CN115044509A - Preparation process of growth-promoting microbial agent - Google Patents
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Abstract
The invention discloses a preparation process of a growth-promoting microbial agent, belonging to the technical field of organic fertilizer and microbial fertilizer preparation. The invention screens and compounds three microbial strains, firstly cultures streptomyces corchorusii, then adds erwinia amyloliquefaciens and bacillus licheniformis for mixed culture to form the core component of the functional microbial inoculum. The microbial agent can effectively promote the absorption and utilization of nutrient substances in soil by crops, promotes 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 benefit and economic benefit.
Description
Technical Field
The invention belongs to the technical field of organic fertilizer and microbial fertilizer manufacturing, and particularly relates to a preparation process of a growth-promoting microbial agent.
Background
The rapid development of the fertilizer industry greatly promotes the development of agriculture in China, the grain yield is stably improved year by year, and the fertilizer plays a vital role in guaranteeing the national grain safety. However, the great investment of chemical fertilizers causes the problems of quality reduction of agricultural products, soil hardening, environmental pollution and the like, and the sustainable agricultural development road of green, organic and ecological environmental protection in China is seriously influenced. The microbial fertilizer is concerned with 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 is rapidly developed, and the method gradually transits from small workshops and soil methods to large-scale, standardized and industrialized production. The registration of microbial fertilizer, the identification requirement of agricultural microbial products, production technical rules, effectiveness evaluation, market supervision, biological safety criteria and other related standards are gradually improved. The microbial fertilizer is also developed into a compound type bacterial fertilizer from a bacterial fertilizer with single function, and a base fertilizer, an organic-inorganic compound bacterial fertilizer, a genetic engineering bacterial fertilizer, a biological organic fertilizer and the like appear in succession. At present, the microbial fertilizer in China can be divided into 3 types such as powder, granules, liquid and the like according to dosage forms, wherein the powder has a longer shelf life, but is easy to agglomerate when being moistened; the granular bacterial manure has a longer shelf life, but the processing mode can greatly reduce or even kill the activity of microorganisms; the liquid bacterial manure has the most effective live bacteria, but is not suitable for transportation and storage. The common microbial preparation in China is powder. The usage amount of the microbial fertilizer in China is increased rapidly, but a large gap still exists compared with the usage amount of the chemical fertilizer, which is related to the insufficient attention degree of the microbial fertilizer in China. The microbial fertilizer has higher market price, uneven product quality, bad property of used strains and easy influence of the environment on the activity, and farmers lack knowledge about the microbial fertilizer and are unscientific in operation in the application process, thus influencing the popularization of the microbial fertilizer in production.
Disclosure of Invention
The invention provides a preparation process of a biotype microbial agent, aiming at the problems in the prior art, the biotype microbial agent is in a solid particle shape and is prepared by mixing and granulating microbial agent microorganisms and carriers, and the obtained microbial agent has high biological activity, is stable and lasting, can continuously and effectively play a growth promoting role and has good inhibiting effect on part of pathogenic bacteria.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
a preparation process of growth-promoting microbial agent comprises the following steps:
(1) activating the strain: respectively activating Erwinia amyloliquefaciens, Streptomyces corchorusii and Bacillus licheniformis by using culture media, and culturing at 28-30 ℃ for 12-18h to obtain activated strains;
(2) preparing a seed solution: respectively inoculating the activated strains into culture bottles filled with liquid culture media, carrying out shake culture at 28-32 ℃ and a rotation speed of 160-;
(3) and (3) amplification culture: inoculating the streptomyces corchorusii seed liquid into a fermentation culture medium according to the volume ratio of 5-8%, wherein the fermentation temperature is 28-30 ℃, the rotating speed is 200-; then simultaneously inoculating the seed liquid of the Erwinia amyloliquefaciens and the Bacillus licheniformis to a fermentation culture medium for mixed culture at the fermentation temperature of 28-30 ℃, the rotation speed of 200-300r/min and the fermentation time of 24-36 h; after fermentation, obtaining mixed bacteria liquid, and spray drying to obtain mixed bacteria powder;
(4) preparing a biochar carrier: crushing waste biological straws, drying, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring to a tubular 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 fabric in 5mol/L hydrochloric acid solution for 1-2h, and repeatedly washing the fabric with deionized water until the pH value is unchanged; finally, placing the obtained powder in a vacuum drying oven at 105 ℃ for drying for 24h to obtain biomass charcoal;
(5) uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, then granulating by using a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min to fully and uniformly mix the materials, uniformly spraying 5% of polydiene alcohol aqueous solution and a proper amount of distilled water to granulate the materials, and drying after granulation is finished to obtain the growth-promoting microbial agent.
