WO2009011645A1 - Co-fermentation auto-immobilisée - Google Patents

Co-fermentation auto-immobilisée Download PDF

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Publication number
WO2009011645A1
WO2009011645A1 PCT/SE2008/050781 SE2008050781W WO2009011645A1 WO 2009011645 A1 WO2009011645 A1 WO 2009011645A1 SE 2008050781 W SE2008050781 W SE 2008050781W WO 2009011645 A1 WO2009011645 A1 WO 2009011645A1
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WO
WIPO (PCT)
Prior art keywords
fermentation process
process according
filamentous
organism
micro
Prior art date
Application number
PCT/SE2008/050781
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English (en)
Inventor
Lars Edebo
Original Assignee
Lars Edebo
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.)
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Publication date
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Publication of WO2009011645A1 publication Critical patent/WO2009011645A1/fr

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    • 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
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/16—Enzymes or microbial cells immobilised on or in a biological cell
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/12—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing sulfite waste liquor or citrus waste
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a process for fermenting complex substrates such as lignocellulosic hydrolysates which contain mixtures of sugars and toxic substances, by using at least two different micro-organisms at high concentrations e.g. for ethanol production.
  • pentoses are common. Since they are not fermented by the ordinary yeast Saccharomyces cerevisiae, great efforts have been devoted to finding alternative microbes or inserting xylose- fermenting genes into Saccharomyces. Other fungi such as zygomycetes have demonstrated high capacities for fermenting xylose. In the chemical depolymerisation and delignification process toxic compounds like furfurals and organic acids, which are antagonistic to the microbes employed, are produced such that no or poor growth occurs, when most fungi are inoculated directly into concentrated lignocellulosic hydrolysates such as dilute sulphuric acid hydrolysate (DAH) and spent sulphite liquor (SSL).
  • DHA dilute sulphuric acid hydrolysate
  • SSL spent sulphite liquor
  • microbes By tradition pure cultures are usually employed in microbiology and biotechnology in order to be able to control the reactions. For complex media one microbe might not be sufficient to reduce the harmful effects of the toxic compounds and to ferment a mixture of sugars. Therefore two or more microbes may be combined.
  • Patil ef a/. Enzyme Microb. Technol. 1990, 12: 141-1478 added fungal mycelium to S. cerevisiae for accelerating ethanol production and several species were tested alone or in combination such as Aspergillus niger, A. oryzae, Fusarium tricinctum, Neurospora sitophila, Penicillium chrysogenum, Sclerotium rolfsii, Sporotrichum pulverulentum, and Trichoderma reesii. Holmgren and Sellstedt (WO2004SE01786 20041201 ) have primarily used the ascomycetes Chalara and Trametes in combinations with S.
  • the present invention solves the above problem by providing a process for the fermentation of complex and toxic substrates such as lignocellulosic hydrolysates into for example ethanol by using at least two different micro-organisms at high concentrations.
  • One organism usually a fungus, has a natural filamentous shape, and is capable of forming a continuous or pellet-like network wherein yeast cells can be enclosed and immobilized. This process is favourable both with respect to high fermentative turnover and inactivation of toxic compounds.
  • the present invention relates to a fermentation process wherein a filamentous micro- organism and yeast and/or bacterial cells are co-operating by fermenting lignocellulosic hydrolysates which contain mixtures of both sugars and toxic substances characterized by a two step process wherein
  • the filamentous micro-organism is aerobically cultivated in a medium and produces mycelium network; and - in the second step the yeast and/or bacterial cells are immobilized with said mycelium network and incubated under oxygen-limited conditions.
  • the filamentous micro-organism produces the mycelium network in the form of pellets.
  • the mycelium network is retained in the fermentor by a screen.
  • the filamentous micro-organism is able to ferment pentose sugars.
  • the filamentous micro-organism is able to ferment xylose.
  • the filamentous micro-organism is a zygomycetes filamentous fungus.
  • the filamentous micro-organism is a fungus of the family Mucoraceae.
  • the filamentous micro-organism is a fungus of the genus Mucor.
  • the filamentous micro-organism is a fungus of the species Mucor indicus.
  • the filamentous micro-organism is the fungus Mucor indicus CCUG 22424.
  • yeast and/or bacterial cells are of the yeast genera.
  • yeast cells are of the genus Saccharomyces.
  • yeast is Saccharomyces cerevisiae.
  • yeast genera are used together with Saccharomyces.
  • bacteria are used in lieu of or together with yeasts.
  • the mycelium network production and immobilization of the non-filamentous micro-organism takes place in the presence of multivalent cations.
  • the multivalent cations are chosen from the group comprising calcium and/or magnesium.
  • the ethanol is produced in the second step from a medium containing lignocellulosic hydrolysates.
  • the lignocellulosic hydrolysate is spent sulphite liquor.
  • the invention also relates to a tube-formed vertical fermentor characterized in that it has an inlet for substrate at the bottom and recovery of ethanol-rich medium at the top, continuous or semi-continuous addition of fresh cells at the top, and release of used cells at the bottom such that concentration gradients become established with respect to sugars, other nutrients including oxygen as well as ethanol.
  • the fermentor is provided with a mixing unit for the biomass consisting of a rotating vertical axis provided with horizontal or nearly horizontal paddles or rods fitting in between rods protruding from the wall of the fermentor.
