Strain of bacteria bacillus megaterium, mobilising of phosphorus v and silicon from objects of lithosphere and resistant to poly (hexamethylene guanidine)

FIELD: agricultural microbiology; geochemistry.

SUBSTANCE: strain Bacillus megaterium var. phosphaticum "ВКМ В-2357 Д" is capable to leach phosphorus and silicon from objects of lithosphere and it is resistant to poly (hexamethylene guanidine).

EFFECT: strain is perspective for reception of the bacterial preparation raising a survival in soil and productivity of crops, for recultivation of technogenic polluted earths, and also in biotechnological works in the field of geochemistry.

15 dwg, 1 ex, 2 tbl

The invention relates to agricultural Microbiology, Geochemistry and the receipt of a new strain of microorganisms capable of leaching phosphorus and silicon of the objects of the lithosphere and resistant to polyhexamethyleneguanidine.

Currently, the main criteria of farming systems remain economic efficiency and environmental safety. The cultivation of high-yielding varieties require high doses of mineral fertilizers and pesticides, leading to depletion of soil fertility and decline in the quality of agricultural products. Among the modern technologies of agriculture biologization, along with the zoning of new varieties, crop rotation, cultivation techniques are different biologics, activating plant-microbe interactions. The use of living microorganisms reduces the geological cycle due to their ability to translate metabolically inactive, associated chemical elements available for plants.

It is proved that phosphorus is in the soil composition of chemical compounds, the absorption of which is unavailable to plants. Such a fixed, non-phosphorus in the soil contains a lot of up to 5-6 tons per each hectare. Bacteria B. megaterium Bacillus var. phosphaticum live in the soil, decompose organic matter and release the contained phosphorus, PureVideo in soluble salts of phosphoric acid. Formed in further compounds of phosphoric acid become available for uptake by plants. It is established that the use of the bacterial preparation is especially effective on fertile soils. Because these soils are on our planet has remained a bit, it is especially important to distinguish strains of microorganisms that are potentially active in the mobilization of nutrients not only organic, but also from non-organic component of soil, such as secondary minerals.

Analysis of mineral phosphate fertilizers has shown that a simple superphosphate contains only 18-21% R₂O₅80% balance - this impurity, the fifth part of which is aluminum, and related elements - pollutants: fluorine, iron and rare earth elements hafnium, zirconium, scandium. Double superphosphate contains about 45-46% R₂O₅on pollution accounts for about 50%. In Russia phosphate fertilizers are made from natural olivine bauxite at Angarsk aluminum plant, so after their application in the fields remains a large amount of aluminum. This item is dangerous not only for people but also for the development of plants increased number of undesirable because aluminum is harmful because it strongly affects the hydrolytic soil acidity, i.e. binds cations of potassium, sodium and other essential is the fatigue items. The introduction of live bacteria B. megaterium Bacillus var. phosphaticum in "sapotaceae" soil is 2-3 times to reduce the consumption of mineral phosphate fertilizers and to improve the quality of agricultural products.

There is a problem soils for agricultural purposes organo-mineral components to support sustainable agriculture. This task is solved in different ways, positively affecting the soil fertility, including amendments fertilizer mixtures. For several reasons, due to physico-chemical and microbial properties of soils mobilization plants useful components is ineffective. At the present time drastically reduced the use of phosphate fertilizers, is therefore a promising technology is the use of silicon-containing compounds for the mobilization of soil phosphates. Found that the change of phosphate regime of soils after making silicon fertilizers is one of the reasons for the increase crop yield. It is known that the mineral skeleton of the compounds of silicon destroyed by the action of silicate bacteria at the expense of broccoli and enzyme silicate [1].

Since the beginning of the last century developed and implemented for agrobiotechnology using bacterial preparations improving relations between soil and plants [2]. Set, h is about main moment in the mechanism of action of microbial agents is their Association to the root zones of plants, as well as the ability to mobilize useful components not only from fertilizer complexes deposited in the soil, but the soil [3]. In this regard, the actual issue is the identification and selection of microorganisms with high activity in the processes of mobilization of nutrients for enhanced plant growth.

