DE2164626B2 - Microbiological process for the simultaneous production of a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and an (omega-1) -keto fatty acid - Google Patents

Description

fc are acrylic acid, salts thereof or Anionenaus-Sifauschharze or mixtures of acrylic acid and / 'or salts thereof with an anion exchange resin * "" 5 is preferably the culture medium in which encryption -'"" ^a ung added, the

_β dicarboxylic acids, omega

and (omega-l) -ketocarboxylic acids L value increased compared to that Additives is used. Normal paraffins with at least 10 coal ends Microorganisms-Si

Recycle it as the only source of carbon.

The strain Corynebacteriu- - * "" "" ■

(MC-1-l-Stanan) was also

at the Fermentation Research Institute,

a

deposited

and include

' ^s deposited.

«- usually 10 to 100 g, preferably 15 to 80 g, and in particular 20 to 70 g per liter of culture medium.

Any nitrogen source that is suitable for growing micro-reanisms can be used as a nitrogen source in the method of the invention. Examples of nitrogen sources are: alkali metal nitrates, sodium nitrate or potassium nitrate; Ammonium salts of inorganic acids, such as ammonium nitrate, ammonium sulfate, ammonium chloride or ammonium phosphate; Ammonium salts of organic acids, ammonium acetate, ammonium lactate or ammonium tartrate; Urea; Ammonia; Yeast extracts; Schextracts and corn steep liquor. These compounds can be used alone or in the form of a mixture of nitrogen sources. The amount of nitrogen source used is usually between about 0.1 and 10 g and preferably between 0.5 and 8 g per liter culture medium. 'ßSiele for mineral salts ^ ₅ R, in the method of the invention can be used, S ₂ PO ₁ Na ^ HPO _4, Na ₃ PO _4, KH ₂ PO ₄ are K ₂ HPO _4, ν PO CaHPO _4, and other phosphates. They are used either singly or in mixtures used may also magnesium, iron, manganese, CaI-m Kuofer- zinc, molybdenum and boron salts are used by the ion rganischen acids ⁸ metals Beta .. They are ^^ ^ g ^ g

^d d ^e MtfO 7 ⁿ f ₄ 0 Test ^ 7to Cacl SS) ^ a (OH) ₂ ⁴ MnSO? - ' ₅ H ₂ O, ¹ CuSO ₄ - 5 H ₂ O, CuCl ZnSO ₁ -THA MoO ₃ , H ₃ BO ₃ and sea

of the invention by incorporating at least Corynebacterium dioxydans nov. sp. Strain MC-1-1 (ATCC 21766)

1. Rods, 0.6 to 1.2 by 3.0 to 9.5 microns. Cell branching and coke-like shapes are determined.

No spore formation. Not able to move by itself. Gram positive.

2. Nutrient agar colony: circular, smooth, undivided, convex raised, pale pink to orange.

3. Nutrient agar slope: moderate growth, filiform, shiny, reddish pink.

4. Nutrient broth: fragile membrane, cloudy, excess sediment.

5. Nutrient gelatinous: no liquefaction.

6. Litmus milk: alkaline, not peptonized.

7. B.C.P.-Müch: alkaline, not peptonized.

8. No indole formation.

9. No formation of nitrite from nitrate.

10. Hydrogen sulfide formation.

11. Starch is not hydrolyzed.

12. Methyl red test: negative.

13. No acetylmethylcarbinol formation.

14. Catalase: positive.

15. No ammonia formation.

16. Citrate is used as the only source of carbon.

17. Aerobic growth.

18. Optimal temperature 30 to 35 ⁰ C.

19. Growth at pH 5.0 to 9.0.

20. Acid formation from glycerine, glucose, fructose ¹ and mannitol.

Neither acid nor gas formation from xylose, lactose, sucrose, starch, maltose, galactose, mannose, Arabinose and cellulose.

The above-mentioned microbiological properties were examined according to the classification standards described in Bergey's Manual of Determinative Bacteriology, 7th Edition, and it was established- "* ~ - - ¹ -"""""'nnts strain to the genus

or calcium salt, una nmuuunu. "

preferably by incorporation of at least acrylic acid or one of its salts and at least one Anion exchange resin, increased in the culture medium

will.

