US3383289A

US3383289A - Microbiological oxidation of alkylbenzenes

Info

Publication number: US3383289A
Application number: US509621A
Authority: US
Inventors: Richard L Raymond; Virginia W Jamison
Original assignee: Sun Oil Co
Current assignee: Sunoco Inc
Priority date: 1965-11-24
Filing date: 1965-11-24
Publication date: 1968-05-14
Anticipated expiration: 1985-05-14
Also published as: GB1111309A; BE690154A; NL6616545A; DE1593460A1; FR1505650A

Description

United States Patent 3,383,289 MICROBIOLOGICAL OXIDATION OF ALKYLBENZENES Richard L. Raymond, Wilmington, Del., and Virginia W.

Jamison, Prospect Park, Pa., assignors to Sun Oil Company, Philadelphia, Pa., a corporation of New Jersey No Drawing. Filed Nov. 24, 1965, Ser. No. 509,621 20 Claims. (Cl. 195-28) ABSTRACT OF THE DISCLOSURE C7-C1o organic acids which are methyl-substituted muconic acids and/or 2,3-dihydroxybenzoic acids are prepared by microbiological oxidation of C -C methylbenzenes having 1-4 methyl groups and at least two consecutive ring carbon atoms by the action of orthodihydroxylating and non-decarboxylating strains of Nocardia.

This invention relates to the fermentation of methylsubstituted benzene hydrocarbons under conditions resulting in the production of either or both of two types of organic acids. More specifically the invention pertains to the microbiological oxidation of methylbenzenes having 7-10 carbon atoms per molecule by means of specifically acting strains of microorganisms of the genus Nocardia. The strains used in accordance with the invention are characterized by their ability to produce from C C methylbenzenes either a methyl-substituted muconic acid or a dihydroxybenzoic acid or both as hereinafter described.

The microbiological oxidation of aromatic hydrocarbons by means of various types of microorganisms has been considered heretofore by several investigators. Numerous prior art reports dealing with this subject matter have been published. Davis and Raymond in an article appearing in Applied Microbiology, vol. 9, No. 5, September 1961, pages 383388, refer to various prior art publications on this subject. These authors therein and also in their United States Patent No. 3,057,784 have described the oxidation of alkylbenzenes by means of certain cultures of Nocardia. In all cases oxidation occurred only on the alkyl group, so that acidic products such as benzoic acid, phenyl acetic acid and phenyl acrylic acid were obtained depending upon the alkylbenzene selected. When the alkyl substituent had an odd number of carbon atoms, the principal oxidation product generally was benzoic acid, while for an even number of carbon atoms it was phenyl acetic acid. The results reported gave no indication that any strains of Nocardia could effect a direct hydroxylation of the benzene ring.

A recent review of microbiological oxidations of hydrocarbons, including alkylbenzenes, appears in the textbook Advances in Enzymology, vol. 27, pages 469-546 (Interscience Publishers, 1965). On page 496 the review authors point out that heretofore in microbiological oxidation of alkylbenzenes generally, most if not all microorganisms do not initially attack the phenyl ring but rather attack an alkyl substituent. In one instance it is reported that toluene was converted to catechol (1,2-dihydroxybenzene) by Pseudo'monas aeruginosa but the pathway involved initial oxidation of the methyl group leading to the formation of benzoic acid, followed by decarboxylation of the carboxyl radical and 1,2-dihydroxylation. No case is reported in which dihydroxylation of the phenyl ring of an alkylbenzene occurred without prior formation of the carboxyl radical from the alkyl substituent followed by decarboxylation thereof. Only in the case of benzene itself has it been found that some microorganisms are capable of effecting direct dihydroxylation of the ring.

