seed liquid: respectively inoculating the activated strains into culture bottles filled with liquid culture medium, shake culturing at 28-32deg.C and rotation speed of 160-200r/min to logarithmic phase, and taking out as seed solution;
(3) And (3) performing expansion culture: inoculating Streptomyces corchorusii seed liquid into a fermentation culture medium according to a volume ratio of 5%, wherein the fermentation temperature is 28-30 ℃, the rotation speed is 200-300r/min, and the fermentation time is 12 hours; simultaneously inoculating the Erwinia amylovora and the Bacillus paratungensis seed solution into a fermentation medium, and performing mixed culture at 28-30deg.C at 200-300r/min for 24 hr; the fermentation is completed to obtain mixed bacterial liquid, and the mixed bacterial powder is obtained by spray drying;
(4) Preparing a biochar carrier: crushing and drying waste biological straws, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring the mixture into a tube furnace in a nitrogen atmosphere, heating to 600 ℃ at the heating rate of 5 ℃/min, and keeping for 1h for carbonization and activation; then, soaking the mixture in 5mol/L hydrochloric acid solution for 1h, and repeatedly washing the mixture with deionized water until the pH value is unchanged; finally, placing the obtained powder into a vacuum drying oven at 105 ℃ for drying for 24 hours to obtain biomass charcoal;
(5) Uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, granulating by a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min, fully and uniformly mixing the materials, uniformly spraying 5% polydienol aqueous solution and a proper amount of distilled water, granulating the materials, and drying after granulating to obtain the growth-promoting microbial agent.
The preservation number of the Erwinia amylovora in the step (1) is CGMCC 1.7276, the Erwinia amylovora is purchased from China general microbiological culture collection center (CGMCC), the North Chen West Lu No. 1, no. 3 in the Korean area of Beijing city, and the preservation time is 2008 3 months and 1 day; the preservation number of the streptomyces corchondri is CGMCC 4.7392, and the streptomyces corchondri is purchased from China general microbiological culture collection center (CGMCC), the North western road No. 1 and No. 3 of the Korean region of Beijing, and the preservation time is 2016, 11, 5 and 5; the preservation number of the bacillus vice is CGMCC 1.15832, and the bacillus vice is purchased from China general microbiological culture collection center (CGMCC), and is 1 # 3 in North west way of the Korean area of Beijing, and the preservation time is 2016, 9, 20 days
The liquid culture medium in the step (2) comprises 2-6% of corn flour, 2-6% of beef extract, 0.01-0.05% of magnesium sulfate, 0.01-0.03% of calcium chloride and the balance of water.
The fermentation medium in the step (3) comprises the following components: glucose 1%, starch 1.5%, bean cake powder 1.5%, yeast powder 1.8%, peptone 0.2%, calcium carbonate 0.3%, magnesium sulfate 0.03%, and water in balance.
The added volume ratio of the Erwinia amylovora seed liquid to the Bacillus paratungensis seed liquid and the Streptomyces cannabinus seed liquid in the step (3) is 1:1:1.
The waste biological straw in the step (4) is wheat straw.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method is as follows: based on the conventional fertilization, 3-5 kg/mu of microbial agent is applied during soil preparation.
