CN111718876A - Compound microbial agent and preparation method and application thereof - Google Patents
Compound microbial agent and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a compound microbial agent containing microorganisms with antagonistic and degrading functions, and a preparation method and application thereof. The invention discloses an antagonistic and degradable microorganism combination, which comprises the following components: lactobacillus plantarum (Lactobacillus plantarum), Bacillus subtilis (Bacillus subtilis), Aspergillus niger (Aspergillus niger), Trichoderma viride (Trichoderma viride), Saccharomyces cerevisiae (Saccharomyces cerevisiae). The antagonistic microbe activity bacterial sludge is obtained by inoculating the microbial strains after the expanded culture into a fermentation tank for mixed culture, and then filtering and centrifugally separating. And further, uniformly mixing biochar obtained by pyrolyzing and carbonizing agricultural organic waste with the active bacterial sludge in proportion, and then drying and granulating at low temperature to obtain the compound microbial agent suitable for application of a base fertilizer. The compound microbial agent disclosed by the invention is used for repairing polluted soil and improving the soil micro-ecological environment, and has a high utilization value.
Description
Technical Field
The invention relates to a soil micro-ecological regulation and control technology in the field of agricultural production, and relates to preparation and application of a compound microbial agent with a soil microbial population regulation and control function. Mainly shows the comprehensive application of soil antagonism, degradation of microorganisms and biochar.
Background
Soil is a complex ecosystem which has a certain degree of purification capacity. However, with the increase of population and economic development in recent years, the number, types and pollution speed of waste pollutants generated in human activities far exceed the self-purification capacity of soil, so that the internal environment quality of the soil is deteriorated, the balance in the soil is broken, the accumulation of the pollutants is dominant, and the soil loses normal ecological restoration function. Soil pollution is harmful to the health of animals and human beings through various direct or indirect ways, and the problem of soil safety is attracting increasing attention of human beings.
The essence of soil microecological regulation is microbial interaction, biodegradation or biotransformation, i.e. the action of microorganisms and their activity products on the vital activities of other microorganisms, the decomposition of organic pollutants and the inactivation of inorganic pollutants. The metabolic function of indigenous microorganisms in the soil is utilized, or the engineering culture microbial community with the degradation and transformation capacity is supplemented, and the microbial metabolic function is promoted or enhanced by creating a suitable environmental condition, so that the aims of degrading and finally eliminating pollutants are fulfilled. The micro-ecological regulation and control technology is increasingly paid social attention.
Common microorganisms for degrading and transforming pollutants in soil environment are bacteria, fungi and actinomycetes, which absorb, precipitate, oxidize and reduce pollutants through life activity processes or products to reduce the toxicity of pollutants in soil, such as: acetic acid, citric acid, lactic acid and the like generated by specific microorganisms in the activity can promote the dissolution of organic pollutants and lead, can also enable phosphorus elements adsorbed and fixed by soil to be released again, improves the utilization rate of phosphorus by plants, and can also effectively reduce the bioavailability of lead by released phosphorus. Meanwhile, the microorganisms can also regulate the activity of soil enzymes, thereby being beneficial to enriching and storing nutrient elements. Humic acid generated by microorganisms in the activity can enhance the respiration intensity of plants and achieve the effect of promoting growth and development.
Along with the wide planting of economic crops and the generation of a large amount of agricultural wastes, the traditional treatment method not only pollutes the environment, but also wastes resources. The biomass of the organic waste is pyrolyzed into biochar under the low oxygen state, the biochar not only contains rich organic matters and has larger specific surface area and developed pore structure, but also has the synergistic effect of improving the physical structure of soil, increasing the content of organic carbon in the soil and increasing the soil fertility, and can provide living space for microorganisms. At present, global resources are increasingly deficient, and the problem of soil environmental pollution is becoming serious, biomass charcoal is a renewable energy source, and has the advantages of being green and sustainable in development.
The combination of the antagonistic microbe activity bacterial sludge and the biochar is used as a repairing material for manufacturing a microbial agent, is used for repairing polluted soil and improving the micro-ecological environment of the soil, and has high utilization value.
Disclosure of Invention
According to the research progress of antagonistic microorganisms in the aspect of soil microecological regulation, the invention screens out the optimal symbiotic strain combination from the antagonistic microorganisms and screens out high-yield strains through independent culture. The screened high-yield strains are as follows: lactobacillus plantarum (Lactobacillus plantarum), Bacillus subtilis (Bacillus subtilis), Aspergillus niger (Aspergillus niger), Trichoderma viride (Trichoderma viride), Saccharomyces cerevisiae (Saccharomyces cerevisiae).