Any commercially available anion exchange resin e.g. B. a quaternary ammonium salt, a primary Amine, a secondary amine, a tertiary amine

or has a polyamine as an exchange group and the teriology, 7th edition ,untersucm, unu ^

Carries out the reaction of the anion exchange, it can be established that the above-mentioned strain can be used for the genus. The anion exchange resin may belong to Corynebacterium. The morphological and z. B. in the hydroxyl form, the chloride form, the -'-- ■ - = ~ i ~~; ^ »« »n F.ie features of the ^above & ™phia ^ weak acid form, such as phosphoric acid, citric -

provided that the above

Belongs to Corynebacterium. The morphological and physiological characteristics of the above Tribal were made with the descriptions in Bergeys

^ · -Lrrnbioloffschen properties of the above-

Manual, but no identical JJ ^^^ ims ^ den according to Bergey's Manual of

Strain that would be described therein can be found. fP ^ SüveBacterioiogy, 7th edition examined,

DTabove mentioned trunk differs from Detemunative ^^ ^ _{da obe? G} ^ tribe

Corynebacterium glutamieum nov. sp., Corynebac- and es wura ^ Corynebactenum belongs D, e

teriumlUium, nov. sp., Corynebacterium petrophflum 5 qm ) £ * and physiological properties

nov. sp. and Corynebacterium aceioglutaimcum nov. ^ gSKitai tribe were labeled with the

sp. in size, different breeding properties as _{compared to the above manual} , it could ^ e-

ten, of the effect on milk, of the formation of Schwe- $ ™ fJj ^ "Scher tribe, which then dies

hydrogen or the fermentation of carbohydrates ^ S found.

Furthermore, the above-mentioned stem also differs from "ware. JJ * ¹ ? ^ Differs from Corynebacte-

of CorynebRCterium hydrocarbodastus nov sp. in J ^^^ ätam nov. sp., Corynebactenum aceto-

the size, the breeding characteristics, the ability "" f? "™ ^P _{n0V sp} , Corynebactenum hhum nov. sp.

NTtmzu7eduzieren, and ^ erFälugkeitKomenh _y drate ^^^ Seriunx aStoacidophilum nov. _Sp . in

to ferment. Furthermore, sie ^ _Γ ^ Γ _ν οη differentiates between carbohydrates and the BJdung von

the above-mentioned strain of Corynebactenum au- 15 g ^ poor ^ stoff. _{£ r is also different} from

rantiacum nov. sp. and Corynebacterium roseum nov. f ^ i ^ him hydrocarbodastus nov sp., Coryne-

sp. in size, the breeding characteristics of the ^^^ t _aTa3iae hy ^ m nov. sp. and Corynebacte-

Ability to reduce nitrate, ^ r Khigke.t Schwe- ^ actenum paraa. ^ _{nov sp d} fermentation of

to form hydrogen, or the ability to produce carbon- "™ 2 ° £ ^ _the effect on milk, the reduction

to ferment hydrates. Consequently ^ became the 20 ^ ^onl ^ ^y J, _itrat 'etc., and from Corynebacterium au-

MC-1-l strain (FERM P No. 690; ATCC 21766) as a tion. _{Corvnebacterium} acetoph.lum

to a new species of the genus Corynebactenum to- ™ * ™ °. _Cory nebacterium roseum nov. sp. in the

well respected. This became Corynebactenum di- nov ^ u ^ _ohlenhydrat en, the decomposition of oxydans nov. sp. called.

able to move by itself. Gram positive. _special on $ 6? £££ & £

2. Nährstoffagarkolonien: smooth circular, un- usually carries 20 ^ Müc _the divided to wavy, convex increased orange 35 37 C Wi d ^ e ^ particular ι ^ _{u obtained.} , _turns to reddish-orange. ψ ^d ™ ** ^ £ f _{c Zvi} hen about 23 and 30 ^e C forthcoming

3. Nährstoffagarschräge: _bet overgrowth, FIU ^ ^ ι ν _{breeding time} is usually about rägt shape, shiny, reddish-pink. ₂ * * _If desired, breeding can be more than

