US3784445A - Culture medium and process for the biological production of alpha, omega-alkanedioic acid - Google Patents

Abstract

An aqueous culture medium comprising (1) 3 to 35 grams per liter of alkali metal, calcium nitrate, magnesium nitrate, strontium nitrate and/or beryllium nitrate; (2) 0.3 to 10 grams per liter of alkali metal phosphate; (3) 1 to 15 grams per liter of an alkali metal acetate, calcium acetate, magnesium acetate, strontium acetate and/or beryllium acetate; and (4) a nutrient source; and a process for the production of an alpha,omegaalkanedioic acid of 10 to 16 carbon atoms comprising (1) introducing a mutated strain of Corynebacterium into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of an n-alkane of 10 to 16 carbon atoms and a complemental amount of the culture medium above; (2) maintaining the composition at a temperature between 20* and 45*C. for 48 t0 96 hours; and (3) separating the alpha,omegaalkanedioic acid from the resulting mixture.

Description

United States Patent [191 Dahlstrom et al.

[11.] 3,784,445 [451 Jan. 8, 1974 1 CULTURE MEDIUM AND PROCESS FOR THE BIOLOGICAL PRODUCTION OF ALPHA, OMEGA-ALKANEDIOIC ACID [75] Inventors: Robert Victor Dahlstrom; James Herbert Jaehnig, both of Manitowoc, Wis.

[73] Assignee: E. I. duPont de Nemours and Company, Wilmington, Del.

22 1 Filed: Dec. 30, 1971 21 Appl. No.: 214,379

[52] US. Cl 195/28 R, 195/30, 195/100 [51] Int. Cl Cl2b 1/00 [58] Field of Search 195/114, 3 H, 28 R,

[56] References Cited OTHER PUBLICATIONS A Compilation of Culture Media for Cultivation of Microorganisms, M. Levine & H. W. Schoenlein, Williams & Wilkins Co., 1930, pp. 31 & 32.

Primary Examiner-Lionel M. Shapiro Assistant Examiner-R. B. Penland Attorney-William A. Hoffman [57] ABSTRACT An aqueous culture medium comprising (1) 3 to 35 grams per liter of alkali metal, calcium nitrate, magnesium nitrate, strontium nitrate and/or beryllium ni trate; (2) 0.3 to 10 grams per liter of alkali metal phosphate; (3) l to 15 grams per liter of an alkali metal acetate, calcium acetate, magnesium acetate, strontium acetate and/or beryllium acetate; and (4) a nutrient source; and a process for the production of an alpha,omega-alkanedioic acid of 10 to 16 carbon atoms comprising (1) introducing a mutated strain of Corynebacterium into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent volume of an n-alkane of rotate carbon atoms and a complemental amount of the culture medium above; (2) maintaining the composition at a temperature between 20 and 45C. for 48 t0 96 hours; and (3) separating the alpha,omega-alkanedioic acid from the resulting mixture.

5 Claims, No Drawings CULTURE MEDIUM AND PROCESS FOR THE BIOLOGICAL PRODUCTION OF ALPHA, OMEGA-ALKANEDIOIC ACID This invention relates to the preparation of al pha,omega-alkanedioic acid from n-alkanes through the use of bacteria. Particularly, this invention relates to the preparation of alpha,omega-alkanedioic acid of to 16 carbon atoms from n-alkanes of 10 to 16 carbon atoms by the use of a mutant of Corynebacterium. More particularly, this invention relates to a culture medium for use in the preparation of alpha,omegaalkanedioic acid from n-alkanes of 10 to 16 carbon atoms and a mutant of Corynebacterium and an improved process which utilizes the culture medium in the preparation of alpha,omega-alkanedioic acid of 10 to 16 carbon atoms.

lt was known that long chain alkanes could be oxidized utilizing Corynebacterium 7ElC (ATCC-l9067) to form their respective acids. However, only low yields and conversions were obtained in such processes. Culture media, of course, were utilized but none of these allowed for high yields and conversions of the alkanes to their respective alpha,omega-alkanedioic acid. Therefore, an improved culture medium which would allow for such improved yields and conversions was sought. An improved process was also sought to further improve the yields and conversions of the alkanes to the alpha,omega-alkanedioic acid.

