SU695230A1 - Process for producing citric acid - Google Patents

Description

The invention relates to the microbiological industry, and in particular to a method for producing citric acid.

Citric acid is obtained mainly by culturing specially selected 5 strains of molds on media containing carbohydrates (sucrose, molasses, starch, etc.) as the main carbon source.

There is a method of producing citric acid by cultivating the producing yeast of the species Candida lipolytica under conditions of aeration on a nutrient medium containing normal paraffins as a carbon source, nitrogen source 15, mineral salts, followed by isolation of the target product from the culture fluid [1].

However, the known method provides a sufficiently high yield of citric acid - 20 lots.

The aim of the invention is to increase the concentration and yield of citric acid.

The goal is achieved by the fact that Candida lipolytica strain Candida lipolytica HMM-187 is used, while cultivation is carried out at a concentration of producer cells of 10-30 g / l dry matter, pH 5.5-6.5 and an oxygen concentration of 50– 80% saturation.

LEMONIC ACID

Characterization of Candida lipolytica HMM-187 strain.

The strain of Candida lipolytica HMM-187 was obtained by treating Candida lipolytica IBPM u-160 (704) cells with nitrosomethylurea and subsequent selection.

The mutant strain is stored in the Bioenergy Division of the IBPM Academy of Sciences of the USSR.

The strain of Candida lipolytica NMM-187 has the following characteristics.

Young culture on liquid and agarized wort is represented by round, oval and slightly elongated cells measuring 2.0-5.0-3.0-10.0 microns in size. Strongly elongated cells 2.4–11.0–16.0 microns in size, as well as chains of

3-4 cells.

When growing on liquid wort at 25 ° C on the eighth day, a thin film forms on the surface of the medium and a small dense precipitate at the bottom of the tube. On the 15-16th day the film becomes thicker, the precipitate increases.

The wort-agar colonies (10 days) are whitish-cream, smooth, dull, convex, the center of the colony is slightly raised, the edges are even. A giant cream-colored colony along the edges is yellowish-green, slightly wrinkled, flat, the center is raised, the edges are slightly lobed,

The growth on the line is flat, smooth, matte, cream-colored, the edges are even.

Culture on glass on potato agar: pseudomycelium is poorly developed. Strict aerob. Sugar does not ferment. Assimilates glucose, ethanol, acetate, glycerin. Weak growth on galactose.

Does not assimilate lactose, maltose, sucrose, raffinose, arabinose, xylose, starch.

Uses (NH ₄ ) ₂ SO ₄ and NH ₄ CI as a nitrogen source.

It does not grow on nitrates.

Milk peptones completely.

The optimum growth temperature is 26-30 ° C, and the maximum is 33 ° C.

It is stored on an agar medium Reader with paraffin and in ampoules in a lyophilized state.

The described strain of Candida lipolytica HMM-187 is characterized by the ability to produce predominantly one citric acid upon growth on media with n-alkanes; isolimonic acid practically does not accumulate.

To assess the stability of the Candida lipolytica HMM-187 mutant, it was subcultured for 4 months on fresh medium with paraffin (cultivation in flasks on a rocking chair); a total of 40 consecutive passages were performed. During frequent reseeding, the culture not only does not lose the ability to produce predominantly citrate, but the ratio of citrate: isocytrate in the medium shifts even more towards the predominant accumulation of citrate (from 8: 1 to 12: 1).

Storage of the mutant strain for 4 years on an agar medium with paraffin also does not impair the properties of this culture.

The mutant strain Candida lipolytica HMM-187 in all tests shows the stability of the main practically important property - the predominant synthesis of citrate on media with α-alkanes.

