DE2202701C3 - Process for the biotechnological production of citric acid - Google Patents

Description

The invention relates to a process for the biotechnological production of citric acid by cultivation of Cmdida oleophila ATCC 20177 in a pH range from 2 to 8 under aerobic conditions in one Culture fluid containing an assimilable carbon source and common neutralizing agents in conjunction with other inorganic and organic components.

The process is characterized in that η-paraffins with 5-35 carbon atoms are used as the carbon source be used.

In known processes for the biotechnological production of citric acid, which are practical in technology molds, particularly Aspergillus niger, are used and as a carbon source saccharides, especially molasses, are used for the culture medium used in fermentation. In addition, fermentation is generally carried out using surface culture. The submerged door but has recently become more and more practical.

However, the submerged culture of Aspergillus niger presents certain difficulties. So requires the submerse Breeding, for example, requires a lot of technical effort to regulate the fermentation process.

DE-OS 18 12 710 already discloses a process for the biotechnological production of citric acid known from a strain of microbes of the genus Candida in sugar-containing nutrient substrates. Here, Candida oleophila ATCC 20177 - which is also used in the present case - is used as the microorganism is - under aerobic conditions in a culture liquid, the assimilable carbohydrates and common neutralizing agents, such as calcium cafbonate, in conjunction with other inorganic and organic ones Contains ingredients.

The yields obtained in this process

but left a lot to be desired.

Surprisingly, it has now been found that Candida oleophila ATCC 20177 is not just carbohydrates assimilated, but also during cultivation in a η-paraffins containing 5 - 35 carbon atoms Culture medium capable of producing citric acid in large quantities.

This is all the more surprising as the microbial strain used according to the invention, for example ίο Use of kerosene as a source of carbon, which is known to be a mixture of the a wide variety of hydrocarbons including aromatic, isocyclic, and branched hydrocarbons with 10-16 carbon atoms and a boiling point of 175-325 ° C, practically none Forms citric acid

Other Candida species are known from DE-OS 20 02 048 and 20 50 361, the paraffinic hydrocarbons i.a. ferment to citric acid, but this in low yield and after a long period of cultivation.

In contrast to this, citric acid is formed in particularly high levels in the process according to the invention Yields that are even superior to those obtained according to DE-OS 18 12 710.

In the process according to the invention, n-paraffins with 9-20 carbon atoms are preferred Carbon source used.

The culture liquid contains preferably 5 - 20, more preferably 10-15% by volume, based on the culture liquid, n-paraffins.

Those for the taxonomy of the strain of the genus Candida used in the method according to the invention olephila ATCC 20177 characteristic properties are the following:

1) Growth in malt extract

After 3 days of growth at 25 ° C, oval or elongated oval cells were formed, their size (2.5 - 5.0 μ) χ (3.0 - 15.0 μ). On the surface of the culture medium has a heavy, expanded ring or a corresponding skin educated. This ring or this skin is smooth or wrinkled and light brownish cream in color. It will no ascospores. instead a pseudomycelium is formed.

2) Spread culture on malt agar

After 3 days of growth at 25 ° C., the spread culture is smooth and light brownish-cream colored. The shape of the cells is the same as in the case of the mosquito extract. There will be no ascospores, but a pseudomycelium is formed.

3) plate culture

The pseudomycelium develops well on potato agar.

4) Physiological properties

i) optimal growth conditions: pH 5.2;

Temperature 30 ⁰ C; aerobic conditions;

υ) Assimilation of potassium nitrate: none

iii) coagulation of milk: none;

tv) osmophueal property
(in 10% NaCI medium): none;

v) liquefaction of gelatin: none;

vi) vitamin requirement: none;

vii) Formation of carotene pigments: none,

5) Assimilable nitrogen source
Peptone, ammonium sulfate and urea

6) Assimilable carbon source

Glucose +

Galactose +

Sucrose +

Maltose -

Lactose -

Strength -

7) Ethanol as the only carbon source
Good growth

8) Kerosene as the only source of carbon
Good growth

9) fermentations of glucose, galactose, sucrose,
Maltose, and lactose

No

lOjArbutin-Spailung
No

The microbial strain used according to the invention thus differs significantly from other strains of the genus Candida.

A living organism culture is registered with the American Type Culture Collection under registration no. 20177 was deposited on November 13, 1968.

The n-paraffins used as hydrocarbons can be used alone or in a mixture will.

The nitrogen sources which are used in the culture medium used according to the invention include for example ammonia, urea, inorganic or organic ammonium salts such as ammonium chloride, Ammonium sulfate or ammonium acetate, peptone, meat extract, yeast extract, maize steeping water, Fish meal, a processed product of the same, soybean cake or a processed product the same and the like more. These nitrogen sources can be used alone or in admixture.