In those cases where benzene itself has been oxidized microbiologically (see pages 506-510 of the aforesaid textbook) catechol is formed. It has been found that the metabolic mechanisms of a few microorganisms are capable of causing further oxidation of the catechol while also bringing about ring splitting between the adjacent hydroxyl groups. This has resulted in the formation of muconic acid usually in the form of the cis,cis-isorner. The overall conversion can be represented as follows:

0 O O H benzene catechol ois,cis-muconic acid While a few microorganisms have been shown to effect the conversion of benzene in this manner, no analogous conversion of an alkylbenzene appears to have been disclosed. Hence the preparation from an alkylbenzene of a l-carboxy-2,3-dihydroxybenzene having the same number of carbon atoms as the parent hydrocarbon or of an alkylsubstituted muconic acid appears to have been unknown heretofore.

We have now discovered that there are strains of microorganisms of the genus Nocardia which can in a novel manner oxidize methyl-substituted benzenes that have at least two consecutive unsubstituted ring carbon atoms. These hydrocarbon substrates used in the present invention are the C7-C10 methyl-substituted benzenes having 14 methyl groups and at least two adjacent ring carbon atoms containing no substituents. In one novel aspect a methyl group attached to a carbon atom next to the two unsubstituted carbons will convert to a carboxyl group while also diorthohydroxylation of the ring at the two adjacent open carbon atoms will occur. Further this will take place without decarboxylation occurring. The result of these metabolic reactions is the formation of 2,3-dihydroxybenzoic acid or homologues thereof depending upon the starting methylbenzene used. For example, from pxylene suitable strains of Nocardia can form 2,3-dihydroxy-p-toluic acid (referred to 'for convenience herein as DHPT). In such cases it appears that p-toluic acid (referred to herein as PTA) is a precursor in the metabolic sequence to DHPT and hence substantial amounts of PTA generally are accumulated whenever DHPT is produced. This is particularly true in later stages of the fermentation. Analogous accumulations of other non-hydroxylated benzoic acids also usually take place concurrently with the production of other 2,3-dihydroxybenzoic acids in this embodiment of the invention.

We have further discovered that some strains of Nocardia exhibit still another novel feature of oxidation for C -C methylbenzenes having at least two consecutive unsubstituted ring carbon atoms. These strains are capable of diorthohydroxylating the ring without any attendant oxidation of the methyl substituent and then causing ring cission between the hydroxylated carbon atoms. This ring splitting is the result of further oxidation which converts the hydroxylated carbons to carboxyl groups. Thus dimethyl-muconic acid (referred to herein for convenience as DMMA) can be produced by biological oxidation as illustrated by the following:

6 f V f m i H l o o o H O O O H O H I l O C O p-xylene 3,6-dimethyla,a-dimethylcatechol muconic acid (DMMA) The substituted catechol shown in the equation is a transitory intermediate in the microbiological reaction and generally does not appear in large amounts in the fermentation broth, although in some cases small amounts may accumulate and be present in the final product. As indicated, the DMMA obtained from the fermentation of p-xylene is in the form of its cis,cis-isomer. This isomer can readily be isomerized to the cis,trans-isomer and/ or the trans,trans-isomer under appropriate isomerization conditions.

It has been found that from C- -C methylbenzenes suitable strains of Nocardia will produce either 2,3-dihy droxybenzoic acids or a homologue of muconic acid or both. Either type acid will have the same number of carbon atoms as the parent hydrocarbon. The muconic acid homologue will also have the same number of methyl substituents as the parent hydrocarbon while the dihydroxybenzoic acid will have one less methyl group. By way of example, from p-xylene some strains of Nocardia will produce DHPT but no DMMA while some will produce both DHPT and DMMA. Among the latter strains some can be made to produce DMMA to the substantial exclusion of DHPT under selected fermentation conditions. As previously stated, whenever DHPT is produced, a substantial amount of PTA generally is also formed.

Suitable types of Nocardia for practicing the present invention are herein referred to as orthodihydroxylating and non-decarboxylating strains. By the term orthodihydroxylating is herein meant that the microorganism is capable of forming on the ring of the methylbenzene two hydroxyl groups which are ortho to each other and one of which is ortho to a substituted carbon atom of the benzene ring. This term, of course, does not indicate that the product ultimately formed in the fermentation necessarily contains any hydroxyl groups, as the muconic acid homologues in fact do not. The term non-decarboxylating" signifies that the microorganism does not cause destruction of carboxyl groups, which have been formed during the oxidation, by releasing carbon dioxide therefrom. Thus it is characteristic of fermentations practiced according to the present invention that any carboxyl group formed remains intact throughout the fermentation.