Such a culture medium has been found. It is aqueous and comprises 1) 3 to 35 grams'per liter of a member selected from the class consisting of alkali metal nitrate, calcium nitrate, magnesium nitrate, strontium nitrate, beryllium nitrate and mixtures thereof; (2) a 0.3 to 10 grams per liter of alkali metal phosphate; (3) l to grams per liter of an alkali metal acetate, calcium acetate, magnesium acetate, strontium acetate, beryllium acetate and mixtures thereof; and (4) a nutrient source.

The improved process for the production of an alpha,omega-alkanedioic acid of 10 to 16 carbon atoms comprises (1) introducing a mutated strain of Corynebacterium capable of producing at high yield and conversion alpha,-omega-alkanedioic acid of 10 to 16 carbon atoms into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of an n-alkane of 10 to 16 carbon atoms and a complemental amount of the culture medium above; (2) maintaining the composition at a temperature of between about 20 to 45C. for 48 to 96 hours; and (3) separating the alpha,omega-alkanedioic acid from the resulting mixture.

Some preferred mutated strains of the Corynebacterium are on file with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852. These strains are referred to throughout by their ATCC number. The organism tentatively assigned to the genus Corynebacterium was first isolated by J. W. Foster (J. Bacteriology 85, 859 1963). The original culture was pink but after a series of mutations with chemical and physical mutagens the culture lost the capability of producing a pigment. The organism is a non-motile, gram rod with metachromatic granules. Snapping division produces angular and palisade arrangement of cells. Older cells often lose gram characteristic with granules still evident. The bacterium is an obligate aerobe and utilizes nitrates as sole source of nitrogen. The bacterium rods are 0.3 to 0.4 by 0.7 to 0.8 micron. They grow well on most common laboratory media, but grow better on the culture medium of this invention. Colonies of bacteria on tryptone glucose agar are dry, granular and small. Optimum temperature of growth is 37C.

The amount of metal nitrate utilized in the culture medium depends on the mutant of Corynebacterium which is used. A relatively large amount of nitrate is required for the bacterium utilizes it as the sole source of nitrogen. Preferably 6 to 30 grams per liter of the metal nitrate is present in the culture medium. The preferred nitrates are the alkali metal nitrates, in particular sodium or potassium. The most preferred concentration of the metal nitrate in the culture medium is 12 to 25 grams per liter.

The alkali metal phosphate is used as a buffer compo nent in the culture medium. A sufficient amount of the phosphate is present in the culture medium to cause it to have a pH of from 6 to 9. Therefore, the amount utilized will vary according to the other components in the culture medium. The preferred amount of alkali metal phosphate in the culture medium is l to 6 grams per 1i ter. Preferred phosphates are K IHOP. and NaH PO H O.

The metal acetate is critical as a component in the culture medium in that it significantly increases the yield of the alpha,omega-alkanedioic acid when the culture medium is used in the process of this invention. The preferred concentration for the metal acetate is from 2.5 to about 10 grams per liter. Preferred acetates are the alkali metal acetates, in particular, sodium and potassium.

The type of nutrient source in the culture medium is not critical. Examples of useful nutrient sources are tomato juice, broth solids, molasses plus yeast extract or cottonseed meal. The first two types of nutrient source are preferred. The amount of nutrient source utilized is normally from 1 to 5 grams per liter, preferably around about 2 grams per liter of the culture medium.

The balance of the culture medium is made up of water. This can be tap water or distilled water. Other components can be present in the culture medium if they do not impede the function of the main components. Some of such components are MgSO '7H O, CaCl (neither of these two should be used in large amounts or they made cause the phosphate to precipitate), FeSO, with ethylenediamine tetraacetic acid chelating agent, MnSO -H O, H BO Na MoO -2H O, KI, and ZnSO '7H O. It is preferred that no ammonium compounds be present for they erode the ability of the me dium to cause the bacterium to accumulate acid. Copper compounds can be included in the medium. However, they tend to give no advantage but instead result in reduced acid production The culture medium can contain emulsifier. This is not required but does normally render the culture medium more conducive to manipulation. Useful emulsifiers are substances such as Ethofats (Armour and Company, Chicago, Illinois) which is monofatty or rosin acid esters of polyoxyethylene glycols having the general formula Examples of other nutrient type additives which can be included in the culture medium are amino acids, vitamins, glucose, sugar, adipic acid, butyric acid and glutaric acid.

