WO2010031793A2 - Bactérie de fermentation thermophile produisant du butanol et/ou de l’hydrogène à partir de glycérol - Google Patents

Bactérie de fermentation thermophile produisant du butanol et/ou de l’hydrogène à partir de glycérol Download PDF

Info

Publication number
WO2010031793A2
WO2010031793A2 PCT/EP2009/062022 EP2009062022W WO2010031793A2 WO 2010031793 A2 WO2010031793 A2 WO 2010031793A2 EP 2009062022 W EP2009062022 W EP 2009062022W WO 2010031793 A2 WO2010031793 A2 WO 2010031793A2
Authority
WO
WIPO (PCT)
Prior art keywords
glycerol
fermentation
strain
butanol
bio
Prior art date
Application number
PCT/EP2009/062022
Other languages
English (en)
Other versions
WO2010031793A3 (fr
Inventor
Zuzana Mladenovska
Slawomir Dabrowski
Original Assignee
Technical University Of Denmark
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 Technical University Of Denmark filed Critical Technical University Of Denmark
Publication of WO2010031793A2 publication Critical patent/WO2010031793A2/fr
Publication of WO2010031793A3 publication Critical patent/WO2010031793A3/fr

Links

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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • 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
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • 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
    • 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
    • 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
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • the present invention pertains to microbiology and biotechnology. More particularly, the present invention relates to a microorganism capable of producing alcohol and/or hydrogen, such as a thermophilic bacterium capable of catalyzing fermentation of glycerol to e.g. butanol and/or hydrogen. Furthermore, methods and uses concerning such a microorganism are disclosed.
  • bio-fuel such as bio-diesel, bio-ethanol, bio-butanol and bio- methane. Due to increasing interest in bio-fuel and bio-energy, the world- wide production of bio-fuel is increasing. Today, mainly bio-ethanol, bio- diesel, and bio-gas, such as bio-methane, are produced in industrial scale.
  • First generation technology produces (bio-)alcohol predominantly from a carbohydrate or sugar, comprising e.g the monosaccharides glucose, disac- chahdes such as sucrose, also called saccharose (i.e. a dimer of the mono- saccharides glucose and fructose), and starch (polysaccharides of glucose).
  • Second generation technology provides (bio-)alcohol from lignocellulosic materials such as straw, wood and agricultural residues, which are often considered as wastes. Consequently, these kinds of materials are often cheap, but the process technology is more advanced than simply converting sugar and starch.
  • lignin which binds together pectin, protein and the two polysaccharides, cellulose and hemicel- lulose, in lignocellulosic biomass.
  • Lignin is believed to give a plant resistance to e.g. microbial attack, as well as to add strength to the plant.
  • pre- treatment is used to render the biomass more susceptible to microbial con- version. This often implies degrading the lignocellulosic structure and releasing the polysaccharides.
  • pre-treatment is followed by treatment with enzymes capable of hydrolyzing cellulose and hemicellulose.
  • the cellulose fraction releases glucose (C ⁇ monosaccharide - sugar with six carbon atoms) and the hemicellulose fraction releases pentoses (C 5 monosaccha- rides - sugar with five carbon atoms) such as xylose, arabinose, galactose, mannose, rhamnose.
  • pentoses C 5 monosaccha- rides - sugar with five carbon atoms
  • xylose is believed to be the second most abundant sugar after glucose.
  • glucose which is easily fermented into e.g. ethanol by many different microorganisms, xylose is not as readily fermented.
  • Bio-diesel is a renewable bio-fuel produced from e.g. plant oils and animal fats.
  • the large-scale bio-diesel production process often comprises a chemical reaction, such as trans-esterification, converting the branched triglycerides of neutral lipids into straight-chain molecules of esters of the long chain fatty acids.
  • the triglycerides can be converted in the presence of a strong alkaline homogenous catalyst and methanol into a mixture of methyl esters and glycerol.
  • the main product, methyl esters are separated from the glycerol fraction and after purification used as a bio-fuel.
  • Glycerol is an unavoidable by-product of e.g. bio-diesel production.
  • the crude glycerol stream leaving the separator is commonly a heavy alkaline aqueous emulsion of 50% of glycerol and soap, methanol and catalyst. Due to the content of methanol, this by-product is regarded as hazardous, difficult to dispose and has a very low value. Therefore in continuation, the glycerol stream can be treated by inorganic acid whereby soaps are split into salts (which remain together with the glycerol) and free fatty acids, which are returned to the transesterification reactor. After removal of methanol, e.g. by vacuum flashing, glycerol of technical quality (-85% purity) is obtained. This can e.g. be sold to glycerol refiner, who is e.g. providing glycerol with a purity of around 99.7%.
  • the glycerol fraction can represent approximately 10% of the reaction mixture volume and is usually processed further for production of glycerol-dehved products with a variety of applications in food- and pharmaceutical industry, cosmetics, textiles, waxes, synthetic rubber, etc.
  • the glycerol by-product from bio-diesel production has been considered as an asset, but due to their value contributing to the reduction of the bio-diesel production costs as the production price of bio-diesel seems far from being competitive with the production price of conventional, petrochemical diesel.
  • accelerated global production of bio-diesel reveals now a surplus of glycerol which is reflected negatively on its market price.
  • bio-gas (bio-methane) production by anaerobic digestion seems to be bio-gas (bio-methane) production by anaerobic digestion.
  • Production of bio-methane is a well-known technology implemented world- wide for treatment of organic waste, such as sewage sludge, household waste, manure and industrial waste. The process is usually operated either in a mesophilic range, at 33-37°C, or in a thermophilic range at 50-55 0 C.
  • Bio- methane can be produced from organic matter in a fermentation process carried out by a complex microbial community consisting of a variety of non- methanogenic and methanogenic microorganisms, bacteria and archaea, respectively.
  • Complete conversion of organic matter into methane is believed to proceed as a cascade of reactions, involving one or more specific microbial group(s) in different steps of the degradation/fermentation process.
  • the degradation process is believed to be initiated by the action of hydrolytic and fermentative bacteria converting the organic compounds, such as carbohydrates, proteins and lipids into acetate, short-chained fatty acids longer than acetate (i.e. propionate, butyrate, iso-butyrate, valerate, iso-valerate, etc.), alcohol(s), lactate and hydrogen and carbon dioxide.
  • Acetate and hydrogen carbon dioxide are major intermediates in the degradation chain and they can be converted directly into methane by two different groups of archaea.
  • the minor pool of carbon comprised in the short-chained fatty acids with three and more carbon atoms is converted into methane by a syntrophic action of the volatile fatty acid oxidizing bacteria and the two type of methanogenic archaea. If the activity of the complex microbial community is in balance, no accumulation of volatile fatty acids is monitored and maximum possible methane yield is achieved. However, under certain conditions, such as overloading or partial inhibition by incoming substrate, an imbalance may arise which can e.g. be seen as accumulation of volatile fatty acids and a decreased methane production. In case of a severe inhibition, the methane production may stop and no accumulation of the volatile fatty acids occurs.
  • thermophilic anaerobic digestion of glycerol has not been explored yet. Only, Holm-Nielsen et al. (2007) reported recently, that the thermophilic anaerobic digestion process is in balance and stable as long as glycerol levels are kept around 5-7 g/l.
  • glycerol is a special physiological feature characteristic of few anaerobic bacteria.
  • the most intensively studied glycerol-fermenting bacteria are mesophile bacteria from the group of enterobacteria - the genera Klebsiella, Enterobacter, Citrobacter, and further Lactobacilli and the Clostridia - such as Clostridium butyricum and Clostridium pasteurianum [Biebl et al. (1999),].
  • glycerol dissimilation involves two pathways, one serving for glycerol oxidation and the other one for the consumption of ex- cess of reducing equivalents.
  • Typical products of the first pathway are bio- mass and acetate, while reduced products such as ethanol, butanol and lactate are generated in the second pathway.
  • Clostridium pasteurianum and Clostridium acetobutylicum are two of the very few fermentative bacteria capable of producing significant amounts of butanol from glycerol, and a mixture of glycerol and glucose, respectively. Additional products are ethanol and organic acids, such as acetate, lactate and bu- tyrate. C. pasteurianum produced 1.3-propanediol as well. [Biebl (2001 ); Andrade and Vasconcelos (2003)].
  • Glycerol appears to be an unavoidable by-product of the bio-diesel production.
  • technologies and methods relating to processes for converting glycerol, e.g. the glycerol-by-product from bio-diesel production into a valuable product.
  • Such technologies and methods may solve or reduce the problem of waste disposal and also bring new, attractive, high-value products on the market contributing to the improvement of the overall economy of e.g. the bio-diesel production.
  • glycerol As a sole source of carbon and energy in fermentation and produce e.g. butanol and or hydrogen.
  • glycerol has never been used as a feedstock in a large-scale industrial fermentation process.
  • Two moderate thermophiles capable of glycerol fermentation have been reported up to date.
  • the strain AT1 [Wittlich P et al. (2001 )] and a new species CaIo- ramator viterbensis (Seyfried M et al. 2002) were isolated from a non- specified habitat and an inclined hot spring, respectively. Both bacteria produce primarily 1 ,3-propanediol from glycerol.
  • Beside 1 ,3- propanediol the other reported by-products of the glycerol fermentation by strain AT1 were butyrate and ethanol, while the type strain of the species Caloramator viter- bensis JW/MS-VS5 T co-produced 1 ,3-propanediol with acetate and hydrogen.
  • Clostridia in a conventional fermentation, including industrial fermentations, possesses limitations.
  • some Clostridia are po- tential pathogens, and thus classified as members of the Risk Class 2 Group.
  • Bio-catalysts for industrial bio-butanol production today are mesophilic species, such as Clostridium acetobutylicum and Clostridium beijerinckii capable of fermenting carbohydrates of molasses, corn, rye or plant biomass hydro- lysates [Jones and Woods, 1986; Zverlov et al. 2006].
  • these butanol producers are not capable of using glycerol as a sole carbon source.
  • thermophilic microorganism suitable for, adaptable to, or adapted to e.g. an industrial fermentation providing e.g. a bio-fuel and/or one or more bio-chemical(s), such as production of butanol and/or hydrogen from glycerol and/or a carbohydrate.
  • microorganism or bio-catalyst suitable for, adapt- able to, or adapted to e.g. a sustainable management of e.g. one or more of bio-fuel(s), bio-chemical(s), bio-diesel and/or glycerol.
  • thermophilic microorganism which is capable of overcoming the above mentioned obstacles, and/or which possesses the above-mentioned desired properties, e.g. for production of bio-fuel(s), bio-gas, and/or bio-chemical(s), such as butanol and/or hydrogen.
  • the inventors identified a strain of a thermophilic bacterium which surpris- ingly and unexpectedly is capable of fermenting glycerol to e.g. butanol and/or hydrogen.
  • This novel microorganism is believed to possess a novel pathway, thereby providing conversion of a substrate comprising glycerol and/or a carbohydrate to e.g. butanol and/or hydrogen by fermentation.
  • a strain according to the invention classified as Thermoanaerobacterium, was deposited on July 11 2008 in concordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) as Thermoanaerobacterium sp. strain 260, and was given deposition number DSM 21660.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • two mutant strains derived from said strain 260 i.e. Thermoanaerobacterium sp. strain 260-M4 and Thermoanaerobacterium sp. strain 260-M9 have been deposited with the deposition numbers DSM 21694 and DSM 21695, respectively.
  • the current invention concerns a thermophilic bacterial strain capable of producing butanol and/or hydrogen from glycerol, and/or co-producing butanol and hydrogen from glycerol.
  • glycerol can be essentially the sole carbohydrate source.
  • a second aspect concerns a starter culture consisting of, or comprising, a strain according to the first aspect of the invention.
  • a third aspect of the invention pertains to the use of a bacterial strain according to the first aspect of the invention, or a starter culture according to the second aspect of the invention in a fermentation providing one or more of biomass, bio-fuel, bio-gas, bio-chemical.
  • a fourth aspect of the invention relates to a method of producing one or more of biomass, bio-fuel, bio-gas, and/or biochemical - said method comprising the step(s) of providing a fermentable substrate comprising a carbo- hydrate and/or glycerol, and subjecting said fermentable substrate to a fermentation, wherein said fermentation comprises a bacterial strain according to the first aspect, or a starter culture according to the second aspect.
  • a method for isolating a ther- mophilic bacterial strain comprising the steps of: (a) providing a sample from a man-made habitat or a natural habitat harboring a microbial community; (b) providing an enrichment microbial culture by cultivation of the sample from step (a) under anaerobic conditions in a glycerol-containing media (liquid or solid) preferably at a temperature or at a temperature range suitable for a thermophilic microorganism; (c) selection of a butanol- and/or hydrogen-positive strain by isolation of a pure culture from step (b) e.g.
  • Figure 1 A phase-contrast photomicrograph of exponentially growing cells of strain 260.
  • the cultivation medium contained 10 g/l glycerol and 2 g/l yeast extract.
  • Figure 2. Phylogenetic dendrogram based on a comparison of the 16S rRNA gene sequence of strain 260 and the related species. Scale bare represents 2 substitutions per 100 nucleotides.
  • the related strains are: Thermoanaero- bacte ⁇ um thermosaccharolyticum DSM 571 ⁇ , Thermoanaerobacterium thermosaccharolyticum GD17, Thermoanaerobacterium thermosaccharo- lyticum W 16, Thermoanaerobacterium thermosaccharolyticum D 120-70, Thermoanaerobacterium aotearoense JW/SL-NZ613 T , Thermoanaerobacterium thermosulfurigenes ATCC 33743, Thermoanaerobacterium aciditoler- ans 761 -119, Thermoanaerobacterium islandicum AK17, Clostridium ther- mocellum DSM 1237, Caloramator viterbiensis DSM 13723 T , Clostridium beijerinckii NCIMB 8052, Clostridium pasteurianum DSM 525 T ,
