WO1998016624A1 - Procede de culture de biomasse - Google Patents

Procede de culture de biomasse Download PDF

Info

Publication number
WO1998016624A1
WO1998016624A1 PCT/DE1997/002277 DE9702277W WO9816624A1 WO 1998016624 A1 WO1998016624 A1 WO 1998016624A1 DE 9702277 W DE9702277 W DE 9702277W WO 9816624 A1 WO9816624 A1 WO 9816624A1
Authority
WO
WIPO (PCT)
Prior art keywords
methanol
cultivation
stage
cultivation stage
concentration
Prior art date
Application number
PCT/DE1997/002277
Other languages
German (de)
English (en)
Inventor
Olaf Lehmann
Christiane Berndt
Klaus-Dieter Menzel
Dominik Driesch
Christian Wulfes
Toralf Gliem
Arnulf Christner
Matthias Hilliger
Karl Miersch
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
Priority to EP97910254A priority Critical patent/EP0932660A1/fr
Priority to JP10517891A priority patent/JP2000514313A/ja
Publication of WO1998016624A1 publication Critical patent/WO1998016624A1/fr

Links

Classifications

    • 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
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6

Definitions

  • the invention is used in the cultivation of methylotrophic microorganisms on the carbon substrate methanol and can be used both in research and in industry.
  • Methanol as a carbon source is an inexpensive substrate, which is also a waste product.
  • methanol has a growth-inhibiting effect if it acts on the microorganisms in higher concentrations and / or over a long period. The reason for this presumably lies in the formation of toxic intermediates, e.g. B. formaldehyde, and their accumulation with excess methanol. Concentrations greater than 0.5 g / l must already be considered as excess methanol.
  • Conventional fed-batch processes in which a larger amount of the C substrate is placed in the culture medium and added or supplemented after it has been consumed, can therefore hardly be used.
  • Methylobacterium it is not possible to cultivate Methylobacterium at a permanent methanol concentration of 0.5 - 10 g / l for a longer period than 20 - 30 h without stagnating growth.
  • Methods are therefore known in which the concentration of the methanol is determined by constant measurement and is kept at values below 0.5 g / l by cyclic or continuous additions. These processes require a high level of human and technical effort.
  • the invention has for its object to develop a method for growing methylotrophic microorganisms on the substrate methanol with a large increase in mass of the starting material (inoculated preserves, shake cultures).
  • the cultivation is to be carried out in stirred and ventilated fermenters and the biomass is to be used to form cellular products or storage materials.
  • the microorganisms are grown in stirred and aerated bioreactors in a k-stage fed-batch process, the average methanol concentration decreasing from stage to stage by the cultivation stage (k-2) with excess methanol, the cultivation stage (k-1) in first section with excess methanol and then with methanol rate limitation and the cultivation step (k) from the beginning under methanol rate limitation.
  • Rate limitation is a state in which the rate of the inflowing methanol is equal to the rate of the methanol consumed by the culture and there is no accumulation of methanol in the culture medium.
  • the cultivation method according to the invention can be carried out in such a way that, before the cultivation stage (k-2), further cultivation stages are carried out, which are carried out according to the same cultivation principle of the cultivation stage (k-2). Consequently In the case of multi-stage cultivation, the cultivation process always takes place in the last three cultivation stages - in those with the highest mass increase - according to the process steps according to the invention.
  • the average methanol concentration in the cultivation stage (k-2) is 2 to 6, preferably 2.5 g / l, in the cultivation stage (k-1) 0.5 to 2, preferably 1 g / l and in the cultivation stage (k ) 0 to 0.5 g / l.
  • the cultivation is carried out in all stages while avoiding growth oxygen limitation; a decrease in the dissolved oxygen concentration below 10% of the saturation concentration is avoided by known measures.
  • methanol is added to the culture medium before the inoculation in an amount of 2 to 10, preferably 3 to 6 g / l. Then the inoculation is carried out with a microorganism culture from shake cultures, canned vaccines or with culture solution, which was taken from one of the subsequent stages.
  • the cultivation is carried out at a constantly regulated pH with stirring and aeration until a biomass concentration sufficient for the inoculation of the cultivation stage (k-1) is reached.
  • proportioning to the alkali metered in via the pH control is metered in in a ratio of 7 to 10, preferably 8.5, mol of methanol to 1 mol of alkali.
  • An alkali can be, for example, NaOH, KOH or NH 4 OH, although divalent alkalis can also be used.
  • This ratio dosage can also be achieved by carrying out the titration with a mixture which contains 7 to 10 moles of methanol and at least 0.3 liters of water per mole of lye. It has been found that the concentration of methanol in the culture medium is almost constant or slightly decreasing over a longer period of time. The cultivation is stopped before the methanol reaches a very low, growth-limiting concentration. Thus, the cultivation in this stage is carried out continuously with methanol shot at a maximum specific growth rate corresponding to the other cultivation conditions (temperature, pH, etc.).
  • methanol is added to the culture medium before the inoculation in an amount of 2 to 10, preferably 3 to 6 g / l. Then the inoculation is carried out with the complete culture solution from the cultivation stage (k-2), or with part of it.
  • the cultivation is carried out at a constantly regulated pH with stirring and aeration until a biomass concentration sufficient for the inoculation of the following cultivation stage (k) is reached.