The culture medium which is discussed above is the medium which is used in the process for preparing the alpha,omega-alkanedioic acid in the full scale fermentation but is normally not used in the pregrowth of the bacteria. Pregrowth of the bacteria can be accomplished by placing a lyophile culture of freeze-dried bacteria on a tomato juice agar slant which contains proteins and carbohydrates, i.e., tomato juice extract and glucose. The bacteria grows for from 24 to 48 hours at 37C. on the slant. A loopful of the bacteria is then transferred to a large flask such as a 125 ml. Erlenmyer for pregrowth. In the pregrowth stage the bacteria normally grows on a solution comprising 5 weight- /volume percent molasses, preferably cane, 0.5 weight- /volume percent yeast extract, 0.15 weight/volume percent K HPO 0.06 weight/volume percent Nail- P0," H and water. Weight/volume percent means grams/- milliliters in percent terms, i.e., 5 grams in 100 mls. is 5 weight/volume percent.

The pregrowth flask is maintained at about 37C. and vibrated at 220 rpms on a shaker with a 1 inch throw for 24 hours. The volume of the material in the initial pregrowth flask is normally 25 ml. although this is not critical. The pregrowth medium plus bacteria are built up by successive spiking of larger and larger volumes. When a pregrowth volume is obtained which is about percent of the total volume of materials to be used to make the alpha, omega-alkanedioic acid, the pregrowth stage is completed. At this point the pregrowth medium should contain at least 1 X 10 organisms per ml. Normally, all the pregrowth medium is used to inoculate the culture medium and n-alkane.

The culture medium described above is useful in growing bacteria which produce acids from hydrocarbons. in particular, the culture medium is useful in preparing alpha,omega-alkanedioic acids from n-alkanes of 10 to 16 carbon atoms by using mutants of Corynebacterium. The process produces higher levels of conversion and yield on the n-alkanes of 10 to 14 carbon atoms. It is most useful on dodecane which produces l,l2-dodecanedioic acid. The n-alkane utilized can be one n-alkane or a mixture of such. A mixture of acids then can be produced by the process of this invention.

In the recitation of the process above it is stated that the Corynebacterium mutant is capable of producing at high yields and conversions the alpha,omegaalkanedioic acid. When the process of this invention is utilized, this statement means to produce alpha,omegaalkanedioic acid at yields and conversions so that the percentage of the n-alkane converted to the alpha,omega-alkanedioic acid is at least 6.8 mole percent; preferably at least 10 mole percent. This is calculated by multiplying the yield times the conversion. Conversion is defined as the percentage amount in moles of the n-alkane converted to other than the nalkane without consideration as to what it is converted to while yield means the percentage amount in moles of the n-alkane converted which is in the form of the alpha,omega-alkanedioic acid. Normally, when conversion is high, yield is lower and vice versa. Therefore, these can vary up and down but the product of the two is always greater than the above recited percentages.

The composition inoculated by the bacteria in the process normally comprises 1 to 50 percent by volume of the n-alkane with 5 to 20 percent by volume being preferred. The remainder of the composition is the culture medium as discussed above. Normally, the Corynebacterium mutant is added to the composition as part of an inoculum. This inoculum usually represents about 5 to 20 percent by volume of the composition plus the inoculum.

The pH of the composition, i.e., the culture medium and n-alkane, should be from 6 to 9 initially with the preferred range being from 7 to 8. The most preferred range is from 7 to 7.5. The temperature of the composition should be from 20 to 45C. both at the time the bacterium is added and during fermentation. The preferred range is 25 to 35C. Contact of the bacteria and the composition is for up to 96 hours. Higher yields and conversions are not obtained until about 48 hours and after 72 hours diminishing returns set in.

One method of separating the alpha,omegaalkanedioic acid from the resulting mixture is to (1) remove the cells and excess hydrocarbon from the aqueous phase by phase separation, (2) adjust the pH of the aqueous layer to 2.0 with concentrated sulfuric acid, (3) filter out the precipitate (alpha,omega-alkanedioic acid), and (4) wash the precipitate with water. The purity of the acid is normally 50 to 65 percent by weight when this procedure is followed.

Mutants of the Corynebacterium which are useful in the process can be prepared by the standard techniques of (1) high energy radiation; (2) chemical mutation; (3) lethal synthesis and combinations thereof. The two former techniques are preferred. The mutants referred to herein were prepared by one or a combination of the three methods.

The culture medium and process are useful in preparing the alpha,omega-alkanedioic acid. The acids are useful as intermediates in the preparation of polyamides which have low water absorption and good stiffness and which can be formed into fibers.

The following Examples are meant to illustrate but not to limit the invention. Parts and percentages are by weight unless otherwise specified.