It is known that the basis of any of the bacterial preparation is a classic scheme of aseptic biotechnological production. However, the cultivation of each individual strain implies the fulfilment of specific conditions necessary for maximum manifestation of the strain of his commercial properties. It is especially important to preserve the properties of the original matrix culture after the permissible period of retention of the drug. In modern industrial biotechnological production of many biological products are manufactured on the basis of different strains of bacteria of the genus Bacillus sp. One of the significant negative effects of such production is fagales bacterial culture, especially B. megaterium Bacillus. Fagales - effect destruction (lizirovania) bacterial cells under the action of bacterial viruses - bacteriophages that may get in the cage outside, or can be activated under certain conditions, in the form of provirus embedded in the bacterial GE is om. The main ways of dealing with lishennymi viruses bacteria are maintaining strict sterility of the original matrix culture for the production of the preparation and selection of resistant to bacteriophage strains.

Known strains of B. megaterium Bacillus var. phosphaticum de Crowbar (W-B208), having the property to mobilize phosphorus. Strains equivalents are deposited in PMBC (all-Russian collection of industrial microorganisms). Data on these strains obtained from the Catalog of strains of the all-Russian Collection of Industrial Microorganisms, 1997"

Closest to the obtained strain is Bacillus B. megaterium de Bary 1884 [4], has the property to mobilize phosphorus. The disadvantages of this strain is its limited functional ability - leaching phosphorus and greater susceptibility to lysis of colonies on the sloped agar medium.

While the invention has set itself the task of obtaining a culture of the strain, with phosphate and silicon mobilizing properties of the objects of the lithosphere and resistant to polyhexamethyleneguanidine.

This task is solved by a new strain of Bacillus B. megaterium var. phosphaticum WCMW-D, which is obtained from the laboratory strain B. megaterium Bacillus by its gradual adaptation to polyhexamethyleneguanidine.

The resulting strain B. megaterium Bacillus var. phosphaticum Pl-04 deposited in Russian national collection is promyshlennykh microorganisms (VKPM) Institute of biochemistry and physiology of microorganisms them. Gscreen (Moscow) 24.05.2005 no VKM B-D. The strain deposited with the scientific description and passport.

The resulting strain B. megaterium Bacillus var. phosphaticum P1-04 ACM-D selected from the laboratory strain B. megaterium Bacillus by the original method by gradual adaptation to polyhexamethyleneguanidine. The adaptation is made by multi-stage concentration increases with targeted incubation of cells.

Polyhexamethylene guanidine belongs to the class of biocidal cationic polyelectrolytes, which in addition to flocculation has a high and a variety of biocidal activity: bactericidal, fungicidal, virucidal, algicides. Speculating action of guanidine is ensured by the presence in each of its elementary link of the cationic guanidine grouping, the same factor and makes it a highly effective biocide. In industry it is used as a flocculant for simultaneous cleaning and disinfection of natural waters and wastewaters, as well as to prevent biobrane industrial heat-exchange equipment, conduits and other equipment in contact with water. Salt of guanidine hydrochloride and phosphate are flocculation disinfectants with integrated non-oxidizing action, not volatile, stable, low toxicity, stable and safe when it is wound, do not destroy or discolor materials, their aqueous solutions are colorless and odorless, explosion and fire, do not accumulate in the body.

The strain B. megaterium Bacillus var. phosphaticum P1-04 ACM-D characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural morphological traits

Pedigree strain: a group of gram-positive rods and cocci that form endospores; Genus Bacillus; B. megaterium Bacillus.

Cells of strain - straight bacilli with rounded ends. Are single or in pairs, sometimes form a chain. The size of cells: 0.5 to 1.5 μm. If the gram stain (crystal violet and then treated with a solution of Lugol's solution, alcohol and Magenta) received a positive response.

