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The Soil Thiobacterii Isolation and Determination of Their Bio-Oxidation Activity

21 May 2015
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Authors: Aida Kistaubaeva

I.                 INTRODUCTION


THE  fact that saline alkaline-soils cover wide area of Kazakhstan results in reduction  of crops from 15 to 45 %. This necessitates the constant search for the most promising methods for their melioration and chemical reclamation by adding plaster, phosphogypsum, pyrite, sulfuric acid, sulfur and industrial waste. Traditional soda-saline soils chemical melioration technologies (adding plaster, phosphogypsum, pyrite, sulfuric acid) are characterized by low efficiency and high costs [1], [2]. The elemental sulfur is one of the most promising methods of melioration of saline alkaline-soils [3]. However, sulfur due to the inertia as meliorant is practically not used and the problem of increasing its melioration efficiency has almost not been recognized [4]. Therefore, there are demands in the development of additional techniques which can increase sulfur oxidation rates in the soil [5], [6].

The product of oxidation of the soil by widely distributed in it Thiobacteria is predominately elemental sulfur.  These bacteria form sulfuric acid which acidifies the soil, helping to transfer some important for plants elements into appropriate forms. In this regard, a new direction in the field of land amelioration of solonetz and alkaline soils- a joint introduction

of elemental sulfur and Thiobacteria oxidizing sulfur during their life appeared [7].

High efficiency of oxidizing (sulfur oxidation to 84% for 12 weeks) culture of Thiobacillus thioparus [8].

To identify the possibilities of using sulfur-oxidizing bacteria to improve soil fertilizer, because sulfur serves not only as meliorant but also as an element of nutrition for plants.

The study found that the sulfur in the soil sulfur-oxidizing microorganisms with the scheme

S → SO2 → SO3 slowly enters its sulfur di- and trioxside forms, where the latter combines with water to form sulfuric acid as a perfect agent for saline alkaline-soils. Thus, melioration process unlike the soil drench solution of sulfuric acid takes place in the "mild" conditions [9].

The purpose of this research is to obtain pure cultures of sulfur-oxidizing bacteria, to determine their sulfur-oxidizing activity and the level of biomass accumulation on selective medium.

Thus, oxidation rates of elemental sulfur can be introduced into the soil by biological products with soil sulfur-oxidizing microorganisms. Moreover, the use of microorganisms isolated from soda-saline soils of the area to endogenous microflora provides their existence and functioning in the ecological niche.

Soil association of sulfur-oxidizing bacteria was obtained by selection from soda-saline soils. To isolate the individual strains and to study  physiological and biochemical culture properties  was used Armbruster nutrient of the following composition: g/l: Na2S2O3 – 10; KH2PO4– 4;  MgSO4 – 0, 8; NH4Cl– 0,4; CaCO3– 5;  solution of microelements – 10 мл/л. Solution of microelements (300 g/ml distillated water): EDТА-15; ZnSO4 - 0, 66; CaCl2  - 1, 66; MnCl2 -0, 75; CoCl2 - 0, 15; (NH4)6 Mo7O24- 0, 15; FeSO4  - 1,5; CuSO4 - 0, 06; NaOH - 3, 3. Nutrients рН 6, 8 – 7, 0 were obtained with the help of 10% solution of NaHCO3.

Cultivation was performed on the isolates Baalsruda medium of the following composition (g / l):Na2S2O2– 5, 0; KNO3 - 2, 0; NH4Cl – 0, 5; NaHCO3 – 1, 0; MgCl2 – 0, 5; K2PO4 – 2, 0; FeSO4 – 0, 01; H2O – 1l; pH 7, 0. Growth of sulfur-oxidizing microorganism’s biomass was evaluated by the change of protein content in the culture broth by the Lowry method and by counting the number of cells.

Biooxidation process of the model substrate (thiosulphate) while the development of sulfur-oxidizing microorganisms was carried out under batch culture flasks in a thermostatic shaker. Changes in the concentration of substrate - thiosulfate and bio-oxidation products – sulfates were under control. Thiosulfate was determined by cyanolized. Sulphate was determined by liquid chromatography Biotronik LC 500. To control the chemical oxidation of reduced sulfur concentration of the main substrate was simultaneously analyzed without introducing microbial environment.