EXAMPLE I F reeze-dried mutant of Corynebacterium sp. G 1 l-25, ATCC 21744 was grown on a tomato juice agar slant. A loopful of the Corynebacterium was transferred to 50 mls. of molasses-yeast broth in a 300 ml. shake flask (Erlenmyer). The broth had been previously autoclaved for 15 minutes at 121C. The composition of the molasses-yeast broth was as follows:

0.15 weight/volume percent K HPO.,,

0.06 weight/volume percent NaH PO -H O,

5 weight/volume percent cane molasses,

0.5 weight/volume percent yeast extract, and

remainder water. its pH had been adjusted to 7.0 to 7.1 with NaOH. The broth containing the Corynebacterium mutant was then incubated for 24 hours at 37C. in a 1 inch throw rotary shaker at 220 rpms. 25 Mls. of this broth was then transferred to 250 mls. of fresh molasses-yeast broth prepared as above in a 1 liter shaker flask. This broth was incubated for 24 hours at 37C. in a 1 inch throw rotary shaker at 240 rpms. This broth was then used as an inoculum for the main fermentation media test flasks.

The main fermentation test flasks were prepared as three days in a rotary shaker at 220 rpms. with a l inch follows: throw.

1. A salts and buffer medium was prepared in dis- After the incubation period, all flasks were adjusted tilled water and contained the following ingredients to the initial volume with distilled water. Ml. samin their respective concentrations: 5 ples were centrifuged for separation of the residual hy- 0.04 weight/volume percent MgSO -7H 0 drocarbon, cells, and aqueous fractions. The cells set- 0.3 weight/volume percent K HOR, tled between the oil and aqueous phases. The 1,12- 0.12 weight/volume percent Nal-l POrH O dodecanedioic acid assay of the aqueous layer was 5 weight/volume percent NaNO done by thin layer chromatography. Twenty-two milli- 0.003 weight/volume percent CaCl 0 .moles of l,l2-dodecanedioic acid were produced per liter of the materials in the fermentation test flasks at Ithe end of the fermentation. The percent (mole) yield Stock Solution Concentration .Tlf 3. E l Pid i l.l.?.'?lfi9lll?l9lf 0.2 volume percent of stock solution i L FeSO. ethylene- EXAMPLE n di i h i H tetraacetic acid 0.125 grams/100 mls. percent. Following the procedure of Example l, 1,12-

stock solution MnSO 'H,O so mgs/lOO mls. 2O dodecanedioic acid was produced from n-dodecane M i f: from a Corynebacterium sp. G 1 1-25, ATCC 21744.

0c 1. 3 13: 5 "figs/100 mg The amounts of sodium nitrate and sodium acetate volume t of were varied in the culture medium as shown in Table STOC SO [1011 Y I NZMMOOZHzO 43 mgslloo I. The results of the acid analysis are also shown. When 0.2 volume percent of the nitrate was being varied, the amount of sodium ace- 01 20 mes/100 .tate was 0.50 Weight/volume percent while when the stock solution acetate was being varied, the amount of sodium nitrate Znsoflmo 80 "ms/100 was 2.50 weight/volume percent.

TABLE I l,l2-Dodeca nedioic acid l: ConvermM/ Yield sion MEDIA VARIABLES liter (mole) (mole) l. laNaNO;=0.3l3% (weight/volume) 38 I8 72 2. %NaNO =0.625% (weight/volume) 38 I6 84 a. %NaN0,--1.25% (weight/volume) 1s 76 4. %NaNO,='-2.50% (weight/volume) 36 21 62 5. %NaC,H,-,O 0.O% (standard) I2 20 20 6. %NaC,H,0 =0.25% (weight/volume) 3| 22 7. %Nac,H,0,=0.50% (weight/volume) 34 25 54 8. %NaC,H,O,--l.00% (weight/volume) 20 2| 36 mM millimoles The pH of the salts and buffer medium was adjusted. EXAMPLE Ill] 9. 2.1. to .7 W. .N f M Following the procedure of Example l, 1,12-

dodecanedioic acid was prepared from dodecane using 6 Salts and buffer medium was dlluted with (115- the various Corynebacterium strains shown in Table ll.

tilled Water to 65 Percent of its Original Concentra' The weight/volume percent of sodium nitrate utilized To the diluted Salts and buffer medium was was 2.5 rather than that of Example I. The results of the added 0.3 weight/volume percent tomato juice and run are shown in Table IL 0.5 weight/volume percent sodium acetate. 20 Mls.

of this solution were added into a 300 ml. shaker TABLE H flask with 0.1 ml. Ethofats (mono-fatty or rosin acid esters of polyoxyethylene glycols) to form the STRAIN g figa g g gii culture medium. 2.5 Mls. dodecane were also. Liter (mole) sion (mole) added to the shaker flask. The flasks were gauze Covered o t lit figtqlovot Corynebaclerium sp.

