WO2011133984A2 - Nouvelle bactérie pour la production de produits chimiques et ses recombinants - Google Patents

Nouvelle bactérie pour la production de produits chimiques et ses recombinants Download PDF

Info

Publication number
WO2011133984A2
WO2011133984A2 PCT/US2011/033832 US2011033832W WO2011133984A2 WO 2011133984 A2 WO2011133984 A2 WO 2011133984A2 US 2011033832 W US2011033832 W US 2011033832W WO 2011133984 A2 WO2011133984 A2 WO 2011133984A2
Authority
WO
WIPO (PCT)
Prior art keywords
product
isolated
bacterium
fermentation end
producing
Prior art date
Application number
PCT/US2011/033832
Other languages
English (en)
Other versions
WO2011133984A3 (fr
Inventor
Tim Bowman
Muhammad Abdul Ali
Kexue Huang
Matthias Schmalisch
Original Assignee
Qteros, Inc.
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 Qteros, Inc. filed Critical Qteros, Inc.
Publication of WO2011133984A2 publication Critical patent/WO2011133984A2/fr
Publication of WO2011133984A3 publication Critical patent/WO2011133984A3/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
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • polysaccharide containing materials include cellulosic, lignocellulosic, and hemicellulosic material; pectin containing material; starch; wood; corn stover; switchgrass; paper; and paper pulp sludge.
  • Ethanol fermentation from biomass including cellulosic, lignocellulosic, pectin, polyglucose and/or polyfructose containing biomass can provide much needed solutions for the world energy problem.
  • Species of yeast, fungi and bacteria have been reported to be able to convert cellulosic biomass of its monomeric sugars to ethanol.
  • many of these microorganisms grow slowly and/or produce ethanol only to low concentrations.
  • This limitation can be due to a general lack of tolerance to ethanol by the organism, or a feedback inhibition or suppression mechanism present in the organism, or to some other mechanism as well as some combination of these mechanisms.
  • Such ethanol production limitations can, in addition to affecting the ethanol titer, can also affect the ethanol productivity.
  • Thermoanaerobacter ethanolicus Clostridium thermocellum, Clostridium beijerinickii, Clostridium acetobutylicum, Clostridium tyrobutyricum, Clostridium thermobutyricum, Thermoanaerobacterium saccharolyticum, Thermoanaerobacter thermohydrosulfuricus , and Saccharomyces cerevisiae, Clostridium acetobutylicum, Moorella ssp., Carboxydocella ssp., Zymomonas mobilis, recombinant E.
  • Coli Klebsiella oxytoca and Clostridium beijerickii as well as other microorganisms. Difficulties in using these or other microorganisms for industrial scale alcohol production can include undesired products and slow fermentation.
  • An organism that can hydrolyze polysaccharides and grow rapidly during the fermentation process while producing ethanol or other desirable products from various feedstocks with high yield and productivity is a highly sought after organism.
  • control of solventogenesis in Clostridium sp. is genetically linked to the control of sporulation (Ravagnani, et al. 2000 Mol. Microbiol. 37:1172-85); Harris, et al. 2002 J. Bact.184:3586- 97 and control of solventogenesis and sporulation can be genetically uncoupled in at least one species, C acetobutylicum (U.S. Patent No. 7,432,090 B2). Sporulation often leads to the termination of solvent production in bacterial fermentations as the cell goes into a dormant state. Therefore, strains of microorganisms that do not sporulate can be useful for increased production of fermentation end products in large-scale continuous fermentations.
  • an isolated novel Gram-positive Clostridium bacterium wherein the bacterium is an anaerobic, cellulolytic and xylanolytic mesophile that produces colonies that are beige pigmented, wherein the bacterium can use carbonaceous biomass as a carbon source and can ferment sugars into ethanol, biofuels, and other chemicals. Also provided are recombinant strains of several Clostridium species wherein sporulation is disrupted. Further provided are methods of using the bacterium and strains to degrade organic material and for use in industrial processes.
  • an isolated Clostridium bacterium (e.g. Clostridium sp. Q.D.) contains a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13.
  • an isolated Clostridium bacterium contains a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13.
  • An isolated Clostridium bacterium can also contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • An isolated Clostridium bacterium can contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90% identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13 and one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequence encodes for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13; and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90% identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium is deposited under NRRL Accession Numbers NRRL B-50361, NRRL B-50362, or NRRL B-50363.
  • an isolated Clostridium bacterium is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • an isolated Clostridium bacterium can hydrolyze hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment cellulosic and/or lignocellulosic material.
  • an isolated Clostridium bacterium can utilize cellulose or xylose as its sole carbon source.
  • Also provided herein is a method of producing a fermentation end-product involving the steps of: contacting a carbonaceous biomass with an isolated Clostridium bacterium in a medium, wherein said isolated Clostridium bacterium comprises a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 %> identity to SEQ ID NO: 13; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the fermentation end-product.
  • a method of producing a fermentation end- product involves the steps of: contacting a carbonaceous biomass with an isolated Clostridium bacterium in a medium, wherein said isolated Clostridium bacterium comprises a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the fermentation end-product.
  • a method of producing a fermentation end-product involves the steps of:
  • said isolated Clostridium bacterium comprises one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the fermentation end-product.
  • a method of producing a fermentation end-product involves the steps of: contacting a carbonaceous biomass with an isolated Clostridium bacterium in a medium, wherein said isolated Clostridium bacterium comprises one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the fermentation end-product.
  • a method of producing a fermentation end-product involves the steps of: contacting a carbonaceous biomass with an isolated Clostridium bacterium in a medium, wherein said isolated Clostridium bacterium comprises: a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13, and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequence encodes for a protein or protein fragment; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the fermentation end-product.
  • a method of producing a fermentation end-product involves the steps of: contacting a carbonaceous biomass with an isolated Clostridium bacterium in a medium, wherein said isolated Clostridium bacterium comprises: a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13, and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%) identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the fermentation end-product.
  • a method of producing a fermentation end-product involves the steps of: contacting a carbonaceous biomass with an isolated Clostridium bacterium in a medium, wherein said isolated Clostridium bacterium is deposited under NRRL Accession Numbers NRRL B-50361, NRRL B-50362, or NRRL B-50363; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the fermentation end-product.
  • an isolated Clostridium bacterium used in the methods of production disclosed herein, is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • an isolated Clostridium bacterium can hydrolyze hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment cellulosic and/or lignocellulosic material.
  • an isolated Clostridium bacterium can utilize cellulose or xylose as its sole carbon source.
  • a carbonaceous biomass used in the methods disclosed herein, comprises woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, sorghum, bamboo, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, bagasse, poplar, or algae.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers Dried Grains with Solubles
  • a carbonaceous biomass contains cellulosic or lignocellulosic materials.
  • a carbonaceous biomass is pretreated to make the polysaccharides more available to the bacterium.
  • ac arbonaceous biomass is pretreated by acid, steam explosion, hot water treatment, alkali, catalase, or a detoxifying or chelating agent.
  • a fermentation end-product is a chemical.
  • a fermentation end- product is a fuel.
  • a fermentation end-product is an alcohol.
  • a fermentation end product comprises 1,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3 -hydroxybutyro lactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, a terpene, ethanol, propanol, isopropanol, n-butanol, hydrogen, formic acid, lactic acid acetic acid, formate, lactate or acetate.
  • a fermentation end-product is ethanol.
  • Also provided herein are methods for the production of a fermentation end product involving the steps of: culturing a medium comprising an isolated Clostridium bacterium for a period of time under conditions suitable for production of the fermentation end-product by said isolated Clostridium bacterium, wherein said isolated Clostridium bacterium comprises a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13; and, harvesting the fermentation end-product from the medium.
  • a method of producing a fermentation end-product involves the steps ofculturing a medium comprising an isolated Clostridium bacterium for a period of time under conditions suitable for production of the fermentation end-product by said isolated Clostridium bacterium, wherein said isolated Clostridium bacterium comprises a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13; and, harvesting the fermentation end-product from the medium.
  • a method of producing a fermentation end-product involves the steps of: culturing a medium comprising an isolated Clostridium bacterium for a period of time under conditions suitable for production of the fermentation end-product by said isolated Clostridium bacterium, wherein said isolated Clostridium bacterium comprises one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment; and, harvesting the fermentation end-product from the medium.
  • a method of producing a fermentation end-product involves the steps of: culturing a medium comprising an isolated Clostridium bacterium for a period of time under conditions suitable for production of the fermentation end-product by said isolated Clostridium bacterium, wherein said isolated Clostridium bacterium comprises one or more nucleotide sequences comprising at least 400 nucleotides with at least 90% identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment; and, harvesting the fermentation end-product from the medium.
  • a method of producing a fermentation end-product involves the steps of: culturing a medium comprising an isolated Clostridium bacterium for a period of time under conditions suitable for production of the fermentation end-product by said isolated Clostridium bacterium, wherein said isolated Clostridium bacterium comprises: a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13, and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequence encodes for a protein or protein fragment; and, harvesting the fermentation end-product from the medium.
  • a method of producing a fermentation end-product involves the steps of: culturing a medium comprising an isolated Clostridium bacterium for a period of time under conditions suitable for production of the fermentation end-product by said isolated Clostridium bacterium, wherein said isolated Clostridium bacterium comprises: a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13, and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment; and, harvesting the fermentation end-product from the medium.
  • a method of producing a fermentation end-product involves the steps of: culturing a medium comprising an isolated Clostridium bacterium for a period of time under conditions suitable for production of the fermentation end-product by said isolated Clostridium bacterium, wherein said isolated Clostridium bacterium is deposited under NRRL Accession Numbers NRRL B-50361, NRRL B-50362, or NRRL B-50363; and, harvesting the fermentation end-product from the medium.
  • an isolated Clostridium bacterium used in the methods of production disclosed herein, is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • an isolated Clostridium bacterium can hydrolyze hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment cellulosic and/or lignocellulosic material.
  • an isolated Clostridium bacterium can utilize cellulose or xylose as its sole carbon source.
  • a carbonaceous biomass used in the methods disclosed herein, comprises woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, sorghum, bamboo, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, bagasse, poplar, or algae.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers Dried Grains with Solubles
  • a carbonaceous biomass contains cellulosic or lignocellulosic materials.
  • a carbonaceous biomass is pretreated to make the polysaccharides more available to the bacterium.
  • ac arbonaceous biomass is pretreated by acid, steam explosion, hot water treatment, alkali, catalase, or a detoxifying or chelating agent.
  • a fermentation end-product is a chemical.
  • a fermentation end- product is a fuel.
  • a fermentation end-product is an alcohol.
  • a fermentation end product comprises 1 ,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3 -hydroxybutyro lactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, a terpene, ethanol, propanol, isopropanol, n-butanol, hydrogen, formic acid, lactic acid acetic acid, formate, lactate or acetate.
  • a fermentation end-product is ethanol.
  • Also provided herein are methods of producing ethanol involving the steps of: contacting a carbonaceous biomass with an isolated Clostridium bacterium in a medium, wherein said isolated Clostridium bacterium comprises a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13; and, incubating the carbonaceous biomass, medium, and Clostridium bacterium for a sufficient amount of time to produce the ethanol.
  • an isolated Clostridium bacterium e.g. Clostridium sp.
  • Q.D. contains a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13.
  • an isolated Clostridium bacterium contains a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13.
  • An isolated Clostridium bacterium can also contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • Clostridium bacterium can contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90% identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13 and one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequence encodes for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13; and,one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%) identity to SEQ ID NO: 1 , wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium is deposited under NRRL Accession Numbers NRRL B-50361 , NRRL B-50362, or NRRL B-50363.
  • an isolated Clostridium bacterium is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • an isolated Clostridium bacterium can hydrolyze hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment cellulosic and/or lignocellulosic material.
  • an isolated Clostridium bacterium can utilize cellulose or xylose as its sole carbon source.
  • a system for producing a fermentation end-product involving: a fermentation vessel; a carbonaceous biomass; an isolated Clostridium bacterium, wherein said isolated Clostridium bacterium comprises a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13; and, a medium.
