DD232309C2 - PROCESS FOR THE PRODUCTION OF CITRONIC ACID BY YEAST - Google Patents

Description

1. Morphological properties

a) Colonies (malt agar, 48 hours) Size: 2 mm

Color: cream Shape: round-irregular Shape cross-section: terraced Edge: wavy, filamentous by mycelium Surface: dull and rough (mycoides growth) Consistency: buttery later stages:

- on Malagar shaped cross-section: raised surface: hairy and beaded in the center

- onglucoseagar Shape cross-section: raised surface: beaded

b) cells

Size: 3-4 um χ 5-8 um, elongated up to 20 um and longer Form: ovoid or elongated Pseudomycelium: yes septate hyphae: yes

2. Physiological parameter

a) Assimilation of the substrates

D-galactose variable L-sorbose + positive D-ribose variable L-arabinose - negative D-arabinose - negative L-rhamnose - negative sucrose - negative maltose - negative trehalose - negative cellobiose - negative salicin - negative arbutin - negative melibiose - negative lactose - negative raffinose - negative melezitose - negative inulin - negative Strength - negative erythritol + positive Ribit - negative galactitol - negative D-glucitol variable m-inositol - negative gluconolactone + positive DL-lactate variable citrate variable without vitamins - negative b) fermentation - negative

In the following examples, the invention is explained in more detail:

example 1

61 medium of the following composition

NH ₄ CI 3.0 g / l

KH ₂ PO ₄ 0.7 g / l

MgSO ₄ 0.35 g / l

CUSO ₄ .5 H ₂ O 0.001 g / l

MnSO ₄ .4H ₂ O 0.015 g / l

ZnCl ₂ 0.01 g / l

Glucose 75.0 g / l

filled with tap water, were at 120 ° C for 30 min. sterilized. After cooling, 6 mg / l of thiamine hydrochloride (dissolved in 10 ml of distilled water and cold sterilized by filtration) and 300 ml of a culture of the yeast Saccharomycopsis lipolytica H 181 ZIMET 43720 grown for 24 hours were added as inoculum.

Medium for inoculum :.

NH ₄ CI 3.0 g / l

KH ₂ PO ₄ 0.7 g / l

MgSO ₄ 0.35 g / l

CuSO ₄ .5 H ₂ O 0.001 g / l

MnSO ₄ .4H ₂ O 0.015 g / l

ZnCl ₂ 0.01 g / l

COSO ₄ .7 H ₂ O 0.004 g / l

H ₃ BO ₃ 0.04 g / l

Yeast extract 2.0 g / l

Glucose 20.0 g / l

solved using tap water.

The fermentation was carried out in a 101 stirred fermentor with 41 filling volumes under sterile conditions. In each case after 50 and 80 hours, a total of 500 g of glucose were added in the form of a sterile 75% solution.

Fermentation conditions:

Temperature 30 ± 1 ⁰ C, air volume 200 l / h, agitation 188 rpm, defoamer addition, pH regulation during the growth phase to pH 4.5 with 20% NaOH, pH regulation during production phase to pH 4.0 with 40% NaOH.

After 144 hours, the fermentation was stopped. The concentration of citric acid and isocitric acid in the fermentation solution was 100 g / l. The proportion of isocitric acid was 6%. After taking into account the volume correction (volume increase by Lauge- and Substratnachdosierung) resulted in a production capacity corresponding to a final concentration of 140g / l acid.

Example 2

In each case 50 ml of a mineral salt medium according to Example 1 were placed in a toothed shake flask, admixed with 4 g / l CaCO ₃ and 6 ml paraffin paraffin (chain lengths Сщ-Сго., Preferably Cir-Cie) and at 121 ° C for 20 min. autoclaved. After cooling, 0.05 mg of thiamine hydrochloride in the form of a sterile aqueous solution and 5 ml of inoculum, which was cultivated as described in Example 1, but containing 20 g / l paraffin instead of glucose, were added to the flask.

The flask was shaken at 30 ° C on a rotary shaker for 90 hours. The pH was maintained between 3.0-6.5 by CaCO ₃ addition. After completion of the fermentation, the content of citric acid was 57 g / l. The isocitric acid content was 2.3 g / l.

Example 3

The fermentation was carried out as in Example 2, but instead of the paraffin, 5 g of triglyceride in the form of animal fat were added to lard. Cultivation of the inoculum was carried out as in Example 1, but instead of the glucose, 25 g / l lard was added. After a shaking period of 116 hours, the concentration of citric acid and isocitric acid was 65 g / l. The isocitric acid content was 12%.

Example 4

Stability test and semi-continuous fermentation

According to Example 2, a fermentation was carried out for 4 days, then 9/10 of the volume were filled under aseptic conditions and then fermented for 4 days. This procedure was repeated 10 times.

The following results were achieved:

Final concentration of citric acid + isocitric acid (g / l)

Sentence 1 55 5

Sentence 10 54 6

It is within the meaning of this invention to carry out the fermentation under batchwise, semi-continuous and continuous conditions and to effect an increase in the product formation rate by increasing the biomass concentration.

Match 5

The cultivations were carried out according to Example 1 both with glucose and with paraffin (150 g / l). For inoculation, however, was a vaccination of the invention described strain from a stock, in which the strain was kept for 51 months by the following method.

Slant culture (medium composition as given in Example 1 for the inoculum). Vaccination two to three weeks. The results with regard to the achieved final concentration of total acid, as well as the proportion of isocitric acid, corresponded to those which were achieved at the beginning of the stocking:

Glucose 150 g / l, duration 110 h, total acid 84 g / l, of which 3.5 g / l isocitric acid.

Paraffin 150g / l, duration 110h, total acid 120g / l, of which 8g / l isocitric acid.

