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INVENTION
Patent of the Russian Federation RU2125549
METHOD OF OBTAINING BIODEGRATION
The name of the inventor: Kiseleva N.I . ; Zharikov GA; Galkina N.N .; Kolumbet LV; Borovik RV; Dyadishchev N.R.
The name of the patent holder: Research Center for Toxicology and Hygienic Regulation of Biologicals
Address for correspondence: 142283 Moscow Region. Serpukhov district, settlement Dashkovka, Lenina 102A, SIC TBP
Date of commencement of the patent: 1997.12.29
The invention relates to the microbiological industry, to the production of biofertilizers and crop protection agents. The method is that a substrate based on organic waste from agriculture and industry is vermicomposted using the new industrial line of earthworms "Obolensky hybrid" obtained by crossing the "Red Californian hybrid" with the Russian population of earthworms Eisenia foetida at 16-24 o C in For 4-6 months. From the biohumus obtained, worms are separated. It is advisable to keep the vermicompost after natural wool separation for ripening. In vermicompost after the separation of worms or after ripening, microorganisms with fungicidal activity are introduced. As a microorganism, a Bac strain is introduced. Subtilis IPM-215 at concentrations of 1 × 10 9 -1 × 10 12 spores per kg or the culture of the Mycophilus fungus Trichoderma viride Pers ex SF Cray N 16 in concentrations of 1 × 10 4 -1 × 10 8 colony forming units per 1 kg. Biofertilization obtained by the proposed method improves the agrochemical parameters of the soil, has fungicidal and growth-stimulating properties for plants.
DESCRIPTION OF THE INVENTION
The invention relates to the microbiological industry, to the production of biofertilizers and crop protection agents.
To improve the quality of the soil, biohumus is used. There are known methods for obtaining biohumus from organic waste by vermicomposting [1, 2]. Recycling of waste is carried out with the help of a special industrial line of earthworms "Red Californian hybrid." The obtained product has high fertilizing properties, but does not significantly affect soil phytopathogens.
Various preparations based on microorganisms such as Bacillus subtilis [3, 4], fungi of the genus Trichoderma [5] are used to protect plants against pathogens.
The closest to the present invention is a method for producing a compost into which a suspension of microorganisms, phytopathogen antagonists: Trichoderma hamatum fungi and Pseudomonas maltophilia or Flavobacterium bacteria, is introduced [6]. However, this compost has low fertilizing properties and does not stimulate plant growth.
The object of the invention is to provide a biofertilizer that improves agrochemical soil characteristics, which has fungicidal and growth-stimulating properties for plants.
The goal is achieved by introducing microorganisms with fungicidal activity into the biohumus obtained from a mixture of agricultural and industrial wastes with the help of a new line of earthworms "Obolensky hybrid".
The essence of the invention lies in the fact that the substrate is processed on the basis of organic waste from agriculture and industry (manure of animals, poultry manure, plant residues, sewage sludge of sewage treatment plants, waste from food processing enterprises) with the help of a new industrial line of earthworms "Obolensky hybrid" By crossing the "Red Californian Hybrid" with the Russian population of earthworms Eisenia foetida. At the final stage of production of vermicompost or in the finished product is introduced or a strain of bacteria Bac. Subtilis IPM-215 [7] at concentrations of 1 × 10 6 to 1 × 10 12 spores per 1 kg, or to the culture of the Mycophilus fungus Trichoderma viride Pers ex SF Gray N 16 at concentrations of 1 × 10 4 to 1 × 10 8 colony forming units per 1 Kg.
Example 1. Production of biohumus.
To produce biohumus, we use the new industrial line of earthworms "Obolensky hybrid", obtained by us crossing the "Red Californian hybrid" with the Russian population of earthworms Eisenia foetida. The new hybrid is adapted to the climatic conditions of Russia, recycles a wide variety of organic waste. As a substrate for vermicomposting, various organic wastes from agricultural and industrial production are used: animal manure, poultry manure, straw, plant residues, substandard vegetables, sewage sludge of treatment plants, etc. Mixed wastes in equal amounts are pre-composted at t = 30- 60 ° C and humidity 60-85% for 20 days. In the resulting substrate, after adjusting pH = 6.5-7.5, earthworms are introduced. Vermicomposting is carried out at the optimum t = 16-24 o C and humidity 40-80% to 6.0 months (Table 1).
At the end of composting, worms are separated from biohumus. The obtained substrate is dried under natural conditions (the ripening stage) for two weeks and sieved through a sieve.
