Nutritive medium for cultivation of phosphate-mobilising and nitrogen-fixing microorganism consortium

FIELD: biotechnologies.

SUBSTANCE: nutritive medium contains potassium dihydrophosphate, potassium hydrophosphate, magnesium sulphate heptahydrate, sodium chloride, calcium sulphate dihydrate, sodium molybdate, iron (II) sulphate, saccharose, nano sapropel and distilled water with a defined component ratio.

EFFECT: enhanced growth rate of phosphate-mobilising and nitrogen-fixing microorganisms.

1 tbl, 15 ex

 

The invention relates to the field of biological products on the basis of individual microorganisms and their combinations (consortia) and can be used in Microbiology and agriculture.

Known nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms, containing glucose, asparagine, potassium sulfate, corn steep liquor, calcium chloride, sodium phosphate and water [1]. The disadvantage of this nutrient medium is a relatively low growth rate phosphatability of microorganisms, as well as the virtual absence of growth of nitrogen-fixing microorganisms, which makes it unsuitable for growing their consortium.

Known nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms containing potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, sodium chloride, calcium carbonate, sucrose and water [2]. The disadvantage of this nutrient medium is the virtual absence of growth phosphatability of microorganisms, which also makes it unsuitable for growing their consortium.

Also known nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms containing potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, Chloe�Eid sodium, calcium sulfate, sodium molybdate, iron(II) sulphate, sucrose and water, is described in [3]. The disadvantage of this nutrient medium is also relatively low growth rate phosphatability of microorganisms.

The closest to the claimed us the object a set of attributes and the achieved technical effect is a nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms containing potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, sodium chloride, calcium sulfate, sodium molybdate, iron(II) sulphate, sucrose, mineral Supplement - sapropel and water, is described in [4]. The disadvantage of this nutrient medium, which in connection with the just-noted circumstance we selected the prototype object is also a relatively low growth rate phosphatability of microorganisms.

The purpose of this invention is to increase the speed of growth phosphatability microorganisms by culturing consortium phosphatability and nitrogen-fixing microorganisms.

This object is achieved in that the nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms containing potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, sodium chloride, calcium sulfate, molybde� sodium, the iron(II) sulphate, sucrose, mineral Supplement, and water, as mineral supplements contains nanoprobing with the following ratio of ingredients (g/l):

The potassium dihydrogen phosphate0.60-0.70
The potassium hydrophosphate0.12-0.20
Magnesium sulfate is heptahydrate0.15-0.25
Sodium chloride0.15-0.25
Calcium sulfate dehydrate0.02-0.06
Molybdate sodium0.0005-0.0007
Ferrous sulfate(II)0.002-0.004
Sucrose18.0-22.0
Nanoprobing0.5-1.5
Distilled waterTo 1 liter

As a result of the use of this nutrient mixture growth rate phosphatability of microorganisms increases in 7-8 times compared to the same indicator of the nutrient medium for the prototype [4].

So far in the literature are not described nutrient medium for cultiv�of the consortium phosphatability and nitrogen-fixing microorganisms containing the above combination of ingredients in General and nanoprobing in particular; moreover, the use of the latter in the composition of nutrient medium for microorganisms not known at all. This fact allows us to conclude that the claimed us the object to the first set of patent legislation of the Russian Federation criterial feature of the invention - novelty. Comparison of the known symptoms of the nutrient medium-prototype [4] and distinctive features that characterize our inventive object (namely the introduction of nanoprobes), is a priori not possible to predict the appearance of his new compared to the prototype properties, namely the above sharp increase in the rate of growth phosphatability microorganisms (while reducing the rate of growth of nitrogen-fixing microorganisms) that are part of the consortium. This point gives us reason to believe that the claimed object is not obvious from the known in the art; therefore, it falls under the second of the established by the legislation of the Russian Federation criterial features of the invention - inventive step. The claimed us the nutrient medium can be easily obtained on an industrial scale, and its use for growing consortium phosphatability and isothecium�x microorganisms not associated with any serious technical problems; in this regard, we may assert that the given object is inherent in the third set by the legislation of the Russian Federation criterial symptom of the invention, namely industrial applicability.