Example 1. The yeast Candida lipolytica HMM-187 is supported on an agarized mineral medium Reader with normal paraffins (Ci ₂ —C ₁₉ ), having the following composition, g / l:

Normal paraffins 10.0 (C12-C ₁₉₎ (ΝΗ ₄ ) ₂ Ο ₄ 3.0 MgSO ₄ -7H ₂ O 0.7 NaCl 0.5 Ca (NO ₃ ) ₂ 0.4 KN ₂ RO ₄ 1,0 K ₂ NRA ₄ 0.1 Yeast autolysate 2.0 ml Trace elements Burkholder * Agar agar 25.0

* A set of trace elements according to Burkholder includes the following components (mg / l of medium): KJ 0.1; B 0.01; MP ² + 0.01;

Zn ² + 0.01; Sc ² + 0.01; Mo ⁶ + 0.01; Fe ² + 0.05.

Yeast is grown on a joint for 3 days at 28. — 30 ° C. The cell suspension from the agar medium is transferred to 750 ml flasks containing 50 ml of seed medium.

The composition of the seed medium, g / l:

Normal paraffins 48.0 (NH ₄ ) ₂ SO ₄ 3.0 MgSO ₄ -7H ₂ O 0.7 NaCl 0.5 Ca (NO3) 2 0.4 KN ₂ RO ₄ 1,0 K2NRO4 0.1 Yeast autolysate 4.0 Trace elements over Burkholder.

After 48 hours of incubation on a rocking chair, the resulting seed culture was transferred to a new batch of flasks with the seed medium, inoculum volume 6% of the volume of the medium.

The second batch of flasks is also placed on a rocking chair at 28-30 ° C; during cultivation, which lasted 50 hours, the pH of the medium is maintained at 6.0-6.5 by the introduction of a sterile 10% solution of KaOH.

500 ml of the grown culture is transferred

into the fermentation medium. 3.5 liters Volume of medium The composition of the fermentation Wednesday, g: Normal paraffins 600 (C12-C ₁₉₎ (NH ₄ ) ₂ SO ₄ 20,0 MgSO ₄ -7H ₂ O 2.8 g NaCl 1,5 Ca (NO ₃ ) ₂ 1,6 KH ₂ PO ₄ 4.0 K ₂ HPO ₄ 0.4 Yeast autolysate 8.0 ml Fermentation is carried out in fermentation nom apparatus ANKUM-2 10 liters

The process is carried out at 29-30 ° С under conditions of intensive mixing and aeration of the medium with air. The oxygen concentration in the medium is supported by 75–85% of saturation.

The pH of the medium is regulated by the introduction of NaOH solution at the level of 6.0-6.5.

The maximum concentration of producer cells on the third day of fermentation reaches 13 g / L.

After 144 hours of cultivation, a concentration of citric acid of 217 g / L is reached, at the same time isolimonic acid accumulates in the medium, its concentration of 6 g / L.

In the apparatus, the total content of anhydrous citric acid is 868 g or 949.2 g of monohydrate. The fermentation yield of anhydrous citric acid by weight is 145% and 158% of the added paraffins monohydrate.

Example 2. Seed (10% of the volume of medium), prepared analogously to example 1, propagated sequentially in fermenters with a capacity of 63 l and 400 l on a medium of the following composition, g / l: 5

Normal paraffins 48 (С12 — Сю)

(NH ₄ ) ₂ SO ₄ 6.0 MgSO ₄ -7H ₂ O 0.7 NaCl 0.5 Ca (NO ₃ ) ₂ 0.4 KH ₂ PO ₄ 1,0 K ₂ HPO, 0.1 Yeast autolysate 2.0 Trace elements over Burkholder.

The concentration of producer cells (by dry weight) reaches 30 g / l. The duration of the process in each fermenter is 20 48 hours at a temperature of 28-30 ° C and a pH of 5.0-5.5.

The main fermentation is carried out in a 3000 L fermenter. The fill factor of the apparatus is 0.6. Seed is introduced into the fermenter in an amount of 25% of the medium volume.