4. Nutrient broth: fragile membrane, clear for _{8 days} , for example 10 days to 2% of excess sediment. SfehTat "bSdnSΑη ^ ΛΓ _ε Αΐ

5. Nutrient gelatin stick: no liquefaction. wihnheh ™ Autovermietung _γ ^e ^ _{h the education and} accumulative

6. Litmus milk: unchanged. _{tion d} i _{ese 'r} 3 acids in the culture medium in full environmental

7. B-CP. Milk: unchanged. 45 ^^^^ _{in every} known way

8. No indole formation. be performed. A culture medium similar to the above

from NM, .us NU, _b ** -% »% £% £ & £

10. Hydrogen sulphide formation. Value and then through compress

11. Starch is not hydrolyzed. _th water vapor at a temperature of beispids

13. No acetylmethylcarbinol formation. _{Step Example} else _may performed also in the manner _SW i

14. Catalase: positive. ₅₅ be that one for several days at 100 ° C for

. _A. , .... iew »Us sterilized intermittently for 1 hour. Attach

15. No ammomak formation. J ^ - _{becomes one of the above} named tribes in the

16. Citrate is inoculated as the only carbon source in the _{culture medium} and at the temperature devalued above under aerobic conditions (both

17 Aerobic growth 60 ₅ as a shaking culture or as a culture that

17. Aerobic growth. P ^ air supply is mixed) bred.

18. Optimal temperature: 30 to 35 C. _{As stated} above, the common

19. Growth at pH 6.0 to 9.0. Use of at least one of the compounds

«4

Sterilize and add them to the culture medium before or after 2 to 3 times higher in the event that an anion degenerates Inoculation of one of the above-mentioned strains or exchange resin is added and more than about Smal to be added during breeding. The preferred higher in the case that acrylic acid or its salts

Concentration of these additives is about 0.1 and an anion exchange resin is added together

up to 5 g acrylic acid per liter of culture medium. In particular, 5 will be.

the other will be a concentration between about 0.5 and

1.5 g of acrylic acid per liter of culture medium are preferred. play explained in more detail. In a special embodiment of the method

according to the invention, in which only acrylic acid or examples to 4 and comparative example 1

whose salts are added, a suitable io

Amount of acrylic acid or its salts 10 minutes 1 g ammonium nitrate, 0.6 g monopotassium phosphate, Sterilized at 120 ° C and after the start of cultivation 4 g of dipotassium phosphate, 0.5 g of magnesium sulfate, 10 mg processing, for example 1 to 48 hours after the start of iron (II) sulfate, 30 mg calcium chloride, 10 / ig manganese cultivation, admitted. chloride and 100 mg of yeast extract were in one liter

If only an anionic exchange resin or acrylic ts tap water is dissolved and the solution is brought to a pH

acid or its salts and an anionic exchange value of 7.0. 50 ml portions of the obtained

resin is added, the cultivation can be carried out in the same way.

be performed. pour. 1.5 ml of n-tridecane was added to each flask as

After the cultivation is complete, the culture carbon source is introduced and. the solution 20 Mi-

The liquid is made acidic, and the fat grooves obtained are sterilized at 120 C.

acids from the culture liquid using As an anionic exchangeable that the culture

an organic solvent such as ether or medium was added, a weakly basic

Benzene, extracted. The extracted fatty acids are used as an anion exchange resin. The resin

then with a sodium hydroxide solution or was first converted to the hydroxy form by

extracted from another alkali solution, with a strong a $ 5 times the volume of a 1N sodium hydroxide

ken acid, such as hydrochloric acid or sulfuric acid solution, was used and then with the

acidified and then washed with an organic lo- lOfold volume of distilled water

solvent such as ether or benzene extracted. After that it was. It became 5 times the amount of a 5 "/ igen

the solvent is removed by evaporation, phosphate buffer solution (pH 7.0), which is made of monopotassium

where the above-mentioned three fatty acids consisted of 30 phosphate and disodium phosphate, added,

will. to convert the resin to the phosphate form. That

If an anion exchange resin is used alone or as- Resin was then combined with ten times its volume used with acrylic acid or its salts, washed amount of distilled water. 10 ml each the culture medium after the end of the cultivation parts of the resin were separated into a 100 mlvation 35 Erlenmeyer flasks introduced from the ion exchange resin by filtration and during one separates. The exchange resin is then intermittently at 100'C for a period of 3 days a mixture of an aqueous solution of a strong sterilized for 30 minutes each. The sterilized Lö-Acid, such as hydrochloric acid or sulfuric acid, with a solution were the culture medium before the one-water miscible organic solvents, such as inoculation, added aseptically.

