CN113004084A - Biological bacterial fertilizer and preparation method thereof - Google Patents
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Abstract
A biological bacterial fertilizer and a preparation method thereof belong to the technical field of soil fertilizers and agricultural biology and aim to solve the problems in the prior art. The biological bacterial fertilizer comprises, by weight, 3-5 parts of tertiary diatomite, 20-30 parts of straws, 8-15 parts of protein residues, 40-60 parts of chicken manure and 5-10 parts of a mixed microbial inoculum; the mixed microbial inoculum is prepared from bacillus subtilis, aspergillus oryzae and aspergillus niger in a mass ratio of 1-5:1: 1. The preparation method comprises the following steps: crushing the straws into fragments; uniformly mixing 20-30 parts of fragments with 3-5 parts of tertiary diatomite, 8-15 parts of protein residues and 40-60 parts of chicken manure according to parts by weight, and adjusting the water content to 50-60% to obtain a mixture; decomposing and sterilizing the mixture for 20-40 days at 50-60 deg.C to obtain microbial inoculum carrier; the mixed microbial inoculum is inoculated into a microbial inoculum carrier according to the inoculation amount of 5-10 percent, and the mixture is uniformly mixed, the culture temperature is 20-30 ℃, and the culture time is 10-15 days; drying at 35-45 ℃ until the water content is less than or equal to 30 percent to obtain the biological bacterial fertilizer.
Description
Technical Field
The invention belongs to the technical field of soil fertilizers and agricultural biology, and particularly relates to a biological bacterial fertilizer based on three-level diatomite and a preparation method thereof.
Background
The total reserve of diatomite is the second place in the world in China, but the grade of diatomite is generally low. When the diatomite ore is developed, most of the low-grade diatomite ore, namely the tertiary diatomite, is not effectively utilized, but is directly abandoned, so that resources are wasted, and meanwhile, the ecological environment is polluted.
For a long time, while the single, continuous and large-scale application of chemical fertilizers in agriculture in China improves crop yield, a series of negative effects are caused, so that the physicochemical properties of soil are changed, the biological system is poor and the functions are degraded, and further the further improvement of the crop yield is limited. In addition, the utilization rate of the fertilizer in the season is low, most of the fertilizer is lost through volatilization, leaching and soil fixation, so that environmental pollution is caused, and the threat and challenge are formed on the safety of agricultural products and the sustainable development of agriculture. A large number of researches prove that the microbial fertilizer has the effects of improving the utilization efficiency of the fertilizer, improving the physical and chemical properties of soil, absorbing heavy metals and residual pesticides in the soil, inhibiting soil-borne diseases and the like, thereby playing an irreplaceable role in improving the quality of agricultural products and the safety of foods. The liquid bacterial fertilizer in the microbial fertilizer is not as widely applied as the solid bacterial fertilizer due to the defects of inconvenient long-distance transportation, low viable bacteria number in the microbial agent, easy pollution and the like. The survival of beneficial bacteria in the solid bacterial manure is influenced by a plurality of factors, such as temperature, humidity, water content and the like, and the selection of a carrier is one of the most critical factors, can provide a suitable environment for the survival and release of microorganisms, and plays a decisive role in the quality of the bacterial manure. However, at present, people lack sufficient knowledge on the importance of the carrier, and the carrier is very random to select, so that the use requirement cannot be well met.
Disclosure of Invention
The invention aims to provide a biological bacterial fertilizer and a preparation method thereof, and solves the problems of inconvenience in long-distance transportation of liquid bacterial fertilizers, low viable bacteria in the fertilizer, easiness in pollution and the like in the prior art and the problem of low survival rate caused by serious influence of beneficial bacteria in solid bacterial fertilizers on the outside. Provides a carrier with certain nutrients and low cost, realizes the improvement of the soil ecological environment and simultaneously meets the preservation requirement of the microbial agent.
