SU412247A1 - - Google Patents

Description

one

This invention relates to a method for producing itaconic acid by means of microorganisms used to make polyacrylonitrileotope nitron fiber.

A method of producing itaconic acid is known, which involves deep cultivation of an inoculum — a culture of Aspergillus lerreus — an acid producer on a nutrient medium containing carbohydrates, for example, food sugar, raw sugar, molasses, corn extract, essential mineral salts, basic fermentation on the same nutrient medium the subsequent production of itaconic acid crystals from the fermentation solution (culture liquid), the clarification of the solutions with activated carbon, filtration, evaporation and crystallization oi

The purpose of the invention is to increase the yield of itaconic acid.

This is achieved by the fact that from the terreus species the strain G-232 is used, and the growth of the seed material is carried out on a nutrient medium containing predominantly 3% carbohydrate at a pH of 2.3-2.5.

The main fermentation is carried out at a pH of 5.5.5, while during the main fermentation after the growth of the mycelium, the medium is fed twice (through

two and four days) to the concentration of carbohydrate in the environment of 90-100 g / l.

The proposed strain Aspergillus terreus G-232 is obtained by treating the natural strain 718/9 Aspergillus terreus with ultraviolet rays and ethyleneimine. Cultural properties of the strain and growth pattern were studied on nutrient media used for the cultivation of Aspergillus terreus — MoyerCoghil modified medium, Chapek-Dox medium, wort agar (malt medium), potatoes, and special media used to produce itaconic acid.

The structure of a giant colony on the fifth day was studied on a modified Moyer – Coghil medium.

This strain has the following characteristics.

1. Morphological properties. Moyer-Koghil modified environment. The composition of the medium, g:

Lactose10

Sugar (sand) 5

Calcium phosphate monosubstituted (KIGRO) 0,06

Magnesium sulphate

(MgSO4) 0.05

Kalny chloride (KC1) 0.1 Sodium chloride (NaCl) Potassium nitrate (KNOa) Corn extract Agar Water up to 1 l. In the three-day culture, the conidial heads are slightly elongated, 27–54 microns in diameter. Bubbles (visicules) of pear-shaped form, 12-15 microns in diameter. 2/3 of the visicles are covered with sterigmas. Double-layer sterigma. First-order sterigm length is 5 microns, the second is 4 microns. Conidia are smooth, round, their average diameter is 2.2 microns. The length of the conidium is 60-105 microns, the diameter is 3-4 microns. 2. Cultural properties. The daily giant colony of strain G-232, enriched with a modified medium de Moyer-Coghil at 32 ° C, has a circular shape with a diameter of 4.9 cm. The colony edge is even, the substrate mycelium is thin, smooth at the beginning of development is colorless, then light brown colors. Koiidiophores iizkie. The surface is covered with conidial sand-colored heads. In the center of the colony there is a thick condition of 0.5 cm in diameter. The outer side of the colony is 0.3 cm wide and is asporogenic. The duration of conidia maturation is 12-14 days at 29-30 ° C. The modified environment of Čapek-Doksa. The composition of the medium, g: Sucrose30 Sodium nitrate (NaNOs) 2.0 Phosphate potassium disubstituted (K2HPO4) 1.0 Magnesium sulphate (MgS04 7 NaO) 0.5 Potassium chloride (KC1) 0.5 Ferrous sulphate (FeS04 - 7 НЗО) 0 , 01 Agar20 l Vodado 1 l. Substrate mycelium of medium thickness, dense, folded. At the beginning of development it is colorless, then brown with a slightly yellowish tinge, in the substrate forms a black-brown pigment. There is aerial mycelium. Conidia sand color. Wort agar (malt medium). Substrate mycelium thin, colorless. Aerial mycelium high. Conidial heads are formed at a considerable distance from one another. J kaptofeliy slice. Substrate mycelium of medium thickness, dense, pale yellow, slightly folded, sand-colored conidia. There is aerial mycelium. 3. Antagonistic properties. Aspergillus terreus G-232 is a weak antagonist against Bacterium SOI, Bacillus mesentericus. Bacillus megathericus. 4. Biochemical characteristics. The selected strain of Aspergillus terreus G-232 well ferments sucrose in the deep mode of cultivation. The activity of the biosynthesis of itacoic acid strain Aspergillus terreus G-232 on different nutrient media is given in the table. The method is as follows. Growing seed is carried out in seed fermentors on nutrient media with a 3% carbohydrate content, pH of the medium is 5.0-5.5. The nutrient medium is sown with a suspension of dry conidia at the rate of 0.7 g of conidia per 1 m of medium. The duration of the growing cycle at: 34-36 ° C is 130-36 hours. Then, seed in an amount of 6-10% by volume is transferred to the basic medium (carbohydrate concentration 6-7%, pH 2.3-2.5) for biosynthesis. The duration of the main fermentation cycle is 60-72 hours. When growing seed and main fermentation, the aeration mode consists of the following ratios: 0.5 volume of air per 1 volume of nutrient medium in lag phase and 0.8 volume of air per; 1 volume of nutrient medium in exponential and stationary phases of growth. The fermentation cycle is completed when the content of carbohydrate unassimilated in the medium is 0.1-0.2%. After the fermentation cycle is completed, the mycelium is separated by filtration from the culture fluid. The resulting solution and washings from the mycelium are clarified with activated charcoal, filtered and evaporated to a concentration of itaconic acid of 35-38%. One stripped off solution is crystallized and get "technical crystals with the content of itaconic acid up to 96%. Duplex crystallization is required to produce a pure reactive acid. The yield of crystalline itaconic acid is 80-85% of itaconic acid in enzyme solutions. The following is illustrated by examples. Example 1. Growing seed crops are carried out in a medium of the following composition, l: Food sugar or raw sugar 30.0 Magnesium sulphate 3.0 Ammonium nitrate 2.7