  • an isolated Clostridium bacterium e.g. Clostridium sp. Q.D.
  • an isolated Clostridium bacterium contains a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13.
  • An isolated Clostridium bacterium can also contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • An isolated Clostridium bacterium can contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90% identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13 and one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 %> identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequence encodes for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13; and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium is deposited under NRRL Accession Numbers NRRL B-50361, NRRL B-50362, or NRRL B-50363.
  • an isolated Clostridium bacterium is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • an isolated Clostridium bacterium can hydrolyze hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment hexose or pentose sugars.
  • an isolated Clostridium bacterium can hydrolyze and ferment cellulosic and/or lignocellulosic material.
  • an isolated Clostridium bacterium can utilize cellulose or xylose as its sole carbon source.
  • a fermentation vessel is configured to house said medium and said microorganism, and wherein said carbonaceous biomass comprises a cellulosic and/or lignocellulosic material.
  • a carbonaceous biomass used in the methods disclosed herein, comprises woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, sorghum, bamboo, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, bagasse, poplar, or algae.
  • a carbonaceous biomass contains cellulosic or lignocellulosic materials.
  • a carbonaceous biomass is pretreated to make the polysaccharides more available to the bacterium.
  • ac arbonaceous biomass is pretreated by acid, steam explosion, hot water treatment, alkali, catalase, or a detoxifying or chelating agent.
  • a fermentation end-product is a chemical.
  • a fermentation end-product is a fuel.
  • a fermentation end-product is an alcohol.
  • a fermentation end product comprises 1 ,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, a terpene, ethanol, propanol, isopropanol, n- butanol, hydrogen, formic acid, lactic acid acetic acid, formate, lactate or acetate.
  • 1 ,4 diacid succinic, fumaric or malic
  • 2,5 furan dicarboxylic acid 3 hydroxy propionic acid
  • a fermentation end-product is ethanol.
  • a fuel plant containing a fermentation vessel configured to house a medium and an isolated Clostridium bacterium, wherein said fermentation vessel comprises a cellulosic and/or lignocellulosic material, wherein said isolated Clostridium bacterium comprises a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13.
  • a cellulosic and/or lignocellulosic material is pretreated
  • an isolated Clostridium bacterium e.g. Clostridium sp.
  • Q.D. contains a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13.
  • an isolated Clostridium bacterium contains a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13.
  • An isolated Clostridium bacterium can also contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • Clostridium bacterium can contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90% identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13 and one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequence encodes for a protein or protein fragment.
  • an isolated Clostridium bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90% identity to SEQ ID NO: 13; and,one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%) identity to SEQ ID NO: 1 , wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Clostridium bacterium is deposited under NRRL Accession Numbers NRRL B-50361 , NRRL B-50362, or NRRL B-50363.
  • composition comprising an isolated gram-positive bacterium, wherein the bacterium is an anaerobic, obligate mesophile that produces beige-pigmented colonies, wherein the bacterium can use hexose or pentose sugars as a carbon source and can ferment said sugars into ethanol, organic acids, biofuels and other chemical products.
  • an isolated Gram-positive bacterium e.g. Clostridium sp. Q.D.
  • an isolated Gram-positive bacterium contains a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13.
  • An isolated Gram-positive bacterium can also contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • An isolated Gram-positive bacterium can contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1 , wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Gram-positive bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13 and one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 %> identity to SEQ ID NO: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42, wherein said nucleotide sequence encodes for a protein or protein fragment.
  • an isolated Gram-positive bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13; and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1 , wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Gram-positive bacterium is deposited under NRRL Accession Numbers NRRL B-50361 , NRRL B-50362, or NRRL B-50363.
  • an isolated Gram- positive bacterium is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • the bacterium is recombinant.
  • the bacterium can utilize cellulose or xylose as its sole carbon source.
  • the bacterium produces alcohol dehydrogenase, wherein the alcohol dehydrogenase reduces acetaldehyde into ethanol.
  • the bacterium produces ethanol at greater than 90% theoretical yield from biomass.
  • the bacterium does not sporulate.
  • an isolated gram-positive bacterium designated Clostridium sp. Q.D, wherein the bacterium is an anaerobic, obligate mesophile that produces beige- pigmented colonies, wherein the bacterium can ferment C5 and C6 carbohydrates into ethanol, organic acids, biofuels and other chemicals, comprising culturing the bacterium at a temperature and in a medium effective to promote growth of the bacterium.
  • an isolated Gram-positive bacterium e.g. Clostridium sp.
  • Q.D. contains a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 99.8 % identity to SEQ ID NO: 13.
  • an isolated Gram-positive bacterium contains a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13.
  • An isolated Gram-positive bacterium can also contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • An isolated Gram-positive bacterium can contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Gram-positive bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13 and one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequence encodes for a protein or protein fragment.
  • an isolated Gram-positive bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13; and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Gram-positive bacterium is deposited under NRRL Accession Numbers NRRL B-50361, NRRL B-50362, or NRRL B-50363.
  • the temperature is from about 30°C to about 40° C; in another embodiment, the temperature is from about 35°C to about 39°C.
  • culturing is at a pH from about 6.0 to about 7.5.
  • the bacterium uses a carbonaceous biomass as a major carbon source.
  • an isolated Gram-positive bacterium e.g. Clostridium sp. Q.D.
  • an isolated Gram-positive bacterium contains a 16S rRNA nucleotide sequence encoded in part by SEQ ID NO: 13.
  • An isolated Gram-positive bacterium can also contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • An isolated Gram-positive bacterium can contain one or more nucleotide sequences comprising at least 400 nucleotides with at least 90% identity to SEQ ID NO: 1 , wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Gram-positive bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13 and one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 %> identity to SEQ ID NO: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42, wherein said nucleotide sequence encodes for a protein or protein fragment.
  • an isolated Gram-positive bacterium can contain both a 16S rRNA nucleotide sequence encoded in part by a nucleotide sequence with greater than 90%> identity to SEQ ID NO: 13; and, one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%> identity to SEQ ID NO: 1 , wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated Gram-positive bacterium is deposited under NRRL Accession Numbers NRRL B-50361 , NRRL B-50362, or NRRL B-50363.
  • an isolated microorganism comprising one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 %> identity to SEQ ID NO: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated microorganism contains one or more nucleotide sequences comprising at least 400 nucleotides with at least 90%) identity to SEQ ID NO: 1 , wherein said nucleotide sequences encode for a protein or protein fragment.
  • an isolated microorganism is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • an isolated microorganism is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous
  • microorganism can hydrolyze hexose or pentose sugars. In another embodiment, an isolated microorganism can hydrolyze and ferment hexose or pentose sugars. In another embodiment, an isolated microorganism can hydrolyze and ferment cellulosic and/or lignocellulosic material. In another embodiment, an isolated microorganism can utilize cellulose or xylose as its sole carbon source. In one embodiment, an isolated microorganism is a Clostridium bacterium.
  • Also provided herein are methods of producing a fermentation end-product involving the steps of: contacting a carbonaceous biomass with an isolated microorganism in a medium, wherein said isolated microorganism comprises one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment; and, incubating the carbonaceous biomass, medium, and said
  • an isolated microorganism for a sufficient amount of time to produce the fermentation end-product.
  • an isolated microorganism is genetically modified, for example to express one or more heterologous genes, to enhance the activity of one or more endogenous enzymes, or to inhibit expression of one or more endogenous genes.
  • an isolated microorganism can hydrolyze hexose or pentose sugars.
  • an isolated microorganism can hydrolyze and ferment hexose or pentose sugars.
  • an isolated microorganism can hydrolyze and ferment cellulosic and/or hgnocellulosic material.
  • an isolated microorganism can utilize cellulose or xylose as its sole carbon source.
  • carbonaceous biomass comprises woody plant matter, non-woody plant matter, cellulosic material, hgnocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, sorghum, bamboo, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, bagasse, poplar, or algae.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers Dried Grains with Solubles
  • a carbonaceous biomass comprises hemicellulosic or hgnocellulosic materials.
  • a carbonaceous biomass is pretreated to make the polysaccharides more available to the bacterium.
  • a carbonaceous biomass is pretreated by acid, steam explosion, hot water treatment, alkali, catalase, or a detoxifying or chelating agent.
  • a fermentation end-product is a chemical.
  • a fermentation end-product is a fuel.
  • a fermentation end-product is an alcohol.
  • a fermentation end product comprises 1,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, a terpene, ethanol, propanol, isopropanol, n-butanol, hydrogen, formic acid, lactic acid acetic acid, formate, lactate or acetate.
  • a microoraganism is a Clostridium bacterium.
  • the fermentation end-product is ethanol.
  • a system for producing a fermentation end-product containing: a fermentation vessel; a carbonaceous biomass; an isolated microorganism, wherein said isolated microorganism comprises one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment; and, a medium.
  • an isolated microorganism is genetically modified.
  • a microorganism is a Clostridium bacterium.
  • a fuel plant comprising a fermentation vessel configured to house a medium and isolated microorganism wherein said fermentation vessel comprises a cellulosic and/or lignocellulosic material, wherein said isolated microorganism comprises one or more nucleotide sequences comprising at least 400 nucleotides with at least 90 % identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, wherein said nucleotide sequences encode for a protein or protein fragment.
  • a microorganism is genetically modified.
  • a microorganism is a Clostridium bacterium.
  • Figure 1 shows a phylogram of the 16S rRNA gene sequences of members of the Clostridiaceae.
  • Figure 2 depicts Clostridium sp. Q.D BLAST comparisons of 16 rRNA sequences to that of three other Clostridia (SEQ ID NOS 11-16, respectively, in order of appearance).
  • Figure 3 shows the morphology of Clostridium sp. Q.D at different growth phases.
  • Figure 4 shows growth curves for Clostridium sp. Q.D.
  • Figure 5 shows Clostridium sp. Q.D ethanol and acetic acid production on crystalline cellulose over time.
  • Figure 6 shows Clostridium sp. Q.D (D) colony morphology compared to strains of C.
  • Figure 7 shows Clostridium sp. Q.D (D) colony morphology on high levels of glucose.
  • Figure 8 shows hydrolysis of different sugars by Clostridium sp. Q.D.
  • Figure 9 is a map of the plasmid pGEMspo2DS12EmJoriT used to transform Clostridium sp. Q.D.
  • Figure 10 shows sequences comprising SpoIID and the promoter incorporated in the vector in FIG. 9.
  • Figure 11 shows sequences comprising the EMR gene incorporated in the vector in FIG. 9.
  • Figure 12 shows the morphology of Clostridium sp. Q.D compared to Clostridium sp. Q.D-5 and Clostridium sp. Q.D-7.
  • Figure 13 compares the growth of the sporulation mutants to that of non-recombinant
  • Figure 14 compares ethanol and acetic acid production of the sporulation mutants to that of non- recombinant Clostridium sp. Q.D.
  • Figure 15 illustrates a method for producing fermentation end products from biomass by first treating biomass with an acid at elevated temperature and pressure in a hydrolysis unit.
  • Figure 16 illustrates a method for producing fermentation end products from biomass by using solvent extraction or separation methods.
  • Figure 17 illustrates a method for producing fermentation end products from biomass by charging biomass to a fermentation vessel.
  • Figure 18 illustrates pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either fermented separately or together.
  • Figure 19 illustrates results from fermentation of cellobiose with Clostridium sp. Q.D while maintaining a pH of 6.5 for 48 hours.
  • Figure 20 illustrates results from fermentation of cellobiose with Clostridium sp. Q.D while maintaining a pH of 5.5.
  • Figure 21 illustrates results from fermentation of cellobiose with Clostridium sp. Q.D while not adjusting pH.
  • Figure 22 illustrates synergistic characteristics of Clostridium sp. Q.D.
  • Figure 23 illustrates a plasmid map for pIMPl .
  • Figure 24 illustrates a plasmid map for pIMCphy.
  • Figure 25 illustrates a plasmid map for pCphyP3510.
  • Figure 26 illustrates a plasmid map for pCphyP3510-l 163.
  • Figure 27 illustrates the plasmid pQInt.