  • FIG. 3 Growth of strain 260 during fermentation of glycerol with concomitant butanol production.
  • the cultivation medium contained 10 g/l glycerol and 2 g/l yeast extract. Legend: glycerol (circle open), OD (tringel open), butanol (square closed).
  • Figure 4 Production of butanol, acetate, ethanol and lactate during growth of strain 260 in medium with 10 g/l glycerol and 2 g/l yeast extract. Legend: glycerol (circle open), acetate (diamond closed), butanol (square closed), lactate (triangle closed), ethanol (dash).
  • Figure 5 The yields of biomass and non-gaseous fermentation products after growth of strain 260 in medium with 10 g/l glycerol and 2 g/l yeast extract.
  • Figure 6 Effect of increasing concentration of glycerol on the yield of fer- mentation products produced by strain 260.
  • the medium was supplemented with 1 g/l yeast extract.
  • the results are means of triplicates with standard deviations.
  • Figure 7 Effect of various concentration of yeast extract on the product yields of strain 260. Fermentation was carried out in a medium with 20 g/l glycerol. The results are means of triplicates with standard deviations.
  • Figure 8 Effect of incubation temperature on the specific growth rate of strain 260 in medium with 10 g/l glycerol and 1 g/l yeast extract. The results are means of triplicates with standard deviations.
  • Figure 9 Effect of incubation temperature on the butanol yield of strain 260 in medium with 10 g/l glycerol and 1 g/l yeast extract. The results are means of triplicates with standard deviations.
  • Figure 10 Effect of pH on the specific growth rate of strain 260 in medium with 10 g/l glycerol and 1 g/l yeast extract. The results are means of triplicates with standard deviations.
  • Figure 11 Effect of pH on the butanol yield of strain 260 in medium with 10 g/l glycerol and 1 g/l yeast extract. The results are means of triplicates with standard deviations.
  • Figure 12 Product yields of strain 260 after growth in medium with 10 g/l glucose and 1 g/l yeast extract. The results are means of duplicates.
  • Figure 13 Product yields of strain 260 after growth in medium with 5 g/l xylose and 1 g/l yeast extract. The results are means of duplicates.
  • Figure 14 Composition of diluted grass hydrolysate (day 0) and product formation after 7 days of fermentation by strain 260. The results are means of duplicates.
  • Figure 15. Product yields of the mutants after growth in media with 10 g/l glycerol and 1 g/l yeast extract. The results are means of duplicates.
  • the medium contained 10 g/l of carbon compounds selected and 2 g/l yeast extract.
  • GIu- cose diamond open
  • xylose square open
  • glycerol circle open
  • acetate diamond closed
  • butanol square closed
  • lactate triangle closed
  • butyrate circle closed
  • ethanol dash
  • 1 ,3-PDO cross
  • Figure 17 Comparison of butanol yields from the fermentations with the wild- type strain 260 and with the mutant strain 260-M9.
  • the medium contained 10 g/l of carbon compounds selected and 2 g/l yeast extract.
  • Figure 18 Schematic representation of the product pattern according to the invention, and possibilities for conversion/appreciation/valorization of one or more (by-) products by methanogenesis.
  • Figure 20 Growth of strain 260, substrate consumption and product formation during batch fermentation without pH control in the lab-scale bioreactor. Legend: pH (cross), glycerol (circle open), butanol (square closed), acetate (diamond closed), ethanol (dash) and capacitance (triangle open).
  • Figure 21 Yields of the fermentation products of strain 260 from the batch fermentation without pH control in the medium with 30 g/l glycerol and 5 g/l tryptose.
  • Figure 22 Growth of strain 260, substrate consumption and product formation during batch fermentation with pH control in the lab-scale bioreactor kept at 0.2 bar overpressure.
  • (A) pH (cross), capacitance (trian- gel open), production rate of gas impulses (thangel closed).
  • Figure 23 Yields of the fermentation products of strain 260 from the batch fermentation with pH control at pH 6.0, and at two different gas overpressures. Fermentation at the 0.2 bar overpressure was done in the medium with 20 g/l glycerol and 5 g/l tryptose, while the fermentation with 1.5 bar overpressure was done in the medium with 30 g/l glycerol and 5 g/l tryptose. Detailed description of the invention
  • composition comprising a chemical compound may thus comprise additional chemical compounds.
  • replicates and/or derivatives of the deposited strains or any other strain according to the invention are encompassed by the inven- tion.
  • the term “replicate” refers to a biological material that is substantially similar and/or identical, e.g. a copy of the material, such as material produced by growth of micro-organisms, e. g. growth of bacteria in culture media.
  • the term “derivative” refers to material created from the biological material and which is substantially modified to have new properties, for example caused by heritable changes in the genetic material. These changes can either occur spontaneously or be the result of applied chemical and/or physical agents (such as mutagenesis agents) and/or by recombinant DNA techniques as known in the art.
  • bio-fuel is meant to com- prise e.g. "bio-diesel”, “bio-ethanol”, “bio-butanol”, bio-hydrogen and “bio- methane”.
  • Bio-fuel is also meant to comprise one or more of “bio-diesel”, “bio-ethanol”, “bio-butanol”, “bio-hydrogen” and/or “bio-methane”, including any combination thereof.
  • bio may indicate that the product is derived from, provided by, and/or comprises a bio-based, environmentally, and/or CO2-"friendly” alternative to e.g. fossil-fuels, and/or non-renewable energy sources.
  • bio may also indicate that the alcohol is derived from conversion or fermentation of a carbon-comprising feedstock, such as an agricultural feedstock, e.g. comprising one or more materials, parts, compounds or composition of or derived from animal-, plant-, or microbial origin, including any combination thereof.
  • a feedstock can be considered re- newable, because it is produced by e.g. means of energy derived directly or indirectly from e.g. the sun using photosynthesis, geothermal energy, etc. provided that all minerals required for growth (such as nitrogen and phosphorus) are returned to the land.
  • Bio-fuels can be produced from a variety of feedstocks comprising or derived from one or more of e.g.
  • sugar cane ba- gasse, miscanthus, sugar beet, sorghum, sorghum, switchgrass, barley, hemp, kenaf, potatoes, sweet potatoes, cassava, sunflower, fruit, molasses, corn, stover, grain, wheat, straw, cotton, etc., but also from other biomass, as well as e.g. many types of cellulose waste and harvestings/harvested material, but also animal carcasses and/or e.g. waste material, by products, or any material from animal production, slaughter houses and the like.
  • modification of a microorganism or "a (genetically) modified microorganism” in the context of the current invention are meant to comprise e.g. any type or form or combination of recombinant DNA techniques, such as any type, form or length of DNA insertion, deletion, mutation, transformation with e.g. one or more plasmid(s), bacterial artificial chromosome(s), phage(s) etc. known in the art. Further included are e.g.
  • alcohol is meant to com- prise one or more, and any combination of chemical compound(s) comprising one or more hydroxyl (OH) groups, such as methanol, ethanol, propanol, bu- tanol, ethylene glycol, propanediol, butanediol, etc., including any isomer and/ or enantiomer.
  • OH hydroxyl
  • methanol also abbreviated “MeOH” indicates an alcohol with the formula CH 3 OH.
  • ethanol and “bio-ethanol” can be used interchangeably and are meant to comprise ethyl alcohol with the formula CH 3 CH 2 OH, also abbrevi- ated EtOH.
  • propanol comprises the isomers of propanol, namely propan-1 -ol, also called n-propanol (CH 3 CH 2 CH 2 OH), and propan-2-ol, also called isopro- pyl alcohol, or isopropanol (CH 3 ) 2 CHOH.
  • butanol in the context of the present invention is meant to comprise an alcohol with the formula C 4 Hi 0 O, including any isomer(s), such as 1 - butanol (n-butanol), 2-butanol (sec-butanol), 2-methyl-1 -propanol (isobu- tanol), and tert-butanol, (2-methyl-2-propanol), and any combination thereof.
  • This four carbon alcohol has been proposed as a new, advanced fuel of the next generation of bio-fuels [U.S. Department of Energy. 2006].
  • ethylene glycol is meant to comprise a diol (i.e. a chemical compound comprising two hydroxyl groups) with the formula 02H 4 (OH) 2 , also called monoethylene glycol (MEG), 1 ,2-ethanediol, or ethane-1 ,2-diol.
  • propanediol is meant to comprise a diol with the formula CsH 8 O 2 , and comprises the two isomeric compounds propane-1 ,2-diol (also called 1 ,2-propanediol, or propylene glycol), and propane-1 ,3-diol (also called 1 ,3- propanediol, or trimethylene glycol), and any combination thereof.
  • butanediol is meant to comprise a diol with the formula C 4 Hi 0 O 2 , and comprises the isomeric compounds 1 ,2-butanediol, 1 ,3-butanediol, 1 ,4- butanediol, and 2,3-butanediol, and any combination thereof.
  • Glycerol is meant to comprise propane-1 ,2,3-thol.
  • Glycerol can be of different qualities and purities, e.g. "low grade” or “crude glycerol”, such as around 65% pure (volume/volume; weight/volume; or mol/mol); “technical quality”, commonly around 87 % pure (volume/volume; weight/volume; or mol/mol); “pure”, e.g. around or more than 99%, 99.5%, or 99.7 % pure (volume/volume; weight/volume; or mol/mol); or “pro analyst', such as at least or more than or 99.9% pure (volume/volume; weight/volume; or mol/mol).
  • "Low grade” or “crude” glycerol can e.g. be obtained from a trans-estehfication process comprising a plant oil, such as rape-seed oil or e.g. animal fats.
  • sole carbohydrate source are meant to indicate -100% (mol/mol; or g/g), or more than 99% (mol/mol, or g/g) of the total carbohydrate(s); "essentially (as) the sole carbohydrate source” indicates e.g. -95-99% (mol/mol, or g/g) of the total carbohydrate(s); and "predominant carbon source” indicates e.g. more than 70% (mol/mol, or g/g), 80% (mol/mol, or g/g), or more than 90% (mol/mol, or g/g) of the total carbohydrate(s).
  • the term "mesophile” is meant to comprise an organism that grows/lives/thrives best in moderate temperature, neither too hot nor too cold, such as at a temperature or optimal growth temperature between 15-40 0 C, such as around 15°C, 20 0 C, 25°C, 30 0 C, 35°C, 37°C, and or 40 0 C.
  • thermophile is meant to comprise an organism which thrives at relatively high temperatures, such as between 40 0 C and 70 0 C, or more.
  • Thermophiles can be classified into obligate and fac- ultative thermophiles: obligate thermophiles (also called extreme thermophiles) require high temperatures for growth, while facultative thermophiles (also called moderate thermophiles) can thrive at high temperatures but also at lower temperatures (e.g. below 50 0 C).
  • Hyperthermophiles are particularly extreme thermophiles for which the optimal temperatures are above 80 °C. Many thermophiles are classified as archaea, also called archae or archaea- bacteha (outdated term).
  • Thermophiles can e.g. be found in various geothermally heated regions of the Earth such as hot springs like those in Yellowstone National Park or in Iceland, as well as deep sea hydrothermal vents, as well as decay- ing plant matter such as peat bogs and compost, digestive tracts of animals and human, man-made systems for treatment of organic waste and wastewater. As a prerequisite for their survival, it is believed that thermophiles contain enzymes that can function at these elevated temperatures.
  • thermophilic bacteria are classified to be of Risk Class 1 Group, since none of them have yet been characterized as pathogens or potential pathogens.
  • the literature on thermophilic glycerol-fermentative bacteria is surprisingly limited, indicating that the thermophilic glycerol fermentation is yet an unexplored field.
  • the strain according to the invention is a low Risk Class Group, such as Group 1.
  • identity or “identical” often refer to two or more sequences, such as nucleic acid or polypeptide sequences, that are the same or that have a specified percentage of nucleic acids or amino acids that are the same, when compared and aligned for maximum correspondence over a comparison win- dow, as measured e.g. using one of the sequence comparison algorithms listed herein, or by manual alignment and visual inspection.
  • Substantially identical can refer to e.g. two nucleic acid or polypeptide sequences that have at least about 60%, about 65%, about 70%, about 80%, about 90%, or about 95% or more nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the sequence comparison algorithms given herein, or by manual alignment and visual inspection. This definition also refers to the complement of the test sequence with respect to its substantial identity to a reference sequence.
  • a comparison window refers to any one of the number of contiguous positions in a sequence (being anything from between about 20 to about 600, most commonly about 100 to about 150) which may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment can e.g. be achieved using computerized implementations of alignment algorithms (e. g. GAP, BESTFIT, FASTA, TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis. USA), or BLAST analyses available e.g. at www.ncbi.nlm.nih.gov or www.ebi.ac.uk).
  • alignment algorithms e.g. GAP, BESTFIT, FASTA, TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis. USA
  • BLAST analyses available e.g. at www.ncbi.nlm.nih.gov or www.ebi.ac.uk.
  • the current invention concerns a thermophilic bacterial strain capable of producing butanol and/or hydrogen from glycerol, and/or co- producing butanol and hydrogen from glycerol.
  • thermophilic bacterial strain is capable of producing butanol and/or hydrogen from a carbohydrate, glycerol, or a combination of glycerol and carbohydrate.
  • glycerol is the sole carbohydrate source, or essentially the sole carbohydrate source. According to another embodiment, glycerol is the predominant carbon source.
  • the strain apart from (co-)producing butanol and/or hydrogen, is also capable of growing/proliferation using glycerol as the major/predominant (e.g. more than 70% (mol/mol), 80% (mol/mol), or more than 90% (mol/mol) of the total carbohydrate(s)), or sole carbohydrate source (100% (mol/mol), or at least 99%(mol/mol), or more than 99% (mol/mol) of the total carbohydrate(s)).
  • glycerol e.g. more than 70% (mol/mol), 80% (mol/mol), or more than 90% (mol/mol) of the total carbohydrate(s)
  • sole carbohydrate source e.g. more than 70% (mol/mol), 80% (mol/mol), or more than 90% (mol/mol) of the total carbohydrate(s)
  • sole carbohydrate source e.g. more than 70% (mol/mol), 80% (mol/mol), or more than 90% (mol/mol) of the total carbo
  • the bacterial strain according to the invention belongs to the genus Thermoanaerobacterium.
  • the strain can also be at least 90%, 95%, 97%, 98% or more identical to Thermoanaerobacterium thermosaccharolyticum based on 16S rRNA gene analysis.
  • a particulate embodiment of a strain according to the invention is strain 260 (DSM 21660), or a derivative of strain 260 (DSM 21660), such as strain 260-M4 (DSM 21694), or strain 260-M9 (DSM 21695), or a derivative of 260-M4 (DSM 21694), or strain 260-M9 (DSM 21695).
