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
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
  • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic 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
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/10—Process efficiency
  • Y02P20/129—Energy recovery, e.g. by cogeneration, H2recovery or pressure recovery turbines
• • 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

Definitions

• the invention relates to the microbial synthesis of polyhydroxyalkanoic acids [PHA (B)] from substrates which are potentially ecotoxic and therefore have to be detoxified and which, if they can be used as a source of carbon and energy for microorganisms for growth and reproduction, the phenomenon of Show excess substrate inhibition.
• PHA polyhydroxyalkanoic acids
• PHB polyhydroxybutyric acid
• PHB (A) is accumulated as a result of imbalances in the nutrient supply, with batch cultivations in the stationary phase. Due to emerging and progressing shortages e.g. nitrogen, oxygen or phosphorus (in the form of phosphate) reduces the rate of propagation and initiates PHB formation.
• nitrogen, oxygen or phosphorus in the form of phosphate
• EP 0149 744 AI describes a continuous process. It clearly relies on the special property of Alcaligenes latus, which is able to grow and complete to synthesize optimal nutrient supply from sugar PHB under unlimited growth conditions. This process enables either by constant, periodic feeding of substrate (fed-batch regime) or by continuous operation, in which fresh nutrient solution is fed to the culture in a constant stream, and on the other hand, a quantity of biomass-containing culture medium is removed from the fermenter , very good PHB enrichments.
• Methylobacterium rhodesianum also has to be arranged in a special metabolic / regulatory manner, which can be used to produce PHB continuously [Ackermann, J.-U., Babel, W. (1997) Appl. Microbiol. Biotechnol. _47, 144-149].
• sugar is also a raw material for PHB synthesis in M. rhodesianum (among other substrates).
• PHB can alternatively also be produced from potentially ecotoxic substrates and has developed a process regime which enables the synthesis of PHB from waste products from the chemical industry and agriculture.
• the method according to the invention thus uses inexpensive carbon sources such as e.g. Phenol or Benzoate and enables problematic disposal with simultaneous synthesis of valuable substances.
• potentially ecotoxic substrates are used which show the phenomenon of excess substrate inhibition and thus in the conventional sense for the synthesis of products of overflow metabolism, ie also from PHB, are not suitable. They are neither suitable for batch operation, nor for the single-stage carbon-substrate-limited chemical ostatic process, since the conditions which actually prevent growth and multiplication and favor PHB synthesis are not realized.
• Such substrates are aromatics, including phenols, benzoic acid and benzaldehyde.
• the latter are known with regard to their bacteriocidal (bacteriostatic) effect and are often noteworthy components in industrial waste water.
• a method has been developed with which PHB can be produced from substrates which show growth inhibition in the case of excess substrate by chemostatically increasing corresponding microorganisms which utilize these substrates in such a way that the heat production, based on the substrate throughput rate, gives a maximum.
• the growth of the cells is monitored calorimetrically and the maximum amount of heat that an ax.
• PHB formation is initiated and controlled by increasing the substrate throughput rate with a small volume exchange.
• microorganism strains used according to the invention are known PHB formers and preferably belong to the genera Coma onas, preferably Comamonas acidovarans and Comamonas testosteroni, Ralstonia
• the substrates in particular are water-soluble Aromatics, preferably phenols, benzoic acid and benzaldehyde.
• Variovorax paradoxus JMP 116 is propagated at benzoate throughput speeds between 0.3 to 1.0 g / l # h at speeds between 0.07 and 0.4 h_1 .
• Ralstonia eutropha JMP 134 is propagated at phenol throughput speeds between 0.3 to 0.6 g / l »h at speeds between 0.05 and 0.2 IT 1 . It is also preferred to multiply Ralstonia eutropha JMP 134 at benzoate throughput rates between 0.25 to 0.7 g / l * h at rates between 0.04 to 0.21 h "1.