In order to achieve the purpose, the biological bacterial fertilizer comprises, by weight, 3-5 parts of tertiary diatomite, 20-30 parts of straw, 8-15 parts of protein residue, 40-60 parts of chicken manure and 5-10 parts of a mixed microbial inoculum;
the mixed microbial inoculum is prepared from bacillus subtilis, aspergillus oryzae and aspergillus niger in a mass ratio of 1-5:1: 1.
The ratio of the bacillus subtilis to the aspergillus oryzae to the aspergillus niger in the mixed microbial inoculum is 5:1: 1.
A preparation method of the biological bacterial fertilizer comprises the following steps:
the method comprises the following steps: crushing the straws into fragments;
step two: uniformly mixing 20-30 parts of the fragments obtained in the step one with 3-5 parts of tertiary diatomite, 8-15 parts of protein residues and 40-60 parts of chicken manure according to parts by weight, and adjusting the water content to 50-60% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step two for 20-40 days at 50-60 ℃ to obtain a microbial inoculum carrier;
step four: respectively carrying out activation and propagation on three preserved microorganisms, namely bacillus subtilis, aspergillus oryzae and aspergillus niger to obtain three propagation bacteria liquids;
step five: preparing three expanded culture bacteria liquids obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 1-5:1: 1;
step six: inoculating the mixed microbial inoculum obtained in the step five into the microbial inoculum carrier obtained in the step three according to the inoculation amount of 5-10%, and uniformly mixing, wherein the culture temperature is 20-30 ℃, and the culture time is 10-15 days; drying at 35-45 ℃ until the water content is less than or equal to 30 percent to obtain the biological bacterial fertilizer.
The expanding culture in the fourth step specifically comprises the following steps:
b, expanding culture of the bacillus subtilis: firstly, inoculating bacillus subtilis into a sterilized LB culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 37 ℃ for 24h at the rotation speed of 200 rpm; then transferring the strain into a new sterilized LB culture medium, wherein the strain inoculation amount is 10%, and performing shake culture at the culture temperature of 37 ℃ for 36 h;
expanding culture of aspergillus oryzae: firstly, inoculating aspergillus oryzae in a sterilized PDA culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
expanding culture of aspergillus niger: firstly, inoculating Aspergillus niger in a sterilized PDA culture medium, and performing shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
the LB culture medium is: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
the PDA culture medium is as follows: 200 g of potato, 20 g of glucose, 15-20 g of agar and 1000 ml of distilled water.
The crushing length of the straws is less than or equal to 5 cm.
The weight parts of the raw materials in the step two are as follows: 5 parts of tertiary diatomite, 30 parts of straw, 10 parts of protein slag and 55 parts of chicken manure; the moisture content was adjusted to 50%.
And the decomposing and sterilizing time in the third step is 30 days.
In the fifth step, the ratio of the bacillus subtilis, the aspergillus oryzae and the aspergillus niger is 5:1: 1.
The invention has the beneficial effects that:
1) the utilization rate of the fertilizer is improved, the investment of the fertilizer is reduced, and the environmental pollution is reduced.
2) Activating and promoting the absorption of the plant to the nutrient elements. Microorganisms in the fertilizer generate various organic acids and inorganic acids, the pH value of the environment is reduced, insoluble phosphate is degraded into effective phosphorus, chelated phosphorus is released, silicate minerals in soil are decomposed, and insoluble potassium, phosphorus, silicon and the like are converted into soluble substances.
3) The microorganisms in the fertilizer produce substances such as phytohormones, acidic substances, vitamins and the like, and stimulate and regulate the growth of plants. Promoting the rooting and seedling emergence of crops, advancing the maturity and improving the stress resistance, yield and quality of the crops.
4) Improve the physical and chemical properties of the soil and improve the content of organic matters in the soil.
Drawings
FIG. 1 is a flow chart of a preparation method of a biological bacterial fertilizer.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings.
