RU2795797C1 - Universal nutrient medium for cultivating microorganisms - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium for cultivation of Pseudomonas fluorescens AP-33, Bacillus subtilis B-12079, Bacillus subtilis B-2896 or Azotobacter chroococcum is proposed. The medium contains the following initial components, g/l: molasses 10, potassium phosphate 2.5, magnesium sulfate 1, iron (III) sulfate 1, ammonium nitrate 1, suspension obtained by mixing 80 g of polished peas with water, brought to a liter, by boiling mixture for 30 minutes and infusion for one hour, followed by cooling to room temperature and filtering twice through two layers of gauze, distilled water to a liter.

EFFECT: invention allows optimizing the technological process for the production of biotechnological preparations, which contain microorganisms of different taxonomic groups.

1 cl, 3 tbl, 3 ex

Description

Technical field

The invention relates to biotechnology, microbiology, and agriculture and can be used to obtain highly effective drugs - plant protection products.

Modern plant protection products are consortiums of microorganisms. Cultivation of each strain on a specific nutrient medium is a laborious and expensive process.

Known nutrient medium for the cultivation of Pseudomonas fluorescens AP-33, consisting of the following components, at their ratio, g/l:

Molasses 15, Potassium phosphate disubstituted trihydrate 0.5, Magnesium sulfate heptahydrate 0.2, Polished peas, pre-treated by autoclaving 250, Succinic acid 0.05, Laprol or agar, respectively 5 or 18, Distilled water up to 1 l [1]. This medium is not intended for the cultivation of Bacillus subtilis and Azotobacter chroococcum.

Known nutrient medium for the cultivation of Bacillus subtilis L-broth. It is prepared as follows: composition (g/l): yeast extract - 5, enzymatic peptone - 15, sodium chloride - 5, distilled water - up to 1 l (pH 6.8-7.0). Sterilization was carried out at a pressure of 1.1 atm. within 40 minutes [2].

The disadvantage of this medium is that when cultivating Bacillus subtilis VKPM B-12079, the maximum number of organisms is not higher than 5×10 ⁹ CFU/ml. Not intended for the cultivation of Pseudomonas fluorescens AP-33 and Azotobacter chroococcum.

Known nutrient medium for the cultivation of Azotobacter chroococcum: peas (decoction) - 100.0 ml; glucose - 10.0 g; (NH ₄ ) ₂ SO ₄ - 1.0 g; KH ₂ PO ₄ H ₂ O - 0.5 g; K ₂ HPO ₄ - 0.5 g; MgSO ₄ 7H ₂ O - 0.3 g; Ca CO ₃ - 1.0 g, then making a working culture on a medium containing (g/l): glucose - 20.0; corn extract - 10.0; yeast extract - 0.25; KH ₂ PO ₄ H ₂ O - 0.5; K ₂ HPO ₄ - 0.5; MgSO ₄ 7H ₂ O - 0.3; Ca CO ₃ - 1.0 [3].

The disadvantage of the environment is a multicomponent composition, not intended for cultivation of Bacillus subtilis, Pseudomonas fluorescens.

The aim of this study is to create an inexpensive universal nutrient medium for the cultivation of microorganisms of various taxonomic groups, using the example of Pseudomonas fluorescens AP-33 Bacillus subtilis 27 D, Bacillus subtilis B-12079, Bacillus subtilis B-2896, Azotobacter chroococcum. The proposed nutrient medium helps to optimize the technological process and reduce the cost of the finished product when a consortium of microorganisms is used in one preparation.

The essence of the technical solution is explained as follows.

Culture medium preparation technology

To use polished peas, its pre-treatment is required. Polished peas, in the amount of 80 g, rinse with tap water, mix in a container, and bring to a liter with water. Then cook on the stove for 30 minutes and infuse for 1 hour. After cooking, cool to room temperature and strain twice through two layers of gauze. Combine the resulting suspension with components (g/l): molasses 10, potassium phosphate 2.5, magnesium sulfate 1, iron(III) sulfate 1, ammonium nitrate 1, bring to 1 liter with distilled water. The pH value should correspond to 7.5-7.6 units. pH. Then sterilize in an autoclave for 30 minutes at 1.0 bar. The resulting medium has a light yellow color, a "clean" smell and a slight precipitate. Cultivation of microorganisms Pseudomonas fluorescens AP-33 Bacillus subtilis 27 D, Bacillus subtilis B-12079, Bacillus subtilis B-2896 is carried out for 20-24 hours, temperature - 30±2°C, forced aeration with a rotation speed of 125 rpm, cultivation of Azotobacter chroococcum after 72 hours without forced aeration.

Control over the number of microorganisms is carried out by the standard method of tenfold dilutions.

Example 1. Comparison of the results of cultivation on nutrient media Pseudomonas fluorescens AP-33

Table 1 shows that the number of Pseudomonas fluorescens AP-33 on the studied nutrient medium is higher.

Example 2. Comparison of the results of cultivation on nutrient media Bacillus subtilis 27 D, Bacillus subtilis B-12079, Bacillus subtilis B-2896

Table 2 shows that the number of microorganisms, Bacillus subtilis 27 D and Bacillus subtilis B-2896 is comparable to the growth on L-broth, and the number of Bacillus subtilis B-12079 is higher.

Example 3. Comparison of the results of cultivation on nutrient media Azotobacter chroococcum

Table 3 shows that the number of Azotobacter chroococcum on the studied nutrient medium is higher.

Thus, the proposed nutrient medium for the cultivation of various strains of the bacterium Pseudomonas fluorescens AP-33, Bacillus subtilis 27 D, Bacillus subtilis B-12079, Bacillus subtilis B-2896, Azotobacter chroococcum is universal, contains inexpensive and affordable raw materials, is incompetent, easy to prepare. , does not require additional equipment for cultivation (there is no need for temperature control of cultures), which allows you to optimize the technological process.

Bibliography

1. NUTRIENT MEDIUM FOR CULTIVATION OF PSEUDOMONAS FLUORESCENS AR-33 Yatsenko E.S., Shirmanov M.V., Evdokimov I.Yu., Boyko S.S., Durnikin D.A. Patent for invention RU 2678133 C1, 01/23/2019.

2. NUTRIENT MEDIUM FOR CULTIVATION OF BACILLUS SUBTILIS. Ryzhenkov N.S., Yatsenko E.S., Mikushina I.V., Shirmanov M.V., Evdokimov I.Yu. Patent for invention RU 2668178 C1, 09/26/2018.

3. Khotyanovich A.V. Methods for cultivating nitrogen-fixing bacteria, methods for obtaining and using preparations based on them (Methodological recommendations), Leningrad, 1991.

Claims (2)

Universal nutrient medium for cultivation of microorganisms of different taxonomic groups Pseudomonas fluorescens AP-33, Bacillus subtilis B-12079, Bacillus subtilis B-2896 or Azotobacter chroococcum containing molasses, potassium phosphate, magnesium sulfate, iron (III) sulfate, ammonium nitrate, suspension, obtained by mixing 80 g of polished peas with water brought to a liter, boiling the mixture for 30 minutes and infusing for one hour, followed by cooling to room temperature and filtering twice through two layers of gauze, and distilled water at the given ratios of the initial components, g /l: molasses 10, potassium phosphate 2.5, magnesium sulfate 1, iron (III) sulfate 1, ammonium nitrate 1, suspension obtained by mixing 80 g of polished peas with water brought to a liter, boiling the mixture for 30 minutes and infusing for one hour, followed by cooling to room temperature and filtering twice through two layers of gauze, distilled water to 1 liter.