  • the bacterial strain is strain 260 (DSM 21660).
  • the strain is a derivative of strain 260 (DSM 21660).
  • the strain is 260-M4 (DSM 21694), or strain 260-M9 (DSM 21695).
  • the strain is a derivative of 260-M4 (DSM 21694), or strain 260-M9 (DSM 21695).
  • the strain is a derivative of strain 260 (DSM 21660), strain 260-M4 (DSM 21694), or strain 260-M9 (DSM 21695).
  • a bacterial strain according to the invention can be capable of producing one or more (bio-)alcohols, including e.g. methanol, ethanol, propanol, butanol, ethylene glycol, propanediol, butanediol, including any isomer and/ or enanti- omer, and any combination thereof.
  • a bacterial strain according to the invention may be producing, or be capable of producing butanol, such as one or more of n-butanol, sec-butanol, isobutanol, and tert-butanol, and any combination thereof.
  • the strain according to the invention is capable of producing at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or more mol butanol per mol glycerol, wherein said butanol is one or more of n- butanol, sec-butanol, isobutanol, and tert-butanol, and any combination thereof.
  • at least 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80 %, 85%, 90%, 95%, 98%, 99%, 99.5% (mol/mol) of the total alcohol(s) produced by a bacterial strain according to the invention is butanol.
  • at least 50% or more; 60 % or more; 70% or more; 80 % or more; or 90% or more (mol/mol) of the total alcohol(s) produced from glycerol by said bacterial strain is butanol.
  • the bacterial strain is capable of producing 0.25, 0.30, 0.35 or more mol butanol per mol glycerol.
  • Said butanol can be one or more of n-butanol, sec-butanol, isobutanol, and tert-butanol, and any combination thereof.
  • a bacterial strain according to the invention can also be involved in production of one or more (bio-)gases, including e.g. methane and hydrogen, includ- ing any combination thereof.
  • a bacterial strain may also be able to (co-)produce hy- drogen from glycerol.
  • the bacterial strain is capable of producing at least 0.1 ; 0.12; 0.14, 0.17; 0.2; 0.3; 0.4; 0.5 or more mol hydrogen per mol glycerol.
  • said strain is capable of producing at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or more mol hydrogen per mol glycerol.
  • the bacterial strain may not be producing significant amounts of one or more undesired byproducts.
  • An example of an unde- sired byproduct is butyrate.
  • the strain is not producing significant amounts of butyrate (i.e. less than 0.1 , or 0.05 g/l), e.g. when grown on glycerol as sole carbon source.
  • a bacterial strain according to the invention may possess one or more pH optimum/optima for production of one or more products, such as one or more bio-gas(es), such as hydrogen, bio-alcohol(s), such as butanol, including any combination thereof.
  • a pH-optimum is in the range of pH 4.0-10; pH 5.0 to 9.0; pH 6.0 to 8.0; pH 6.5 and 7.5; pH 6.8 and 7.2, or around pH 7.
  • a pH-optimum is below pH 4.0, around pH 4.0; around pH 5.0; around pH 6.0; around pH 7.0; around pH 8; around pH 9.0; around pH 10.0; around pH 11.0, or above pH 11.0.
  • a pH-optimum is in the range of around pH 4.0 to 5.0; pH 5.0 to 6.0; pH 6.0 to 7.0; pH 7.0 to 8.0; pH 8.0 to 9.0; pH 9.0 to pH 10.0; pH 10.0 to pH 11.0.
  • the pH optimum for butanol and/or hydrogen production is in the range of pH 5.0 to 8.0; pH 6.5 to 7.5; pH 6.8 to 7.2; or around pH 7.
  • a bacterial strain according to the invention may possess one or more temperature optimum/optima for production of one or more products, such as one or more bio-gas(es) (e.g. hydrogen), and/or one or more bio-alcohol(s) (e.g. butanol), including any combination thereof.
  • a temperature optimum is in the range of around 30 to 90 0 C; 40 to 80 0 C; 50 to 65°C; 55 to 63°C, or around 60 0 C.
  • a temperature optimum is around 30°C; 35°C; 40 0 C; 45°C; 50 0 C; 55°C; 56°C; 57°C; 58°C; 59°C; 60 0 C; 61 0 C; 62°C; 63°C; 64°C; 65°C; 66°C; 67°C; 68°C; 69°C; 70 0 C; 71 0 C; 72°C; 73°C; 74°C; 75°C; 76°C; 77°C; 78°C; 79°C; 80 0 C; 85°C; 90 0 C; or more than 90 0 C.
  • a temperature optimum is in the range of around 30-35°C; 35-40°C; 40-45 0 C; 45- 50°C; 50-55°C; 55-60 0 C; 60-65°C; 65-70°C; 70-75 0 C; 75-80°C; 80-85°C; or 85-90 0 C.
  • the temperature optimum for butanol and/or hydrogen production is in the range of 35 to 70 °C; 50 to 65 °C; 55 to 63 0 C, or around 60 0 C.
  • a bacterial strain according to the invention can be able to ferment one or more of crude glycerol (e.g. 65% weight/volume or more), glyc- erol of technical quality (e.g. 85 % weight/volume or more); pure glycerol (e.g. 99 % weight/volume or more); pro analysii (p. a.) glycerol (e.g. 99.9 % weight/volume or more).
  • the glycerol can also be a by-product, or derived from a by-product from the production of a bio-fuel, such as glycerol derived from production of biodiesel or bioethanol.
  • the strain is capable of fermenting glycerol provided or derived from the production of a bio-fuel.
  • the bacterial strain is capable of fermenting glycerol derived from production of biodiesel or bioethanol.
  • the bacterial strain is capable of producing one or more of biomass, alcohol, methanol, ethanol, propanol, isopropanol, propanediol, butanol, bio-fuel, bio-gas, hydrogen, and any combination thereof.
  • the bacterium is capable of producing alcohol, such as one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, ethylene glycol, 1 ,2- propanediol, 1 ,3-propanediol, 1 ,2-butanediol, 1 ,3-butanediol, 1 ,4-butanediol, 2,3-butanediol, and any combination thereof.
  • alcohol such as one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, ethylene glycol, 1 ,2- propanediol, 1 ,3-propanediol, 1 ,2-butanediol, 1 ,3-butanediol, 1
  • a process, use or method comprising e.g. a fermentation comprising a bacte- rial strain according to the invention could be carried out in a medium containing a carbon substrate to be converted into a bio-fuel and/or bio-gas, such as e.g. butanol and hydrogen.
  • a fermentable substrate may comprise one or more carbon compounds which could be a source of carbon and/or energy, and/or the sole source of carbon and energy.
  • the carbon compound(s)/composition(s)/carbohydrate(s) could be one or more of glycerol, monosaccharide (such as one or more of arabinose, fructose, glucose, galactose, mannose, xylose), disaccharide (such as cellobiose, lactose, maltose, sucrose), polysaccharides (such as arabinoxylan, cellulose, pectin, starch, xylan).
  • the glycerol is used as a major, or sole source of carbon and/or energy, e.g. in a defined, mineral medium.
  • such a defined medium could comprise minerals, salts, buffers, vitamins, co-factors, suitable for growth of the cultures and production of the butanol.
  • a suitable medium for butanol production from glycerol and/or a carbohydrate source by strain 260 is given in the experimental section Examples.
  • a fermentation with a strain according to the invention could be performed under aerobic, anaerobic, and/or microaerobic conditions.
  • a fermentation is performed under anaerobic conditions, such as under an atmosphere of argon, nitrogen, carbon dioxide, a mixture of nitrogen and carbon dioxide.
  • Water used for preparation of media could be boiled/autoclaved, medium could e.g. be flushed with nitrogen, nitrogen/carbon dioxide, or carbon dioxide to eliminate oxygen. If necessary, traces of oxygen could be removed from the media by addition of reducing agents commonly used for cultivation of anaerobic bacteria such as one or more of e.g. sodium sulfide, sodium sulfite, cysteine hydrochloride, sodium thioglycolate, titanium citrate.
  • thermophilic strain accord- ing to the invention belongs to the genus Thermoanaerobacter, Thermoan- aerobacte ⁇ um, or Clostridium. According to a further embodiment, said strain is known to produce alcohol.
  • the strain is a Thermoan- aerobacter
  • the strain belongs to the following species (including subspecies and strains, as well as strains derived from said genus, species, and subspecies): Thermoanaerobacter acetoethylicus, Thermoanaerobacter brockii, Thermoanaerobacter brockii subsp. finnii, Thermoanerobacter brockii subsp.
  • lactiethylicus Thermoanaerobacter cellu- lolyticus, Thermoanaerobacter ethanolicus, Thermoanaerobacter inferii, Thermoanaerobacter italicus, Thermoanaerobacter keratinophilus, Thermoanaerobacter kivui, Thermoanaerobacter mathranii, Thermoanaerobacter mathranii subsp.
  • Thermoanaerobacter pseude- thanolicus Thermoanaerobacter siderophilus, Thermoanaerobacter subter- raneus, Thermoanaerobacter sulfurigignens, Thermoanaerobacter sulfurophi- lus, Thermoanaerobacter tengcongensis, Thermoanaerobacter thermoco- priae, Thermoanaerobacter thermohydrosulf uncus, Thermoanaerobacter uzonensis, Thermoanaerobacter wiegelii, Thermoanaerobacter yonseiensis.
  • the strain according to the invention is a Thermoanaerobacterium, and optionally said strain belongs to the following species (including subspecies and strains, as well as strains derived from said genus, species, and subspecies): Thermoanaerobacterium aciditoler- ans, Thermoanaerobacterium aotearoense, Thermoanaerobacterium bryan- tii,Thermoanaerobacterium fijiensis, Thermoanaerobacterium islandicum, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium sac- charolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermo- anaerobacterium thermosulfurigenes, Thermoanaerobacterium xylanolyti- cum, Thermoanaerobacterium ze
  • the strain according to the invention is a Clostridium, and optionally said strain belongs to the following species (including subspecies and strains, as well as strains derived from said genus, species, and subspecies): Clostridium thermolacticum, Clostridium thermoaceticum, Clostridium thermoalcaliphilum, Clostridium thermoamylolyticum, Clostridium thermoautotrophicum, Clostridium thermobutyricum, Clostridium thermocel- lum, Clostridium thermopalmarium, Clostridium thermosuccinogenes, Clostridium thermosulfurogenes.
  • species including subspecies and strains, as well as strains derived from said genus, species, and subspecies
  • a bacterial strain according to the inven- tion is identical to, substantially identical to, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical to one or more of: Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium thermosaccharolyticum DSM 571 ⁇ , Thermoanaerobacterium thermosaccharolyticum GD17, Thermoanaerobacterium thermosaccharolyticum W16, Thermoanaerobacterium thermosaccharolyticum D120-70, Thermoanaerobacterium aotearoense JW/SL-NZ613 T , Thermoanaerobacterium thermosulfurigenes ATCC 33743, Thermoanaerobacterium aciditolerans
  • the 16S rRNA of a strain according to the invention is identical to, substantially identical to, or more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to the 16S rRNA of strain 260 (DSM 21660), strain 260-M4 (DSM 21694), or strain 260-M9 (DSM 21695).
  • the 16S rRNA of a strain according to the invention is encoded by a DNA sequence which is substantially identical to, or more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to SEQ ID 1 , SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11 , SEQ ID 12, SEQ ID 13, SEQ ID 14, or SEQ ID 15.
  • a bacterial strain according to the invention is strain 260 (DSM 21660), strain 260-M4 (DSM 21694), or strain 260-M9 (DSM 21695), or a derivative of any one of said strains.
  • a derivative may comprise one or more mutation(s), wherein said mutation(s) can be selected e.g. from the group consisting of one or more of point mutation, deletion, insertion, cross-over, transformation, and any combination thereof.
  • a derivative may comprise a sin- gle or more mutation(s)/change(s) compared to the strain the derivative is derived from no more than 1 ; 2 to 5; 6 to 10; 11 -20; 21 -50; or 51 -100 mutations/changes, e.g. in 1 ; 2 to 5; 6 to 10; 11-20; 21 -50; or 51-100 different genes in the genome (chromosome and/or plasmid).
  • a mutation/change comprises a plasmid, such as e.g. transformation with a plasmid, providing a (stable) replicating plasmid, such as a single, low or multi-copy plasmid, and/or loss of a plasmid.
  • a bacterial strain according to the invention is not capable of producing methane.
  • strain 260 is not able to produce methane, it is believed that such a bacterial stain e.g. in co- culture with a methanogenic archaea, such as one or more archae, will be able to produce methane, as the hydrogen made by strain 260 will be converted to methane by archaea.
  • a bacterial strain may be capable of fermenting glycerol of different qualities/purities, such as one or more of crude glycerol (65% weight/volume or more), glycerol of technical quality (85 % weight/volume or more); pure glycerol (99 % weight/volume or more); pro analysii ⁇ p. a.) glycerol (99.9 % weight/volume or more); glycerol comprising one or more impurities (e.g. glycerol derived from or produced by e.g. a bio- fuel; bio-diesel(), and any combination thereof.
  • the glycerol can be e.g.
  • low grade such as around less than, or around 65% pure (volume/volume; weight/volume; or mol/mol); "technical quality", commonly between 80 and 95%, such as around 87 % pure (volume/volume; weight/volume; or mol/mol); "pure”, e.g. around or more than 95%, 96, 97, 98, 99%, 99.5%, or 99.7 % pure (volume/volume; weight/volume; or mol/mol); or "pro analyst' (p. a.), such as at least or more than 99.7, 99.8, 99.9%, 99.95%, 99.99% pure (volume/volume; weight/volume; or mol/mol).
  • "Low grade" glycerol can e.g.
  • glycerol is of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95%, 97%, 98%, 99%, 99%, 99.5%, 99.7%, 99.8%, 99.9% purity.
  • said glycerol is a by- or waste-product from bio-diesel production, and/or from a trans-estehfication process using a plant oil or fat.
  • the glycerol comprises a mixture of one or more glycerols of different quality/purity.
  • the bacterial strain can be used in a fermentation, wherein no, or essentially no other microorganism(s) are present.
  • This can e.g. be a fermentation, where the bacterial strain ac- cording to the invention is predominant (e.g. more than 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, or 99.999999% of all microorganisms are members of the thermophilic strain according to the invention).
  • Common techniques known in the art could e.g.
  • said fermentation comprises co-fermentation with one or more microorganism(s) different from said thermophilic bacterium.
  • one or more microorganism ⁇ ) different from said thermophilic bacterium provide the glycerol used by said thermophilic bacterium. This can e.g. be during co-fermentation with one or more of such a different/additional microorganism, or in a separate step, such as a different fermentation.
  • a strain according to the first aspect of the invention is provided by the use of a method according to the fifth aspect of the invention.
  • a second aspect concerns a starter culture comprising or consisting of a strain according the invention, such as a bacterial strain according to the first aspect, or a bacterial strain provided according to the fifth aspect of the invention.
  • An embodiment relates also to one or more of e.g. production, packaging, transportation, conservation, viability testing, testing of shelf-life, resuscitating, dosing, cfu determination, purity testing/determination, etc. commonly known in the art, including any means thereto.
