JPS58138387A - Production of citric acid - Google Patents

Abstract

PURPOSE:In the production of citric acid from n-paraffin by a a fermentation process, the concentration of the oxygen dissolved in the culture medium is kept in a certain range to enable the efficient production of citric acid for a long period of time. CONSTITUTION:In the production of citric acid from n-paraffin utilizing a yeast in Saccharomycopsis or Candida, the concentration of the oxygen dissolved in the culture medium is kept in the range from 10 to 45% based on the concentration of the oxygen sautrated in the water kept at the cultivation temperature. In order to realize the condition, oxygen gas or a gas containing the same, such as air, is fed continuously or intermittently during the cultivation. Thus, high productivity is maintained in citric acid production.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing citric acid from n-paraffin as a raw material by an aerobic fermentation method using yeast of the genus Satucharomycopsis or yeast of the genus Candida. , relates to a method for maintaining high citric acid productivity by maintaining the dissolved oxygen concentration of a culture medium within a specific range.

JP-A-Sho, 119-711 discloses that citric acid is produced from the culture by aerobically cultivating yeast of the genus Sasikalomyfbussis or genus Candida in a medium containing n-paraffin.
No. 93 and Japanese Patent Publication No. 53-19676.

The present inventors further investigated a method for efficiently mass producing citric acid over a long period of time using the above method, and extracted part or all of the aqueous phase containing the produced citric acid from the culture tank, and created a new medium. As a result of continuing the culture by repeatedly using the cells by adding citric acid, the production efficiency was initially good, but as the operation was repeated, the productivity of citric acid gradually decreased and it became difficult to maintain high productivity. The problem was that I couldn't do it.

Therefore, as a result of further investigation, we found that even if the amount of oxygen supplied to the culture medium is too high or too low, it will have a negative effect on the rate of citric acid production. We have discovered that it is important to adjust the temperature within a specific range during the period, and have arrived at the present invention.

On the other hand, JP-A No. 48-10286 describes a method for separating bacterial cells from a petroleum fermentation solution when producing so-called petroleum proteins using n-paraffin. Taking advantage of the fact that the specific gravity of the fermentation liquid is higher than that of other components, a part of the culture in the paraffin-water-bacteria emulsion state in the continuous fermentation device is extracted into a separation tank.
Leave it to stand still, and the bacterial cells that do not contain paraffin are placed in the bottom layer.
A method is disclosed in which unassimilated paraffin is separated into an upper layer, the upper paraffin layer is returned to the continuous fermentation device, and the final product (petroleum protein) is obtained from the bacterial cells in the lower layer.

However, the above publication only describes a method for separating bacterial cells (petroleum protein) from a culture, but does not describe or suggest a method for producing citric acid using n-paraffin as a raw material. Furthermore, in order to produce citric acid with high productivity over a long period of time using yeast of the genus Satucharomycopsis, etc.
Of course, there is no description or suggestion regarding a preferable dissolved oxygen concentration as in the present invention.

That is, the method of the present invention provides a method for maintaining high productivity of citric acid over a long period of time. In the method for producing citric acid from n-paraffin, the dissolved oxygen concentration of the medium in the culture tank is maintained in the range of 10 to 45% of the saturated dissolved oxygen concentration of water at the culture temperature. Relating to a manufacturing method.

The strain used in the present invention is a yeast of the genus Candida or Satucharomycopsis, and examples thereof include the strains disclosed in the above-mentioned publications and publications, and NRRLY7580 disclosed in German Published Patent Application No. 2356970.

N-paraffins with various return prime numbers are used, and those with return prime numbers of about 9 to 19 are usually used.

In addition to n-paraffin, the medium components include a nitrogen source, an inorganic salt, and other appropriate nutrient sources as required, and examples thereof include those disclosed in the above-mentioned publications or publications.

Nitrogen sources thus include inorganic nitrogen sources such as, for example, ammonium nitrate, ammonium sulphate, ammonium chloride or ammonium phosphate, or organic nitrogen sources such as, for example, peptone, casamino acids, uric acid or constituent liquor; inorganic salts include, for example, potassium phosphate. ,
Sodium phosphate, magnesium sulfate, manganese sulfate,
These include zinc sulfate and calcium chloride. Other nutritional sources that may be added if necessary include yeast extract, cornstarch liquor, meat extract, and vitamins, from which an appropriate one may be selected and used.

