RU2286324C1 - METHOD FOR PRODUCTION OF BACTERIAL FERTILIZER BASED ON BACTERIUM OF GENUS Azotobacter - Google Patents

Abstract

FIELD: agriculture microbiology, microbiological industry.

SUBSTANCE: claimed method includes culturing of genus Azotobacter bacteria on dense Ashby medium for 24-28 h at 26-28°C. Then obtained cell mass in cultured in liquid Burk medium with addition of feed yeast extract, and obtained accumulation culture is introduced into working capacity of Burk medium with addition of triptophane and feed yeast extract. After culturing finished product is stored in refrigerator.

EFFECT: increased cell growth activity.

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Description

The invention relates to the field of agricultural microbiology and can be implemented in the microbiological industry to obtain various bacterial fertilizers.

The use of azotobacter strains in fertilizer production is widely known, for example, the Azotobacter chroococcum T12 strain, the Azotobacter chroococcum B-3173 strain deposited at the All-Russian Scientific Research Institute of Genetics and Genetics (see US Pat. No. 2074159, C 05 F 11/08, 1997). The disadvantage of these strains is the lack of a positive effect in increasing the yield of green crops, as well as a rather high nitrate content in them, if bacterial fertilizer based on this strain is used for their cultivation.

It is known that tryptophan is added to the nutrient medium to stimulate bacterial growth (see RF Patent No. 2035442, C 05 F 11/08, 1992). Use it in combination with Muromtsev's medium for Achromobacter bacteria.

As a prototype, a method for synthesizing the bacterial strain Azotobacter chroococcum VKPM B 6010 used to produce bacterial fertilizer for green cultures can be considered (see RF patent No. 2074159, C 08 F 11/08, 1997). The strain is grown in a fermenter on a Burke culture medium to a cell concentration of 10 ⁸ -10 ⁹ cells / ml.

Synthesis products on Burka nutrient medium (after 4 days of incubation at 28-30 ° C): biotin - 0.07, pyridoxine - 0.59, thiamine - 1.9 mg / l, heteroauxin - 65.0 mg / l. Nitrogen fixation - 0.26 μg nitrogen / ml per hour.

The strain Azotobacter chroococcum B35 has the ability to attach to the root surface of a number of green crops, in particular celery (Apium), parsley (Petrocelinum) and lettuce (Lactuca sativa), where it functions for 5 months after inoculation. In this case, the yield of green crops increases and the content of nitrates decreases.

However, the known method does not always allow to obtain a rapid increase in the biomass of bacteria due to the physiological activity of a particular strain. The composition of the known medium for the cultivation of nitrogen fixers does not include compounds - precursors of the synthesis of phytohormones (indolyl-3-acetic acid (IAA)), as well as growth factors - biomass growth stimulants (vitamins, amino acids). To create a bacterial fertilizer, a specific strain is used, which manifests its activity under certain conditions and is used to grow certain plants.

The task at hand is the expansion of the possibilities of using a wide range of strains of bacteria of the genus Azotobacter for the preparation of bacterial fertilizers.

EFFECT: increased growth activity of bacterial cells and improved adaptation of fertilizers to specific conditions.

This technical result is achieved by the fact that in the method of preparing a bacterial fertilizer based on bacteria of the genus Azotobacter, including the cultivation of bacteria on Burke's nutrient medium, the bacteria are cultured previously on Ashby's solid medium for 24-28 hours, at a temperature of 26-28 ° C; then they inoculate Burke's liquid medium with the addition of fodder yeast extract (ECD) in an amount of 0.3-0.7 g / l (to obtain cell biomass in the exponential phase), incubated in an incubator at the same temperature for 20-24 hours at stirring the resulting accumulative culture is introduced into the working volume of fresh Burke medium with the addition of tryptophan 0.2-1.0 g / l and EKD, cultivated for 40-48 hours at the same temperature; then placed in the refrigerator for further storage.

An accumulative culture of azotobacter strains is obtained enriched with the presence of indolyl-3-acetic acid.

Pre-cultivation of bacteria on Ashby activates nitrogen-fixing ability.

Burke has a richer mineral composition.

Fodder yeast extract (ECD) contains a complex of vitamins and amino acids, which has a stimulating effect on the rate of reproduction and nitrogen-fixing activity. Using EKD allows you to get a significant increase in biomass in the first 4-6 hours of cultivation, regardless of the amount of culture introduced. Tryptophan stimulates the synthesis of IAA to amounts of 40 μg / ml of culture fluid, however, the maximum amount depends on the characteristics of the strain.

DESCRIPTION OF THE TECHNOLOGICAL PROCESS

1. The cultured strain is seeded on a solid Ashby medium and cultured for 24-28 hours, at a temperature of 26-28 ° C for the accumulation of a large mass of actively dividing nitrogen-fixing cells.

2. The resulting mass of cells is washed off the surface of a dense medium with 2-5 ml of liquid Burke medium and inoculated in 0.5 liters of Burke medium with the addition of 0.3-0.7 g / l ECD to obtain a population of cells with a concentration of 10 ⁹ -10 ¹¹ cells / ml Cultivate for 20-24 hours at the same temperature.

3. The resulting suspension is added to the working volume of Burke's medium (10-50 liters) with an ECD and with the addition of tryptophan (0.2-1.0 g / l). Tryptophan is introduced for the intensive accumulation of IAA in the working volume of the medium. Cultivate for 40-48 hours, at a temperature of 26-28 ° C.