  • Figure 28 illustrates the plasmid pQIntl .
  • Figure 29 illustrates the plasmid pQInt2.
  • an isolated Gram-positive bacterium wherein the bacterium is an obligate anaerobic, mesophilic, cellulolytic organism that produces colonies that are beige pigmented, wherein the bacterium can use polysaccharides as a sole carbon source and can oxidize glucose into ethanol or one or more organic acid as its fermentation product.
  • An example of the bacterium is designated Clostridium sp. Q.D, having the NRRL patent deposit designation NRRL B- 50361.
  • "obligate” means required or compulsory.
  • a "mesophilic” is a bacterium that preferentially ferments a carbon source at about 30-40° C. Clostridium sp. Q.D consists of motile rods that form slightly subterminal spores.
  • Percent identity values for Q.D bacterium compared with representative members of the Clostridium sp. ranged from 99.8% with Clostridium xylanolyticum to 99.7% with Clostridium algidixylanolyticum. While C. algidixylanolyticum is described as making ethanol as a fermentation end product, C. xylanolyticum is not. Also C. xylanolyticum has terminal endospores whereas C. algidixylanolyticum has subterminal endospores.
  • the bacterium comprises a 16S rRNA nucleic acid sequence which is unique as compared to a parental strain. In another aspect, the bacterium comprises a 16S rRNA nucleic acid that is greater than about 98% similar to the nucleic acid identified as Clostridium algidixylanolyticum strain SPL73. In another aspect, the bacterium comprises a 16S rRNA nucleic acid that is greater than about 99% similar to the nucleic acid identified as Clostridium algidixylanolyticum strain SPL73. In yet another aspect, the bacterium comprises a 16S rRNA nucleic acid identified in FIG. 2 (SEQ ID NO: 13).
  • the bacterium is an obligate anaerobic mesophile that can ferment biomass or carbonaceous material into ethanol, organic acids and other fermentation end products.
  • the bacterium can degrade cellulose and/or xylose into ethanol and acetic or lactic acid.
  • the phrase “the medium can optionally contain glucose” means that the medium may or may not contain glucose as an ingredient and that the description includes both media containing glucose and media not containing glucose.
  • enzyme reactive conditions refers to environmental conditions ⁇ i.e. , such factors as temperature, pH, or lack of inhibiting substances) which will permit the enzyme to function. Enzyme reactive conditions can be either in vitro, such as in a test tube, or in vivo, such as within a cell.
  • gene refers to a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. , introns, 5' and 3' untranslated sequences).
  • host cell includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide of the invention.
  • Host cells include progeny of a single host cell, and the progeny can not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells transfected, transformed, or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention.
  • a host cell which comprises a recombinant vector of the invention is a recombinant host cell, recombinant cell, or recombinant microorganism.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it in its native state.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it in its native state.
  • polynucleotide refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • isolated polypeptide and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, i.e., it is not associated with in vivo substances.
  • An “increased” amount is typically a "statistically significant” amount, and can include an increase that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (including all integers and decimal points in between, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by an unmodified microorganism or a differently modified
  • operably linked means placing a gene under the regulatory control of a promoter, which then controls the transcription and optionally the translation of the gene.
  • the genetic sequence or promoter is positioned at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting; i.e. the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function.
  • a regulatory sequence element can be positioned with respect to a gene to be placed under its control in the same position as the element is situated in its in its natural setting with respect to the native gene it controls.
  • constitutive promoter refers to a polynucleotide sequence that induces transcription or is typically active, (i.e., promotes transcription), under most conditions, such as those that occur in a host cell.
  • a constitutive promoter is generally active in a host cell through a variety of different environmental conditions.
  • inducible promoter refers to a polynucleotide sequence that induces transcription or is typically active only under certain conditions, such as in the presence of a specific transcription factor or transcription factor complex, a given molecule factor (e.g., IPTG) or a given environmental condition (e.g., CO 2 concentration, nutrient levels, light, heat). In the absence of that condition, inducible promoters typically do not allow significant or measurable levels of transcriptional activity.
  • a specific transcription factor or transcription factor complex e.g., IPTG
  • a given environmental condition e.g., CO 2 concentration, nutrient levels, light, heat
  • low temperature-adapted refers to an enzyme that has been adapted to have optimal activity at a temperature below about 20°C, such as 19 °C, 18 °C, 17 °C, 16 °C, 15 °C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, - 7°C, -8°C, -9°C, -10°C, -11°C, -12°C, -13°C, -14°C, or -15°C.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, rRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • a polynucleotide sequence can include genomic sequence, extra-genomic and plasmid-encoded sequence and a smaller engineered gene segment that express, or can be adapted to express, proteins, polypeptides, peptides and the like. Such segments can be naturally isolated, or modified synthetically by the hand of man.
  • Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • additional coding or non- coding sequences can, but need not, be present within a polynucleotide of the present invention, and a polynucleotide can, but need not, be linked to other molecules and/or support materials.
  • Polynucleotides can comprise a native sequence (i.e., an endogenous sequence) or can comprise a variant, or a biological functional equivalent of such a sequence.
  • Polynucleotide variants can contain one or more base substitutions, additions, deletions and/or insertions, as further described below.
  • a polynucleotide variant encodes a polypeptide with the same sequence as the native protein.
  • a polynucleotide variant encodes a polypeptide with substantially similar enzymatic activity as the native protein.
  • a polynucleotide variant encodes a protein with increased enzymatic activity relative to the native polypeptide. The effect on the enzymatic activity of the encoded polypeptide can generally be assessed as described herein.
  • a polynucleotide can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably.
  • the maximum length of a polynucleotide sequence which can be used to transform a microorganism is governed only by the nature of the recombinant protocol employed.
  • polynucleotide variant and “variant” and the like refer to polynucleotides that display substantial sequence identity with any of the reference polynucleotide sequences or genes described herein, and to polynucleotides that hybridize with any polynucleotide reference sequence described herein, or any polynucleotide coding sequence of any gene or protein referred to herein, under low stringency, medium stringency, high stringency, or very high stringency conditions that are defined hereinafter and known in the art. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide.
  • polynucleotide variant and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered
  • polynucleotide retains the biological function or activity of the reference polynucleotide, or has increased activity in relation to the reference polynucleotide (i.e., optimized).
  • Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51%> to at least 99% and all integer percentages in between) sequence identity with a reference polynucleotide described herein.
  • polynucleotide variant and “variant” also include naturally- occurring allelic variants that encode these enzymes.
  • naturally- occurring variants include allelic variants (same locus), homologs (different locus), and orthologs (different organism).
  • Naturally occurring variants such as these can be identified and isolated using well-known molecular biology techniques including, for example, various polymerase chain reaction (PCR) and hybridization-based techniques as known in the art.
  • Naturally occurring variants can be isolated from any organism that encodes one or more genes having a suitable enzymatic activity described herein (e.g., C-C ligase, diol
  • dehydrogenase pectate lyase, alginate lyase, diol dehydratase, transporter, etc.
  • Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or microorganisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions.
  • non-naturally occurring variants can have been optimized for use in a given microorganism (e.g., E. coli), such as by engineering and screening the enzymes for increased activity, stability, or any other desirable feature.
  • the variations can produce both conservative and non- conservative amino acid substitutions (as compared to the originally encoded product).
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a reference polypeptide.
  • Variant polynucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a biologically active polypeptide.
  • variants of a reference polynucleotide sequence will have at least about 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, 90% to 95% or more, and even about 97% or 98% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a variant polynucleotide sequence encodes a protein with substantially similar activity compared to a protein encoded by the respective reference polynucleotide sequence.
  • substantially similar activity means variant protein activity that is within +/- 15% of the activity of a protein encoded by the respective reference polynucleotide sequence.
  • a variant polynucleotide sequence encodes a protein with greater activity compared to a protein encoded by the respective reference polynucleotide sequence.
  • the nucleic acid can be detected with a probe capable of hybridizing to the nucleic acid of a cell or a sample.
  • This probe can be a nucleic acid comprising the nucleotide sequence of a coding strand or its complementary strand or the nucleotide sequence of a sense strand or antisense strand, or a fragment thereof.
  • the nucleic acid can comprise the nucleic acid comprising nucleotide sequence of the bacterium, for example the nucleic acid of SEQ ID NO: l , or fragments thereof.
  • the probe can be either DNA or RNA and can bind either DNA or RNA, or both, in the biological sample.
  • the nucleic acid can be SEQ ID NO: l , or any of the other disclosed nucleic acids, and fragments thereof, can be utilized as probes or primers to detect nucleic acids of the disclosed bacterium
  • a polynucleotide probe or primer comprising at least 15 contiguous nucleotides can be utilized to detect a nucleic acid of the disclosed bacterium.
  • the polynucleotide probe or primer can be at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or at least 200 nucleotides in length.
  • nucleic acid probe refers to a nucleic acid fragment that selectively hybridizes under stringent conditions with a nucleic acid comprising a nucleic acid set forth in a sequence listed herein. This hybridization must be specific. The degree of complementarity between the hybridizing nucleic acid and the sequence to which it hybridizes should be at least enough to exclude hybridization with a nucleic acid encoding an unrelated protein.
  • washing conditions refers to the washing conditions used in a hybridization protocol.
  • the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5° C. to 20° C. below the calculated Tm of the nucleic acid hybrid under study.
  • the denaturation temperature is approximately 5° C, 6° C, 7° C, 8° C, 9° C, 10° C, 1 1 ° C, 12° C, 13° C, 14° C, 15° C, 16° C, 17° C, 18° C, 19° C, or 20° C. below the calculated Tm of the nucleic acid hybrid under study.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or polypeptide-coding nucleic acid of interest and then washed under conditions of different stringencies.
  • the Tm of such an oligonucleotide can be estimated by allowing 20° C for each A or T nucleotide, and 4 °C. for each G or C. For example, an 18 nucleotide probe of 50% G+C would, therefore, have an approximate Tm of 54° C.
  • Stringent conditions are known to one of skill in the art. See, for example, Sambrook et al. (2001). The following is an exemplary set of hybridization conditions and is not limiting: [0095] Very High Stringency
  • Hybridization 5XSSC at 65° C. for 16 hours. Wash twice: 2XSSC at room temperature (RT) for 15 minutes each. Wash twice: 0.5XSSC at 65° C. for 20 minutes each.
  • Hybridization 5X-6XSSC at 65°C.-70° C. for 16-20 hours. Wash twice: 2XSSC at RT for 5-20 minutes each. Wash twice: 1XSSC at 55° C.-70° C. for 30 minutes each.
  • Hybridization 6XSSC at RT to 55° C. for 16-20 hours. Wash at least twice: 2X-3XSSC at RT to 55° C. for 20-30 minutes each.
  • variants of nucleic acids and polypeptides herein disclosed can typically have at least, about 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent similarity, or homology, to the stated sequence or the native sequence.
  • the similarity can be calculated after aligning the two sequences so that the similarity is at its highest level.
  • a sequence recited as having a particular percent similarity to another sequence refers to sequences that have the recited similarity as calculated by any one or more of the calculation methods described above.
  • a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent similarity to the second sequence as calculated by any of the other calculation methods.
  • a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent similarity to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods.
  • a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using each of the calculation methods (although, in practice, the different calculation methods will often result in different calculated similarity percentages).
  • amino acids can be substituted for other amino acids in a protein sequence without appreciable loss of the desired activity. It is thus contemplated that various changes can be made in the peptide sequences of the disclosed protein sequences, or their corresponding nucleic acid sequences without appreciable loss of the biological activity.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Amino acids have been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (- 0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
  • amino acids can be substituted by other amino acids having a similar hydropathic index or score and result in a protein with similar biological activity, i.e., still obtain a biologically-functional protein.
  • substitution of amino acids whose hydropathic indices are within +/-0.2 is preferred, those within +/-0.1 are more preferred, and those within +/-0.5 are most preferred.
  • leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • an amino acid can be substituted by another amino acid having a similar hydrophilicity score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein.
  • substitution of amino acids whose hydropathic indices are within +/-0.2 is preferred, those within +/-0.1 are more preferred, and those within. +/-.0.5 are most preferred.