To determine the activity of enzymes sulfur metabolism there were used bacteria of an exponential growth end. Cells were separated from the medium by centrifugation for 20 min at 10,000 g washed twice with fresh medium containing no source of energy then they were washed with 0, 05 M Tris- HCl buffer, pH 7, 4. Cells were disrupted by sonication at 22 kHz in a buffer for 3 min at 1 min intervals for cooling. The homogenate was centrifuged for 25 min at 40,000 g and the supernatant was used to determine enzymes by spectrophotometric methods or colorimetric spectrophotometer «Hitachi 200-20» (Japan).

Tiosulfatdehydrogene was measured according to the rate of ferricyanide recovery at 420 nm in the presence of sulphite or thiosulphate. Tiosulfatreductase activity was measured by the rate of hydrogen sulfide formation from thiosulfate at the presence of recovered glutathione and ditiotrietola.

II.                Results and Discussion

Isolation chemolithoautotrophic, sulfur-oxidizing bacteria associations of soil (genus Thiobacillus) of soda- saline soils.

Referred to as "sulfur-oxidizing" microorganisms if they are capable of model substrate thiosulfate oxidation a nutrient medium comprising to sulfate (S6 +).

Elective conditions for the development of sulfur-oxidizing microorganisms were created by incorporation into the culture medium reduced sulfur source, thus the pH conditions in the range of neutral and slightly alkaline values. Microorganisms have been cultivation out in flasks on a mixing device at a temperature of 28 °C.

 The sterile nutrient media formed a stable association representing after several passages collecting a culture of bacteria capable of oxidation of reduced sulfur compounds.

Sulfur-oxidizing microorganisms obtained Association HC1 – NC-23 is representatives of biocenosis soda- saline soils.

It is known that the development of sulfur-oxidizing microorganisms related to oxidation of reduced sulfur compounds to the final product - sulfates or partially oxidized to form , for example polythionates, accompanied by a change in pH environment [7]. In order to determine the bio-oxidation activity of the microorganisms were investigated associations biooxidation processes model substrate (thiosulfate).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE I

Change in Ph of the Culture Medium during

Cultivation Associations Sulfur-Oxidizing Microorganisms

Cumulative culture

рН

Cultivation’s time, day

 

0

5

7

10

18

NK-1

7,1

7,4

7,6

7,3

7,0

NK-2

6,4

6,7

6,4

6,2

6,1

NK-3

6,4

5,8

5,1

4,8

4,3

NK-4

7,0

7,9

8,2

8,2

8,1

NK-5

6,4

6,7

6,3

6,3

5,6

NK-6

7,1

7,5

7,5

7,4

7,1

NK-7

7,0

7,8

8,1

8,2

8,1

NK-8

6,4

6,7

6,2

6,1

6,0

NK-9

6,4

5,8

5,1

4,7

4,5

NK-10

6,4

6,7

6,3

6,3

5,7

NK-11

6,4

5,8

5,1

4,9

4,3

NK-12

7,1

7,4

7,5

7,2

7,2

NK-13

6,4

6,7

6,4

6,1

5,8

NK-14

6,4

6,7

6,3

6,2

6,2

NK-15

6,4

5,8

5,1

4,7

4,4

NK-16

6,4

5,7

5,1

4,8

4,3

NK-17

6,4

6,7

6,4

6,2

6,1

NK-18

7,1

8,0

7,5

7,1

7,3

NK-19

6,4

6,7

6,4

6,1

6,0

NK-20

6,4

5,8

5,1

4,7

4,1

NK-21

7,0

7,8

8,3

8,1

7,9

NK-22

6,4

5,8

5,1

4,6

4,3

NK-23

6,5

6,8

6,4

6,3

5,8

 

 

Based on data obtained, the culture of the association of  NK -1 , NK- 4, NK- 6, NK - 7 , NK - 12, NK - 18, NK - 21, are able to oxidize thiosulfate only to polythionates that accompanied alkalinity of the nutrient medium.

Development of microorganisms includes to the cumulative culture of NK -2, NK - 5, NK - 8, NK - 10, NK - 13, NK - 14, NK - 17, NK- 19, NK - 23 is associated with an initial increase of the pH to 7, 0-8, 0 units and its further reduction to 6,2-5,8, which is a consequence of the accumulation of sulfate as the final oxidation product [6].