Corn extract:

for sugar 6,0

for raw sugar 3.0

pH of 5.0-5.5.

(The growing process is carried out at a temperature of 34-36 ° C for 30-36 hours. The seed in the amount of 5-10% by volume, taken at the end of the exponential growth phase and containing 22-24 g / l dry mycelium, translated t to basic medium for biosynthesis.

Medium for biosynthesis contains, g / l: Food sugar or raw sugar 70-80.0 Magnesium sulphate 3.0

Ammonium nitrate2.0

Corn extract: for sugar environment2,3

for raw sugar, add

pH of 2.3-2.6.

After seeding, the medium contains 2-3 g / l of dry mycelium. The biosynthesis of itaconic acid is carried out at a temperature of 134-35 ° C for 60-72 hours. After this period, the culture solution contains 30–40 g / l of itaconic acid, 0.1–0.2% sugar unassimilated by the fungus, 9–11 g / l of dry mycelium.

After filtering the mycelium, the solutions are sent for chemical processing.

Example 2. Molasses medium for growing seed is characterized by the following indicators, g / l:

Sugar molasses 30,0

Ammonium Nitrate1-2

RP 5.0-5.5.

The growing conditions for the inoculum are similar to those in Example 1.

The medium for the main fermentation contains, g / l:

Sugar molasses 30-35

Ammonium Nitrate1-1.5

pH of 2.3-2.5.

A characteristic feature of itaconic acid biosynthesis on molasses media is

feeding submerged culture during the cycle.

Usually, two supplements are made. The first - on the second day after the growth of the mycelium, the second - on the fourth day. In both cases, in order to increase the yield of itaconic acid, the sugar concentration is adjusted in the medium to 9-10%. Biosynthesis is carried out at a temperature of 34-35 ° C. The duration of the whole fermentation cycle is 6-7 days.

At the end of the fermentation cycle, the culture solution contains 40-50 g / l of itacanoic acid.

Subject invention

Claims (3)

1. A method for producing itaconic acid, which involves the in-depth cultivation of seed - the culture of Aspergillus terreus - a producer of acid on a nutrient medium containing carbohydrates, such as edible sugar, raw sugar, molasses, corn extract, essential mineral salts, basic fermentation on the same nutrient medium followed by obtaining crystals of itaconic acid from the fermentation solution (culture liquid), clarification of the solutions with activated carbon, filtration, evaporation and crystallization, characterized in that, in order to increase the yield of acid, terraus is used from the G-232 strainer, and the seed is grown on a nutrient medium containing predominantly 3% carbohydrate at a pH of 2.3- 2.5 2. A method according to claim 1, characterized in that the main fermentation is carried out at an RP of 5-5.5. 3. The method according to paragraph.n. 1-2, characterized in that during the main fermentation after the growth of the mycelium, the medium is fed twice - two and four days before the concentration of carbohydrate in the medium 90-100 g / l.