  • amino acid substitutions can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take any of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Changes which are not expected to be advantageous can also be used if these resulting proteins have the same or improved characteristics, relative to the unmodified polypeptide from which they are engineered.
  • a polynucleotide comprises codons in its coding sequence that are optimized to increase the thermostability of an mRNA transcribed from the polynucleotide. In one embodiment this optimization does not change the amino acid sequence encoded by the polynucleotide.
  • a polynucleotide comprises codons in its protein coding sequence that are optimized to increase translation efficiency of an mRNA from the polynucleotide in a host cell. In one embodiment this optimization does not change the amino acid sequence encoded by the polynucleotide.
  • a method for which uses variants of full-length polypeptides having any of the enzymatic activities described herein, truncated fragments of these full-length polypeptides, variants of truncated fragments, as well as their related biologically active fragments.
  • biologically active fragments of a polypeptide can participate in an interaction, for example, an intra-molecular or an inter-molecular interaction.
  • An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken).
  • Bioly active fragments of a polypeptide/enzyme an enzymatic activity described herein include peptides comprising amino acid sequences sufficiently similar to, or derived from, the amino acid sequences of a (putative) full-length reference polypeptide sequence.
  • biologically active fragments comprise a domain or motif with at least one enzymatic activity, and can include one or more (and in some cases all) of the various active domains.
  • a biologically active fragment of a an enzyme can be a polypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 600 or more contiguous amino acids, including all integers in between, of a reference polypeptide sequence.
  • a biologically active fragment comprises a conserved enzymatic sequence, domain, or motif, as described elsewhere herein and known in the art.
  • the biologically-active fragment has no less than about 1%, 10%, 25%, or 50%> of an activity of the wild-type polypeptide from which it is derived.
  • exogenous refers to a polynucleotide sequence or polypeptide that does not naturally occur in a given wild-type cell or microorganism, but is typically introduced into the cell by a molecular biological technique, i.e., engineering to produce a recombinant microorganism.
  • exogenous polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein or enzyme.
  • endogenous refers to naturally- occurring polynucleotide sequences or polypeptides that can be found in a given wild-type cell or microorganism.
  • certain naturally- occurring bacterial or yeast species do not typically contain a benzaldehyde lyase gene, and, therefore, do not comprise an "endogenous" polynucleotide sequence that encodes a benzaldehyde lyase.
  • a microorganism can comprise an endogenous copy of a given polynucleotide sequence or gene
  • the introduction of a plasmid or vector encoding that sequence such as to over-express or otherwise regulate the expression of the encoded protein, represents an "exogenous" copy of that gene or polynucleotide sequence.
  • Any of the of pathways, genes, or enzymes described herein can utilize or rely on an "endogenous” sequence, or can be provided as one or more "exogenous" polynucleotide sequences, and/or can be used according to the endogenous sequences already contained within a given microorganism.
  • sequence identity for example, comprising a “sequence 50%> identical to,” as used herein, refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” can be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg,
  • sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides can each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more)
  • polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window can comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window can be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et ah
  • FASTA FASTA
  • TFASTA TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA
  • Best alignment i.e., resulting in the highest percentage homology over the comparison window
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Aus
  • transformation refers to the permanent, heritable alteration in a cell resulting from the uptake and incorporation of foreign DNA into the host-cell genome. This includes the transfer of an exogenous gene from one microorganism into the genome of another microorganism as well as the addition of additional copies of an endogenous gene into a microorganism.
  • recombinant refers to an organism that genetically modified to comprise one or more heterologous or endogenous nucleic acid molecules, such as in a plasmid or vector. Such nucleic acid molecules can be comprised extrachromosomally or integrated into the chromosome of an organism.
  • non-recombinant means an organism is not genetically modified.
  • a recombinant organism can be modified to overexpress an endogenous gene encoding an enzyme through modification of promoter elements (e.g., replacing an endogenous promoter element with a constitutive or highly active promoter).
  • a recombinant organism can be modified by introducing a heterologous nucleic acid molecule encoding a protein that is not otherwise expressed in the host organism.
  • vector refers to a polynucleotide molecule, such as a DNA molecule. It can be derived, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector can contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini- chromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self- replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • Such a vector can comprise specific sequences that allow recombination into a particular, desired site of the host chromosome.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • a vector can be one which is operably functional in a bacterial cell, such as a cyanobacterial cell.
  • the vector can include a reporter gene, such as a green fluorescent protein (GFP), which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
  • inactivate or “inactivating” as used herein for a gene, refer to a reduction in expression and/or activity of the gene.
  • inactivate or “inactivating” as used herein for a biological pathway, refer to a reduction in the activity of an enzyme in a the pathway. For example, inactivating an enzyme of the lactic acid pathway would lead to the production of less lactic acid.
  • wild-type and wild- occurring are used interchangeably to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.
  • a wild type gene or gene product e.g., a polypeptide
  • a wild type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or "wild-type” form of the gene.
  • fuel or “biofuel” as used herein has its ordinary meaning as known to those skilled in the art and can include one or more compounds suitable as liquid fuels, gaseous fuels, biodiesel fuels (long-chain alkyl (methyl, propyl or ethyl) esters), heating oils (hydrocarbons in the 14-20 carbon range), reagents, chemical feedstocks and includes, but is not limited to, hydrocarbons (both light and heavy), hydrogen, methane, hydroxy compounds such as alcohols (e.g. ethanol, butanol, propanol, methanol, etc.), and carbonyl compounds such as aldehydes and ketones (e.g. acetone, formaldehyde, 1 - propanal, etc.).
  • hydrocarbons both light and heavy
  • hydrogen methane
  • hydroxy compounds such as alcohols (e.g. ethanol, butanol, propanol, methanol, etc.)
  • carbonyl compounds such as aldehydes and ketones
  • fixation end-product or “end-product” as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biofuels, chemical additives, processing aids, food additives, organic acids (e.g. acetic, lactic, formic, citric acid etc.), derivatives of organic acids such as esters (e.g. wax esters, glycerides, etc.) or other functional compounds.
  • biofuels chemical additives
  • processing aids e.g. acetic, lactic, formic, citric acid etc.
  • derivatives of organic acids such as esters (e.g. wax esters, glycerides, etc.) or other functional compounds.
  • end-products include, but are not limited to, an alcohol, ethanol, butanol, methanol, 1 , 2-propanediol, 1 , 3 -propanediol, lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid, enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases and can be present as a pure compound, a mixture, or an impure or diluted form.
  • fixation end-product or “end-product” as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biofuels, chemical additives, processing aids, food additives, organic acids (e.g. acetic, lactic, formic, citric acid etc.), derivatives of organic acids such as esters (e.g. wax esters, glycerides, etc.) or other functional compounds.
  • biofuels chemical additives
  • processing aids e.g. acetic, lactic, formic, citric acid etc.
  • derivatives of organic acids such as esters (e.g. wax esters, glycerides, etc.) or other functional compounds.
  • end-products include, but are not limited to, an alcohol, ethanol, butanol, methanol, 1 , 2-propanediol, 1 , 3 -propanediol, lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid, enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases and can be present as a pure compound, a mixture, or an impure or diluted form.
  • end-products can be produced through saccharification and fermentation using enzyme- enhancing products and processes.
  • These end-products include, but are not limited to, an alcohol, ethanol, butanol, methanol, 1 , 2-propanediol, 1 , 3 -propanediol, lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid, enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases and can be present as a pure compound, a mixture, or an impure or diluted form.
  • external source as it relates to a quantity of an enzyme or enzymes provided to a product or a process means that the quantity of the enzyme or enzymes is not produced by a microorganism in the product or process.
  • An external source of an enzyme can include, but is not limited to an enzyme provided in purified form, cell extracts, culture medium or an enzyme obtained from a commercially available source.
  • plant polysaccharide as used herein has its ordinary meaning as known to those skilled in the art and can comprise one or more carbohydrate polymers of sugars and sugar derivatives as well as derivatives of sugar polymers and/or other polymeric materials that occur in plant matter.
  • exemplary plant polysaccharides include lignin, cellulose, starch, pectin, and hemicellulose. Others are chitin, sulfonated polysaccharides such as alginic acid, agarose, carrageenan, porphyran, furcelleran and funoran.
  • the polysaccharide can have two or more sugar units or derivatives of sugar units.
  • the sugar units and/or derivatives of sugar units can repeat in a regular pattern, or non-regular pattern.
  • the sugar units can be hexose units or pentose units, or combinations of these.
  • the derivatives of sugar units can be sugar alcohols, sugar acids, amino sugars, etc.
  • the polysaccharides can be linear, branched, cross-linked, or a mixture thereof. One type or class of polysaccharide can be cross-linked to another type or class of polysaccharide.
  • fermentable sugars as used herein has its ordinary meaning as known to those skilled in the art and can include one or more sugars and/or sugar derivatives that can be used as a carbon source by the microorganism, including monomers, dimers, and polymers of these compounds including two or more of these compounds. In some cases, the microorganism can break down these polymers, such as by hydrolysis, prior to incorporating the broken down material.
  • Exemplary fermentable sugars include, but are not limited to glucose, xylose, arabinose, galactose, mannose, rhamnose, cellobiose, lactose, sucrose, maltose, and fructose.
  • sacharification has its ordinary meaning as known to those skilled in the art and can include conversion of plant polysaccharides to lower molecular weight species that can be used by the microorganism at hand. For some microorganisms, this would include conversion to monosaccharides, disaccharides, trisaccharides, and oligosaccharides of up to about seven monomer units, as well as similar sized chains of sugar derivatives and combinations of sugars and sugar derivatives. For some microorganisms, the allowable chain-length can be longer (e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units or more) and for some microorganisms the allowable chain-length can be shorter (e.g. 1, 2, 3, 4, 5, 6, or 7 monomer units).
  • biomass comprises organic material derived from living organisms, including any member from the kingdoms: Monera, Protista, Fungi, Plantae, or Animalia.
  • Organic material that comprises oligosaccharides e.g., pentose saccharides, hexose saccharides, or longer saccharides
  • Organic material includes organisms or material derived therefrom.
  • Organic material includes cellulosic, hemicellulosic, and/or lignocellulosic material.
  • biomass comprises genetically-modified organisms or parts of organisms, such as genetically-modified plant matter, algal matter, animal matter.
  • biomass comprises non-genetically modified organisms or parts of organisms, such as non-genetically modified plant matter, algal matter, animal matter
  • feedstock is also used to refer to biomass being used in a process, such as those described herein.
  • Plant matter comprises members of the kingdom Plantae, such as terrestrial plants and aquatic or marine plants.
  • terrestrial plants comprise crop plants (such as fruit, vegetable or grain plants).
  • aquatic or marine plants include, but are not limited to, sea grass, salt marsh grasses (such as Spartina sp. or Phragmites sp.) or the like.
  • a crop plant comprises a plant that is cultivated or harvested for oral consumption, or for utilization in an industrial, pharmaceutical, or commercial process.
  • crop plants include but are not limited to corn, wheat, rice, barley, soybeans, bamboo, cotton, crambe, jute, sorghum, high biomass sorghum, oats, tobacco, grasses, (e.g., Miscanthus grass or switch grass), trees (softwoods and hardwoods) or tree leaves, beans rape/canola, alfalfa, flax, sunflowers, safflowers, millet, rye, sugarcane, sugar beets, cocoa, tea, Brassica sp.
  • Plant matter also comprises material derived from a member of the kingdom Plantae, such as woody plant matter, non- woody plant matter, cellulosic material, lignocellulosic material, or hemicellulosic material.
  • Plant matter includes carbohydrates (such as pectin, starch, inulin, fructans, glucans, lignin, cellulose, or xylan).
  • Plant matter also includes sugar alcohols, such as glycerol.
  • plant matter comprises a corn product, (e.g. corn stover, corn cobs, corn grain, corn steep liquor, corn steep solids, or corn grind), stillage, bagasse, leaves, pomace, or material derived therefrom.
