EP0972065A1 - Procede microbien de production de polyhydroxyalcanoates - Google Patents

Procede microbien de production de polyhydroxyalcanoates

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Publication number
EP0972065A1
EP0972065A1 EP98906847A EP98906847A EP0972065A1 EP 0972065 A1 EP0972065 A1 EP 0972065A1 EP 98906847 A EP98906847 A EP 98906847A EP 98906847 A EP98906847 A EP 98906847A EP 0972065 A1 EP0972065 A1 EP 0972065A1
Authority
EP
European Patent Office
Prior art keywords
microbial
dsm
culture medium
fermentation
medium
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.)
Withdrawn
Application number
EP98906847A
Other languages
German (de)
English (en)
Inventor
Ernst-Joachim Bormann
Mike Leissner
Christiane Berndt
Klaus Metzner
Arnulf Christner
Matthias Hilliger
Rudolf Geuther
Karin Perlet
Martin Roth
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.)
Dow Olefinverbund GmbH
Original Assignee
Buna Sow Leuna Olefinverbund GmbH
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 Buna Sow Leuna Olefinverbund GmbH filed Critical Buna Sow Leuna Olefinverbund GmbH
Publication of EP0972065A1 publication Critical patent/EP0972065A1/fr
Withdrawn legal-status Critical Current

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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
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • 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; 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/05—Alcaligenes