Methanol or acetone, treated to make the fatty acids, 40 was then Corynebacterium dioxydans adsorbed on the ion exchange resin, out-ATCC 21766 20 hours in the above to solve. After removing the organic Lö- culture medium grown, and the resulting solution in solvent is aseptically inoculated from this liquid by evaporation of an amount of 5% each, It was shaken culture at 30 ° C with 98 Wecheinem in a similar manner as above organic solvents such as ether or ben- «be run per minute (amplitude 70 mm), and zoL extracted, and after removing the solution after 24 hours, 50 ml of 10 minutes at by means of sodium acrylate sterilized in the raw state at 120 ° C, the three fatty acids are converted into 50 ml of des obtain. Raw fatty acids can also be added aseptically through the culture medium. Filtering off the fatty acids from the aqueous phase. The cultivation was carried out 120 hours after the maintenance are broken off after the removal of the organ- 50 vapor from the cells. The culture medium, see solvent remaining. to which neither the ion exchange resin nor the sodium

The crude fatty acids are added with a suitable acrylate (Example 1) and that

Alcohol, such as methanol, is esterified and added through a culture medium to which only sodium acrylate is added

known separation process, e.g. B. by distillation (Example 2), were concentrated with

under reduced pressure, made strongly acidic in the dimethyl ester of 55 sulfuric acid and 3 times with the

straight-chain dicarboxylic acid, a methyl ester of the amount of ether corresponding to the culture medium

omega-hydroxy fatty acid and extracted in a methyl ester of the. The extracts were combined and 3 times with

(omega-l) -keto fatty acid separated. extracted from a 1N sodium hydroxide solution. The Na-

The procedures for obtaining the above-mentioned trium hydroxide-containing solution were then

Acids from the culture media are not made acidic with concentrated sulfuric acid

the invention. and then extracted with ether. The ether

The addition of acrylic acid or its salts and was using. dehydrated and anhydrous sodium sulfate

an anion exchange resin to the culture medium, after which the ether is removed by evaporation,

the method according to the invention are preferred. The crude acids were obtained.

Compared with the case that these compounds were not 65 On the other hand, the culture medium became to which only

are added, the amounts of crude fat- the ion exchange resin had been added, and

acids about 1.5 to 2 times higher in the event that the culture medium to which both the ion exchange

Acrylic acid or its salts are added, such as resin as well as the sodium acrylate have been added

409533/339

was (Example 4), by suction filtering into the culture liquid and the ion exchange resin separately. The resin was placed in a column, and that 40 times its volume of a mixture of equal amounts of 2N hydrochloric acid and methanol were through the The column flowed to determine the fatty acids absorbed on the resin; Was the methanol out Removed from this eluate by evaporation, the eluted fatty acids became insoluble. The extraction with Ether provided the raw acids.

For comparison purposes, the procedure of Example 4 was repeated except that Corynebacterram 7EIC strain (ATCC 13767) used. The results are given under Comparative Example 1.

The crude acids so obtained were weighed and the results are shown in Table I.

The acids were methylated with diazomethane, and the content of undecane-l, ll-dicarboxylic acid, 13-hydroxytridecanoic acid and 12-ketotridecanoic acid was measured. The results are given in Table I. The proportion of monocarboxylic acid (tridecanoic acid) was very little.