Strains of Nocardia which are orthodihydroxylating and non-decarboxylating have been found among various species occurring in nature, including species classified in accordance with Bergeys Manual as Nocardia corallz'na, Nocardia salmonicolor and Nocardia minima. For the present purpose appropriate strains of Nocardia corallina generally are preferred. Numerous attempts have been made to find among other known hydrocarbon-consuming genera strains which have similar orthodihydroxylating and non-decarboxylating characteristics. Among the genera tried are species of Brevibacterium, Pseudomonas, Streptomyces, Candida and Bacillus. Thus far, however, none of these has shown the desired characteristics that are exhibited by suitable strains of Nocardia used in practicing the present invention.

In accordance with the invention a methylbenzene of the C -C range is converted to an organic acid product by means of a Nocardia microorganism having the abovedescribed properties. The starting hydrocarbon can be any mono-, di-, trior tetramethylbenzene which has at least two consecutive unsubstituted ring carbon atoms. More particularly the following methylbenzenes can be used: toluene; 0-, mor p-xylene; pseudocumene; hemirnellitene; and prehnitene. The product acid is either a methyl-substituted higher homologue of muconic acid or a 2,3-dihydroxybenzoic acid or both. When a 2,3-dihydroxybenzoic acid is produced, it is generally associated with the corresponding non-hydroxylated benzoic acid which, as previously indicated, seems to be the metabolic precursor for the dihydroxylated product. The conversion is effected by subjecting the methyl-substituted benzene in the presence of a nutrient medium and under fermentation conditions to the action of an orthodihydroxylating and non-decarboxylating strain of Nocardia. After the desired fermentation oxidation has occurred, at least one of the acids of the foregoing types is recovered from the fermentation broth. For some of the microorganism strains both a muconic acid homologue and a 2,3-dihydroxybenzoic acid are produced and recovered, whereas in other cases substantially only one or the other of the product acid types is made.

The following table shows specifically the hydrocarbon substrates which can be used in practicing the invention and the acid products obtainable therefrom. The table lists the substrates and product acids both by name and by formula.

OBTAINABLE PRODUCT ACIDS Hydrocarbon Aromatic Acid Substituted Muconic Acid Substrate (I) (I) OH 2 r OH HOOOC=C-C=C-COOH I I C G (p-xylene) (2,3-dihydroxy- (a,a-dimcthyl muconic acid) p-toluic acid) (I) (I O OH O O OH r 6 OH HOOC-C=OJJ=C-COOH (Dseudocumcne) (2,3-dihydroxy-4,6-di- (u,a,5-trimethyl muconic acid) methyl benzoic acid) O- OH (2,3-dihydroxy4,5-dimethyl muconic acid) (I) C O OH C- C OH I i F 1 C- O OH HOOCC=CC=CC OOH (hemimellitene) (2,3-dihydroxy-5,6-di- (01,8,5' trimethyl muconic acid) methyl-benzoic acid) 0 C O OH 0 C OH I l 0 0 OH HOOC-C=CC=C-COOH (prchnitene) (2,3-dihydroxy-4,5,6- (a,a,/3,B-tetramethyl muconic acid) trimethyl-benzoic acid) From the table it can be seen that the dihydroxylating and non-decarboxylating strains of Nocardia used in practicing the invention can convert each of the methylbenzenes listed to an aromatic acid which is a 2,3-dihydroxybenzoic acid and/or to a muconic acid homologue which has one methyl substituent at the alpha position and which may or may not have other methyl groups depending upon which hydrocarbon substrate is used. For all substrates except pseudocumene only one dihydroxylated aromatic acid and/or one muconic acid homologue results from the metabolic reactions shown by these Nocardia strains. In the case of pseudocumene there are two dihydroxylated aromatic acid isomers which may appear, which isomers differ only in the position of one methyl group. It can be seen that all of the acid products have the same number of carbon atoms as the starting hydrocarbon, that the muconic acid type product has the same number of methyl substituents as the substrate and that the aromatic type acid has one less methyl substituent, the latter having been converted to a carboxyl group.