  • the present invention discloses a process for obtaining an improved ethanol yield from lignocellulosic hydrolysates by the use of a combination of one or more non-filamentous micro-organisms, preferably highly hexose-fermentative micro-organisms chosen from the group comprising bacteria, or yeast genera, preferably of the genus Saccharomyces and most preferably Saccharomyces cerevisiae with a filamentous, pellet-forming, xylose- fermentative micro-organism chosen from the group comprising zygomycetes filamentous fungi, preferably from a fungus of the family Mucoraceae , more preferably from a fungus of the genus Mucor, most preferably a fungus of the species Mucor indicus which also has high capacity to consume toxic compounds like furfural, hydroxymethylfurfural, and acetic acid.
  • non-filamentous micro-organisms preferably highly hexose-ferment
  • filamentous microbes growing in liquid media may form pellets which can be kept in the fermentor by using a standard, relatively coarse mesh. Aerobic conditions favour growth and production of fungal biomass.
  • pellet-like units consisting of intertwined hyphae form.
  • the pellets are often spherical, similar to the beads used for chromatography and ion-exchange.
  • the diameter is often one or a few millimetres.
  • this pellet network is utilized to immobilize and retain non-filamentous cells in its meshes. Retention is enhanced by adsorption to the filaments and aggregation. Adsorption and aggregation are favoured by multivalent cationic compounds.
  • microbes When more than one kind of microbes is growing in the same medium, their reciprocal concentrations and activities may change over time. However, when growth is limited, as it is in toxic media, this effect is smaller. Thus, if the microbes are first grown separately outside the fermentor, and then mixed and attached to each other for a second fermentation step, the balance between them can be controlled, such that their different capacities can complement each other. Pellet formation as well as yeast aggregation and attachment are influenced by the presence of divalent cations such as calcium and magnesium. Furthermore such ions exerted protection of the cells in the toxic media.
  • the high fermentative capacity for hexoses by Saccharomyces cerevisiae yeast cells can be enclosed in the above filamentous network and immobilized at high concentrations which are favourable both with respect to high fermentative turnover and inactivation of toxic compounds.
  • hexose-fermentative bacteria or yeast cells of different yeast genera are used in lieu of, or together with Saccharomyces.
  • the fermentation of xylose into ethanol can be performed by other xylose-fermentative micro-organisms in cooperation with the filamentous, pellet- forming, xylose-fermentative micro-organism, such as for example by xylose-fermentative yeast (e.g. Pichia stipitis, Candida shehafae) or bacteria (e.g. genetically engineered Corynebacterium glutamicum, E.coli, Zymomonas mobilis).
  • xylose-fermentative yeast e.g. Pichia stipitis, Candida shehafae
  • bacteria
  • the different micro-organisms are chosen such that they do not interfere negatively with each other, and supplement each other with respect to inactivation of toxic compounds and fermentation. Neither of the micro-organisms survived when inoculated separately at low concentrations, but when they act together in high concentrations of cells, they succeed in fermenting the hexoses as well as xylose, and survive well for more than a week of mixed culture.
  • fresh cells can be supplied throughout the fermentation process which may proceed by a continuous flow of medium.
  • the second step involves 'anaerobic' (oxygen-limited), immobilized, co-operative fermentation by M. indicus and S. cerevisiae in undiluted Na-SSL.
  • Step 1 Aerobic cultivation
  • Step 2 Co-operative fermentation by M. indicus and S. cerevisiae Fifty grams of the wet mycelium obtained in step 1 , containing ca 0.9 g dry weight mycelium was mixed with 8.9 g baker ' s yeast (Saccharomyces cerevisiae) containing ca 2.5 g dry weight yeast and 41.1 g 25% Na-SSL medium as above. The mixture was kept in a 250 ml flask with baffles, sealed with aluminium foil kept with a rubber band, and incubated on a reciprocating shaker table (100 rpm) at ca 30°C.
  • baker ' s yeast Sacharomyces cerevisiae
  • a synergistic and concentrated metabolic capacity is accomplished by aggregation and adsorption of yeast cells into and onto mycelial pellet particles. Such pellets are suitable for immobilization and containment in a fermentor.
  • the metabolic capacity encompasses consumption of sugars and toxic compounds present in the liquid to become fermented as well as production of ethanol. Production of biomass is mainly accomplished in a process which is aerobic at least for the mycelium and precedes fermentation. No growth is required in the fermentation step which makes possible fermentation of fairly toxic media.
  • the liquid may stream into a tube with a high concentration of fungal biomass which is arranged similarly to a chromatography column and is working like a catalyzer with fermenting cells immobilised as pellets rather than a vessel for cultivation of fungal biomass.
  • a gradient of toxicity is created with greatest toxic effect at the bottom, such that the cells become most damaged there.
  • the toxicity is decreasing as the medium is flowing upwards. Notwithstanding this effect fresh cells may be added at the top to achieve maximum productivity of ethanol, and damaged cells removed at the bottom.
  • the fermentor is provided with a mixing unit for the biomass consisting of a rotating vertical axis provided with horizontal or nearly horizontal paddles or rods fitting in between rods protruding from the wall of the fermentor.
  • a mixing unit for the biomass consisting of a rotating vertical axis provided with horizontal or nearly horizontal paddles or rods fitting in between rods protruding from the wall of the fermentor.
  • the paddles or rods are rotating slowly.