The composition of the fermentation medium, g / l:

Normal paraffins 100 (Ci ₂ —C ₁₉ ) (NH ₄ ) ₂ SO ₄ 5,0 MgSO ₄ -7H ₂ O 1.4 NaCl 0.5 Ca (NO ₃ ) ₂ 0.8 KH ₂ PO ₄ 2.0 K ₂ HPO ₄ 0.2 Yeast autolysate 4.0 Trace elements

over Burkholder.

Fermentation is carried out at 28-30 ° C. 40 pH of the medium is maintained at a level of 5.6-6.0 by the introduction of a sterile 20% NaOH solution.

The oxygen concentration in the fermentation medium was maintained on the first day 45-50-60%, the second or fourth day increased to 80% and on the fifth day reduced to 50%.

During the fermentation process on. on the second or fourth day, with an interval of one hour, normal paraffins are added to the fermentation medium; each portion was 2% of the medium volume.

The maximum concentration of producer cells on the second or third day of fermentation is 20 g / L.

The duration of the main fermentation is 120 hours

By the end of fermentation, the total content of citric acid in the apparatus is 368 kg. The fermentation yield of anhydrous acid is 128% by weight of the added paraffins.

Using the proposed method allows to establish the production of citric acid from non-food raw materials - normal paraffins - with a higher yield than by known methods. The cost of citric acid obtained by this method is significantly lower than the cost of this acid obtained from molasses.

Claims (2)

