US3562110A - Production of amino acids - Google Patents

Production of amino acids Download PDF

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Publication number
US3562110A
US3562110A US700580*A US3562110DA US3562110A US 3562110 A US3562110 A US 3562110A US 3562110D A US3562110D A US 3562110DA US 3562110 A US3562110 A US 3562110A
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Prior art keywords
amino acids
cellulose
fermentation
aliphatic hydrocarbon
production
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Expired - Lifetime
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US700580*A
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English (en)
Inventor
John D Douros Jr
Lars A Naslund
William J Lahl
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ExxonMobil Technology and Engineering Co
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Exxon Research and Engineering Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/26—Processes using, or culture media containing, hydrocarbons
    • C12N1/28—Processes using, or culture media containing, hydrocarbons aliphatic
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino 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
    • Y10S435/803—Physical recovery methods, e.g. chromatography, grinding
    • 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
    • Y10S435/859—Micrococcus

Definitions

  • n-aliphatic hydrocar- While the Present lnveljltlon pp i to a broad bon be the sole source of carbon in the process, preferred scope of operfflble bactfllal ml'cfoofganlsms, there are li h h d b f d b i comprised i il a number of microorganisms WhlCll are especially suitable f CircaJ ffi for hydrocarbon assimilation, These microorganisms are tabulated hereinbelow, along with their corresponding ATCC registration numbers which were secured by depositing samples with the American Type Culture Collec- Thus, the present invention is broadly concerned with tion, 212 M Street, Northwest, Washington 7, DC, or a biosynthesis fermentation process for the production of other designated numbers.
  • the invention is concerned with the use of C -C n-aliphatic hydrocarbon feed, particularly C -C n-parafiins as the primary source of carbon in the process.
  • C C n-paraffins are contacted with a microorganism under fermentation conditions in the presence of cellulose to produce high yields of extracellular amino acids. This process may be carried out continuously or batchwise.
  • Colonies on defined agar are small (1 mm.), circular convex, having entire edge. Colonies on nutrient agar are larger (2 to mm.), raised mucoid, generally round.
  • the growth media comprise an aqueous mineral salt medium and excess oxygen.
  • Oxygen is supplied to the cultivation substrate medium or broth in any form capable of being assimilated readily by the inoculant microorganism.
  • Oxygen-containing compounds may also be used to supply oxygen as long as they do not adversely affect microorganism cell growth and the conversion of the oxidized hydrocarbon feed to microorganism cells.
  • Oxygen may be supplied as an oxygen-containing gas, such as air at atmospheric or elevated pressure or oxygenenriched air wherein the oxygen concentration may be up to 70% to 90%. In general, between about 0.1 and about 10, preferably between about 0.8 and about 2.5 volumes per minute of air are supplied to the reactor per volume of liquid in the fermentor.
  • the source of nitrogen may be any organic or inorganic nitrogencontaining compound which is capable of releasing nitrogen in a form suitable for metabolic utilization by the growing microorganism.
  • Suitable organic nitrogen compounds are, for example, proteins, acid-hydrolyzed proteins, enzyme-digested proteins, amino acid, yeast extract, asparagine, and urea.
  • Suitable inorganic nitrogen compounds are ammonia, ammonium hydroxide, nitric acid or salts thereof, such as ammonium phosphate, ammonium citrate, ammonium sulfate, ammonium nitrate and ammonium acid pyrophosphate.
  • a very convenient and satisfactory method of supplying nitrogen to the process is to employ ammonium hydroxide, ammonium phosphate or ammonium acid phosphate, which can be added as the salt per se or which can be produced in situ in the aqueous fermentation media by bubbling ammonia gas or gaseous ammonia through the broth or injecting aqueous ammonium hydroxide into the broth to which phosphoric acid was previously added, thereby forming ammonium acid phosphate.
  • the desired pH range of about 3.0 to 8.5 is maintained and the requisite nitrogen supplied.
  • the microorganism comprises a yeast the preferred pH is in the range of 3.0 to 7.5 such as 4.0 to 5.0.
  • the microorganism comprises a bacteria the desired pH is in the range of 5.0 to 8.5, such as about 7.0.
  • Ammonium hydroxide may be supplied to the biosynthesis bath in amounts of between about 0.08 and about 0.20, preferably between about 0.1 and about 0.15, gram of nitrogen per gram of dried cells produced. This amounts to between about 0.01 and about 1.0 wt. percent, preferably between about 0.1 and about 0.15 wt. percent, nitrogen based on the total biosynthesis bath.
  • the carbon source preferably the sole carbon source, for the fermentation process is an n-aliphatic hydrocarbon feed.
  • the n-aliphatic hydrocarbon feed contains from potassium phosphate, potassium sulfate, potassium citrate,
  • Component Can use use use use C11Cin n-aliphatic hydroearbom 4-120 5-80 10-50 Cellulose" 260 2. 5-40 5-25 K2HPO4 0. 5l5 l-lO 2-8 (NH4) 2111 04- 5-15 7-13 8-13 8804 0. 1-1. 0 0. 2-0. 9 0.3-0. 5 F0304 7 H20. 0 002-0. 5 0. 005-0. 04 0 0l-0. 02 MgSOr? H2O 0. l-0. 7 0. 2-0. 0. 3-0. 3 M11804- 7 H2O. 0. 002-0. 05 0. 005-0. 04 0. 01-0. 03 NaCl 0. 002-0. 05 0. 005-0. 04: 0. 01-0. 08 Water Remainder to equal wt. percent Cellulose is preferably in stri s such as about V to 2 Wide and to 6 long. Desirable strips are wide and 1 long.
  • Concentratioln (milligrams per iter) Usually Preferably Component Can use use use use use ZHSOJ'II2O 0-0. 4 0-0. 3 0-0. 2 0-0. 06 0-0. 05 0-0. 04 0-1. 2 0-0. 06 O-0. 2 0-0. 12 0-0. 005
  • the essential and optional nutrients may be supplied in the form of other salts or acids than those tabulated hereinabove.
