A diploid strain of aspergillus niger producing citric acid

 

The strain can be used as a producer of citric acid by fermentation deep way carbohydrate-containing environments. A diploid strain of Aspergillus niger VKPM F-817 obtained by the selection method of polyploidie. The fermentation of a new strain output of citric acid from sugar amounts: diluted molasses medium with a sugar concentration of 30 g/DM3- 87,0%, concentrated molasses medium with concentration of sugar 130 g/DM3- 66%, Saharsa-mineral medium with concentration of sugar 150 g/DM3- 95,6%. The technical result of the invention is to obtain a new diploid strain of Aspergillus niger with resistance to morphological variability during storage and consistently high productivity. table 2.

The invention relates to the microbiological industry, and refers to a diploid strain of the fungus Aspergillus niger is the product of citric acid fermentation deep way carbohydrate-containing environments.

A known strain of the fungus Aspergillus niger VKPM F-681, which was obtained under the influence of UV rays by the method of stepwise selection of chernoochene mutant fungus 163/9. To stabilize the properties of the selected mutants were selected sustainable with the cell strain was allowed to use it as the product of citric acid by fermentation of Saharsa-mineral media, hydrolyzed starch. The yield of citric acid were respectively: % entered from sugar 89,0 and 92,5, and in g per 1 g of air-dried mycelium 18,7 and 12.0 /1/.

However, when storing strain VKPM F-681 marked increase in morphological variability of colonies of up to 7%; spontaneous variants of this strain was observed large fluctuations in the yield of citric acid.

Long-term studies on the application of the method of mutagenesis showed that a significant increase in productivity in the mutant strains achieved in the first stages of selection, in subsequent stages of selection mutagenesis method less effective. Therefore, for the breeding of new producers of citric acid was used non-applicable currently in the selection of Aspergillus niger method of polyploidy.

The prototype of the invention is mitotic diploid strain Asp.niger 2B-1-10 /2/ selected method of polyploidy under the influence masnago poison d-camphor from haploid strain VKPM F-681. First, under the influence of camphor was obtained polymorphic culture D-4. Selection diploid culture of the population polymorphic culture was performed stepwise. To enrich the culture of diploid cells was used filtration method and radiation lethal to japanska colony on wort-agar folded, pubescent, conidial heads are bigger than the original strain. Konditorei black, rich.

The diameter of the conidial heads of 200-300 microns.

The diameter of the bubbles canadianese 39-47 microns.

Length sterigma first layer 21-39 microns.

Length sterigma second layer of 9 μm.

The diameter of the conidia of 5 ám.

The number of conidia in 1 g of inoculum 11,4109.

Weight 1 conidia 8,810-8mg.

The microscopic structure of a collection larger than the original strain in 1,5-2,0 times.

The mass of a single conidia more than 1.7 times.

In the work of authors /2/ data on the activity of diploid and original haploid strains in the experiments on the fermentation of Saharsa-mineral environment and hydrolyzed starch. In these environments, diploid conidia swell, increasing in diameter to 10-27 μm, the original strain to 6-9 μm. Germ tubes from diploid 1.5-2.0 times larger in diameter than the original strain. From germinated conidia are formed mycelial pellets. The original strain granules with dense long peripheral hyphae; diploid granules large loose with short peripheral hyphae.

When fermentation on Saharsa-mineral environment through 5 days output lemon Cimislia the diploid less than in haploids and is 0.5-0.3 g of air-dried mycelium (HSR). A slight increase in mitotic diploid on Saharsa-mineral environment does not allow to obtain a sufficiently stable results in the release of citric acid.

Mitotic diploid after breeding were kept in the form of air-dry conidia at 20oC. After 1.5 years of storage only 70% of the colonies retained the original phenotype.

In addition, the article does not mention information concerning education for citric acid fermentation by a collection of molasses media.

The technical result of the invention is a new strain of the fungus Aspergillus niger is the producer of citric acid, with consistently high productivity.

Method of breeding a new strain. The source for obtaining strain served as a famous mitotic diploid strain Asp.niger 2B-1 to 10.