Definitions

  • the invention relates to a microbial process for the production of polyhydroxyalkanoates, preferably polyhydroxybutyric acid, by fermentation.
  • the process can be used to prepare polyhydroxyalkanoates.
  • Polyhydroxyalkanoates are biosynthesized by numerous microorganisms (Babel, W., Riis, V. and Hainich, E .: Plaste und Kautschuk [1990] 37, 109).
  • the biopolymers gained economic interest due to their thermoplastic properties and their biodegradability.
  • High cellular product concentrations are known especially in the bacterial genera Alcaligenes, Azotobacter, Pensdomonas and Bacillus (Anderson, A.J. and Dawes, E.A .: Microbial Review [1990] 54, 450).
  • the heteropolymer polyhydroxybutyric acid-polyhydroxyvaleric acid is produced industrially (Hartley, P .: Energy [1991] ⁇ 3, 48). Biotechnologically relevant processes use inorganic nitrogen sources and glucose, sucrose, molasses, methanol and hydrocarbons as carbon sources. Patent application DE 196 30 175.0 describes a process for the fermentative production of polyhydroxybutyric acid by cultivating a strain of Methylobacterium on glycerol as a source of carbon and energy. Supplementation with ingredients of technical raw materials or protein hydrolyzates or amino acids in low concentrations has been described (Fujita, M., Nakamura, K, Kuroki, H., Yoshie, N.
  • the invention has for its object to develop an economically effective cultivation process for the production of polyhydroxyalkanoates with a shortening of the fermentation time and simplification of the cultivation conditions.
  • the object is achieved in that the cultivation process with the microorganism strain Ralstonia eutropha ⁇ Alcaligenes eutrophus) DSM 11348 is carried out in a medium with nitrogen and carbon sources as well as mineral salts and trace elements.
  • the strain is a mutant of Ralstonia eutropha ⁇ Alcaligenes eutrophus) DSM 531.
  • the strain DSM 11348 was deposited on December 19, 1996 with the German strain collection of microorganisms and cell cultures GmbH. (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, according to the "Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure".
  • the microorganism strain Ralstonia eutropha DSM 11348 according to the invention differs from the starting strain Ralstonia eutropha ⁇ Alcaligenes eutrophus) DSM 531 as follows:
  • the mutative change in the DNA of DSM 11348 compared to the parent strain DSM 531 is detected as follows: The total DNA of the strains is fragmented with the restriction endonuclease Sspl (recognition sequence in the DNA: AAT. ATT). This results in DNA fragments with a size of about 10 to 500 kb (kb: kilobases), which are separated by means of pulse field gel electrophoresis. In the size range between 100 and 145 kb Parent strain DSM 531 DNA fragments of the following size detectable (kb): 100; 106; 117.5; 127.4; 135.2; 140; 144.7.
  • the bacterial strain DSM 11348 has the following changes compared to the parent strain DSM 531: The fragment 135.2 kb in size is missing. In addition 5 there is a DNA fragment with a size of 112.4 kb.
  • the bioprocess can be carried out on the media with a complex nitrogen source with the addition of glucose or glycerol or acetic acid as the carbon source.
  • the bioprocess regime based on this medium principle extends consistently from the stock keeping to the emerse and
  • the fermentation takes place after a volume-appropriate pre-cultivation in the submerged main culture under aerobic and contamination-free conditions by the strain Ralstonia eutropha ⁇ Alcaligenes eutrophus) DSM 11348 in a liquid medium at temperatures of 20 to 37 ° C and an acidity of pH 6.0 to 8 , 0 for the duration of 24 to 120
  • Glycerol preserves of the above-mentioned strain, which are deposited at -18 ° C and contain bacterial cell material in a concentration of 6 to 10 g bio-dry substance (TS) / 1, serve as inoculation material for the first submerged passage in
  • the inoculum contains peptones or protein hydrolyzates, glucose or glycerol, KH 2 PO and / or K 2 HPO 4 as well as a trace salt mixture.
  • the substrate combination is qualitative across all fermentation stages maintained. After 42 to 72 hours of cultivation at 28 to 32 ° C on a rotary table with 180 to 220 rpm, the biological material has reached a concentration of 6 to 12 gTS / 1. After the first liquid culture has matured, a second submerged cultivation passage is optionally added in an amount of 5 to 10% of the starting volume.
  • the second liquid passage is cultivated either in 2.5 liter steep breast bottles with 400 ml of the protein hydrolyzate medium or in stirred and ventilated stirred tank reactors with a fermentation volume of 5 to 25 liters with the usual biotechnological equipment level for stirring, aeration and pH-stating.
  • the cultivation time is 20 to 40 hours at a temperature of 30 to 32 ° C. NaOH (lower pH limit) and H 2 SO 4 (upper pH limit) are used for the acidity correction.
  • the main culture in a stirred tank reactor with the usual biotechnical equipment level is inoculated in an amount of 5 to 10% of the medium volume with the mature preculture and for a period of 24 to 120 hours with stirring, aeration, pH adjustment and subsequent dosing of glucose or glycerol or acetic acid.
  • Protein hydrolyzates as peptones or amino acid mixtures from vegetable products (soy protein), animal products (casein, gelatin, meat) or microbial biomass are suitable as organic nitrogen sources.
  • the protein is digested using known technical processes by mineral or enzymatic hydrolysis.
  • the protein hydrolyzates with an organic nitrogen content of 8 to 15% are used in the medium in concentrations of 10 to 50 g / 1.
  • Either glucose or glycerol or acetic acid is used as the carbon source. Only one of the first two substrates mentioned is added to the medium in concentrations of 10 to 100 g / l after separate sterilization. During the fermentation, when 10 to 20 g glucose / 1 or 10 to 20 g glycerol / 1 is reached, an amount of 20 to 50 g / 1 of the substrate in question is replenished.