Example 2
The preparation method of the growth-promoting microbial agent comprises the following steps:
(1) Activating the strain: respectively activating Erwinia amylovora, streptomyces corchomicus and bacillus paralicheniformis by using a culture medium, and culturing for 15 hours at 28-30 ℃ to obtain an activated strain;
(2) Preparing seed liquid: respectively inoculating the activated strains into culture bottles filled with liquid culture medium, shake culturing at 28-32deg.C and rotation speed of 160-200r/min to logarithmic phase, and taking out as seed solution;
(3) And (3) performing expansion culture: inoculating the streptomyces corchondri seed liquid into a fermentation culture medium according to the volume ratio of 6%, wherein the fermentation temperature is 28-30 ℃, the rotation speed is 200-300r/min, and the fermentation time is 12h; simultaneously inoculating the Erwinia amylovora and the Bacillus paratungensis seed solution into a fermentation medium, and performing mixed culture at 28-30deg.C at 200-300r/min for 30 hr; the fermentation is completed to obtain mixed bacterial liquid, and the mixed bacterial powder is obtained by spray drying;
(4) Preparing a biochar carrier: crushing and drying waste biological straws, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring the mixture into a tube furnace in a nitrogen atmosphere, heating to 600 ℃ at the heating rate of 5 ℃/min, and keeping for 1h for carbonization and activation; then, soaking the mixture in 5mol/L hydrochloric acid solution for 1h, and repeatedly washing the mixture with deionized water until the pH value is unchanged; finally, placing the obtained powder into a vacuum drying oven at 105 ℃ for drying for 24 hours to obtain biomass charcoal;
(5) Uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, granulating by a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min, fully and uniformly mixing the materials, uniformly spraying 5% polydienol aqueous solution and a proper amount of distilled water, granulating the materials, and drying after granulating to obtain the growth-promoting microbial agent.
The preservation number of the Erwinia amylovora in the step (1) is CGMCC 1.7276, the Erwinia amylovora is purchased from China general microbiological culture collection center (CGMCC), the North Chen West Lu No. 1, no. 3 in the Korean area of Beijing city, and the preservation time is 2008 3 months and 1 day; the preservation number of the streptomyces corchondri is CGMCC 4.7392, and the streptomyces corchondri is purchased from China general microbiological culture collection center (CGMCC), the North western road No. 1 and No. 3 of the Korean region of Beijing, and the preservation time is 2016, 11, 5 and 5; the preservation number of the bacillus vice is CGMCC 1.15832, and the bacillus vice is purchased from China general microbiological culture collection center (CGMCC), and is 1 # 3 in North west way of the Korean area of Beijing, and the preservation time is 2016, 9, 20 days
The liquid culture medium in the step (2) comprises 2-6% of corn flour, 2-6% of beef extract, 0.01-0.05% of magnesium sulfate, 0.01-0.03% of calcium chloride and the balance of water.
The fermentation medium in the step (3) comprises the following components: glucose 1%, starch 1.5%, bean cake powder 1.5%, yeast powder 1.8%, peptone 0.2%, calcium carbonate 0.3%, magnesium sulfate 0.03%, and water in balance.
The added volume ratio of the Erwinia amylovora seed liquid to the Bacillus paratungensis seed liquid and the Streptomyces cannabinus seed liquid in the step (3) is 1:1:1.
The waste biological straw in the step (4) is corn straw.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method is as follows: based on the conventional fertilization, 3-5 kg/mu of microbial agent is applied during soil preparation.
Example 3
The preparation method of the growth-promoting microbial agent comprises the following steps:
(1) Activating the strain: respectively activating Erwinia amylovora, streptomyces corchomicus and bacillus paralicheniformis by using a culture medium, and culturing for 18 hours at 28-30 ℃ to obtain an activated strain;
(2) Preparing seed liquid: respectively inoculating the activated strains into culture bottles filled with liquid culture medium, shake culturing at 28-32deg.C and rotation speed of 160-200r/min to logarithmic phase, and taking out as seed solution;
(3) And (3) performing expansion culture: inoculating streptomyces corchondri seed liquid into a fermentation culture medium according to the volume ratio of 8%, wherein the fermentation temperature is 28-30 ℃, the rotation speed is 200-300r/min, and the fermentation time is 12h; simultaneously inoculating the Erwinia amylovora and the Bacillus paratungensis seed solution into a fermentation medium, and performing mixed culture at 28-30deg.C at 200-300r/min for 36 hr; the fermentation is completed to obtain mixed bacterial liquid, and the mixed bacterial powder is obtained by spray drying;
(4) Preparing a biochar carrier: crushing and drying waste biological straws, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring the mixture into a tube furnace in a nitrogen atmosphere, heating to 600 ℃ at the heating rate of 5 ℃/min, and keeping for 2 hours for carbonization and activation; then, soaking the mixture in 5mol/L hydrochloric acid solution for 2 hours, and repeatedly washing the mixture with deionized water until the pH value is unchanged; finally, placing the obtained powder into a vacuum drying oven at 105 ℃ for drying for 24 hours to obtain biomass charcoal;
(5) Uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, granulating by a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min, fully and uniformly mixing the materials, uniformly spraying 5% polydienol aqueous solution and a proper amount of distilled water, granulating the materials, and drying after granulating to obtain the growth-promoting microbial agent.