• the strains used are generally accessible in culture collections.
• a constant amount of heat is withdrawn from the fermentor via a helical heat exchanger.
• the mass flow of coolant through the heat exchanger and the temperature difference between the inlet and outlet are kept constant.
• An electric heater is controlled so that the reactor temperature remains constant. The difference between the current electrical heating output and that before inoculation corresponds to the heat production of the microorganisms.
• the Ralstonia eutropha JMP 134 strain is used. The cultivation takes place in a 2.2 1, thermally insulated fermentor at pH 7.0 and 30 ° C. All media used in this and the other examples contain 1.14 g / 1 NH 4 C1, 1.7 g / 1 KH 2 Po 4 , 2.18 g / 1 K 2 HP0 4 and trace salts (in mg / 1) MgS0 4 »7H 2 0 (712), CaCl 2 ⁇ 2H 2 0 (37), FeS0 4 » 7H 2 0 (50), CuS0 4 « 5H 2 0 (7.8), MnS0 4 » lH 2 0 (6, 1), ZnS0 4 • 7H 2 0 (4.4), NaMo0 4 »2H 2 0 (2.5).
• the fermentor is filled with 1.7 1 nutrient medium with 100 mg / 1 phenol.
• a cooling medium continuously withdraws a constant output and the reactor temperature is maintained with the help of an electric heater. It is gassed with 100 Nl / h humid air and homogenized by stirring at 900 rpm.
• Incoming medium, IN NaOH to maintain the pH and the air are brought to the reactor temperature using a heat exchanger.
• 100 ml of a preculture, the biomass of which was brought to about 500 mg / l by fed-batch cultivation, are used to inoculate the fermenter.
• the heating power required to keep the reactor temperature constant is reduced by the heat production of the microorganisms as soon as they start to use the phenol.
• the fermentor is then fed from a well-stirred mixing container (200 rpm), which contains a medium with 1 g / 1 phenol, at 200 ml / h.
• a medium containing 10 g / 1 phenol flows to the mixing container at 100 ml / h.
• Heat production rises linearly until a break point indicates the start of PHB production.
• the PHB content then increases until the heat production reaches a maximum of 2.7 W / 1. This happens with a substrate throughput of approx. 0.69 g / (l ⁇ h).
• the culture medium contains residual phenol ⁇ 0.1 mg / 1 and a biomass concentration of 2.9 g / 1 with a PHB content of 17% of the dry bacterial mass.
• the Ral stonia eutropha JMP 134 strain is cultured analogously to Example 1.
• the media contain 0.88 g / 1 sodium benzoate in the mixing container and 12 g / 1 sodium benzoate in the storage bottle and the inflow to the fermenter is 100 ml / h.
• the pH is kept constant by titration of 0.5 N hydrochloric acid.
• the measurement of Heat production, the preheating of the media, the acid and the humid air takes place as described in Example 1.
• the maximum heat production of approx. 0.94 W / 1 is achieved with a substrate throughput of approx. 0.353 g / (lh).
• the culture medium contains a ret concentration of sodium benzoate of 25 mg / 1 and 4.2 g / 1 biomass with a PHB content of 25%.
• the Varivorax paradoxus DSM 4065 strain is continuously propagated as indicated in Example 1.
• the media contain 1.2 g / 1 sodium benzoate in the mixing container and 9.5 g / 1 sodium benzoate in the storage bottle with otherwise the same composition of the medium as in Example 1.
• the determination of the heat flow, the temperature of the media, the humid air and of the titrant corresponds to Example 1.
• the inflow to the fermentor is 240 ml / h and the pH is kept constant by adding 0.5 N HCl.
• the maximum heat production of approx. 3.9 W / 1 is achieved with a substrate throughput of 1.14 g (l * h).
• the culture medium contains a residual concentration of sodium benzoate of 45 mg / 1 and 3.6 g / 1 biomass with a PHB content of 21%.

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

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Citations (2)

• 1998
  • 1998-04-30 DE DE19820168A patent/DE19820168C2/de not_active Expired - Fee Related
• 1999
  • 1999-04-26 WO PCT/EP1999/002803 patent/WO1999057298A1/de not_active Ceased
  • 1999-04-26 AU AU39287/99A patent/AU3928799A/en not_active Abandoned
  • 1999-04-26 US US09/787,580 patent/US6395520B1/en not_active Expired - Fee Related
  • 1999-04-26 JP JP2000547251A patent/JP4416947B2/ja not_active Expired - Fee Related

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