  • a very satisfactory medium is prepared as follows:
  • the temperature of the biosynthesis bath may be varied between about 20 C. and about 55 C. depending upon the specific microorganism being grown, but preferred temperatures when using bacteria are between about 25 C. and about 45 C. such as about 35 C.
  • the pH is preferably in the range from 5.5 to 8.5 such as about 7.0. be supplied by any technique but are preferably supplied by their water-soluble salts.
  • Potassium may be supplied as potassium chloride, about 1 to 30 carbon atoms in the molecule preferably 11 to 30 carbon atoms.
  • a desirable n-parafiin hydrocarbon feed contains from about 11 to 20 carbon atoms in the molecule, such as a feed of the following composition.
  • the harvesting of the microbial cells and the amino acids accumulated in the fermentation broth can be done by suitable means.
  • the cells may first be separated from the fermentation broth by centrifugation (e.g. closed bowl, liquid cyclones or hydroclones, evaporation (e.g. falling film, wiped film), filtration (e.g. micropore, dialysis, reverse osmosis), flocculation, settling and decanta tion (e.g.
  • the separated, concentrated cells may then be dried by spray drying, drum drying, freeze drying, vacuum drying, tray drying, oven drying or any other drying procedure or combination of procedures to obtain a final product having extremely high protein content and no impurities detrimental to humans or animals.
  • the extracellular amino acids accumulated in the broth may be recovered by fractional crystallization or evaporation (e.g. falling film, wiped film), spray drying, drum drying, freeze drying, vacuum drying, tray drying, oven drying. Another general method of recovery would be the adsorption of the amino acids on ion exchange resins, followed by selective elution.
  • the cellulose is preferably cellulose acetate strips or cellulose bags which are prepared by treating wood pulp with acetic acid, acetic anhydride and sulfuric acid as a are secured by the addition of the cellulose strips or cellulose bags to the fermentation process in that the production of extracellular amino acids is markedly increased.
  • a container or flask 10 contains a two hole stopper 1. Glass tubing 3 is inserted into the stopper 1, to which is attached at the upper end a dispo-plug 2 and at the lower end a cellulose bag 4. A concentrated P salt solution 5 is positioned within the cellulose bag 4 which is positioned within the saline solution plus substrate A6.
  • EXAMPLE 1 A sterile aerobic fermentation process was conducted in the apparatus illustrated in the drawing. 20 ml. of a sterile 5 concentrate of P salts medium were added into the cellulose bag. 80 ml. of a sterile 0.85% saline solution containing 2.0 ml. of substrate A was introduced into the flask. The saline solution was inoculated with a 1% inoculum of Micrococcus cerificans ATCC No. 14987, which had been grown on substrate A for 24 hours, and the fermentation was conducted on a rotary mechanical shaker at 300 rpm. at 30 C. for 72 hours.
  • the fermentation broth was periodically sampled, at which time the bacterial cells and other impurities were removed by centrifugation, and the amount of extracellular amino acids in the clear centrifugate was determined by the following assay methods.
  • One such series of assay methods is described in the second edition of Microbiological Assay of the Vitamin B-Complex and Amino Acids (1952) by E. C. Barton Wright, Pitman Publishing Corp., New York, NY.
  • Another assay method used was the Technicon Auto Analyzer which provides an automatic analytical system based upon the ion exchange chromatography system established by Spackman, Moore and Stein.
  • Inoeulum M. cerificans 0 1.0 1. 0 1.0 1.0 1.0 1. 0 1. 0
  • Substrate A (mL) 2. 0 0 2. 0 2. 0 2. O 2. 0 2. O 2. 0
  • Cellulose bag Yes Yes Yes No Yes No Yes No Yes Fermentation time (days) 3 3 1 2 2 3 3 Noru.-P Present trace amount.
  • EXAMPLE 2 catalyst The cellulose is fully acetylated (three acetate groups per glucose unit) and, at the same time, the sulfuric acid causes degradation of the cellulose polymer so that the product contains only about 200 300 glucose units per polymer chain. At this point in the process the ce lulose acetate is partially hydrolyzed by the addition of water until an average of 22.5 acetate groups per glucose unit remain. This product is a thermoplastic.
  • Additional aerobic fermentation operations were conducted using cellulose strips.
  • a series of three sterile fermentations was carried out in 500 ml. fermentation flasks containing 100 ml. of P medium, 2.0 ml. substrate A and 1.0 gram of cellulose strips A x 1").
  • the medium was inoculated with a 1% inoculum of Micrococcus cerificans ATCC :No. 14987.
  • the fermentations were conducted As pointed out heretofore, unexpected, desirable results 75 on a rotary mechanical shaker at 300 rpm. at 30 C.
  • the fermentation flasks were periodically removed at 1, 2 and 3 days and the broth harvested and assayed as in Example 1. The results obtained are set forth in Table III.
  • Aerobic fermentation process for the production of extracellular amino acids which comprises incubating a fermentation broth with Micrococcus cerificans ATCC No. 14987, said broth comprising an aqueous inorganic salt growth medium, an oxygen-containing gas and a liquid petroleum hydrocarbon fraction as a primary source of carbon, conducting the fermentation process in the presence of cellulose acetate and under conditions adapted to promote cell growth whereby a high yield of extracellular amino acids is secured.
  • cellulose is selected from the class consisting of cellulose acetate strips and cellulose acetate bags.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
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  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
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  • General Engineering & Computer Science (AREA)
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US700580*A 1968-01-25 1968-01-25 Production of amino acids Expired - Lifetime US3562110A (en)