  • an initially constant methanol dosage of 0.5 to 1.5, preferably 0.8 grams of methanol per liter of culture solution and hour is started.
  • the flow rate of this dosage can be increased by a factor of 1.5 to 10, preferably 3 to 4, until the end of the second cultivation stage. It was found in this procedure that there is an excess of methanol until about 8 to 15 hours after the inoculation and then that the methanol is consumed by reducing its concentration to a growth-limiting value close to 0. Furthermore, the growth of the culture is limited in methanol, that is to say that the amount of methanol flowing in is used up immediately and there is no accumulation of methanol in the culture medium. The amount of the methanol inflow rate determines the growth rate and the achievable biomass concentration.
  • the inoculation is carried out with the complete culture solution from the cultivation stage (k-1) or with a part thereof.
  • the cultivation is carried out at a constantly controlled pH with stirring and aeration until a sufficient biomass concentration is reached.
  • the methanol supply of the last cultivation stage (k) is carried out from the beginning as a continuous dosage, the inflow rate of the methanol being so is chosen that a flow equilibrium called rate limitation is established.
  • methanol addition of 0.05 to 1.05, preferably 0.2 to 0.4 g of methanol per g of biomass (dry matter) and hour.
  • a dosage can be implemented, for example, by increasing the rate of the methanol feed in the same time units by the same factor in the range of approximately 1.1 to 1.7 per hour (exponential profile).
  • the methanol dosage is only increased to such an extent that, given the maximum values for aeration, stirrer speed and fermenter pressure, the dissolved oxygen value does not fall below 10% of the saturation concentration.
  • Figures 1 - 3 show the course of the biomass growth, the alkali / methanol mixture or the methanol dosage, the methanol concentration and the p ⁇ 2 value in the 3 cultivation stages described.
  • Figure 1 1st cultivation level (k-2)
  • Figure 2 2nd cultivation level (k-1)
  • Figure 3 3rd cultivation level (k)
  • a biomass of approx. 42 kg dry matter is required on the C-substrate methanol in order to initiate the product formation process at a biomass concentration of approx. 20 g / l (dry matter) in a 4000 liter fermenter with a 2100 liter filling volume.
  • a mineral salt medium is used as the culture medium, which in the 1st stage contains all required mineral salts and trace elements for biomass growth up to 5 g / l (dry matter), in the 2nd stage all required mineral salts and trace elements except nitrogen for biomass growth up to 10 g / l l (dry matter) and in level 3 contains all required mineral salts and trace elements except nitrogen for biomass growth up to 30 g / l (dry matter).
  • 0.1 ml / l polypropylene glycol (PPG) is added to the medium before sterilization, further additions of this defoamer are carried out during the fermentation if necessary.
  • the cultivation takes place in all stages at a temperature of 31 - 32 ° C, an aeration rate of 0.4 to 0.6 vvm and an overpressure of 200 - 600 mbar as well as a constantly regulated pH value of 6.7 to 6.8 .
  • the first cultivation stage in the 30 l fermenter begins with the inoculation of the medium from a shaking culture carried out for 25 hours at 32 ° C., consisting of 6 shaking bottles with a filling volume of 400 ml each. A biomass starting concentration of 0.1 g / l (dry mass) is thus achieved. Before the inoculation, an amount of 60 g of methanol corresponding to 3 g / l is added to the culture medium. A mixture consisting of 75 ml of 45% sodium hydroxide solution, 427.5 ml of methanol and 475 ml of fully demineralized water is used to regulate the pH. Increasing the stirrer speed prevents the pO value from dropping below 20% of the saturation concentration.
  • the cultivation is ended at a biomass concentration of approx. 4 g / l (dry matter).
  • the methanol concentration at the end of the cultivation is 2 g / l.
  • the second cultivation stage begins with a biomass starting concentration of approx. 0.4 g / l (dry matter).
  • an amount of 600 g of methanol corresponding to 3 g / l is added to the culture medium.
  • 25% ammonium hydroxide solution is used to regulate the pH.
  • Increasing the stirrer speed prevents the pO 2 value from dropping below 20% of the saturation concentration.
  • the third cultivation stage begins with a biomass starting concentration of approx. 1 g / l (dry matter). 25% ammonia solution is used to regulate the pH.
  • the culture is now supplied with methanol exclusively via continuous metering.
  • the methanol dosage starts with an inflow rate of 600 g / h corresponding to 0.3 g / l and h immediately after the inoculation. From the second hour of cultivation, this inflow rate is increased continuously by a factor of 1.136 per hour. The increase is controlled by a computer every minute, by 0.2134% each.
  • the pO 2 value drops steadily and reaches a value of 30% around the 9th hour of cultivation.
  • the pO 2 value is kept in a range from 30 to 50% by gradually increasing the stirrer speed from 100 to 200 rpm. After reaching the upper stirrer speed of 200 rpm, the pO 2 value drops to 20% by the 23rd cultivation hour. From this point in time, when the inflow rate of the methanol has reached an amount of about 8100 g / h, the pO 2 value is regulated in a range of 10-20% of the saturation concentration via the methanol dosage. The dosage is reduced when the pO 2 value drops below 15% and increased when the pO 2 value rises above 15%.
  • the culture is in the state of methanol rate limitation, as in the second half of stage 2. Only in this way is it possible to react immediately to an increase in the pO 2 value or to reduce the methanol flow rate and thus to avoid oxygen limitation of growth.

