CA1105862A - Manufacture of fatty acids having straight and long carbon chains using a microorganism - Google Patents

Abstract

MANUFACTURE OF FATTY ACIDS HAVING STRAIGHT
AND LONG CARBON CHAINS USING A MICROORGANISM
Abstract of the Disclosure A method for the oxidation of hydrocarbons to mono-carboxylic acids and thence to dicarboxylic acids by the aerobic resting cell reaction of the organism Debaryomyces phaffii ATCC 20499.

Description

This in~ention concerns the production of dicarboxylic acids or dicarboxylic acids along with monocarboxylic acids corresponding to hydrocarbons having straight and long carbon chains and/or monocarboxylic acids having straight and long carbon chains as substrate using a microorsanism.
Monocarboxylic acids having straight and long carbon chains are useful raw materials of surfactants, detergents, stabilizers, and the like. However, their use has been limited since natural fats, such as beef fat and palm oil, have been mostly employed for the preparation of the above-mentioned chemicals.
Dicarboxylic acids having straight and long carbon chains are useful raw materials for the preparation of plastisizers, synthetic resins, synthetic lubricants, oils, perfumes, and the like. The establishment of a method of the manufacture of dicarboxylic acids with varied carbon numbers on an industrial scale from petroleum derived feedstocks has been desired.
Microbial production of monocarboxylic acids and dicarboxylic acids is well known. In these reported reactions, normal paraffins contained in petroleum distillate are used as substrate for corresponding mono- and di-carboxylic acid.
Further, the use of natural as well as synthetic monocarboxylic acids as raw materials for microbial conversion to dicarboxylic acids has been reported: VanderLinden and Thijsse, "The ': ' .
.

Mechanisms o~ Microbial Oxidations o~ Petroleum Hydxocarbons";
~dvances in Enzymology, ~ol. 27, p. 469 ~1~651; ~.M~nura, U.S.
3,793,153, 6. Akabor~, et al., U.S. 3,843,466.
Commercially advantageous methods have not yet been establishe`d.
It has no~ been ~ound that a yeast strain which belongs to De~aryomyces genus can produce dicarboxylic acids or di-carboxylic acids along w~th monoaarboxylic acids by oxidizing h~drocarbons or mixtures of h~drocarbons and mono-car~oxylic acids having straig~t and long car~on chains.
As described herein, this invention is characterized ~y using a yeast stra;n belonging to the Debaryomyces genus, Debaryomyces p~affii; in order to produce di-carboxylic acids or di-car~oxylic acids along w~th mono-carboxylic acids ad~antageously.
Thus according to the present invention there is provided in the production of C8-C18 mono-and di-carboxylic acids from, in the case of mono-carboxylic acids, straight chain hydrocarbons wherein the hydrocarbon and the resulting

2~ acid have the same number of carbon atoms in the skeletal chain, and in the case of di-carboxyl~c acids, from straight chain hydrocarbons and mono-carboxylic acids wherein the hydrocarbon and the mono-carboxylic acid have the same number of carbon atoms in the skeletal chain, by aerob;cally cultivating the hydrocaxbon or mono-carboxylic with a micro-organism in a ; ~' 5 ~ ~

nutrient medium, the improvement which comprise~ u~ing as the micro-organism De~ar~o~ycës ph~f;~:i A~CC 2~499.
Utiliz~ng the m~.cro-or~an~sm, this in~ention produces, ~l~ di-carboxylic acids or a mixture of di-car~oxylic ac~ds and mono-car~oxylic acids correspond~ng to t~e hydrocarbons having str-aig~t and long car~on chain~ which have ~een used as the substrate, C21 di-car~oxyl~c ac~ds corresponaing to natural as well as synthetic monocar~oxyl~c acids used as raw material and (3~. di-car~oxylic acids corresponding to a mixture o hydro-car~ons.having long, straight carbon chains and mono-carboxylic acids of similar chain length ~en used as substrate.

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The carboxylic acid producin~ microorganisms, Debaryomyces phaffii, employed in this invention was collected from the soil near a petroleum refinery in Akita Prefecture and was isolated for use. Said microorganism was identified as Debaryomyces phaffii from the following micrological properties.
The strain named BR-151 of said microorganism has been deposited with the Fermentation Research Institute, the Agency of ; Industrial Trade and Industry, Japan with the accession under FERM-P No. 4300 as well as with the American Type Culture Collection with the accession number ATCC-20499.
~ The properties of said BR-151 are described below:
1. Shape and si~e:
Growth in malt extract: After 3 days at 25C the cells are oval or long-oval, (1.5-5.5)x(3.5-13.5)~; single or in pairs.
; A sediment and a thin, dull, creeping pellicle are formed.
Growth on malt agar: After 7 days at 25C the colony is whitish or dark yellowish, dull to shiny, smooth with a - slightly sinuous margin.
Slide culture on potato- and corn meal agar:
The formation of pseudomycelium is lacking.
2. Formation of ascospores:
Ascospores are formed on /8M Van't Hoff's gypsum blocks and V 8 agar. The spores are spherical with a warty wall and an oil drop inside.
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3. Fermentation of sugars:
Glucose Galactose +(weak or slow~
Sucrose +
Maltose +
Lactose