The production method of the antagonistic composite micro-activity bacterial sludge comprises the following steps:
a. slant culture: respectively inoculating lactobacillus plantarum, bacillus subtilis, aspergillus niger, trichoderma viride and saccharomyces cerevisiae original strains on a solid culture medium under the aseptic condition, culturing the aspergillus niger, the trichoderma viride and the saccharomyces cerevisiae for 2-6 days at the temperature of 15-38 ℃, and culturing the lactobacillus plantarum and the bacillus subtilis for 1-3 days at the temperature of 15-42 ℃.
In a preferred embodiment of the present invention, the seed culture obtained by slant culture in step a is scraped by a sampler or by an inoculating loop, and further washed into the culture medium with sterile water as a seed solution in step b and seed culture.
b. First-order seed culture (shake flask culture stage): and (b) inoculating 1mL of seed solution cultured in the step (a) into a 250mL triangular flask filled with 20mL of liquid culture medium under the aseptic condition. Carrying out shake cultivation for 2-6 days at the temperature of 15-38 ℃ for Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae at a speed of 100-200 r/min; and (3) statically culturing the lactobacillus plantarum and the bacillus subtilis for 1-3 days at 15-42 ℃, and stopping culturing when the OD600 value of single-strain liquid culture is between 3.0 and 4.0 to prepare first-grade seeds.
c. Secondary seed culture: and respectively inoculating the first-class seeds into a seed fermentation tank according to the inoculation amount of the liquid culture medium with the volume ratio of 5-20%. Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae are stirred at a speed of 100-200/min at 15-38 ℃, the ventilation rate is 1: 0.5-1.5, the culture is carried out for 2-6 days, and Lactobacillus plantarum and Bacillus subtilis are cultured at 15-42 ℃ for 1-3 days, so as to prepare secondary seeds.
d. Main body fermentation: and respectively inoculating the secondary seeds into a fermentation tank according to the inoculation amount of the liquid culture medium with the volume ratio of 5-20%, and performing high-density fermentation culture to obtain the microbial inoculum.
Wherein, the culture medium used by the aspergillus niger, the trichoderma viride and the saccharomyces cerevisiae in the steps a, b and c is a PDA culture medium, and the culture medium used by the lactobacillus plantarum and the bacillus subtilis is an MRS culture medium. The solid medium is different from the liquid medium in that a proper amount of agar is added in the preparation process of the solid medium.
Wherein the formula of the culture medium used in the step d comprises the following components in percentage by mass: 3-20% of molasses, 0.5-10% of ammonium sulfate, 0.5-5% of peptone, 0.5-3% of calcium carbonate and the balance of water.
The high-density fermentation culture can adopt a fed-batch culture (FBC) mode, wherein a fed-batch carbon source is any one of glucose, glycerol, sucrose and molasses or a mixture thereof; the nitrogen sources are: any one of ammonium sulfate, beef extract, peptone or mixture thereof.
And the conditions of fermentation pH, dissolved oxygen, stirring speed, temperature and the like are regulated and controlled in stages in the culture process. Specifically, the fermentation culture process comprises an aerobic culture stage, wherein aeration is performed at intervals within 0-24 hours at the beginning, fermentation is performed under aerobic conditions, the aeration amount is 1: 1-1.5, the fermentation dissolved oxygen is regulated and controlled to be 5-15%, the stirring speed is 150-200r/min, the stirring interval time is 1-3 hours, the stirring is performed for 1-3 minutes, and the temperature is 25-35 ℃; micro dissolved oxygen and anaerobic culture stage: and (3) 24-96 h, keeping the upper layer of the fermentation liquor in a slightly oxygen-soluble state, performing static culture, stirring at intervals, wherein the stirring interval time is 3-5 h, stirring for 3-5 min, and adjusting the pH value to be kept stable between 3.8-4.5 by using calcium carbonate at the temperature of 25-35 ℃.
e. Centrifugal separation: and d, performing conventional filtration and centrifugal separation on the fermentation liquor in the step d to obtain antagonistic microbe activity bacterial sludge.
