KR810000896B1 - Process for manufacturing citric acid from olefins by fermentation - Google Patents

Abstract

Citric acid was produced by culturing Candida on a medium contg. olefins (C8-C40). Thus, C. tropicalis was cultured aerobically at 30oC for 3 days in a medium contg. octadecene-1 10, NH4Cl 0.3, KH2PO4 0.05, MgSO47H2O 0.03%, FeSO47H2O 5, MnSO4H2O 0.06, ZnSO4H2O 5, biotin 50, and thiamine-HCl 100 mg/L. The culture contained 59.5 g citric acid/L.

Description

Method for producing citric acid by fermentation from olefins

The present invention relates to a method for producing citric acid by fermentation from olefins.

It is well known to produce citric acid by its assimilation using normal paraffins of C ₁₂ -C ₁₈ as carbon sources for culturing microorganisms.

Moreover, the method of using paraffin wax as a carbon source for fermenting hydrocarbon is also tried.

By the way, since paraffin wax becomes a solid state at the fermentation temperature which is 25-35 degreeC normally, it is necessary to disperse | distribute or dissolve paraffin wax in a particulate form in a medium for a fermentation process.

Thus, over the years, many researchers have used various surfactants to improve the dispersibility of solid paraffin waxes.

However, their dispersibility only appears when the concentration of the surfactant in the medium is 3% (by weight), since the surface active agent often interferes with the productivity of citric acid in the culturing process and decreases the dispersibility of the surface active agent by rapid assimilation. The use of surface active agents did not produce sufficient effects.

The inventors have prepared citric acid by assimilation of olefins based on the fact that the melting point of olefins is lower than normal paraffins with the same carbon number and the dispersibility of olefins is also better than that of normal paraffins (particularly better when carbon number is 8 or more). Research and development of fermentation process.

Currently, olefins having 13 or less carbon atoms are widely used as raw materials for plasticizers and synthetic detergents, and olefins having 14 or more carbon atoms are not in demand.

The present invention provides an advantageous method of effectively utilizing C ₁₄ or more olefins that are not in demand.

It is a feature of the present invention to use a mixture of raw olefins as the carbon source in the medium.

As a carbon source, a mixture of raw olefins produced from pyrolysis of raw paraffin wax can be used, thereby reducing the cost of the carbon source.

Therefore, the use of raw olefins in the process of the present invention is advantageous in reducing the cost of producing citric acid.

It is a main object of the present invention to provide a method for producing citric acid in which an olefin assimilable microorganism is cultured in an aerobic state in a medium containing olefin.

This object can be achieved by culturing microorganisms selected from the group of Candida Tropicalis, Candida Intermediate and Candida Blumpy in a medium containing C ₈ -C ₄₀ olefins as the carbon source.

In particular, the optimum microorganisms to be used in the preparation method of the present invention are strains belonging to Candidatropicis and its nutritive mutants and variants thereof.

Moreover, you may use the mixture of an olefin as a carbon source in the fermentation process of this invention.

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The above mixture is brought to a liquid state at a fermentation temperature of about 30 ° C., which has good dispersibility in the medium.

The concentration of olefins in the medium is 1-20% by weight, with 5-15% by weight preferred.

As the nitrogen source of the medium, inorganic or organic ammonium salts such as ammonium chloride and ammonium acetate or various nitrogen compounds may be used or mixed.

As inorganic nutrients in the medium, conventional inorganic salts such as phosphate, sulfate, hydrochloride, potassium salt, sodium salt, magnesium salt, iron salt, manganese salt, copper salt and zinc salt can be used.

The pH in the medium is adjusted using calcium carbonate and alkaline compounds.

As the organic nutrients, trace amounts of biotin and thiamine are used or mixed, respectively, or natural substances such as yeast extract and corn steep liquor containing biotin and thiamine are used.

When biotin and thiamine are used separately, biotin or thiamine of 1000 µg / l or less is sufficient to increase citric acid production.

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When biotin and thiamine are added together to the medium, the total amount of biotin and thiamine is 1000 µg / L, preferably 50-100 µg / L, which is sufficient for culturing.

In the process of the present invention, an α-olefin or a mixture of olefins may be used as the carbon source.

Since these substances are liquid at fermentation temperature, no surfactant is required to disrupt the microbial culture.

The present invention is described in detail as follows.

Microorganisms are cultured in aerobic state.

The culture temperature is in the range of 25˚-40 ℃ and preferably about 30 ℃, and the pH value of the medium is 3-10, preferably 4-6, and the pH value of the medium is added with alkali or salt such as sodium carbonate or calcium carbonate. Adjust

Incubation is usually carried out for 50-150 hours, with 80-100 hours being preferred.

Citric acid accumulated in the medium is isolated in the form of calcium citrate by a normal method such as filtration or centrifugation, and purified by chromatographic method or ion exchange method.

Citric acid production methods for culturing microorganisms in a medium containing normal paraffin (nC ₁₂ -C ₁₈ ) as a carbon source are well known.

Therefore, in order to understand the assimilation of olefins by well-known microorganisms, the culture of microorganisms was observed in an inorganic slope medium containing olefins.

The aforementioned inorganic slope medium is composed of the following components.

Microorganisms were incubated at 30 ° C. for 3 days in the aforementioned slope medium.