Specific microorganisms which have been used for the present purpose include the following:

(1) A wild-type strain obtained from soil in Alabama, having characteristics approximating those set forth for Nocardia coraltina in Bergeys Manual and hence classified as such species. A culture of this strain has been deposited with the American Type Culture Collection in Washington, D.C., under the number ATCC 19,070. Colonies of this microorganism have an orange color.

(2) A reddish colored mutant obtained by ultraviolet irradiation of ATCC No. 19,070. The mutant has also been deposited with the American Type Culture Collection and has been designated as ATCC No. 19,071.

(3) A strain isolated from Pennsylvania soil and likewise classified as Nocardia corallina. This microorganism is orange colored like the first-mentioned wild-type specimen but shows distinct differences in enzymatic oxidative characteristics as hereinafter described. A culture deposit of this strain has been designated as ATCC No. 19,148.

(4) A soil isolate having characteristics approximating those given in Bergeys Manual as Nocardia salmonicolor and hence so classified. A culture deposit has been identified as ATCC No. 19,149.

(5) Another soil isolate classified as per Bergeys Manual as Nocardia minima and designated as ATCC No. 19,150.

Conc., g./l. of H 0 2 MgSO -7H O Na CO 0.1 CaCl -2H O 0.01 MnSO -H O 0.02 FeSO 7H O 0.005 Na KPO 3.0 KH PO 2.0 Urea 2.0

This mineral salt composition normally would have a pH of about 7.1. When it is desired to carry out the fermentation at a pH below 7, as is the case when the object is to maximize the production of the muconic acid homologue, the amount of KH PO relative to Na HPO can be increased to reduce the pH to a lower level.

The process of the invention is generally carried out at a temperature within the range of -40 C. and preferably at 28-32" C. under aerobic conditions with agitation. The nutrient medium should have a pH in the range of 4 to 9 and more desirably 6-8. When the Nocardia strain in one capable of effecting ring splitting to form a muconic acid homologue, production of such acid can be maximized by maintaining the pH in the range of 6-7, with a pH of about 6.8-7.0 usually being best. When a dihydroxybenzoic acid is the preferred product, its formation can be favored by operating at a pH in the range of 7-8 and a level of about 7.8 is generally preferred.

In preparing a Nocardia culture for use in the present process, a sample of a suitable Nocardia strain from a slant is transferred to a shake flask containing mineral salts solution and a suitable carbon source for growth. The carbon source can be a suitable hydrocarbon such as hexadecane. saturates derived from kerosene or toluene, or a carbohydrate or hydrolyzed protein. Preferably the carbon source material is added periodically in small amounts during incubation. In some cases it may be desirable also to have growth-stimulating materials such as peptone, beef extract or yeast extract present, although this is often not necessary. In the case of the mutant ATCC No. 19,071 referred to above, such material should be supplied since this oragnism, unlike the parent wildtype ATCC No. 19,070, requires a source of the vitamin, p-aminobenzoic acid, at least for initial growth and such material can provide this growth factor. The mixture is incubated at 30 C. and hexadecane (or other carbon source material) is added from time to time as the cell growth takes place, preferably being added in increasing amounts. After an incubation period typically of 24 hours, the cells can then be used for purpose of the invention.

The fermentation can be carried out by subjecting the methylbenzene substrate in the presence of the nutrient medium to action of the Nocardia organism under either growth or non-growth conditions. When growth conditions are employed, a sample of the inoculum prepared as above described is added to a mineral salts medium in a fermentor and the cells are first grown at 30 C. on hexadecane, for example, for about 24 hours without any addition of the methylbenzene substrate. After good growth has been obtained, periodic additions of the methylbenzene, along with additional amounts of hexadecane to sustain growth, are made and the fermentation is continued until maximum yield of the desired dihydroxybenzoic acid and/or muconic acid homologue is obtained. A total fermentation time of 96 hours usually is typical for obtaining maximum product yield.