Preparing biochar:
the biochar is prepared from agricultural organic wastes through a closed internal circulation low-temperature anoxic pyrolysis carbonization technology. The closed internal circulation low-temperature oxygen-deficient pyrolysis carbonization is that agricultural organic waste is crushed, dried and then placed in a closed carbonization furnace, and is heated by an external heat source under the condition of oxygen deficiency, so that the temperature of materials in the furnace reaches 400-450 ℃ for pyrolysis carbonization. The anoxic condition is that the carbonization furnace is sealed in the heating process of an external heat source, and oxygen or air is not introduced into the carbonization furnace in the heating process. The internal circulation is in the heating process, the carbonization furnace is closed, and materials and gas in the carbonization furnace circulate in the furnace and are not communicated with the external environment. The pyrolysis carbonization time of the materials in the carbonization furnace is 1.5 to 16 hours, and in a preferred embodiment, the carbonization time is 5 to 8 hours.
The agricultural waste includes, but is not limited to, waste having organic matter, such as branches, leaves, platycodon grandiflorum, wood chips, and the like of plants.
The preparation method of the compound microbial agent comprises the following steps:
and e, mixing the antagonistic microbe activity bacterial sludge obtained by centrifugal separation in the step e and the biochar. And further mixing the biochar with the antagonistic microbe active bacterial sludge according to the mass ratio of 10:1-4, uniformly stirring, drying at 35-40 ℃ and granulating to obtain the composite microbial agent particles with the particle size of 2-4 mm. The prepared compound microbial agent can be directly applied and also can be used as a fermentation microbial agent of an organic fertilizer.
In the prepared compound microbial agent, lactobacillus plantarum 1.5 × 108～1.0×1010cfu/g, bacillus subtilis 1.0 × 108～5.0×1010cfu/g, Aspergillus niger 1.0 × 108～1.0×1010cfu/g, Trichoderma viride 1.0 × 108～1.0×1010cfu/g, Saccharomyces cerevisiae 5.0 × 108～1.0×1010cfu/g。
Preferably, the lactobacillus plantarum strain is 5.0 × 108～1.0×1010cfu/g, particularly preferably 1.0 × 109～6.0×109cfu/g, bacillus subtilis 1.5 × 108～5.0×1010cfu/g, particularly preferably 5.0 × 108～5.0×109cfu/g, Aspergillus niger 5.0 × 108～5.0×109cfu/g, green wood 5.0 × 108～5.0×109cfu/g, Saccharomyces cerevisiae 1.6 × 109～6.0×109cfu/g。
In a particularly preferred embodiment of the invention, Lactobacillus plantarum 1.5 × 109cfu/g, bacillus subtilis 1.0 × 109cfu/g, Aspergillus niger 1.0 × 109cfu/g, Trichoderma viride 1.0 × 109cfu/g, Saccharomyces cerevisiae 1.5 × 109cfu/g。
The amount of each species is adjusted by one skilled in the art by techniques conventional in the art. In a preferred embodiment, in step d, the ratio of each strain in the prepared bacterial sludge is adjusted by adjusting the addition amount of each strain.
The bacterial amount of the complex microbial inoculum is measured based on the finally obtained complex microbial inoculum. The amount of each strain is based on the content of each strain in the composite microbial agent per unit mass.
The composite microbial inoculum disclosed by the invention has the proportion of biochar of 65-95% based on the total mass of the composite microbial inoculum.
In the using method of the compound microbial inoculum, the compound microbial inoculum is applied in a bottom application mode, and is applied once in spring or once in spring planting and autumn harvest, and the application amount is 400 kilograms per mu every year.
The beneficial technical effects of the invention are as follows:
the product of the invention can provide various nutrient elements necessary for the growth and development of agricultural, forest and urban greening plants, can also increase the activity of beneficial microorganisms in soil, and overcomes the defects caused by excessive application and unbalanced fertilization of chemical fertilizers. The invention also takes the role of soil organic matter humus transformation, increases soil granular structure, improves water and fertilizer retention capacity, activates nutrients solidified by soil, improves fertilizer utilization rate, and simultaneously beneficial microorganisms in the compound microbial agent secrete various beneficial substances to the soil, effectively promotes nutrient transformation, lightens soil-borne diseases, repairs polluted soil, is an ideal material for agricultural planting, landscaping planting and seedling cultivation, and has wide prospect in agricultural sustainable development.
Drawings
FIG. 1: a closed internal circulation low-temperature anoxic pyrolysis carbonization technical schematic diagram.
Detailed Description
The invention is illustrated below with reference to examples, but not intended to limit the scope of the invention.