The preservation number of the Erwinia amylovora in the step (1) is CGMCC 1.7276, the Erwinia amylovora is purchased from China general microbiological culture collection center (CGMCC), the North Chen West Lu No. 1, no. 3 in the Korean area of Beijing city, and the preservation time is 2008 3 months and 1 day; the preservation number of the streptomyces corchondri is CGMCC 4.7392, and the streptomyces corchondri is purchased from China general microbiological culture collection center (CGMCC), the North western road No. 1 and No. 3 of the Korean region of Beijing, and the preservation time is 2016, 11, 5 and 5; the preservation number of the bacillus vice is CGMCC 1.15832, and the bacillus vice is purchased from China general microbiological culture collection center (CGMCC), and is 1 # 3 in North west way of the Korean area of Beijing, and the preservation time is 2016, 9, 20 days
The liquid culture medium in the step (2) comprises 2-6% of corn flour, 2-6% of beef extract, 0.01-0.05% of magnesium sulfate, 0.01-0.03% of calcium chloride and the balance of water.
The fermentation medium in the step (3) comprises the following components: glucose 1%, starch 1.5%, bean cake powder 1.5%, yeast powder 1.8%, peptone 0.2%, calcium carbonate 0.3%, magnesium sulfate 0.03%, and water in balance.
The added volume ratio of the Erwinia amylovora seed liquid to the Bacillus paratungensis seed liquid and the Streptomyces cannabinus seed liquid in the step (3) is 1:1:1.
The waste biological straw in the step (4) is wheat or corn straw.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method is as follows: based on the conventional fertilization, 3-5 kg/mu of microbial agent is applied during soil preparation.
Comparative example 1
The preparation method of the growth-promoting microbial agent comprises the following steps:
(1) Activating the strain: respectively activating Erwinia amylovora, streptomyces corchomicus and bacillus paralicheniformis by using a culture medium, and culturing for 18 hours at 28-30 ℃ to obtain an activated strain;
(2) Preparing seed liquid: respectively inoculating the activated strains into culture bottles filled with liquid culture medium, shake culturing at 28-32deg.C and rotation speed of 160-200r/min to logarithmic phase, and taking out as seed solution;
(3) And (3) performing expansion culture: inoculating streptomyces corchondri seed liquid into a fermentation culture medium according to the volume ratio of 8%, wherein the fermentation temperature is 28-30 ℃, the rotation speed is 200-300r/min, and the fermentation time is 12h; simultaneously inoculating the Erwinia amylovora and the Bacillus paratungensis seed solution into a fermentation medium, and performing mixed culture at 28-30deg.C at 200-300r/min for 36 hr; the fermentation is completed to obtain mixed bacterial liquid, and the mixed bacterial powder is obtained by spray drying;
(4) Preparing a biochar carrier: crushing and drying the waste biological straws, transferring the straw powder into a tube furnace in a nitrogen atmosphere, heating to 600 ℃ at a heating rate of 5 ℃/min, and keeping for 2 hours for carbonization and activation; then, soaking the mixture in 5mol/L hydrochloric acid solution for 2 hours, and repeatedly washing the mixture with deionized water until the pH value is unchanged; finally, placing the obtained powder into a vacuum drying oven at 105 ℃ for drying for 24 hours to obtain biomass charcoal;
(5) Uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, granulating by a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min, fully and uniformly mixing the materials, uniformly spraying 5% polydienol aqueous solution and a proper amount of distilled water, granulating the materials, and drying after granulating to obtain the growth-promoting microbial agent.