Diatomaceous earth is a porous biogenetic silicalite. Its main chemical composition is amorphous SiO2And contains a small amount of Al2O3、Fe2O3CaO, organic matter, and the like. Due to the porous structure of the diatomite, the diatomite has good adsorption and slow release effects on various ions, can prevent fertilizer from caking and caking, avoids leaching loss and fixation of nutrients, improves the utilization rate, and has good fertilizer retention and supply effects. Researches show that the diatomite can adsorb partial nitrogen, phosphorus and potassium elements in soil and slowly release the elements in the growth period of crops, the slow release effect of the diatomite is better than that of the soil without the diatomite, and the utilization rate of the nitrogen, phosphorus and potassium is improved. Further, diatomaceous earth is mainly composed of SiO2The silicon is activated by a physical and chemical method, so that the effective silicon content is improved, and the silicon can be directly absorbed by plants. The organic fertilizer produced by using the organic fertilizer as a raw material is particularly suitable for rice planting, can promote the vegetative growth of rice plants, improve tillering and disease resistance, increase the grain number per spike and the thousand grain weight, and improve the yield. The total reserve of diatomite is the second place in the world in China, but the grade of diatomite is generally low. In the development of diatomite mineralsMost of the low-grade diatomite ores, namely the tertiary diatomite, are not effectively utilized but are directly abandoned, so that resources are wasted, and meanwhile, the ecological environment is polluted. The raw materials applied in the application are the part of low-grade diatomite ore.
The biological bacterial fertilizer comprises, by weight, 3-5 parts of tertiary diatomite, 20-30 parts of straws, 8-15 parts of protein residues, 40-60 parts of chicken manure and 5-10 parts of a mixed microbial inoculum;
the mixed microbial inoculum is prepared from bacillus subtilis, aspergillus oryzae and aspergillus niger in a mass ratio of 1-5:1: 1.
The ratio of the bacillus subtilis to the aspergillus oryzae to the aspergillus niger in the mixed microbial inoculum is 5:1: 1.
Referring to the attached figure 1, the preparation method of the biological bacterial fertilizer comprises the following steps:
the method comprises the following steps: crushing the straws into fragments;
step two: uniformly mixing 20-30 parts of the fragments obtained in the step one with 3-5 parts of tertiary diatomite, 8-15 parts of protein residues and 40-60 parts of chicken manure according to parts by weight, and adjusting the water content to 50-60% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step two for 20-40 days at 50-60 ℃ to obtain a microbial inoculum carrier;
step four: respectively carrying out activation and propagation on three preserved microorganisms, namely bacillus subtilis, aspergillus oryzae and aspergillus niger to obtain three propagation bacteria liquids;
step five: preparing three expanded culture bacteria liquids obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 1-5:1: 1;
step six: inoculating the mixed microbial inoculum obtained in the step five into the microbial inoculum carrier obtained in the step three according to the inoculation amount of 5-10%, and uniformly mixing, wherein the culture temperature is 20-30 ℃, and the culture time is 10-15 days; drying at 35-45 ℃ until the water content is less than or equal to 30 percent to obtain the biological bacterial fertilizer.
The expanding culture in the fourth step specifically comprises the following steps:
b, expanding culture of the bacillus subtilis: firstly, inoculating bacillus subtilis into a sterilized LB culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 37 ℃ for 24h at the rotation speed of 200 rpm; then transferring the strain into a new sterilized LB culture medium, wherein the strain inoculation amount is 10%, and performing shake culture at the culture temperature of 37 ℃ for 36 h;
expanding culture of aspergillus oryzae: firstly, inoculating aspergillus oryzae in a sterilized PDA culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
expanding culture of aspergillus niger: firstly, inoculating Aspergillus niger in a sterilized PDA culture medium, and performing shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
the LB culture medium is: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
the PDA culture medium is as follows: 200 g of potato, 20 g of glucose, 15-20 g of agar and 1000 ml of distilled water.