Preparation method of composite microbial agent
Example 1:
a. slant culture: respectively inoculating lactobacillus plantarum, bacillus subtilis, aspergillus niger, trichoderma viride and saccharomyces cerevisiae original strains on a solid culture medium under the aseptic condition, culturing the aspergillus niger, the trichoderma viride and the saccharomyces cerevisiae for 3 days at the temperature of 30 ℃, and culturing the lactobacillus plantarum and the bacillus subtilis for 2 days at the temperature of 37 ℃.
b. First-order seed culture, and a shake flask culture stage: and (b) inoculating 1mL of seed solution cultured in the step (a) into a 250mL triangular flask filled with 20mL of liquid culture medium under the aseptic condition. Carrying out shake culture at the temperature of 30 ℃ for 3 days at 150r/min by using aspergillus niger, trichoderma viride and saccharomyces cerevisiae; standing and culturing Lactobacillus plantarum and Bacillus subtilis at 37 deg.C for 2 days, and stopping culturing when OD600 value of single strain liquid culture is 3.0-4.0 to obtain first-class seed.
c. Secondary seed culture, and a seeding tank culture stage: inoculating the first-class seeds into seed fermenters respectively according to the inoculation amount of 10% of the volume ratio of the liquid culture medium. Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae are stirred at a speed of 180/min at 30 ℃, the ventilation rate is 1:1, the culture is carried out for 4 days, and Lactobacillus plantarum and Bacillus subtilis are cultured for 2 days at 37 ℃ to prepare secondary seeds.
d. Main body fermentation: inoculating the second-level seeds into a fermentation tank according to the inoculation amount of 15% of the volume ratio of the liquid culture medium, and performing high-density fermentation culture to obtain the microbial inoculum. Wherein the high-density fermentation culture adopts a fed-batch culture mode, and the formula of the used culture medium comprises the following components in percentage by mass: molasses 8%, ammonium sulfate 6%, peptone 2.5%, calcium carbonate 2%, and water in balance.
The high-density fermentation culture can adopt a fed-batch culture (FBC) mode, wherein the fed-batch carbon source is any one of glucose, glycerol, sucrose and molasses or a mixture thereof; the nitrogen sources are: any one of ammonium sulfate, beef extract, peptone or mixture thereof.
The fermentation culture process comprises the following steps: aerobic culture stage: ventilating at intervals within 0-24 hours from the beginning, fermenting under aerobic condition, wherein the ventilation amount is 1:1.2, the fermentation dissolved oxygen is regulated and controlled to be 10%, the stirring speed is 180r/min, the stirring interval time is 2 hours, the stirring time is 2 minutes, and the temperature is 30 ℃; micro dissolved oxygen and anaerobic culture stage: keeping the upper layer of the fermentation liquor in a slightly oxygen-soluble state for 60 hours, statically culturing, stirring at intervals of 4 hours and 4 minutes, adjusting the pH value by using calcium carbonate, keeping the pH value stable at about 4, and keeping the temperature at 30 ℃.
e. Centrifugal separation: and d, performing common centrifugal separation on the fermentation liquor in the step d to obtain antagonistic microbe activity bacterial sludge.
f. Preparing a compound microbial agent: the biochar prepared by crushing, drying and dehydrating agricultural wastes and antagonistic microbe active bacteria mud are mixed according to the mass ratio of 10:1, are uniformly stirred and are dried and granulated at 40 ℃, and the composite microbial agent particles with the particle size of 2-4mm are obtained, and the water content is less than or equal to 15%.
Example 2: preparation method of compound microbial agent
a. Slant culture: respectively inoculating lactobacillus plantarum, bacillus subtilis, aspergillus niger, trichoderma viride and saccharomyces cerevisiae original strains on a solid culture medium under the aseptic condition, culturing the aspergillus niger, the trichoderma viride and the saccharomyces cerevisiae for 2 days at the temperature of 25 ℃, and culturing the lactobacillus plantarum and the bacillus subtilis for 1 day at the temperature of 25 ℃.
b. First-order seed culture (shake flask culture stage): and (b) inoculating 1mL of seed solution cultured in the step (a) into a 250mL triangular flask filled with 20mL of liquid culture medium under the aseptic condition. Carrying out shake cultivation for 2 days at the temperature of 25 ℃ at 100-200 r/min by Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae; standing and culturing Lactobacillus plantarum and Bacillus subtilis at 25 deg.C for 1 day, and stopping culturing when OD600 value of single strain liquid culture is 3.0-4.0 to obtain first-class seed.