Conidia of the original strain 2B-1 to 10 were subjected to UV-rays at a dose of 6 kJ/m, in which the survival of conidia in chernoochene diploid strain is 0.1%; chernoochene haploid strain VKPM F-681 - 0,02%. The exposure helped to eliminate from the population of the original strain is haploid cells and get monomorphic diploid culture.

After irradiation Aravali from 5,18 to 7.80 g of citric acid to the flask, the yield of air-dried mycelium in the flask was 0.29 to 0.55 g, the productivity of mycelium (12,2-18,8) g LK/g HSR.

From the population of recombinants first generation bred morphologically homogeneous culture. Its growth on Saharsa-mineral environment has improved and was 0.82 g of HSR in the flask; the productivity of the mycelium was 10.2 g LK/g high-speed rail. New selected strain of Aspergillus niger in the all-Russian collection of industrial microorganisms assigned a collection number VKPM F-817.

Cultural and morphological properties of Aspergillus niger strain VKPM F-817. 2 days of growth on wort-agar is formed colony diameter of 16 mm, white fluffy aonidiella. In 5 days the total number of birds 81 mm Konditorei dark gray, sterile region of 7.5 mm On day 10 are formed conidial head size 182-334 μm. The diameter of the bubbles canadianese 36-47 μm. Length sterigma first layer 22-43 μm; a second layer of 9.0 μm. The average diameter of the conidia of 4.9 μm, and the amount of conidia of 61.6 μm3.

Compared with the original strain 2B-1-10 recombinant following exposure and selection decreased the size of conidia and their weight.

Comparative characteristics of the strains are presented in table. 1.

A genetic feature. Strain VKPM F-817 SL is education.

Add in Saharsa-mineral environment of sugar beet molasses in the amount of 17 g/DM3environment improves the formation of granules of fungal mycelium; adding to the wort-agar medium of Baker's yeast autolysate at a dose of 50 cm3/DM3environment enhances the maturation of conidia. Yeast autolysate is a source of vitamins and nucleic bases, which suggests that the new strain VKPM F-817 is partially deficient.

Physiological and biochemical properties. Asp.niger strain VKPM F-817 aerobe, the temperature of cultivation 28-32oC, pH 5.5 and 7.5.

The sources of carbon assimilates glucose, fructose, sucrose, maltose, dextrins; to a lesser extent, mannitol, malic and succinic acids, aqueous solutions of tannin.

The sources of nitrogen: absorbs nitrogen organic compounds such as protein, peptone, ammonium salts, nitrates, amino acids, yeast autolysate.

Stability during storage. Diploid strain Asp.niger VKPM F-817 selected in 1998. When stored in the form of air-dry conidia with large sterile quartz sand retains its properties. Withstands subcultures on wort-agar medium: after two subcultures variability in colony morphology is 0.4%, last is montannah variants of typical colonies.

Diploid strain VKPM F-817 is a highly active producer of citric acid by fermentation of carbohydrate-containing environments - from beet molasses, Saharsa-mineral and hydrolyzed starch.

The usefulness of recombinant mitotic diploid illustrated by several examples.

Example 1 Test per diluted environment from beet molasses is subjected to seed culture mitotic diploid 2B-1-10, recombinant diploid VKPM F-817 and haploid strain VKPM F-681.

For the fermentation of beet molasses is prepared seed material in the form of air-dry conidia containing 15109conidia in 1 g of inoculum. Seed is grown on the medium composition (g/DM3): beer wort nikolenka 8oBLG, sodium chloride 10, urea 1, copper sulfate pentahydrate 0,02, agar 20, pH of 5.6.

The medium is sterilized at 0.07 MPa for 30 min in a sterile box filled with a layer height of 11 mm in a pre-sterilized with 0.1 MPa of the cell.

Sowed the cuvette is placed in a thermostat with a temperature of 32oC and relative humidity 60%. Within 8 d are formed Mature conidia and dried film of the fungus. On 8-9 day collection kondh conditions on the rocking chair AVA-50p with the number of oscillations 160 per minute in flasks with a capacity of 750 cm3when the temperature in thermostat 32oC.