The sporulation: form spherical endospores. The type of division: simple.

Motility: motile cells. Pigmentation of colonies/ biomass.

On mesopatamia agar (MPA) cell strains form colonies off-white color. Do not release the pigments in a nutrient medium.

Mastopathy agar (31°C, 48 h) forms a sharply edged colonies are off-white. Old colony yellow.

Physiological and biochemical characteristics

Aerobe.

Optimum temperature for growth +31°

The optimum pH 7.0

Produces acid from glucose

Catalogoplantillas.

Well kept in glass tubes on the sloped agar medium (MPA with the addition of MnSO₄10 mg/l)at a temperature of +4°C.

The strain is not zoopathogenic or phytopathogenic not constitute a hazard for other reasons.

Additional properties: for strain P1-04 B. megaterium Bacillus var. phosphaticum has increased capacity for production indolylacetic acid and gibberellins.

Strain VKPM no VKM B-D identified in the "Determinant" of Berga (1974) as the type strain B. megaterium BACILLUS.

The strain B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D differs from the strain of the prototype Bacillus B. megaterium de Bary 1884 ability to leaching of phosphorus and silicon, an inorganic substrate and resistance to polyhexamethyleneguanidine.

The invention is illustrated in the following specific example of the use of the strain B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D.

Example

For growing seed culture of Bacillus B. megaterium var. phosphaticum Pl-04 No. WCMW-D in test tubes using a nutrient medium of the following composition:

Sodium chloride - 5 g;

Peptone - 5-10 g;

Meat extract 3 g;

Manganese sulphate 10 mg;

Agar - 15 g;

Polyhexamethylene guanidine - 1.5 g;

Tap water to 1000 ml.

For cultivation of strain in liquid culture using culture media the following composition:

-Nutrient broth for cultivation of microorganisms dry pH of 7.2±0,2 - 15.5,

Manganese sulfate is 10 mg.

-Water water - 1000 ml, the optimal range of pH: 7,0-7,5.

Optimum temperature for growth plus 31±1°C. it is Recommended to grow the strain by the method of deep cultivation in flasks on a shaker at 200 Rev/min

The strain should be stored in test tubes on the sloped agar medium at a temperature of plus 5°C. the Addition of guanidine in a nutrient agar medium reduces lithogenetic culture and increases the shelf life without reseeding for the new slanted agar medium for up to 6 months.

All salt is dissolved in a small amount of water on a magnetic stirrer, add peptone and meat extract. Then the solution is brought to a specified amount of tap water. Immediately bring the pH of the medium is 40% sodium hydroxide solution or a 10% solution of hydrochloric acid to a pH of 6.0 to 6.2, so that after sterilization the pH was 6.5 and 7.5. After adjusting the pH of the agar medium was added and melt it in a water bath. Agar medium is poured into test tubes and sterilized in an autoclave for 40 minutes at 1.0 at.

Because the strain B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D obtained for the first time and has not previously been used in industrial technology, it is possible to draw a conclusion on compliance with its criteria of "novelty" and "from breathalyse level.

Below the results of the research study microbiological characteristics and mobilization of silicon and phosphorus bacteria strain Bacillus B. megaterium var. phosphaticum Pl-04 No. WCMW-D

The purpose of the tests:

1. to determine the purity and abundance of bacteria in liquid culture of B. megaterium Bacillus var. phosphaticum strain P1-04 No. VKM B-D.

Abracelet the presence of pathogenic organisms.

3. To determine the potential geochemical activity of the bacteria Bacillus B. megaterium var. phosphaticum strain P1-04 No. VKM B-D in the mobilization of silicon and phosphorus from the objects of the lithosphere.

Materials and methods research

Bacterial culture of Bacillus B. megaterium var. phosphaticum strain P1-04 No. VKM B-D.