c. Secondary seed culture: inoculating the first-class seeds into seed fermenters respectively according to the inoculum size of 15% of the volume ratio of the liquid culture medium. Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae with stirring speed of 100/min at 25 deg.C and ventilation amount of 1:0.5, culturing for 2 days, and culturing Lactobacillus plantarum and Bacillus subtilis at 25 deg.C for 1 day to obtain secondary seed.
d. Main body fermentation: inoculating the second-level seeds into a fermentation tank according to the inoculation amount of 15% of the volume ratio of the liquid culture medium, and performing high-density fermentation culture to obtain the microbial inoculum. Wherein the high-density fermentation culture adopts a fed-batch culture mode, and the fed-batch carbon source comprises: any one of glucose, glycerol, sucrose, molasses or mixtures thereof; the nitrogen sources are: the fermentation culture process comprises the aerobic culture stage, wherein the fermentation is carried out at intervals within 0-24 hours from the beginning, the aeration amount is 1:1, the fermentation dissolved oxygen is regulated and controlled to be 5%, the stirring speed is 150r/min, the stirring interval is 1 hour, the stirring is carried out for 1 minute, and the temperature is 25 ℃; micro dissolved oxygen and anaerobic culture stage: keeping the upper layer of the fermentation liquor in a slightly oxygen-soluble state for 24 hours, statically culturing, stirring at intervals of 3 hours and 3 minutes, adjusting the pH value by using calcium carbonate to keep the pH value stable between 3.8 and keeping the temperature at 25 ℃.
e. Centrifugal separation: and d, performing common centrifugal separation on the fermentation liquor in the step d to obtain antagonistic microbe activity bacterial sludge.
f. Preparing a compound microbial agent: the biochar prepared by crushing, drying and dehydrating agricultural wastes and antagonistic microbe active bacteria mud are mixed according to the mass ratio of 5: 1, are uniformly stirred and are dried and granulated at 40 ℃, and the composite microbial agent particles with the particle size of 2-4mm are obtained, and the water content is less than or equal to 15%.
Example 3: preparation method of compound microbial agent
a. Slant culture: respectively inoculating lactobacillus plantarum, bacillus subtilis, aspergillus niger, trichoderma viride and saccharomyces cerevisiae original strains on a solid culture medium under the aseptic condition, culturing the aspergillus niger, the trichoderma viride and the saccharomyces cerevisiae for 3 days at the temperature of 35 ℃, and culturing the lactobacillus plantarum and the bacillus subtilis for 3 days at the temperature of 35 ℃.
b. First-order seed culture (shake flask culture stage): and (b) inoculating 1mL of seed solution cultured in the step (a) into a 250mL triangular flask filled with 20mL of liquid culture medium under the aseptic condition. Carrying out shake cultivation for 2-6 days at the temperature of 35 ℃ by Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae at a speed of 100-200 r/min; standing and culturing Lactobacillus plantarum and Bacillus subtilis at 35 deg.C for 3 days, and stopping culturing when OD600 value of single strain liquid culture is 3.0-4.0 to obtain first-class seed.
c. Secondary seed culture: inoculating the first-class seeds into seed fermenters respectively according to the inoculation amount of 10% of the volume ratio of the liquid culture medium. Stirring at 200/min under 35 deg.C with Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae at a rate of 1:1.5, culturing for 5 days, and culturing for 3 days at 35 deg.C with Lactobacillus plantarum and Bacillus subtilis to obtain secondary seed.
d. Main body fermentation: inoculating the second-level seeds into a fermentation tank according to the inoculation amount of the liquid culture medium with the volume ratio of 20%, and performing high-density fermentation culture to obtain the microbial inoculum. Wherein the high-density fermentation culture adopts a fed-batch culture mode, and the fed-batch carbon source comprises: any one of glucose, glycerol, sucrose, molasses or mixtures thereof; the nitrogen sources are: the fermentation culture process comprises the aerobic culture stage, wherein the fermentation is carried out at intervals within 0-24 hours from the beginning, the aeration amount is 1:1, the fermentation dissolved oxygen is regulated and controlled to be 15%, the stirring speed is 200r/min, the stirring interval is 3 hours, the stirring is carried out for 3 minutes, and the temperature is 35 ℃; micro dissolved oxygen and anaerobic culture stage: keeping the upper layer of the fermentation liquor in a slightly oxygen-soluble state for 60h, performing static culture, stirring at intervals of 5 h and 5 min, adjusting the pH value by using calcium carbonate, keeping the pH value stable between 4.5 and keeping the temperature at 35 ℃.