The growth along the stroke is flat, smooth, dull, cream-colored, the edge is even. Culture on glass on potato agar: pseudomycelium is poorly developed. Strong aerobic. Sahara does not ferment. Assimilates glucose, ethanol, acetate, glycerin. Weak growth on galactose. Does not assimilate lactose, maltose sucrose, raffinose, arabinose, xylose starch. Uses (NH4) 2S04 and NH4CI as a nitrogen source. On nitrates is not growing. Milk is very strong. The optimum growth temperature is 26-30 ° C, maximum - 33 ° C. Stored on agar medium Reader with paraffin and in ampoules in a diophilized state. The described M.M.M.-187 Candida lipolytica strain is characterized by the ability to produce predominantly one citric acid on medium with m-alkanes, but the single-monoacid acid practically does not accumulate. To assess the stability of the Candida lipolytica mutant HMM-187, it is subcultured for 4 months on fresh paraffin-containing medium (cultured in flasks on a rocking chair), in general, it has 40 consecutive passages. During frequent reseeding, the culture not only does not lose the ability to produce mainly citrate, but the ratio of cyTraT: isocitrate in the medium shifts further towards the predominant accumulation of citrate (from 8: 1 to 12: 1). Storage of the mutant strain for 4 years on agar medium with paraffin also does not impair the properties of this culture. In all tests, the mutant strain of Candida lipolytica HMM-187 reveals the stability of the main practically important property — preferential synthesis of citrate on media with K-alkanes. Example 1. Candida lipolytica HMM-187 yeast is maintained on Reader's agar mineral medium with normal paraffins (Ci2-Cig), having the following composition, g / l: Normal paraffins 10.0 (Ci2C e) (NH4) 2043.0 MgSO4-7H2O0, 7 NaCl0.5 Ca (UOz) 20.4 KH2PO41.0 K2HRO40.1 Yeast autolysate 2.0 ml Burkholder trace element set Agar-Agar Burkholder trace element set includes the following components (mg / l of medium): KJ 0.1; B 0.01; Mp 0.01; Zn2 + 0.01; 0.01; Mo 0.01; Fe2 + 0.05. The yeast is grown on a spit for 3 days at 28 .. The cell suspension from the agar medium is transferred to 750 ml flasks containing 50 ml of the inoculum. The composition of the inoculum, g / l: Normal paraffins 48.0 (NH4) 2S043.0 MgS04-7H200.7 NaCl0.5 Ca (NO) 20.4 KH2P041.0 K2HP040.1 Burnt-neck microelement 4.0 ml A set of trace elements according to Burkholder. After 48 h of incubation on a rocking chair, the obtained seed culture is transferred to a new batch of flasks with a seed medium, the inoculum volume is 6% of the volume of the medium. The second batch of flasks is placed on a rocking chair at 28-30 ° C; during cultivation, which lasted 50 hours, the pH of the medium is maintained at 6.0-6.5 by the introduction of a sterile 10% solution of NaOH. 500 ml of the grown culture is transferred to a fermentation medium. Volume - 3.5 liters. The composition of the fermentation medium, g: Normal paraffin 1 600 (Ci2-Cig) (NH4) 2S0420.0 MgS04-7H2O2.8 g NaCl1.5 Ca (NOC) 21.6 KH2PO44.0 K2HP040.4 Yeast autolysate 8.0 ml Fermentation wire t in fermetation apparatus ANKUM-2 volume of 10 liters. The process is carried out at 29-30 ° C in the words of intensive mixing and aeration of the medium with air. The concentration of the acid in the medium is maintained at 75–85% saturation. pH is regulated by the introduction of iNaOH solution at the level of 6,, 5. The maximum concentration of procent cells on the third day of fermentation is 13 g / l. After 144 hours of cultivation, the concentration of citric acid is 217 g / l, and at the same time isoimonic acid accumulates in the medium, its concentration is 6 g / l. In the apparatus, the total content of citric acid anhydrous is 868 g or monohydra 949.2 g. The fermentation yield with anhydrous citric acid is 45% by weight and 158% monohydrate from the added arafins. Example 2. Seed material i, iu7o from the volume of the medium), prepared similarly to example 1, is kneaded successively in fermenters with a capacity of 63 liters and 400 liters on the medium of the following composition, g / l: Normal paraffins48 (0) 2Cjg) (IP4) 25046.0 MgSOi-THaO0.7 NaCl0.5 Ca (MO3) 20.4 KP2P041.0 K2HP040.1 Yeast autolysate2.0 Burkgolder trace elements. The concentrate, and the producer cells (dry weight) reaches 30 g / l. The duration of the process in each fermenter 48 hours at a temperature of 28-30 ° C and pH 5.0-5.5. The main fermentation is carried out in a 3000 liter fermenter. The fill factor of the apparatus is 0.6. The seed is introduced into the fermenter in an amount of 10% of the volume of the medium. The composition of the fermentation medium, g / l: Normal paraffins (Ci2-Cig) (NH4) 2S04 MgS04-7H2O Ca (NY) 3 KH2P04 K2HPO4 Yeast autolysate Burkholder trace elements. Fermentation is carried out at 28-30 ° C. pH is maintained at the level of 5.6-6.0 by the introduction of sterile 20% aqueous solution of NaOH. The oxygen concentration in the fermentation medium was maintained on the first day by 50–60%, the second to fourth days was increased to 80%, and on the fifth day it was reduced to 50%. During the fermentation process on. the second to fourth, with an interval of 24 hours, normal paraffins are added to the fermeite medium, each portion being 2% of the volume of the medium. The maximum concentration of producer cells on the second or third day of fermentation is 20 g / l. The duration of the main fermentation is 120 hours. By the end of the fermentation, the total content of citric acid in the apparatus is 368 kg. The fermentation yield of anhydrous acid is 128% by weight of paraffin added. The use of the addition method allows the production of citric acid from non-edible raw materials — normal paraffins — with a higher yield than by known methods. The cost of citric acid obtained by this method is significantly lower than the cost of this acid, obtained from molasses. The invention of the method for producing citric acid by cultivating the yeast Candida lipolytica species producing it under aeration conditions on a nutrient medium containing normal paraffins as a carbon source, a source of nitrogen, mineral salts, followed by separation of the target product from the culture fluid, characterized in that In order to increase the yield of citric acid from Candida lipolytica species, Candida lipolytica NMM-187 is used, while the culture of the producer cells is 10-30% for dry matter, pH 5, 5-6.5 and an oxygen concentration of 50-80% saturation. Sources of information taken into account during the examination 1. USSR Patent No. 448652, cl. C 12D 1/04, pub. 1974.