The preservation number of the Erwinia amylovora in the step (1) is CGMCC 1.7276, the Erwinia amylovora is purchased from China general microbiological culture collection center (CGMCC), the North Chen West Lu No. 1, no. 3 in the Korean area of Beijing city, and the preservation time is 2008 3 months and 1 day; the preservation number of the streptomyces corchondri is CGMCC 4.7392, and the streptomyces corchondri is purchased from China general microbiological culture collection center (CGMCC), the North western road No. 1 and No. 3 of the Korean region of Beijing, and the preservation time is 2016, 11, 5 and 5; the preservation number of the bacillus vice is CGMCC 1.15832, and the bacillus vice is purchased from China general microbiological culture collection center (CGMCC), and is 1 # 3 in North west way of the Korean area of Beijing, and the preservation time is 2016, 9, 20 days
The liquid culture medium in the step (2) comprises 2-6% of corn flour, 2-6% of beef extract, 0.01-0.05% of magnesium sulfate, 0.01-0.03% of calcium chloride and the balance of water.
The fermentation medium in the step (3) comprises the following components: glucose 1%, starch 1.5%, bean cake powder 1.5%, yeast powder 1.8%, peptone 0.2%, calcium carbonate 0.3%, magnesium sulfate 0.03%, and water in balance.
The added volume ratio of the Erwinia amylovora seed liquid to the Bacillus paratungensis seed liquid and the Streptomyces cannabinus seed liquid in the step (3) is 1:1:1.
The waste biological straw in the step (4) is wheat or corn straw.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method is as follows: based on the conventional fertilization, 3-5 kg/mu of microbial agent is applied during soil preparation.
This comparative example was the same as example 3 except that the straw was not activated with sodium carbonate.
Comparative example 2
The comparative example step (3) was carried out by adding the Erwinia amylovora seed solution, the Bacillus paratlicheniformis seed solution and the Streptomyces cannabinus seed solution in a volume ratio of 1:1:2, and the other raw materials and the preparation method were the same as in example 3.
Comparative example 3
The comparative example step (3) was carried out by adding the Erwinia amylovora seed solution, the Bacillus paratlicheniformis seed solution and the Streptomyces cannabinus seed solution in a volume ratio of 1:2:1, and the other raw materials and the preparation method were the same as in example 3.
Comparative example 4
The comparative example step (3) was carried out by adding 2:1:1 by volume of Erwinia amylovora seed solution, bacillus licheniformis seed solution and Streptomyces cannabinus seed solution, and the other raw materials and preparation method were the same as in example 3.
Comparative example 5
The comparative example step (3) of the Erwinia amylovora seed solution and the Bacillus paratlicheniformis seed solution are added in a volume ratio of 1:1, and the rest raw materials and the preparation method are the same as those of the example 3.
Comparative example 6
The comparative example step (3) was carried out by adding the Erwinia amylovora seed solution and the Streptomyces cannabinus seed solution in a volume ratio of 1:1, and the other raw materials and the preparation method were the same as in example 3.
Comparative example 7
The comparative example step (3) was carried out with a volume ratio of 1:1 of the seed solution of Bacillus licheniformis and the seed solution of Streptomyces cannabinus, and the other raw materials and preparation methods were the same as in example 3.
Comparative example 8
In the step (3) of the comparative example, only Streptomyces cannabinus seed liquid is added, and inoculated into a fermentation medium according to the volume ratio of 8%, and the other raw materials and the preparation method are the same as those in the example 3
Comparative example 9
In the step (3) of the comparative example, only the seed liquid of the auxiliary bacillus licheniformis is added, and the seed liquid is inoculated into a fermentation medium according to the volume ratio of 8%, and the rest raw materials and the preparation method are the same as those in the example 3.
Comparative example 10
In the step (3) of the comparative example, only Erwinia amylovora seed liquid is added, and the Erwinia amylovora seed liquid is inoculated into a fermentation medium according to the volume ratio of 8%, and the other raw materials and the preparation method are the same as those in the example 3
Performance testing
Determination of phosphate solubilizing Activity
Qualitative detection: taking the mixed bacterial solutions of the steps (3) of the examples 1-3 and the comparative examples 2-10, inoculating the mixed bacterial solutions on a Meng Jinna solid culture medium, culturing for 7D at 30 ℃, and preliminarily determining the phosphate dissolving capacity according to the ratio of the diameter (D) of the phosphate dissolving ring to the diameter (D) of the bacterial colony.