When the Nocardia organism is used under non-growth conditions for practicing the invention, cells grown as previously described are separated from the broth by centrifuging and washed with phosphate buffer solution and then are resuspended in phosphate buffer solution. The suspension is maintained at say 30 C. and the methylbenzene substrate is added periodically in incremental amounts or continuously while the mixture is being aerated and stirred. Addition of the substrate is continued until the fermentation has given an optimum yield of the desired acid product.

After the fermentation has been completed, the cells are separated from the broth by centrifugation and the clear broth can then be processed in any suitable manner for recovery of the acid products. In cases where a muconic acid homologue (e.g., DMMA) has been produced it can be separately recovered by acidifying the broth with a mineral acid (e.g., HCl) to a pH of say 2, whereupon the DMMA will selectively precipitate from solution and can be separated by filtration and then purified by water washing. Any DHPT and PTA present in the broth will remain in the aqueous solution and can be removed therefrom by extraction with a suitable solvent such as ether, dioxane or amylacetate. The DI-IPT and PTA can thereafter be separated from each other chromatographically employing an anion exchange resin. As an alternative procedure, the acidified aqueous solution obtained upon filtering out the precipitated DMMA can be evaporated to obtain a concentrate of DHPT and PTA and these products can then be separated from each other by extraction of the concentrate with a suitable selective solvent.

A preferred microorganism for practicing the invention to produce the muconic acid type of derivative is Nocardia corallina ATCC No. 19,070 mentioned above. Cultural and physiological characteristics which identify and distinguish this microorganism are as follows.

Staining characteristics:

Age24 to 168 hours Gram-gm+, granules Cell morphology:

Form-rod with branching in young culture (0-48 hrs.) Motilitynon-rnotile Size 24 hours1l.5 by 3-20 microns, branching 48 hours-0.5-1.5 by 1-5 microns, some branch ing 72 hours-1 by 1-2 microns Agar colonies:

Age-72 hours Form-Circular Elevationconvex Surface-butyrous Marginentire Chromogenesis-orange Agar stroke:

Age-72 hours Form-filamentous Consistency-butyrous Chromogenesis-light orange Nutrient broth:

Surface growthflocculent Subsurface growthnone Amountfair growth Sediment-granular, orange Gelatin stab:

Liquefaction-none Growth-none Potato dextrose agar:

Age72 hours Growthnone Potato slant:

Age-72 hours Chromogenesis-deep orange Consistency-butyrous Glucose agar:

Age-72 hours Chromogenesis-light orange, cream Consistency-butyrous Action on sugars:

'Maltose-no acid, no gas, very good growth Sorbitolno acid, no gas, very good growth Dextrose-no acid, no gas, growth Mannito1no acid, no gas, slight growth Lactose-slight alkaline, no gas, good growth L-arabinose-slight alkaline, no gas, growth Saccharose-no acid, no gas, growth Levulose-no acid, no gas, good growth Inosito1--slight alkaline, no gas, good growth Action on milk: Reaction-none; ring growth, orange Other characteristics:

Nitrites from nitrates Hydrogen su-lfide not produced Indole not produced No phenol or naphthalene utilized Starch not hydrolyzed Utilizes sodium and amonium salts as nitrogen source The mutant previously mentioned and identified as ATCC No. 19,071, like ATCC No. 19,070, also has ringsplitting characteristics and produces the muconic acid type of product. This mutant has the same identifying properties as tabulated above but, unlike the parent organism, requires a specific vitamin for growth, namely, paminobenzoic acid. In the fermentation of p-xylene the mutant is more prone to give an accumulation of PTA and DHPT in the broth than is ATCC No. 19,070, although it does not necessarily produce these products and in many cases will show substantial accumulation of DMMA only.

