Nutritive medium for growth 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, nanobentonite and distilled water.

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

1 tbl, 14 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 growing consortium phosphatability and nitrogen-fixing microorganisms containing potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, sodium chloride, calcium carbonate, sucrose and water [1]. The disadvantage of this nutrient medium is a relatively low rate of growth on it nitrogen-fixing microorganisms and the virtual absence of growth phosphatability of microorganisms, which also makes it unsuitable for growing their consortium.

Also known nutrient medium for growing 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 and water (so-called Wednesday Burke) [2]. The disadvantage of this nutrient medium is also relatively low rate of growth of nitrogen-fixing and phosphatability of microorganisms, and therefore, their consortium as a whole.

The closest to the claimed us the object a set of attributes and the achieved technical effect is IIN�makes nutrient medium for growing 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 additive - bentonite and water [3]. The disadvantage of this nutrient medium, which in connection with the just-noted circumstance we selected the prototype object, is also relatively low growth rate phosphatability of microorganisms.

The purpose of the present invention is to increase the speed of growth phosphatability microorganism on a nutrient medium while maintaining practically unchanged the rate of growth of nitrogen-fixing microorganisms in the process of growing consortium based on them.

The declared objective is achieved in that in a nutrient medium for growing 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, and water, as the mineral additives used nanoantenna with the following ratio of ingredients (g/l):

Magnesium sulfate is heptahydrate
The potassium dihydrogen phosphate0.60-0.70
The potassium hydrophosphate0.12-0.20
0.15-0.25
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
Nanoantenna0.7-1.5
Distilled waterTo 1 liter

Using this nutrient mixture growth rate phosphatability of microorganisms increases by 25-30% in comparison with that nutrient medium for the prototype [3], the rate of growth of nitrogen-fixing microorganisms remains almost unchanged.

So far in the literature are not described nutrient medium for growing consortium of nitrogen-fixing and phosphatability microorganisms containing the above combination of ingredients and nanoantenna in particular; moreover, the use of nanoantenna in the composition of nutrient media for cultivation of microorganisms not known at all. This fact gives us reason to believe that tawlae�th us the object corresponds to the first criterial feature of the invention, established patent law of the Russian Federation, - the novelty. A comparison of the known characteristics of the nutrient medium-prototype [3] and distinctive features that characterize the claimed us the object (namely, the substitution contained in the nutrient medium prototype of bentonite on nanoantenna), does not allow to predict a priori the appearance of his new compared to the prototype properties, namely the above-mentioned increase in the rate of growth phosphatability microorganisms that are part of the above consortium, while maintaining virtually unchanged the rate of growth of nitrogen-fixing. This fact allows us to conclude that the claimed us the object is not obvious from the known in the art level, and therefore, corresponds to the second set by the legislation of the Russian Federation criterial feature of the invention is an inventive step. Finally, our proposed nutrient medium can be rather easily obtained on an industrial scale and its use for growing consortium phosphatability and nitrogen-fixing microorganisms is achieved without any problems, so the claimed us the object is inherent in the third set by the legislation of the Russian Federation criterial symptom of the invention and industrial applicability.

The use of the claimed on the subject of izobreteny� nutrient medium for growing consortium phosphatability and nitrogen-fixing microorganisms is illustrated by the following examples.

Example 1

(preparation of nanoantenna)

Natural bentonite from tarn-Warski field (Nurlat district of the Republic of Tatarstan) is ground into flour and mixed with distilled or deionized (demineralized) water at the rate of 20 g of bentonite in 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-bentonite suspension with a particle size of bentonite from 5 to 100 nm. The thus prepared suspension of nanoantenna further used as one of the components of the nutrient medium for growing consortium phosphatability and nitrogen-fixing microorganisms.

Example 2

Prepare a nutrient medium for growing 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 chloride0.1
Calcium sulfate dehydrate0.02
Molybdate sodium0.0005
Ferrous sulfate(II)0.002
Sucrose18.0
Nanoantenna0.7
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 (Sphingo bacteriummultivorum, Registration number VKPM b-10385) and (Pseudomonas brassicacearum, Registration number VKPM b-10388), respectively. For this pre-grown nitrogen-fixing microorganisms on agar medium Ashby, and phosphatability - on agar medium Muromtseva, then both 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 day), when the process is stopped. To determine the population of microorganisms immediately carry out planting of the consortium on agar nutrients such a�environment setting parameters (Wednesday Ashby in the case of nitrogen-fixing and Wednesday Muromtseva - in case phosphatability) and determine the average rate of growth in (mlnt-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). Information on the growth rate of phosphatability and nitrogen-fixing microorganisms for the above nutrient medium are presented in Table 1.

Example 3

Carried out as Example 2, but for growing 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 sodium0.0006
Ferrous sulfate(II)0.003
Sucrose20.0
Nanoantenna1.0
Distilled waterTo 1 liter

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

Example 4

Carried out in the same manner as that of Example 2, but for growing consortium phosphatability and nitrogen-fixing microorganisms use 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
Nanoantenna1.5
Distilled waterTo 1 liter

Results on the determination of the growth rate named above microorganisms for this case, see Table 1.