  • Acetic acid is used as a 10 to 90% aqueous solution by recursively feeding it into the bioprocess via the dosage regimen for pH stating. It serves to compensate for the pH increase that is adequate for growth, which is caused by the NHt release from the organic nitrogen source and by the absorption of the acid anions in exchange for hydroxyl ions.
  • the fermentation batches based on the dry biomass, reach product concentrations of 50 to 85% polyhydroxyalkanoate as polyhydroxybutyric acid after 45 hours.
  • the space-time yield on a peptone / glucose medium on a 2 liter scale is 0.91 g of product / 1 hour.
  • the product is isolated from the biomass in a known manner with or without cell disruption.
  • the trace salt solution used in the examples has the following composition (g / 1):
  • DSM 11348 is resuspended with 1 ml of physiological NaCl solution and transferred to 100 ml of growth medium of the following composition in 500 ml steep breast bottles (g / 1): glucose 20; Bactotrypton 10; KH 2 PO 4 1; CaCl 2 0.015; Trace salt solution 2.5 ml; Aqua dest. ad 1 1; pH 6.8 to 7.0; Sterilization at 120 ° C for 20 min. The culture cultures are shaken for 36 hours at 30 ° C on a rotary table (200 rpm).
  • a ripening criterion they show a dry biomass concentration of 6.5 to 7.5 g TS / 1 and an acidity of pH 6.7 to 6.8. In an amount of 10% they serve as inoculation material for 2 1 main culture medium in a 2.5 1 bioreactor with the usual technical equipment for stirring, aeration, temperature peration and pH-stating.
  • the medium has the following composition (g / 1): glucose 100; Bactotrypton 20; KH 2 PO 4 0.5; CaCl 2 0.015; Trace salt solution 2.5 ml; Aqua dest. ad 1 1; pH 6.8 to 7.0; Sterilization at 120 ° C for 20 min.
  • the speed of the 6-bladed disk stirrer is 850 rpm, the ventilation rate is 0.5: 1 wm, the temperature is set to 30 ° C.
  • the culture is regulated to an acidity range from pH 6.8 to 7.1 with 4 N NaOH (lower limit) and 6 NH 2 SO 4 (upper limit). Glucose is added in solid form at a concentration of 20 g / l for the 45th hour. At the time of harvest after 55 hours, the culture reaches a dry biomass concentration of 55 g TS / 1 and a product concentration of 91% in the biomass.
  • DSM 11348 is resuspended with 1 ml of physiological NaCl solution and in an amount of 1 ml in 100 ml of culture medium of the following composition (g / 1): glycerol 30; Soy peptone 10; KH 2 PO 4 1; CaCl 2 0.015; Trace salt solution 2.5 ml; Aqua dest. ad 1 1; pH 6.8 to 7.0; Sterilization at 120 ° C for 20 min.
  • the 500 ml culture flasks are shaken for 28 hours at 30 ° C on a rotary table.
  • this first cultivation culture serves as inoculation material for 1 1 medium of the second cultivation culture in a 1.5 1 bioreactor with the usual technical standard for stirring, aeration, temperature control and pH-stating.
  • the medium has the following composition (g / 1): glycerol 30; Casamino acids 20; KH 2 PO 4 1; CaCl 2 0.030; Trace salts 5 ml; Aqua dest ad 1 1; pH 6.8 to 7.0; Sterilization at 120 ° C for 20 min.
  • the speed of the 4-blade disc stirrer is 400 rpm, the ventilation rate is 0.5: 1 wm, the temperature is set to 30 ° C.
  • the acidity of the culture is regulated with 4 N NaOH (lower limit range) and 6 NH 2 SO (upper limit range) from pH 6.8 to 7.1. After a fermentation time of 22 hours, the culture has reached 6.5 g TS / 1 dry biomass and is in an amount of 10% by volume on 2 1 of the main culture medium in a 2.5 1 bioreactor with the usual technical standard for stirring, Be - Transfer ventilation, temperature control and pH stating.
  • the medium has the following composition (g / 1): glycerol 50; Casamino acids 20, KH 2 PO 4 0.5; CaCl 2 0.030; Trace salt solution 5 ml; Aqua dest. ad 1 1; pH 6.8 to 7.0; Sterilization at 120 ° C for 20 min.
  • the speed of the 6-blade stirrer is 850 rpm, the ventilation rate is 0.5: 1 wm, the temperature is set to 30 ° C.
  • the acidity of the culture is regulated to pH 6.8 to 7.1 with 4 N NaOH (lower limit) and H 2 SO (upper limit). 50 g of glycerol 1 are added at the 23rd hour. At the time of harvest, the fermentation batch reached a dry matter concentration of 27 g TS / 1 and a product concentration of 65% in the biomass after 47 hours.
  • DSM 11348 is resuspended with 1 ml of physiological NaCl solution and 1 ml of this is transferred to 100 ml of growth medium of the following composition (g / 1): glucose 20; Bactotrypton 10; KH 2 PO 4 1, CaCl 2 0.015; Trace salt solution 2.5 ml; Aqua dest. ad 1 1; pH 6.8 to 7.0; Sterilization at 120 ° C for 20 min.
  • the 500 ml culture flasks are shaken for 40 hours at 30 ° C on a rotary table (200 rpm).
  • a ripening criterion they have a bio-dry matter concentration of 6.5 g TS / 1 and an acidity of pH 6.8. In an amount of 10% of the volume, they serve as inoculation material for 1 1 main culture medium in a 1.5 1 bioreactor with the usual technical equipment for stirring, aeration, temperature control and pH stating.
  • the medium has the following composition (g / 1): soy peptone 20; KH 2 PO 4 0.5; CaCl 2 0.015; Trace salt solution 2.5 ml; Aqua dest. ad 1 1; pH 6.8 to 7.0; Sterilization at 120 ° C for 20 min.
  • the speed of the 4-blade disc stirrer is 400 rpm, the ventilation rate is 0.5: 1 wm, the temperature is set to 30 ° C.
  • the acidity of the culture is regulated to pH ⁇ 7.05 with 50% acetic acid. After a fermentation time of 42 hours, the culture reaches a dry biomass concentration of 15 g TS / 1 and a product concentration of 50% PHB in the biomass.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un nouveau procédé microbien de production de polyhydroxyalcanoates par fermentation aérobie stérile d'un micro-organisme bactérien sur un milieu de culture liquide, procédé qui se caractérise en ce que l'on utilise, comme micro-organisme, la souche Ralstonia eutropha (Alcaligenes eutrophus) DSM 11348.
EP98906847A 1997-02-04 1998-01-27 Procede microbien de production de polyhydroxyalcanoates Withdrawn EP0972065A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19704045A DE19704045A1 (de) 1997-02-04 1997-02-04 Mikrobielles Verfahren zur Herstellung von Polyhydroxyalkanoaten
DE19704045 1997-02-04
PCT/DE1998/000233 WO1998033931A1 (fr) 1997-02-04 1998-01-27 Procede microbien de production de polyhydroxyalcanoates