4. The finished product is stored in a refrigerator at a temperature of 0 to 10 ° C.

EXAMPLE OF IMPLEMENTATION OF THE METHOD.

1. The cultured strain is seeded on a solid Ashby medium and cultured for 26 hours at a temperature of 27 ° C to accumulate a large mass of actively dividing nitrogen-fixing cells.

Reducing the time of cultivation does not allow to obtain a sufficient mass of cells, and an increase leads to an increase in the timing of preparation of the final product. Cultivation at temperatures below 26 ° C prolongs the development of microbial cells, an increase in temperature can lead to premature death of some bacteria.

2. The resulting cell mass is washed off from the surface of a dense medium with 2-5 ml of Burke’s liquid medium and inoculated in 0.5 liters of Burke’s medium with the addition of 0.5 g / L EKD to obtain a cell population with a concentration of 10 ⁹ -10 ¹¹ cells / ml. Cultivate for 22 hours. Reducing the timing of cultivation does not allow to obtain sufficient concentrations of cells, and the extension of the terms leads to an overall increase in time for preparing the final product. Reducing the amount of introduced extract of fodder yeast less than 0.3 g / l does not allow to obtain sufficient concentrations of bacteria in the medium, and the introduction of excess quantities of the extract increases the cost of the final product, without giving significant advantages to the drug.

Table 1 shows the cell concentrations of different strains of Azotobacter bacteria in ml of Burke medium. All of them gave a significant increase in biomass.

3. The resulting suspension is added to the working volume of Burke's medium (10-50 liters) with an ECD, with the addition of tryptophan (0.6 g / l). Cultivate for 44 hours at a temperature of 27 ° C. An increase in the dose of tryptophan introduced increases the cost of the process, a decrease does not create favorable conditions for the accumulation of a phytostimulator. Cultivation of less than 40 hours does not allow to accumulate sufficient concentrations of phytohormone in the final product, an increase in time leads to a rise in the cost of the process. The previously selected temperature regime of cultivation is most optimal for the development of azotobacter cells.

Table 2 shows the concentrations of IAA accumulated in the Burke medium by various strains of Azotobacter bacteria. All of them produced fairly high concentrations of IAA.

4. After 44 hours, the resulting finished product is transferred to a refrigerator for storage.

To justify the parameters given in the formula, tests were carried out at their extreme and transcendental values, on the basis of which the above conclusions were made.

The seeds obtained from different cultures were treated with the obtained preparations and the activity of their germination was observed, as well as the growth of biomass in the grown 3-week seedlings of different plant species. The research results are shown in tables 3, 4 (table 3 - seed germination, table 4 - increase in biomass).

Thus, a method for producing an accumulative culture for preparing a preparation from azotobacter strains living in the soil of a particular territory and adapted to these environmental conditions is proposed.

The proposed method allows the use of different strains of Azotobacter bacteria as the basis for the preparation of fertilizer. It was found that they are most active if they are introduced into the same soil from which they were isolated. The increase in yield can be judged by the germination of seeds and the effective accumulation of plant biomass (tables 3, 4).

Table 1 The studied strains The number of cells in 1 ml of Burke's medium: without ECD with ECD. Az.chroococcum 66 2.510 ⁶ 20.410 ⁸ Az.chroococcum 11 1.5 · 10 ⁶ 13.0 · 10 ⁸ Az.chroococcum 12 1.410 ⁶ 13.510 ⁹ Az.chroococcum 1 2.010 ⁶ 18.0 · 10 ⁸ Az.chroococcum 6 15.0 · 10 ⁶ 60.0 · 10 ⁷ table 2 The studied strains The number of IAA in the environment of Burke ... (μg / ml) without tryptophan with tryptophan Az.chroococcum 6 0-traces 12.3 Az.chroococcum 11 0-traces 32,0 Az.chroococcum 12 0-traces 32,2 Az.chroococcum 1 0-traces 30.6 Az.chroococcum 66 0-traces 35.8 Table 3 The studied strains The number of seeds sprouted from 100 ... (%) goatskin ... beans ... neobr. arr neobr. arr Az.chroococcum 66 61 79 84 96 Az.chroococcum 11 69 76 87 95 Az.chroococcum 12 67 78 83 98 Az.chroococcum 1 71 79 89 98 Az.chroococcum b 51 72 91 97

Table 4 The studied strains The mass of 100 3-week-old seedlings ... (g) goatskin ... beans ... neobr. arr neobr. arr Az.chroococcum 66 4.2 11,4 275 315 Az.chroococcum 11 4.8 10.9 246 304 Az.chroococcum 12 4.3 10.5 239 308 Az.chroococcum 1 4.9 11.2 264 302 Az.chroococcum 6 4.4 10.8 283 311

Claims (1)

A method of preparing a bacterial fertilizer based on bacteria of the genus Azotobacter by cultivating them on Burke nutrient medium, characterized in that the bacteria are cultured previously on Ashby solid medium for 24-28 hours at a temperature of 26-28 ° C, then they are inoculated into Burke liquid with the addition of fodder yeast extract in an amount of 0.3-0.7 g / l, kept in a thermostat at the same temperature for 20-24 hours with stirring, the resulting accumulative culture is introduced into the working volume of fresh Burke medium with the addition of tripts Ofana 0.2-1.0 g / l and extract of fodder yeast, after culturing for 40-48 hours at the same temperature, the finished product is placed in a refrigerator for storage.