Declare on the subject of the invention, the nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms may be illustrated by the following examples.

Example 1

Natural sapropel deposits near lake White (Tukai area of the Republic of Tatarstan) is ground into flour and mixed with distilled or deionized (demineralized) water at the rate of 20 g of sapropel per 100 ml of water. The resulting mixture is treated with ultrasound in an ultrasonic disperser the TGS-0,25 a power of 80 W at a frequency of 18.5 kHz with amplitude of the ultrasonic waveguide is 5 μm for (5-20) minutes at room temperature, resulting in a water-sapropelic suspension with particle sizes of sapropel from 5 to 100 nm. The thus prepared suspension of nanoprobes further used as one of the components of the nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms.

Example 2

Prepare a nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms of the composition, g/l:

The potassium dihydrogen phosphate0.60
The potassium hydrophosphate0.12
Magnesium sulfate is heptahydrate0.15
Sodium chloride0.15
Calcium sulfate dehydrate0.02
Molybdate sodium0.0005
Ferrous sulfate(II)0.002
Sucrose18.0
Nanoprobing0.5
Distilled waterTo 1 liter

Make up the consortium phosphatability and nitrogen-fixing microorganisms with a 1:1 ratio by the number of colony forming units on the basis of the collection (deposited) of named strains of microorganisms (Sphingobacterium multivorum, Registration number VKPM b-10385) and (Pseudomonas brassicacearum, Registration number VKPM b-10388), respectively. For this first cultivated phosphatability microorganisms on agar medium Muromtseva, azotfiksiruyushchie� - on agar medium Ashby; further, these crops are sown on nutrient medium of the above composition. The cultivation was conducted for that period of time in which there is an increase in their numbers (4.5 days), after which the process is stopped. To determine the population of microorganisms immediately carry out planting of the consortium on Aga-rysowanie nutrient medium (Wednesday Muromtsev in case phosphatability microorganisms and the environment Ashby in the case of nitrogen-fixing) and determine the average rate of growth in (mln·g-1·day-1) as the quotient from dividing the number of microorganisms (millions of units) on a lot of the nutrient medium (in g) and the growing period (in days). Data on the rate of growth phosphatability and nitrogen-fixing microorganisms for the above nutrient medium are presented in Table 1.

Example 3

Carried out as Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms take nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.64
The potassium hydrophosphate0.16
Magnesium sulfate is heptahydrate0.20
The sodium chloride� 0.20
Calcium sulfate dehydrate0.05
Molybdate sodium0.0006
Ferrous sulfate(II)0.003
Sucrose20.0
Nanoprobing1.0
Distilled waterTo 1 liter

Data on growth rate of microorganisms for this case are presented in Table 1.

Example 4

Perform as Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms take nutrient medium composition, g/l:

Ferrous sulfate(II)
The potassium dihydrogen phosphate0.70
The potassium hydrophosphate0.20
Magnesium sulfate is heptahydrate0.25
Sodium chloride0.25
Calcium sulfate dehydrate0.06
Molybdate sodium0.0007
0.004
Sucrose22.0
Nanoprobing1.5
Distilled waterTo 1 liter

Information on the growth rate of these microorganisms for this case are shown in Table 1.

Example 5 (comparative)

Carried out in the same manner as that of Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms prepared nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.64
The potassium hydrophosphate0.16
Magnesium sulfate is heptahydrate0.20
Sodium chloride0.20
Calcium sulfate dehydrate0.05
Molybdate sodium0.0005
Ferrous sulfate(II)0.003
Sucrose20.0
N�notepaper 0.3
Distilled waterTo 1 liter

Information about the rate of growth of the above microorganisms for this case is presented in Table 1.

Example 6 (comparative)

Perform according to the General procedure of Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms use nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.64
The potassium hydrophosphate0.16
Magnesium sulfate is heptahydrate0.20
Sodium chloride0.20
Calcium sulfate dehydrate0.05
Molybdate sodium0.0005
Ferrous sulfate(II)0.003
Sucrose20.0
Nanoprobing2.0
Distilled waterTo 1 liter

The velocity growth� of microorganisms for this case, see in Table 1.