Further, the collection number of the starch-degrading Erwinia amylovora in the step (1) is CGMCC 1.7276, which is purchased from China general microbiological culture Collection center (CGMCC), No. 3 of Xilu No. 1 of Beijing, Chaoyang, North Chen, and the collection time is 2008, 3 months and 1 day; the preservation number of the streptomyces corchorusii is CGMCC 4.7392, and the streptomyces corchorusii is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu, North Cheng Yang area, Beijing, and the preservation time is 2016, 11 and 5 days; the Bacillus licheniformis has a preservation number of CGMCC 1.15832, and is purchased from China general microbiological culture Collection center (CGMCC), No. 3 of Xilu No. 1 of Beijing Korean district, with a preservation time of 2016, 9 years, 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: 1% of glucose, 1.5% of starch, 1.5% of bean cake powder, 1.8% of yeast powder, 0.2% of peptone, 0.3% of calcium carbonate, 0.03% of magnesium sulfate and the balance of water.
Further, the volume ratio of the Erwinia amylovora seed liquid, the Bacillus licheniformis seed liquid and the Streptomyces nummulosus seed liquid in the step (3) is 1:1: 1.
Further, the waste biological straws in the step (4) are wheat straws or corn straws.
Furthermore, the growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method comprises the following steps: on the basis of conventional fertilization, 3-5 kg/mu of microbial inoculum is fertilized in soil preparation.
Advantageous effects
Aiming at the defects of low activity, single function, unstable action and the like of microbial inoculum fertilizers in the prior art, the invention screens and compounds three microbial strains, firstly cultures streptomyces corchorusii, and then adds erwinia amyloliquefaciens and bacillus licheniformis for mixed culture to form the core component of the functional microbial inoculum. The streptomyces corchorusii can produce organic acidic substances such as succinic acid, lactic acid and the like, and can acidolyze insoluble phosphorus in soil to release phosphate ions; meanwhile, hydroxyl and carboxyl in the organic acid are combined with metal ions in the mineral substance to destroy the crystal structure of the mineral substance and cause the mineral substance to be decomposed, so that potassium element in the indissolvable mineral substance in the soil is released for the growth and development of plants. The Erwinia amylovora decomposes 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 plants on phosphorus elements is improved; the bacillus licheniformis can generate 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 the fruiting rate. The three bacteria cooperate with each other and act synergistically to promote the growth of crops.
In order to improve the biological activity and stability of active bacteria, the biomass charcoal is prepared from the waste biological straws, the internal porosity of the biomass charcoal is improved by pre-activating the biomass charcoal with sodium carbonate, the strain activity can be stimulated, and microbial strains are adsorbed and protected to continuously and effectively play a role, so that on one hand, the recycling of waste biological resources is realized, and on the other hand, the biomass charcoal is used as a carrier of a microbial agent, so that the microorganisms are effectively protected, the 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, realizes the recycling of waste biological straws, and has wide social and economic benefits.
Drawings
FIG. 1 is SEM images of the internal structures of the microbial inoculum particles obtained in example 3 of the invention and comparative example 1.
Detailed Description
The technical solution of the present invention is further described below with reference to specific embodiments, but is not limited thereto.