Identification of potential activity of bacteria in the mobilization of silicon and phosphorus were conducted by the method of EP 52.24.3.-82 (SiO₂and GOST 18309-72 (PO₄)²using the material-technical base of scientific-research laboratory of hydrogeochemistry of the Institute of Geology, oil and gas Tomsk Polytechnic University, registered in the System of analytical laboratories of Gosstandart of Russia, №ROSS RU. 0001.511901 from 12.08.2002, the concentration of phosphorus and silicon is performed using mass spectrometry method with inductively coupled plasma (ICP-MS) instruments company Perkin-Elmer ELAN-DRC-e by method No. 480-X MNR "Method of determining element is the composition of natural and drinking waters by ICP-MS" (Moscow, 2002), developed by the Institute of microelectronics technology and high purity materials RAS and certified in accordance with GOST 8.563-96 and EAST 41-08-205-99.

It was delivered three series of experiments as a test object leaching used phosphorite ore "djeroy" Sardara deposits in Uzbekistan established composition, %: P₂O₅- 13,6; Cao - 44,75; MgO - 1,05; CO₂- to 20.88; Fe₂About₃- 3,03;. SiO₂- 2,7; C_org- 0,11. The methodology of the research was to conduct a series of experimental and control options according to the following scheme.

Experienced options. Hitch sterile environment was filled in sterile nutrient medium containing 0.1 ml daily suspension obtained from the corresponding bacterial drug by aseptic cultivation 100 times [5]. The ratio of solid and liquid components was 1:3. Control options set in accordance with the prototype, but without the addition of bacteria.

The total period of experience was 30 days, which were divided into repetition in 5, 10 and 30 days. After each time step, the solution was poured into a clean sterile vessel to determine the number of bacteria to this date, and the amount of leached silicon and phosphorus.

Nutrient medium for cultivation and accounting bacteria had the following composition: NH₄Cl - 0,2 is; MgSO₄·7H₂O, 0.1 g; NaCl, 0.2 g; MnSO₄- traces; FeSO₄·7H₂O - traces; glucose 20 g; pH 7,0; distilled water - 1000 ml

Sterilization of phosphate rock, pulverized to a size of 0.2 mm was carried out in the experimental flasks in the oven at a temperature of 130°daily for 20 minutes for three days. Other required materials, and nutrient medium were sterilized in an autoclave at a temperature of 120°C for 30 minutes. Glucose was sterilized at 100°With 10 minutes daily for three days.

Counting of bacteria in the experimental solutions were implemented at the end of each time method 10-fold dilutions, followed by plating on agar medium corresponding composition but not containing phosphorus compounds in liquid and solid forms [6].

Results

Pathogenic microflora environment (Endo) in all the studied drugs were not found. Test drugs on the number of living cells and the purity of the culture was performed by counting cells in suspension by the method of dilution and seeding in Petri dishes in culture medium type mycopathologia agar.

All tested cultures were clean, the prison uniform. Visually looked like opaque suspension sand color. Bacteria B. megaterium Bacillus var. phosphaticum form the issue is small colonies: small, with smooth edges, round, matte, smooth, without mucus, opaque, yellowish-white. The total titer of 1.8×10⁹/ml. the Results of the study on the mobilization of phosphorus and silicon bacteria B. megaterium Bacillus var. phosphaticum strain P1-04 are presented in table 1. Leaching (mobilization) silica (SiO₂) and phosphorus (PO₄)²from phosphate ore with bacteria

In figures 1-12 shows electron micrograph of the surface of the crushed ore (rock) after 30 days of incubation with bacteria or without them at different magnifications. To compare experimental and control options selected similar large rounded particles.

Figure 1. Control option after incubation with culture medium without bacteria. Increase X300.

Figure 2. Control option after incubation with culture medium without bacteria. A fragment of the surface of the particle 1. Increase ×2000.