Landscapes

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

Abstract

La culture de micro-organismes méthylotrophes sur le substrat de carbone méthanol s'effectue de manière connue en soi à l'aide de procédés de mesure et d'alimentation ultérieure, afin de limiter la concentration en méthanol à des taux très bas. L'invention concerne un procédé de mise en culture à plusieurs étapes de culture, dans des bioréacteurs agités et ventilés, selon lequel des méthodes d'alimentation appropriées permettent de diminuer la concentration moyenne en méthanol d'étape en étape. A cet effet, l'alimentation en méthanol dans l'étape de culture (k-2) est proportionnelle à la lessive alcaline dosée lors de l'ajustement du pH, dans l'étape de culture (k-1), sous forme de dosage spécifique, et dans la dernière étape de culture (k), d'abord sous forme de dosage spécifique, puis sous forme de dosage ajusté par pO2. Ce procédé évite l'accumulation d'intermédiaires toxiques. Une mesure et un ajustement de la régulation en méthanol ne sont plus nécessaires pendant le processus global de culture. Ce procédé contribue de ce fait à réduire considérablement les coûts lorsqu'il est appliqué à l'échelle industrielle.
PCT/DE1997/002277 1996-10-12 1997-10-04 Procede de culture de biomasse WO1998016624A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97910254A EP0932660A1 (fr) 1996-10-12 1997-10-04 Procede de culture de biomasse
JP10517891A JP2000514313A (ja) 1996-10-12 1997-10-04 バイオマスを培養する方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1996142105 DE19642105A1 (de) 1996-10-12 1996-10-12 Verfahren zur Anzucht von Biomasse
DE19642105.5 1996-10-12

Publications (1)

Publication Number Publication Date
WO1998016624A1 true WO1998016624A1 (fr) 1998-04-23

Family

ID=7808550

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1997/002277 WO1998016624A1 (fr) 1996-10-12 1997-10-04 Procede de culture de biomasse

Country Status (4)

Country Link
EP (1) EP0932660A1 (fr)
JP (1) JP2000514313A (fr)
DE (1) DE19642105A1 (fr)
WO (1) WO1998016624A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19910143C2 (de) * 1999-02-26 2001-03-15 Saechsisches Inst Fuer Angewan Verfahren zur kontinuierlichen biotechnischen Herstellung von Poly-beta-hydroxybuttersäure