, G 11-25, ATCC 21744 46 40 4t Into the mam fermentation test flasts prepared above 41 28 49 were added 5 mls. of the inoculum broth prepared as 2? g2 22 above. The flasks were then incubated at 43 sg 7 so TABLE ll-Continued l, l Z-DODECANEDlOlC AClD mM millimoles We claim:

1. A process for the production of an alpha, omegaalkanedioic acid of to 16 carbon atoms which comprises (l) introducing a mutated strain of Corynebacterium capable of producing at high yield and conversion alpha, omega-alkanedioic acid of 10 to 16 carbon atoms into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of nalkane of 10 to 16 carbon atoms and from 50 to 99 percent by volume of an aqueous culture medium comprising (a) 3 to 35 grams per liter of a member selected from the group consisting of alkali metal nitrate, calcium nitrate, magnesium nitrate, strontium nitrate, beryllium nitrate and mixtures thereof, (b) 0.3 to 10 grams per liter of alkali metal phosphate, (c) l to grams per liter of a member selected from the group swea n fallsal nmst l. a ate a i m t magnesium acetate, strontium acetate, beryllium acetate and mixtures thereof and (d) a nutrient source; (2) maintaining the composition at a temperature of between about 20C. and 45C. for 48 to 96 hours and (3) separating the alpha, omega-alkanedioic acid from the resulting mixture.

2. The process of claim 1 wherein the n-alkane is from 10 to 14 carbon atoms, the amount of n-alkane in the composition is 5 to 20 percent by volume, and the amount of culture medium is to percent by volume.

3. A process for the production of an alpha, omegaalkanedioic acid of 10 to 16 carbon atoms which comprises (l) introducing a mutated strain of Corynebacterium capable of producing at high yield and conversion alpha, omega-alkanedioic acid of 10 to 16 carbon atoms into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of nalkane of 10 to 16 carbon atoms and from 50 to 99 percent by volume of a culture medium comprising (a) 6 to 30 grams per liter of a member of the group consisting of sodium nitrate and potassium nitrate, (b) l to 6 grams per liter of an alkali metal phosphate, (c) 2.5 to 10 grams per liter of a member selected from the group consisting of sodium acetate and potassium acetate and (d) a nutrient source; (2) maintaining the composition at a temperature of between about 20C. and 45C. for 48 to 96 hours and (3) separating the alpha, omegaalkanedioic acid from the resulting mixture.

4. The process of claim 3 wherein the n-alkane is from 10 to 14 carbon atoms, the volume of the nalkane in the composition is 5 to 20 percent by volume, and the amount of culture medium is 80 to 95 percent by volume.

5. The process of claim 4 wherein the n-alkane contains 12 carbon atoms.

Claims (4)

1. 2. The process of claim 1 wherein the n-alkane is from 10 to 14 carbon atoms, the amount of n-alkane in the composition is 5 to 20 percent by volume, and the amount of culture medium is 80 to 95 percent by volume.
2. 3. A process for the production of an alpha, omega-alkanedioic acid of 10 to 16 carbon atoms which comprises (1) introducing a mutated strain of Corynebacterium capable of producing at high yield and conversion alpha, omega-alkanedioic acid of 10 to 16 carbon atoms into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of n-alkane of 10 to 16 carbon atoms and from 50 to 99 percent by volume of a culture medium comprising (a) 6 to 30 grams per liter of a member of the group consisting of sodium nitrate and potassium nitrate, (b) 1 to 6 grams per liter of an alkali metal phosphate, (c) 2.5 to 10 grams per liter of a member selected from the group consisting of sodium acetate and potassium acetate and (d) a nutrient source; (2) maintaining the composition at a temperature of between about 20*C. and 45*C. for 48 to 96 hours and (3) separating the alpha, omega-alkanedioic acid from the resulting mixture.
3. 4. The process of claim 3 wherein the n-alkane is from 10 to 14 carbon atoms, the volume of the n-alkane in the composition is 5 to 20 percent by volume, and the amount of culture medium is 80 to 95 perCent by volume.
4. 5. The process of claim 4 wherein the n-alkane contains 12 carbon atoms.