Optionally, an inorganic material such as a phosphate, a magnesium salt or a manganese salt, and a vitamin such as thiamine or nicotinic acid can be incorporated into the culture medium.

To prevent the pH, value of the culture medium fluctuates, it is advisable to add calcium carbonate or sodium carbonate to the culture medium beforehand, to neutralize the citric acid formed and accumulated during cultivation. the Salts or calcium hydroxide or sodium hydroxide can also be added to the culture medium during the Breeding can be added.

The culture medium used in the process according to the invention is prepared in such a way that an aqueous solution of the above nutrients is sterilized and then the hydrocarbon or adding the hydrocarbons. But you can in the event that a sterilization of the hydrocarbon it is necessary to subject the hydrocarbon to a sterilization by heating or filtration beforehand * fen. The hydrocarbon or hydrocarbons are generally added to the culture medium at the beginning added, but they can also be added to the culture medium in portions or continuously during cultivation to be set.

Then there is normally a watery deposit of calcium carbonate sterilized by heating and the culture medium as a neutralizing agent admitted.

Aerobic cultivation is used in the method according to the invention. It is beneficial from one Subtle breeding method with vortexing by blowing in air with subsequent lively Stir for good dispersion of the carbon

! 0 to make use of hydrogen in the culture medium. The amount of air that is blown into the culture medium for this purpose is Vj to 2 times the volume of the culture medium per minute, and the pressure in the fermentation tank can be normal pressure or a

! 5 be increased pressure.

The cultivation is preferred at temperatures of about 20 to 35 ° C and at a pH of 2 to 8, preferably 4 to 8, and carried out for a period of about 3 to 7 days.

The citric acid formed is at the end of the culture in the fermentation liquid when using calcium carbonate as a neutralizing agent! as calcium citrate before, that of the fermentation liquid by filtering it off together with the remaining yeast, offset the Solids after washing with a certain amount of sulfuric acid to free the citric acid and to bring into solution, and then filtering off the undissolved solid substances that result from Calcium sulfate and yeast are made up.

JO The filtrate is treated with activated charcoal or ion exchange resins for the purpose of decolorization and then concentrated, whereby the citric acid is precipitated and collected. You can also go to Add sulfuric acid directly to the fermentation liquid, remove the yeast and calcium sulfate by filtering and concentrate the filtrate - if necessary after appropriate cleaning - to add the citric acid Bring out crystallization.

The advantages associated with the present invention can be summarized as follows:

1) Compared with the usual case of using saccharides as a raw material, it is when Process according to the invention not required, ferrocyanide, methanol and the like for the culture medium and pretreatment operations such as desalting and cleaning of the Saccharide is unnecessary as a raw material, and citric acid is obtained in higher yield.

2) The acidifying ability of the microbial strain ATCC 20177 of the present invention is highly stable, and the microorganisms are easy to handle.
3) Fermentation has fewer concerns about bacterial or yeast contamination, even if the fermentation equipment, culture medium, and air have not been carefully sterilized. Furthermore, in comparison with other methods using molasses and the like as raw materials, the formation of foam during cultivation is less in the method of the present invention, whereby the method of the present invention can be made particularly economical both in terms of its practical implementation and in terms of its efficiency on the saving of antifoam agents, In addition, the method according to the invention is more economical in terms of the

operational implementation of the Fermentalion compared to other known methods.

4) Since the fermentation time in the process according to the invention is, for example, about 74 hours can be reduced, it is possible that the ratio in throughput to the plant size is extraordinary cheap to design. Furthermore, the method according to the invention can be carried out batchwise as well as continuously.

5) The process according to the invention allows citric acid to be obtained more economically Way using a hydrocarbon as a carbon source by means of a submerged culture.

6) In the method according to the invention, the η-paraffin is fermented in a high concentration, which is 5-20%, in a culture medium. The n-paraffin can also be added to the culture medium discontinuously or continuously during the Fermentation can be added.

7) According to the process according to the invention, citric acid is obtained in an astonishingly high yield of 150% by weight, based on the n-paraffin. obtain.

In order to demonstrate the superiority of the method according to the invention over the known method show, the following experiments were carried out.