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4166004A (en) * 1976-07-24 1979-08-28 Hoechst Aktiengesellschaft Process for the preparation of single cell protein using Methylmonas clara ATCC 31226
US4229543A (en) * 1973-05-11 1980-10-21 Agency Of Industrial Science & Technology Process for culturing methanol-utilizing yeasts
EP0144474A1 (fr) * 1983-12-09 1985-06-19 Fabriques De Tabac Reunies S.A. Procédé de fermentation en continu
EP0237002A1 (fr) * 1986-03-10 1987-09-16 Phillips Petroleum Company Fermentation de bactéries à productivité élevée
US5434062A (en) * 1992-11-23 1995-07-18 National Research Council Of Canada Process for the preparation of poly-beta-hydroxybutyric acid polymers

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD293138A5 (de) * 1990-03-02 1991-08-22 Chemie Ag Bitterfeld-Wolfen,De Verfahren zur herstellung von ubichinon-10 mittels bakterien

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4229543A (en) * 1973-05-11 1980-10-21 Agency Of Industrial Science & Technology Process for culturing methanol-utilizing yeasts
US4166004A (en) * 1976-07-24 1979-08-28 Hoechst Aktiengesellschaft Process for the preparation of single cell protein using Methylmonas clara ATCC 31226
EP0144474A1 (fr) * 1983-12-09 1985-06-19 Fabriques De Tabac Reunies S.A. Procédé de fermentation en continu
EP0237002A1 (fr) * 1986-03-10 1987-09-16 Phillips Petroleum Company Fermentation de bactéries à productivité élevée
US5434062A (en) * 1992-11-23 1995-07-18 National Research Council Of Canada Process for the preparation of poly-beta-hydroxybutyric acid polymers

Also Published As

Publication number Publication date
EP0932660A1 (fr) 1999-08-04
JP2000514313A (ja) 2000-10-31
DE19642105A1 (de) 1998-04-16

Similar Documents

Publication Publication Date Title
DE102005029306B4 (de) Verfahren zum Betreiben einer Feststofffermenteranlage sowie Vorrichtung hierzu
EP1123409B1 (fr) Procede de preparation de gamma-decalactone
EP0141784B1 (fr) Procédé pour la décomposition de dérivés de s-Triazine dans les solution aqueuses
DE2452720A1 (de) Verfahren zur kontinuierlichen kultivierung von hefe
EP0434035A1 (fr) Procédé de fabrication de l'acide 6-hydroxynicotinique
WO1998016624A1 (fr) Procede de culture de biomasse
DE3650395T2 (de) Gärung zur Gewinnung von d-Milchsäure.
EP0829541A2 (fr) Procédé de fermentation contrÔlé par osmose pour la préparation d'acarbose
DE2747510A1 (de) Verfahren zur herstellung von coenzym q
WO2009013066A1 (fr) Procédé de fermentation de cultures cellulaires
DE19619084A1 (de) Verfahren zur Herstellung von Poly-ß-hydroxybuttersäure und Copolymeren
DE69432240T2 (de) Verfahren zur Herstellung von bakterien Zellen, welche poly-3-hydroxy Buttersäure enthalten
EP0556465A2 (fr) Procédé microbiologique pour la préparation d'acides hétérocycliques aromatiques hydroxylés
DE2818668C3 (de) Mikrobiologisches Medium
DD297444A5 (de) Verfahren und vorrichtung fuer eine folgesteuerung bei aeroben bioprozessen
DE2924868A1 (de) Verfahren zur vermehrung von myxococcus fulvus dsm 1368
EP0758398B1 (fr) Procede de biotransformation d'acides carboxyliques en presence d'un micro-organisme
DE2300478C3 (de) Verfahren zur aeroben Züchtung von Hefen in synthetischen Medien
DE4025597C1 (en) Cultivating microorganisms to give conc. cell suspension - ŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸŸ by growing microorganisms in fermentation vessel, passing suspension through tangential throughflow liq
WO2013045368A1 (fr) Procédé de production de biogaz
DD256149A1 (de) Verfahren und vorrichtung zur regelung der geloest-sauerstoff-konzentration
DD283737A7 (de) Verfahren zur kultivierung von methanotrophen bakterien
DE1442188A1 (de) Verfahren zur Gewinnung von L-Glutaminsaeure durch Vergaerung
DE2300478B2 (de) Verfahren zur aeroben zuechtung von hefen in synthetischen medien
DD206684A3 (de) Verfahren zur zuechtung von mikroorganismen

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1997910254

Country of ref document: EP

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1998 517891

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 09284536

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1997910254

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997910254

Country of ref document: EP