DE974991C - Process for the production of citric acid by submerged mold fermentation - Google Patents

Description

ISSUED JUNE 29, 1961

M 21307 IVaJOb

Mold fermentation

The invention relates to the production of citric acid from carbohydrates Substances produced by submerged fermentation by means of molds, namely Aspergillus niger, and relates in particular the use of copper in ionized form as part of the nutrient substrate to to increase the yield of citric acid.

It is known from previous work, some of which was considered in U.S. Patent 2,492,667, that small amounts of iron in the carbohydrate medium cause large amounts of such cells to form which do not lead to citric acid formation. It has also been shown that one can reduce the undesirable effect of iron by using Zn ⁺⁺ if the Fe ⁺ ++ concentration is kept within 0.2 to 0.8 mg per liter. The removal of the Fe ions, which mostly occur in much larger quantities in the usual nutrient media, up to this concentration, as well as other cations, was achieved through the use of cation exchangers.

It has now been found, surprisingly, that the harmful effect of the high iron ion concentration in the nutrient medium can be neutralized by adding copper in ionized form. According to the invention, the substrate solution containing not less than about 0.36 mg iron ions per liter is added copper in ionized form in an amount of about 5 to 500 mg per liter. This addition of copper ions, e.g. B. in the form of copper salts, such as copper sulfate, is essential

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higher than the copper concentration in the nutrient solution, which is necessary for cell growth is equivalent to. The development of unproductive cells that occurs at higher iron concentrations will now pushed back or their development practically prevented and the formation of citric acid in fermentation batches whose iron ion concentration is above the necessary small trace amounts, promoted. This effect could not be foreseen, ίο after it was rather known that copper in larger Concentrations towards microorganisms are generally toxic.

It is no longer necessary to remove iron from the nutrient medium. In special cases, in the process according to the invention, the concentration of the cations before the addition of the copper can be reduced by using cation exchangers in a manner known per se.

The effect of small amounts of Cu ⁺⁺ on the development of spores and the vegetative growth of fermentation fungi has already been the subject of extensive studies. So one has copper as a trace element alongside others, e.g. B. in an amount of 0.48 mg per liter of nutrient substrate, used to obtain optimal growth. About the specific effects of copper ions on the growth and metabolism of fungi under conditions that are foreign to their natural growth, such as: B. in the submerged KuI-doors, is little known. In these submerged conditions, the normal cell morphology in particular is severely disturbed. This directs biochemical reactions in certain ways. Some illustrative examples are given below in order to explain the iron-antagonistic effect of adding copper in ionized form. It is not intended to limit the invention to any particular application.

Fermentation conditions and methods of analysis are the same as those in U.S. Patent 2,492,667. As can be seen, the cellular structure of a selected strain of Aspergillus niger as the basis for establishing a relationship to determine the effects of the Used copper. The typical or optimal growth characteristics are those found in in the aforementioned patent.

The copper can advantageously be added as copper sulfate ^ o , but it can also be added in the form of any salt that is soluble in the solvents, which salt, however, must not have a toxic or otherwise undesirable anion. The amounts of the various cationic constituents in the description refer to milligrams of copper, iron or zinc per liter of medium.

example 1

Corn syrup (corn sugar) with various ash and iron contents, which had not been freed from cations, was subjected to fermentation. In addition to the specified variable amounts of Cu ⁺ + and Fe ⁺⁺⁺ , 50 mg Zn ⁺⁺ per liter were added to the fermentation media, which had the following solid components:

The medium was adjusted to a _pH value from 2.5 to 2.65 with concentrated H ₂ SO _4, sterilized in an autoclave at 0.7 at 10 minutes and was introduced into a Gärsäule. Acidity levels were calculated as citric acid monohydrate and sugar conversions were based on the initial concentration of fermentable carbohydrates determined by the Somogyi sugar analysis method. ₁₀ ,

Ammonium carbonate ... 0.2% Monopotassium phosphate .. 0.014% Magnesium sulfate 0.1 «/» Carbohydrates 12.0 to 15.0% Not from cations freed water , 4000 ecm

Table I.

ash Fe +++ Cu ++ Acidity conversion Cell structure mg / 1 mg / 1 G 0/0 Ο, Ο 0.0 0.0 *
Filiform, no acid generating 200, O 400.0 77.0 Growth typical O, I5 0.91 < dense aggregates 300.0 307.0 59.0 Heavily inhibited, compact Aggregates f 100.0 379.0 73.0 Growth typical 0.19 0.73 y 200.0 412.0 79.2 Growth typical r 0.0 0.0 0.0 Thread-like, non-acidic O, 2O 0.36 j 100.0 402.0 77.2 forming
Growth typical I. 200.0 385.0 74.2 Growth typical