Example 6

According to Example 2, a fermentation with a mixed substrate was carried out by predetermining simultaneously with 6 ml of paraffin 2y-sriucose per 50 ml of mineral salt medium. After 90 hours fermentation time, the content of citric acid was 77 g / l. The isocitric acid content was 5.6 g / l.

Claims (1)

Invention claim: A process for the production of citric acid by yeast, characterized in that the acid-tolerant yeast strain Saccharomycopsis lipolytica H 181 ZIMET 43720 at a pH of <6.5, preferably 3.0 to 5.0 and a temperature of <40 ⁰ C, preferably 28 to 36 ⁰ C, cultured under aerobic, submerged conditions in a known manner. Field of application of the invention The invention relates to a process for the production of citric acid by yeasts under aerobic, submerged conditions in media with different carbon sources. Characteristic of the known technical solutions Processes for the microbial recovery of citric acid have been practiced for decades. In this case, a strain of Aspergillus is used predominantly and as fermentation process, the shell method - also called surface process - applied. A particular advantage of this method is that the fermentation in the production phase is autosterly, since the Aspergillus cultures can carry out the production of citric acid even at pH values between 2 and 3. The submerged method, which works with more favorable space-time yields, could be used because of the more sensitive strains selected for submerged fermentation (Röhr, M. et al .: An improved method for characterization of citrate production by Conidiaof Aspergillus niger, BIOTECHN. LETTERS 1 282 286 (1979). and the higher energy requirement (Schierholt, J .: Fermentation Processes for the Production of Citric Acid, PROCESS BIOCHEMISTRY 11, 20-21 (1977) do not completely displace the surface process.) In addition, the raw material molasses must be treated with regard to heavy metal content fungus Aspergillus niger, that it can only produce citric acid in large quantities from hexose- or hexose-containing substrates such as glucose, invert sugar or sucrose In recent decades, numerous patents and academic publications have appeared on the use of yeasts as citric acid producers (summary literature: Suzuki, T. akeo: Production of organic acids, HAKKO TO KOGYO 35 (6) 459-71 (1977) and Stottmeister, U. and. a. The use of paraffins and other non-carbon hydrate sources for microbial citric acid synthesis, Z. Allgemein. Microbiol. 22 (6) 399-424 (1982). The advantage of the yeasts is their ability to grow submerged without complication and to form citric acid from a variety of assimilable carbon sources such as glucose, invert sugars, fats, oils, n-alkanes, acetic acid and ethanol. The disadvantage is that depending on the carbon source and other parameters of the fermentation more or less isocitric acid is excreted. Thus, about 10-20% of the total acid is excreted as isocitric acid by the strain Candida (Saccharomycopsis) lipolytica EH 59 ZIMET H134 with glucose as the C source and about 50% of the total acid with n-alkanes as the C source. Similarly, almost all citric acid described in the literature behave as secreting yeast wild strains. The performance of these strains are very stable, the acid concentrations reached high (in summary in: Stottmeister, U. et al .: The use of paraffins and other non-carbon hydrate sources ..., Z. General.Microbiol. 22 (6) 399-424 (1982). However, the isocitrate content is disadvantageous in the conventional way of working up by calcium precipitation A citric acid-isocitric acid mixture is difficult to crystallize In some technical applications of citric acid, a proportion of isocitric acid is undesirable due to the poorer complexing properties of the latter Citric acid leaving strains of interest, which achieve a very high proportion of citric acid in the total acid, have now succeeded in altering strains that separate citric acid / isocitric acid in such a way that, especially when using n-alkanes as the C source, almost only ci excreted. In the statements to be found in the literature about the strains with the specific power to form almost only citric acid, information about the stability and constancy of this performance is lacking. Many mutants are well known to undergo uncontrollable back mutations. The pathway through the mutation followed by selection to obtain strains with specific benefits involves a great deal of work and the success rate of such work is uncertain. The disadvantages of the previous methods are the use of Aspergillus sp. in the additional expenses for submerged fermentation and in the utilization of yeasts, that strains with a specific performance are obtained mutatively and that information about the stability of the strains thus obtained is lacking. Furthermore, most of the mutants still have the distinct characteristic of forming isocitric acid (e.g., DE-OS 2264763 Benckiser GmbH 1972). Object of the invention The object of the invention is to substantially accumulate only citric acid by a fermentation process in large quantities and with high productivity with a yeast from different carbon sources. Essence of the invention The invention has for its object to select a yeast strain that is able to produce in an aerobic, submersed fermentation process, predominantly citric acid. According to the invention the object is achieved in that the yeast strain Saccharomycopsis Lipolytica H 181 ZIMET43720 in known media at a pH of <6.5, preferably at 3.0 to 5.0 and at a temperature <40 ⁰ C, preferably at 28 cultured to 36 ⁰ C. The strain is characterized by the fact that it assimilable carbon sources such as. As glucose, glucose-containing hydrolysates, invert sugar, η-paraffins, triglycerides such as lard, beef tallow, foot oil, etc., ethanol, glycerol, acetic acid or acetate in the presence of a mineral nutrient solution and growth substances (such as Hefeautolysat, thiamine) grows and with Stable performance large amounts of citric acid with a very low isocitric acid content precipitates. Another essential feature of the strain is that it uses different C sources such. B. η-paraffin and glucose can be used simultaneously in a mixture or that you use different C sources at different times, such. For the growth phase paraffin and for the product formation phase glucose or vice versa. After the exhaustion of the nitrogen source of the mineral salt medium, which is associated with an adjustment of replicatorischen growth, in addition to a Reservestoff formation, the citric acid excretion. The strain retains its ability to form citric acid at a high rate at achievable high final concentrations and low isocitric acid content, unchanged over a long period of time.
Subsequently, the new strain is characterized.