  • a third aspect of the invention pertains to a use of a bacterial strain and/or starter culture in a fermentation to produce one or more of biomass, methanol, ethanol, propanol, isopropanol, propanediol, butanol, bio-fuel, bio-gas, hydrogen, methane, and any combination thereof.
  • a bacterial strain or starter culture is used in a fermentation to produce or providing a biofuel and/or a biochemical.
  • a bacterial strain or starter culture is used in a fermentation to produce or providing one or more of biomass, bio-fuel, bio- gas, methanol, ethanol, propanol, isopropanol, propanediol, butanol, hydrogen, acetate, lactate, and any combination thereof.
  • this may comprise the use of a fermentable substrate according to the invention, e.g. a substrate comprising glycerol and/or a carbohydrate.
  • said glycerol is of a quality/purity, as described elsewhere, such as in the first aspect.
  • This may include a use, wherein the glycerol is a by-product, a by-product produced during bio-fuel production, or a by-product produced during bio-diesel pro- duction, or glycerol derived, e.g. purified from a by-product, such as a byproduct produced during bio-fuel production, or a by-product produced during bio-diesel production.
  • a use of a bacterial strain or starter culture according to the invention may comprise one or more fermentation(s), wherein said fermentation(s) essentially comprise(s) only said strain and/or starter culture, or said fermentation is a co-fermentation comprising one or more microorganism(s) different from said strain and/or starter culture.
  • a co-fermentation with a strain / starter culture according to the invention provides a higher yield of one or more of biomass, biofuel(s), and biogas(es), including any combination thereof, compared to e.g. a fermentation without said strain and/or starter culture.
  • a co-fermentation with a strain / starter culture according to the invention could provide a higher yield of one or more of methanol, ethanol, propanol, isopropanol, pro- panediol, butanol, hydrogen, methane, and any combination thereof, compared to e.g. a fermentation without said strain and/or starter culture.
  • product inhibition of a fermentation yielding glycerol as a product or by-product is reduced by the strain/starter culture according to the invention.
  • the invention in a fourth aspect, relates to a method of producing one or more of biomass, bio-fuel(s), bio-gas(es), and/or biochemical (s), including any combination thereof, comprising the steps of providing a fermentable substrate comprising a carbohydrate and/or glycerol, and subjecting said fermentable substrate to a fermentation, wherein said fermentation comprises a bacterial strain according to the first aspect, or a starter culture ac- cording to the second aspect of the invention.
  • the method concerns producing one or more of methanol, ethanol, propanol, isopropanol, propanediol, butanol, hydrogen, acetate, lactate, and any combination thereof from a carbohydrate and/or glycerol comprising the steps of providing a fermentable substrate comprising a carbohydrate and/or glycerol, and subjecting said fermentable substrate to a fermentation, wherein said fermentation comprises a bacterial strain according to the first aspect, or a starter culture according to the second aspect of the invention.
  • the glycerol can be of a quality/purity as described elsewhere, such as in the first aspect. This may include a use, wherein the glycerol is a by-product, a by-product produced during bio-fuel production, or a by-product produced during bio-diesel production, or glycerol derived, e.g. purified from a by- product, such as a by-product produced during bio-fuel production, or a byproduct produced during bio-diesel production.
  • the glycerol is crude glycerol (i.e. 65% weight/volume or more), glycerol of technical quality (i.e. 85 % weight/volume or more); pure glycerol (i.e.
  • glycerol i.e. 99.9 % weight/volume or more
  • the glycerol is solely or at least in part derived from bio-fuel production.
  • the glycerol is a waste product from bio-diesel and/or bio-ethanol production.
  • the fermentation essentially comprises only said strain (i.e. a strain according to the first aspect), or said fermentation is a fermentation comprising in addition to said strain one or more microorgan- ism(s) that are different from said strain.
  • said fermentation is a co-fermentation comprising one or more microorgan- ism(s) different from said strain.
  • such a (co-)fermentation provides a higher yield of one or more of biomass, bio- fuel, bio-gas, and/or biochemical, including any combination thereof, compared to a fermentation without a bacterial strain according to the first aspect.
  • said (co-)fermentation provides a higher yield of one or more of methanol, ethanol, propanol, isopropanol, pro- panediol, butanol, hydrogen, acetate, lactate, and any combination thereof, compared to a fermentation without said strain.
  • the fermentation comprises one or more of batch fermentation, fed-batch fermentation, semi-continuous fermentation, fermentation with immobilized culture, and continuous fermentation, and any combination thereof.
  • a method is provided of producing one or more of biomass, biofuel(s), and biogas(es), including any combination thereof from a carbohydrate and/or glycerol comprising the step(s) of providing a fermentable substrate comprising e.g. a carbohydrate and/or glycerol, and subjecting said fermentable substrate to a fermentation, wherein said fermentation comprises a bacterial strain and/or a starter culture, such as a bacterial strain according to the first aspect of the invention.
  • said biomass, .biofuel(s) and/or biogas(es) can be provided or produced from glycerol, and/or predominantly glycerol.
  • one or more of methanol, ethanol, propanol, isopropanol, propanediol, butanol, hydrogen, methane, and any combination thereof can be provided or produced from a carbohydrate and/or glycerol comprising the step(s) of providing a fermentable substrate comprising e.g. a carbohydrate and/or glycerol, and subjecting said fermentable substrate to a fermentation, wherein said fermentation comprises a bacterial strain and/or a starter culture according to the invention.
  • biofuel(s) and/or biogas(es) can be produced from glycerol, and/or predominantly glycerol.
  • Such a method may comprise a fermentable substrate, e.g. comprising glycerol and/or one or more carbohydrate(s).
  • said glycerol is of a quality/purity, as described elsewhere, such as in the first aspect.
  • This may include a method, wherein the glycerol is a by-product, a by-product produced during bio-fuel production, such as during bio-ethanol and/or bio-diesel production, or glycerol derived, e.g. purified from a byproduct, such as a by-product produced during bio-fuel production, such as during bio-ethanol and/or bio-diesel production.
  • a method according to the invention comprises one or more fermentation ⁇ ), wherein said one or more fermentation(s) essentially comprise(s) only said strain and/or starter culture, or said one or more fermentation(s) is a co-fermentation comprising one or more microorganism(s) different from said strain and/or starter culture.
  • a co-fermentation with said strain / starter culture provides a higher yield of one or more of biomass, methanol, ethanol, propanol, isopropanol, propanediol, butanol, bio-fuel, bio- gas, hydrogen, methane, and any combination thereof, compared to a fer- mentation without said strain and/or starter culture.
  • a fermentation according to the invention may comprises one or more of batch fermentation, fed-batch fermentation, semi-continuous fermentation, fermentation with immobilized culture, and continuous fermentation, and any combination thereof.
  • a method according to the invention relates to a co-fermentation, wherein said co-fermentation with a strain/starter culture according to the invention provides a higher yield of one or more of biomass, methanol, ethanol, propanol, isopropanol, propanediol, butanol, bio-fuel, bio- gas, hydrogen, and any combination thereof, compared to e.g. a fermentation without said strain. This can e.g.
  • a fermentation may comprise or be one or more of batch fermentation, fed-batch fermentation, semi-continuous fermen- tation, fermentation with immobilized culture, and continuous fermentation, and any combination thereof.
  • a fermentation broth comprising glycerol is provided, either alone, or together with one or more carbohy- drate(s).
  • Suitable glycerol concentrations can be e.g. less than 0.5 g/L, ⁇ 0.5g/L, 0.5-1 g/L, ⁇ 1 g/L, 1 -2g/L, ⁇ 2g/L, 2-5g/L, ⁇ 5g/L, 5-10g/L, ⁇ 10g/L, 10- 15g/L, ⁇ 15g/L, 15-25g/L, ⁇ 25g/L, 25-50g/L, ⁇ 50g/L, 50-75g/L, ⁇ 75g/L, 75- 100g/L, ⁇ 100g/L, 100-150g/L, ⁇ 150g/L, 150-250g/L, ⁇ 250g/L, or more than 250g/L.
  • alcohol production is increased when providing a glycerol concentration of ⁇ 0.5g/L, 0.5-1 g/L, ⁇ 1 g/L, 1 -2g/L, ⁇ 2g/L, 2-5g/L, ⁇ 5g/L, 5-10g/L, ⁇ 10g/L, 10-15g/L, ⁇ 15g/L, 15-25g/L, ⁇ 25g/L, 25-50g/L, ⁇ 50g/L, 50-75g/L, ⁇ 75g/L, 75-100g/L, ⁇ 100g/L, 100-150g/L, ⁇ 150g/L, 150-250g/L, ⁇ 250g/L, or more than 250g/L, compared to when a glycerol concentration is provided that is 2 x higher, or 2 x lower.
  • Bio-ethanol is the only alcohol produced today by large scale fermentation processes. This first generation production technology mainly based on microbial fermentation of C6-sugars from e.g. sugar cane, corn, grain (such as barley, wheat, rye, rice, sorghum, etc.) and sugar beets. In an attempt to ex- ploit all available sugars/carbohydrates in the plant biomass, scientific and technological development is driven in the direction of complex, cellulosic biomass exploitation for bio-ethanol production. According to one embodiment of the invention, glycerol derived from or associated with bio-ethanol production is converted to butanol.
  • the bacterial strain is suitable for -, or used in -, or comprised in a use or method comprising a first generation bio-alcohol fermentation.
  • the bacterium is suitable for, or used in, or comprised in a use or method comprising a second generation bio-alcohol fermentation.
  • a novel thermophilic bacterial strain is provided with surprising abilities such as the possibility of producing one or more bio-fuels, such as butanol and/or hydrogen, from a variety of carbon sources including glucose and xylose- but also glycerol.
  • the strain produces ethanol, lactic acid and acetic acid in addition to butanol but the yield of butanol is in accordance with the best strains previously described. Seemingly, the pathway used for conversion of glycerol into butanol has not previously been described.
  • the use of glycerol as substrate makes the strain interesting as large amounts of glycerol are produced from bio-diesel production.
  • a bacterial strain according to the invention has the potential of making a useful high-value product out of a fraction, which is more or less regarded as a waste-stream today, such as glycerol.
  • a strain according to the invention is capable of fermentation in a broth with a dry matter content of around, or at least 1 , 2, 5, 10, 15, 20, 25, 30% (w/w), or more.
  • Example 1 microbiology methods have been used to discover and characterize the new bacterial strain. Analysis of the 16S rRNA has been used to determine the relationship of the new microbe.
  • Example 9 shows results of strain improvements using chemical mutagene- sis. Several mutants strains were found with an improved butanol yield compared to the "wild type" strain 260.
  • the invention also provides a bio-catalyst for production of e.g. bio-fuel(s) and/or bio-chemical(s), which is suitable for e.g. conventional bio-refineries and/or for converting a waste product (glycerol) into a valuable fuel/biochemical.
  • bio-fuel(s) and/or bio-chemical(s) which is suitable for e.g. conventional bio-refineries and/or for converting a waste product (glycerol) into a valuable fuel/biochemical.
  • thermophilic bacterial strain capable of producing hydrogen and/or butanol from glycerol, comprising the steps of:
  • step (b) providing an enrichment microbial culture by cultivation of the sample from step (a) under anaerobic conditions in a glycerol-containing media (Nq- uid or solid) at a temperature suitable for a thermophilic microorganism;
  • step (c) selection of a butanol- and/or hydrogen-positive strain by isolation of a pure culture from step (b) e.g. by growing microorganisms from diluted bio- mass on solidified glycerol-containing medium; and optionally
  • step (d) analysis of the culture from step (c) for glycerol consumption and hydro- gen and/or butanol formation.
  • the glycerol-containing media in step (b), step (c), or steps (b) and (c) may comprise glycerol as the sole carbohydrate source, essentially as the sole carbohydrate source, or predominantly as the sole carbohydrate source.
  • a suitable habitat is believed to be a habitat exhibiting anaerobic degradation of complex organic matter, and/or a habitat comprising glycerol as a free compound or in a bound form, e.g. as lipid.
  • the man-made habitat can e.g. be selected from one or more of manure, sewage sludge, oil waste, fish waste, slaughter house waste, or slaughter house-derived waste, plant-oil containing agricultural residues, bioreactor content, biogas reactor content, content of a bioreactor comprising glycerol, optionally co-digested with other type of organic matter or waste.
  • the natural habitat can e.g. be selected from e.g. one or more of digestive tract of animals and human, excrements, soil, sediment from lakes, hot springs, and seas, optionally comprising decaying algal biomass.
  • a temperature suitable for growing one or more thermophilic microorganism in step (b), step (c), or steps (b) and (c) can be a temperature or a temperature range similar to the temperature (range) of the habitat, from which a sample was provided from in step (a).
  • a temperature suitable for growing one or more thermophilic microorganism can also be a temperature (range) similar or identical to the temperature optimum/optima for production of one or more products, such as one or more temperature (ranges) disclosed in the first aspect of the invention.
  • Said screening and/or isolation method is believed to be suitable for providing a bacterial strain according to the first aspect of the invention, or a starter culture according the second aspect.
  • a strain isolated and/or identified according to the isolation method is also believed to be suitable for a use according to the third aspect, and/or a method according to the fourth aspect.
  • the screening and/or isolation method provides a bacterial strain according to the first aspect of the invention, and/or a starter culture according to the second aspect of the invention.
  • a bacterial strain and/or starter culture can e.g.
  • biomass bio-fuel, bio-gas, methanol, ethanol, propanol, iso- propanol, propanediol, butanol, hydrogen, acetate, lactate, and any combination thereof.
  • Glycerol is a by-product produced during bio-fuel production.
  • the amounts of glycerol produced are increasing on the world market- and the invention therefore has the potential of producing value out of this product.
  • One of the new alternative technologies for glycerol processing can be biological conversion of glycerol into liquid bio-fuels, green chemicals and bio- energy on the basis of fermentation processes. This may comprise and/or require one or more steps, such as:
  • Expected achievements may comprise one or more of: 1. Characterization of potential and limitation of anaerobic degradation of glycerol feedstocks;
  • results on a regional, national, multinational, global scale may comprise one or more of e.g.:
  • a full or almost full conversion of glycerol feedstock into one or more of e.g. bio-fuel(s), bio-energy, bio- chemical, and high-value green chemical(s), including any combination thereof, is achieved e.g. within a bio-fuel or bio-diesel production plant.
  • a pathway leading to an undesired side-/by-product is modified by e.g. deleting, silencing, and/or up-regulating one or more genes leading to said side-/by-product.
  • This can be achieved by the use of common techniques known in the art, such as molecular biology techniques including, PCR, DNA sequencing, transformation, crossing over, insertion/deletion, point mutation, etc.
  • Table I Genes and enzymes related to butanol production pathway.
  • Butanol production is believed to be linked to NADH consumption. Therefore, e.g. a mutation/alteration in one or more genes encoding NADH consuming enzymes is believed to be leading to improvement of butanol productivity.
  • targets genes and encoded enzymes which could be deleted or altered are listed in Table I. Examples of inactivating the acetate kinase were reported by Liu et al (2006), elimination of lactic acid production via gene knock-out of L-lactate dehydrogenase was described by Liu et al. (2006), and the impact of the site-directed mutagenesis on the catalytic activity of alcohol dehydrogenase was pub- lished by Bogin et al. (1997).
  • Complete deletion and/or inactivation of a gene can lead to the lethal mutation, if such a gene/gene product is essential for an organism.
  • enzymes generating energy factors i.e. ATP
  • energy factors i.e. ATP
  • the strength of down-regulation could be tuned by the use of different types of promoters, including induced, constitutive or synthetic. This can be achieved by methods commonly known in the art.
  • Target gene 5' and 3' parts and its flanks could be amplified by PCR and inserted into e.g. an E. coli vector (i.e.pUC19).
  • a marker gene e.g. an antibiotic resistance marker gene or auxotrophic marker gene like ura3
  • rbs sequence i.e. AAGGAG
  • methylated and/or unmethylated DNA could be use for transformation protocol.
  • Mutants/transformant can be selected based on their marker gene, e.g. by antibiotic exposure or recovery of an auxotrophic mutation. PCR could be use to further investigate/verify transformants.
  • Antisense RNA gene to a target gene could be constructed by synthesis or PCR amplification with the appropriate pair of primers.
  • An antiRNA gene is cloned downstream of different type(s)/strength(s) of promoter(s) using common DNA techniques known in the art.
  • Resulted constructs, called antiRNA expression cassette is then introduced into e.g. the middle, or another position of non-coding or non-important region of genomic DNA of the strain 260 inserted into the plasmid DNA.
  • the marker gene is introduced.
  • the whole construct is then used for transformation as described above.
  • the use of induced promoters of the strain 260 like P/ac, i.e. lactose promoter
  • the wild-type strain has the ability to form spores, which could imply certain limitation for the production process, since the cells in different growth stages could be present together in a bioreactor. It has been shown that it is advantageous to create a non-sporulating solventogenic mutants [Jones and Woods, 1986]. According to one embodiment of the invention, a non- sporulating solventogenic mutant of strain 260 is provided.
  • Another appropriate tool for creation of mutants with a modified metabolic pathway can be chemical mutagenesis with N-methyl-N'-nitro-N- nitrosoguanidine,NMMG, in combination with use of a selection compound in the cultivation media, such as chloro-2-butyrate, chloro-acetate, chloro- ethanol, optionally in combination with elevated butanol concentrations.
  • NMMG was also reported to be used for isolation of a butanol resistant mutant with improved butanol productivity [Jones and Wood, 1986].
  • genes in the respective pathways are members of a multi-gene family of 2 or more genes. In such a case, it may be necessary to delete/modify/up- or downregulate more than 1 , such as 2 or more, or all genes in said multi-gene family.
  • one or more than one pathway is altered.
  • one or more pathways leading to e.g. one or more of lactic acid, acetate, ethanol, and hydrogen is/are altered.
  • the present invention provides e.g. a strain/biocatalyst, such as strain 260.
  • a strain according to the invention can e.g. be used as the sole fermentative microorganism in the process of converting glycerol and/or carbohydrate(s)/saccharide(s) into e.g. one or more of bu- tanol, acetate, lactate, ethanol and hydrogen/carbon dioxide.
  • such a strain can be provided in a defined co-, tri-or multi culture together with other fermentative bacteria to ferment e.g. complex lignocellu- losic biomass with and without e.g. preceding thermal or thermochemical pre- treatment and enzymatic hydrolysis.
  • a strain is provided in a co-, tri- or multi culture together with e.g. methanogenic archaea of the trophic groups of acetate-utilizing methanogens and/or hydrogen/carbon dioxide-utilizing methanogens, for conversion of glycerol, glycerol and carbohydrate ⁇ ), or carbohydrate(s) to methane.
  • a strain is provided suitable for bioaugmentation of a complex microbial community producing methane. This could be particularly useful in case when e.g. the inherent population of the glycerol-fermenting bacteria does not have a sufficient potential or lacks the capacity/ability to convert glycerol, such as a concentrated or highly concentrated glycerol stream.
  • the present invention may comprise a fermentation process comprising a strain/biocatalyst according to the invention in a fermentation medium.
  • a suitable fermentation medium often comprises water, a carbon source, macro- and micronuthents necessary for growth of the culture, and formation of fermentation product(s), such butanol and/or hydrogen.
  • An appropriate carbon source is e.g. glycerol, either alone or in combination with another carbon source, such as one or more carbohydrate(s).
  • Glycerol can be derived from trans-esterification of th-glycerides of plant-, animal- and algal ori- gin, as well as glycerol generated by microbial activity, such as fermentation.
  • Other appropriate carbon sources can be carbohydrate(s), e.g.
  • refined substances in form of refined substances, or e.g. as complex substrates containing e.g. one or more of glucose, xylose, arabinose, mannose; sucrose, cellobiose, maltose, lactose; dextrin, starch, hydrolysates of pectin, hydrolysate of hemicellulose, hydrolysate of cellulose, hydrolysate of lignocellulose.
  • complex substrates e.g. one or more of glucose, xylose, arabinose, mannose; sucrose, cellobiose, maltose, lactose; dextrin, starch, hydrolysates of pectin, hydrolysate of hemicellulose, hydrolysate of cellulose, hydrolysate of lignocellulose.
  • Suitable carbon substrates could e.g. be provided by processing sugar beet and sugar cane such refined sugar and molasses, from grain and corn starch, milk whey, algal biomass, and
  • a fermentation product according to the invention such as a bio-alcohol could be produced in fermentation media from mixtures of glycerol with refined and/or complex sugar-containing raw materials described above.
  • Nitrogen can be provided in form of e.g. one or more inorganic compound(s), such as pure ammonia, ammonia salts, urea etc.
  • a suitable organic nitrogen source may comprise one or more of e.g. defined individual amino acid(s), mixture of several amino acids - such as casaminoacids, protein hydrolysates, e.g.
  • Suitable source of phosphorus are e.g. phosphates.
  • Phosphate and minerals could e.g. be provided in form of vinasse - a residual stream from fermentation of molasse derived from the sugar beet-and sugarcane- based ethanol production, oil seeds meal. Minerals and trace metals could e.g. be added as chemicals.
  • Demand for growth factors and vitamins could e.g. be met by addition of one or more of yeast extract, wheat germ meal, beef extract, corn steep liquor.
  • the fermentation media is ster- ile/autoclaved, e.g. for an application comprising a strain according to the invention, such as strain 260 in monoculture or in defined co-culture, tri- culture or multi-component culture.
  • a strain according to the invention such as strain 260 in monoculture or in defined co-culture, tri- culture or multi-component culture.
  • the fermentation medium does not need to be sterile.
  • glycerol could be diluted into the fermentation media, such as a stream of stillage from starch-based ethanol production, vinasse from sugar beet- or sugar cane based ethanol production, wine vinasse, plant juice, manure, sewage sludge or household waste.
  • a fermentation process according to the invention could be carried out e.g. as a batch fermentation, for example when a strain/culture is inoculated into a closed container containing sterile fermentation medium and incubated under appropriate conditions, such as conditions described above or elsewhere.
  • a fermentation could also be carried out as fed-batch fermentation, or as a semi-continuous or as a continuous process, such as a chemostat, under appropriate conditions.
  • a batch fermentation can be operated without pH control. Often it will stop when the level of an inhibitory product, such as a fermentation product, such as an alcohol or acid produced, reaches a critical level (e.g. an alcohol concentration higher than ⁇ 8g/l) or a nutrient is exhausted.
  • a fermentation can also be carried out as batch fermentation with pH regulation, where a pH drop caused by accumulation of acid is counteracted by addition of a base. Fermentation with a pH control can be carried out at one fixed pH set point. Alternatively, especially when the pH range for alcohol/butanol production is broader than the pH interval for growth, two and/or several pH set points could be chosen for the pH regulated fermentation.
  • the first pH set-point should then be chosen as optimal for formation of cell biomass, while the following pH set-points should be optimal for alcohol/butanol production.
  • the lower limit of product formation could be lower than 5.0, i.e. the pH min for growth.
  • the upper limit for product formation is most probably identical with pHmax for growth.
  • a continuous fermentation process is operated as fermentation without pH control, or fermentation with pH control.
  • Fermentation process can be also be carried out at one, two or more defined, temperatures/temperature ranges, facilitating reduction of the operational temperature after completing the exponential growth of the biomass during continuing fermentation and butanol production. This could be particularly important for a media comprising e.g. a high substrate concentration.
  • the first temperature set point could e.g. correspond to, or be near the optimum for growth, while the following temperature set points could correspond to, or be near the optimum for alcohol/butanol production.
  • the temperature for alcohol/butanol production could e.g. be the same or similar to T op t for growth. Alternatively, it could also be set below the growth Topt of the production strain, e.g. in order to counteract toxicity of a fermentation product, such as alcohol/butanol [Carnarius, 1940; Glassner et al. 1991].
  • a fermentation according to the invention could also be performed with sus- pended-cells systems and/or as a combination of suspended-cells systems with cell recycling to the fermenter.
  • systems with immobilized cells and immobilized spores of the production strain are believed to be suitable, e.g. when applied in packed columns, up-flow anaerobic sludge blanket reactor, or fluidized bed bioreactors [Jones and Woods, 1986, Ahring et al. (1992)]. Fermentation could also be carried out with use of various head- space gasses such as nitrogen, carbon dioxide, nitrogen/carbon dioxide, hy- drogen/carbon dioxide, carbon monoxide.
  • head- space gasses such as nitrogen, carbon dioxide, nitrogen/carbon dioxide, hy- drogen/carbon dioxide, carbon monoxide.
  • alcohol such as butanol
  • alcohol/butanol can be purified by the above mentioned techniques in a product recovery process carried out simultaneously or parallel with a fermentation.
  • Alcohol/butanol recovery can e.g. be carried out as in situ extraction or as alcohol/butanol recovery from a recycled fermentation broth outside the fermenter.
  • Products and/or by-products obtained or obtainable by fermentation of glycerol and/or carbohydrates with strain 260 are shown in Figure 18.
  • Hydrogen can e.g. be used in fuel cells to generate electricity and heat, in biological methane production for production of heat and electricity, or in methanol synthesis.
  • Carbon dioxide is commonly used for production of dry ice.
  • Lactate can be purified and used for food and feed preservation, in cosmetics, or as a building block for production of biodegradable plastics.
  • Acetate is a chemical composition/reagent and can be used for production of synthetic fibers, beverage and food application.
  • Ethanol can be used as a solvent and a feedstock for synthesis of other products, or e.g. as fuel for heat and internal combustion engines.
  • the residual cell biomass from fermentations can be used as a source of single cell protein for feed additive, and for extraction of vitamin Bi 2 .
  • Another way of increasing the value of one or more by-products after alco- hol/butanol recovery is e.g. implementation of an anaerobic digestion stage for biological treatment of effluents and residues from the butanol fermentation.
  • the organic matter comprising residual substrate, proteins and metabo- lites, could e.g. be converted into bio-methane and used for production of heat and electricity.
  • bio-energy is produced during the effluent treatment.
  • the treated effluent can potentially be re-used as the process water.
  • Strain 260 (DSM deposition number 21660) was isolated from a slurry of a thermophilic, lab-scale, completely stirred bio-gas reactor.
  • the bio-gas reactor was bio-augmented with Methanosarcina sp. V- 1 P and operated at 55°C for a period of 3 months under continuous supply of a mixture of cattle manure and glycerol trioleate [Mladenovska (1997)].
  • the basal medium used for enrichment and isolation was prepared essen- tially as previously described [Angelidaki et al. (1990)] with following modifications: concentration of sulfide was increased to 0.5 g/l, the vitamin solution, 10 ml/I, was that of the DSMZ medium no. 141.
  • the basal medium was supplemented with 3 g/l glycerol (99% purity, Sigma, USA) and 0.5 g/l yeast extract (Difco, USA) during the enrichment and isolation procedure.
  • the media were autoclaved at 140 0 C for 20 minutes. For the purpose of routine cultivation and if other not stated, the medium was omitted for cysteine hydrochlo- ride, and the concentration of glycerol and yeast extract were increased to 10 g/l and 2 g/l, respectively.
  • the cells were grown in 27-ml Hungate tubes (Hungate RE; 1969) or 30-ml serum vials with 10 ml media under an atmosphere of N2/CO2
  • Enrichment cultures were obtained by dilution of one ml of digested slurry in