Table I continued

ash Fe +++
mg / 1 Cu ++
mg / 1 Acidity
G conversion
Vo Cell structure 0.21 O.55 { 100.0
200.0 423.O
382.0 8l, 5
73.5 Growth typical
A little inhibited O, 25 2.91 100.0
200.0 400.0
382.0 77.O
73.2 Growth typical
Growth typical
somewhat inhibited 0.29 2.79 \ 100.0
200.0
300, O 361.0
396.O
393.0 69.4
76.2
75.6 Abundant growth, for
Tending to thread formation
Growth typical
Growth typical

In these experiments, the most important thing is shown in experiment No. 3, in which the iron content is only 0.36 mg per liter, which clearly inhibits the fermentation of a medium with a medium ash content. Developed in the absence of copper in ionized form In the Aspergillus Niger strain, there is a thread-like, non-acid-forming cell structure. With the possible exception of approach no. 6, in which 100 mg copper per liter a growth with Reduced acidity tendency, all approaches were in between 100 and 200 mg per liter fermented satisfactorily with copper.

Example 2

In another series of experiments with non-cationic corn syrup, allowed the concentration of Cu ++ and Fe +++ vary mutually. The results of such experiments on a sugar medium as used in Example 1 are listed in Table II.

Table II

approach
No. Fe ++ +
mg / 1 Cu ++
mg / 1 Acidity
G At first
sugar
S. Around
Wall
lung
»/ 0 20 y
-I
22 y ΙΟ, Ο
5O, O
ΙΟΟ, Ο
150.0
10.0
50.0
100.0
150.0
10.0
50.0
100.0
150.0 50.0
50.0
50.0
50.0
100.0
100.0
100.0
100.0
200.0
200.0
200.0
200.0 393.0
349.0
256.0
62.0
380.0
317.0
272.0
149.0
375.0
341.0
181.0
68.0 5O5, O
505.0
505.0
505.0
505.0
5OO, O
505.0
500.0
500.0
500, O
5OO, O
500.0 77.8
69.1
50.7
14.2
77.4
65.4
53.9
29.8
75, o
68.2
36.2
13.6

Table II continued

approach
No. ί 2.
23 ι Fe +++ Cu ++ Acidity At first
sugar Around
Wall
lung ί mg / 1 mg / 1 G G ° / o 2Λ < ΙΟ, Ο 3ΟΟ, Ο 378.0 5OO, O 75.6 T * 1 5ο, ο 3ΟΟ, Ο 344.Ο 500.0 68.8 ί ΙΟΟ, Ο 300.0 274, Ο 5OO, O 54.8 150.0 300.0 133.Ο 500.0 26.6 ΙΟ, Ο 5ΟΟ, Ο 37Ο, Ο 5OO, O 74.Ο 5ο, ο 5ΟΟ, Ο 327, Ο 5OO, O 65.4 ΙΟΟ, Ο 5ΟΟ, Ο 298.0 5OO, O 60.6 ΐ5ο, ο 5ΟΟ, Ο 138.0 500.0 27.6

This table clearly shows the counteracting effect of copper over a wide range of Fe ⁺ ++ - concentration. Cu ⁺⁺ in amounts of 50 to 100 mg per liter made Fe +++ in concentrations of 50 mg per liter inactive with regard to the development of a satisfactory amount of acid. It should be noted, however, that although were 500 mg per liter of Cu ⁺ + used, the Fe ⁺⁺⁺ quantities of 100 to 150 mg per liter were not regulated up to a point that a Säurebildüng was obtained in normal technical scores. Nevertheless, better under these conditions were obtained as 5o ^e / o conversion of the fermentable carbohydrates output when 50 to 500 mg per liter Cu ^++, together with 100 mg per liter of Fe +++ are used.

Example 3

As has been stated earlier, it was a common experience in the art of fermentation that undesirable effects of iron by the use of Zn ⁺⁺ may be reduced if the Fe ⁺ + quantities within the limits of about 0.2 to 0 .8 mg per liter can be maintained. Despite this fact, the lack of equivalence between Cu ⁺ + and Zn ++ is shown as antagonists to iron by the following:

Table III

Approach no. Cu ++ Zn ++ Acidity At first
sugar conversion Cell structure Typical: dense aggregates, mg / 1 mg / 1 0 ·
ö G % above average 3D Ο, Ο Ο, Ο 0.0 507 31 Ο, Ο 5.O 0.0 507 - yield 32 Ο, Ο ΙΟ, Ο 0.0 507 - 33
34 Ο, Ο
Ο, Ο 2O, O
40.0 0.0
0.0 507
507 35 Ο, Ο 8o, O 0.0 507 - 36 Ο, Ο 160.0 0.0 507 - 37 0.0 320.0 66.0 507 l8, O 38 5> o Ο, Ο 213.0 507 42.0 Not typical: thread-forming, 39 10.0 Ο, Ο 207.0 507 40.0 non-acidifying 40 20.0 0, O 367.0 507 72.4 41 40.0 Ο, Ο 339> above 502 67.5 42 80.0 Ο, Ο 34 ⁶ > ° 502 69.2 Not typical: thread-like 43 160.0 Ο, Ο 407.0 502 81.1 Aggregates \ Dense to thread-like J aggregates I. } Typical: dense aggregates J