e. Centrifugal separation: and d, performing common centrifugal separation on the fermentation liquor in the step d to obtain antagonistic microbe activity bacterial sludge.
f. Preparing a compound microbial agent: the biochar prepared by crushing, drying and dehydrating agricultural wastes and antagonistic microbe active bacteria mud are mixed according to the mass ratio of 5: 2, are uniformly stirred and are dried and granulated at 40 ℃, and the composite microbial agent particles with the particle size of 2-4mm are obtained, wherein the water content is less than or equal to 15%.
Second, composition of compound microbial inoculum
By the preparation method of the above examples 1-3, the complex microbial inoculum as shown in table 1 can be obtained by adjusting the amount of the active strains added in the main fermentation process in the step d.
Table 1: examples of complex microbial constitution schemes are, the unit: cfu/g.
The bacterial amount of the complex microbial inoculum is measured based on the finally obtained complex microbial inoculum.
Application method of compound microbial agent
The fertilizer is directly applied in a bottom application mode, the application amount is 200 kilograms per mu per year, and the fertilizer is applied once in spring according to artificial conditions (fruit tree planting or planting in spring and harvesting in autumn), or is applied in a ring opening and ditch filling mode and is applied around the trees, the application amount is determined according to the tree vigor, generally about 5 kilograms of fertilizer is applied to each plant of a big tree in terms of the tree age, and the number of small fruits or young trees can be halved. Is used for improving the microbial population structure of soil, increasing the soil fertility, degrading toxic and harmful substances and the like.
Fourth, utilization experiment of composite microbial inoculum
The demonstration work of the compound microbial inoculum on landscaping is carried out in a new Pudong area in 2019, the demonstration area is originally a domestic garbage treatment center and a centralized stacking point, the land is reclaimed to be a littleleaf boxwood nursery land, 2-month-old sand bed seedlings are selected for transplanting, the observation period is 2 years, and the demonstration area is 10 mu. In the demonstration process, the experimental group and the control group are consistent with the conventional management levels except that 200 kg of the compound microbial agent in the scheme 3 is applied to the experimental group in the first spring acre of the second year of nursery preparation and seedling transplantation. The experimental group adopts the biological agent prepared by the scheme 3 to carry out experiments. The control group had no applied biological agent. According to the following data, the compound microbial agent can effectively improve soil physicochemical properties such as landscaping and the like, promote the growth and the propagation of beneficial microorganisms, inhibit the breeding and the propagation of harmful microorganisms and improve the granular structure; mineral substances and organic pollutants in the soil are effectively decomposed, and meanwhile, the effect of fixing heavy metals in the soil is achieved; the root system of the crops is developed, the branches and leaves are flourishing, the disease resistance of the plants and the incidence rate of plant diseases and insect pests are enhanced, and the use frequency of chemical fertilizers and pesticides is reduced.
Soil activity:
TABLE 2 root microbial Activity of Buxus microphylla
TABLE 3 root soil enzyme Activity of different treatments of Buxus microphylla
Physical and chemical properties:
TABLE 4 physicochemical Properties of root soil of Buxus microphylla treated differently
Growth performance
TABLE 5 growth record
| Group of | Root of herbaceous plant | Stem of a tree | Leaf of Chinese character | Tree shaped | Frequency of diseases and insect pests |
| Demonstration group | Deep root and many lateral roots | Rough and strong | Dense, dark green | Spherical and mellow | 2 |
| Control group | Root system is general | Thin and weak | Relatively thin and yellow | Irregularity | 5 |
Claims (10)
1. A compound microbial agent, which is characterized by comprising antagonistic microbe active bacterial sludge and biochar; the antagonistic antimicrobial active bacterial sludge comprises a combination of Lactobacillus plantarum (Lactobacillus plantarum), bacillus subtilis (bacillus subtilis), Aspergillus niger (Aspergillus niger), Trichoderma viride (Trichoderma viride), Saccharomyces cerevisiae (Saccharomyces cerevisiae); the biochar is a product prepared by crushing and drying agricultural wastes and then performing closed internal circulation low-temperature anoxic pyrolysis and carbonization.