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

Abstract

L'invention porte sur un processus de fermentation selon lequel un micro-organisme filamenteux et des cellules de levure coopèrent à la production par fermentation d'éthanol à partir d'hydrolysats ligno-cellulosiques contenant des mélanges de sucres et de substances toxiques. Le processus de fermentation comporte deux étapes : 1) culture aérobie du micro-organisme filamenteux dans un milieu et production d'un réseau mycélien, et 2) immobilisation du micro-organisme filamenteux dans le réseau mycélien, et son incubation dans des conditions de rareté d'oxygène. L'invention porte également sur un fermenteur optimisé pour le processus de fermentation de l'invention.
PCT/SE2008/050781 2007-07-13 2008-06-26 Co-fermentation auto-immobilisée WO2009011645A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE0701731 2007-07-13
SE0701731-2 2007-07-13

Publications (1)

Publication Number Publication Date
WO2009011645A1 true WO2009011645A1 (fr) 2009-01-22

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK109693A (da) * 1993-09-28 1995-03-29 Finn Gemynthe Madsen Fremgangsmåde og anlæg til omsætning af biomasse ved hjælp af mikroorganismer
EP1352953A2 (fr) * 2002-03-27 2003-10-15 Biovitis S.A. Inoculum fongique, procédés de préparation dudit inoculum et procédés de mise en oeuvre dans le traitement d'effuents riches en matière organique
WO2004029240A1 (fr) * 2002-09-25 2004-04-08 Universidad de Córdoba Procede d'obtention de biocapsules de levures, biocapsules ainsi obtenues et leurs applications
WO2005054487A1 (fr) * 2003-12-01 2005-06-16 Swetree Technologies Ab Procede de fermentation, culture de depart et milieu de croissance

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK109693A (da) * 1993-09-28 1995-03-29 Finn Gemynthe Madsen Fremgangsmåde og anlæg til omsætning af biomasse ved hjælp af mikroorganismer
EP1352953A2 (fr) * 2002-03-27 2003-10-15 Biovitis S.A. Inoculum fongique, procédés de préparation dudit inoculum et procédés de mise en oeuvre dans le traitement d'effuents riches en matière organique
WO2004029240A1 (fr) * 2002-09-25 2004-04-08 Universidad de Córdoba Procede d'obtention de biocapsules de levures, biocapsules ainsi obtenues et leurs applications
WO2005054487A1 (fr) * 2003-12-01 2005-06-16 Swetree Technologies Ab Procede de fermentation, culture de depart et milieu de croissance

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AURANGZEB M. ET AL.: "Ethanol fermentation of raw starch", PAKISTAN JOURNAL OF SCIENTIFIC AND INDUSTRIAL RESEARCH, vol. 35, no. 4, April 1992 (1992-04-01), pages 162 - 164 *
KIM S.W. ET AL.: "Effect of aeration and agitation on the production of mycelial biomass and exopoly-saccharides in an enthomopathogenic fungus Paecilomyces sinclairii", LETTERS IN APPLIED MICROBIOLOGY, vol. 36, 2003, pages 321 - 326 *
MILLATI R. ET AL.: "Performance of Rhizopus, Rhizomucor, and Mucor in ethanol production from glucose, xylose, and wood hydrolyzates", ENZYME AND MICROBIAL TECHNOLOGY, vol. 36, 2005, pages 294 - 300, XP004698764 *
PALMQVIST E. ET AL.: "Simultaneous detoxification and enzyme production of hemicellulose hydrolysates obtained after steam pretreatment", ENZYME AND MICROBIAL TECHNOLOGY, vol. 20, 1997, pages 286 - 293, XP007904791 *

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