One suitable example for the culture medium is 10-400 g of n-paraffin, 1-20 g of a nitrogen compound (e.g. ammonium sulfate).
, o, i~3g potassium hydrogen phosphate, 0.1~5
g of disodium hydrogen phosphate, 0.1 to 1 g of magnesium sulfate heptahydrate, and 0.1 to 20 mg each of zinc sulfate.
Heptahydrate, manganese sulfate tetrahydrate, calcium chloride, sodium chloride and copper sulfate, and 100
Prepared by adding ~1500 mg of yeast extract or cornstarch liquor to 11 g of distilled water.

After sterilizing the medium at 120°C for about 110 to 15 minutes,
The bacterial strain is inoculated and cultured in a culture tank. Cultivation.

The pH at the time of cultivation is suitably 5 to 9, and since the pH decreases with the production of citric acid, it is desirable to neutralize with aqueous ammonia or the like. Culture temperature is approximately 20-35℃
Usually, the culture is carried out under aerobic conditions such as shaking culture or stirring culture.

In the present invention, the dissolved oxygen concentration in the culture medium is about 10 to about 45 times higher than the saturated dissolved oxygen concentration of water at the same temperature as the culture temperature.
It is necessary to set the range of about 25%, especially about 25%.
It is preferable to maintain the concentration in the range of ~35%, and therefore, it is desirable to continuously or intermittently supply oxygen-containing gas such as oxygen or air to the culture medium during the culture period.

Dissolved oxygen concentration can be measured by various methods, but continuous measurement of dissolved oxygen concentration in a medium using an electrode is convenient for controlling culture conditions.

The value of dissolved oxygen concentration is the value of dissolved oxygen concentration of water at the same temperature (
For example, the relative value of the dissolved oxygen concentration of the medium (7.5 ppm at 60° C.) is expressed as (6).

In the present invention, if the dissolved oxygen concentration of the medium in the culture tank is less than about 10% or more than about 45%, the productivity of citric acid decreases as the culture continues for a long period of time, making it impractical to use. The production rate of enoic acid cannot be maintained.

The method of the present invention can be applied to any of the continuous culture method, semi-continuous culture method, and batch culture method, and part of the culture is transferred from one culture tank to another tank while the culture continues. Although there is a continuous culture method in which only the n-paraffin layer containing the bacterial cells that has been extracted and allowed to stand still for phase separation is recycled into a culture tank, the following semi-continuous culture method is particularly suitable.

That is, when the culture was carried out to some extent and the desired amount of citric acid was produced, in order to separate the aqueous phase and the oil phase of n-paraffin in the medium, the medium was kept under non-stirring conditions, that is, stirring was substantially interrupted. The culture medium is allowed to stand still to promote separation, forming an aqueous layer that does not substantially contain n-paraffin and an n-paraffin layer that contains bacterial cells. Part or all of the former is removed and quenched. A semi-continuous culture method in which acid is collected, a new medium is added to the culture tank, and agitation is restarted to continue the culture. Since the inside of the system is not contaminated by other microorganisms and is a simple system, efficient production can be maintained for a long period of time.

The semi-continuous culture method will be further explained below.

When the culture is allowed to stand still, it separates into an oil layer and an aqueous layer, and since citric acid-producing yeast of the genus Candida or Satucharomycopsis exhibits lipophilic properties, most of them are contained in the oil layer.

Of the separated culture medium, part or all of the lower aqueous layer is removed, and citric acid in this component is collected by a conventional method such as extraction. From the viewpoint of production efficiency, the ratio of the water layer extracted is preferably about 1/2 to 8/9 of the water layer formed.

Fresh medium is added to the remaining medium, stirring is resumed, and culture continues.

The above operations are repeated once the desired amount of citric acid has been produced, and usually the stirring of the medium is interrupted, the medium is allowed to stand, the medium is removed and added, and then stirring is started again. The time required for this depends on the capacity of the culture tank, but when using a culture tank with a capacity of about 0.5 to 501, it is usually about 30 minutes to about 1 hour.