In the process of bio-oxidation was observed accumulation of intermediates products- polythionates that formed in the oxidation of thiosulfate and molecular sulfur, in that case, evidenced by the increasing pH of the culture medium from 6, 6 to 8, 1 units on the 5th day of cultivation.

Polythionates accumulation in the medium, firstly is associated with the microbiological oxidation of thiosulfate to tetrathionate, which is accompanied by alkalinization of the medium by the reaction:

 

2S2O32- +  ½ O2 + H2O → 2S4O32- + 2OH-

 

However, it must be considered and ways of chemical oxidation of thiosulfate ions, and in chemical polythionates disproportionation of sulphite and thiosulphate reciprocal action [5]. First accumulation polythionates replaced by the reaction of decreasing concentration in the medium that may be associated with the partial reduction of microbial sulfur to form thiosulfate, and the oxidation of sulphite to sulphate. Alternately increasing and decreasing concentrations of the intermediates in the process of bio-oxidation characterizes the equilibrium nature of the transitions and polythionates thiosulfate.

Sulfur-oxidizing bacteria for associations that make up the savings culture NK- 3, NK- 9 , NK- 11 , NK- 15 , NK- 16 , NK-20, NK-22, isolated from soda-saline soils in the metabolism of thiosulfate is a direct education sulfate without accumulation polythionates, as evidenced by a decrease of pH from 6,4 to 4,3 without alkalizing.

It should be noted that the association of NK- 2, NK- 5, NK- 8, NK- 10, NK- 13, NK- 14, NK- 17, NK- 19, NK- 23, the importance of economic education sulfate ratio YR p/ s power 0, 86, which is the result of accumulation in the environment of products of incomplete oxidation of reduced sulfur compounds ( polythionates, molecular sulfur, sulfites ).

Association of NK- 3, NK- 9, NK -11, NK- 15, NK- 16, NK-20, NK-22 is a complex of sulfur-oxidizing microorganisms involved in the complete oxidation of thiosulphate to sulphate, as evidenced by high value:

YR p/ s = 1, 47.

Importance of the economic factor Yx p/ s for these associations was more typical for the bacteria Thiobacillus ferooxidans, which vary in the range of 2, 2-6, 6 x 10- 3 g or 3, 5-5, 3 × 10- 3 ASB/Fe2 +. Odds Comparison of microbial growth and development in this case is very conditional, since the values ​​for pure cultures cannot be compared with the values ​​for the association of microorganisms.

Thus, the final product of the bio-oxidation of reduced sulfur compounds for associations NK- 2, NK- 5, NK- 8, NK- 10, NK- 13, NK- 14, NK- 17, NK- 19, NK- 23, and NK -3, NK-9, NK- 11, NK- 15, NK- 16, NK- 20, NK- 22 are sulfates, as evidenced by their accumulation in the culture medium, leading to a decrease in pH to 5, 6 at day 18 units culturing. However, it should be noted that increasing the concentration started on day 10 and cultivation was then continued throughout the experiment. This may mean simply that the data consist of the association of microorganisms on their various physiological characteristics and bio-oxidation. Isolation of pure cultures and their development processes Trials will confirm this assumption.

Microscopic studies have yielded their association microbiological characteristics was performed using preparations of living cells on a microscope equipped with phase-contrast device. Type flagellation was determined by the nature of the motion of bacterial cells in the microscope.

Below are presented the morphological properties investigated associations:

  • The community of bacterial cells, which is dominated by small straight rod-shaped bacteria (1, 2 x0, 4 mic), moreover found curved rod-shaped bacteria (1, 6 x0, 8 mic), round (cocci) sticks 0, 4 mic;
  • Dominated by the bacterial cells in the form of rod (2, 0 x 0, 8 mic) with and fixed polar flagellation. In association with them rod detected size of 0, 4 mic;
  • The association rod-shaped bacteria. Dominate the cells sizes: 5, 0 x1, 2 mic and 1, 2 x0, 8 mic; noted the presence of cocci rod 0, 4 mic;
  • Rod-shaped bacteria, which dominated of thin elongated bacilli 2, 2 x 0, 8 mic). Rod-shaped bacterium found 0, 1-2, 4 and 3, 0 mic x 0, 4 mic, and curved rod-shaped cells;
  • The bacterial cells predominate in the form coccoid rods size of 0, 4 mic with a polar flagellation. In association with them, rod-shaped bacterium found two types: fixed (1, 6 x0, 4 mic), and cells capable of movement, the dimensions of 2, 0 x0, 8 mic;
  • The association represented rod-shaped bacteria. It is noted the presence of cell size: 1, 0 x0, 4 mic; 2, 5 x0, 6 mic, 1, 2 x0, 8 mic and 0, 4 microns coccoid rods. Are the dominant bacteria, which have the form of small rods (1, 2 x0, 4 mic);
  • Rod-shaped bacteria: small (1, 2 x0, 4 mic) and cocci (0, 4 mic) with polar flagellation, still elongated sticks. When the microscope fixed cells stained preparations were found Gram-positive filamentous forms.