A culture medium was used that, in principle

urea
KH ₂ PO ₄
MgSO ₄ · 7 H ₂ O
MnSO ₄ ■ η H ₂ O
Corn steeping water
CaCO ₁

0.10 wt% 0.02 wt% 0.02% by weight 0.025% by weight 1.0% by weight 10.0% by weight were closed, and 15 mm. Sterilized at 120 ° C. the Flasks were filled with 2.5 ml inoculum of Candida oleophi'a ATCC 20177 in a Heie-Malzkuliur (0 3% Yeast extract, 0.3% malt extract. 1% glucose. 0.5% peptone) inoculated and in case a) 4 days and in cases b) and c) shaken for 7 days at 28 ° C. at 135 rpm.

Then 50 ml of water and 25 ml of 2N HCl were added to the contents of the flask, and the precipitates were added of calcium salts in solution on a water bath

ίο brought. It was quickly cooled and the solution made up to 250 ml with water. The fluids obtained were based on the number of microorganisms (Thoma-Zeiss counting chamber after dilution) Centrifugation and neutralization for residual sugar according to Schaffer-Somogy (only in case a)) and on Citric acid by precipitation of excess Ca using NH2SO4 and determination using pentabromoacetone checked.

The following results were obtained:

and either a) 15% by weight glucose or b) 10% by volume kerosene or c) 10% by volume Cio-Cu-n-paraffin im Mixture. Remainder of water.

25 ml each of this culture medium were poured into 500 ml shake flasks, which were ver with cotton stoppers

attempt number of Sub. Jt- Zirconia (Substrate) Ml ¹ MU- Säüf ~ CdüS- orgamsmen- prey. be cells pulled up used Substrate a) glucose 8 χ 103 98% 65-68% b) kerosene 10 χ 103 more substantial not be unchanged tunable does he need rest c) n-paraffins 14 χ 10 « almost quanti 120% relative

It turns out that in experiment c) (according to the invention) a considerably higher yield of citric acid than in test a) (according to DT-OS 18 12 710) combined with a stronger microorganism multiplication occurred, no significant citric acid formation during experiment b) despite relatively good growth of the microorganisms.

The following examples illustrate the invention

example

After filling in 100 ml of a culture medium containing 1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3% Malt extract (the percentages are calculated as weight per volume) in a 500 ml bottle and Sterilizing the culture medium by boiling, the inoculum was prepared by incubating Candida oleophila ATCC 20177 in the culture medium and cultivation by shaking the Culture medium at 28 ° C. From the inoculum obtained by this procedure, 400 ml were used Inoculation of the fermentation medium used.

In about 9 liters of water were 120 g of glucose and 60 g of peplon. 36 g yeast extract and 36 g malt extract dissolved, and the solution thus obtained was sterilized by 15 minutes long boiling at 120 ⁰ C in. A fermentor of 301 and then cooled to 28 ° C. Then, 1.2 liters of a mixture of η-paraffins having 11-15 carbon atoms which had been previously sterilized by filtration and 600 g of CaCO ₃ which had been sterilized by heating were added to the solution in the fermenter, and also sterilized water added to Gemisdi to adjust the volume of the mixture to 11.6 liters. This was followed by thorough stirring.

The culture medium obtained in this way was inoculated with the inoculum prepared as indicated above. In the fermentation medium obtained in this way, the initial concentration of the n-paraffins was 10 Vol- ⁰ Zb and the initial pH was 5.5.

Immediately after inoculation, fermentation was started and aeration speed was set of 12 liters of air / minute, a stirring speed of 500 rpm and a temperature of 28 ° C adhered to. Under these conditions, fermentation was completed after 96 hours.

After completion of fermentation, the amount of citric acid that had formed was 1320 g, and the microorganism population was 8.5 ⁸ cells per ml. The amount corresponded to a citric acid yield of 152% by weight.

The resulting fermentation broth became calcium citrate, the microorganisms and excess calcium carbonate separated and collected by filtration, and after washing the filtered mixture

sulfuric acid was added to this mixture in order to liberate the citric acid. After removing the microorganisms and calcium sulfate by filtration, the remaining filtrate was concentrated and crystallized in a crystallizer to thereby obtain citric acid crystals.

Example 2

_{24 g of NH 4} Cl and 2.4 g of KH ₂ PO ₄ were added to about 9 liters of water. 2.4 g MgSO ₄ · 7 H ₂ O. 3.0 g MnSO ₄ · HlI ₂ O and 120 g corn steeping water dissolved, the solution was sterilized by boiling for 15 minutes at 120 ° C in a BO-liter shaking fermenter and then on 28 "C. To the solution thus obtained were added 1.2 liters of a mixture of η-paraffins having 11-15 carbon atoms, which had previously been sterilized by heating, and 600 g of powdered calcium carbonate, which had also been sterilized by heating. The 2μ volume of the mixture was then adjusted to 11.6 liters by adding sterilized water, followed by thorough stirring, and 400 ml of the inoculum prepared according to the procedure of Example 1 was inoculated into the culture medium thus obtained. In the fermentation medium obtained in this way, the initial concentration of η-paraffins was 10% by volume and the pH was 5.5.