Example 7 (comparative)

Carried out in the same manner as that of Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms use nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.50
The potassium hydrophosphate0.09
Magnesium sulfate is heptahydrate0.10
Sodium chloride0.15
Calcium sulfate dehydrate0.015
Molybdate sodium0.0003
Ferrous sulfate(II)0.001
Sucrose14.0
Nanoprobing1.0
Distilled waterTo 1 liter

Indicators of growth rate of microorganisms for this case, see Table 1.

Example 8 (comparative)

Carried out the General scheme of Example 2, but the cultivation of the above consortium of microorganisms is carried out on mittelmassige composition, g/l:

The potassium dihydrogen phosphate0.90
The potassium hydrophosphate0.30
Magnesium sulfate is heptahydrate0.30
Sodium chloride0.35
Calcium sulfate dehydrate0.09
Molybdate sodium0.0010
Ferrous sulfate(II)0.006
Sucrose28.0
Nanoprobing1.0
Distilled waterTo 1 liter

Information on the growth rate of microorganisms for this case, see Table 1.

Example 9 (comparative)

Perform in the same manner as Example 2, but for growing consortium of microorganisms use nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.50
The potassium hydrophosphate0.09
Magnesium sulfate is heptahydrate0.10
Sodium chloride0.15
Calcium sulfate dehydrate0.015
Molybdate sodium0.0003
Ferrous sulfate(II)0.001
Sucrose14.0
Nanoprobing2.0
Distilled waterTo 1 liter

The values of the growth rate of microorganisms for this case are shown in Table 1.

Example 10 (comparative)

Operates on the same General scheme as that of Example 2, but the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms is carried out on a nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.90
The potassium hydrophosphate0.30
Magnesium sulfate is heptahydrate0.30
Sodium chloride0.35
Calcium sulfate dehydrate0.09
Molybdate sodium0.0010
Ferrous sulfate(II)0.006
Sucrose28.0
Nanoprobing2.5
Distilled waterTo 1 liter

Indicators of growth rate of microorganisms for this case are given in Table 1.

Example 11 (according to the prototype [4])

Operates on the same General scheme as that of Example 2, but the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms is carried out on a nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.64
The potassium hydrophosphate0.16
Magnesium sulfate is heptahydrate0.20
Sodium chloride0.20
Calcium sulfate dehydrate0.05
Molybdate sodium0.0006
Ferrous sulfate(II)0.003
Sucrose20.0
Sapropel1.0
Distilled waterTo 1 liter

Indicators of growth rate of microorganisms for this case are given in Table 1.

Example 12 (according to the prototype [4])

Operates on the same General scheme as that of Example 2, but the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms is carried out on a nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.70
The potassium hydrophosphate0.20
Magnesium sulfate is heptahydrate0.25
Sodium chloride0.25
Calcium sulfate dehydrate0.06
Molybdate sodium0.0007
Ferrous sulfate(II)0.004
Sucrose22.0
Sapropel1.5
Distilled waterTo 1 liter

Indicators of growth rate of microorganisms for this case are given in Table 1.

Example 13 (the analogue of [3])

Perform the same process scheme as that of Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms use nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.64
The potassium hydrophosphate0.16
Magnesium sulfate is heptahydrate0.20
Sodium chloride0.20
Calcium sulfate dehydrate0.05
Molybdate sodium0.0005
Ferrous sulfate(II)0.003
Sucrose20.0
Distilled waterTo 1 liter

Data on growth rate of microorganisms for this case, see Table 1.

Example 14 (n� the analogue [1])

Perform the same process scheme as that of Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms use nutrient medium composition, g/l:

Potassium sulphate0.20
Calcium chloride hexahydrate3.30
Phosphate sodium dodecahydrate3.80
Corn steep liquor0.20
Glucose10.0
Asparagine1.00
Distilled waterTo 1 liter

Data on growth rate of microorganisms for this case are shown in Table 1.