Figure 3. Large particle ore after a 30-day incubation with complex bacteria natural biocenosis, selected from the gray forest soil Tomsk region. Increase X300.

Figure 4. A fragment of the larger particles of ore after a 30-day incubation with complex bacteria natural biocenosis, selected from the gray forest soil Tomsk region. Increase ×5000.

Figure 5. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B. The smooth surface of the ore particles figure 6. Increase ×5000.

Figure 6. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B-2357. Smooth particle ore. Increase X300.

Figure 7. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B-2357. "Eaten" after leaching of elements in bacteria the surface of the ore particles. Increase ×2000.

Figure 8. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B-2357. "Eaten" after leaching of elements in bacteria the surface of the ore particles. Increase ×2000.

Figure 9. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B-2357. Loose particle of the ore after the destructive action of bacteria. Increase ×500.

Figure 10. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B-2357. The fragment surfaces of loose particles of ore Fig.9. Increase ×2000.

Figure 11. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B-2357. Loose particle of the ore after the destructive action of bacteria. Increase ×2000.

Figure 12. A prototype after a 30-day incubation with B. megaterium Bacillus P1-04 No. VKM B-2357. The fragment surfaces of loose particles of ore 11 Increase ×5000.

Discussion of results

It is now known that the translation of phosphorus and silicon in a soluble state from the soil once the ranks groups of microorganisms. This phenomenon has found practical application in the production of biological products, promoting mobilization of these elements from the solid state to the soluble form, which is available for cultivated plants. Thus, with the help of bacteria may increase soil fertility.

The data in table 1 show that bacteria obtained strain capable of mobilizing silicon and phosphorus from the objects of the lithosphere, in this case from phosphate rock. Analyzing quantitative characteristics of the experiment, draws attention to the existence of a large potential activity of the obtained strain of bacteria in the mobilization of silicon and phosphorus, which is clear from a large number of leached elements per bacterial cell.

It should be noted that the resulting strain active against recalcitrant object of the lithosphere - rock. Geochemical activity of bacteria obtained strain is more intense and significant in relation to the soil.

Here are the results of electron microscopic studies of the surface ore after 30 days of incubation with bacteria B. megaterium Bacillus var. Phosphaticum strain P1-04 No. VKM B-D.

In parallel with the determination of the rate of leaching of silicon and phosphorus carried out electron microscopic study of rocks of th the 30-day incubation with bacteria B. megaterium Bacillus var. phosphaticum strain P1-04 No. VKM B-D. For this object table was covered with a thin layer of glue, he poured the analyzed powder. After drying, the object table was placed in the vacuum post JEE-4B, where the surface was applied a thin (20-30 nm) layer of silver by the method of thermal evaporation in vacuum. The resulting preparation was investigated in a raster console ASID-4D ("JEOL", Japan) electron microscope "JEM-100 SHP" ("JEOL", Japan) at an accelerating voltage of 20 kV.

For a comparative study of the direct destructive action of bacteria on inorganic substrate ore "djeroy" were selected the same type of ore particles rounded. Control served as a nutrient medium without bacteria (Figure 1 -2), as a natural biocenosis used complex bacteria isolated from the gray forest soil Tomsk region (Fig.3-4), experienced option served bacteria strain Bacillus megateriun var. phosphaticum P1-04 No. VKM B-D.

(Figure 5-12). Here at high magnification, it is evident that it was the version with a pure culture of bacteria (experience) the surface of the particles is irregular, "eaten" and more loose, which is a consequence of the leaching of nutrients bacteria. The images indicate a direct nutrient effect of the strain B. megaterium Bacillus var. phosphaticum on mineral substrate.

It was a study of the effect of pH of the medium n is the phosphorus content in the culture medium of the strain B. megaterium Bacillus var. phosphaticum Pl-04 No. BKMB-D.

During the experience was measured concentration of the biomass of the studied microorganisms, the phosphate content and the pH of the culture media. Each variant was performed in three analytical replicates. Control was liquid medium, cultivated microorganisms.

