RU2080372C1 - Strain of fungus aspergillus niger - a producer of citric acid - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: new strain-producer of citric acid, Aspergillus niger VKPM F-678, is obtained from early used strain Aspergillus niger L-1 by method of step-by-step selection. Under conditions of industrial culturing the conversion of sugar to citric acid by this strain is 90.1%. EFFECT: new strain indicated above, increased yield of product. 4 tbl

Description

The invention relates to the microbiological industry and for obtaining a strain of the fungus Aspergillus niger producer of citric acid for deep cultivation on molasses media. The selected strain Aspergillus niger F-678 has a high activity of formation of citric acid during deep cultivation in diluted and sugar-concentrated molasses media, producing up to 10.9 kg / m ³ per day of citric acid. In this case, the yield of citric acid from reducing substances reaches 90.1%
A known strain of the fungus Aspergillus niger CCF-1599, selected by Czechoslovak researchers and capable of deep cultivation on media containing 10 to 20% sugar substrate, synthesizes up to 75% of citric acid from added sugar within 5-7 days of fermentation.

The disadvantage of strain CCF-1599 is its low industrial efficiency [1]
Technical and economic indicators of known strains used in the CIS countries in the production of citric acid by the method of deep cultivation are presented in table. one.

The disadvantage of these strains is the short duration of the period of active acid formation, high consumption of raw materials and low acid yield from sugar [2 4]
The closest to the proposed technical essence and the achieved positive effect is the strain of the fungus Aspergillus niger VKPM F-326 - a producer of citric acid with a high activity of citric acid formation during deep cultivation on molasses diluted in sugar, forming up to 9.9 kg / m ³ per day of citric acid. In this case, the yield from reducing substances is 88.2%. However, the technical and economic indicators of the prototype are inferior to the new strain [4]
The aim of the invention is to obtain a new universal strain having a higher activity for the formation of citric acid both under short cycle conditions and during prolonged fermentation of diluted and sugar-concentrated molasses media.

 A new strain of Aspergillus niger F-678 fungus was obtained by stepwise selection based on mutagenesis using mutagens of chemical and physical nature from the previously used industrial strain Aspergillus niger L-1 with subsequent stabilizing selection of spontaneous variants.

 Strain Asp. niger F-678 is deposited in the All-Union Collection of Industrial Microorganisms (VKPM) VNII Genetics.

 Strain Asp. piger F-678 is characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural and morphological features
After 5 days of cultivation, a new strain on wort agar forms colonies with a diameter of 6.7 cm. Coloring of ivory colonies. Aerial mycelium is poorly developed. Conidia is plentiful. The reverse side of the colony is smooth. The conidial heads are round, with a diameter of 135.4 ± 12.5 microns. Swelling of conidiophores spherical in size (37.5 ± 3.9) • (35.8 ± 3.7) microns. Sterigmas are two-layer. The length of the sterigm I is of the order of 9.1 ± 0.9 μm, of the second 5.9 ± 0.4 μm. The diameter of conidia is 3.9 ± 0.1 μm. The conidia membrane is ivory colored. Conidiophores are straight, colorless, 942 ± 282 μm long and 12.9 ± 0.3 μm wide.

Physiological and biochemical characteristics
Aerobe.

The temperature range of growth is 22 38 ^o C.

The optimum growth temperature is 32 ± 1 ^o C.

 Optimum pH 5.6 7.2.

 Attitude to nitrogen sources: assimilates nitrogen of organic compounds, for example peptone, protein, amino acids, yeast autolysate, urea, and also assimilates nitrogen of mineral salts, preferably ammonium salts, nitrates.

 Attitude to carbon sources; it is well absorbed and grows on maltose, glucose, sucrose, fructose, D-xylose, D-mannose, is less absorbed by dextrin, sorbitol, starch, D-raffinose and grows poorly on lactose.

 It is well stored in the form of dried conidia (up to 5 10% residual moisture) or in the form of a culture grown on beveled wort agar.

The cultivation temperature is 32 ± 1 ^o C.

