RU2203322C2 - Diploid strain of aspergillus niger as producer of citric acid - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: strain can be used as producer of citric acid in fermentation of carbohydrate-containing media by submerged method. Diploid strain Aspergillus niger VKPM F-817 is obtained by selection using polyploidy method. In fermentation with the novel strain the yield of citric acid of sugar added is: in diluted molasses medium with sugar concentration 30 g/g/dm³, 87.0%; in concentrated molasses medium with sugar concentration 130 g/g/dm³, 66% and in sucrose-mineral medium with sugar concentration 150 g/g/dm³, 95.6%. Invention provides preparing the novel diploid strain of Aspergillus niger showing resistance against morphological variability in storage and stably high productivity. EFFECT: valuable properties of strain, increased yield of citric acid. 2 tbl, 3 ex

Description

 The invention relates to the microbiological industry and relates to a diploid strain of the fungus Aspergillus niger, a producer of citric acid for fermentation by a deep method of carbohydrate-containing media.

 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 from a black-colored mutant of the fungus 163/9. To stabilize the properties of selected mutants, the selection of stable spontaneous variants was carried out. The stability of the morphological-cultural and physiological-biochemical properties of the selected strain made it possible to use it as a producer of citric acid in the fermentation of sucrose-mineral media and starch hydrolyzate. The yield of citric acid was respectively:% of the introduced sugar 89.0 and 92.5, and in g per 1 g of air-dried mycelium 18.7 and 12.0 / 1 /.

 However, during storage of the VKPM strain F-681, an increase in the morphological variability of the colonies to 7% was noted; spontaneous variants of this strain showed large fluctuations in the yield of citric acid.

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

The prototype of the invention is the mitotic diploid strain Asp.niger 2B-1-10 / 2 /, selected by polyploidy under the influence of mitotic d-camphor venom from the haploid strain VKPM F-681. Initially, under the influence of camphor, a polymorphic culture of D-4 was obtained. Selection of a diploid culture from a polymorphic culture population was carried out stepwise. To enrich the culture with diploid cells, the filtration method and irradiation with a lethal dose of UV rays for the haploid strain (6 kJ / m ² ) were used.

 The morphology of the colony of mitotic diploid 2B-1-10.

 The giant colony on the wort agar is folded, pubescent, the conidial heads are larger than the original strain. Conidiorection is black, plentiful.

 The diameter of the conidial head is 200-300 microns.

 The diameter of the swelling of the conidiophores is 39-47 microns.

 The length of the sterigm of the first layer is 21-39 microns.

 The length of the sterigm of the second layer is 9 μm.

 The diameter of conidia is 5 microns.

The number of conidia in 1 g of seed 11.4 • 10 ⁹ .

The mass of 1 conidia is 8.8 • 10 ^-8 mg.

 The microscopic structures of the diploid are 1.5-2.0 times larger than the initial strain.

 The mass of one conidia is 1.7 times greater.

 The authors' work / 2 / presents data on the activity of the diploid and initial haploid strains in experiments on the fermentation of a sucrose-mineral medium and starch hydrolyzate. In these media, the conidia of the diploid swell, increasing in diameter to 10-27 microns, in the original strain to 6-9 microns. The diploid germ tubes are 1.5-2.0 times larger in diameter than the original strain. From sprouted conidia, mycelial granules are formed. In the initial strain, the granules are dense with long peripheral hyphae; in diploid, granules are large, loose with short peripheral hyphae.

 When fermentation on a sucrose-mineral medium after 5 days, the yield of citric acid in diploid is higher on average by 18% than in haploid. The conversion of sugar to citric acid is 91-100%. The growth of mycelium in diploid is less than that of haploids and is 0.5-0.3 g of air-dry mycelium (SCM). A slight increase in mitotic diploid on a sucrose-mineral medium does not allow to obtain sufficiently stable results on the yield of citric acid.

After selection, the mitotic diploid was stored in the form of air-dried conidia at 20 ^o C. After 1.5 years of storage, only 70% of the colonies retained the original phenotype.

 In addition, the article does not mention information regarding the formation of citric acid during diploid fermentation of molasses media.

 The technical result of the invention is a new strain of the fungus Aspergillus niger - a producer of citric acid having a stably high productivity.

 The method of selection of a new strain. The initial mitotic diploid strain Asp.niger 2B-1-10 served as the starting strain.