Was analyzed the relationship between pH and concentration of PO₄ ^3-. It is shown that with increasing acidity increases the phosphorus content in the culture medium, which corresponds to literature data (Fig).

On Fig shows the dependence between pH environment of the strain B. megaterium Bacillus var. phosphaticum P1-04 No. BKM B-D and the concentration of phosphorus (PO₄ ^3-joining solution. The graph shows a negative linear relationship specified parameters.

Control experiments served environment where grown studied microorganisms. When measuring the amount of phosphorus in the control detected that its content increases over time.

On Fig presents the results of the measurement of acidity in the advanced options.

Where

You pH change in the acidity of the culture fluid of strain

B. megaterium Bacillus var. Phosphaticum P1-04 No. VKM B-D

Nb pH change in the acidity of the culture fluid of strain

Pseudomonas sp. In - 6798

Is the pH change in the acidity of the culture fluid

fo formability isolate VK D

For pH - control

To pH change in the acidity of the control

Based on the research we can conclude that in all variants is offset from the pH in the acidic side.

The decrease in pH was observed in the control. Along with the change of acidity in the control there was a shift in the ion solution RHO₄ ^3-(Fig). Analysis of the composition of the medium showed that the environment is the natural acidification, as the source of nitrogen is ammonium chloride (NH₄Cl). Are the salt of the weak base and a strong acid, so the solution hydrolysis occurs:

NH₄Cl→NH₄ ⁺+Cl^-

The acidification of the environment in the control, in turn, affects the dissolution of CA₃PO₄. Apparently, a similar effect is possible in soils with the application of fertilizers containing ammonium.

In the literature there are data suggesting that the bacteria B. megaterium Bacillus are K-strategists with inert, gradual growth and "smoothed" dynamics [6]. This property of the strain B. megaterium Bacillus var. Phosphaticum BKM-2357 E confirmed by the data of experiments.

In order to avoid the effect of mobilization of phosphate by hydrolysis was replaced with a nitrogen source with ammonium chloride to urea (0,43 g/l). In the second series of experiments as a control strain of bacteria was taken Escherichia coli Eschericia coli In-899, which is not a soil microorganism.

After replacement of the medium composition, it was found that during the experiment in the variants with all studied strains pH environment, as well as in the first case, acidified (Fig). The accumulation of phosphate in the medium with urea under the influence of the strain B. megaterium Bacillus P1-04 No. BKM B-D and control, where:

You pH change in the acidity of the culture fluid of strain B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D.

Nb pH change in the acidity of the culture fluid of strain

Pseudomonas sp. In-6798

Is the pH change in the acidity of the culture fluid

phosphate-mobilizing isolate VK D

Ec pH change in the acidity of the culture fluid strain

Escherichia coli In-889

To pH change in the acidity of the control

The analysis of the content of soluble phosphate in the course of experience has shown the following: the content of phosphate in the control does not increase, as it does not change the pH of the acidic side, i.e. there is no natural acidification of the environment. The first 12 hours the content of soluble phosphate in the control variant has decreased sharply, then within the next 12 hours, the concentration of phosphate increased and amounted to 0.12 mg RHO₄ ^3-/L. figure 3 shows that the concentration of soluble phosphate in the control on the second day began to decline, this process continued for the next 24 hours. The concentration of the phosphate in the control amounted to 0.02 mg RHO ₄ ^3-/HP

In the variant with the strain B. megaterium Bacillus P1-04 No. VKM B-D accumulation of phosphate in the first 12 hours was increased, the concentration of RHO₄ ^3-was 0,42 mg P/l, compared with an initial value of 0.32 mg P/L. the Next 10 hours, the concentration of phosphate was slightly reduced to 0.38 mg P/L. the Reduction of soluble phosphate is associated with its consumption by the bacteria Bacillus B. megaterium, as at this time there is bacteria growth. On the second day of the experiment the concentration of phosphate increased and amounted 0,42 mg/l, this process continued for the rest of the time.