Example 15 (the analogue of [2])

Perform the same process scheme as that of Example 2, but for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms use nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.10
The potassium hydrophosphateMagnesium sulfate is heptahydrate0.20
Sodium chloride0.20
Calcium carbonate5.00
Sucrose20.0
Distilled waterTo 1 liter

Data on growth rate of microorganisms for this case also, see Table 1.

Table 1
No. exampleThe content of nano-sapropel in the nutrient mixture, g/lThe average rate of growth phosphatability microorganisms {Sphingobacterium multivorum), mln·g-1·day-1The average rate of growth of nitrogen-fixing microorganisms (Pseudomonas brassicacearum), mln·g-1·day-1
20.5373.34.7
31.0391.14.9
41.5382.2 4.9
5 (comparative)0.3284.46.7
6 (comparative)2.0311.15.3
7 (comparative)1.0351.17.1
8 (comparative)1.0344.46.7
9 (comparative)2.0337.85.8
10 (comparative)2.0328.95.3
11 (the prototype [4])-46.715.1
12 (the prototype [4])-47.114.9
13 (by the analogue of [3])-38.98.0
14 (by the analogue of [1])- 40.02.7
15 (the analogue of [2])-1.312.5

As you can see from the Table 1 data, the use of the claimed nutrient medium containing nanocapsule in the amount of (0.5-1.5) g/l, significantly (7-8 times) to increase the rate of growth phosphatability (Sphingobacterium multivorum) of microorganisms by reducing the rate of growth of nitrogen-fixing Pseudomonas brassicacearum} of microorganisms within their consortium compared with those for nutrient medium prototype [4] (and to an even greater extent compared to the media-analogues [1], [2] and [3]). In this case the claimed number of us nanoprobes in the nutrient mixture are important when increasing its sverhuspeshnogo top of the proposed limit (1.5 g/l) further increase in the rate of growth phosphatability microorganisms already happening, if you reduce the same below the bottom of the claimed value (1.0 g/l) notes a decrease in the rate of their growth (although it remains significantly higher compared to the one that occurs on a nutrient medium [4]).

Similar to the above results were obtained in our and other cultures of nitrogen-fixing and phosphatability microorganisms (in particular, Azotobacter chroococcum, Register�operating room VKPM b-10387 and Achromobacter xylosoxidans, Registration number VKPM b-10386).

LITERATURE

[1] Basic microbiological and biochemical methods of soil investigation (guidelines) / ed. by Y. M. Wozniakowski. - L.: ARRIAM, 1987. S. 31.

[2] a Guide to practical classes in Microbiology. 3rd revised edition, ed. by N. With. Egorova. M: Publishing house of Moscow University. 1995. P. 204.

[3] the Patent of the Russian Federation 2.177.466 (2001), IPC C05F 11/08, C12N 1/20.

[4] the Application for invention of the Russian Federation No. 2012145906 from 26.10.2012, IPC C12N 1/00, C12N 1/20, C12N 1/22 (prototype).

Nutrient medium for the cultivation of the consortium phosphatability and nitrogen-fixing microorganisms containing potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate is heptahydrate, sodium chloride, calcium sulphate dehydrate, sodium molybdate, iron(II) sulphate, sucrose, mineral additive and distilled water, characterized in that as a mineral Supplement it contains nanoprobing in the following ratio of components, g/l:

the potassium dihydrogen phosphate0.60-0.70
the potassium hydrophosphate0.12-0.20
magnesium sulfate is heptahydrate0.15-0.25
sodium chloride0.15-0.5
calcium sulfate dehydrate0.02-0.06
molybdate sodium0.0005-0.0007
ferrous sulfate(II)0.002-0.004
sucrose18.0-22.0
nanoprobing0.5-1.5
distilled waterto 1 liter



 

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EFFECT: invention enables to remove petroleum hydrocarbons from water and bottom deposits with low oxygen concentration in the water and in high latitude conditions.

3 tbl, 2 ex

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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