Sulfur-oxidizing bacteria studied associations are Gram-negative, represented by single cells or form clusters of two or more units.

 

TABLE II

CULTURAL ASSOCIATIONS A PROPERTY

OF SULFUR-OXIDIZING MICROORGANISMS

Associated

Type

of

colony

Description colonies

Forms

size

optical

properties

color

Type 1

1

Round

d=2 mm

translucent

White

2

Round

point

translucent

without

color

3

Round

point

matt

white

4

Round

d=2 mm

opaque

grey

Type 2

1

Round

point

translucent

without

 color

2

round with

wavy edge

d=2 mm

matt

white

3

Irregularly

shaped inner

part thickened, surroundedby translucentfringe

d=3-4 mm

opaque

white

and grey

Type 3

1

Round

d=1,5 mm

matt

white

2

Round

point

opaque

white

Type 4

1

Round

d=2 mm

opaque

white

and

yellow

Type 7

1

Round

point

opaque

white

 

 

Thus, the examined associations are represented by rod-shaped bacteria mainly differing in the size of cells and the presence of flagella.

Pure cultures of sulfur-oxidizing bacteria were obtained from collecting soil microbial associations at their growth on medium with thiosulfate (sulfur source) and NH4CI (nitrogen source). In the result, there were isolated 36 strains of sulfur-oxidizing bacteria. According to the results of the phenotypic identification  bacteria were assigned to 6 species: Thiobacillus thioparus; Thiobacillus thiooxidans; Thiobacillus denitrificans; Thiobacillus thiocyanoxidans; Thiobacillus novellus; Thiobacillus ferrooxidanas.

The development of all the strains on  nutrient medium was characterized by its full thiosulfate to form disposing sulfate on the 8 day. 10 strains  were accumulated up to 3900-4300 mg / dm 3900-4300 mg/ CFU/sulfata in liquid culture. In this case the substrate is not detected on the third day of cultivation.

To quantify bio-oxidation activity of  Thiobacillii strains there was performed  a special study of their enzymatic activity  (Table III) .

Enzymes of dehydrogenase which belong to  class of oxidoreductases participate in the process of thiosulfate biooxidation. Tiosulfatdehydrogenase  and tiosulfatreductase are two of the key enzymes of energy metabolism sulfur-oxidizing microorganisms [9].

The highest value of dehydrogenase activity in the disposal of sulfate was found in 7 strains. (1, 5 , 10, 11, 16 , 24, 31 - for them to do shading in the table). High values ​​of enzyme activity were achieved on the third day of cultivation, when the bacteria were in the logarithmic growth phase , which is characterized by intensive consumption of the substrate, and hence a superior enzymatic activity.

Cells harvest in those same seven strains was twice higher than that of the middle-strains dehydrogenase activity. It is also found that the yield of cells depends on the oxygen regime of cultivation. The oxidation of sulfur compounds in increasing the oxygen content to 20 % is accompanied by two-fold increase in cell concentration.

 

TABLE III

Sulfur Metabolism Enzyme Activity of Thiobacterii After

72 Hour Batch Cultivation

Strain

Protein

concentration

mg/ml

Enzyme activity ,

nmol/(min * mg protein)

Thiosulfate

dehydrogenase

Thiosulfate

reductase

1

130

1532

1834

5

100

1547

1509

10

150

1611

2762

11

105

1509

1956

13

60

929

1150

15

90

1207

1497

16

110

1394

2003

24

120

1592

2398

25

70

1023

1248

31

145

1603

2679

 

 

The presence of reduced compounds of sulfur in the culture medium also contributes significantly stability and increase in the harvest of culture cells in 1, 2 times.

