CS250277B1 - Process for the production of gluconic acid and its salts - Google Patents

Abstract

The method of producing gluconic acid and its salts in a submerged culture of the fungus Aspergillus foetidus var. pailidus by conversion of glucose1 substrate is addressed. The advantage of this method is that with the help of the high-production strain Aspergillus foetidus var. pailidus AN 166, higher yields are achieved and highly concentrated sugar substrates (up to 40% by weight) can be processed.

Description

The present invention relates to a process for the preparation of gluconic acid and its salts in a submersible culture of Aspergillus foetidus var. pallidus by converting the sugar substrate.

Gluconic acid and its salts are increasingly used, for example as anticorrosive agents for metal surface treatment, as well as in cleaning, polishing and galvanizing baths for metal objects, for removing deposits in containers and pipes, as additives in the paint industry, concrete additives, in pharmacy and many other applications.

The biological oxidation of glucose and some other carbohydrates containing bound glucose has been known for a long time. Bacteria of the genera Pseudomonas and Acetobacter were used for this purpose [Humphrey AE, Reilly PJ, Biotechnol. Bioeng. 7: 229 (1965); Nyeste et al., Eur. J. Appl. Microbiol. Biotechnol. 10:87 (1980); Currie J. N., U.S. Patent No. 1896,811], but fungi, in particular Aspergillus and Penicillium, are widely used [Currie J. N. et al., U.S. Patent No. 1,893,819 (1930); Zlffer J. et al., DE 18 17 907 (1968); Porges N. et al., Ind. Eng. Chem. 33: 1065 (1941)], since they do not further oxidize gluconic acid and thereby reduce its yield.

It is known that the concentration of dissolved oxygen, or its rate of transfer to the fermentation medium, the concentration of hydrogen ions in the medium, and the quality of the nitrogen source, among other less significant factors, are influenced by the rate of gluconic acid formation and yield.

There are some problems affecting the rate of oxygen transfer to the fermentation medium. Increasing the stirrer frequency is limited by the mechanical strength of the microorganism used, increasing the aeration rate is limited by the foaming of the culture medium. Increasing oxygen solubility by increasing system pressure [Herrick Η. T. et al., Ind. Eng. Chem. 27: 681 (1935)] leads to technical and design difficulties.

The effect of the concentration of hydrogen ions is known and there is a close optimum when the synthesis of gluconic acid is the fastest.

In fungal cultures, gluconic acid is formed by the action of the enzyme glucose oxidase (β-D-glucose: Oz coxidioireductase, E. C. 1. 1. 3. 4.) on glucose in the presence of oxygen. Thus, for optimum process performance, the microorganism used must produce the highest relative amount of glucose oxidase.

The essence of the process for the preparation of the gluconic acid and its salts according to the invention is to cultivate a mutant of Aspergillus foetidus var. pallidus AN 166, whose spore inoculum is either directly inoculated into the production fermenter or previously in an inoculation tank so that the resulting spore suspension concentration corresponds to an extinction constant E420 = 0,005 to 0,02, after which the mutant is cultured at 25 to 35 ° C at concentration of hydrogen ions, maintained by the addition of alkaline reacting solutions, in particular NaOH, KOH, NH 3 OH, Na 2 CO 3, at a pH of 5.0 - 7.0 in a solution of 5 to 40 wt. glucose substrate.

A mutant of this microorganism is deposited in the CCF Collection of Microorganisms under number 1889 and is the subject of the present invention. This mutant generates a large amount of the glucose oxidase enzyme under appropriate culture conditions.

After fermentation, the cell mass of the microorganism is separated from the liquid portion of the culture medium and reused to convert the glucose substrate.

The nitrogen source is the most important influence on the production rate of gluconic acid and its yield.

By changing the qualitative composition and the total amount of nitrogen source, the process was controlled to be optimal for two basic types of gluconic acid preparation:

(a) for the single fermentation of glucose solutions, whereby as little cell matter as possible with the highest specific glucose oxidase activity is required because of the maximum yield of gluconic acid;

b) for re-fermentation of separated cell mass, where a higher concentration of cell mass is required for a rational process; losses of the carbon source due to the formation of cell mass are relatively lower.

Spore or vegetative inoculum is used to inoculate fermentation vessels. Its amount does not depend on the mass amount of the glucose source, but only on the volume of the fermentation medium.