The crushing length of the straws is less than or equal to 5 cm.
The weight parts of the raw materials in the step two are as follows: 5 parts of tertiary diatomite, 30 parts of straw, 10 parts of protein slag and 55 parts of chicken manure; the moisture content was adjusted to 50%.
And the decomposing and sterilizing time in the third step is 30 days.
In the fifth step, the ratio of the bacillus subtilis, the aspergillus oryzae and the aspergillus niger is 5:1: 1.
The protein residue used in the invention is corn protein residue, and the used diatomite specifically refers to discarded low-grade diatomite when the diatomite ore is mined and is used.
Example 1
The biological bacterial fertilizer comprises, by weight, 3 parts of tertiary diatomite, 20 parts of straw, 8 parts of protein residue, 40 parts of chicken manure and 5 parts of a mixed microbial inoculum;
the mixed microbial inoculum is prepared from bacillus subtilis, aspergillus oryzae and aspergillus niger in a mass ratio of 1:1: 1.
Example 2
The biological bacterial fertilizer comprises, by weight, 5 parts of tertiary diatomite, 30 parts of straws, 15 parts of protein residues, 60 parts of chicken manure and 10 parts of a mixed microbial inoculum;
the mixed microbial inoculum is prepared from bacillus subtilis, aspergillus oryzae and aspergillus niger in a mass ratio of 5:1: 1.
Example 3
The biological bacterial fertilizer comprises, by weight, 4 parts of tertiary diatomite, 25 parts of straw, 12 parts of protein residue, 50 parts of chicken manure and 10 parts of mixed microbial inoculum;
the mixed microbial inoculum is prepared from bacillus subtilis, aspergillus oryzae and aspergillus niger in a mass ratio of 3:1: 1.
Example 4
A preparation method of the biological bacterial fertilizer comprises the following steps:
the method comprises the following steps: crushing the straws into fragments with the length of 5 cm;
step two: uniformly mixing 30 parts of the fragments obtained in the step one with 5 parts of tertiary diatomite, 15 parts of protein residues and 60 parts of chicken manure in parts by weight, and adjusting the water content to 60% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step two for 40 days at the temperature of 60 ℃ to obtain a microbial inoculum carrier;
step four: respectively carrying out activation and propagation on three preserved microorganisms, namely bacillus subtilis, aspergillus oryzae and aspergillus niger to obtain three propagation bacteria liquids;
step five: preparing three expanded culture bacteria liquids obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 5:1: 1;
step six: inoculating the mixed microbial inoculum obtained in the step five into the microbial inoculum carrier obtained in the step three according to the inoculation amount of 10%, and uniformly mixing, wherein the culture temperature is 30 ℃, and the culture time is 15 days; drying at 45 ℃ until the water content is less than or equal to 30 percent to obtain the biological bacterial fertilizer.
The expanding culture in the fourth step specifically comprises the following steps:
b, expanding culture of the bacillus subtilis: firstly, inoculating bacillus subtilis into a sterilized LB culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 37 ℃ for 24h at the rotation speed of 200 rpm; then transferring the strain into a new sterilized LB culture medium, wherein the strain inoculation amount is 10%, and performing shake culture at the culture temperature of 37 ℃ for 36 h;
expanding culture of aspergillus oryzae: firstly, inoculating aspergillus oryzae in a sterilized PDA culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
expanding culture of aspergillus niger: firstly, inoculating Aspergillus niger in a sterilized PDA culture medium, and performing shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
the LB culture medium is: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
the PDA culture medium is as follows: 200 g of potato, 20 g of glucose, 20 g of agar and 1000 ml of distilled water.