<> Example 5

(comparative)

Carried out in the same manner as that of Example 2, but for growing 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
Nanoantenna0.4
Distilled waterTo 1 liter

The rate of growth of the above microorganisms for this case are given in Table 1.

Example 6

(comparative)

Perform according to the General procedure of Example 2, but for growing consortium of phosphate-mobilizing and nitrogen-fixing microorgani�MOU 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
Nanoantenna2.0
Distilled waterTo 1 liter

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

Example 7

(comparative)

Carried out in the same manner as that of Example 2, but for growing 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
Nanoantenna1.2
Distilled waterTo 1 liter

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

Example 8

(comparative)

Perform as Example 2, but for growing consortium of the above-mentioned 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 chloride 0.35
Calcium sulfate dehydrate0.09
Molybdate sodium0.0010
Ferrous sulfate(II)0.006
Sucrose28.0
Nanoantenna1.2
Distilled waterTo 1 liter

The rate of growth of each of the aforementioned types of microorganisms for this case are given in Table 1.

Example 9

(comparative)

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

Ferrous sulfate(II)
The potassium dihydrogen phosphate0.50
The potassium hydrophosphate0.09
Magnesium sulfate is heptahydrate0.10
Sodium chloride0.15
Calcium sulfate dehydrate0.015
Molybdate sodium0.0003
0.001
Sucrose14.0
Nanoantenna2.0
Distilled waterTo 1 liter

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

Example 10

(comparative)

Perform as 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
Nanoantenna 2.0
Distilled waterTo 1 liter

Indicators of growth rate of microorganisms for this case is presented in Table 1.

Example 11

(the prototype [3])

Carried out in the same flowchart as that of Example 2, but for growing 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 sodium0.0006
Ferrous sulfate(II)0.003
Sucrose20.0
Bentonite1.4
Distilled waterTo 1 liter

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

Example 12

(the prototype [3])

Carried out in the same manner as that of Example 2, but for growing consortium phosphatability and nitrogen-fixing microorganisms take 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
Bentonite1.8
Distilled waterTo 1 liter

Results on the determination of the growth rate named above microorganisms for this case, see Table 1.

Example 13

(by the analogue of [2])

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

The potassium dihydrogen phosphate0.64
The potassium hydrophosphate 0.16
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 are shown in Table 1.

Example 14

(by the analogue of [1])

Perform the same process scheme as that of Example 1, but for growing consortium phosphatability and nitrogen-fixing microorganisms take nutrient medium composition, g/l:

The potassium dihydrogen phosphate0.10
The potassium hydrophosphate0.20
Magnesium sulfate is heptahydrate0.20
Sodium chloride0.20
Calcium carbonate 5.00
Sucrose20.0
Distilled waterto 1 liter

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

Table 1
No. exampleThe content of nanoantenna in the nutrient mixture, g/lThe average rate of growth phosphatability microorganism Sphingobacterium multivorum, mlnt-1·day-1An average speed
growth of nitrogen-fixing microorganisms Pseudomonas brassicacearum, mlnt-1·day-1
20.7395.022.0
31.0413.024.0
41.5405.55 (comparative)0.4360.023.0
6 (comparative)2.0375.021.0
7 (comparative)1.2362.522.0
8 (comparative)1.2370.021.5
9 (comparative)2.0340.021.0
10 (comparative)2.0357.522.5
11 (the prototype [3])-320.024.0
12 (the prototype [3])-332.524.5
13 (by the analogue of [2])-49.09.0
14 (by the analogue of [1])1.514.0

As you can see from the Table 1 data, the use of the claimed nutrient medium containing nanoantenna in the amount of (0.7-1.5) g/l, allows approximately 25-30% increase in the rate of growth phosphatability (Sphingobacterium multivorum) of microorganisms and to maintain practically constant the rate of growth of nitrogen-fixing Pseudomonas brassicacearum) microorganisms within their consortium compared with those for nutrient medium-prototype [3] and analogues [1] and [2]. In this case the claimed number of us nanoantenna in the nutrient mixture are important when you increase it beyond the top of the proposed limit further increase in the rate of growth of those and other microorganisms is not observed (and even slight decrease), while decreasing the same below the bottom of the claimed limit of a decreasing growth rate.

We note in conclusion that similar results were obtained in our and other cultures of nitrogen-fixing and phosphatability microorganisms (in particular, Azotobacter chroococcum, Registration number VKPM b-10387 and Achromobacter xylosoxidans, Registration number VKPM b-10386).

LITERATURE

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

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

[3] the Application for invention of the Russian Federation No. 2012145904 from 26.10.2012, IPC C12N 1/00, C12N 1/20, C12N 1/22 (prototype).

Nutrient medium for growing 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 nanoantenna 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
sucrose 18,0-22,0
nanoantenna0,7-1,5
distilled waterto 1 liter



 

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