In the case of a spore inoculum, an amount of the spores of the microorganism used rinsed with 0.1% Tween 80 solution is used such that their concentration in the medium at the start of the fermentation measured as extinction at 420 nm reaches E420 = 0.005-0.02.

The vegetative inoculum prepared by germination of spores at a concentration corresponding to E420 of - 0.05 to 0.2 is dosed into the fermentation medium in an amount of 3 to 10% of the total volume, corresponding to the initial dry matter at fermentation of 0.05 to 0.1 mg / ml.

The preparation of the gluconic acid according to the process of the invention is further illustrated in the following examples.

Example 1

Preparation of inoculum:

A spore of Aspergillus foetidus var. pallidus AN 166, growing for 7 days on wort agar on 15 cm diameter Petri dishes, rinsing with 50 ml of 0.1 wt. surfactants based on este250277 fatty acids (Tween 80). 150 ml of the medium in a fermentation tank is seeded with these spore suspension. The number of dishes is selected so that the volume of the spore inoculum is 2-4% of the volume of the fermentation medium. After twelve hours of low aeration (0.1 volume of air per volume of medium per minute) when the spores germinate at 30 ° C, the physical conditions for the fermentation itself are adjusted, ie the aeration is increased and the agitator is started.

Preparation of fermentation medium:

Prepare a sterile fermentation medium of the following composition (in% by weight):

% glucose

0.025% KCl,

0.025% KH2PO4

0.025% MgSO4. 7 H2O,

0.1% Ca (NO3) 2.4 H2O

0.056% (NH4) 2SO2.

The pH correction during fermentation is performed by adding 20% NaOH or Na 2 CO 3 solution.

The fermentation itself is carried out in a stainless steel tank with a centrally placed turbine-type mechanical stirrer. Fermentation volume 150 l, stirring 400 rpm, aeration 75 l / min, temperature 30 ° C, pH is continuously adjusted to 6.0. The fermentation time, including the germination phase, is 40 to 45 hours. The fermentation, resulting in the sodium salt of gluconic acid, allows a yield of 90 to 95% by weight to be achieved. gluconic acid to the sugar introduced. Other organic acids are not formed.

Example 2

Preparation of inoculum:

Malt agar spores were inoculated with propagation tanks (medium volume of 2.5 L) containing the medium of the composition given in Example 1, in which only the glucose percentage was reduced to 5% by weight. It is cultured at pH 6.0, 30 ° C, 1000 rpm and aeration 1 L / min per 1 L of medium. The vegetative inoculum thus grown inoculates the fermentation tank itself. The amount of inoculum used is 5-10% by volume. All fermentation parameters, medium composition and yield are the same as in Example 1. The fermentation time is 28-30 hours.

Example 3

Preparation of inoculum:

Both methods described in Examples 1 and 2 can be used.

Preparation of fermentation medium:

Sterile soil contains:

0.2 wt. Ca (NO3) 2.4 H2O

0.11% (NH4) 2SO4, other chemicals are dosed as in Example 1. The amount of inoculum is 10% v / v.

After the fermentation is complete, the mycelium is separated and reused to convert glucose to gluconic acid in a mineral medium of the composition (wt.%):

% glucose

0.025% KH2PO4 a

0.1% CaCl2 under the same physical conditions.

In all cases, glucose as a carbon source may be replaced by some other sugar substrate (e.g., sucrose, maltose, and the like).

Claims (2)

A process for the production of gluconic acid and its salts in a submerged culture of Aspergillus foetidus var. pallidus by conversion of a glucose substrate characterized by culturing a mutant of this microorganism AN 166 whose spore inoculum in an amount corresponding to the volume of the fermentation medium is either directly inoculated into the production fermenter or previously into the Inoculation Tank so that the resulting spore suspension concentration corresponds to the extinction constant E420 OF THE INVENTION equals 0.005 to 0.02, after which the mutant is cultured at a temperature of 25 to 35 ° C and a pH of 5.0 to 7.0 in the range of 5 to 40% by weight. glucose substrate. 2. A method according to claim 1, wherein after fermentation, the cell mass of the microorganism is separated from the liquid portion of the culture medium and reused for the conversion of the glucose substrate.