The following examples specifically illustrate embodiments of the invention. Product analyses for these runs were done by U.V. spectroscopy. Identification of the specific products obtainable had previously been ascertained employing elemental analyses and U.V., LR. and mass spectral procedures.

EXAMPLE I Nocardia corallina ATCC No. 19,070 was used to prepare a,u'-DMMA from p-xylene in a 40 1. fermentor operated in continuous manner as a vortexing system. A mineral salt solution of the approximate composition listed above was used and n-hexadecane was employed as the growth substrate. The mixture was inoculated with the organism and was stirred vigorously at about 30 C. while being aerated by suction of air into the vortex formed by the stirred mixture. First the organism was allowed to grow in the presence of the hexadecane together with trace amounts of p-xylene until about 5-6 grams of cells had accumulated. This required about 24 hours. Thereafter the fermentation was continued at 30 C. while continuously introducing a mixture of (by volume) 90% p-xyleue and n-hexadecane at a rate such that the p-xylene addition rate was in the range of 20-40 mL/hr. This maintained the p-xylene concentration in the fermentation broth in the range of 80-350 ppm. The pH during the run varied somewhat but generally was within the range of 6.5 to 7.0. Samples of the broth were taken at various times during the fermentation and were analyzed for contents of DMMA, DHPT and also PTA. No significant amount of either of the .latter two compounds was detected. Th DMMA results were:

10 Hours from start: a,oc'-DMMA, g./l. of broth 30 1.8 41 4.5 56 9.8 65 11.6

Under the conditions used the DMMA was essentially the only product accumulated. This shows that the organism ATCC No. 19,070 can be highly selective in making the muconic acid type product under appropriate conditions.

EXAMPLE II Another run was made under similar conditions as in Example I except that a lower rate of stirring was used and the fermentation was allowed to proceed for 106 hours. Analysis of the broth at the end of that time gave the following results:

a,a'-DMMA g./liter 13.4

PTA g./liter 1.1

DHPT Negligible EXAMPLE III pH: Max. yield of DMMA, g./l. 6.0 4.4 6.5 8.1 7 O 11.6 8 0 0.06

These results indicate that best production of the muconic acid homologue is obtained at a pH of the order of 7 and that product formation is markedly decreased at higher pH levels for this partciular microorganism. The same is true for ATCC No. 19,070.

EXAMPLE IV Nocardia salmonicolor ATCC No. 19,149 was employed in the fermentation of p-xylene with the objective of producing DHPT. The 40 l. fermentor was used with generally the same procedure as described in Example I. For about the first 36 hours the organism was grown on n-hexadecane at a pH of about 7.0 and thereafter the pH was kept in the range of 7.5-8.0 and a :10 mixture of p-xylene:hexadecane was continuously introduced at a rate that maintained the p-xylene concentration in the broth at 50-200 ppm. The products in this case consisted of DHPT and PTA only and their concentrations in the broth for three sampling times were as follows:

Cone, g

DHPT PTA Time from Start, hrs:

EXAMPLE V Three runs were made under conditions generally similar to those used in Example IV and again using ATCC No. 19,149, and the p-xylene concentration in the broth following the growth stage on n-hexadecane was controlled at levels of about 50, and 250 mg./l., respectively. The initial production rates of DHPT and Initial Production Yld. at 96 hrs., g./l.

Rate, g./l./hr.

DI-IIT PTA DHPT PTA The data show that increasing the xylene concentration within the limits tried suppressed the formation of PTA and increased the maximum yield of DHPT.