2. The complex microbial inoculant according to claim 1, wherein the complex microbial inoculant comprises Lactobacillus plantarum 1.5 × 108～1.0×1010cfu/g, bacillus subtilis 1.0 × 108～5.0×1010cfu/g, Aspergillus niger 1.0 × 108～1.0×1010cfu/g, Trichoderma viride 1.0 × 108～1.0×1010cfu/g, Saccharomyces cerevisiae 5.0 × 108～1.0×1010cfu/g。
3. The compound microbial inoculant according to claim 1, wherein the closed internal circulation low-temperature anoxic pyrolysis carbonization is that agricultural organic waste is placed in a carbonization furnace, heated by an external heat source under a closed condition, and pyrolyzed and carbonized at 400-450 ℃.
4. A method for preparing the complex microbial inoculant of any one of claims 1-3, comprising the following steps:
a. slant culture: respectively inoculating lactobacillus plantarum, bacillus subtilis, aspergillus niger, trichoderma viride and saccharomyces cerevisiae strains on a solid culture medium under an aseptic condition and carrying out strain culture;
b. first-order seed culture: respectively inoculating the strains obtained by the culture in the step a into triangular flasks filled with liquid culture media under aseptic conditions for culture, wherein aspergillus niger, trichoderma viride and saccharomyces cerevisiae are cultured by a shaking table, and lactobacillus plantarum and bacillus subtilis are statically cultured to obtain first-grade seeds;
c. secondary seed culture: respectively inoculating the primary seeds obtained in the step b into a fermentation tank for further fermentation to obtain secondary seeds;
d. main body fermentation: inoculating the secondary seeds into a fermentation tank, and performing mixed culture, wherein the mixed culture is to mix the secondary seeds and perform high-density fermentation culture to obtain fermentation liquor;
e. centrifugal separation: filtering and centrifugally separating the fermentation liquor obtained in the step d to obtain microbial active bacterial sludge;
f. mixing and granulating: and e, mixing the microbial active bacterial sludge obtained in the step e with biochar, uniformly stirring, drying and granulating to obtain the antagonistic compound microbial agent.
5. The method for preparing a composite microbial inoculant according to claim 4, wherein in the step a, the slant culture is performed by culturing Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae at 30 ℃ for 3 days, and culturing Lactobacillus plantarum and Bacillus subtilis at 37 ℃ for 2 days.
6. The method according to claim 4, wherein in step b, said shake cultivation is performed at 30 ℃ for 3 days at 150r/min for Aspergillus niger, Trichoderma viride, Saccharomyces cerevisiae; the static culture is static culture of lactobacillus plantarum and bacillus subtilis for 2 days at 37 ℃; stopping culturing when the OD600 value of single strain liquid culture of each strain is between 3.0 and 4.0.
7. The method for preparing a composite microbial inoculant according to claim 4, wherein in the step c, the secondary seed culture is performed by culturing Aspergillus niger, Trichoderma viride and Saccharomyces cerevisiae at 30 ℃ in a fermentation tank at a stirring speed of 180/min and a sterile air aeration ratio of 1:1 for 4 days; culturing lactobacillus plantarum and bacillus subtilis in a fermentation tank at 37 ℃ for 2 days; the aeration ratio is the ratio of the aeration quantity of sterile air to the volume of the fermentation liquid.
8. The method for preparing a complex microbial inoculant according to claim 4, wherein in step d, the second-level seeds are mixed and inoculated into a fermentation tank at an inoculum size of 15% by volume of the liquid culture medium for high-density fermentation culture, wherein the high-density fermentation culture is fed-batch culture, and the fed-batch carbon source is one or a mixture of glucose, glycerol, sucrose and molasses; the supplementary nitrogen source is one or the mixture of ammonium sulfate, beef extract and peptone.
9. The preparation method of the composite microbial inoculant according to claim 4, wherein the step f is to mix the biochar and the antagonistic microbial activity bacteria mud according to the mass ratio of 10:1-4, uniformly stir the mixture, and dry and granulate the mixture at 35-40 ℃ to obtain composite microbial inoculant particles with the particle size of 2-4mm, wherein the water content of the particles is less than or equal to 15%.
10. Use of the complex microbial inoculant according to any one of claims 1 to 3, wherein: the compound microbial agent is applied in a bottom application mode, and is applied once in spring or once in spring planting and autumn harvest, wherein the application amount is 400 kilograms per mu every year.
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