Dimethylundecane l.ll -dicarboxylate, methyl-13-hydroxytridecanate and methyl 12-ketotridecanate were obtained by gas chromatography, and the Melting points, mass spectra and the infrared absorption spectra are determined. These esters were saponified to form undecane-l.ll-dicarboxylic acid, 13-hydroxytridecanoic acid and 12-ketotr decanoic acid. The melting points, mass spectra and infrared spectra were measured. These measured values corresponded to those of standard products.

Table I.

Amount of formed ¹ ohen

(g / l culture medium)

Salary, determined by

Gas chromatography
Undecane-l.ll-dicarboxylic acid (g / l culture medium)

13-hydroxytridecanoic acid
(g / l culture medium)

12-ketotridecanoic acid
(g / l culture medium)

Example 1 without sodium

acrylate, without ion exchange resin

Example 2

Sodium acrylate added

Example 3

Ion exchange resin
admitted

Example 4
Sodium acrylate

plus ion exchange resin

admitted

3.32

0.72 1.48 0.44

5.05

1.01 2.08 0.68

9.60

2.04
4.20
1.16

16.20

3.85
7.24
1.80

Comparative example 1

Sodium acrylate

admitted plus

Ion exchange resin

2.43

0.73
0.24

. was used in an amount of 150 mg per 100 ml of culture

B eis ρ ι e 1 e 5 to 8 _{45 medium in 50 mg parts} at the same time as the addition

λ e ammonium chloride, 1 g of monopotassium phosphate, of the anion exchange resin were added. All in all

3 5ε disodium phosphate, 0.5 g magnesium sulfate, was shaken for 150 hours at 25 ° C

ICI me EisenennD-sulfate, 30 mg calcium chloride and 98 changes per minute (amplitude 70 mm) cultivated

100 me of meat extract were in one liter of conduit after the breeder's termination became the same

dissolved in water to form a culture medium. The 50 treatment as described in Examples 1 to 4,

βΉ value of the 'Kvuturmediums was carried out to 7.5 and the crude acids obtained. These

Then 100 ml parts in 500 ml acids were weighed. The results obtained

Shake flask inserted. Table Π shows each of these flasks. The acids were using

wSE3 mI n-octadecane are added and methylated at 120 ⁰ C diazomethane, and then the content

Sterilized for Ts minutes. Then 5 g of 55 hexadecane-UO-dicarboxylic acid, 18-hydroxystea-

Penton 2 5 ε meat extract, 2.5 g yeast extract and ric acid and 17-ketostearic acid determined. Which he-

1 liter of tap water with the formation of a culture product is shown in table Π. The amount

mediums with a pH of 7.0 mixed cory- an monocarboxylic acid (stearic acid) was very low.

nehacteriZ dioxydans ATCC 21766 was 10 hours- dimethylhexadecane-RO-di-carboxylate, methyl-18-hy-

The solution obtained with the cells 60 droxystearate and methyl 17-ketostearate were cultured by

«> ™ -Λ in Hi> flask separated in an inoculation lot by gas chromatography and recovered. the

^ l 2 ° / inoculated melting points, mass spectra and infrared spectra

^je ^ L ^S ct _a rV hasic anion exchange resin was measured in were. These esters were formed

λ ifvhen manner as described in Example 3, of hexadecane-UO-dicarboxylic acid, 18-hydroxystea-

sterilized and 5 ml of the resin were _{saponified in 3 portions 6 5} ric acid and 17-ketostearic acid, and the

Sch 24 48 or 72 hours to 100 ml culture melting points, mass spectra and infrared spectra medium 'given. Potassium acrylate, in the same
Sterilized like sodium acrylate in example 2,

these acids were determined. These values correspond to those of standard synthesized products.

