CN106883998B - Preparation method of multifunctional microbial fertilizer and fermentation medium composition - Google Patents
Preparation method of multifunctional microbial fertilizer and fermentation medium composition Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention discloses a preparation method of a microbial multifunctional fertilizer and a fermentation culture medium composition, belongs to the technical field of microbial fertilizer preparation, and particularly relates to a preparation method of a microbial fertilizer. The preparation method of the microbial multifunctional fertilizer comprises the following operation steps: (1) preparing a culture medium; (2) activating strains; (3) preparing liquid seeds; (4) liquid state fermentation; (5) preparing seed mixing fertilizer and leaf fertilizer; (6) and (4) preparing a ground fertilizer. The fermentation medium comprises the following components: 15000g of glucose, 10000g of peptone, 5000g of yeast extract and KH2PO41300g，K2HPO41000g，MgSO4·7H2O 500g，MnSO4·H2O 100g，FeSO4·7H2O 100g，CaC12·2H2O1500g, water is added to 1000L, and the pH value is 7.0-7.5. The preparation method is simple to operate and low in cost, and the prepared microbial fertilizer can be used for preventing and treatingThe plant disease can promote the growth of plants, reduce the dosage of chemical fertilizers and pesticides, and effectively solve the problems of pesticide pollution, land hardening and the like.
Description
Technical Field
The invention relates to the technical field of microbial fertilizer preparation, in particular to a preparation method of a microbial multifunctional fertilizer and a fermentation medium composition.
Background
China has a large population and a small cultivated land area per capita, and has important significance in improving crop yield. Soil fertilization is a common way to increase yield. Currently, fertilizers used in agriculture include microbial fertilizers and chemical fertilizers. The long-term application of chemical fertilizers can cause soil hardening; and at present, based on the health concepts of 'green' and 'environmental protection', the demand of people on pollution-free, safe and sanitary green food is increasing day by day, so that the microbial fertilizer is favored. In addition, the application of the microbial fertilizer can reduce the application of chemical fertilizer, improve soil and improve the quality of agricultural products; the microbial fertilizer has the characteristics of long acting, no toxicity, no pollution, resource saving, low cost and the like, and can make up for the defects of chemical fertilizers.
The ground fertilizer is an important way for plants to absorb nutrient components, and the foliar fertilizer is an important supplement for the plants to absorb the nutrient components. Along with the aggravation of the influence of various plant diseases and insect pests in the growth process of plants, if the microbial fertilizer capable of promoting the growth of the plants and antagonizing the plant diseases is applied, the yield and the income of the plants and the crops can be increased, and the economic benefit of farmers can be improved. Therefore, the developed excellent bacterial strains can be utilized to ferment, and the obtained fermentation product can be prepared into multifunctional microbial fertilizers such as ground fertilizers, leaf fertilizers, seed dressing fertilizers and the like.
Disclosure of Invention
The invention relates to a method for preparing bacillus subtilisBacillus subtilis) The method for preparing the microbial fertilizer by the BS501a and the culture medium formula for producing the microbial fertilizer by fermentation can prepare the microbial multifunctional fertilizer which can prevent and control plant diseases and promote plant growth, and solves the problems in the prior art.
The invention is realized by the following technical scheme:
a preparation method of a microbial multifunctional fertilizer comprises the following operation steps:
(1) preparing a culture medium: respectively preparing a strain activation culture medium, a seed culture medium and a fermentation culture medium, and sterilizing at high temperature for later use;
the strain activation culture medium comprises: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, and adding water to 1L, wherein the pH value is 7.0-7.5;
the seed culture medium is as follows: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, and adding water to 1L, wherein the pH value is 7.0-7.5;
the fermentation medium is as follows: 15000g of glucose, 10000g of peptone, 5000g of yeast extract and KH2PO41300g，K2HPO41000g，MgSO4·7H2O 500g， MnSO4·H2O 100g，FeSO4·7H2O 100g，CaC12·2H2O1500g, adding water to 1000L, and adjusting the pH value to 7.0-7.5;
(2) activating strains: picking slopes for preservationBacillus subtilisBS501a is inoculated in the strain activation culture medium prepared in the step (1), and shaking table shaking culture is carried out for 16-24h at 35-37 ℃ and 180-;
(3) preparing liquid seeds: inoculating the activated bacillus subtilis liquid obtained in the step (2) into the seed culture medium prepared in the step (1) according to the inoculation amount of 5-10% (volume percentage), and introducing sterile air to culture for 16-24h at 35-37 ℃ to obtain liquid seeds;
(4) liquid state fermentation: inoculating the liquid seed prepared in the step (3) into the fermentation culture medium prepared in the step (1) according to the inoculation amount of 5-10% (volume percentage), culturing for 48h at 30 ℃ by introducing sterile air, counting by using flat plate bacteria, and enabling the number of viable bacteria to reach 1 multiplied by 109 - 9×109CFU/mL to obtain viable bacteria culture;
(5) preparing a leaf fertilizer and a seed dressing fertilizer: and (4) standing the live bacteria culture prepared in the step (4) to precipitate bacteria. Removing thallus to obtain microbial foliar fertilizer and seed dressing fertilizer;
(6) preparation of a ground fertilizer: and (4) ventilating and drying the live bacteria culture obtained in the step (4) to obtain the ground fertilizer.