Quantitative detection: 1mL of the mixed bacterial liquid is inoculated into 50mL of Meng Jinna liquid culture medium, and a blank control group is inoculated into 1mL of sterilized bacterial suspension in Meng Jinna liquid culture medium. Each treatment was repeated 3 times while culturing at 28-30deg.C 180r/min for 1-7d. Taking the bacterial suspension every day at regular time from day 1, centrifuging at 4 ℃ for 10min at 10000r/min, taking the supernatant, and measuring the content of soluble phosphorus in the supernatant by using a molybdenum-antimony colorimetric method. The phosphate dissolving amount of the strain to be detected is the difference value of the soluble phosphorus content of the experimental group and the blank control group.
Measurement of growth promoting Properties
1) IAA production ability measurement. The Salkowski method is used for measuring the secretion capacity of the bacterial liquid auxin. Qualitative detection, namely determining whether the color of the color mixture has the capability of secreting IAA by taking the color change of the color mixture after the color mixture is added into a colorimetric solution and is kept stand at room temperature for 15min as a judgment basis. And (3) quantitatively detecting, namely adding a diploid volume colorimetric solution into a supernatant obtained after the centrifugation of the bacterial suspension, developing at a dark place at room temperature for 30min, measuring an OD value at 530nm, and calculating the auxin production amount of the bacterial solution to be detected by referring to an auxin standard curve.
The experimental results are shown in table 1:
TABLE 1 phosphorus dissolution test results
Test group D/d Phosphate solubilizing amount (mg/L) IAA production (μg mL) -1 )
Example 1 1.45 52.36 7.12
Example 2 1.46 53.21 7.26
Example 3 1.46 53.55 7.31
Comparative example 2 1.33 45.36 6.12
Comparative example 3 1.31 41.25 6.23
Comparative example 4 1.33 44.69 6.02
Comparative example 5 1.25 36.25 4.21
Comparative example 6 1.28 38.69 3.25
Comparative example 7 1.25 36.14 4.09
Comparative example 8 1.22 30.22 0.65
Comparative example 9 1.06 8.69 1.3
Comparative example 10 1.15 29.96 0.71
Blank control 0 0.23 0
From the data in the table, we can see that after three strains of the embodiment of the invention are mixed according to the equal volume, the phosphate dissolving amount reaches 52mg/L, the IAA yield reaches 7 mug/mL, and the synergistic effect between the three strains is weakened or eliminated according to the comparative examples of the changed strain proportion and the strain number of 2-10, so that the phosphate dissolving capacity and the growth promoting capacity are reduced. For further verification, the invention also performed a planting test.
Planting test
Experimentally: 2021, 4-6 months, three-party chemical planting test base in the city of Dahurian county in Yi-city, soil in the test base, fertility and the like
Test crop: vegetable, such as butter cabbage
And (3) fertilization: the microbial agents obtained in examples 1-3 and comparative examples 1-10 of the present invention were applied in an amount of 3 kg/mu based on the conventional fertilization, respectively, while the conventional fertilization group was used as a control CK.
The planting test effect is shown in table 2:
TABLE 2 experiment results of cabbage planting
Test group Individual weight/g Plant height/cm 667m 2 Yield/kg Yield increase/%
Example 1 101.2 26.3 2812.3 27.12
Example 2 105.3 26.9 2825.9 27.74
Example 3 106.9 27.5 2899.4 31.06
Comparative example 1 93.8 25.6 2655.5 20.03
Comparative example 2 91.3 24.3 2601.2 17.58
Comparative example 3 91.5 25.3 2623.2 18.57
Comparative example 4 90.1 24.1 2585.9 16.89
Comparative example 5 80.3 23.2 2436.8 10.15
Comparative example 6 81.6 23.1 2444.8 10.51
Comparative example 7 78.2 22.5 2401.3 8.54
Comparative example 8 61.2 22.9 2323.5 5.03
Comparative example 9 62.3 21.2 2315.9 4.68
Comparative example 10 60.8 21.3 2289.3 3.48
Conventional fertilizing CK 58.1 20.6 2212.3 -
From the data in the table, the microbial inoculum has remarkable effect on promoting the growth of the cabbages, remarkably improves the yield of the cabbages, changes the comparative examples 2-10 of the strain composition and the comparative example 1 without activated charcoal, and slightly reduces the planting effect. The internal surface morphology electron microscopy observation of the microbial agent particles obtained in the embodiment 3 and the comparative example 1 also shows that the biomass charcoal of the invention is in a porous and loose state, and can provide good protection for microorganisms, so that nutrients are slowly released and fertilizer efficiency is prolonged.