Example 1 Ammonium chloride a g % dipotassium hydrogen phosphate 0. Ig, potassium hydrogen phosphate 0.1g, magnesium sulfate heptahydrate 0.5g, yeast extract 1gs sodium chloride 100mg
, calcium chloride 10 mg, zinc sulfate heptahydrate 5 mg,
Manganese sulfate tetrahydrate 1 mg, steel sulfate pentahydrate 0.2 m
g, 0.1 mg of sodium borate decahydrate was dissolved in 14 distilled water and adjusted to pH 7. 900mg of this liquid and n-
Paraffin blend 2 (C130, 3%, C1450, 0%,
Pour 60J of C154B, 5%, C161, 2%) into a 21-volume mini jar fermenter and heat at 120°C for 20 minutes.
Sterilized for minutes. After cooling, L. suboritica NT 1-33 (N
RRL Y75'80) suspension s, om6 was inoculated,
Culture was started at a culture temperature of 30° C. and a stirring blade rotation speed of 500 rpm.

However, in order to maintain the pH of the culture medium at approximately 6.5, a device that automatically injected an ammonium aqueous solution was used. In addition, the dissolved oxygen concentration of the culture medium was measured using s
- Continuously measured with a type 1 dissolved oxygen electrode, and the value is
Oxygen gas was continuously or intermittently supplied into the culture medium so that the saturated dissolved oxygen concentration was in the range of about 25% to about 30% of the saturated dissolved oxygen concentration of water at 30°C.

After culturing for 70 hours, the rotation of the stirring blade was stopped and about
Leave to stand for 50 minutes to 1 hour to separate into an oil layer and an aqueous layer, and aseptically extract 500 ml of the aqueous layer containing produced enoic acid from the lower layer, and add 460 ml of a separately sterilized medium with the above composition.
and n-) 40 ml of Rhizocane was supplied aseptically, and the rotation of the stirring blade was restarted to continue the culture. That is, semi-continuous culture was performed in which the bacterial cells were reused.

Thereafter, when the culture was continued for 24 hours, the culture was allowed to stand still in the same manner as above, and the operations of removing the aqueous layer containing the produced enoic acid and supplying a new medium (second time) were repeated.

The above operation was repeated, and the concentration of citric acid in the aqueous layer extracted each time was measured.

The results are shown in Table 1. However, the value 6 is the concentration of citric acid in 500 tng of the water layer extracted per 24 hours of culture time.

The average productivity of citric acid for the three times was 0.87 g/l·hr.

Examples 2 and 3 The same procedure was carried out except that the dissolved oxygen concentration was set to about 15 to about 18% (Example 2) or about 27 to about 40% (Example 5).

Example 4 Satucharomycopsis lipolyteica MT1 as a microorganism
002 (Microorganism storage entrustment application form acceptance number 1610, N
RRL y7576) was used in the same medium as in Example 1, and while controlling dissolved oxygen in the same manner, semi-continuous culture was performed in which the bacterial cells were reused. As a result, the citric acid concentration was sb, 2g/lz for the first time, and 3s, sg/l for the second time.
, 3rd time 36.5g/j? Met.

Example 5 The culture was continued for 90 hours using the same medium as in Example 1 and controlling dissolved oxygen in the same manner. That is, normal batch culture was performed. As a result, 54 g/l of citric acid was produced. Citric acid is 0.6g/1-hr (54g/
A'・90hr).

Comparative Example 1 and Comparative Example 2 Dissolved oxygen concentration was approximately 4% to approximately 7% (Comparative Example 1) and approximately 50%.
% (Comparative Example 2).

Table 1

Claims (2)

[Claims] (1) In a method for producing citric acid from n-paraffin by an aerobic fermentation method using yeast of the genus Satucharomycopsis or yeast of the genus Candida, the dissolved oxygen concentration of the medium in the culture tank is determined at the culture temperature. A method for producing citric acid, which comprises maintaining the saturated dissolved oxygen concentration in water within a range of 10 to 45%. (2) Form an aqueous layer that does not substantially contain n-paraffin and an n-paraffin layer that contains bacterial cells in the culture tank, take out part or all of the former to collect citric acid, and culture The method for producing citric acid according to claim 1, characterized in that the culture is continued by adding a new medium to the tank.