  • plant matter comprises distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, pits, fermentation waste, skins, straw, seeds, shells, beancake, sawdust, wood flour, wood pulp, paper pulp, paper pulp waste streams, rice or oat hulls, bagasse, grass clippings, lumber, or food leftovers.
  • These materials can come from farms, forestry, industrial sources, households, etc.
  • plant matter comprises an agricultural waste byproduct or side stream.
  • plant matter comprises a source of pectin such as citrus fruit (e.g., orange, grapefruit, lemon, or limes), potato, tomato, grape, mango, gooseberry, carrot, sugar-beet, and apple, among others.
  • plant matter comprises plant peel (e.g., citrus peels) and/or pomace (e.g., grape pomace).
  • plant matter is characterized by the chemical species present, such as proteins, polysaccharides or oils.
  • plant matter is from a genetically modified plant.
  • a genetically-modified plant produces hydrolytic enzymes (such as a cellulase, hemicellulase, or pectinase etc.) at or near the end of its life cycles.
  • a genetically-modified plant encompasses a mutated species or a species that can initiate the breakdown of cell wall components.
  • plant matter is from a non-genetically modified plant.
  • Animal matter comprises material derived from a member of the kingdom Animaliae (e.g., bone meal, hair, heads, tails, beaks, eyes, feathers, entrails, skin, shells, scales, meat trimmings, hooves or feet) or animal excrement (e.g., manure).
  • animal matter comprises animal carcasses, milk, meat, fat, animal processing waste, or animal waste (manure from cattle, poultry, and hogs).
  • Algal matter comprises material derived from a member of the kingdoms Monera (e.g.
  • Cyanobacteria or Protista ⁇ e.g. algae (such as green algae, red algae, glaucophytes, cyanobacteria,) or fungus-like members of Protista (such as slime molds, water molds, etc).
  • Algal matter includes seaweed (such as kelp or red macroalgae), or marine microflora, including plankton.
  • Organic material comprises waste from farms, forestry, industrial sources, households or municipalities.
  • organic material comprises sewage, garbage, food waste ⁇ e.g., restaurant waste), waste paper, toilet paper, yard clippings, or cardboard.
  • carbonaceous biomass as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biological materials that can be converted into a biofuel, chemical or other product.
  • Carbonaceous biomass can comprise municipal waste (waste paper, recycled toilet papers, yard clippings, etc.), wood, plant material, plant matter, plant extract, bacterial matter ⁇ e.g. bacterial cellulose), distillers' grains, a natural or synthetic polymer, or a combination thereof.
  • biomass does not include fossilized sources of carbon, such as
  • hydrocarbons that are typically found within the top layer of the Earth's crust ⁇ e.g., natural gas, nonvolatile materials composed of almost pure carbon, like anthracite coal, etc.).
  • polysaccharides, oligosaccharides, monosaccharides or other sugar components of biomass include, but are not limited to, alginate, agar, carrageenan, fucoidan, floridean starch, pectin, gluronate, mannuronate, mannitol, lyxose, cellulose, hemicellulose, glycerol, xylitol, glucose, mannose, galactose, xylose, xylan, mannan, arabinan, arabinose, glucuronate, galacturonate (including di- and tri- galacturonates), rhamnose, and the like.
  • broth has its ordinary meaning as known to those skilled in the art and can include the entire contents of the combination of soluble and insoluble matter, suspended matter, cells and medium, such as for example the entire contents of a fermentation reaction can be referred to as a fermentation broth.
  • productivity has its ordinary meaning as known to those skilled in the art and can include the mass of a material of interest produced in a given time in a given volume. Units can be, for example, grams per liter-hour, or some other combination of mass, volume, and time. In fermentation, productivity is frequently used to characterize how fast a product can be made within a given fermentation volume. The volume can be referenced to the total volume of the fermentation vessel, the working volume of the fermentation vessel, or the actual volume of broth being fermented. The context of the phrase will indicate the meaning intended to one of skill in the art.
  • Productivity ⁇ e.g. g/L/d) is different from "titer" ⁇ e.g. g/L) in that productivity includes a time term, and titer is analogous to concentration.
  • conversion efficiency or “yield” as used herein have their ordinary meaning as known to those skilled in the art and can include the mass of product made from a mass of substrate. The term can be expressed as a percentage yield of the product from a starting mass of substrate. For the production of ethanol from glucose, the net reaction is generally accepted as:
  • the theoretical maximum conversion efficiency or yield is 51% (wt). Frequently, the conversion efficiency will be referenced to the theoretical maximum, for example, "80% of the theoretical maximum.” In the case of conversion of glucose to ethanol, this statement would indicate a conversion efficiency of 41% (wt.).
  • the context of the phrase will indicate the substrate and product intended to one of skill in the art.
  • the theoretical maximum conversion efficiency of the biomass to ethanol is an average of the maximum conversion efficiencies of the individual carbon source constituents weighted by the relative concentration of each carbon source.
  • the theoretical maximum conversion efficiency is calculated based on an assumed saccharification yield.
  • the theoretical maximum conversion efficiency can be calculated by assuming saccharification of the cellulose to the assimilable carbon source glucose of about 75% by weight.
  • lOg of cellulose can provide 7.5g of glucose which can provide a maximum theoretical conversion efficiency of about 7.5g * 51% or 3.8g of ethanol.
  • the efficiency of the saccharification step can be calculated or determined, i.e., saccharification yield.
  • Saccharification yields can include between about 10-100%, about 20-90%, about 30-80%, about 40-70% or about 50-60%, such as about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
  • the saccharification yield takes into account the amount of ethanol, and acidic products produced plus the amount of residual monomeric sugars detected in the media.
  • the ethanol figures resulting from media components are not adjusted in this experiment. These can account for up to 3 g/1 ethanol production or equivalent of up to 6g/l sugar as much as +/- 10%- 15% saccharification yield (or saccharification efficiency). For this reason the saccharification yield % can be greater than 100% for some plots.
  • a term "phytate” as used herein has its ordinary meaning as known to those skilled in the art can be include phytic acid, its salts, and its combined forms as well as combinations of these.
  • pretreatment refers to any mechanical, chemical, thermal, biochemical process or combination of these processes whether in a combined step or performed sequentially, that achieves disruption or expansion of a biomass so as to render the biomass more susceptible to attack by enzymes and/or microorganisms.
  • pretreatment can include removal or disruption of lignin so as to make the cellulose and hemicellulose polymers in the plant biomass more available to cellulolytic enzymes and/or microorganisms, for example, by treatment with acid or base.
  • pretreatment can include the use of a microorganism of one type to render plant polysaccharides more accessible to microorganisms of another type.
  • pretreatment can also include disruption or expansion of cellulosic and/or hemicellulosic material.
  • Steam explosion, and ammonia fiber expansion (or explosion) (AFEX) are well known thermal/chemical techniques. Hydrolysis, including methods that utilize acids and/or enzymes can be used. Other thermal, chemical, biochemical, enzymatic techniques can also be used.
  • fed-batch or “fed-batch fermentation” as used herein has its ordinary meaning as known to those skilled in the art and can include a method of culturing microorganisms where nutrients, other medium components, or biocatalysts (including, for example, enzymes, fresh microorganisms, extracellular broth, etc.) are supplied to the fermentor during cultivation, but culture broth is not harvested from the fermentor until the end of the fermentation, although it can also include “self seeding” or "partial harvest” techniques where a portion of the fermentor volume is harvested and then fresh medium is added to the remaining broth in the fermentor, with at least a portion of the inoculum being the broth that was left in the fermentor.
  • nutrients, other medium components, or biocatalysts including, for example, enzymes, fresh microorganisms, extracellular broth, etc.
  • a fed-batch process might be referred to with a phrase such as, "fed-batch with cell augmentation.”
  • This phrase can include an operation where nutrients and microbial cells are added or one where microbial cells with no substantial amount of nutrients are added.
  • the more general phrase "fed-batch” encompasses these operations as well. The context where any of these phrases is used will indicate to one of skill in the art the techniques being considered.
  • sugar compounds as used herein has its ordinary meaning as known to those skilled in the art and can include monosaccharide sugars, including but not limited to hexoses and pentoses; sugar alcohols; sugar acids; sugar amines; compounds containing two or more of these linked together directly or indirectly through covalent or ionic bonds; and mixtures thereof. Included within this description are disaccharides; trisaccharides; oligosaccharides; polysaccharides; and sugar chains, branched and/or linear, of any length.
  • xylano lytic refers to any substance capable of breaking down xylan.
  • cellulolytic refers to any substance capable of breaking down cellulose.
  • compositions and methods are provided for enzyme conditioning of feedstock or biomass to allow saccharification and fermentation to one or more industrially useful fermentation end- products.
  • biocatalyst as used herein has its ordinary meaning as known to those skilled in the art and can include one or more enzymes and microorganisms, including solutions, suspensions, and mixtures of enzymes and microorganisms.
  • this word will refer to the possible use of either enzymes or microorganisms to serve a particular function, in other contexts the word will refer to the combined use of the two, and in other contexts the word will refer to only one of the two.
  • the context of the phrase will indicate the meaning intended to one of skill in the art.
  • fatty acid comprising material has its ordinary meaning as known to those skilled in the art and can comprise one or more chemical compounds that include one or more fatty acid moieties as well as derivatives of these compounds and materials that comprise one or more of these compounds.
  • Common examples of compounds that include one or more fatty acid moieties include triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, lysophospholipids, free fatty acids, fatty acid salts, soaps, fatty acid comprising amides, esters of fatty acids and monohydric alcohols, esters of fatty acids and polyhydric alcohols including glycols (e.g.
  • a fatty acid comprising material can be one or more of these compounds in an isolated or purified form. It can be a material that includes one or more of these compounds that is combined or blended with other similar or different materials.
  • Solid forms include whole forms, such as cells, beans, and seeds; ground, chopped, slurried, extracted, flaked, milled, etc.
  • the fatty acid portion of the fatty acid comprising compound can be a simple fatty acid, such as one that includes a carboxyl group attached to a substituted or un-substituted alkyl group.
  • the substituted or unsubstituted alkyl group can be straight or branched, saturated or unsaturated.
  • Substitutions on the alkyl group can include hydroxyls, phosphates, halogens, alkoxy, or aryl groups.
  • the substituted or unsubstituted alkyl group can have 7 to 29 carbons and preferably 1 1 to 23 carbons (e.g., 8 to 30 carbons and preferably 12 to 24 carbons counting the carboxyl group) arranged in a linear chain with or without side chains and/or substitutions.
  • Addition of the fatty acid comprising compound can be by way of adding a material comprising the fatty acid comprising compound.
  • pH modifier as used herein has its ordinary meaning as known to those skilled in the art and can include any material that will tend to increase, decrease or hold steady the pH of the broth or medium.
  • a pH modifier can be an acid, a base, a buffer, or a material that reacts with other materials present to serve to raise, lower, or hold steady the pH.
  • more than one pH modifier can be used, such as more than one acid, more than one base, one or more acid with one or more bases, one or more acids with one or more buffers, one or more bases with one or more buffers, or one or more acids with one or more bases with one or more buffers.
  • a buffer can be produced in the broth or medium or separately and used as an ingredient by at least partially reacting in acid or base with a base or an acid, respectively. When more than one pH modifiers are utilized, they can be added at the same time or at different times.
  • media components such as a carbon source or a nitrogen source serve as a pH modifier; suitable media components include those with high or low pH or those with buffering capacity.
  • exemplary media components include acid- or base- hydrolyzed plant polysaccharides having residual acid or base, ammonia fiber explosion (AFEX) treated plant material with residual ammonia, lactic acid, corn steep solids or liquor.
  • AFEX ammonia fiber explosion
  • compositions and methods are provided for enzyme conditioning of feedstock or biomass to allow saccharification and fermentation to one or more industrially useful fermentation end- products.
  • Microorganisms useful in compositions and methods of the invention include, but are not limited to bacteria, or yeast.
  • bacteria include, but are not limited to, any bacterium found in the genus of Clostridium, such as C. acetobutylicum, C. aerotolerans, C. beijerinckii, C.