Example 5
The method comprises the following steps: crushing the straws into fragments, wherein the length of each fragment is 3 cm;
step two: uniformly mixing 20 parts of the fragments obtained in the step one with 3 parts of tertiary diatomite, 8 parts of protein residues and 40 parts of chicken manure in parts by weight, and adjusting the water content to 50% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step two for 20 days at the temperature of 50 ℃ to obtain a microbial inoculum carrier;
step four: respectively carrying out activation and propagation on three preserved microorganisms, namely bacillus subtilis, aspergillus oryzae and aspergillus niger to obtain three propagation bacteria liquids;
step five: preparing three expanded culture bacteria liquids obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 1:1: 1;
step six: inoculating the mixed microbial inoculum obtained in the step five into the microbial inoculum carrier obtained in the step three according to the inoculation amount of 5%, and uniformly mixing, wherein the culture temperature is 20 ℃, and the culture time is 10 days; drying at 35 ℃ until the water content is less than or equal to 30 percent to obtain the biological bacterial fertilizer.
The expanding culture in the fourth step specifically comprises the following steps:
b, expanding culture of the bacillus subtilis: firstly, inoculating bacillus subtilis into a sterilized LB culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 37 ℃ for 24h at the rotation speed of 200 rpm; then transferring the strain into a new sterilized LB culture medium, wherein the strain inoculation amount is 10%, and performing shake culture at the culture temperature of 37 ℃ for 36 h;
expanding culture of aspergillus oryzae: firstly, inoculating aspergillus oryzae in a sterilized PDA culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
expanding culture of aspergillus niger: firstly, inoculating Aspergillus niger in a sterilized PDA culture medium, and performing shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
the LB culture medium is: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
the PDA culture medium is as follows: 200 g of potato, 20 g of glucose, 15-20 g of agar and 1000 ml of distilled water.
Example 6
A preparation method of the biological bacterial fertilizer comprises the following steps:
the method comprises the following steps: crushing the straws into chips, wherein the length of each chip is 1 cm;
step two: uniformly mixing 25 parts by weight of the fragments obtained in the step one with 4 parts by weight of tertiary diatomite, 11 parts by weight of protein slag and 50 parts by weight of chicken manure, and adjusting the water content to 55% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step two for 30 days at 55 ℃ to obtain a microbial inoculum carrier;
step four: respectively carrying out activation and propagation on three preserved microorganisms, namely bacillus subtilis, aspergillus oryzae and aspergillus niger to obtain three propagation bacteria liquids;
step five: preparing three expanded culture bacteria liquids obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 3:1: 1;
step six: inoculating the mixed microbial inoculum obtained in the fifth step into the microbial inoculum carrier obtained in the third step according to the inoculation amount of 8%, uniformly mixing, wherein the culture temperature is 25 ℃, and the culture time is 13 days; drying at 40 ℃ until the water content is less than or equal to 30 percent to obtain the biological bacterial fertilizer.
The expanding culture in the fourth step specifically comprises the following steps:
b, expanding culture of the bacillus subtilis: firstly, inoculating bacillus subtilis into a sterilized LB culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 37 ℃ for 24h at the rotation speed of 200 rpm; then transferring the strain into a new sterilized LB culture medium, wherein the strain inoculation amount is 10%, and performing shake culture at the culture temperature of 37 ℃ for 36 h;
expanding culture of aspergillus oryzae: firstly, inoculating aspergillus oryzae in a sterilized PDA culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
expanding culture of aspergillus niger: firstly, inoculating Aspergillus niger in a sterilized PDA culture medium, and performing shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
the LB culture medium is: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
the PDA culture medium is as follows: 200 g of potato, 20 g of glucose, 15-20 g of agar and 1000 ml of distilled water.
Example 7
A preparation method of the biological bacterial fertilizer comprises the following steps:
the method comprises the following steps: crushing the straws into fragments, wherein the length of the fragments is less than or equal to 5 cm;
step two: uniformly mixing 5 parts of tertiary diatomite, 30 parts of straws, 10 parts of protein residues and 55 parts of chicken manure according to a proportion, and adjusting the water content to 50% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step S2 for 35 days, and controlling the temperature to be 50-60 ℃ to obtain a microbial inoculum carrier;
step four: and respectively carrying out activation and propagation on the three preserved microorganisms.
