US3383289A - Microbiological oxidation of alkylbenzenes - Google Patents

Microbiological oxidation of alkylbenzenes Download PDF

Info

Publication number
US3383289A
US3383289A US509621A US50962165A US3383289A US 3383289 A US3383289 A US 3383289A US 509621 A US509621 A US 509621A US 50962165 A US50962165 A US 50962165A US 3383289 A US3383289 A US 3383289A
Authority
US
United States
Prior art keywords
acid
nocardia
atcc
fermentation
xylene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US509621A
Other languages
English (en)
Inventor
Richard L Raymond
Virginia W Jamison
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sunoco Inc
Original Assignee
Sun Oil Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sun Oil Co filed Critical Sun Oil Co
Priority to US509621A priority Critical patent/US3383289A/en
Priority to GB43413/66A priority patent/GB1111309A/en
Priority to DE19661593460 priority patent/DE1593460A1/de
Priority to FR84787A priority patent/FR1505650A/fr
Priority to NL6616545A priority patent/NL6616545A/xx
Priority to BE690154D priority patent/BE690154A/xx
Application granted granted Critical
Publication of US3383289A publication Critical patent/US3383289A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/872—Nocardia

Definitions

  • 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.
  • DMMA dimethyl-muconic acid
  • DMMA 3,6-dimethyla,a-dimethylcatechol muconic acid
  • 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.
  • 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.
  • Suitable types of Nocardia for practicing the present invention are herein referred to as orthodihydroxylating and non-decarboxylating strains.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • microorganisms which have been used for the present purpose include the following:
  • 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.
  • This mineral salt composition normally would have a pH of about 7.1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Nocardia organism 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.
  • 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.
  • a muconic acid homologue e.g., DMMA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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:
  • the acids that can be prepared by the present invention are valuable products having various applications of commercial interest.
  • 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.
  • 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.
  • strain is a member of the species Nocardia corallina, Nocardia salmonicolor or Nocardia minima.
  • strain is a member of the species Nocardia corallz'na, Nocardia salmonicolor or Nocardia minima.
  • strain is Nocardia corallina ATCC No. 19,070 or ATCC No. 19,071.
  • methylbenzene is a xylene and a monomethyl-Z,3-dihydroxybenzoic acid, a dimethylmuconic' acid or both are recovered from the fermentation mixture.
  • strain is a member of the species Nocara'ia corallina, Nocardia salmonicolor or Nocardia minima.
  • strain is ATCC No. 19,070, ATCC No. 19,071, ATCC No. 19,148, ATCC No. 19,149 or ATCC No. 19,150.
  • 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.
  • strain is of the species Nocardza corallina and said conditions include a. pH in the approximate range of 6-7.

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
US509621A 1965-11-24 1965-11-24 Microbiological oxidation of alkylbenzenes Expired - Lifetime US3383289A (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US509621A US3383289A (en) 1965-11-24 1965-11-24 Microbiological oxidation of alkylbenzenes
GB43413/66A GB1111309A (en) 1965-11-24 1966-09-28 Microbiological oxidation of alkylbenzenes
DE19661593460 DE1593460A1 (de) 1965-11-24 1966-11-11 Verfahren zum mikrobiologischen Oxydieren von Alkylbenzolen
FR84787A FR1505650A (fr) 1965-11-24 1966-11-24 Oxydation d'alcoyl-benzènes par des micro-organismes
NL6616545A NL6616545A (enrdf_load_stackoverflow) 1965-11-24 1966-11-24
BE690154D BE690154A (enrdf_load_stackoverflow) 1965-11-24 1966-11-24

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US509621A US3383289A (en) 1965-11-24 1965-11-24 Microbiological oxidation of alkylbenzenes

Publications (1)

Publication Number Publication Date
US3383289A true US3383289A (en) 1968-05-14

Family

ID=24027416

Family Applications (1)

Application Number Title Priority Date Filing Date
US509621A Expired - Lifetime US3383289A (en) 1965-11-24 1965-11-24 Microbiological oxidation of alkylbenzenes

Country Status (6)

Country Link
US (1) US3383289A (enrdf_load_stackoverflow)
BE (1) BE690154A (enrdf_load_stackoverflow)
DE (1) DE1593460A1 (enrdf_load_stackoverflow)
FR (1) FR1505650A (enrdf_load_stackoverflow)
GB (1) GB1111309A (enrdf_load_stackoverflow)
NL (1) NL6616545A (enrdf_load_stackoverflow)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3458399A (en) * 1966-08-31 1969-07-29 Sun Oil Co Fermentation of hydrocarbons
US3518196A (en) * 1967-12-18 1970-06-30 Sun Oil Co Ultraviolet stabilized petroleum hydrocarbons
US3523894A (en) * 1966-07-18 1970-08-11 Sun Oil Co Corrosion inhibitors
US3629072A (en) * 1970-11-09 1971-12-21 Texaco Inc Microbiological process for preparation of internal monoalkenes
US3645847A (en) * 1968-02-08 1972-02-29 Sun Oil Co Microbiological hydroxylation of aromatic acids
US3991112A (en) * 1970-06-23 1976-11-09 Sun Research And Development Co. Methylated muconic acid hydrazides
US4355107A (en) * 1981-07-27 1982-10-19 Celanese Corporation Production of muconic acid
US4588688A (en) * 1981-07-27 1986-05-13 Celanese Corporation Process for the production of muconic acid
US4731328A (en) * 1981-07-27 1988-03-15 Celgene Corporation Process for the production of muconic acid
US4929396A (en) * 1983-01-13 1990-05-29 Celgene Corporation Production of hexamethylenediamine muconate salt
US5213973A (en) * 1990-06-06 1993-05-25 Lonza Ltd. Microbiological process for oxidation of methyl groups