  • clostridioforme C. colicanis, C. difficile, C. fallax, C. formicaceticum, C. histolyticum, C. innocuum, C. Ijungdahlii, C. laramie, C. lavalense, C. novyi, C. oedematiens, C. paraputrificum, C. perfringens, C. phytofermentans (including NRRL B-50364 or NRRL B-50351), C. piliforme, C. ramosum, C.
  • yeast that can be utilized in co-culture methods of the invention include but are not limited to, species found in Cryptococcaceae, Sporobolomycetaceae with the genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon,
  • Rhodotorula and Sporobolomyces and Bullera the families Endo- and Saccharomycetaceae, with the genera Saccharomyces, Debaromyces, Lipomyces, Hansenula, Endomycopsis, Pichia, Hanseniaspora, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolitica, Emericella nidulans, Aspergillus nidulans, Deparymyces hansenii and Torulaspora hansenii.
  • a microorganism in another embodiment can be wild type, or a genetically modified strain.
  • a microorganism can be genetically modified to express one or more polypeptides capable of neutralizing a toxic by-product or inhibitor, which can result in enhanced end-product production in yield and/or rate of production.
  • modifications include chemical or physical mutagenesis, directed evolution, or genetic alteration to enhance enzyme activity of endogenous proteins, introducing one or more heterogeneous nucleic acid molecules into a host microorganism to express a polypeptide not otherwise expressed in the host, modifying physical and chemical conditions to enhance enzyme function ⁇ e.g., modifying and/or maintaining a certain temperature, pH, nutrient concentration, or biomass concentration), or a combination of one or more such modifications.
  • Described herein are also methods and compositions for pre-treating biomass prior to extraction of industrially useful end-products.
  • more complete saccharification of biomass and fermentation of the saccharification products results in higher fuel yields.
  • a Clostridium species for example Clostridium sp. Q.D or a variant thereof is contacted with pretreated or non-pretreated feedstock containing cellulosic, hemicellulosic, and/or lignocellulosic material.
  • Additional nutrients can be present or added to the biomass material to be processed by the microorganism including nitrogen-containing compounds such as amino acids, proteins, hydrolyzed proteins, ammonia, urea, nitrate, nitrite, soy, soy derivatives, casein, casein derivatives, milk powder, milk derivatives, whey, yeast extract, hydrolyze yeast, autolyzed yeast, corn steep liquor, corn steep solids, monosodium glutamate, and/or other fermentation nitrogen sources, vitamins, and/or mineral supplements.
  • one or more additional lower molecular weight carbon sources can be added or be present such as glucose, sucrose, maltose, corn syrup, lactic acid, etc.
  • Such lower molecular weight carbon sources can serve multiple functions including providing an initial carbon source at the start of the fermentation period, help build cell count, control the carbon/nitrogen ratio, remove excess nitrogen, or some other function.
  • aerobic/anaerobic cycling is employed for the bioconversion of cellulosic/lignocellulosic material to fuels and chemicals.
  • the anaerobic microorganism can ferment biomass directly without the need of a pretreatment.
  • the anaerobic microorganism can hydrolyze and ferment a biomass without the need of a pretreatment.
  • feedstocks are contacted with biocatalysts capable of breaking down plant- derived polymeric material into lower molecular weight products that can subsequently be transformed by biocatalysts to fuels and/or other desirable chemicals.
  • pretreatment methods can include treatment under conditions of high or low pH.
  • High or low pH treatment includes, but is not limited to, treatment using concentrated acids or concentrated alkali, or treatment using dilute acids or dilute alkali.
  • Alkaline compositions useful for treatment of biomass in the methods of the present invention include, but are not limited to, caustic, such as caustic lime, caustic soda, caustic potash, sodium, potassium, or calcium hydroxide, or calcium oxide.
  • suitable amounts of alkaline useful for the treatment of biomass ranges from O.Olg to 3g of alkaline (e.g. caustic) for every gram of biomass to be treated.
  • suitable amounts of alkaline useful for the treatment of biomass include, but are not limited to, about O.Olg of alkaline (e.g.
  • caustic 0.02g, 0.03g, 0.04g, 0.05g, 0.075g, O. lg, 0.2g, 0.3g, 0.4g, 0.5g, 0.75g, lg, 2g, or about 3g of alkaline (e.g. caustic) for every gram of biomass to be treated.
  • alkaline e.g. caustic
  • pretreatment of biomass comprises dilute acid hydrolysis.
  • Example of dilute acid hydrolysis treatment are disclosed in T. A. Lloyd and C. E Wyman, Bioresource
  • pretreatment of biomass comprises pH controlled liquid hot water treatment. Examples of pH controlled liquid hot water treatments are disclosed in N. Mosier et al, Bioresource Technology, (2005) 96, 1986, incorporated by reference herein in its entirety.
  • pretreatment of biomass comprises aqueous ammonia recycle process (ARP). Examples of aqueous ammonia recycle process are described in T. H. Kim and Y. Y. Lee, Bioresource Technology, (2005)96, incorporated by reference herein in its entirety.
  • the above-mentioned methods have two steps: a pretreatment step that leads to a wash stream, and an enzymatic hydrolysis step of pretreated-biomass that produces a hydrolyzate stream.
  • the pH at which the pretreatment step is carried out increases progressively from dilute acid hydrolysis to hot water pretreatment to alkaline reagent based methods (AFEX, ARP, and lime pretreatments).
  • Dilute acid and hot water treatment methods solubilize mostly hemicellulose, whereas methods employing alkaline reagents remove most lignin during the pretreatment step.
  • the wash stream from the pretreatment step in the former methods contains mostly hemicellulose-based sugars, whereas this stream has mostly lignin for the high-pH methods.
  • the subsequent enzymatic hydrolysis of the residual feedstock leads to mixed carbohydrates (C5 and C6) in the alkali-based pretreatment methods, while glucose is the major product in the hydrolysate from the low and neutral pH methods.
  • the enzymatic digestibility of the residual biomass is somewhat better for the high-pH methods due to the removal of lignin that can interfere with the accessibility of cellulase enzyme to cellulose.
  • pretreatment results in removal of about 20%, 30%, 40%, 50%, 60%, 70% or more of the lignin component of the feedstock.
  • the microorganism e.g., Clostridium phytofermentans, Clostridium, sp. Q.D or a variant thereof
  • the microorganism is capable of fermenting both five-carbon and six-carbon sugars, which can be present in the feedstock, or can result from the enzymatic degradation of components of the feedstock.
  • a two-step pretreatment is used to partially or entirely remove C5 polysaccharides and other components. After washing, the second step consists of an alkali treatment to remove lignin components.
  • the pretreated biomass is then washed prior to saccharification and fermentation.
  • One such pretreatment consists of a dilute acid treatment at room temperature or an elevated temperature, followed by a washing or neutralization step, and then an alkaline contact to remove lignin.
  • one such pretreatment can consist of a mild acid treatment with an acid that is organic (such as acetic acid, citric acid, malic acid, or oxalic acid) or inorganic (such as nitric, hydrochloric, or sulfuric acid), followed by washing and an alkaline treatment in 0.5 to 2.0% NaOH.
  • This type of pretreatment results in a higher percentage of oligomeric to monomeric saccharides, is preferentially fermented by an microorganism such as Clostridium, sp. Q.D or a variant thereof.
  • pretreatment of biomass comprises ionic liquid pretreatment.
  • Biomass can be pretreated by incubation with an ionic liquid, followed by extraction with a wash solvent such as alcohol or water.
  • the treated biomass can then be separated from the ionic liquid/wash-solvent solution by centrifugation or filtration, and sent to the saccharification reactor or vessel.
  • wash solvent such as alcohol or water.
  • Examples of pretreatment methods are disclosed in U.S. Patent No. 4600590 to Dale, U.S. Patent No. 4644060 to Chou, U.S. Patent No. 5037663 to Dale.
  • the feedstock contains cellulose, hemicellulose, soluble oligomers, simple sugars, lignins, volatiles and/or ash.
  • the parameters of the pretreatment can be changed to vary the concentration of the components of the pretreated feedstock. For example, in some embodiments a pretreatment is chosen so that the concentration of hemicellulose and/or soluble oligomers is high and the concentration of lignins is low after
  • parameters of the pretreatment include temperature, pressure, time, and pH.
  • the parameters of the pretreatment are changed to vary the concentration of the components of the pretreated feedstock such that concentration of the components in the pretreated stock is optimal for fermentation with a microorganism, e.g. Clostridium sp. Q.D or a variant thereof.
  • a microorganism e.g. Clostridium sp. Q.D or a variant thereof.
  • the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is about l%-99%, such as about 1-10%, 1-20%, 1- 30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% 1-99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5- 60%, 5-70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15-30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-80%, 15- 90% 15-99%, 20-10%, 20-20%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20- 99%, 25-10%, 25-20%, 25-30%, 25-40%, 25-50%, 25-60%, 25-70%
  • the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is 5% to 30%. In some embodiments, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is 10% to 20%.
  • the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is about l%-99%, such as about 1-10%, 1-20%, 1-30%, 1- 40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% 1-99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5- 70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15-30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-80%, 15-90% 15- 99%, 20-10%, 20-20%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20-99%, 25- 10%, 25-20%, 25-30%, 25-40%, 25-50%, 25-60%, 25-7
  • the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), or 99%.
  • the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is 5% to 40%>.
  • the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is 10% to 30%.
  • the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is about l%>-99%>, such as about 1 -10%, 1 -20%, 1 -30%, 1 -40%, 1 -50%, 1 -60%, 1 -70%, 1 -80%, 1 -90% 1 -99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15-30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-80%, 15-90% 15- 99%, 20-10%, 20-20%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20-99%, 25- 10%, 25-20%,
  • the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), or 99%).
  • concentration of soluble oligomers in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), or 99%).
  • soluble oligomers include, but are not limited to, cellobiose and xylobiose.
  • the parameters of the pretreatment are changed such that
  • concentration of soluble oligomers in the pretreated feedstock is 30% to 90%>.
  • the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is 45% to 80%.
  • the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is 45% to 80% and the soluble oligomers are primarily cellobiose and xylobiose.
  • the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is about l%-99%, such as about 1-10%, 1-20%, 1-30%, 1- 40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% 1-99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5- 70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15-30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-80%, 15-90% 15- 99%, 20-10%, 20-20%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20-99%, 25- 10%, 25-20%, 25-30%, 25-40%, 25-50%, 25-60%, 25-70%
  • the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%). In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 20%. In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 5%. Examples of simple sugars include, but are not limited to monomers and dimers.
  • the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is 0% to 20%.
  • the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is 0%> to 5%>.
  • the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is less than 1%> to 2%). In some embodiments, the parameters of the pretreatment are changed such that the concentration of phenolics is minimized.
  • the parameters of the pretreatment are changed such that concentration of furfural and low molecular weight lignins in the pretreated feedstock is less than 10%>, 9%, 8%, 7%>, 6%), 5%), 4%), 3%), 2%), or 1%>. In some embodiments, the parameters of the pretreatment are changed such that concentration of furfural and low molecular weight lignins in the pretreated feedstock is less than l% to 2%.
  • the parameters of the pretreatment are changed such that concentration of accessible cellulose is 10%> to 20 %>, the concentration of hemicellulose is 10%> to 30%>, the concentration of soluble oligomers is 45%> to 80%>, the concentration of simple sugars is 0%> to 5%>, and the concentration of lignins is 0%> to 5% and the concentration of furfural and low molecular weight lignins in the pretreated feedstock is less than 1%> to 2%>.
  • the parameters of the pretreatment are changed to obtain a high concentration of hemicellulose (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher) and a low concentration of lignins (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%).
  • the parameters of the pretreatment are changed to obtain a high concentration of hemicellulose and a low concentration of lignins such that concentration of the components in the pretreated stock is optimal for fermentation with a microorganism such as a member of the genus Clostridium, for example Clostridium sp. Q.D or variants thereof.
  • pretreatment feedstock can be cooled to a temperature which allows for growth of the microorganism(s).
  • pH can be altered prior to, or concurrently with, addition of one or more microorganisms.
  • Alteration of the pH of a pretreated feedstock can be accomplished by washing the feedstock (e.g., with water) one or more times to remove an alkaline or acidic substance, or other substance used or produced during pretreatment. Washing can comprise exposing the pretreated feedstock to an equal volume of water 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more times.