These experiments with regard to the comparative, iron ion counteracting effects of Cu ++ and Zn ++ were mixed with a non-cationic medium with corn syrup, such as it was used in Example ι, obtained with 0.8 mg per Fe +++ per liter. As can be seen, are Zinc amounts of up to 160 mg per liter are insufficient to control the undesired cell structure that is caused by Fe +++ and other, unknown trace elements introduced with the raw sugar substrate will come from. There is a tendency towards the development of an acid-forming cell material 320 mg Zn ++ per liter. However, it is of interest to note that copper is effective in production a completely typical cell structure in a concentration of only 20 mg per liter.

Example 4

In cases where, as has been found, it is desirable to work with a depleted, carbohydrate-containing raw material in order to take advantage of a reduced ash content achieved by ion removal, there is still an inhibitory effect of copper clear. The following table shows the antagonistic effect of Cu ⁺ and Zn ++ + to iron in such from. Cations released sugar medium, the. 10 mg per liter of the Fe +++ were added after deionization. The special, cation-depleted corn syrup medium used to compare the antagonistic effects of copper and zinc with iron had the following composition:

95 ammonium carbonate .. 0.2%

Monopotassium phosphate. 0.014 ¹ vol

Magnesium sulfate 0.1%

Calcium chloride 6.0 parts per million

Molybdic acid 50, o parts per million

H ₂ O 6.0 parts per million

Morpholine 500.0 parts per million

Carbohydrates (from

Cations free) ... 12.0 to 15.0%

Water (of cations

exempt) 4000 ecm.

'

This medium was adjusted to a _pH value from 2.5 to 2.65 with concentrated hydrochloric acid, sterilized in an autoclave at 0.7 atmospheres for 10 minutes and then introduced into the Gärsäule.

The results of the testing of this cation-depleted medium are given in Table IV and are comparable to those in Table III for de-cationized corn syrup given are. At any normal concentration, the zinc failed to produce of good acid-forming cell material, while an almost typical cell structure with a copper concentration of only 25 parts per million began to appear.

Table IV

Approach no. Cu ++
mg / 1 Zn ++
mg / 1 Acidity
G At first
sugar
G conversion
° / o Cell structure 50 Ο, Ο Ο, Ο 27.0 493 5.6 Thread-like, very little
Acid-producing cell material 51 5, O 0.0 I45.O 493 29.4 Thread-like up together
balling, little acid
forming cell material 52 25.0 0.0 258.0 493 52.3 Balled up to thread
shaped, moderately acidic
of the cell material 53 50.0 0.0 385, O 493 78.1 Typical cell material 54 100.0 0.0 361.0 493 73.2 Inhibited, tight aggregates,
good acid-producing cell
material 55
56
57
58 0.0
0.0
0.0
0.0 5.0
25.0
50.0
100.0 31.O
29.0
II5.0
107.0 493
493
493
493 6.3
5.3
23.3
21.7 Filiform, very little
Acid-producing cell material

The examples show that the use of copper ions in submerged fermentations results in sugary fermentations Nutrient solutions that have not been freed from cations, as well as nutrient media that have not been freed from cations were released, can be fermented to citric acid in good yield. The size Significance of this discovered effect of ionized copper lies in the fact that by its application, the deionization stage of the nutrient medium containing carbohydrates can be avoided can. This deionization treatment has hitherto been necessary to remove most of the iron ions to remove and reduce their concentration in the solution to be fermented. About that In addition, the established antagonistic effect of copper in relation to iron allows it to be used of technically pure substrates, which were previously due to their relatively high content of Heavy metals, especially iron, could not be used. Besides, you need now for these culture solutions in terms of the type of container and the devices and the Pay little attention to the length of the storage time in such facilities, because the metal impurities, which are absorbed as inhibitors, in the presence of copper have only a minor influence on the type of cell structure in which citric acid is formed.

Claims (2)

Patent claims: 1. Process for the production of citric acid by submerged fermentation of carbohydrate and containing iron ions. Nutrient substrates by Aspergillus niger, characterized in that that of the substrate solution containing no less than about 0.36 mg of iron ions per liter Copper is added in ionized form in an amount of about 5 to 500 mg per liter. 2. The method according to claim 1, characterized in that that the carbohydrate-containing nutrient substrate is subjected to a tail-wise deionization before the addition of the copper. Considered publications:
U.S. Patent No. 2,492,673;
Ind. Eng. Chem., 40, p. 1203, panel 1 (1948);
"Results of Enzyme Research" by R. Weidenhagen, Vol. 11, pp. 226 to 229, 1950. 109 60S / 4 6.61