The application method of the microbial multifunctional fertilizer comprises the following steps: when the fertilizer is used for dressing seeds, the fertilizer is diluted by 100 times of water and used for dressing seeds; when the foliar fertilizer is sprayed on the leaf surfaces, the foliar fertilizer is diluted by 100 times of water, and is sprayed on the leaf surfaces of the seedling stage and the fruit stage of crops; the ground fertilizer is spread on the ground when being cultivated.
The invention has the beneficial effects that:
1. the bacillus subtilis has the functions of promoting plant growth and antagonizing plant pathogenic bacteria, and the microbial multifunctional fertilizer prepared by fermentation has the double functions of promoting crop growth and preventing and treating plant leaf diseases and fruit diseases, and can reduce the use amount of chemical fertilizer and pesticide, improve soil, improve farmland environment quality and improve the quality of agricultural products. The bacillus subtilis can successfully colonize plant rhizosphere, body surface or in vivo, secretes antibacterial substances to inhibit the growth of pathogenic bacteria, has a biocontrol effect, and has a strong inhibiting effect on pathogenic fungi of various plants and crops;
2. in the preparation method, the fermentation medium can increase the number of bacillus subtilis in the fermentation product; the secretion of antagonistic metabolites is improved through microbial metabolism, the growth of plants is promoted by the living bacteria, and the effective biological bactericide effect can be ensured to be exerted in the whole growth cycle of the plants or crops, so that the disease resistance of the plants or crops is enhanced. The culture medium is rich in nitrogen, phosphorus, potassium and various mineral substances, and can enhance soil fertility and reduce soil fertilizing amount.
Detailed Description
Example 1 optimized production of fermentation Medium by orthogonal Experimental method
To improveB. subtilisThe antagonistic capacity of the BS501a fermentation product optimizes the composition of the fermentation medium and the fermentation conditions by adopting a single-factor test and an orthogonal test, and the optimal medium composition is obtained as follows: 1.5% of glucose, 1% of peptone, 0.5% of yeast extract and KH2PO4 0.13%, K2HPO4 0.1%, MgSO4·7H2O 0.05%, MnSO4·H2O 0.01%, FeSO4·7H2O 0.01% , CaC12·2H20.15% of O and 7.0 of pH; the optimal fermentation temperature is 30 ℃, and the optimal fermentation time is 48 h.
Example 2 antibacterial Spectrum of Bacillus subtilis BS501a fermentation broth
The antibacterial spectrum of the bacillus subtilis BS501a is tested by adopting a face-off test. Because the growth speeds of the plant pathogenic fungi are different, in order to accurately judge the antagonistic activity of the bacillus subtilis BS501a, a method for culturing the bacillus subtilis BS501a and the plant pathogenic fungi in stages or in the same phase is adopted. For plant pathogenic fungi with higher growth speed, firstly, diagonally inoculating bacillus subtilis BS501a on a fresh PDA plate, culturing for 24h at 26 ℃, transferring a fungus cake to be tested in the center of the plate, then, continuously culturing for 48h, and measuring the width of an antagonistic zone. For plant pathogenic fungi with medium growth speed, inoculating a fungus cake of the tested fungi in the center of a fresh PDA plate, simultaneously inoculating a bacillus subtilis BS501a strain around the diagonal line of the fungus cake, culturing for 72h at 26 ℃, and measuring the width of an antagonistic zone. For plant pathogenic fungi with slow growth speed, inoculating a fungus cake of the tested fungi in the center of a fresh PDA plate, culturing at 26 ℃ for 24h, then inoculating bacillus subtilis BS501a on the plate in a diagonal manner, continuously culturing for 72h, and measuring the width of an antagonistic zone. The results are shown in Table 1.