It should be emphasized that in the second series of experiments was to exclude the possibility of influence of low pH on the dissolution of calcium phosphate. Based on the obtained data, it was concluded that the accumulation of phosphate in the culture fluid of Bacillus B. megaterium var. phosphaticum P1-04 No. VKM B-D fully connected with the life processes of bacteria of the studied strain.

Selection of microorganisms Bacillus B. megaterium var. phosphaticum resistant polyhexamethyleneguanidine

Resistance to polyhexamethyleneguanidine determined during seeding bacteria strain Bacillus B. megaterium var. phosphaticum P1-04 No. VKM B-D on beveled mastopathy agar. To do this in the unconsolidated mastopathy agar was added polyhexamethylene guanidine following Konzentrat the nd: 50 µl to 10 ml of medium; 100 µl to 10 ml of medium and 150 ál per 10 ml of medium. As initial solution used industrial 20% aqueous solution of guanidine hydrochloride, which represents a transparent liquid colorless to yellow (pH 7-7-9,5). Then gave the agar to harden and seeded beveled mastopathy agar bacteria using microbial loop. Table 2 presents the results of five independent experiments, each conducted in seven analytical replicates.

The tubes were placed in a thermostat at a temperature of +31°C. two days have pointed to the growth of colonies of Bacillus B. megaterium var. phosphaticum on the jambs respectively added to the nutrient medium, the amount of guanidine. Grown colonies visually looked like a folded, rounded, large, 6-7 mm in diameter, whitish-creamy education. When you re-seeding shoals containing 100 μl of guanidine in 10 ml of medium, observed the pigmentation of the colonies in the form of staining to pinkish-cream color.

It was noted that during storage slanted agar medium with a culture of B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D in the refrigerator at a temperature of +5°C is lizirovania bacterial colonies after 4-8 weeks. However, in those tubes where to mesopotania the agar was added polyhexamethylene the one, colonies of B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D remained characteristic of this strain signs - the original shape, color and texture. Viability grown with the addition of guanidine bacteria remained for six months, which is three times the period, which is usually necessary for the preservation of colonies of B. megaterium Bacillus to maintain morphological and physiological properties of the specific source of pure culture of bacteria.

Thus, the conclusion was made about the viability of using guanidine as a stabilizer, allowing you to save and keep for a long time functionally active viability and the declared properties of the obtained strain B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D.

Addition of guanidine in agar nutrient medium used for culturing the strain increases at three times the lifespan of uterine cultures, contributes to the creation of conditions for long-term and stable maintenance of commercial properties, reduce the complexity of the process and to increase its economic efficiency.

Also the resulting strain B. megaterium Bacillus var. phosphaticum P1-04 No. VKM B-D is promising for obtaining bacterial drug that increases survival in soil and agricultural productivity is ultor, for remediation of anthropogenic contaminated land, as well as in biotechnological activities in the field of Geochemistry.

Sources of information

1. Samsonov N.E. // Agrochemistry. 2005. No. 8.

2. Degens B.P. et al. // Soil Biol. Biochem. 2000. V.32. No. 2.

3. Hristovska T.V. microorganisms as a component of the ecosystem in question. M.: Nauka, 1984.

4. In-847: INMI, VKM B-847; VNIISHM, 49. Received as: "B. megaterium Bacillus subsp. phosphaticum" Menkina 1950 (prototype).

5. Bellanova N.P., Karavaiko G.N., Avakian Z.A. // Microbiology. 1985. No. 1.

6. Kalinin C.V., Kozhevin P.A., D. G. Zvyagintsev // Microbiology. 1999, t, No. 2.

Bacterial strain B. megaterium Bacillus var. phosphaticum BKM-D mobilizing phosphorus and silicon of the objects of the lithosphere and resistant to polyhexamethyleneguanidine.