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US70058068A 1968-01-25 1968-01-25

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US (1) US3562110A (fr)
BE (1) BE727371A (fr)
CH (1) CH506620A (fr)
DE (1) DE1903162A1 (fr)
FR (1) FR2000751A1 (fr)
GB (1) GB1230365A (fr)
NL (1) NL6901218A (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3660235A (en) * 1969-08-22 1972-05-02 Ajinomoto Kk Method for producing phenylalanine by fermentation
US3713977A (en) * 1970-01-22 1973-01-30 Kyowa Hakko Kogyo Kk Process for producing l-histidine
US3791925A (en) * 1970-05-20 1974-02-12 Kyowa Hakko Kogyo Kk Process for producing l-histidine
US3943038A (en) * 1973-08-16 1976-03-09 Ajinomoto Co., Inc. Method for producing amino acids by culturing hydrogen-oxidizing bacteria

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3660235A (en) * 1969-08-22 1972-05-02 Ajinomoto Kk Method for producing phenylalanine by fermentation
US3713977A (en) * 1970-01-22 1973-01-30 Kyowa Hakko Kogyo Kk Process for producing l-histidine
US3791925A (en) * 1970-05-20 1974-02-12 Kyowa Hakko Kogyo Kk Process for producing l-histidine
US3943038A (en) * 1973-08-16 1976-03-09 Ajinomoto Co., Inc. Method for producing amino acids by culturing hydrogen-oxidizing bacteria

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GB1230365A (fr) 1971-04-28
NL6901218A (fr) 1969-07-29
CH506620A (de) 1971-04-30
DE1903162A1 (de) 1969-08-28
FR2000751A1 (fr) 1969-09-12
BE727371A (fr) 1969-07-24

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