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2734843A1 (fr) * 1995-06-02 1996-12-06 Centre Nat Rech Scient Nouveau procede de bioconversion microbienne.

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3301766A (en) * 1964-09-23 1967-01-31 Exxon Research Engineering Co Fermentation process for preparing cinnamic acid and 5-phenyl valeric acid
US3326770A (en) * 1963-12-03 1967-06-20 Mobil Oil Corp Growing microorganisms on volatile hydrocarbons

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3326770A (en) * 1963-12-03 1967-06-20 Mobil Oil Corp Growing microorganisms on volatile hydrocarbons
US3301766A (en) * 1964-09-23 1967-01-31 Exxon Research Engineering Co Fermentation process for preparing cinnamic acid and 5-phenyl valeric acid

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3523894A (en) * 1966-07-18 1970-08-11 Sun Oil Co Corrosion inhibitors
US3458399A (en) * 1966-08-31 1969-07-29 Sun Oil Co Fermentation of hydrocarbons
US3518196A (en) * 1967-12-18 1970-06-30 Sun Oil Co Ultraviolet stabilized petroleum hydrocarbons
US3645847A (en) * 1968-02-08 1972-02-29 Sun Oil Co Microbiological hydroxylation of aromatic acids
US3991112A (en) * 1970-06-23 1976-11-09 Sun Research And Development Co. Methylated muconic acid hydrazides
US3629072A (en) * 1970-11-09 1971-12-21 Texaco Inc Microbiological process for preparation of internal monoalkenes
US4355107A (en) * 1981-07-27 1982-10-19 Celanese Corporation Production of muconic acid
US4588688A (en) * 1981-07-27 1986-05-13 Celanese Corporation Process for the production of muconic acid
US4731328A (en) * 1981-07-27 1988-03-15 Celgene Corporation Process for the production of muconic acid
US4929396A (en) * 1983-01-13 1990-05-29 Celgene Corporation Production of hexamethylenediamine muconate salt
US5213973A (en) * 1990-06-06 1993-05-25 Lonza Ltd. Microbiological process for oxidation of methyl groups

Also Published As

Publication number Publication date
NL6616545A (enrdf_load_stackoverflow) 1967-05-25
FR1505650A (fr) 1967-12-15
DE1593460A1 (de) 1970-09-03
BE690154A (enrdf_load_stackoverflow) 1967-05-24
GB1111309A (en) 1968-04-24

Similar Documents

Publication Publication Date Title
US3383289A (en) Microbiological oxidation of alkylbenzenes
US4508822A (en) Biochemical process
Haynes et al. Pseudomonas aureofaciens kluyver and phenazine α-carboxylic acid, its characteristic pigment
USRE30872E (en) Process for producing 2-keto-L-gulonic acid
US3907639A (en) Method for producing 2-keto-L-gulonic acid
HK147796A (en) Fermentation process for producing 2-keto-l-gulonic acid
US4879229A (en) Method for preparing 2,5-diketo-D-gluconic acid
US3823070A (en) Process for producing a straight chain dicarboxylic acid,an omega-hydroxy fatty acid,and an omega-1-keto fatty acid
EP0074169B1 (en) Micro-biological production of muconic acid
Davis et al. Metabolism of p-and m-xylene by species of Pseudomonas
van Ravenswaay Claasen et al. Substrate specificity of the paraffin hydroxylase of Pseudomonas aeruginosa
EP0032830B1 (en) Preparation of 2-keto-l-gulonic acid
US4588688A (en) Process for the production of muconic acid
US4654303A (en) Construction of novel mutant microorganisms
US3553081A (en) Process of microbiological oxidation
Sebek et al. The oxidation of the stereoisomeric 2, 3-butanediols by Pseudomonas
US3796630A (en) Microbial production of dicarboxylic acids
US4962027A (en) Production of 3-hydroxypropionaldehyde from glycerol by Klebsiella pneumoniae
US3748231A (en) Microbiological oxidation of alkyl-substituted cyclic compounds
EP0274146A1 (en) Process for the preparation of 2-arylpropionic acids
US3645847A (en) Microbiological hydroxylation of aromatic acids
US4673646A (en) Production of 2-hydroxymuconic semialdehyde
US3458399A (en) Fermentation of hydrocarbons
US3340155A (en) Microbiological oxidation of substituted naphthalenes
US4925798A (en) 3-hydroxydicarboxylic acids and process for their production