TABLE 1 Bacillus subtilis BS501a antibiogram
| Test fungi | Width/mm of antagonistic band |
| Magnaporthe grisea | （7.4±0.23）a |
| Fusarium culmorum | （6.98±0.21）a |
| Pythium gramineum | （3.06±0.15）b |
| Blight of watermelon | （5.25±0.34）c |
| Blight of hot pepper | （4.73±0.30）c |
| Fusarium moniliforme | （3.50±0.21）d |
| Bacterial wilt of corn | （4.25±0.16）cd |
| Root rot of wheat | （8.58±0.47）a |
| Corn leaf spot germ | （7.18±0.32）b |
| Wheat scab germ | （5.38±0.36）c |
| Rot of apple | （5.00±0.20）c |
| Ring rot of apple | （4.72±0.38）c |
| Cucumber gray mold | （8.03±0.71）ab |
| Rhizoctonia cerealis | （9.10±0.12）a |
| Early blight of tomato | （8.65±0.43）ab |
| Blight of cotton | （7.10±0.21）bc |
| Verticillium dahliae of cotton | （7.25±0.09）b |
Note: the English letters are the same and mean that the difference is not obvious, and the English letters are different and mean that the difference is obvious.
The inhibition effect is strong when the width of the antagonistic band is 4.25mm as a critical point, the inhibition effect is strong when the width of the antagonistic band is more than 4.25mm, and the inhibition effect is weak when the width of the antagonistic band is less than 4.25 mm. The results of the confrontation tests show that the bacillus subtilis BS501a fermentation liquor has strong inhibiting effect on rhizoctonia solani, rhizoctonia rot, fusarium graminearum, early blight of tomato, gray mold of cucumber, fusarium oxysporum, verticillium wilt of cotton, fusarium wilt of pepper, rhizoctonia solani, microsporum maydis, fusarium oxysporum, apple rot and ring rot of apple; has weak inhibition effect on maize ralstonia solanacearum, maize fusarium moniliforme and pythium graminearum.
Example 3 Bacillus subtilis BS501a fermentation broth biosafety assay
According to pharmacopoeia, an acute toxicity experiment is adopted to detect the biological safety of the bacillus subtilis BS501a fermentation liquor. And (3) freeze-drying the bacillus subtilis BS501a fermentation liquor to prepare freeze-dried powder. Sequentially diluting lyophilized powder with normal saline to 5 × 10-2g/mL，5×10-3g/mL，5×10-4g/mL，5×10-5g/mL，5×10-6g/mL, etc. Feeding 48 Kunming mice of 3 weeks old for 7 days for observation, wherein the mice freely feed and drink water in the observation period. After the observation period, the mice were randomly divided into 6 groups of 8 mice each. Mice were fasted prior to dosing, but not water deprived. After overnight, it was weighed. Taking 5 groups of mice, and per mouse, per orally intragastrically irrigating 0.2ml bacillus subtilis BS501a fermentation liquid freeze-dried powder solution according to the weight, so that the dosages of the bacillus subtilis BS501a fermentation liquid freeze-dried powder of the 5 groups of mice are respectively 500mg/kg, 50 mg/kg, 5 mg/kg, 0.5 mg/kg and 0.05 mg/kg. Group 6 mice were set as a blank control group and were gavaged with 0.2ml of physiological saline through the mouth. Post-administration timeThe food was taken every 2h and continuously observed for 7 d. And observing the appearance, mental performance, ingestion condition and weight change of the mice in the feeding process. 7d, dissecting and observing the pathological change of the viscera.
According to the national technical supervision bureau GB15670-1995 acute oral toxicity grading standard, the LD50 value of the bacillus subtilis BS501a fermentation liquid freeze-dried powder is more than 500mg/kg, which proves that the bacillus subtilis BS501a fermentation liquid freeze-dried powder belongs to low-toxicity grade and meets the requirements of green biopesticides.
Example 5 prevention and control Effect of Bacillus subtilis BS501a fermentation liquid potting
Culturing rice for about 3 weeks, spraying 5mL (adding 0.5% Tween 80) of Pyricularia oryzae spore suspension to leaves and stems of the rice by using a spray can when most of the rice grows to three leaves and one heart, spraying the suspension for 24h, and spraying the fermentation supernatant (adding 0.5% Tween 80) of Bacillus subtilis BS501a to leaves to prevent and treat the rice 1 time per day and 5mL each time. The control was treated with deionized water. And (4) alternately culturing for 6d (counting time from spraying of rice blast fungus spore suspension) under 100% illumination for 14h and dark for 10h at the temperature of 28 ℃, and investigating the rice infection condition. Survey grading standards are in units of leaves (IRRI):
grade 0 is disease-free
Grade 1, less than 5 leaf spots and less than 1cm in length
Grade 3 is 6-10 leaf spots, and the length of part of the spots is more than 1cm
The 5-grade has 11-25 leaf spots, and parts of the spots are connected into a tablet and account for 10-25 percent of the area
The 7-grade has more than 26 leaf spots, and parts of the 7-grade are connected into pieces and occupy 26 to 50 percent of the area
The 9 grades are that the scabs of the leaves are connected into slices, which account for more than 50 percent of the area of the leaves or are completely withered.