 The cultivation duration is 7 days.

Storage temperature + 18 ± 1 ^o C.

 Genetic features of prototroph, osmophil.

Strain Asp. niger F-678 identified by the Identifier of fungi of the genus Aspergillus [5]
Strain F-678 differs from the parent strain L-1 by morphological and cultural characteristics. In contrast to the parent strain, it has smaller conidia, sterigma of the first and second orders and swelling of the conidiophores. The conidial membrane is painted in ivory, i.e. practically devoid of pigment. The parent strain has a dark beige color.

In addition, the breeding strain has a high germination energy of conidia, i.e. germination time of conidia of strain F-678 in the wort (7 ^o B for sugar) 5 hours, while the parent strain 7 hours

 During the cultivation of seed mycelium (inoculum) in a selected strain, the accumulation of biomass due to the rapid germination of conidia is more intense, which makes it possible to reduce the inoculum preparation time by 5–7 hours.

The new strain on the first day of the process synthesizes mainly citric acid. So, on the third day in the composition of the synthesized acids, citric acid accounts for 90 percent or more, and for the parent strain L-1 about 80%
The invention is illustrated by the following specific examples of the use of strain Asp. niger F-678.

Example 1. The cultivation of acid-forming mycelium and the synthesis of citric acid was carried out under laboratory conditions on a shaker with a number of swings 160 ± 5 min ^-1 in flasks with a capacity of 700 cm ³ at 32 ± 1 ^o C. To grow seed mycelium, molasses solution with a concentration of sugar 30 was prepared g / dm ^{3 of the} following composition, g / dm ³ :
Molasses 60.0
Sodium Carbonate 0.08
Potassium hexacyanoferroate hydrate 0.14
Ammonium oxalate hydrate (for complete precipitation of calcium and magnesium salts based on CaO) 2.13
Potassium dihydroorthophosphate 0.16
Magnesium Sulfate Hydrate 0.25
Zinc sulfate, hydrate 0.005
Water tap the rest up to 1 dm ³
pH 6.7
For fermentation, molasses medium containing 30 g / dm ³ sugar was prepared according to the recipe described above, but without the addition of magnesium sulfate. The composition of the medium to feed the culture, g / DM ³ :
Molasses 497.0
Potassium hexacyanoferroate hydrate 1.18
Water tap the rest up to 1 dm ³
pH 6.0
The cultivation of seed mycelium was carried out in 50 cm ^{3 of} molasses medium by inoculating it with a homogeneous suspension of conidia at the rate of 0.01 g / m ³ . The volume of the conidial suspension is 10 cm ³ . The cultivation of seed mycelium was 24 hours. The inoculum thus prepared served as seed for inoculation of the fermentation medium. Then in the medium prepared for fermentation, in a volume of 50 cm ³³ , 10 cm ^{3 of} seed mycelium were introduced.

In the fermentation process, the culture was fed with molasses solution containing 250 g / dm ³ sugar in two doses: the first feed after 24 hours of fermentation in an amount of 10 cm ³ , the second after 32 hours of fermentation in an amount of 9 cm ³ . The process was completed after 5 days.

As a control, a well-known production strain F-326 was used. The test results are presented in table 2. Data table. 2 show that the selection strain increases the mass fraction of citric acid in the composition of the synthesized acids by 5.4%; I will remove citric acid by 13.7% and the acid yield from sugar by 7.8%
Example 2. The conditions for conducting and preparing the culture media are the same as in Example 1. But the culture recharge scheme is changed: the first gravy after 24 hours of fermentation in the amount of 10 cm ³ , the second after 30 hours of fermentation in the amount of 9 cm ³ , and then 4 cm ³ on the 5th, 6th and 7th day of fermentation. The process was conducted for 9 days. As a control (prototype) used the same domestic strain F-326. The results of the experiments are presented in table. 2 show that, during long cycles, the selected strain F-678 increases the mass fraction (up to 98.2%) of citric acid in the total amount of synthesized acids. The formation of citric acid with 1 m ³ per day (eat) in a new strain under these conditions increases compared to strain F-326 by 8.4% and its yield from sugar by 5.0%, respectively.