Example 1
A preparation process of growth-promoting microbial agent comprises the following steps:
(1) activating the strain: respectively activating Erwinia amyloliquefaciens, Streptomyces corchorusii and Bacillus licheniformis by using culture media, and culturing at 28-30 ℃ for 12h to obtain activated strains;
(2) preparing a seed solution: respectively inoculating the activated strains into culture bottles filled with liquid culture media, carrying out shake culture at 28-32 ℃ and a rotation speed of 160-;
(3) and (3) amplification culture: inoculating the streptomyces corchorusii seed liquid into a fermentation culture medium according to the volume ratio of 5 percent, wherein the fermentation temperature is 28-30 ℃, the rotating speed is 200-; then simultaneously inoculating the seed liquid of the Erwinia amyloliquefaciens and the Bacillus licheniformis to a fermentation culture medium for mixed culture at the fermentation temperature of 28-30 ℃, the rotation speed of 200-300r/min and the fermentation time of 24 h; after fermentation, obtaining mixed bacteria liquid, and spray drying to obtain mixed bacteria powder;
(4) preparing a biochar carrier: crushing waste biological straws, drying, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring to a tubular 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 fabric in 5mol/L hydrochloric acid solution for 1h, and repeatedly washing the fabric with deionized water until the pH value is unchanged; finally, placing the obtained powder in a vacuum drying oven at 105 ℃ for drying for 24h to obtain biomass charcoal;
(5) uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, then granulating by using a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min to fully and uniformly mix the materials, uniformly spraying 5% of polydiene alcohol aqueous solution and a proper amount of distilled water to granulate the materials, and drying after granulation is finished to obtain the growth-promoting microbial agent.
The collection number of the starch-degrading Erwinia amylovora is CGMCC 1.7276, and the Erwinia amylovora is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu-Chen in the south of Chaozhou, Beijing, and the collection time is 2008, 3 months and 1 day; the preservation number of the streptomyces corchorusii is CGMCC 4.7392, and the streptomyces corchorusii is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu, North Cheng Yang area, Beijing, and the preservation time is 2016, 11 and 5 days; the Bacillus licheniformis has a preservation number of CGMCC 1.15832, and is purchased from China general microbiological culture Collection center (CGMCC), No. 3 of Xilu No. 1 of Beijing Korean district, with a preservation time of 2016, 9 years, 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: 1% of glucose, 1.5% of starch, 1.5% of bean cake powder, 1.8% of yeast powder, 0.2% of peptone, 0.3% of calcium carbonate, 0.03% of magnesium sulfate and the balance of water.
And (3) adding the Erwinia amylovora seed liquid, the Bacillus licheniformis seed liquid and the Streptomyces nummulosus seed liquid in a volume ratio of 1:1: 1.
And (4) the waste biological straws are wheat straws.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method comprises the following steps: on the basis of conventional fertilization, 3-5 kg/mu of microbial inoculum is fertilized in soil preparation.
Example 2
A preparation process of growth-promoting microbial agent comprises the following steps:
(1) activating the strain: respectively activating Erwinia amyloliquefaciens, Streptomyces corchorusii and Bacillus licheniformis by using culture media, and culturing for 15h at 28-30 ℃ to obtain activated strains;
(2) preparing a seed solution: respectively inoculating the activated strains into culture bottles filled with liquid culture media, carrying out shake culture at 28-32 ℃ and a rotation speed of 160-;
(3) and (3) amplification culture: inoculating the streptomyces corchorusii seed liquid into a fermentation culture medium according to the volume ratio of 6 percent, wherein the fermentation temperature is 28-30 ℃, the rotating speed is 200-; then simultaneously inoculating the seed liquid of the Erwinia amyloliquefaciens and the Bacillus licheniformis to a fermentation culture medium for mixed culture at the fermentation temperature of 28-30 ℃, the rotation speed of 200-300r/min and the fermentation time of 30 h; after fermentation, obtaining mixed bacteria liquid, and spray drying to obtain mixed bacteria powder;
(4) preparing a biochar carrier: crushing waste biological straws, drying, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring to a tubular 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 fabric in 5mol/L hydrochloric acid solution for 1h, and repeatedly washing the fabric with deionized water until the pH value is unchanged; finally, placing the obtained powder in a vacuum drying oven at 105 ℃ for drying for 24h to obtain biomass charcoal;
(5) uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, then granulating by using a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min to fully mix the materials uniformly, then uniformly spraying 5% polydiene alcohol aqueous solution and a proper amount of distilled water to granulate the materials, and drying after granulation is finished to obtain the growth-promoting microbial agent.