As the carbon source used beet molasses containing 44.67% of sucrose.

The fermentation is carried out in two stages. In the first stage 50 cm3the diluted molasses medium (30 g sugar 1 DM3from conidia of the fungus grown mycelium pellets.

The composition of the medium, g/DM3: Molasses beet - 67,07 Potassium hexacyanoferrate trihydrate - 0.2 Ammonium oxalate monohydrate was 1.43 Potassium dihydrophosphate - 0.16 Magnesium sulfate heptahydrate - 0,25 Through day 10 cm3the nutrient solution with mycelial pellets are transferred into 50 cm3fermentation medium of the same composition, with the exception of magnesium sulfate; dosage of ammonium oxalate was reduced by half.

On the second day topping produce a concentrated solution of molasses (559 g molasses 1 DM3) subjected to processing by hexacyanoferrate potassium and sterilization.

For comparison, in the same conditions grow the original strain VKPM F-681 and the original diploid strain 2B-1-10. During the day conidia swell: from 5.0 μm to 16-20 μm in mitotic diploid 2B-1 to 10, from 4.9 μm to 16-18 microns in recombinant VKPM F-817, from 3.9 μm to 11-16 µm in haploid strain VKPM F-681. The width of the seedlings from diploid 2B-1-10 - 5,3 IWC diploid strains formed a loose granules. The original diploid grain diameter of 800 μm, from recombinant - 700 microns, the haploid strain - 600 microns.

The haploid strain VKPM F-681 dense granules.

The test results on diluted molasses environment are presented in table.2 in example 1.

As you can see from the table the highest yield of citric acid from sugar molasses obtained from diploid recombinant strain VKPM F-817, he was 87%, while that of the original strain 2B-1-10 exit citric acid 78%.

Example 2 Testing the concentrated medium from beet molasses is subjected to seed culture mitotic diploid 2B-1-10, recombinant diploid VKPM F-817 and haploid strain VKPM F-681. Seed material for biochemical experience prepared according to the method described in example 1.

Preparation of medium for fermentation is using molasses medium containing 130 g/DM3sugar in the original environment. In the fermentation process additional dolive molasses environment are not held. Other test conditions are similar to example 1.

The end of the experience made on the 4th day of fermentation.

The test results diploid cultures - mitotic diploid 2B-1-10, recombinant diploid VKPM F-817 and gonnoi acid from sugar reaches 66%, the original diploid strain of 59%.

Example 3 fermentation of Saharsa-mineral environment seed strain VKPM F-817 is grown according to the method described in example 1.

The definition of productivity is the method of submerged culture on the rocking chair AVA-50p.

Fermentation is carried out in two stages. The first stage is grown seed mycelium (granules) in the medium composition (g/DM3): sugar 50, molasses beet 17, ammonium nitrate 2,5, magnesium sulfate heptahydrate 0.25 dihydroorotase potassium 0,16, pH 6.9. The growing mycelium was grown for 2 days.

In the second stage of fermentation used the medium of the same composition as for the cultivation of mycelium pellets, but differing increased to 150 g/DM3sugar content. 55 cm3fermentation environment contribute 10 cm3nutrient medium with mycelial pellets.

The duration of fermentation is 5 days. The results of the experiment are presented in table.2 example 3. As can be seen from the data in the table.2, of two diploid cultures dominated by recombinant VKPM F-817. The yield of citric acid from sugar in diploid 2B-1-10 - 94,8%, the recombinant is 95.6%. More abundant growth of mycelium from the recombinant strain does not reduce the yield of citric acid and creates more stoicheia).

SOURCES of INFORMATION
1. RF patent 2089615, IPC 6 12 P 7/48, With 12 N 18/14, 1997.

2. T. A. Nikiforova, E. I. Shcherbakov. Breeding and biological properties of mitotic diploid Asp. niger/ J. Storage and processing of agricultural products, 1997, 12, S. 51-53.


Claims

A diploid strain of Aspergillus niger VKPM F-817 - producer of citric acid.

 

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1 tbl, 6 ex

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