  • a pH modifier can be added. For example, an acid, a buffer, or a material that reacts with other materials present can be added to modulate the pH of the feedstock.
  • more than one pH modifier can be used, such as one or more bases, one or more bases with one or more buffers, one or more acids, one or more acids with one or more buffers, or one or more buffers.
  • more than one pH modifiers can be added at the same time or at different times.
  • Other non-limiting exemplary methods for neutralizing feedstocks treated with alkaline substances have been described, for example in U.S. Patent Nos. 4,048,341 ; 4, 182,780; and 5,693,296.
  • one or more acids can be combined, resulting in a buffer.
  • Suitable acids and buffers that can be used as pH modifiers include any liquid or gaseous acid that is compatible with the microorganism. Non- limiting examples include peroxyacetic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, and hydrochloric acid.
  • the pH can be lowered to neutral pH or acidic pH, for example a pH of 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, or lower.
  • the pH is lowered and/or maintained within a range of about pH 4.5 to about 7.1, or about 4.5 to about 6.9, or about pH 5.0 to about 6.3, or about pH 5.5 to about 6.3, or about pH 6.0 to about 6.5, or about pH 5.5 to about 6.9 or about pH 6.2 to about 6.7.
  • biomass can be pre-treated at an elevated temperature and/or pressure.
  • biomass is pre treated at a temperature range of 20°C to 400°C.
  • biomass is pretreated at a temperature of about 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 80°C, 90°C, 100°C, 120°C, 150°C, 200°C, 250°C, 300°C, 350°C, 400°C or higher.
  • elevated temperatures are provided by the use of steam, hot water, or hot gases.
  • steam can be injected into a biomass containing vessel.
  • the steam, hot water, or hot gas can be injected into a vessel jacket such that it heats, but does not directly contact the biomass.
  • a biomass can be treated at an elevated pressure.
  • biomass is pre treated at a pressure range of about lpsi to about 30psi.
  • biomass is pre treated at a pressure or about lpsi, 2psi, 3psi, 4psi, 5psi, 6psi, 7psi, 8psi, 9psi, l Opsi, 12psi, 15psi, 18psi, 20psi, 22psi, 24psi, 26psi, 28psi, 30psi or more.
  • biomass can be treated with elevated pressures by the injection of steam into a biomass containing vessel.
  • the biomass can be treated to vacuum conditions prior or subsequent to alkaline or acid treatment or any other treatment methods provided herein.
  • alkaline or acid pretreated biomass is washed (e.g. with water (hot or cold) or other solvent such as alcohol (e.g. ethanol)), pH neutralized with an acid, base, or buffering agent (e.g. phosphate, citrate, borate, or carbonate salt) or dried prior to fermentation.
  • the drying step can be performed under vacuum to increase the rate of evaporation of water or other solvents.
  • the drying step can be performed at elevated temperatures such as about 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 80°C, 90°C, 100°C, 120°C, 150°C, 200°C, 250°C, 300°C or more.
  • the pretreatment step includes a step of solids recovery.
  • the solids recovery step can be during or after pretreatment (e.g., acid or alkali
  • the solids recovery step provided by the methods of the present invention includes the use of a sieve, filter, screen, or a membrane for separating the liquid and solids fractions.
  • a suitable sieve pore diameter size ranges from about 0.001 microns to 8mm, such as about 0.005 microns to 3mm or about 0.01 microns to 1mm.
  • a sieve pore size has a pore diameter of about O.Olmicrons, 0.02 microns, 0.05 microns, 0.1 microns, 0.5 microns, 1 micron, 2 microns, 4 microns, 5 microns, 10 microns, 20 microns, 25 microns, 50 microns, 75 microns, 100 microns, 125 microns, 150 microns, 200 microns, 250 microns, 300 microns, 400 microns, 500 microns, 750 microns, 1mm or more.
  • biomass e.g. corn stover
  • a method of pre-treatment includes but is not limited to, biomass particle size reduction, such as for example shredding, milling, chipping, crushing, grinding, or pulverizing.
  • biomass particle size reduction can include size separation methods such as sieving, or other suitable methods known in the art to separate materials based on size.
  • size separation can provide for enhanced yields.
  • separation of finely shredded biomass e.g.
  • particles smaller than about 8 mm in diameter such as, 8, 7.9, 7.7, 7.5, 7.3, 7, 6.9, 6.7, 6.5, 6.3, 6, 5.9, 5.7, 5.5, 5.3, 5, 4.9, 4.7, 4.5, 4.3, 4, 3.9, 3.7, 3.5, 3.3, 3, 2.9, 2.7, 2.5, 2.3, 2, 1.9, 1.7, 1.5, 1.3, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mm) from larger particles allows the recycling of the larger particles back into the size reduction process, thereby increasing the final yield of processed biomass.
  • a fermentative mixture which comprises a pretreated lignocellulosic feedstock comprising less than about 50% of a lignin component present in the feedstock prior to pretreatment and comprising more than about 60% of a hemicellulose component present in the feedstock prior to pretreatment; and a microorganism capable of fermenting a five-carbon sugar, such as xylose, arabinose or a combination thereof, and a six-carbon sugar, such as glucose, galactose, mannose or a combination thereof.
  • pretreatment of the lignocellulosic feedstock comprises adding an alkaline substance which raises the pH to an alkaline level, for example NaOH.
  • NaOH is added at a concentration of about 0.5%> to about 2%> by weight of the feedstock.
  • pretreatment also comprises addition of a chelating agent.
  • the microorganism is a bacterium, such as a member of the genus Clostridium, for example Clostridium sp. Q.D or a variant thereof.
  • the present disclosure also provides a fermentative mixture comprising: a cellulosic feedstock pre-treated with an alkaline substance which maintains an alkaline pH, and at a temperature of from about 80°C to about 120°C; and a microorganism capable of fermenting a five-carbon sugar and a six- carbon sugar.
  • the five-carbon sugar is xylose, arabinose, or a combination thereof.
  • the six-carbon sugar is glucose, galactose, mannose, or a combination thereof.
  • the alkaline substance is NaOH.
  • NaOH is added at a
  • the microorganism is a bacterium, such as a member of the genus Clostridium, for example Clostridium sp. Q.D or a variant thereof.
  • the microorganism is genetically modified to enhance activity of one or more hydrolytic enzymes.
  • a fermentative mixture comprising a cellulosic feedstock pre-treated with an alkaline substance which increases the pH to an alkaline level, at a temperature of from about 80°C to about 120°C; and a microorganism capable of uptake and fermentation of an oligosaccharide.
  • the alkaline substance is NaOH.
  • NaOH is added at a concentration of about 0.5% to about 2% by weight of the feedstock.
  • the microorganism is a bacterium, such as a member of the genus Clostridium, for example Clostridium sp. Q.D or a variant thereof.
  • the microorganism is genetically modified to express or increase expression of an enzyme capable of hydro lyzing the oligosaccharide, a transporter capable of transporting the oligosaccharide, or a combination thereof.
  • Another aspect of the present disclosure provides a fermentative mixture comprising a cellulosic feedstock comprising cellulosic material from one or more sources, wherein the feedstock is pre-treated with a substance which increases the pH to an alkaline level, at a temperature of from about 80°C to about 120°C; and a microorganism capable of fermenting the cellulosic material from at least two different sources to produce a fermentation end-product at substantially a same yield coefficient.
  • the sources of cellulosic material are corn stover, bagasse, switchgrass or poplar.
  • the alkaline substance is NaOH.
  • NaOH is added at a concentration of about 0.5% to about 2% by weight of the feedstock.
  • the microorganism is a bacterium, such as a member of the genus Clostridium, for example Clostridium sp. Q.D or a variant thereof.
  • a process for simultaneous saccharification and fermentation of cellulosic solids from biomass into biofuel or another end-product comprises treating the biomass in a closed container with a microorganism under conditions where the microorganism produces saccharolytic enzymes sufficient to substantially convert the biomass into oligomers, monosaccharides and disaccharides.
  • the microorganism subsequently converts the oligomers, monosaccharides and disaccharides into ethanol and/or another biofuel or product.
  • a process for saccharification and fermentation comprises treating the biomass in a container with the microorganism, and adding one or more enzymes before, concurrent or after contacting the biomass with the microorganism, wherein the enzymes added aid in the breakdown or detoxification of carbohydrates or lignocellulosic material.
  • the bioconversion process comprises a separate hydrolysis and fermentation (SHF) process.
  • SHF hydrolysis and fermentation
  • the enzymes can be used under their optimal conditions regardless of the fermentation conditions and the microorganism is only required to ferment released sugars.
  • hydrolysis enzymes are externally added.
  • the bioconversion process comprises a saccharification and fermentation (SSF) process. In an SSF embodiment, hydrolysis and fermentation take place in the same reactor under the same conditions.
  • SSF saccharification and fermentation
  • the bioconversion process comprises a consolidated bioprocess (CBP).
  • CBP is a variation of SSF in which the enzymes are produced by the microorganism that carries out the fermentation.
  • enzymes can be both externally added enzymes and enzymes produced by the fermentative microorganism.
  • biomass is partially hydrolyzed with externally added enzymes at their optimal condition, the slurry is then transferred to a separate tank in which the fermentative microorganism (e.g. Clostridium sp. Q.D or a variant thereof) converts the hydrolyzed sugar into the desired product (e.g. fuel or chemical) and completes the hydrolysis of the residual cellulose and hemicellulose.
  • the fermentative microorganism e.g. Clostridium sp. Q.D or a variant thereof
  • pretreated biomass is partially hydrolyzed by externally added enzymes to reduce the viscosity.
  • Hydrolysis occurs at the optimal pH and temperature conditions (e.g. pH 5.5, 50°C for fungal cellulases).
  • Hydrolysis time and enzyme loading can be adjusted such that conversion is limited to cellodextrins (soluble and insoluble) and hemicellulose oligomers.
  • the resultant mixture can be subjected to fermentation conditions.
  • the resultant mixture can be pumped over time (fed batch) into a reactor containing a microorganism (e.g. Clostridium sp. Q.D or a variant thereof) and media.
  • a microorganism e.g. Clostridium sp. Q.D or a variant thereof
  • the microorganism can then produce endogenous enzymes to complete the hydrolysis into fermentable sugars (soluble oligomers) and convert those sugars into ethanol and/or other products in a production tank.
  • the production tank can then be operated under fermentation optimal conditions (e.g. pH 6.5, 35°C). In this way externally added enzyme is minimized due to operation under the enzyme's optimal conditions and due to a portion of the enzyme coming from the microorganism (e.g. Clostridium sp. Q.D or a variant thereof).
  • exogenous enzymes added include a xylanase, a hemicellulase, a glucanase or a glucosidase. In some embodiments, exogenous enzymes added do not include a xylanase, a hemicellulase, a glucanase or a glucosidase. In other embodiments, the amount of exogenous cellulase is greatly reduced, one-quarter or less of the amount normally added to a fermentation by a microorganism that cannot saccharify the biomass. In some embodiments, an exogenous enzyme added is a cellulase. In one example, the cellulase is
  • exogenous enzymes include a ⁇ -glucanase/xylanase mix that contains cellulase and hemicellulase activity.
  • a second microorganism can be used to convert residual carbohydrates into a fermentation end-product.
  • the second microorganism is a yeast such as
  • a process of producing a biofuel or chemical product from a lignin- containing biomass comprises: 1) contacting the lignin- containing biomass with an aqueous alkaline solution at a concentration sufficient to hydrolyze at least a portion of the lignin-containing biomass; 2) neutralizing the treated biomass to a pH between 5 to 9 (e.g.
  • disaccharides, and/or biofuel or other fermentation end-product optionally, introducing a culture of a second microorganism wherein the second microorganism is capable of substantially converting the oligomers, monosaccharides and disaccharides into biofuel.
  • cellulose is useful as a starting material for the production of fermentation end-products in methods and compositions described herein.
  • Cellulose is one of the major components in plant cell wall.