Immediately after the inoculation, fermentation was started and a ventilation speed was set speed of 12 liters of air per minute and a stirring speed of 500 rpm set and one Temperature of 28 ° C maintained. Under these conditions, fermentation was over after 96 hours.

After completion of fermentation, the amount of citric acid formed was 1230 g, which is a citric acid yield of 141% by weight.

By adding sulfuric acid to the fermentation broth thus obtained, the citric acid was in Set free, and the microorganisms and calcium sulfate were removed therefrom by filtration. The filtrate was concentrated and crystallized in a crystallizer to give citric acid crystals to win.

Example 3

In about 9 liters of water, 24 g of NH ₄ Cl. 2.4 g of KH ^ PO ₄ . 2.4 g of MgSO ₄ · 7 H ₂ O, 3.0 g of MnSO ₄ · η H ₂ O and 120 g of corn steep liquor were dissolved, and the solution was poured into a 30 liter fermenter 120 ° C, followed by cooling to 28 ° C.

To the solution thus obtained, 480 ml of a Mixture of η-paraffins with 11-15 carbon atoms, which had previously been sterilized by heating, and 600 g of powdered calcium carbonate which had also been sterilized by heating. given. Then the volume of the mixture by adding sterilized water to 11.6 liters set and stirred. The culture medium obtained in this way was mixed with 400 ml of an inoculum derived from Candida oleophila ATCC 20177 was prepared analogously to Example I, inoculated. This became the initial concentration adjusted to η-paraffins to 4.0 vol .-% and the initial pl I of the culture medium to 5.5.

Immediately after inoculation, fermentation was started and, for this purpose, a ventilation rate of 12 liters l.ufi / min .. a stirring speed of 500 rpm and a temperature of 28 C complied with. After fermentation started. were 2 vol .-% n-paraffins added periodically in three stages and fermentation ceased in 74 hours

The citric acid that comes out after the end of the

Fermentation gebiiuui i'üiiic, uciiug fi'ii'i'igcrii'fiiiuig '1J ¹ JOg, which corresponds to a citric acid bulge of 155% by weight.

Citric acid crystals were obtained from the fermentation broth analogously to Example 1.

Example 4

Three fermenters with a capacity of 30 liters each were connected in series. Then the fermentation of Candida oleophila ATCC 20177 analogous to Example 2 was started in the first fermenter. Thereafter, a sterilized culture medium containing 0.2% (weight / volume) NII ₄ CI. 0.02% (wt. Vol.) KH> PO ₄ , 0.02% (wt. Vol.) MgSO ₄ · 7 H ₂ O, 0.025% (wt. _{7 vol.) MnSO 4} · η H ₂ O. 1.0% (wt Vol .-% η-paraffins with 11-20 carbon atoms and 5.0% (GewVVoI.) CaCOi, continuously added to the first fermenter at a dosage of 12 liters per 24 hours, namely 24 hours after the start of fermentation. The fermentation broth was then continuously conveyed into the second fermenter and then into the third fermenter. After the first, second and third fermenters were brought to the stationary state, the fermentation broth was continuously withdrawn from the third fermenter at a rate of 12 liters per 24 hours, and the citric acid formed was determined.

When fermentation was continued for 20 days, there were no special circumstances regarding fermentation necessary. In addition - after the system had reached the steady state - Citric acid produced in an amount of 107 g / liter. This value corresponds to a citric acid yield of 148% by weight based on the amount of n-paraffins added.

Citric acid crystals were obtained analogously to Example 1 from the fermentation broth obtained in this way.

Claims (5)

Patent claims: 1. Process for the biotechnological production of citric acid by growing Candida aleophila ATCC 20177 in a pH range of 2 to 8 under aerobic conditions in a culture liquid containing an assimilable carbon source and common neutralizing agents in conjunction with others contains inorganic and organic components, characterized in that as Carbon source η-paraffins with 5 - 35 carbon atoms can be used. 2. The method according to claim 1, characterized in that with η-paraffins as the carbon source 9-20 carbon atoms can be used. 3. The method according to claim 1 and 2, characterized in that the culture liquid 5 - 20, preferably 10-15% by volume. based on the culture liquid, contains η-paraffins. 4. The method according to claim 1 to 3, characterized in that the fermentation at a Temperature between about 20-35 ° C carried out will. 5. The method according to claim 1 to 4, characterized in that in the presence of CaCOi breeds as a neutralizing agent.