The collection number of the starch-degrading Erwinia amylovora is CGMCC 1.7276, and the Erwinia amylovora is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu-Chen in the south of Chaozhou, Beijing, and the collection time is 2008, 3 months and 1 day; the preservation number of the streptomyces corchorusii is CGMCC 4.7392, and the streptomyces corchorusii is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu, North Cheng Yang area, Beijing, and the preservation time is 2016, 11 and 5 days; the Bacillus licheniformis has a preservation number of CGMCC 1.15832, and is purchased from China general microbiological culture Collection center (CGMCC), No. 3 of Xilu No. 1 of Beijing Korean district, with a preservation time of 2016, 9 years, 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: 1% of glucose, 1.5% of starch, 1.5% of bean cake powder, 1.8% of yeast powder, 0.2% of peptone, 0.3% of calcium carbonate, 0.03% of magnesium sulfate and the balance of water.
And (3) adding the Erwinia amylovora seed liquid, the Bacillus licheniformis seed liquid and the Streptomyces nummulosus seed liquid in a volume ratio of 1:1: 1.
And (4) the waste biological straws are corn straws.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method comprises the following steps: on the basis of conventional fertilization, 3-5 kg/mu of microbial inoculum is fertilized in soil preparation.
Example 3
A preparation process of growth-promoting microbial agent comprises the following steps:
(1) activating the strain: respectively activating Erwinia amyloliquefaciens, Streptomyces corchorusii and Bacillus licheniformis by using culture media, and culturing at 28-30 ℃ for 18h to obtain activated strains;
(2) preparing a seed solution: respectively inoculating the activated strains into culture bottles filled with liquid culture media, carrying out shake culture at 28-32 ℃ and a rotation speed of 160-;
(3) and (3) amplification culture: inoculating streptomyces corchorusii 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-; then simultaneously inoculating the seed liquid of the Erwinia amyloliquefaciens and the Bacillus licheniformis to a fermentation culture medium for mixed culture at the fermentation temperature of 28-30 ℃, the rotation speed of 200-300r/min and the fermentation time of 36 h; after fermentation, obtaining mixed bacteria liquid, and spray drying to obtain mixed bacteria powder;
(4) preparing a biochar carrier: crushing waste biological straws, drying, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring to a tubular furnace in a nitrogen atmosphere, heating to 600 ℃ at the heating rate of 5 ℃/min, and keeping for 2h for carbonization and activation; then, soaking the fabric in 5mol/L hydrochloric acid solution for 2 hours, and then repeatedly washing the fabric with deionized water until the pH value is unchanged; finally, placing the obtained powder in a vacuum drying oven at 105 ℃ for drying for 24h to obtain biomass charcoal;
(5) uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, then granulating by using a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min to fully and uniformly mix the materials, uniformly spraying 5% of polydiene alcohol aqueous solution and a proper amount of distilled water to granulate the materials, and drying after granulation is finished to obtain the growth-promoting microbial agent.
The collection number of the starch-degrading Erwinia amylovora is CGMCC 1.7276, and the Erwinia amylovora is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu-Chen in the south of Chaozhou, Beijing, and the collection time is 2008, 3 months and 1 day; the preservation number of the streptomyces corchorusii is CGMCC 4.7392, and the streptomyces corchorusii is purchased from China general microbiological culture Collection center (CGMCC), No. 3 of Xilu No. 1 of Beijing market on Chaoyang district, and the preservation time is 2016, 11 and 5 days; the Bacillus licheniformis has a preservation number of CGMCC 1.15832, and is purchased from China general microbiological culture Collection center (CGMCC), No. 3 of Xilu No. 1 of Beijing Korean district, with a preservation time of 2016, 9 years, 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: 1% of glucose, 1.5% of starch, 1.5% of bean cake powder, 1.8% of yeast powder, 0.2% of peptone, 0.3% of calcium carbonate, 0.03% of magnesium sulfate and the balance of water.
And (3) adding the Erwinia amylovora seed liquid, the Bacillus licheniformis seed liquid and the Streptomyces nummulosus seed liquid in a volume ratio of 1:1: 1.