Table II Example 5

without potassium acrylate, without ion exchange resin

Example 6

Potassium acrylate
admitted

Example 7

Ion exchange resin
admitted

Examples 8
Potassium acrylate

plus ion exchange resin

admitted

Amount of crude acids formed

(g / l culture medium) 3.04

Content determined by gas chromatography

Hexadecane-1,16-dicarboxylic acid

(g / l culture medium) 0.66

18-hydroxystearic acid

(g / l culture medium) 1.38

17-ketostearic acid

(g / l culture medium) 0.38

Examples 9 to 12

0.5 g urea, 1 g monopotassium phosphate, 3 g disodium phosphate, 0.5 g magnesium sulfate, 10 mg iron (II) sulfate and 100 mg meat extract were dissolved in one liter of tap water and the pH of the solution was adjusted to 6.5 . Then 50 ml portions were each placed in 500 ml shake flasks, and 1.5 ml of n-pentadecane was added to each of these flasks as a carbon source. Then 20 minutes at 12O ⁰ C was sterilized. A spoonful of Corynebacterium hydrocarbooxydans nov. sp. ATCC 21767, which had previously been cultivated for 24 hours in a slant culture (pH = 7.0) consisting of 10 g peptone, 5 g meat extract, 5 g yeast extract, 20 g agar and 1 liter tap water, inoculated into the flasks.

The ion exchange resin used was a strongly basic anion exchange resin which converted into the hydroxyl form and sterilized in the manner described in Example 3 was used. The addition to that Culture medium was carried out in such a way that after 12 hours after inoculation of the cells, 10 ml of the ion exchange resin was added to each shake flask. Acrylic acid was at 10 minutes 120 ° C and the culture medium in a 4.56

1.02
2.10
0.54

9.46

2.10
4.40
0.98

15.72

3.14
7.10
1.80

Amount of 25 mg per 50 ml of culture medium was added 18 hours after inoculating the cells. It was a shaking culture for 120 hours under shaking conditions of 120 changes per minute

s5 30 ⁰ C carried out.

After the completion of the cultivation, the products were prepared in the same manner as in Examples 1 to 4 described, treated. The obtained crude acids were weighed and the results are in Table III reproduced.

These products were methylated with diazomethane and the content was determined by gas chromatography of tridecane-l ^ -dicarboxylic acid, 15-hydroxypentadecanoic acid and 14-ketopentadecanoic acid measured. The results are shown in Table III. The proportion of monocarboxylic acid (pentadecanoic acid) was very low. Dimethyltridecane-1.lS-dicarboxylate, Methyl 15-hydroxypentadecanate and methyl 14-ketopentadecanate were obtained by gas chromatography and the melting points, mass spectra and Infrared spectra measured. Furthermore, these products were formed with the formation of tridecane-1.lS-dicarboxylic acid, 15-hydroxypentadecanoic acid and 14-ketopentadecanoic acid saponified The melting points, mass spectra and infrared spectra of these acids were measured. The values corresponded to those of synthesized standard products.

Table III Example 9

without acrylic acid, without ion exchange resin

Example 10

Acrylic acid
admitted

Example 11

Ion exchange resin
admitted

Example 12
Acrylic acid plus

Ion exchange resin
admitted

Amount of crude acids formed
(g / l culture medium)

Content determined by gas chromatography
Tridecane-1,13-dicarboxylic acid
(g / l culture medium)

15-hydroxypentadecanoic acid
(g / l culture medium)

14-ketopentadecanoic acid

(g / l culture medium)

2.04

1.04
0.04
0.32

3.15

1.42
0.08
0.48

6.20

3.00
0.12
1.05

10.34

5.40
0.16
1.48

Examples 13 and 14

2 g ammonium acetate, 0.6 g monopotassium phosphate, 4 g disodium phosphate, 0.5 g magnesium phosphate, 10 mg Eixen (II) sulfate, 30 mg calcium chloride, 10 μβ manganese chloride, 100 mg yeast extract and 1 liter of tap water were mixed to form a sewing medium . In each case 15 liters of the nutrient medium were placed in two 30 liter fermentation vessels. After adjusting the pH to 7.0, 450 ml of a mixture of 70% n-pentadecane and around 30% n-hexadecane were added. It was then sterilized with water at 120 ^{° C. for 20 minutes.} Then 5 percent by volume of a cell fluid obtained by preculturing Corynebacterium dioxydans nov. sp. ATCC 21766 was aseptically inoculated into the two 30 liter fermentation vessels.