The finished biohumus has the following chemical composition,%:
- Moisture mass fraction - 54-60
- Organic substances - 37-46 per dry product
- Mineral substances - 54-63
- Humic substances - 24-28
- Total nitrogen - 3.0-4.0
- Total phosphorus (according to P 2 O 5 ) - 1.0-3.0
- Total potassium (according to K 2 O) - 1.0-1.5
- PH 6.5-7.5
- Pathogenic microflora - None
- Eggs of helminths - Not identified
Biohumus contains a large amount of humic substances (up to 28% on dry matter), which provides high fertilizing properties. All nutrients for plants are in the form of mineral, easily digestible salts. Their amount is up to 63% by dry matter. Biohumus does not contain pathogenic microorganisms and heavy metals. Consequently, the biohumus obtained is a highly effective, environmentally friendly organic fertilizer that improves agrochemical soil parameters. After separation of worms or after ripening of vermicompost, microorganisms with fungicidal activity are introduced.
Example 2. Preparation of a fertilizer with different contents of Bac. Subtilis.
Strain of bacteria Bac. Subtilis ИПМ-215 is used for the production of a bacterial bactophyte against plant diseases [8]. The bactophyte preparation is recommended for use in doses of 6 kg / ha with a titer of 2.5 · 10 10 spores / g. At the recommended dose of biohumus 3 t / ha it is necessary to add 2 g of Bac in 1 kg of vermicompost. Subtilis with a titer of 2.5 × 10 10 spores / g, i.e. The initially calculated rate of introduction of bacteria is 2.5 · 10 10 spores / g · 2 g = 5.0 · 10 10 spores / kg vermicompost.
After the separation of worms or the finished biohumus is packed into 1 kg pots and a culture of the Bac strain is introduced. Subtilis ИПМ-215 in 3 different concentrations: initially calculated, 2 times smaller and 2 times larger. The repetition of the experiments is threefold.
The components of biofertilizer are well mixed. After 2 weeks of storage at room temperature (20 ± 5) ° C, fertilizer samples are taken from which suspensions are prepared (10 g for 90 ml of sterile tap water). The prepared suspensions are shaken on a shaker for 40 minutes at (28 ± 1) ° C and a rotation speed of 220-240 rpm. Then prepare a series of tenfold dilutions in saline solution and make seeding from these dilutions in depth on Petri dishes with sucrose-agar agar. Cultivation is carried out at (28 ± 1) o C for 48 hours. At the end of this time, the number of colonies grown is counted, then the recalculation per 1 kg of biofertilizer is carried out (Table 2).
The conducted experiments show the possibility of creating a combined fertilizer with a different concentration of Bac culture. Subtilis. The most optimal is the introduction of Bac. Subtilis in an amount of 2.5 × 10 10 spores / kg. When such a quantity of bacteria is introduced into biohumus, their concentration increases 1.5 times in 2 weeks, reaching the required value for suppression of soil phytopathogens. Therefore, in subsequent experiments we used a combined biofertilizer with an initial titer of 2.5 × 10 10 spores / kg. Considering the multiplication of bacteria, the dose of introducing microorganisms can be reduced in comparison with the calculated one. This significantly reduces the cost of combined biofertilizer and, consequently, the cost of processing soil from phytopathogens.
Example 3 Preparation of biofertilizer with different contents of Trichoderma viride Pers ex SF Gray N 16.
Strain N 16 of the fungus Trichoderma viride Pers ex SF Gray was obtained by L. Kolumbet. As a result of selection work on the basis of productivity and hyperparasitic activity with a natural clone of Trichoderma viride isolated from the soil in the Moscow Region (the strain was deposited with the Central Collection of Microorganisms of the State Concern "BIOPREPARAT" TsKMK "B" on February 15, 1994, registration number - MK F-59M ).
Vermicompost after separation of worms or ready vermicompost is packed in 1 kg pots and culture of Mycophilus fungus Trichoderma viride Pers ex SF Gray N 16 in 3 different concentrations: 1 · 10 4 , 1 · 10 6 , 1 · 10 8 colony forming units CFU) per 1 kg. The repetition of the experiments is threefold.
The components of biofertilizer are well mixed. After 2 weeks of storage at room temperature (20 ± 5) ° C, fertilizer samples are taken from which suspensions are prepared (10 g per 90 ml of sterile tap water). The prepared suspensions are shaken on a shaker for 40 minutes at (26 ± 1) ° C and a rotation speed of 220-240 rpm. Then, a series of tenfold dilutions in saline is prepared and seeded from these dilutions into Petri dishes with a synthetic nutrient medium (Czapek's medium) containing Triton X-100 to limit the growth of the fungal colonies. Cultivation is carried out at (26 ± 1) o C. After three days, the number of colonies grown is counted, then the recalculation per 1 kg of biofertilizer is carried out (Table 3).