B, bacillus subtilis: inoculating into sterilized LB medium, placing at 37 deg.C, shaking and culturing for 24h at 200 rpm. Then, the cells were transferred to a new sterilized LB medium with a inoculum size of 10% and were shake-cultured at 37 ℃ for 36 hours.
Aspergillus oryzae and Aspergillus niger were inoculated in sterilized PDA medium, respectively, and cultured on shaking table at 28 deg.C for 3 days at 150 rpm. Then, the cells were transferred to a new sterilized PDA medium with a inoculum size of 10% and were incubated at 28 ℃ for 3 days with shaking in a shaker at 150 rpm.
LB culture medium: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
PDA culture medium: 200 g of potato, 20 g of glucose, 15-20 g of agar and 1000 ml of distilled water.
Step five: and (4) preparing the three expanded culture solutions obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 5:1: 1.
Step six: and (4) inoculating 10% of the mixed microbial inoculum obtained in the step five into the microbial inoculum carrier obtained in the step S3, uniformly mixing, controlling the temperature to be 20-30 ℃, culturing for 10 days, and drying at low temperature until the water content is less than or equal to 30% to obtain the biological bacterial fertilizer.
Example 8
A preparation method of the biological bacterial fertilizer comprises the following steps:
the method comprises the following steps: crushing the straws into fragments, wherein the length of the fragments is less than or equal to 3 cm;
step two: uniformly mixing 5 parts of tertiary diatomite, 32 parts of straws, 8 parts of protein residues and 55 parts of chicken manure according to a proportion, and adjusting the water content to 50% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step two for 30 days, and controlling the temperature to be 50-60 ℃ to obtain a microbial inoculum carrier;
step four: and respectively carrying out activation and propagation on the three preserved microorganisms.
B, bacillus subtilis: inoculating into sterilized LB medium, placing at 37 deg.C, shaking and culturing for 24h at 200 rpm. Then, the cells were transferred to a new sterilized LB medium with a inoculum size of 10% and were shake-cultured at 37 ℃ for 36 hours.
Aspergillus oryzae and Aspergillus niger were inoculated in sterilized PDA medium, respectively, and cultured on shaking table at 28 deg.C for 3 days at 150 rpm. Then, the cells were transferred to a new sterilized PDA medium with a inoculum size of 10% and were incubated at 28 ℃ for 3 days with shaking in a shaker at 150 rpm.
LB culture medium: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
PDA culture medium: 200 g of potato, 20 g of glucose, 15-20 g of agar and 1000 ml of distilled water.
Step five: and (4) preparing the three expanded culture solutions obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 5:1: 1.
Step six: and (4) inoculating 8% of the mixed microbial inoculum obtained in the step five into the microbial inoculum carrier obtained in the step S3, uniformly mixing, controlling the temperature to be 20-30 ℃, culturing for 15 days, and drying at low temperature until the water content is less than or equal to 30% to obtain the biological bacterial fertilizer.
The fertilizer efficiency comparison condition of the three-stage diatomite biological bacterial fertilizer and the commercial biological bacterial fertilizer is shown in table 1. As can be seen from the table, the nutrient contents of alkaline-hydrolyzable nitrogen, available phosphorus, available potassium and the like in the soil after the biological bacterial fertilizer is applied are higher than those of the commercial biological bacterial fertilizer. The corn yield is also 8% and 7% higher respectively. Therefore, the biological bacterial fertilizer has good effect. Improving the nutrient activity of nitrogen, phosphorus and potassium of soil and the crop yield.
