RU2237048C1 - Method for cultivating nitrogen-fixing bacteria - Google Patents

Abstract

FIELD: agriculture, plant growing.

SUBSTANCE: the present innovation deals with manufacturing, application and storage of bacterial fertilizers and could be applied at cultivating leguminous grasses and other agricultural plants. The method, also, deals with growing a bacterial stock culture upon nutritive medium and preparing a working culture upon corresponding nutritive medium. One should introduce aqueous solution of sodium selenate either into nutritive medium for cultivating a working culture or into nutritive medium for growing a stock culture for the purpose to achieve final concentration of sodium selenate to be 1·10^-4g/l liquid nutritive medium. The present method provides increased viability of fertilizers, broadened assortment of stimulants at decreased financial expenses.

EFFECT: higher efficiency of cultivation.

2 ex, 1 tbl

Description

The invention relates to the field of agriculture, crop production and relates to the production, use and storage of bacterial fertilizers, and can be used in the cultivation of legumes and other agricultural plants.

The unsolved problems of the production and practical use of bacterial preparations in the field are the relatively low reproduction rate of nodule bacteria and their low viability during storage and use. Substances of a phenolic nature contained in the main known carrier, peat, have an adverse effect on nodule bacteria, as a result of which the nitrogen fixation process is weakened.

Known methods of stimulating nitrogen fixation by introducing new technologically beneficial strains of bacteria with increased environmental valency used in the production of bacterial fertilizers [RF Patent No. 2061666, 6 C 05 F 11/08, 1996. Naumyvakin LV, Piruzyan ES, Soloviev V.P., Chernin L.S., Tilba V.A. The strain of bacteria Rhizobium fredii for the preparation of bacterial fertilizers; RF patent №2074159, 6 С 05 F 11/08, 1997. Ozhiganova G.U., Lansky G.P., Chernov I.A. The bacterial strain Azotobacter chroococcum used to obtain bacterial fertilizer for green crops].

It is known to use peat compost as an organic carrier, and the resulting mixture, inoculated with the bacterial component, is incubated with stirring for 3-5 days [RF Patent No. 2084431, 6 C 05 F 11/08, 1997. L. Yudkin, Kovalev N.G., Khotanovich A.V., Temnova O.V. A method of obtaining a bacterial fertilizer]. The disadvantages of these methods are, accordingly, the narrowness of use and, in the latter case, the high complexity and energy costs.

There is a method of introducing a solution of a bacterial preparation in the range of 0.5-5.0 · 10 ¹² tank / ha by foliar method on vegetative plants [RF Patent No. 2193837, A 01 C 21/00, C 05 F 11/08, 2001. Kirov E .I., Maistrenko G.G., Makarov V.I., Kutsenogiy K.P. The method of introducing nitrogen-fixing bacteria into the soil]. However, this method provides additional, remote from the time of sowing events associated with additional costs of materials and energy, and is also dependent on weather conditions.

A known method of stimulating nitrogen-fixing bacteria of legumes [RF Patent No. 2111636, A 01 B 79/02, A 01 C 21/00, 1998. Bekuzarova SA, Abaev AA, Farniev AT A method of stimulating nitrogen-fixing bacteria in legumes]. In this case, as a stimulant, an extract is used - a waste of starch - syrup production, which in the amount of 90-100 kg / ha in the form of an aqueous solution is applied as a top dressing 13-15 days after emergence. The disadvantage of this method is its relative high cost and inaccessibility of the extract, as well as the fact that even a slight excess of the specified dose significantly acidifies the mixture and adversely affects the microflora of the soil.

There is a way to stimulate nitrogen-fixing bacteria in legumes by intensive use of macro- and microelements [G. Pospypanov The effect of intensive cultivation of leguminous herbs on their symbiotic activity, protein collection and increased soil fertility - Vestnik s.kh. Science, No. 9, 1987. S. 62-64]. After biofertilizer is applied to the soil, mineral fertilizers are applied to stimulate the activity of nitrogen fixers. In this method, significant material costs for the application of mineral fertilizers for grass are inevitable, which at the present stage can afford only a small number of farms. Another weak point of the method is the risk of overdose and reduced product quality.