3 liters of a weakly basic anion exchange resin was obtained in the same manner as in Example 3 described, treated, and after 20 minutes of sterilization at 120 ° C added to a fermentation vessel. No anion exchange resin was added to the other fermentation vessel. It became a breed in which was stirred by air supply, carried out at 30 ° C, the air flow rate Was 10 liters per minute and the stirring speed was 250 rpm. 24 hours after inoculation 15 g of sodium acrylate, which had been sterilized for 10 minutes at 120 ° C, were added to the fermentation vessel, in which the ion exchange resin was, added. The cultivation was continued for another 96 hours. The fermentation vessel, which did not contain ion exchange resin and sodium acrylate, became one Subjected to cultivation for 120 hours.

After the completion of the cultivation, the crude acids were weighed. The acids have been methylated and the amounts of tridecane-1,13-dicarbous acid, tetradecane -1,14 - dicarboxylic acid, 15 - hydroxypentadecanoic acid, 16-hydroxypalmitic acid, 14-ketopentadecanoic acid and 15-ketopalmitic acid were measured in a similar manner. The results are in Table IV reproduced. The proportions of monocarboxylic acids, pentadecanoic acid and palmitic acid were very low.

Dimethyltridecane-15-dicarboxylate, dimethyltetradecane -1,14 - dicarboxylate, 15 - hydroxypentadecanate and methyl 16-hydroxypalmitate, 14-methylketopentadecanate and methyl-15-ketopahnitat were obtained by gas chromatography and the melting points, Mass spectra and infrared spectra measured. These esters were saponified to give tridecane-1.13 - dicarboxylic acid, tetradecane -1,14 - dicarboxylic acid, 15 - hydroxypentadecanoic acid, 16 - hydroxypalmitic acid, 14-ketopentadecanoic acid and 15-ketopalmitic acid. The melting points, mass spectra and infrared spectra were measured. These values corresponded to those of synthesized standard pro-

lo ducks.

Table IV

Amount of educated Example 13 Example 14 * o crude acids without sodium Sodium acrylate > 5 (g / 15 1 culture acrylate, plus ions medium) without ions replacement resin Salary, definitely replacement resin admitted by gas chromatography s5 graphics Tridecane-l, 13-di- carboxylic acid 45.1 231.4 (g / 151 culture medium) 30 tetradecane-1.14- dicarboxylic acid (g / 151 culture medium) 15-hydroxypenta 6.8 33.8 35 decanoic acid (g / 151 culture medium) 16-hydroxypal 2.9 14.5 mitic acid 40 (g / 151 culture medium) 14-ketopenta 14.1 70.4 decanoic acid (g / 151 culture 45 medium) 15-ketopalmitine 6.1 30.5 acid (g / 151 Kul turmedium) 4.3 21.5 1.8 8.5

Claims (2)