 Conidia of the original strain 2B-1-10 were exposed to UV rays at a dose of 6 kJ / m, at which the survival of conidia in the black-colored diploid strain is 0.1%; in the black-colored haploid strain VKPM F-681, 0.02%. Irradiation made it possible to eliminate haploid cells from the population of the initial strain and obtain a monomorphic diploid culture.

After irradiation with a dose of 6 kJ / m ² , 30 cultures were selected. During the fermentation of the sucrose-mineral medium, 7 out of 30 cultures produced 5.18 to 7.80 g of citric acid per flask, while the yield of air-dry mycelium on the flask was 0.29-0.55 g, mycelial productivity (12.2 -18.8) g LK / g HSR.

 A morphologically homogeneous culture was selected from the population of recombinants of the first generation. Its growth on the sucrose-mineral medium improved and amounted to 0.82 g of high-speed iron per flask; mycelial productivity was 10.2 g LK / g HSR. The new breeding strain of Aspergillus niger in the All-Russian collection of industrial microorganisms is assigned a collection number VKPM F-817.

Cultural and morphological properties of Aspergillus niger strain VKPM F-817. On the 2nd day of growth, a colony with a diameter of 16 mm, white fluffy aconidial, is formed on wort agar. On day 5, the colony size is 81 mm. Conidiocar wearing dark gray, "sterile" edge of 7.5 mm. On day 10, conidial heads with a size of 182-334 microns are formed. The diameter of the swelling of the conidiophores 36-47 microns. The length of the sterigm of the first layer is 22-43 microns; the second layer is 9.0 μm. The average diameter of conidia is 4.9 μm and the volume of conidia is 61.6 μm ³ .

 Compared with the initial strain 2B-1-10, the recombinant as a result of irradiation and selection slightly decreased the size of the conidia and their mass.

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

 Genetic feature. Strain VKPM F-817 weakly grows on mineral media. The addition of organic additives to the nutrient medium enhances growth and conidia formation.

The addition of beet molasses to the sucrose-mineral medium in an amount of 17 g / dm ^{3 of the} medium improves the formation of fungal mycelium granules; adding to the wort-agar medium autolysate of baking yeast at a dose of 50 cm ³ / dm ^{3 of the} medium 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 aerob, cultivation temperature 28-32 ^o C, pH 5.5-7.5.

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

 Attitude to nitrogen sources: assimilates nitrogen of organic compounds, such as protein, peptone, ammonium salts, nitrites, amino acids, yeast autolysate.

 Storage Stability. The diploid strain Asp.niger VKPM F-817 was selected in 1998. When stored in the form of air-dried conidia with large sterile quartz sand, it retains its properties. Withstands transfers on wort-agar medium: after two transfers, the variability of colony morphology is 0.4%, after four transfers, 2.0%.

 The propagation of conidia in order to obtain seed is preceded by the selection of spontaneous variants from a typical colony.

 The diploid strain VKPM F-817 is a highly active producer of citric acid in the fermentation of carbohydrate-containing media - from beet molasses, sucrose-mineral and starch hydrolyzate.

 The practical utility of the mitotic diploid recombinant is illustrated by a number of examples.

Example 1
Seed culture of mitotic diploid 2B-1-10, diploid recombinant VKPM F-817 and haploid strain VKPM F-681 were tested on a diluted medium from beet molasses.

For fermentation of the medium from beet molasses, seed is prepared in the form of air-dried conidia containing 15 • 10 ⁹ conidia in 1 g of seed. Seeds are grown on a composition medium (g / dm ³ ): unsweetened beer wort 8 ^o Blg, sodium chloride 10, urea 1, copper sulfate pentahydrate 0.02, agar 20, pH 5.6.

 The medium is sterilized at 0.07 MPa for 30 minutes and, in a sterile box, is poured with a layer height of 11 mm into cells previously sterilized at 0.1 MPa.

Inoculated cuvettes are placed in a thermostat with a temperature of 32 ^o With a relative humidity of 60%. Within 8 days, mature conidia are formed and the fungus film is dried. On days 8–9, conidia are collected using a vacuum collector or a spatula.

The fermentation process is carried out in deep conditions on a rocker AVU-50r with a number of vibrations of 160 per minute in flasks with a capacity of 750 cm ³ at a temperature in thermostat 32 ^o C.