  • the pure cultures were maintained by transfer of a 5% inoculum (vol- ume/volume) in the media with 10 g/l glycerol and 2 g/l yeast extract. The cultures were kept for short-term storage at room temperature. For the long- term storage, the exponentially growing cultures were transferred into the basal media with 20% glycerol (50% volume:volume) and kept at -80 0 C.
  • Handling an anaerobic culture can be done in anaerobic glove box, and incubation of the culture can be done as described above in Hungate tubes, in anaerobic jars with oxygen-free headspace, or in Gas Pak systems containing catalysts for oxygen removal. Such methods are well known in the art.
  • the isolate was tested for growth in acidic and alkaline media. Different pH values in media were obtained by varying the concentrations of HCO3 " and CO3 2" in the media and CO2 content of the headspace gas.
  • the pH growth range was determined by cultivation of the cultures at selected pH, while the temperature was maintained at 60°C.
  • the growth temperature interval was determined by cultivation of the culture at pH 7.0 and defined temperatures from a range 35-70 0 C.
  • the strain was always adapted to the new conditions by sub-cultuhng the culture at the respective pH's or temperatures for two times before the pH and temperature response was determined.
  • Substrate tolerance was examined by growth of the pure culture in the media supplemented with glycerol and 1 g/l yeast. Glycerol was tested in an interval of 5 to 100 g/l. Growth was monitored by the OD measurement, analysis of glycerol consumption and product formation.
  • Crude glycerol a by-product of the transestehfication of the vegetable oils from the biodiesel production company Meroco, Slovakia, was tested as a fermentation substrate.
  • the pre-reduced medium was supplemented with 20 g/l of crude glycerol and 5 g/l yeast extract and incubated with strain 260 for a period of 48 hours. Consumption of sub- strate and production of fermentation products was determined.
  • butanol Tolerance to butanol was tested by cultivation of the culture in media with 20 g/l glycerol, 1 g/l yeast extract and exogenously added butanol. Butanol was spiked to the tubes with inoculated medium immediately prior to the start of the incubation. The butanol concentrations tested were 5, 7, 8, 16, 27 and 42 g/l. Growth was monitored by the OD measurement, analysis of glycerol consumption and butanol formation. A control culture without addition of the exogenous butanol was included in the test.
  • Requirement for yeast extract was determined by comparison of the cell growth in basal mineral medium supplemented with vitamins and 20 g/l glycerol, and in parallel, in the same media supplemented further with yeast extract. The effect of adding yeast extract in a concentration range 1 to 5 g/l was investigated.
  • the ability of the culture to utilize various carbon compounds for growth was determined by inoculating cells (5% volume/volume) into basal media con- taining selected substrates at a final concentration 4 g/l.
  • Arabinose, cello- biose, glucose, glucuronic acid, lactose, maltose, mannose, sucrose, xylose, yeast extract were added to the autoclaved, basal medium from the separate, sterile stock solutions. The stock solutions were autoclaved at 120 0 C for 20 minutes.
  • Potato starch, pectine from citrus fruits and insoluble substrates, microcrystalline cellulose Avicel, beech wood xylan were added to the basal media in the final concentration 4 g/l, aliquots of the respective media were distributed in the Hungate tubes, which were autoclaved.
  • the autoclavation temperature of the media with starch and pectine was reduced to 120 0 C for 20 minutes.
  • grass hydrolysate Potential of the culture to grow with a complex carbohydrate substrate was tested with grass hydrolysate.
  • the grass hydrolysate was prepared by ther- mochemical pre-treatement and subsequent enzymatic hydrolysis. 300 g of fresh, shredded garden grass, collected in October in Denmark, was sus- pended in 300 g tap water and pre-treated by wet-oxidation. The conditions for wet-oxidation were as reported previously [Lissens et al. 2004], i.e. 185°C, 12 bars oxygen pressure and no addition of sodium carbonate.
  • the pH of slurry was adjusted to 5.0, and a mixture of enzymes Cellulast (Novozymes, Denmark) and Novozymes 188 (Novozymes, Denmark) in a ratio 3:1 (volume/volume) was added to the pretreated grass.
  • the dosage of Celluclast was 7 FPU/g total solids of the grass.
  • the enzymatic hydrolysis was carried out at 50 0 C with occasional shaking of the bottles with hand. After 3 days of incubation, the hydrolysate was decanted and four milliliters were transferred directly to the 10 ml of the basal media in Hungate tubes. Diluted hydrolysate was inoculated with the pure culture and fermented at 60 0 C for 7 days.
  • Sensitivity of the culture to antibiotics was examined in media with 10 g/l glycerol and 2 g/l yeast, supplemented with one of the following sterile- filtered antibiotics at given final concentrations: D-cyclosehne, 1000 mg/l; chloramphenicol, 500 mg/l ; penicillin, 100 mg/l; tetracycline, 100 mg/l ; 100 mg/l streptomycin, 100 mg/l. Control culture without any antibiotics was included in the test as well. Incubation was carried out for one week at 60°C.
  • Glucose, xylose, cellobiose, lactic acid and ethanol were quantified in culture supernatants by HPLC with refractive index detection.
  • the system was equipped with an Aminex HPX-87H column and operated at 60 0 C with 4 mM sulfuric acid as eluent with a flowrate of 0.6 ml/min.
  • Glycerol, butanol, 1 ,3- propanediol were analysed using the same HPLC set-up and method as for the carbohydrates, except that the analysis time of each sample was prolonged to 40 minutes.
  • 16S rRNA gene sequence determination and analysis 1 ml of exponentially growing cells were harvested from the cultivation media with 10 g/l glycerol and 2 g/l yeast extract and used for extraction of genomic DNA. Extraction procedure was done with Genomic Mini Kit (A&A Biotechnology, Tru) according to the instruction of the manufacturer. The purified DNA was used as template in the PCR reactions. The 16S rRNA gene was amplified with the 27 forward primer and the reverse primer 1492 [Lane et al 1991]. The PCR product was purified and cloned into the pUC19 vector. Three purified plasmids with expected length of the insert were sequenced. The 16S rRNA gene sequence of isolate 260 was aligned against the selected sequences available in the public databases.
  • the alignment was made with MEGA 4.
  • the evolutionary history was inferred using the Neighbor- Joining method [Saitou and Nei, 1987].
  • the optimal tree with the sum of branch length equal to 0.42628301 was shown.
  • the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
  • the evolutionary distances were computed using the Maximum Composite Likelihood method [Tamura et al. (2004)] and are in the units of the number of base substitutions per site. Codon positions included were first, second, third and the non-coding. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 1387 positions in the final dataset.
  • Thermoanaerobacterium thermosaccharolyticum DSM 571 ⁇ M59119
  • Thermoanaerobacterium thermosaccharolyticum GD17 EF680277
  • Thermoanaerobacterium thermosaccharolyticum W16 EU563362
  • Thermoanaerobacterium thermosaccharolyticum D120-70 AF247003
  • Thermoanaerobacterium sulfurigignens JW/SL-NZ826 T AF234164
  • Thermoanaerobacterium thermosulfurigenes ATCC 33743 L09171
  • Thermoanaerobacterium aciditolerans 761 -119 AY350594
  • Thermoanaerobacterium islandicum AK17 (EF088330), Clostridium thermocellum DSM 1237 (NC009012), Caloramator viterbensis DSM 13723 T (AF181848), Clostridium beijerinckii NCIMB 8052 (NC009617), Clostridium pasteurianum DSM 525 T (DQ911268), Clostridium acetobutylicum DSM 792 (U 17030), Clostridium acetobutylicum VKPM B-4786 (AM231184).
  • the basal medium containing 10 g/l glycerol, 2 g/l yeast extract and 0.5 g/l cysteine hydrochloride was used for cultivation of cells through the mutagenesis experiment.
  • the cells of the wild-type strain 260 were harvested from the cultivation media by centhfugation of 2 ml of culture broth in Eppen- dorph tubes for 10 min at 10.000 g. In total, five portions of cell pellets were prepared.
  • the cells were resuspended in 200 ⁇ l of the basal media, and a solution of 500 ⁇ g/ml N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in 1 M sodium citrate buffer with pH 5.0, was added.
  • MNNG N-methyl-N'-nitro-N-nitrosoguanidine
  • the final concentrations of MNNG in the resus- pended cell stocks were 100, 55, 30, and 15 ⁇ g/ml, respectively.
  • the fifth stock of cells served as a control without any addition of MNNG.
  • the cell suspensions were incubated at 60 0 C for 20 minutes.
  • the treated cells were transferred to 10 ml of cultivation media supplemented with an appropriate selection compound. Chloro-acetate, chloro-2-butyrate, a mixture of chloro- acetate and chloro-2-butyrate, chloro-ethanol were chosen as the selection compounds at the final concentration of 5 mM. After observation of the growth, a transfer to a fresh cultivation medium with the same selection compound was made.
  • the actively growing treated cultures were decimally diluted in the liquid media and transferred to the roll-tubes containing solidified cultivation medium supplemented with 5 mM of the selected compound. Following incubation was carried out at 60 0 C. The colony growth was observed in the tubes with the mixture of chloro-2-butyrate and chloro- acetate. The colonies were picked and transferred into the liquid cultivation media with 5 mM chloro-2-butyrate and 5 mM chloro-acetate, and incubated at 60 0 C.
  • Bioreactor experiments Growth of strain 260 was tested in batch fermentations carried out in a lab- scale bioreactor (SGS, France). The 35L bioreactor with a working volume of 10L was used for all experiments.
  • the fermenter was equipped with pH controlling system and oxygen probe (BioController ADI 1030, Aplikon Biotechnology BV) for monitoring of the pH and oxygen level in the media.
  • BioController ADI 1030 Aplikon Biotechnology BV
  • the fermenter was filled with the above described anaerobic medium and the strict anaerobic conditions were created by addition of the reducing agent.
  • the medium was sparged with N2/CO2 until the initial pH of the media reached the desirable pH from a range of 7.0 - 7.7.
  • a 10% (vol/vol) inoculum of a culture grown for 48 hours was used.
  • the bioreactor was kept closed under the N2/CO2 atmosphere.
  • the culture was grown at the temperature 59°C, with constant stirring at 100 rpm.
  • the fermentations were carried out in a batch mode either without- or with the pH control.
  • the pH of the media was controlled by addition of a 2M NaOH in response to the monitored pH in the fermenter.
  • One pH set-point was selected for the investigation, i.e. the pH 6.0.
  • two different strategies of controlling the headspace gas overpressure were tested. In the second case, the inoculated reactor was completely closed and accumulation of the fermentation gasses was allowed until an overpressure of 1.5 bar was achieved.
  • the fermentation gasses were continuously released while a 0.2 bar overpressure was constantly kept in the headspace of the bioreactor.
  • the first test was carried out in the media with 30 g/l glycerol and 5 g/l tryptose, while the second test was done in the media with 20 g/l glycerol and 5 g/l tryptose.
  • the culture was analyzed for substrate consumption, formation of liquid fermentation products, pH and viability of the cells.
  • the fermenter was connected to a gas meter, which was used for registration of the frequency of gas releasing pulses from the gasmeter per time unit. The total volume of gas produced was determined by counting the impulses from the gasmeter (1 impuls ⁇ 130 ml).
  • the strain 260 was isolated from a slurry of a lab-scale biogas reactor producing methane from the mixture of cattle manure and glycerol trioleate.
  • the reactor was operated at 55°C and a HRT of 15 days.
  • the pH of the digested biomass was 8.0, and the level of volatile fatty acids 1800 mg acetate/I. It was estimated by the most probable number technique that the digested slurry contained a mixed microbial population with at least 4x10 6 cells/ml of methanogens producing methane from the conversion of 1 g/l glycerol trioleate.
  • the strain 260 formed colonies.
  • the colonies were 1 mm diameter, white, round and had an entire edge.
  • the surface in the centre of the colonies was smooth and changed towards a more granular surface close to the edge. No pigmentation was observed in any growth phase.
  • the strain 260 grew as single occurring rods with 1.5- 6 ⁇ m length during the exponential growth phase (Figure 1 ). The length of the rods was extended to 3-12 ⁇ m in the stationary growth phase. Moreover, formation of terminal spores was observed in the late exponential- and stationary growth phase. Spores were found to be thermoresistant, able to survive a 10 min exposure of the sporeforming-culture to 95°C, but not auto- clavation at 120 0 C for 20 min.
  • strain 260 is related to Clostridia, more specifically to the genus Thermoanaerobacterium ( Figure 2).
  • Figure 2 The strain 260 clustered together with strains of the species Thermoanaerobacterium thermosaccharolyticum and its 16S rRNA gene sequence shared 97% similarity with the sequence of the type strain DSM 571 ⁇ .
  • Strain 260 was found to be more closely related to Thermoanaerobacterium thermosaccharolyticum W16 and GD17 with a 99.5% homology in their 16S rRNA gene sequence.
  • the similarity level of 98% was determined between isolate 260 and Thermoanaerobacterium is- landicum, Thermoanaerobacterium aotearoense and Thermoanaerobacte- rium acidotolerans.
  • FIG. 3 Growth of the biomass of the strain 260 and concomitant glycerol fermenta- tion is shown in Figure 3.
  • the biomass was formed during the first 24 hours of fermentation. At the point of entering the stationary growth phase, the OD was 0.75 and during following incubation it was reduced by 50%. Reduction in turbidity of the fermentation broth can be related to the formation of spores.
  • Three phases of glycerol consumption were distinguished in Figure 3. The first one associated with growth of cells, the second- and the third phase during the stationary phase indicating different rates of glycerol consumption and butanol production during the period 24 - 75 hours of fermentation time, and 75-115 hours of fermentation time, respectively.
  • Strain 260 converted one third of the total amount of substrate and produced one third of the total amount of butanol in the first, growth-associated phase. The other two thirds of the total amount of the product were generated by the cells of the stationary growth phase.
  • FIG. 4 A more detailed picture of the glycerol conversion by strain 260 into various products is shown in Figure 4. This experiment confirmed that three stages of glycerol conversion can be distinguished during a 90 hours batch fermentation.
  • the main product of glycerol fermentation was butanol, while ethanol, acetate and lactate appeared as the minor products.
  • the short-chained volatile fatty acids such as propionate, butyrate, isobutyrate, valerate, isovalerate were not detected at the end of glycerol fermentation.
  • Formation of 1 ,3- propanediol was not detected. Hydrogen and carbon dioxide were the only two gasses produced.