fatty acid, straight-chain dicarboxylic acid and (omega-1) claims: "Ketocarboxylic acid on an industrial scale from n-paraffins using microorganisms. 1. Microbiological process for simultaneous delivery. gen production of a straight-chain dicarboxylic acid, 5 Accordingly, it is the object of the invention to create a combiner ^ omega-hydroxy fatty acid as well as a (ome- drive, in which at the same time a straight-l) -! keto fatty acid, each of these acids being the chain dicarboxylic acid, an omega -Hydroxyfatty acid has the same number of carbon atoms as that and an (omega-l) -keto fatty acid in high yield n-paraffin substrate in the culture medium, characterized by η-paraffins using an n-paraffin. Characterized that one Corynebacte- io assimilating strain who has the ability to produce this rium dioxydans nov. sp. ATCC 21 766 or Cory acids at the same time, produced willow,
nebacterium hydrocarbooxydans uov. sp. ATCC There were two new η-paraffin assimilating 21767 in a culture medium that contains a stick strain of the genus Corynebacterium, which is the Fastoff source, Mineral salts and an n-paraffin with the ability to at least have straight-chain dicarbons Contains 10 carbon atoms, among 15 acids, omega-hydroxy fatty acids and (omega-1) -aerobes Conditions breeds ketocarboxylic acid to form in large quantities, ge 2. The method according to claim 1, characterized thereby. records that the culture medium also contains a These 2 strains, Corynebacterium dioxydans nov. Additive from (! Er group acrylic acid, its sp.ATCC 21766 and Corynebacterium hydrocarbo salts and anion exchange resins. 20 oxydans ATCC 21767 differ from the ones before known η-paraffin assimilating strains. she have a distinct ability in certain ways have an oxidizing effect (diterminal oxidation) while assimilating η-paraffins as a carbon source, 25 and yield and accumulate large amounts of The invention relates to a microbiological straight-chain dicarboxylic acids, omega-hydroxy fatty acids for the simultaneous production of straight chain acids and (omega-iy-ketocarboxylic acids, which the gen dicarboxylic acid, an omega-hydroxy fatty acid with the same number of carbon atoms as the substrate in the and an (omega-l) keto fatty acid, each of which have culture medium. In particular, these have Acids the same number of carbon atoms possesses 30 2 strains the unique ability to be omega-hydroxy like the n-paraffin substrate in the culture medium. to enrich fatty acids in large quantities. This is It is known that fatty acids from η-paraffins surprisingly, since they have so far been very unstable Microorganisms are formed, e.g. B. were viewed by interim products, and it Yeasts of the genus Pichia (Agr. Biol. Chem. 29, 1009, it was difficult to obtain them in substantial quantities in 1965), by the strain 7EIC (ATCC 13 767) of a 35 brazen. unknown species belonging to the genus Corynebacterium The present invention thus consists in one heard that differ from the two microorganisms - microbiological processes for simultaneous Her-strains, the position in the method according to the invention of a straight-chain dicarboxylic acid, a can be used (see below), with regard to size and omega-hydroxy fatty acid and an (omega-l) -keto form and such properties as the fermentability 4 ° fatty acid, each of these acids having the same number differ from carbohydrates (J. Bacteriol, 85, has carbon atoms like the n-paraffin substrate 859, 1963), by bacteria of the genus Pseudomo- in the culture medium, characterized in that one nas (Nature, 198, 289, 1963) by yeasts of the genus Corynebacterium dioxydans nov. sp. ATCC 21766 Candida (J. Gen. Appl. Microbiol, 12, 119, 1966) and or Corynehacterium hydrocarbooxydans nov. sp. by molds of the genus Botrytis (J. of 45 ATCC 21767 in a culture medium containing a Stick-Japaa. Society of Agricultural Chemistry, 40, 364, stoff quelle, mineral salts and an η-paraffin with we-1966). At least 10 carbon atoms under aerobic. In these publications it is only described conditions breeding. that oiuega-hydroxy fatty acids, dicarboxylic acids or furthermore it was found that when adding Ketocarboxylic acids as metabolic intermediates 5 ° agreed additives, of which it is known that while the assimilation of η-paraffins by them alone can be applied to others Microorganisms can be detected. Products using microorganisms In J. Gen. Appl. Microbiol, 12, 119 (1966) can be obtained from other genera to which, in the case of the prescription, that in the cultivation of Candida rugosa drive according to the invention used culture JF101 in a medium, the 2.5 ml η-paraffin per 55 medium, the above 3 acids in large Containing 80 ml of an inorganic salt solution, 16.7 mg amounts are formed and these acids are equal to one another Dicarboxylic acid formed per liter of culture liquid can be formed early with increased yield, became. It was, however, the general view that it was also found that the common The turn of these particular additives into a particularly high amount of fatty acid, formed by microorganisms from η-paraffin, is very small. In particular, it leads 60 significant improvements
difficult to enrich an omega-hydroxy fatty acid, For example, it was known that dicarboxylic acids, because it is an unstable metabolic intermediate. derived from η-hexane and n-heptane, by resting It was not previously known that an omega-Hy cell from Pseudomonas could be produced using hydroxy fatty acid, a straight-chain dicarboxylic acid and acrylic acid are obtained (Biochim. Biophys. Acta, an (omega-l) -ketocarboxylic acid at the same time in large 65 84, 195, 1964). It is also known that p-toluene amounts are formed. carboxylic acid from p-xylene by Actynomyces under η-paraffins are easily available at low cost, using an ion exchange resin is formed and it is therefore desirable to use omega-hydroxy (Biotechnol. Bioeng. 10, 689,1968).