The closest in technological essence is the method of cultivation of nitrogen-fixing bacteria, providing for the cultivation of the uterine culture of bacteria on an environment of the following composition: peas (decoction) - 100.0 ml; glucose - 10.0 g; (NH ₄ ) ₂ SO ₄ - 1.0 g; KH ₂ PO ₄ · N ₂ O - 0.5 g; To ₂ HPO ₄ - 0.5 g; MgSO ₄ · 7H ₂ O - 0.3 g; Ca CO ₃ - 1.0 g, then the manufacture of a working culture on a medium containing (g / l): glucose - 20.0; corn extract - 10.0; yeast extract - 0.25; KH ₂ PO ₄ · N ₂ O - 0.5; K ₂ HPO ₄ - 0.5; MgSO ₄ · 7H ₂ O — 0.3; Ca CO ₃ - 1.0, after which the implementation of the inoculation of prepared peat - a massive introduction of liquid culture into peat [Khotyanovich A.V. Cultivation methods of nitrogen-fixing bacteria, methods for producing and using preparations based on them (Methodical recommendations) Leningrad, 1991]. The disadvantages of this method are the risk of culture inhibition when using salts of K, Mg, Ca, disturbances in the aeration system, even with a slight change in the reaction of the medium, as well as a narrow range of storage temperatures of the crops.

The proposed method provides an increase in the titer of bacteria in the culture fluid, the viability and activity of the bacterial component of fertilizers, expanding the range of stimulants while reducing material costs. This is achieved by the fact that in the method of culturing nitrogen-fixing bacteria, including growing a uterine culture, then producing a working culture, after which the prepared peat is inoculated - mass introduction of liquid culture into peat, according to the invention, an aqueous solution of selenate is introduced into the nutrient medium for the uterine or for working culture sodium so that the final concentration of sodium selenate is 1-10 ^-4 g / l of a liquid nutrient medium.

The trace element contained in the substance selenium is able to increase the activity of almost all parts of the antioxidant system of microorganisms, limiting their growth and development without negative impact on the enzymatic apparatus. This allows not only to increase the viability and activity of nitrogen fixers, but also significantly reduce the time and increase the temperature range of the cultivation of nitrogen-fixing bacteria.

Another sign that distinguishes the proposed method from the known ones is that the use of ultra-low doses of selenium salt does not require significant economic costs, since the amount of the introduced substance (sodium selenate) used is very small.

Example 1

To solve the problem in a liquid nutrient medium, the following composition:

Peas (decoction) 100.0 ml

Glucose 10.0 g

(NH ₄ ) ₂ SO ₄ 1.0 g

KH ₂ PO ₄ · N ₂ O 0.5 g

K ₂ HPO ₄ 0.5 g

MgSO ₄ · 7H ₂ O 0.3 g

CaCO ₃ 1.0 g

a volume of 200 ml, poured into flasks with a capacity of 750 ml and intended for the cultivation of bacteria, add a solution of sodium selenate (Na ₂ SeO ₄ ), so that its final concentration in the nutrient medium is 1 · 10 ^-4 g / l. The culture medium is sterilized during normal operation. After autoclaving, swabs from test tubes with a strain of certain types of bacteria are placed in a liquid nutrient medium. Flasks are placed in a rocking chair for two days until they reach a certain titer of bacterial cells (the titer is individual for different strains). The temperature of the nutrient medium is 25-28 ° C. The culture fluid from the flasks is placed in working bottles with a capacity of 5-10 l, which are placed for two days on a rocking chair. Thus, a concentrated suspension of bacterial cells is obtained, which is then used to prepare finished products.

Example 2

Liquid nutrient medium of the following composition:

Glucose 20.0

Corn Extract 10.0

Yeast Extract 0.25

KN ₂ PO ₄ · N ₂ O 0.5

K ₂ HPO ₄ 0.5

CaCO ₃ 1.0

poured into bottles with a capacity of 5-10 l and intended for the cultivation of bacteria, sterilize, inoculate with uterine cultures of certain types of bacteria and add a sterile solution of sodium selenate, so that the final concentration is 1 · 10 ^-4 g / l of substance. Flasks are placed in a rocking chair for two days until a certain titer of bacterial cells accumulates in them. The temperature in the rocking chair is 25-28 ° C. This results in a concentrated suspension of bacterial cells, which is then used to prepare the finished preparations.

In all cases, the titer of bacteria in the drug significantly exceeds the control indicators. So when enriched with selenium in a liquid nutrient medium in flasks (Example 1), the final titer exceeded the control by 32% (rhizotorfin) and 33.5% (agrophile).

When sodium selenate was added to the bottles to create a working culture under rocking conditions (Example 2), the final bacterium titer exceeded the control values by 31.7% and 34% for rhizotorfin and agrophile, respectively.

The results of studies of the introduction of sodium selenate in various ways are shown in the table.

Claims (1)

A method of cultivating nitrogen-fixing bacteria, including growing a mother liquor culture of bacteria on a nutrient medium and manufacturing a working culture on an appropriate nutrient medium, characterized in that an aqueous solution of sodium selenate is added to the nutrient medium for growing the mother culture or to the culture medium for cultivating the working culture so that the final concentration of sodium selenate was 1 · 10 ^-4 g / l of liquid nutrient medium.