  • the yield of hydrogen was determined to be 0.14 mol hydrogen/mol glycerol consumed.
  • the yields of the non-gaseous fermentation products expressed on the molar- and mass basis are shown in Figure 5.
  • the cell mass yield was found to correspond to 24% of the sum of the non- gaseous fermentation products and the cell dry weight.
  • strain 260 The ability of strain 260 to grow and ferment elevated concentrations of glyc- erol was investigated. Strain 260 was grown in media containing up to 100 g/l glycerol and the yields of non-gaseous fermentation products, i.e. butanol, ethanol, acetate and lactate are shown in Figure 6. The strain performed well without any apparent sign of substrate inhibition in the whole range of glycerol concentrations tested. The highest- and the lowest butanol yield were 0.347 mol butanol/mol glycerol and 0.261 mol butanol/mol glycerol, obtained with 30 g/l glycerol and 5 g/l glycerol, respectively.
  • the butanol yield in media containing 100 g/l glycerol was comparable with the yield determined in the media with 5g /I glycerol.
  • Acetate yield was found to be constant in the whole range of the glycerol concentrations tested.
  • the lactate yield increased along with increasing concentration of glycerol, while the opposite trend was observed for ethanol. Its yield decreased with increasing concentration of glycerol.
  • the maximum level of butanol produced was 68 mM (5 g/l) in the media with 100 g/l glycerol. At the end of this fermentation 80% of substrate was left and the fermentation was most probably stopped by the pH 4.7.
  • Strain 260 was incubated in the presence of exogenous butanol.
  • the culture was negatively affected by the exogenous butanol and growth with limited glycerol consumption and butanol production occurred in the presence of 4.4 g/l exogenous butanol.
  • 6.6 g/l of the exogenous butanol was critical, since growth was detected only in one tube of the triplicate. No growth at higher butanol concentrations was observed.
  • strain 260 was able to grow on glycerol in defined, mineral media supplemented with vitamins, which proves that the strain 260 was able to utilize glycerol as the sole source of carbon and energy.
  • yeast extract In the absence of yeast extract, the yields of both alcohols were lower and the lactate yield was similar as in the media with yeast extract. In contrast, the yield of acetate was significantly higher in the defined media than in the complex media with yeast.
  • Yeast had a positive effect on the production of butanol when applied at 1 -3 g/l, but addition of 5 g/l yeast extract resulted in reduction of the butanol yield to the level determined in mineral media.
  • Yeast extract had a stimulatory effect for ethanol formation and the ethanol yield was increasing in parallel with increasing yeast concentration. Variation in the concentration of yeast extract did not affected acetate and lactate yield. From the view of butanol production, the optimal concentration of yeast extract was 3 g/l, when the best yield, 0.43 mol butanol/mol glycerol and the highest butanol concentration, 95 mM (7 g/l) was detected in the fermentation broth.
  • the specific growth rate ⁇ in medium with 10 g/l glycerol + 2 g/l yeast extract was calculated to 0.26 h "1 and the corresponding doubling time Td was 2.7 hours.
  • Strain 260 was able to grow with a variety of carbohydrates.
  • the spectrum of fermentation products and product yields after 7 days of growth on glucose and xylose are shown in Figure 12 and 13, respectively.
  • Butanol became the second major fermentation product after acetate.
  • significant amount of lactate was formed.
  • Ethanol was produced only in small amount and its yield corresponded to 6% and 3% of the total yield of the fermentation products from glucose and xylose. Formation of butyrate was not detected.
  • the other carbohydrates supporting good growth and butanol production by strain 260 were: arabinose, cellobiose, lactose maltose, mannose, sucrose, starch. No growth was obtained with microcrys- talline cellulose Avicel, carboxymethylcellulose, beech wood xylan, pectin and rape seeds meal.
  • strain 260 was able to grow and ferment the mixture of sugars in diluted grass hydrolysate. (Figure 14). Conversion of glucose, xylose and cellobiose into fermentation products was monitored. Strain 260 preferred glu- cose and cellobiose to xylose. After 7 days of fermentation, 98% of the initial glucose amount and 86% of the initial cellobiose amount were consumed, while 69% of the initial xylose amount was utilized. The product pattern and product yield were similar as found for pure glucose.
  • Acetate was the major fermentation product with a yield 0.7 mol acetate/mol sugars converted, while butanol and lactate were produced with a lower yield, 0.4 and 0.3 mol prod- ucts/mol sugars converted, respectively. Ethanol was produced in a low amount with the yield 0.06 mol ethanol/mol sugars converted.
  • glycerol When glycerol was present together with the carbohydrates, the sugars were consumed simultaneously with glycerol. However, in the mixture of glucose, xylose and glycerol, the conversion of glycerol and xylose was found to be incomplete both with 260- M9 and 260. In addition, the ability of 260-M9 to utilize glucose in the presence of glycerol was impaired. 55% of the initial glucose amount was left in the mixture of glucose, xylose and glycerol at the end of fermentation, while the wild-type strain completely utilized glucose from this mixture. 260-M9 produced the same fermentation products as the wild-type strain, i.e. acetate, butanol, lactate, ethanol.
  • 260-M9 produced more butyrate than 260.
  • the highest concentration was detected in media with glucose and xylose, 0.5 g/l butyrate and 0.16 g/l for 260-M9 and 260, respectively.
  • At the end of fermentation only traces of butyrate (below 0.1 g/l) were detected in the 260-M9 fermentation media. None butyrate was detected in the media fermented by the wild-type strain 260.
  • the yield in media with combinations of carbohydrates and glycerol were higher when the fermentation was carried out by strain 260.
  • the increase of the yield was 10%, 31 % and 10% for the substrate combinations of both carbohydrates with glycerol, glucose with glycerol, and xylose with glycerol, respectively, after 10 days of incubation.
  • Strain 260 was able to grow with crude glycerol as a substrate. After 48 hours of incubation, the initial glycerol concentration was reduced from 20 g/l to 5 g/l and the products butanol, ethanol and acetate in concentrations 3.4 g/l, 0.8 g/l and 0.3 g/l, respectively, were detected in the media. The product yields are summarized in Figure 19.
  • thermophilic bacterial strain capable of CO- producing hydrogen and butanol from glycerol and subsequent isolation of an axenic culture
  • glycerol fermenting microorganisms Screening based on microbial cultivation is used for detection of glycerol fermenting microorganisms.
  • the glycerol fermenting microorganisms is identified within samples of a complex microbial community, if upon cultivation of the microbial sample in the glycerol containing media, three following criteria are met: 1/ growth of the bacterial cells is observed; 2/ utilization of glycerol is proved and 3/ generation of desired fermentation products, i.e. butanol and hydrogen from glycerol is demonstrated.
  • the first strategy based on establishment of an enrichment culture and subsequent isolation of the pure, axenic culture.
  • the anaerobic, glycerol fermenting bacteria can be enriched and isolated from the complex microbial communities, which undergo anaerobic degradation of the glycerol-containing organic matter.
  • the glycerol substrate must be present either as a free compound or in a bound form as a triglyceride, which is the main constituent of animal fats and vegetable oils.
  • Microbial conversion of glycerol-containing organic matter proceeds in man-made habitats, such as systems for biomethanation of organic waste and wastewater.
  • the man-made biosystems can have different technical levels, ranging from simple anaerobic lagoons to advanced bioreac- tors.
  • the glycerol-containing organic waste treated in these systems can be manure, sewage sludge, household waste, agricultural waste with residues of oil-seed plants, glycerol streams from biodiesel production, and industrial waste and wastewater from food processing industries, particularly from edible oil- and fish processing and slaughterhouses.
  • the natural habitats with microbial conversion of glycerol-containing organic matter can be sediments with decaying algal- and plant biomass in lakes, hot springs, and seas, as well as soils and digestive tract of animals and human.
  • thermophilic, glycerol fermenting anaerobic bacteria can be with the highest probability found in habitats with a temperature of a interval 40-100 0 C. Furthermore, the thermophilic, glycerol fermenting bacteria can be present in habitats with mesophilic temperature, since the minimal temperature for growth of the moderate thermophilic bacteria can be identical with the mesophilic temperature range.
  • the screening is carried out under anaerobic conditions, in anaerobically prepared cultivation medium by means of anaerobic cultivation techniques - see e.g. Hungate (1969) and Bryant (1976).
  • an enrichment culture is established.
  • Complex microbial sample from a selected habitat is used in amounts ranging from 10% to 0.000000001 % (e.g. by serial dilution) for inoculation of an appropriate, sterile, cultivation media with suitable pH values, such as a pH in the range of 4.0 -10.0. It can also be advisable that the pH is similar to the pH of the habitat, where the sample is taken from.
  • Inoculation is done into the medium with glycerol, and in parallel, into the medium containing all medium compounds except glycerol.
  • the medium without glycerol is used for determination of the background microbial growth and exact concentration of the chemical compounds of interest (glycerol, bu- tanol hydrogen), especially when the sample is rich in organic matter.
  • Incuba- tion of the inoculated media is carried out at appropriate thermophilic temperatures, most preferably at temperatures from the interval 50-90 0 C. The incubation period can last from one day to 30- or more days. During the incubation period, the medium with and without glycerol is examined for bacterial growth.
  • Growth of bacteria is determined by measurement of the optical density and turbidity, and by examination of the culture broth by microscopy.
  • Consumption of glycerol is determined by an appropriate analytical method from the difference of the glycerol concentration at the start of the cultivation and at the selected day of the incubation period, corrected for the back- ground concentration of glycerol.
  • Qualitative and quantitative analysis of the fermentation products is carried out at day 0 and at the selected day of the incubation period. The final products of glycerol fermentation will be dependent on the microbial complexity of the enrichment culture.
  • the expected final fermentation products could be hydrogen, carbon dioxide, lactate, volatile fatty acids - like acetate, propionate, butyrate, and alcohols, i.e. ethanol, butanol or 1 ,3-propanediol. Formation of butanol gives the culture characteristic smell which can be noticed under inoculation, when transferring the full-grown culture to the fresh cultivation media.
  • the enrichment culture consists of both fer- mentative bacteria and methanogenic archaea
  • the spectrum of the final product of glycerol fermentation will include methane as well.
  • BESA 2-bromoethanesulfonic acid
  • the glycerol consuming bacteria After incubation of the inoculated solidified media for one to thirty or more days, the glycerol consuming bacteria will yield colonies.
  • the gaseous fermentation products like hydrogen and carbon dixide can be easily detected in the headspace of the closed tubes. Transfer of bacterial cells from the single appearing colonies into the glycerol-containing liquid media, and its subsequent cultivation will result in establishment of the pure, axenic culture.
  • the cultures can be checked for the desired physiological properties, such as glycerol conversion and co-production of butanol and hydrogen by analysis of the culture broth and headspace gas, respectively. Purity of the culture has to be checked by examination of the culture broth by microscopy, analysis of the fermentation products, and by molecular methods analyzing the 16S rRNA gene.
  • the enrichment can be omitted, and the isolation pro- cedure is carried out directly from the complex microbial sample.
  • the complex microbial sample is decimally diluted and inoculated into the glycerol- containing, solidified, anaerobic, sterile cultivation media.
  • the cultivation of the inoculated media is carried out at pH and temperature that are similar to the conditions in the particular habitat. If the original sample contained only bacteria with the glycerol- consuming cells, these will form colonies and formation of the hydrogen in the headspace can be detected. If the complex microbial sample consists of a co- or multiculture of bacteria and hydrogen- consuming archaea, growth of the colonies will be observed and methane formation detected.
  • the colonies will be transferred to the glycerol-containing liquid medium and incubated under the appropriate conditions. If growth is scored as positive, both glycerol utilization and formation of expected prod- ucts has to be verified. Purity of the culture has to be checked by examination of the culture broth by microscopy, analysis of fermentation products, and by molecular methods analyzing the 16S rRNA gene.
  • Thermoanaeobacterium sp., strain 260 is the first thermophilic, glycerol-fermenting bacterium isolated from a man-made ecosystem - a thermophilic biogas reactor treating organic waste. Despite of the phylogenetic relation of the isolate 260 to the strains of the species Thermoanaerobacterium thermosaccharolyticum, its physiological properties were found to be very different from the existing profile of the characterized thermophilic anaerobic bacteria. Thermoanaerobacterium sp. strain 260 is one of the very few thermophiles which is able to grow and ferment glycerol as the sole substrate.
  • thermophiles A major fermentation product from glyc- erol fermentation was butanol, which was surprising, since it was suggested that production of significant concentrations of butanol by thermophiles was unlikely because of its toxic effect on cells in the thermophilic growth range.
  • the other, and minor products of the glycerol fermentation were acetate, lactate, ethanol without significant formation of 1 ,3-propanediol or butyrate.
  • Hy- drogen was co-produced with butanol, acetate, lactate and ethanol.
  • the optimal conditions for butanol production were neutral pH and the temperatures of 57-60 0 C in cultivation media with growth stimulating factors, i.e. yeast extract.
  • the strain 260 was able to utilize a wide array of carbohydrates.
  • thermophilic UASB reactors S ⁇ rensen AH, Winther-Nielsen M, Ahring BK (1991) Kinetics of lactate, acetate, and propionate in unadapted and lactate adapted thermophilic, anaerobic sewage sludge: the influence of sludge adaptation for start-up of thermophilic UASB reactors. Appl Microbiol Biotechnol 34:823-827