4. Assimilation of carbon compounds:
See Table 1

5. Splitting of arbutin: Positive

6. Assimilation of KN03: Negative : 7. Growth in vitamin-free medium: Positive 8. Growth at 37 C: Positive Assimilation of carbon compoun ds Glucose +
Galactose +
L-sorbose +
Sucrose +
~:~ Maltose +
Cellobiose +
Trehalose +
Lactose Melibiose +
:~ Soluble starch +
::~ D-xylose +

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Ethanol +
Glycerol +
Salicin +
Inositol In this invention, hydrocarbons having 8 to 18 carbon atoms are appropriate raw materials (substrate) for the pro-duction of dicarboxylic acids or a mixture of di- and mono-carboxylic acids, especially hydrocarbons of 11 to 16 carbon chain length are desirable. For the selective production of dicarboxylic acids, monocarboxylic acids and hydrocarbons having a skeletal length of 8 to 18 carbon atoms each axe suitable precursors, hydrocarbon skeletal lengths of 11 to 16 carbon numbers are preferred.
The oxidation reaction of this invention is a typical resting cell phenomenon and can be carried out in an aqueous buffer solution, as for example a phosphate buffer solution of pH 7.
The reaction can also be carried out in a growth medium which contains nutrient for the yeast. The reaction then becomes a cultivation-oxidation reaction. In this mode of operation, the media shall contain the usual nutrient material including an assimilable carbon source, nitrogen source, and appropriate vitamins and minerals, all well-known to those skilled in the art.

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Carbon sources appropriate to a growth medium can include for example glucose, sucrose, maltose, the substrate hydrocarbon or monocarboxylic acid, and the like.
Nitrogen sources can include inorganic nitrogen compounds as for example ammonium nitrate, ammonium phosphate, or the like; and organic nitrogen-containing materials as for example peptone, corn steep liquor, and amino acids.
Vitamins and minerals needed for growth can include sodium phosphate, calcium phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate, manganese sulfate as minerals and yeast extract and the like as vitamin-containing additive.
When the oxidation is carried out as a resting cell reaction, it becomes necessary to grow up a healthy cell mass prior to oxidation. This cell mass is best prepared by growing the yeast culture in the above growth medium prior to oxidation.
The whole cell culture including cells and nutrient material can be used in the oxidation reaction, or the cell mass can be removed from the spent nutrient by centrifugation or filtration prior to adding the cell mass to the substrate.
Thus, in this invention, the strain of carboxylic acid producing microorganisms, Deba~om~ces phaffii ATCC 20499, or a culture thereof or cells of the strain cultured previously are added to the medium containing the substrate to carry out the reaction and agitated, aerated through a nozzle, or shaken so that the microorganism can contact the components of the medium thoroughly.

The reaction temperature is kept at 25 to 35C and pH
is controlled at 3 to 9, preferably pH4 to 8. The period of time of reaction depends on the substrate to be used, but usually a reaction takes 24 to 120 hours to finish completely.
We have found it advantageous to use a mixed substrate containing at least some monocarboxylic acid for the reason that the monocarboxylic acid tends to increase solubility of the substrate in the aqueous reaction mixture and to supress accumulation of additional monocarboxylic acid during oxidation.
A readily available source of the monocarboxylic acid for this mixed substrate is from the accumulation of the acid from previous reactions.
When cultivation (reaction) is carried out as described above, a substantial amount of dicarboxylic acids or a mixture of dicarboxylic acids containing monocarboxylic acids is produced and accumulated. These carboxylic acids are separated and purified by a conventional method such as extraction, solid-liquid separation, neutralization-extraction and fractional distillation, and then harvested as monocarboxylic acids and dicarboxylic acids, or a mixture of both.
As illustrated by the preferred embodiment described below, the organism of this invention produces monocarboxylic acids and dicarboxylic acids in high yields by well known cultivation methods using the new microorganism. Therefore, it is believe~ that this invention contributes greatly to the 5~62 production of carboxylic acids with straight and long carbon chains.
Example 1 Medium composition for flask cultivation Sucrose 30g NH4C1 4g KH2P4 2g MgSO4~7H2O 0.6g 4 2 0.01g FeSO4.7H2O 0.01g Mycological peptone 0.5g Yeast extract 0.5g The components for pre-cultivation were dissolved in distilled water to make the total volume 1 liter and adjusted to pH6.5. One hundred milliliters of the resulting media was added to a 500 ml flask and sterilized in an autoclave at 115C for 15 min. Cells of Debaryomyces phaffii BR-151 which had been grown on malt extract agar at 30C for one month were inoculated (3 loopsful) to the above sterile medium and were cultured on a reciprocal shaker at 28C for 29 hours.
Medium composition for fermentation 2 4 10g NH4C1 5g MgSO4.7H2O 0.6g FeSO4.7H2O 0.01g 4 2 0.OQ8g Mycological peptone 0.5g Yeast extract 0.5g - The above-enumerated components for a fermentation medium were dissolved into distilled water to make the total volume 1 liter and sterilized at 115C for 15 minutes; 800ml of the medium and 110g of the appropriate hydrocarbons or carboxylic acids as shown in Table 2 were sterilized in an autoclave at 115C for 15 minutes.
Into a 2 1 fermentor were placed 100ml of the pre-cultivation medium, 800ml of the fermentation medium and 110g of sterilized reactant substrate. The mixture was allowed to reach with aeration and agitation at 30C for 96 to 120 hours at pH 6.5 to pH 7.5. 2N potassium hydroxide was used for pH
adjustment. When foaming was observed during the cultivation, ~,.
small quantities of a solution of 15~ defoaming agent tmanu-factured by Toshiba Silicone Co.; TSA 730) which had previously been autoclaved at 115C for 15 min. were poured into the culture medium. On finishing cultivation, solid potassium hydroxide was added to the culture broth to pH 10, the broth emptied from the fermentor, filtered under the reduced pressure using filter aid, and washed. Products were extracted by ether, methylated with diazomethane after the ether was removed and analyzed by gas chromatography.
The results are shown in Table 2.