And (4) the waste biological straws are wheat straws or corn straws.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method comprises the following steps: on the basis of conventional fertilization, 3-5 kg/mu of microbial inoculum is fertilized in soil preparation.
Comparative example 1
A preparation process of growth-promoting microbial agent comprises the following steps:
(1) activating the strain: respectively activating Erwinia amyloliquefaciens, Streptomyces corchorusii and Bacillus licheniformis by using culture media, and culturing at 28-30 ℃ for 18h to obtain activated strains;
(2) preparing a seed solution: respectively inoculating the activated strains into culture bottles filled with liquid culture media, carrying out shake culture at 28-32 ℃ and a rotation speed of 160-;
(3) and (3) amplification culture: inoculating streptomyces corchorusii 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-; then simultaneously inoculating the seed liquid of the Erwinia amyloliquefaciens and the Bacillus licheniformis to a fermentation culture medium for mixed culture at the fermentation temperature of 28-30 ℃, the rotation speed of 200-300r/min and the fermentation time of 36 h; after fermentation, obtaining mixed bacteria liquid, and spray drying to obtain mixed bacteria powder;
(4) preparing a biochar carrier: crushing waste biological straws, drying, transferring straw powder into a tubular 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 fabric in 5mol/L hydrochloric acid solution for 2 hours, and then repeatedly washing the fabric with deionized water until the pH value is unchanged; finally, placing the obtained powder in a vacuum drying oven at 105 ℃ for drying for 24h to obtain biomass charcoal;
(5) uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, then granulating by using a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min to fully and uniformly mix the materials, uniformly spraying 5% of polydiene alcohol aqueous solution and a proper amount of distilled water to granulate the materials, and drying after granulation is finished to obtain the growth-promoting microbial agent.
The collection number of the starch-degrading Erwinia amylovora is CGMCC 1.7276, and the Erwinia amylovora is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu-Chen in the south of Chaozhou, Beijing, and the collection time is 2008, 3 months and 1 day; the preservation number of the streptomyces corchorusii is CGMCC 4.7392, and the streptomyces corchorusii is purchased from China general microbiological culture Collection center (CGMCC), No. 3 Hospital No. 1 Xilu, North Cheng Yang area, Beijing, and the preservation time is 2016, 11 and 5 days; the Bacillus licheniformis has a preservation number of CGMCC 1.15832, and is purchased from China general microbiological culture Collection center (CGMCC), No. 3 of Xilu No. 1 of Beijing Korean district, with a preservation time of 2016, 9 years, 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: 1% of glucose, 1.5% of starch, 1.5% of bean cake powder, 1.8% of yeast powder, 0.2% of peptone, 0.3% of calcium carbonate, 0.03% of magnesium sulfate and the balance of water.
And (3) adding the Erwinia amylovora seed liquid, the Bacillus licheniformis seed liquid and the Streptomyces nummulosus seed liquid in a volume ratio of 1:1: 1.
And (4) the waste biological straws are wheat straws or corn straws.
The growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method comprises the following steps: on the basis of conventional fertilization, 3-5 kg/mu of microbial inoculum is fertilized in soil preparation.
The comparative example is the same as example 3 except that the straw is not activated by sodium carbonate.
Comparative example 2
In the step (3) of the comparative example, the volume ratio of the Erwinia amylovora seed solution to the Bacillus licheniformis seed solution to the Streptomyces nummulosus seed solution is 1:1:2, and the rest raw materials and the preparation method are the same as those in the example 3.
Comparative example 3
In the step (3) of the comparative example, the volume ratio of the Erwinia amylovora seed solution to the Bacillus licheniformis seed solution to the Streptomyces nummulosus seed solution is 1:2:1, and the rest raw materials and the preparation method are the same as those in the example 3.
Comparative example 4
In the step (3) of the comparative example, the volume ratio of the Erwinia amylovora seed solution to the Bacillus licheniformis seed solution to the Streptomyces nummulosus seed solution is 2:1:1, and the rest raw materials and the preparation method are the same as those in the example 3.