TABLE 1 comparison of fertilizer efficiency of the third-stage diatomite biological bacterial fertilizer and commercial biological bacterial fertilizer
Claims (8)
1. The biological bacterial fertilizer is characterized by comprising 3-5 parts of tertiary diatomite, 20-30 parts of straw, 8-15 parts of protein residue, 40-60 parts of chicken manure and 5-10 parts of mixed microbial inoculum by weight;
the mixed microbial inoculum is prepared from bacillus subtilis, aspergillus oryzae and aspergillus niger in a mass ratio of 1-5:1: 1.
2. The biological bacterial fertilizer as claimed in claim 1, wherein the ratio of bacillus subtilis, aspergillus oryzae and aspergillus niger in the mixed bacterial agent is 5:1: 1.
3. The preparation method of the biological bacterial fertilizer as claimed in claim 1, which is characterized by comprising the following steps:
the method comprises the following steps: crushing the straws into fragments;
step two: uniformly mixing 20-30 parts of the fragments obtained in the step one with 3-5 parts of tertiary diatomite, 8-15 parts of protein residues and 40-60 parts of chicken manure according to parts by weight, and adjusting the water content to 50-60% to obtain a mixture;
step three: decomposing and sterilizing the mixture obtained in the step two for 20-40 days at 50-60 ℃ to obtain a microbial inoculum carrier;
step four: respectively carrying out activation and propagation on three preserved microorganisms, namely bacillus subtilis, aspergillus oryzae and aspergillus niger to obtain three propagation bacteria liquids;
step five: preparing three expanded culture bacteria liquids obtained in the fourth step into a mixed microbial inoculum according to the proportion of bacillus subtilis, aspergillus oryzae and aspergillus niger being 1-5:1: 1;
step six: inoculating the mixed microbial inoculum obtained in the step five into the microbial inoculum carrier obtained in the step three according to the inoculation amount of 5-10%, and uniformly mixing, wherein the culture temperature is 20-30 ℃, and the culture time is 10-15 days; drying at 35-45 deg.C until the water content is less than or equal to 30% to obtain the biological bacterial fertilizer.
4. The preparation method according to claim 3, wherein the expanding culture in step four is specifically:
b, expanding culture of the bacillus subtilis: firstly, inoculating bacillus subtilis into a sterilized LB culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 37 ℃ for 24h at the rotation speed of 200 rpm; then transferring the strain into a new sterilized LB culture medium, wherein the strain inoculation amount is 10%, and performing shake culture at the culture temperature of 37 ℃ for 36 h;
expanding culture of aspergillus oryzae: firstly, inoculating aspergillus oryzae in a sterilized PDA culture medium, and carrying out shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
expanding culture of aspergillus niger: firstly, inoculating Aspergillus niger in a sterilized PDA culture medium, and performing shaking culture on a shaking table at the culture temperature of 28 ℃ for 3 days at the rotation speed of 150 rpm; then transferring the mixture into a new sterilized PDA culture medium, wherein the inoculation amount is 10%, performing shake culture at the culture temperature of 28 ℃ for 3 days, and the rotation speed of a shaking table is 150 rpm;
the LB culture medium is: 10g of tryptone, 5g of yeast extract and 10g of NaCl, shaking the container until solute is dissolved, adjusting the pH to 7.0 by using 5mol/L of NaOH, and fixing the volume to 1L by using deionized water;
the PDA culture medium is as follows: 200 g of potato, 20 g of glucose, 15-20 g of agar and 1000 ml of distilled water.
5. The method as claimed in claim 3, wherein the crushed straw length is not more than 5 cm.
6. The preparation method according to claim 3, wherein the raw materials in the second step are in parts by weight: 5 parts of tertiary diatomite, 30 parts of straw, 10 parts of protein slag and 55 parts of chicken manure; the moisture content was adjusted to 50%.
7. The method according to claim 3, wherein the time for the sterilization by decomposition in the third step is 30 days.
8. The preparation method according to claim 3, wherein in the fifth step, the ratio of the bacillus subtilis to the aspergillus oryzae to the aspergillus niger is 5:1: 1.
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