EXAMPLE VI This example illustrates the use of cells of Nocardia salmonicolor ATCC No. 19,149 under non-growth con- 20 dition in the bio-oxidation of p-xylene. First several batches of the cells were grown on n-hexadecane in a 40 l. fermentor at a pH of about 7 for 34 hours. The cells were recovered from the broth by centrifuging and then were suspended in a phosphate buffer solution containing only 5 Na HPO and KH PO in amounts to maintain pH at about 8. No source materials for nitrogen or trace elements were present. Two batches of the cells in buffer solution were prepared having cell concentrations respectively of about 5 and 15 g./l. A fermentation of each batch at C. was run under vortexing aeration conditions by continuously introducing to the suspension a 90:10 mixture of p-xylenezn-hexadecane at a rate such that the p-xylene concentration in the mixture was maintained at 200-300 p.p.m. In each run samples of the broth were taken at times of 16, 26 and 34 hours after the addition of p-xylene and were analyzed. Results were as follows:

Time from Product c0110., g/l. Addition of p-xylene, hrs. DHPI PTA Cell cone, g./l.:

EXAMPLE VII Shake flask runs were made using three different dihydroxylating and non-decarboxylating strains of Nocardia in the fermentation of p-xylene. Specifically these were the Nocardia salmonicolor ATCC No. 19,149 of Examples IV-VI, another strain of Nocardia corallina identified by ATCC No. 19,148 and a Nocardia mim'ma identified by ATCC No. 19,150. The procedure in these runs involved inoculating 100 ml. of mineral salts solution in a 500 ml. shake flask with the organism, adding 0.05 ml. of n-hexadecane, shaking at 30 C. for 24 hours, thereafter adding small amounts of p-xylene together with n-hexadecane from time to time and shaking for a total time of the order of 96 hours. The fermentation beers were then analyzed by U.V. absorptivity. The following tabulation shows the results obtained for duplicate runs with each strain of microorganism.

Product cone, gl../ Species ATCC N0.

DHPT PTA Salmonicolor 19, 149 0.30 1.15 0. 37 1. 08 Corallina 19, 148 0.27 1.13 0. 19 1. 07 Miuima 19, 150 0.15 0. 15 0. 15 0. 41

The data show that each of the foregoing strains also can dihydroxylate the benzene ring without concurrently causing decarboxylation. However, none of these strains seem to cause production of the muconic acid homologue, at least under the fermentation conditions here employed.

EXAMPLE VIII Two batch runs were made in stirred fermentors using Nocardia cor-allina designated as ATCC No. 19,070 and ATCC No. 19,071, respectively. In each run a mixture of 480 ml. of an anion exchange resin and sufficient mineral salts solution to make a total volume of 3000 ml. were used. The medium also contained 0.2% peptone and 0.1% beef extract and its pH was maintained at about 6.5. Following inoculation the organisms were allowed to grow on n-hexadecane for 36 hours and thereafter p-xylene was added in small amounts from time to time while the mixture was being stirred and aerated at 30 C. Each run was conducted for total hours and 24 ml. of p-xylene total were used. The kinds and amounts of products, including those absorbed on the anion exchange resin as well as those in the broth, were then determined, the combined results being as follows:

Product amount, g./l. of broth Product ATCC No. 19,070 ATCC No. 19,071

DMMA 12. 2 14. 6 O. 7 0. 8 1. 0 0. 8 0. 5 0. 6

3,6-dimethyleatech0l.

From these data it can be seen that both microorganisms, under the conditions used in these runs, can cause accumulation of both the substituted muconic acid product and the substituted 2,3-dihydroxybenzoic acid product. Further, the data also show that under some circumstances it is possible for the substituted catechol inter mediate to accumulate, although the proportion thereof is minor for the specific strains of microorganisms here used.

In the last-described example an anion exchange resin was, as stated, employed in the fermentation mixture during the fermentation. The use of such resin during fermentation causes an unexpected improvement in the yield of desired acid product. However, such use of resin is not claimed herein, as this constitutes the subject matter of the copending Humphrey and Raymond application Ser. No. 512,543, filed Dec. 8, 1965.

The foregoing examples illustrate the preparation of methyl-substituted muconic acids and/or 2,3-dihydroxybenzoic acids from C -C methylbenzenes as herein specified. While the examples are specifically directed to the bio-oxidation of p-xylene, fermentations with other methylbenzenes as herein specified give analogous results. For these other substrate hydrocarbons the fermentation reactions proceed by analogous metabolic paths, so that one or the other or both of these kinds of acids are produced whenever dihydroxylating and non-decarboxylating strains of Nocardia are employed to effect the fermentation.