 Beet molasses containing 44.67% sucrose is used as a carbon source.

Fermentation is carried out in two stages. At the first stage, 50 cm ^{3 of} diluted molasses medium (30 g sugar in 1 dm ³ ) are grown from fungus conidia granules of mycelium.

The composition of the medium, g / DM ³ :
Beet molasses - 67.07
Potassium hexacyanoferrate trihydrate - 0.2
Ammonium oxalate monohydrate - 1.43
Potassium dihydrogen phosphate - 0.16
Magnesium sulfate heptahydrate - 0.25
After 24 hours, 10 cm ^{3 of the} nutrient solution with mycelial granules is transferred to 50 cm ^{3 of a} fermentation medium of the same composition, with the exception of magnesium sulfate; the dose of ammonium oxalate was halved.

On the second day, topped up with a concentrated molasses solution (559 g molasses in 1 dm ³ ), subjected to potassium hexacyanoferrate treatment and sterilization.

 For comparison, the original VKPM F-681 strain and the original diploid strain 2B-1-10 are grown under the same conditions. Conidia swell during the day: from 5.0 μm to 16-20 μm in the mitotic diploid 2B-1-10, from 4.9 μm to 16-18 μm in the recombinant VKPM F-817, from 3.9 μm to 11- 16 μm in the haploid strain VKPM F-681. The width of the seedlings of diploid 2B-1-10 is 5.3 microns; recombinant 4.6 microns; the parent strain VKPM F-681 - 4.0 μm.

 On the fermentation medium for 6 days, loose granules form in diploid strains. The initial diploid has a granule diameter of 800 μm, the recombinant has 700 μm, and the haploid strain has 600 μm.

 The haploid strain VKPM F-681 has dense granules.

 The results of the test on a diluted molasses medium are presented in table 2 in example 1.

 As can be seen from the table, the highest yield of citric acid from molasses sugar was obtained in the diploid recombinant strain VKPM F-817, it was 87%, while in the original strain 2B-1-10, the yield of citric acid was 78%.

Example 2
The seed culture of mitotic diploid 2B-1-10, diploid recombinant VKPM F-817 and haploid strain VKPM F-681 were tested in a concentrated medium from beet molasses. Seeds for biochemical experiments are prepared according to the method described in example 1.

The preparation of the fermentation medium is characterized by the use of molasses medium containing 130 g / dm ³ sugar in the initial medium. In the fermentation process, additional refills of molasses medium are not carried out. The remaining test conditions are similar to example 1.

 The end of the experiment produced on the 4th day of fermentation.

 The results of the verification of diploid cultures — mitotic diploid 2B-1-10, recombinant diploid VKPM F-817 and haploid strain VKPM F-681 are presented in Table 2 of Example 2.

 In a diploid recombinant strain, the yield of citric acid from sugar reaches 66%, in the initial diploid strain, 59%.

Example 3
For fermentation of the sucrose-mineral medium, the seed of the VKPM strain F-817 is grown according to the method described in example 1.

 Productivity is determined by the method of submerged culture on a rocking chair AVU-50r.

Fermentation is carried out in two stages. In the first stage, seed mycelium (granules) is grown on a composition medium (g / dm ³ ): sugar 50, beet molasses 17, ammonium nitrate 2.5, magnesium sulfate heptahydrate 0.25, potassium dihydroorthophosphate 0.16, pH 6.9. Inoculum mycelium was grown for 2 days.

In the second stage of fermentation, a medium of the same composition is used as for growing mycelium granules, but with a sugar content increased to 150 g / dm ³ . In 55 cm ^{3 of} fermentation medium 10 cm ^{3 of} nutrient medium with mycelial granules is added.

 The duration of fermentation is 5 days. The experimental results are presented in Table 2 of Example 3. As can be seen from the data in Table 2, the recombinant VKPM F-817 dominates from two diploid cultures. The yield of citric acid from sugar in diploid 2B-1-10 is 94.8%, in the recombinant 95.6%. The more abundant growth of mycelium in the recombinant strain does not reduce the yield of citric acid and creates greater resistance to changing external factors (relative humidity, temperature fluctuations, quality of raw materials).

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

 2. T. A. Nikiforova, E. Ya. Shcherbakova. Selection and biological properties of mitotic diploid Asp. niger / J. Storage and processing of agricultural raw materials, 1997, 12, pp. 51-53.

Claims (1)

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

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