It should be noted that the above-mentioned embodiments are merely some, but not all embodiments of the preferred mode of carrying out the invention. It is apparent that based on the above embodiments of the present invention, one of ordinary skill in the art would recognize. All other embodiments obtained by the person without making any inventive effort shall fall within the scope of protection of the present invention.

Claims (3)

1. The preparation method of the growth-promoting microbial agent is characterized by comprising the following steps of:
(1) Activating the strain: respectively activating Erwinia amylovora, streptomyces corchomicus and bacillus paralicheniformis by using a culture medium, and culturing for 12-18 hours at 28-30 ℃ to obtain an activated strain;
(2) Preparing seed liquid: respectively inoculating the activated strains into culture bottles filled with liquid culture medium, shake culturing at 28-32deg.C and rotation speed of 160-200r/min to logarithmic phase, and taking out as seed solution;
(3) And (3) performing expansion culture: inoculating streptomyces corchondri seed liquid into a fermentation culture medium according to the volume ratio of 5-8%, wherein the fermentation temperature is 28-30 ℃, the rotation speed is 200-300r/min, and the fermentation time is 12h; simultaneously inoculating the Erwinia amylovora and the Bacillus paratungensis seed solution into a fermentation medium, and performing mixed culture at 28-30deg.C at 200-300r/min for 24-36 hr; the fermentation is completed to obtain mixed bacterial liquid, and the mixed bacterial powder is obtained by spray drying;
(4) Preparing a biochar carrier: crushing and drying the waste biological straws, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring the mixture into a tube furnace in a nitrogen atmosphere, heating to 600 ℃ at the heating rate of 5 ℃/min, and keeping for 1-2h for carbonization and activation; then, soaking the mixture in 5mol/L hydrochloric acid solution for 1-2h, and repeatedly washing the mixture with deionized water until the pH value is unchanged; finally, placing the obtained powder into a vacuum drying oven at 105 ℃ for drying for 24 hours to obtain biomass charcoal;
(5) Uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, granulating by a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min, fully and uniformly mixing the materials, uniformly spraying 5% polydienol aqueous solution and a proper amount of distilled water, granulating the materials, and drying after granulating to obtain the growth-promoting microbial agent;
the preservation number of the erwinia amylovora in the step (1) is CGMCC 1.7276, the preservation number of the streptomyces corchondrillensis is CGMCC 4.7392, and the preservation number of the auxiliary bacillus licheniformis is CGMCC 1.15832;
the composition of the liquid culture medium in the step (2) is as follows: corn flour 2-6%, beef extract 2-6%, magnesium sulfate 0.01-0.05%, calcium chloride 0.01-0.03%, and water in balance;
the fermentation medium in the step (3) comprises the following components: glucose 1%, starch 1.5%, bean cake powder 1.5%, yeast powder 1.8%, peptone 0.2%, calcium carbonate 0.3%, magnesium sulfate 0.03%, and water in balance;
the Erwinia amylovora seed liquid, the Bacillus paratungensis seed liquid and the Streptomyces cannabinus seed liquid are added in a volume ratio of 1:1:1;
the growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria.
2. The method for preparing a growth-promoting microbial agent according to claim 1, wherein the waste biological straw in the step (4) is wheat or corn straw.
3. The method for preparing a growth-promoting microbial agent according to claim 1, wherein the method for applying the growth-promoting microbial agent is as follows: based on the conventional fertilization, 3-5 kg/mu of microbial agent is applied during soil preparation.
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