Comparative example 5
In the step (3) of the comparative example, the volume ratio of the Erwinia amylovora seed solution to the Bacillus licheniformis seed solution is 1:1, and the rest raw materials and the preparation method are the same as those in the example 3.
Comparative example 6
In the step (3) of the comparative example, the volume ratio of the Erwinia amylovora seed liquid to the Streptomyces nummulosus seed liquid is 1:1, and the rest raw materials and the preparation method are the same as those in the example 3.
Comparative example 7
In the step (3) of the comparative example, the volume ratio of the bacillus licheniformis seed liquid to the streptomyces nummulosus seed liquid is 1:1, and the rest raw materials and the preparation method are the same as those in the example 3.
Comparative example 8
In the step (3) of the comparative example, only streptomyces nummulosus seed liquid is added and inoculated in a fermentation medium according to the volume ratio of 8 percent, and the rest 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 Bacillus licheniformis seed solution is added and inoculated in the fermentation medium according to the volume ratio of 8 percent, and the rest raw materials and the preparation method are the same as the example 3.
Comparative example 10
In the step (3) of the comparative example, only the Erwinia amylovora seed solution is added and inoculated in the fermentation medium according to the volume ratio of 8 percent, and the rest raw materials and the preparation method are the same as those in the example 3
Performance testing
Determination of phosphate solubilizing Activity
And (3) qualitative detection: and (3) inoculating the mixed bacterial liquid obtained in the step (3) of the examples 1-3 and the comparative examples 2-10 on a Monkina solid culture medium, culturing at 30 ℃ for 7D, and preliminarily determining the phosphate-solubilizing capacity according to the ratio of the diameter (D) of the phosphate-solubilizing ring to the diameter (D) of the bacterial colony.
And (3) quantitative detection: 1mL of mixed bacterial liquid is inoculated in 50mL of Monkina liquid culture medium, and 1m L sterile bacterial suspension is inoculated in the Monkina liquid culture medium in a blank control group. Each treatment was repeated 3 times, and the cells were incubated at 28-30 ℃ for 1-7 days at 180 r/min. And (3) centrifuging the bacterial suspension at 4 ℃ 10000r/min for 10min from day 1 every day, taking the supernatant, and measuring the content of soluble phosphorus in the supernatant by using a molybdenum-antimony anti-colorimetric method. The phosphorus 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.
Determination of growth promoting Properties
1) And (4) measuring the IAA production capacity. The Salkowski method is used for measuring the secretion capacity of the auxin in the bacterial liquid. And (4) performing qualitative detection, namely determining whether the capability of secreting IAA is available or not by taking color change after the colorimetric solution is added and standing for 15min at room temperature as a judgment basis. And (3) quantitatively detecting, namely adding a colorimetric solution with twice volume to the supernatant obtained after the bacterial suspension is centrifuged, carrying out color development at room temperature in a dark place for 30min, measuring an OD (optical density) value at 530nm, and calculating the auxin production amount of the bacterial solution to be detected according to an auxin standard curve.
The results of the experiment are shown in table 1:
TABLE 1 results of phosphorus dissolution test
| Test group | D/d | Phosphorus amount (mg/L) | Produce IAA (mu g 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, it can be seen that after the three strains are mixed according to the equal volume, the phosphorus dissolving amount reaches 52mg/L, the IAA yield reaches 7 mu g/mL, and the synergistic effect among the three strains is weakened or disappeared in the comparative examples 2-10 with the changed strain proportion and the changed strain number, so that the phosphorus dissolving capacity and the growth promoting capacity are reduced. For further verification, the present invention also performed planting trials.
Planting test
Test site: in 2021, 4-6 months, three-party chemical planting test base in Linyi city Junan county, test soil, medium fertility
Test work: the green vegetables are cream pakchoi
Fertilizing: on the basis of conventional fertilization, the microbial agents obtained in examples 1-3 and comparative examples 1-10 of the invention are respectively applied in an amount of 3 kg/mu, and meanwhile, a conventional fertilization group is taken as a control example CK.