Publications (1)

Publication Number Publication Date
EP0972065A1 true EP0972065A1 (fr) 2000-01-19

Family

ID=7819196

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98906847A Withdrawn EP0972065A1 (fr) 1997-02-04 1998-01-27 Procede microbien de production de polyhydroxyalcanoates

Country Status (5)

Country Link
EP (1) EP0972065A1 (fr)
JP (1) JP3222478B2 (fr)
CA (1) CA2280086A1 (fr)
DE (1) DE19704045A1 (fr)
WO (1) WO1998033931A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3516100A (en) 1999-03-05 2000-09-21 Monsanto Technology Llc Multigene expression vectors for the biosynthesis of products via multienzyme biological pathways
KR20040046678A (ko) * 2002-11-28 2004-06-05 (주) 켐포트 폴리-베타-하이드록시 부틸산을 생산하는 신규의 미생물 및 그를 이용한 고농도 발효 제조방법
CA2848574A1 (fr) * 2011-09-12 2013-03-21 Oakbio Inc. Conversion chimio-autotrophe d'oxydes de carbone dans des dechets industriels en biomasse et produits chimiques

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3343576A1 (de) * 1983-12-01 1985-06-13 Lentia GmbH Chem. u. pharm. Erzeugnisse - Industriebedarf, 8000 München Verfahren zur biotechnologischen herstellung von poly-d(-)-3-hydroxybuttersaeure
JPH03224492A (ja) * 1990-01-29 1991-10-03 Showa Denko Kk 共重合体およびその製造法
NL9201803A (nl) * 1992-10-16 1994-05-16 Inst Voor Agrotech Onderzoek PHB-producerend microorganisme, werkwijze voor het verkrijgen van een PHB-producerend microorganisme, werkwijze voor het produceren van PHB en werkwijze voor het verwijderen van glycerol uit een glycerol bevattend medium.
GB9503174D0 (en) * 1995-02-17 1995-04-05 Zeneca Ltd Polymer production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9833931A1 *

Also Published As

Publication number Publication date
CA2280086A1 (fr) 1998-08-06
WO1998033931A1 (fr) 1998-08-06
JP2000512857A (ja) 2000-10-03
DE19704045A1 (de) 1998-08-06
JP3222478B2 (ja) 2001-10-29

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