Landscapes

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

Abstract

La présente invention concerne une bactérie thermophile capable de produire du butanol et/ou de l’hydrogène à partir de glycérol, y compris son utilisation et des procédés associés à la production de biomasse, de biocarburant, de biogaz, et/ou d’agent(s) biochimique(s).
PCT/EP2009/062022 2008-09-16 2009-09-16 Bactérie de fermentation thermophile produisant du butanol et/ou de l’hydrogène à partir de glycérol WO2010031793A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9731108P 2008-09-16 2008-09-16
EP08164397 2008-09-16
EP08164397.5 2008-09-16
US61/097,311 2008-09-16

Publications (2)

Publication Number Publication Date
WO2010031793A2 true WO2010031793A2 (fr) 2010-03-25
WO2010031793A3 WO2010031793A3 (fr) 2010-07-15

Family

ID=40239695

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/062022 WO2010031793A2 (fr) 2008-09-16 2009-09-16 Bactérie de fermentation thermophile produisant du butanol et/ou de l’hydrogène à partir de glycérol

Country Status (1)

Country Link
WO (1) WO2010031793A2 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013033665A1 (fr) * 2011-09-01 2013-03-07 The Board Of Trustrees Of University Of Alabama, For And On Behalf Of The University Of Alabama, In Huntsville Procédé pour faciliter la bioconversion par des micro-organismes du glycérol brut dérivé du biodiesel
CN103003414A (zh) * 2010-04-19 2013-03-27 札幌啤酒株式会社 新微生物以及使用该新微生物的氢的制造方法、1,3-丙二醇的制造方法和生物柴油废液的处理方法
EP2905340A1 (fr) * 2014-02-07 2015-08-12 Arkema France Coproduction biologique de 1,3-propanediol et de n-butanol
US9249431B2 (en) 2008-02-28 2016-02-02 Green Biologics Limited Production process
CN106995790A (zh) * 2017-06-02 2017-08-01 南京工业大学 一株利用木聚糖为唯一碳源直接生产丁醇的菌株及其应用
CN108328861A (zh) * 2018-02-10 2018-07-27 潘培连 屠宰场水污染控制的方法
US10301652B2 (en) 2016-10-14 2019-05-28 Centre De Recherche Industrielle Du Quebec (Criq) Process for hydrogen production from glycerol
US10801043B2 (en) 2016-10-14 2020-10-13 Centre De Recherche Industrielle Du Quebec (Criq) Process for hydrogen production from glycerol

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5697986A (en) * 1996-04-12 1997-12-16 The United States Of America, As Represented By The Secretary Of Agriculture Fuels as solvents for the conduct of enzymatic reactions

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5697986A (en) * 1996-04-12 1997-12-16 The United States Of America, As Represented By The Secretary Of Agriculture Fuels as solvents for the conduct of enzymatic reactions

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ANDRADE JOSE CARLOS ET AL: "Continuous cultures of Clostridium acetobutylicum: Culture stability and low-grade glycerol utilisation." BIOTECHNOLOGY LETTERS, vol. 25, no. 2, January 2003 (2003-01), pages 121-125, XP009111044 ISSN: 0141-5492 cited in the application *
FREIER-SCHROEDER D ET AL: "BUTANOL FORMATION BY CLOSTRIDIUM-THERMOSACCHAROLYTICUM AT NEUTRAL PH" BIOTECHNOLOGY LETTERS, vol. 11, no. 11, 1989, pages 831-836, XP009111058 ISSN: 0141-5492 *
ITO ET AL: "Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process" JOURNAL OF BIOSCIENCE AND BIOENGINEERING, ELSEVIER, AMSTERDAM, NL, vol. 100, no. 3, 1 September 2005 (2005-09-01), pages 260-265, XP005665599 ISSN: 1389-1723 *
O-THONG ET AL: "Thermophilic fermentative hydrogen production by the newly isolated Thermoanaerobacterium thermosaccharolyticum PSU-2" INTERNATIONAL JOURNAL OF HYDROGEN ENERGY, ELSEVIER SCIENCE PUBLISHERS B.V., BARKING, GB, vol. 33, no. 4, 1 February 2008 (2008-02-01), pages 1204-1214, XP022504693 *
SEYFRIED M ET AL: "CALORAMATOR VITERBENSIS SP. NOV., A NOVEL THERMOPHOLIC, GLYCEROL-FERMENTING BACTERIUM ISOLATED FROM A HOT SPRING IN ITALY" INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 52, no. PART 04, 1 July 2002 (2002-07-01), pages 1177-1184, XP009038872 ISSN: 1466-5026 *
STROOT P G ET AL: "Description of a new butanol-producing thermophile Thermoanaerobacterium strain Mel9" ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 99, 1999, page 510, XP009111030 & 99TH GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY; CHICAGO, ILLINOIS, USA; MAY 30-JUNE 3, 1999 ISSN: 1060-2011 *
SUIHKO M-L ET AL: "Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills" SYSTEMATIC AND APPLIED MICROBIOLOGY, XX, XX, vol. 28, no. 6, 1 August 2005 (2005-08-01), pages 555-561, XP004971439 ISSN: 0723-2020 *
ZHANG T ET AL: "Biohydrogen production from starch in wastewater under thermophilic condition" JOURNAL OF ENVIRONMENTAL MANAGEMENT 20031001 GB, vol. 69, no. 2, 1 October 2003 (2003-10-01), pages 149-156, XP009127449 ISSN: 0301-4797 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9249431B2 (en) 2008-02-28 2016-02-02 Green Biologics Limited Production process
CN103003414A (zh) * 2010-04-19 2013-03-27 札幌啤酒株式会社 新微生物以及使用该新微生物的氢的制造方法、1,3-丙二醇的制造方法和生物柴油废液的处理方法
JP5667174B2 (ja) * 2010-04-19 2015-02-12 サッポロビール株式会社 新規微生物並びにそれを用いた水素製造方法、１，３−プロパンジオール製造方法及びバイオディーゼル廃液の処理方法
US9303274B2 (en) 2010-04-19 2016-04-05 Sapporo Breweries Limited Microorganism, and hydrogen production process, 1,3-propanediol production process and biodiesel liquid waste treatment method each using the microorganism
WO2013033665A1 (fr) * 2011-09-01 2013-03-07 The Board Of Trustrees Of University Of Alabama, For And On Behalf Of The University Of Alabama, In Huntsville Procédé pour faciliter la bioconversion par des micro-organismes du glycérol brut dérivé du biodiesel
EP2905340A1 (fr) * 2014-02-07 2015-08-12 Arkema France Coproduction biologique de 1,3-propanediol et de n-butanol
WO2015117967A1 (fr) * 2014-02-07 2015-08-13 Arkema France Coproduction biologique de 1,3-propanediol et de n-butanol
US10301652B2 (en) 2016-10-14 2019-05-28 Centre De Recherche Industrielle Du Quebec (Criq) Process for hydrogen production from glycerol
US10801043B2 (en) 2016-10-14 2020-10-13 Centre De Recherche Industrielle Du Quebec (Criq) Process for hydrogen production from glycerol
CN106995790A (zh) * 2017-06-02 2017-08-01 南京工业大学 一株利用木聚糖为唯一碳源直接生产丁醇的菌株及其应用
CN106995790B (zh) * 2017-06-02 2019-08-30 南京工业大学 一株利用木聚糖为唯一碳源直接生产丁醇的菌株及其应用
CN108328861A (zh) * 2018-02-10 2018-07-27 潘培连 屠宰场水污染控制的方法
CN108328861B (zh) * 2018-02-10 2020-12-25 邳州泰利恒商贸有限公司 屠宰场水污染控制的方法

Also Published As

Publication number Publication date
WO2010031793A3 (fr) 2010-07-15

Similar Documents

Publication Publication Date Title
Azman et al. Biohydrogen production from de-oiled rice bran as sustainable feedstock in fermentative process
Ma et al. The utilization of acid-tolerant bacteria on ethanol production from kitchen garbage
Lo et al. Cellulosic hydrogen production with a sequencing bacterial hydrolysis and dark fermentation strategy
Sinha et al. Biohydrogen production from various feedstocks by Bacillus firmus NMBL-03
US10724056B2 (en) Method of producing alcohol with a Pichia stipitis strain
WO2010031793A2 (fr) Bactérie de fermentation thermophile produisant du butanol et/ou de l’hydrogène à partir de glycérol
Al-Shorgani et al. Isolation of a Clostridium acetobutylicum strain and characterization of its fermentation performance on agricultural wastes
US8685684B2 (en) Process for the production of bio-fuels and/or bio-chemicals from biomass fermentation
Harun et al. Hydrogen production performance by Enterobacter cloacae KBH3 isolated from termite guts
US20110230682A1 (en) Microorganisms with inactivated lactate dehydrogenase gene (ldh) for chemical production
US20160032324A1 (en) Process for producing fermentation products and fermentation medium compositions therefor
Xia et al. Recent studies on thermophilic anaerobic bioconversion of lignocellulosic biomass
Ai et al. Effect of pH and buffer on butyric acid production and microbial community characteristics in bioconversion of rice straw with undefined mixed culture
EP2758517A1 (fr) Bioconversion en une seule étape de biomasse lignocellulosique en biocarburants au moyen de bactéries thermophiles extrêmes
CN101250561A (zh) 一种发酵生产丁醇和丁二酸的方法
WO2014009273A1 (fr) Procédés et cultures microbiennes pour la conversion améliorée de biomasse lignocellulosique
EP2739722B1 (fr) Fermentation butanolique à l'aide d'une biomasse prétraitée par un acide
KR101599997B1 (ko) 신규한 고온 효모 피키아 길리에르몬디 y-2 및 이의 용도
Lee et al. Biological hydrogen production: dark fermentation
WO2010114481A1 (fr) Procédés pour améliorer la production de biogaz en présence de substrats durs
US20130344555A1 (en) Dsmz 24726 for second generation bio-ethanol production
Rezahasani et al. Sugar fermentation: C4 platforms
Roy Biological gaseous energy recovery from lignocellulosic biomass
Davis et al. Enrichment of amino acids from biomass residuum
Noparat Biohydrogen production from xylose by the newly isolated Clostridium beijerinckii PS-3

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09783095

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09783095

Country of ref document: EP

Kind code of ref document: A2