Example 3. The conditions are the same as in example 1. The cultivation of seed mycelium was carried out on molasses media containing 50 g / DM ³ sugar; fermentation in molasses with a sugar content of 130 g / dm ³ without additional feeding of the fungus.

For the cultivation of seed mycelium used molasses medium composition, g / DM ³ :
Molasses 100.0
Sodium Carbonate 0.13
Potassium hexacyanoferroate hydrate 0.23
Ammonium Oxalate Hydrate 3.5
Magnesium Sulfate Hydrate 0.25
Potassium dihydroorthophosphate 0.16
Zinc sulfate, hydrate 0.01
Water tap the rest up to 1 dm ³
pH 6.5.

For fermentation used molasses medium composition, g / DM ³ :
Molasses 260.0
Sodium carbonate 0.35
Potassium hexacyanoferroate hydrate 0.610
Ammonium Oxalate Hydrate 4.6
Potassium dihydroorthophosphate 0.16
Zinc sulfate, hydrate 0.01
Water tap the rest up to 1 dm ³
pH 6.8.

The duration of fermentation is 5 days. Tests have shown that in sugar-concentrated molasses media with a sugar content of 130 g / dm ^{3, the} selected strain forms 11.4% more citric acid than the known strain F-326 (Table 3). At the same time, the acid yield from sugar increases by 6.1%
Example 4. The cultivation of seed mycelium of a new strain and the synthesis of citric acid was carried out by the deep method in a production environment using technology at the St. Petersburg plant. The control was the production strain F-326.

A molasses solution of the same composition was used to prepare a conidial suspension, cultivate seed mycelium, fermentation and subscribe cultures of both strains. Inoculum mycelium was grown in inoculant fermenters with a capacity of 5 m ³ . The volume of molasses medium is 3 m ³ . The sugar content in it is 30 g / dm ³ . After 24 hours, the grown mycelium was transferred to a 50 m ³ fermenter containing 27 m ³ molasses solution with a sugar concentration of 30 g / dm ³ . After 24 hours of fermentation, the fungus was started topping up by adding to the molasses solution containing 225 g / dm ³ sugar, 1 m ³ every 1.5 hours.

On the fourth and fifth days of fermentation, the weaned solution was weaned in an amount of 5 m ³ . Then 2 m ³ water and 5 m ³ molasses solution with a sugar concentration of 225 g / dm ^{3 were} poured into the fermenter. The duration of the process in the experimental cycles averaged 6.72 days, in the control 6.69 days.

Under the production conditions of the selected strain, the mass fraction of citric acid in the synthesized acids on average from three experiments reached 90.9%; the mass of citric acid with 1 m ^{3 of} fermenter per day was 10.93 kg, the specific consumption of 46% molasses for sugar 1 ton of citric acid - 2412 kg, sugar yield 90.13% (table. 4). In the industrial strain F-326, the mass fraction of citric acid in the synthesized acids on average from three experiments was 89.7%, the productivity by weight of citric acid from 1 m ³ per day was 10.21 kg, the specific consumption of 46% molasses per 1 ton citric acid 2821 kg, citric acid yield from sugar 77.05%
Thus, a new selection strain under production conditions forms 7.0% more citric acid with 1 m ³ per day and gives a molasses savings of 14.5 compared to the production strain F-326.

 Therefore, the strain F-678 has a number of advantages: higher productivity in citric acid and its high yield from sugar to 90% or more causes a decrease in the main consumption of raw materials and auxiliary materials; high germination energy of conidia reduces the risk of secondary infection during the fermentation period; the high adaptability of the new strain makes it possible to increase its competitiveness in the world market.

 The economic effect of the use of strain F-678 in the production of citric acid will be 10,355 rubles per 1 ton of citric acid (1993 prices).

Claims (1)

 The strain of the fungus Aspergillus niger VKPM F-678 producer of citric acid.

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