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- ~ . ., Example 2 Components for the fermentation medium descri~ed in Example 1 as well as 50g. sucrose were dissolved in 1 liter distilled water and adjusted to pH 5.5. Fifty milliliters of the medium were placed in a 500ml shaking flask and inoculated with 2 loopsful of the DebaryomYces Phaffii strain used in Example 1, and growth was maintained at 30C for 26 hours. The obtained culture broth was centrifuged in order to separate the cells (about lg dry weight). A reaction solution was prepared by mixing lOOml of 0.5M phosphate buffer (pH 7.0) with lOml of normal dodecane. A reaction was carried out between this reaction solution and the filtered cells at 30C for 72 hours. The reaction products were basified to pH 10 with potassium hydroxide and were analyzed using the same process as in Example 1. As a result, 5.3mg/L of lauriG acid and 4.lmg/L
of l,10-decane dicarboxylic acids were produced in the reaction medium. For this experiment, the medium, substrate, and the equipment were autoclaved previously so that the experiment could be carried out under sterile conditions.

Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In the production of C8-C18 mono-and di-carboxylic acids from, in the case of mono-carboxylic acids, straight chain hydrocarbons wherein the hydroarbon and the resulting acid have the same number of carbon atoms in the skeletal chain, and in the case of di-carboxylic acids, from straight chain hydrocarbons and mono-carboxylic acids wherein the hydrocarbon and the mono-carboxylic acid have the same number of carbon atoms in the skeletal chain, by aerobically cultivating the hydrocarbon or mono-carboxylic with a micro-organism in a nutrient medium, the improvement which comprises using as the micro-organism Debaryomyces phaffii ATCC 20499.

2. In the production of C8-C18 mono-and di-carboxylic acids from straight chain hydrocarbons wherein the hydrocarbon and the resulting acid have the same number of carbon atoms in the skeletal chain by aerobically cultivating the hydrocarbon with a micro-organism in a nutrient medium, the improvement which comprises using as the micro-organism Debaryomyces phaffii ATCC 20499

3. In the production of C8-C18 dicarboxylic acids from mono-carboxylic acids wherein the mono-carboxylic acid and the resulting di-carboxylic acid have the same number of carbon atoms in the skeletal chain by aerobically cultivating the mono-carboxylic acid with a micro-organism in a nutrient medium, the improvement which comprises using the micro-organism Debaryomyces phaffii ATCC 20499.

4. In the production of C8-C18 di-carboxylic acids from straight chain hydrocarbons and mono-carboxylic acids wherein the hydrocarbon and mono-carboxylic acid and the resulting di-carboxylic acid have the same munber of carbon atoms in the skeletal chain by aerobically cultivating the hydrocarbon and the mono-carboxylic adic with a micro-organism in a nutrient medium, the improvement which comprises using as the micro-organism Debaryomyces phaffii ATCC 20499.

5. In the production of C8-C18 mono-and di-carboxylic acids from straight chain hydrocarbons wherein the hydrocarbon and the resulting acid have the same number of carbon atoms in the skeletal chain by growing a micro-organism in a nutrient medium, separating the cell mass comprising the micro-organism and allowing the cell mass to react aerobically with the hydrocarbon, the improvement which comprises using as the organism Debaryomyces phaffii ATCC 20499.