The culture results are shown in Table 1.

The microorganisms listed in Table 1 are well known as strains for producing citric acid from normal paraffins.

TABLE 1

From the results of the preliminary experiment as described in Table 1, the present inventors have completed the present invention regarding a method for producing citric acid by fermentation from olefins of C ₈ -C ₄₀ .

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the present invention is not limited to the following examples.

Example 1

(1) fermentation

Sterilized for 10 minutes at 115 ℃ and inoculated into a 50 ml three-necked flask containing 30 ml of the medium cooled to 30 ℃.

The above-mentioned seeds include strains belonging to Candida Tropicalis, Candida Bumpty, Candida Polytica, Candida Guillihermandi, Peachia and Devariiomiceshanseni, each containing a quantity of platinum in the medium. Inoculated.

Next, the culture was incubated at 30 ° C. for 3 days in a reciprocating shaker oscillating 120 times per minute with a stroke of 7 cm.

After about 20 hours from the start of the culture, 5% (by weight) of sterilized calcium carbonate was added to the medium to adjust the pH to 5.5.

(2) recovery

After adjusting the pH to 2.5 with hydrochloric acid, the citric acid produced during the cultivation as described above was recovered in the form of calcium salt by filtration of the medium using diatomaceous earth as a filter aid.

The filter cake was washed with water, and the washed water was mixed with the clear filtrate to neutralize the filtrate mixed as caustic alkali and then heated.

The mixed filtrate was cooled and filtered under reduced pressure to recover calcium citrate.

The produced calcium citrate was suspended in 10 times the volume of water, and an aqueous solution of barium chloride was added to the filtrate, and then titrated with 50% aqueous sulfuric acid solution until the sulfuric acid slightly appeared, and then heated in boiling water for 30 minutes. If necessary, the filtrate is decolorized, the resulting solution is filtered while heating, and concentrated to 50-60 ° C. under reduced pressure.

While concentrating the filtrate, the precipitated calcium sulfate was filtered until some thin syrup was obtained and the concentration continued. The thus obtained syrup-like concentrated liquid is cooled at 0 ° C. to obtain citric acid crystals. The amount of citric acid produced is as follows.

Example 2

The culture was carried out by the method of Example 1.

Candy or Tropical IFO-0589 was cultured in a medium containing the following components as a carbon source.

The amount of citric acid produced was 60.7 g per liter of medium.

Example 3

The culture was carried out under the same conditions as in Example 1 in a medium containing a mixture of C _16-30 normal olefins prepared from ethylene polymerization by the Ziegler method.

The medium is the same as in Example 1 except for the vitamin.

In order to test the vitamin requirement in culture, 100 mg of biotin and thiamine were added to 1 L of the medium.

Meanwhile, 100 µg of a mixture of biotin and thiamine-hydrochloride was added to 1 L of medium.

Candida Tropical IFO-0589 was used in this example. Incubation was continued for 140 hours.

The amount of citric acid was 61.5 g per 1 liter of the medium containing only biotin, and the mixture containing biotin and thiamine hydrochloride was 48.4 g per 1 liter of the medium containing only thiamine hydrochloride.

Example 4

10% (by weight) of an olefin mixture of O _10-35 prepared from wax by pyrolysis was added to a medium consisting of the following components.

Candida Tropicalis IFO-0589, IAM-4185 and IAM-12202 were inoculated in the above medium in amounts of 300 platinum each.

The culture was carried out as in Example 1.

The amount of citric acid produced is as follows.

Amount of strain citric acid

Candida Tropical IFO-0589 per badge 61.5g

4 40.5 g per 1 L of IAM-4185 medium

4 45.4 g per 1 L of IAM-12202 medium

The component ratio of the raw material olefin manufactured from the raw material wax by pyrolysis is as follows.

Ingredient ratio

n-α- olefin _{^{(nC = 10 -1-nC =}} 35 -1) 73.38% ( by weight)

Isoolefin and inner olefin 17.10% (weight)

n-paraffin (nC ₂₀ -nC ₃₇ ) 9.52% (weight)

Example 5

It was fermented using Jar in a medium containing a mixture of C _16-30 normal olefins prepared from the polymerization reaction of ethylene by the Ziegler method.

Seeds of Candida Tropical IFO-0589 were incubated at 30 ° C. for 70 hours in a medium as described in Example 1 prior to the method described above.

65 ml of this seed and 163 ml of the α-olefin described above were added to a 2.6 L jar fermentor containing 1072 ml of the medium as described in Example 1.

During the incubation period, an aqueous 10N-caustic soda solution was injected to incubate at 30 ° C. for 30 days at 1000 r.p.m and 0.5 VVM-ventilation while maintaining a pH of 5.0.

The total yield of citric acid was 126 g per liter of medium, and the total yield of citric acid for the α-olefin feed was 126% by weight.

Claims (1)

The culture temperature is about 25 ° C. in an aqueous nutrient medium containing 100 μg / l or less of vitamins selected from the group consisting of normal olefins having 8-40 carbon atoms or a mixture of 8-40 carbon atoms and biotin or biotin and thiamine hydrochloride as the main carbon sources. A method for producing citric acid by fermentation from olefins, wherein the olefin assimilable microorganism belonging to Candida tropicalis is cultured in an aerobic state at a pH of about 3-10.