Thus, the general result of this study is to select the most active strains of thiobacteria. These are: Thiobacillus ferroxidans 10, 16, 24; Thiobacillus thiooxidans 5, 11, 31; Thiobacillus denitrificans 1.

 

 

 

 

 

TABLE IV

Cells Harvest (Lg Cfu / Ml) of Bacteria of the Genus Thiobacillus on Conditions OF BATCH Cultivation in SELECTIVE Culture Medium

Strain

Cultivation time, hours

24

48

72

96

1

1,9+0,1

4,7+0,1

8+0,1

7,9+0,2

5

2+0,2

5+0,2

7,9+0,1

7,9+0,3

10

2+0,3

5+0,1

8+0,2

8,1+0,1

11

1,8+0,1

4,3+0,2

7,7+0,2

7,8+0,3

13

1,5+0,3

3,5+0,2

6+0,1

6,1+0,1

15

1,6+0,1

3+0,1

5,5+0,1

5,6+0,1

16

1,8+0,1

4,6+0,2

7,5+0,1

7,4+0,2

24

1,9+0,1

4,1+0,2

7,6+0,2

7,6+0,1

25

1,5+0,1

3,2+0,1

6,1+0,2

6,2+0,3

31

2+0,2

4,9+0,2

8+0,2

8+0,3

 

 

Cells harvest in those same seven strains was twice higher than that of the middle-strains dehydrogenase activity. It is also found that the yield of cells depends on the oxygen regime of cultivation. The oxidation of sulfur compounds in increasing the oxygen content to 20 %  is accompanied by two-fold increase in cell concentration. The presence of reduced compounds of sulfur in the culture medium also contributes significantly stability and increase in the harvest of culture cells in 1, 2 times.

Thus, the general result of this study is to select the most active strains of thiobacteria. These are: Thiobacillus ferroxidans 10, 16, 24; Thiobacillus thiooxidans 5, 11, 31; Thiobacillus denitrificans 1.

These strains are expected be used for introduction into soil to raise the level of sulfur biooxidation. To do this it is necessary to obtain cells biomass. In this regard, optimal spacing of pH and temperature for cultivation were adjusted. Two of them were chosen as the most active strains: Thiobacillus thiooxidans 10 and Thiobacillus ferroxidans 31 (Table V).

It has been established that the optimum pH range is from 6, 6 to 9 units. The greatest increase in biomass in the stationary phase of growth was observed at pH 7. Maintaining neutral pH is requirement for effective biomass accumulation of sulfur-oxidizing bacteria.

Decrease of pH of the culture medium to 3 units is characterized by minimal microbial growth (25-30 % for 5 -day culture).  It is noted that Thiobacteria development at pH 9 was also accompanied by increase of biomass.

 

TABLE V

Change in the Number of CFU in the Growth of Sulfur-Oxidizing Microorganisms in the Different PH Conditions of Culture Medium

Time

Number of CFU х105/sm3

Strain 10

Number of CFU х105/sm3

Strain 31

рН 3

рН 7

рН 9

рН 3

рН 7

0

5,17±0,72

6,72±1,01

4,48±0,58

5,60±0,78

7,36±0,72

1

5,90±0,77

7,36±0,88

5,92±0,65

6,18±0,87

7,87±0,94

5

6,48±0,71

12,48±0,72

9,78±0,67

7,28±0,82

13,06±0,51

 

 

This allows us to recommend these strains or their associations be implemented as biological products for soda- saline soils.

Seasonal temperature decline down to 7 °C in winter can lead to significant limitation for using the strains in systems of soda-saline soils melioration. Most bacteria capable of oxidation of sulfur compounds are mesophylls. Range of optimal temperatures for sulfur-oxidizing bacteria of genus Thiobacillus is from 15 to 42 °C.

Due to the seasonal fluctuations in soil temperature, growth of strains  Thiobacillus thiooxidans 31 and Thiobacillus ferooxidans 10 was analyzed in the following temperature ranges: 4 - 5 ° C, 23 °C (average soil temperature in summer) and 28 °C (optimal range for  development of sulfur-oxidizing bacteria of  genus Thiobacillus).