The acids that can be prepared by the present invention are valuable products having various applications of commercial interest. For example, the substituted muconic acids, being di-terminal acids, are useful in the preparation of polymers of various types. The dihydroxybenzoic acids have utility as chelating agents, metal deactivators and dye intermediates.

We claim:

1. Method of producing organic acid having 7-10 carbon atoms, said acid being a methyl-substituted muconic acid or a 2,3-dihydroxybenzoic acid or both, which comprises subjecting a C -C methylbenzene having 1-4 methyl groups and at least two consecutive unsubstituted ring carbon atoms in the presence of a nutrient medium and under fermentation conditions to the action of an orthodihydroxylating and nondecarboxylating strain of Nocardia and recovering an acid of at least one of the aforesaid acid types from the fermentation mixture.

2. Method according to claim 1 wherein said fermentation conditions include a pH level above 7 and a 2,3- dihydroxybenzoic acid is recovered.

3. Method according to claim 1 wherein said fermentation conditions include a pH level below 7 and a methylsubstituted muconic acid is recovered.

4. Method according to claim 1 wherein said strain is a member of the species Nocardia corallina, Nocardia salmonicolor or Nocardia minima.

5. Method according to claim 1 wherein said strain is ATCC No. 19,070, ATCC No. 19,071, ATCC No. 19,148, ATCC No. 19,149 or ATCC No. 19,150.

6. Method according to claim 1 wherein said methylbenzene is p-xylene.

7. Method according to claim 6 wherein said strain is a member of the species Nocardia corallz'na, Nocardia salmonicolor or Nocardia minima.

8. Method according to claim 7 wherein 2,3-dihydroxyp-toluic acid is recovered.

9. Method according to claim 7 wherein a,a'-dimethylmuconic acid is recovered.

10. Method according to claim 7 wherein both 2,3-dihydroxy-p-toluic acid and a,a'-dimethylmuconic acid are recovered.

11. Method according to claim 6 wherein said strain is Nocardia corallina ATCC No. 19,070 or ATCC No. 19,071.

12. Method according to claim 1 wherein said methylbenzene is p-xylene, said strain is a member of the species Nocardia corallina, said fermentation conditions include a pH below 7 and u,a'-dimethylmuconic acid is recovered.

13. Method according to claim 1 wherein said methylbenzene is p-xylene, said strain is a member of the species Nocardia salmonicolor, said fermentation conditions include a pH above 7 and 2,3-dihydroxy-p-toluic acid is recovered.

14. Method according to claim 1 wherein said methylbenzene is a xylene and a monomethyl-Z,3-dihydroxybenzoic acid, a dimethylmuconic' acid or both are recovered from the fermentation mixture.

15. Method according to claim 14 wherein said strain is a member of the species Nocara'ia corallina, Nocardia salmonicolor or Nocardia minima.

16. Method according to claim 14 wherein said strain is ATCC No. 19,070, ATCC No. 19,071, ATCC No. 19,148, ATCC No. 19,149 or ATCC No. 19,150.

17. Method of preparing a,a'-dimethylmuconic acid which comprises subjecting p-xylene in the presence of a nutrient medium and under fermentation conditions including a pH level not substantially above 7 to the action of an orthodihydroxylating, non-decarboxylating and ring-splitting strain of Nocardia and recovering said acid from the fermentation broth.

18. Method according to claim 17 wherein said strain is of the species Nocardza corallina and said conditions include a. pH in the approximate range of 6-7.

19. Method according to claim 18 wherein said strain is ATCC No. 19,070 or ATCC No. 19,071.

20. Method according to claim 1 wherein said strain is ATCC No. 19,070 or ATCC No. 19,071.

References Cited UNITED STATES PATENTS LIONEL M. SHAPIRO, Primary Examiner.