The effect of the planting test is shown in table 2:
TABLE 2 Chinese cabbage planting test results
| Test group | Weight per gram of individual plant | 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 fertilization CK | 58.1 | 20.6 | 2212.3 | - |
The data in the table show that the microbial agent has obvious growth promoting effect on the pakchoi, remarkably improves the yield of the pakchoi, changes the comparative examples 2-10 of the strain composition and the comparative example 1 without biochar activation, and slightly reduces the planting effect. The electron microscope observation of the internal surface topography of the microbial agent particles obtained in the embodiment 3 and the comparative example 1 shows that the biomass charcoal is in a porous and loose state, and can provide good protection for microorganisms, thereby slowly releasing nutrients and prolonging the fertilizer efficiency.
It should be noted that the above-mentioned embodiments are only some of the preferred modes for implementing the invention, and not all of them. It will be apparent to those skilled in the art that the above embodiments of the present invention are based on the present invention. All other embodiments obtained by workers without creative work shall fall within the protection scope of the present invention.
Claims (7)
1. A preparation process of a growth-promoting microbial agent is characterized by comprising the following steps:
(1) activating the strain: respectively activating Erwinia amyloliquefaciens, Streptomyces corchorusii and Bacillus licheniformis by using culture media, and culturing at 28-30 ℃ for 12-18h to obtain activated strains;
(2) preparing a seed solution: respectively inoculating the activated strains into culture bottles filled with liquid culture media, carrying out shake culture at 28-32 ℃ and a rotation speed of 160-;
(3) and (3) amplification culture: inoculating the streptomyces corchorusii 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 12 h; then simultaneously inoculating the seed liquid of the Erwinia amyloliquefaciens and the Bacillus licheniformis to a fermentation culture medium for mixed culture at the fermentation temperature of 28-30 ℃, the rotation speed of 200-300r/min and the fermentation time of 24-36 h; after fermentation, obtaining mixed bacteria liquid, and spray drying to obtain mixed bacteria powder;
(4) preparing a biochar carrier: crushing waste biological straws, drying, fully mixing straw powder and sodium carbonate according to the mass ratio of 1:0.5, transferring to a tubular 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 fabric in 5mol/L hydrochloric acid solution for 1-2h, and repeatedly washing the fabric with deionized water until the pH value is unchanged; finally, placing the obtained powder in a vacuum drying oven at 105 ℃ for drying for 24h to obtain biomass charcoal;
(5) uniformly mixing the mixed bacterial powder and the biochar according to the mass ratio of 3:10, then granulating by using a coating machine, setting the rotating speed of the coating machine to be 50r/min, stirring for 30min to fully and uniformly mix the materials, uniformly spraying 5% of polydiene alcohol aqueous solution and a proper amount of distilled water to granulate the materials, and drying after granulation is finished to obtain the growth-promoting microbial agent.
2. The process for preparing growth-promoting microbial agent according to claim 1, wherein the preservation number of the erwinia amylovora in the step (1) is CGMCC 1.7276, the preservation number of the streptomyces corchorusii is CGMCC 4.7392, and the preservation number of the bacillus paracasei is CGMCC 1.15832.
3. The process for preparing a growth-promoting microbial inoculant according to claim 1, wherein the liquid culture medium in 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.
4. The process for preparing growth-promoting microbial agent according to claim 1, wherein the fermentation medium in the step (3) is composed of: 1% of glucose, 1.5% of starch, 1.5% of bean cake powder, 1.8% of yeast powder, 0.2% of peptone, 0.3% of calcium carbonate, 0.03% of magnesium sulfate and the balance of water.
5. The process for preparing growth-promoting microbial agent according to claim 1, wherein the volume ratio of the Erwinia amylovora seed solution, the Bacillus licheniformis seed solution and the Streptomyces nummulosus seed solution added in the step (3) is 1:1: 1.
6. The process for preparing growth-promoting microbial inoculant according to claim 1, wherein the waste biological straw in the step (4) is wheat straw or corn straw.
7. The preparation process of the growth-promoting microbial agent according to claim 1, wherein the growth-promoting microbial agent contains more than or equal to 30 hundred million/g of effective viable bacteria, and the application method comprises the following steps: on the basis of conventional fertilization, 3-5 kg/mu of microbial inoculum is fertilized in soil preparation.
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