After the rice blast germ spore suspension is sprayed for 2-3 days, the rice blast germ infection traces can be seen on the rice leaves. The rice leaves which are not sprayed with the fermented supernatant of the bacillus subtilis BS501a are obviously infected by rice blast germs, most of the leaves have obvious rice blast disease spots, the number is large, the area is large, part of the disease spots are diffused to the whole leaves, and the infected leaves obviously turn yellow like being burnt by fire. The rice sprayed with the fermented supernatant of the bacillus subtilis BS501a has most leaves kept fresh green, and a small number of infected leaf lesions with small area. The specific disease index and the prevention and treatment efficiency are shown in Table 2.
TABLE 2 prevention and treatment efficiency of fermentation supernatant of Bacillus subtilis BS501a
Note:
example 6 growth promoting Effect of Bacillus subtilis BS501a fermentation broth on Rice
Soaking healthy rice seeds in 100 times diluted bacillus subtilis fermentation liquor for 24 hours, sowing the rice seeds in a plastic yarn disc, uniformly covering the surface with spun yarns, simulating natural illumination in a multiband incubator, culturing and growing seedlings, and adding clear water along the edge of the yarn disc as required. Each treated 100 pellets. The control was soaked in clear water. When the rice grows to have two leaves and one heart, the germination percentage and the fresh weight of the seedlings are measured. The result shows that compared with the control, the fresh weight of the rice seedling after the soaking treatment by the fermentation liquor of the bacillus subtilis BS501a is improved by 15.93 percent, but the fermentation liquor has no influence on the germination rate of the rice seed.
Claims (1)
1. A preparation method of a microbial multifunctional fertilizer is characterized by comprising the following steps: the method comprises the following operation steps:
(1) preparing a culture medium: respectively preparing a strain activation culture medium, a seed culture medium and a fermentation culture medium, and sterilizing at high temperature for later use;
the strain activation culture medium comprises: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, and adding water to 1L, wherein the pH value is 7.0-7.5;
the seed culture medium is as follows: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, and adding water to 1L, wherein the pH value is 7.0-7.5;
the fermentation medium is as follows: 15000g of glucose, 10000g of peptone, 5000g of yeast extract and KH2PO41300g，K2HPO41000g，MgSO4·7H2O500g，MnSO4·H2O100g，FeSO4·7H2O100g，CaC12·2H2O1500g, adding water to 1000L, and pH 7.0-7.5;
(2) activating strains: selecting Bacillus subtilis for slant preservationBacillus subtilis) BS501a is inoculated in the strain activation culture medium prepared in the step (1), and shaking table shaking culture is carried out for 16-24h at 35-37 ℃ and 180-;
(3) preparing liquid seeds: inoculating the activated bacillus subtilis liquid obtained in the step (2) into the seed culture medium prepared in the step (1) according to the inoculation amount of 5-10% in percentage by volume, and introducing air to culture for 16-24 hours at 35-37 ℃ to obtain liquid seeds;
(4) liquid state fermentation: inoculating the liquid seed prepared in the step (3) into the fermentation culture medium prepared in the step (1) according to the inoculation amount of 5-10% in volume percentage, culturing for 48 hours at 30 ℃ in sterile air, and counting by flat plate bacteria until the number of viable bacteria reaches 3 multiplied by 109-9×109CFU/mL to obtain viable bacteria culture;
(5) preparing a leaf fertilizer and a seed dressing fertilizer: standing the live bacteria culture prepared in the step (4), precipitating bacteria, and removing the bacteria to obtain a microbial leaf fertilizer and a seed dressing fertilizer;
(6) preparation of a ground fertilizer: and (4) ventilating and drying the live bacteria culture obtained in the step (4) to obtain the ground fertilizer.
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Effective date of registration: 20211210 Address after: 047600 Huang Ya Dong Zhen Shui Yi Cun, Licheng County, Changzhi City, Shanxi Province Patentee after: Licheng Xinwang organic fertilizer Co.,Ltd. Address before: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Patentee before: Liu Jiaojiao Effective date of registration: 20211210 Address after: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Patentee after: Liu Jiaojiao Address before: 450001 science and Technology Department of Henan University of technology, No.100 Lianhua street, Zhengzhou high tech Industrial Development Zone, Henan Province Patentee before: He'nan University of Technology |
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