Development of sulfur-oxidizing microorganisms at temperatures of 23-28 ° C was characterized by maximum biomass growth on the number of CFU (88-122 % on the 5th day of experiment), which suggests the feasibility of cultivation and use of strains and their associations in the specified temperature range.

 

TABLE VI

Change in Number of CFU in the Growth of Studied Sulfur-Oxidizing Microorganisms Associations in Different Temperature Conditions

Time of

cultivation, days

 

Number of CFU х105/sm3

Strain 10

Number of CFUх105/sm3

Strain 31

4°С

23°С

28°С

4°С

23°С

0

5,28±0,74

5,92±0,53

4,67±0,51

4,18±0,45

5,67±0,39

1

5,99±0,72

9,01±0,42

8,21±0,37

4,64±0,33

8,23±0,74

5

6,30±0,71

11,14±0,49

10,37±0,43

5,06±0,35

10,24±0,81

 

Thus, the temperature factor has a significant influence on the development of these organisms and is one of the limiting their effective work in the soil (biotransformation sulfur). In this regard, while using methods of reclamation of soda- saline soils by using sulfur-oxidizing bacteria, the necessary condition to keep the bacteria activity is to maintain the temperature at 23-28 °C.

III.               Conclusion

36 strains of sulfur-oxidizing bacteria were identified in soda-saline soils. According to the results of phenotypic identification they are assigned to the following taxa: Thiobacillus thioparus; Thiobacillus thiooxidans; Thiobacillus denitrificans; Thiobacillus thiocyanoxidans; Thiobacillus novellus; Thiobacillus ferrooxidanas.

 Activity of enzymes of sulfur metabolism of  thiobacteria after 72 hour batch cultivation was determined. Activity Level of tiosulfatdehydrogenese is 929-1611 nmol / (min * mg protein). Tiosulfatdehydrogenese activity ranges from 1150 to 2762 nmol / (min * mg protein). Selected are 7 active strains.

It was found that the greatest increase in biomass strains was observed on the 5th day batch culture in medium Baalsruda at pH 7, 0. The temperature optimum was from 23 to 28°C.

Bibliography and references
[1] Fоllet R.H., Murphу L.S. Fertilizers and soil amendments. Prentice-Hall, Inc., Englewood cliffs –USA, New Jersey, 2001: p. 557. [2] Prather R.J., Goertzen J.O., Rhoades J.D., Frenkel H. Efficient Amendment Use in Sodic Soil Reclamation. Soil Sci. Soc. Am. Journal, 2008, Vol. 42: p. 782-786. [3] Bole J.B. Amelioration of a calcareous solonetzic soil by irrigation, deep ripping, and acidification with elemental sulfur. Canadian Journal of Soil Science, 2006, Vol.66: p. 347-356. [4] Germida J. J., Janzen. H. H. Factors affecting the oxidation of elemental sulfur in soils. Nutrient Cycling in Agroecosystems. Fertilizer research, 2003, Numbers 1-2, Vol. 35: p. 101-114. [5] Nor Y.M., Tabatabai M.A. Oxidation of Elemental Sulfur in Soils. Soil Sci. Soc. Am. Journal, 2007, Vol.41: p. 736-741. [6] Armando G., Sergio R. The effect of chemical oxidation on the biological sulfide oxidation by an alkaliphilic sulfoxidizing bacterial consortium. Elsevier, 2007, № 40: p. 292-298. [7] MсСready R.G.L. Bacterial oxidation of sulfur as a means of reclaiming solonetzic soil. Solonetzic Soils in Alberta (A Progress Report Alberta solonetzic soils working group). Edmonton, Alberta, 2002: p. 13-31. [8] McCready R.G.L., Krouse H.R.. Sulfur isotope fractionation during the oxidation of elemental sulfur by thiobacilli in a solonetzic soil. Canadian Journal of Soil Science, 2002, Vol. 62: p.105-110. [9] Vidyalakshmi R, Paranthaman and Bhakyari R. Sulfur Oxidizing Bacteria and Pulse Nutrition. World Journal of Agricultural Sciences, 2009, Vol.5, № 3: p. 270-278.
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Comments 3 Add comment
10.02.2017 21:48:59
Спасибо за публикацию
10.02.2017 23:07:46
спасибо за публикацию
24.04.2017 22:32:44
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