The conducted experiments show that the introduction of the Mycophilus fungus culture Trichoderma viride into vermicompost increases the biomass of the fungus, which indicates that vermicompost is an appropriate substrate for the growth of the fungal mycelium. In this way, it is possible to create a new form of biofertilizer containing a fungicidal component in the form of Trichoderma viride culture. This allows to reduce the cost of production of biofertilizer, which has fertilizing and fungicidal properties.
Example 4. Investigation of the protective properties of fertilizers.
To study the protective properties of fertilizers, it is introduced into the soil in a ratio of 1: 4 by volume. Experiments are carried out in pots, 1 kg of soil mixture in each. The repetition of the experiments is threefold.
The study is carried out on an artificial infectious background, for which a mixture of phytopathogens is introduced into the soil: Fusarium culmorum, F. moniliforum, F. heterosporum, F. oxysporum, Verticillium dahliae, Rhizoctonia solani, Colletotrichum atraventa, Alternaria brassicae.
In the prepared soil mixtures, seeds of cucumber seeds are pre-sown.
Reduction of infectivity of different soil mixtures is evaluated after 1 month, conducting microbiological analysis of soil samples on various nutrient media. In samples of soil mixtures with fungicidal components (Bac. Subtilis and Trichoderma viride), a significant decrease in the content of phytopathogens was noted. The presence of fungicidal properties of biofertilizer is reflected in morphometric indicators of plant growth (length and weight of green mass, number of leaves, whiskers).
As can be seen from Table. 4, introduction of biofertilization into the soil leads to an increase in the morphometric parameters, which indicates the protective effect of biofertilizer.
Example 5. Study of the growth-stimulating activity of biofertilizer.
Biofertilizer containing culture Bac. Subtilis in a concentration of 2.5 × 10 10 spores / kg, are applied to the soil (1: 4 by volume). As control used: soil sod, soil with introduced culture Bac. Subtilis and soil with biohumus. In the soil samples, seeds of cucumbers and eggplants are sown. The results of plant growth are shown in Table. 5, and 6.
The data show that the culture of Bac. Subtilis and biohumus separately have a weak growth stimulating effect. In the biofertilizer obtained by the proposed method, a synergism of their action appears (see Tables 5, 6).
Thus, biofertilizer containing a bacterial strain Bac. Subtilis ИПМ-215, possesses not only high fertilizing and protective properties, but also has a high growth stimulating activity.
USED BOOKS
1. US Patent No. 4,108,625, Cl. C 05 F 11/08, 1978.
2. Patent of the USSR No. 1724658 A1, cl. C 05 F 11/00, 1989.
3. Patent of France No. 8113118, 1981.
4. Patent of Russia No. 2019966 C1, cl. A 01 63/00, 1991.
5. Patent of France No. 2545099, C 12 N 1/14, 1984.
6. US Patent No. 4,642,131, Cl. C 05 F 11/08, 1985.
7. Authorship of the USSR N 1144377, 1983.
8. State catalog of pesticides permitted in the territory of the Russian Federation. - 1997.
CLAIM
1. A method for producing bio-fertilizer, comprising obtaining vermicompost by vermicomposting agricultural and industrial wastes using earthworms and separating vermicompost from worms, characterized in that worms "Obolensky hybrid" are used from earthworms, obtained by crossing the "Red Californian hybrid" with the Russian The population of earthworms Eisenia foetida vermicomposting is carried out at 16-24 ° C for 4-6 months and microorganisms with fungicidal activity are introduced.
2. A method according to claim 1, characterized in that after vermicomposting, the vermicompost is kept under natural conditions for ripening.
3. A method according to claims 1 and 2, characterized in that microorganisms having fungicidal activity are added after separation of worms or after ripening of vermicompost.
4. Method according to claims 1 to 3, characterized in that a strain of bacteria Bacillus subtilis ИПМ-215 is used from microorganisms with fungicidal activity in concentrations of 1 × 10 9-1 · 10 12 spores per 1 kg of vermicompost.
5. A method according to claims 1 to 3, characterized in that microorganisms with fungicidal activity use the culture of microphilic fungus Trichoderma viride Pers ex SFGray N 16 in concentrations of 1 × 10 4-1 × 10 8 colony forming units per 1 kg of vermicompost.
print version
Date of publication 04.03.2007gg
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