  • Cellulose is a linear condensation polymer consisting of D-anhydro glucopyranose joined together by -l ,4-linkage. The degree of polymerization ranges from 100 to 20,000. Adjacent cellulose molecules are coupled by extensive hydrogen bonds and van der Waals forces, resulting in a parallel alignment. The parallel sheet-like structure renders cellulose very stable.
  • Pretreatment can also include utilization of one or more strong cellulose swelling agents that facilitate disruption of the fiber structure and thus rendering the cellulosic material more amendable to saccharification and fermentation.
  • Some considerations have been given in selecting an efficient method of swelling for various cellulosic material: 1) the hydrogen bonding fraction; 2) solvent molar volume; 3) the cellulose structure.
  • the width and distribution of voids are important as well. It is known that the swelling is more pronounced in the presence of electrostatic repulsion, provided by alkali solution or ionic surfactants.
  • conditioning of a biomass can be concurrent to contact with a microorganism that is capable of saccharification and fermentation.
  • a microorganism that is capable of saccharification and fermentation.
  • other examples describing the pretreatment of lignocellulosic biomass have been published as U.S. Pat. Nos. 4,304,649, 5,366,558, 5,41 1,603, and 5,705,369.
  • pretreatment of biomass comprises enzyme hydrolysis with one or more enzymes from Clostridium sp. Q.D.
  • pretreatment of biomass comprises enzyme hydrolysis with one or more enzymes from Clostridium sp. Q.D, wherein the one or more enzyme is selected from the group consisting of endonucleases, exonucleases, cellobiohydrolases, beta- glucosidases, glycoside hydrolases, glycosyltransferases, lyases, esterases and proteins containing carbohydrate-binding modules.
  • biomass can be pretreated with a hydrolase identified in C. phytofermentans.
  • pretreatment of biomass comprises enzyme hydrolysis with one or more of enzymes listed in Table 1.
  • Table 1 show examples of known activities of some of the glycoside hydrolases, lyases, esterases, and proteins containing carbohydrate-binding modules family members predicted to be present in Clostridia, for example, C. phytofermentans.
  • Known activities are listed by activity and corresponding PC number as determined by the International Union of Biochemistry and Molecular Biology.
  • enzymes that degrade polysaccharides are used for the pretreatment of biomass and can include enzymes that degrade cellulose, namely, cellulases.
  • cellulases include endocellulases (EC 3.2.1.4) and exo-cellulases (EC 3.2.1.91), and hydrolyze beta-1,4- glucosidic bonds.
  • Endocellulases EC 3.2.1.4
  • exo-cellulases EC 3.2.1.91
  • enzymes that degrade polysaccharides are used for the pretreatment of biomass and can include enzymes that have the ability to degrade hemicellulose, namely,
  • Hemicellulose can be a major component of plant biomass and can contain a mixture of pentoses and hexoses, for example, D-xylopyranose, L-arabinofuranose, D-mannopyranose, D- glucopyranose, D-galactopyranose, D-glucopyranosyluronic acid and other sugars.
  • pentoses and hexoses for example, D-xylopyranose, L-arabinofuranose, D-mannopyranose, D- glucopyranose, D-galactopyranose, D-glucopyranosyluronic acid and other sugars.
  • predicted hemicellulases identified in C.
  • phytofermentans used in the pretreatment of biomass include enzymes active on the linear backbone of hemicellulose, for example, endo-beta- 1,4-D-xylanase (EC 3.2.1.8), such as GH5, GH10, GH1 1 , and GH43 family members; 1 ,4-beta-D-xyloside xylohydrolase (EC 3.2.1.37), such as GH30, GH43, and GH3 family members; and beta-mannanase (EC 3.2.1.78), such as GH26 family members.
  • endo-beta- 1,4-D-xylanase EC 3.2.1.8
  • GH5 GH10
  • GH1 1 GH43 family members
  • 1 ,4-beta-D-xyloside xylohydrolase EC 3.2.1.37
  • beta-mannanase EC 3.2.1.78
  • enzymes that degrade polysaccharides are used for the pretreatment of biomass and can include enzymes that have the ability to degrade pectin, namely, pectinases.
  • pectinases enzymes that have the ability to degrade pectin, namely, pectinases.
  • the cross-linked cellulose network can be embedded in a matrix of pectins that can be covalently cross-linked to xyloglucans and certain structural proteins.
  • Pectin can comprise
  • HG homogalacturonan
  • RH rhamnogalacturonan
  • the parameters of the pretreatment are changed to obtain a high
  • the parameters of the pretreatment are changed to obtain a high concentration of hemicellulose and a low concentration of lignins such that concentration of the components in the pretreated stock is optimal for fermentation with a microbe such as Clostridium sp. Q.D.
  • compositions and methods allowing saccharification and fermentation to one or more industrially useful fermentation end-products.
  • Saccharification includes conversion of long-chain sugar polymers, such as cellulose, to monosaccharides, disaccharides, trisaccharides, and oligosaccharides of up to about seven monomer units, as well as similar sized chains of sugar derivatives and combinations of sugars and sugar derivatives.
  • the chain-length for saccharides can be longer ⁇ e.g. 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units or more) and or shorter ⁇ e.g. 1, 2, 3, 4, 5, 6 monomer units).
  • directly processing means that a microorganism is capable of both hydro lyzing biomass and fermenting without the need for conditioning the biomass, such as subjecting the biomass to chemical, heat, enzymatic treatment or combinations thereof.
  • Methods and compositions described herein contemplate utilizing fermentation process for extracting industrially useful fermentation end-products from biomass.
  • the term "fermentation” as used herein has its ordinary meaning as known to those skilled in the art and can include culturing of a microorganism or group of microorganisms in or on a suitable medium for the microorganisms.
  • the microorganisms can be aerobes, anaerobes, facultative anaerobes, heterotrophs, autotrophs, photoautotrophs, photoheterotrophs, chemoautotrophs, and/or chemoheterotrophs.
  • the cellular activity, including cell growth can be growing aerobic, microaerophilic, or anaerobic.
  • the cells can be in any phase of growth, including lag (or conduction), exponential, transition, stationary, death, dormant, vegetative, sporulating, etc.
  • Organisms disclosed herein can be incorporated into methods and compositions of the invention so as to enhance fermentation end-product yield and/or rate of production.
  • One example of such a microorganism is Clostridium sp. Q.D, which can simultaneously hydrolyze and ferment lignocellulosic biomass.
  • Clostridium sp. Q.D is capable of hydro lyzing and fermenting hexose (C6) and pentose (C5) polysaccharides (e.g. carbohydrates).
  • C6 hexose
  • C5 pentose
  • Clostridium sp. Q.D is capable of acting directly on lignocellulosic biomass without any pretreatment.
  • Clostridium sp. Q.D can produce hemicellulases, pectinases, xylansases, or chitinases.
  • the invention provides for modified microorganisms which ferment hexose and pentose polysaccharides which are part of a biomass.
  • a Clostridium hydrolyzes and ferment hexose and pentose polysaccharides which are part of a biomass.
  • Clostridium sp. Q.D or variants thereof hydro lyze and ferment hexose and pentose polysaccharides which are part of a biomass.
  • the biomass comprises lignocellulose.
  • the biomass comprises hemicellulose.
  • Methods of the invention can also included co-culture with an microorganism that naturally produces or is genetically modified to produce one or more enzymes, such as hydrolytic enzymes (such as cellulase(s), hemicellulase(s), or pectinases etc.) or antioxidants (such as catalase, superoxide dismutase or glutathione peroxidase).
  • a culture medium containing such a microorganism can be contacted with biomass (e.g., in a bioreactor) prior to, concurrent with, or subsequent to contact with a second microorganism.
  • biomass e.g., in a bioreactor
  • a first microorganism produces saccharifying enzyme while a second microorganism ferments C5 and C6 sugars.
  • the first microorganism produces saccharifying enzyme while a second microorganism ferments C5 and C6 sugars.
  • the first microorganism produces saccharifying enzyme while a second microorganism ferments C
  • microorganism is C. sp. Q.D.
  • Mixtures of microorganisms can be provided as solid mixtures (e.g., freeze-dried mixtures), or as liquid dispersions of the microorganisms, and grown in co-culture with a second microorganism.
  • Co-culture methods capable of use with the present invention are known, such as those disclosed in U.S. Patent Application Publication No. 20070178569, which is hereby incorporated by reference in its entirety.
  • fuel or “biofuel” as used herein has its ordinary meaning as known to those skilled in the art and can include one or more compounds suitable as liquid fuels, gaseous fuels, biodiesel fuels (long-chain alkyl (methyl, propyl or ethyl) esters), heating oils (hydrocarbons in the 14-20 carbon range), reagents, chemical feedstocks and includes, but is not limited to, hydrocarbons (both light and heavy), hydrogen, methane, hydroxy compounds such as alcohols (e.g. ethanol, butanol, propanol, methanol, etc.), and carbonyl compounds such as aldehydes and ketones (e.g. acetone, formaldehyde, 1- propanal, etc.).
  • hydrocarbons both light and heavy
  • hydrogen methane
  • hydroxy compounds such as alcohols (e.g. ethanol, butanol, propanol, methanol, etc.)
  • carbonyl compounds such as aldehydes and ketones (e
  • fertilization end-product or “end-product” as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biofuels,or chemicals,(such as additives, processing aids, food additives, organic acids (e.g. acetic, lactic, formic, citric acid etc.), derivatives of organic acids such as esters (e.g. wax esters, glycerides, etc.) or other compounds).
  • biofuels e.g. acetic, lactic, formic, citric acid etc.
  • derivatives of organic acids e.g. wax esters, glycerides, etc.
  • end-products include, but are not limited to, an alcohol (such as ethanol, butanol, methanol, 1 , 2- propanediol, or 1, 3 -propanediol), an acid (such as lactic acid, formic acid, acetic acid, succinic acid, or pyruvic acid), enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases and can be present as a pure compound, a mixture, or an impure or diluted form.
  • a fermentation end-product is made using a process or microorganism disclosed herein.
  • production of a fermentation end-product is enhanced through saccharification and fermentation using enzyme- enhancing products or processes.
  • a fermentation end-product is a 1,4 diacid (succinic, fumaric and malic), 2,5 furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3 -hydroxybutyro lactone, glycerol, sorbitol, xylitol/arabitol, butanediol, butanol, isopentenyl diphosphate, methane, methanol, ethane, ethene, ethanol, n-propane, 1 -propene, 1 - propanol, propanal, acetone, propionate, n-butane, 1 -butene, 1 -butanol, butanal, butanoate, isobutanal, isobutanol, 2-methylbutanal, 2-methylbutanol, 3-methylbutanal
  • one or more modification of conditions for hydrolysis and/or fermentation is implemented to enhance end-product production. Examples of such
  • modifications include genetic modification to enhance enzyme activity in a microorganism that already comprises genes for encoding one or more target enzymes, introducing one or more heterogeneous nucleic acid molecules into a host microorganism to express and enhance activity of an enzyme not otherwise expressed in the host, modifying physical and chemical conditions to enhance enzyme function (e.g., modifying and/or maintaining a certain temperature, pH, nutrient concentration, temporal), or a combination of one or more such modifications.
  • Other embodiments include overexpression of an endogenous nucleic acid molecule into the host microorganism to express and enhance activity of an enzyme already expressed in the host or to express activity of an enzyme in the host when the enzyme would not normally be expressed in the naturally- occurring host microorganism.
  • a microorganism can be genetically modified to enhance enzyme activity of one or more enzymes, including but not limited to hydrolytic enzymes (such as cellulase(s), hemicellulase(s), or pectinase(s) etc.).
  • hydrolytic enzymes such as cellulase(s), hemicellulase(s), or pectinase(s) etc.
  • a method is used to genetically modify a microorganism (such as a Clostridium species) that is disclosed in US 20100086981 or
  • an enzyme can be selected from the annotated genome of C. phytofermentans, another bacterial species, such as B. subtilis, E. coli, various Clostridium species, or yeasts such as S. cerevisiae for utilization in products and processes described herein. Examples include enzymes such as L- butanediol dehydrogenase, acetoin reductase, 3-hydroxyacyl-CoA dehydrogenase, cis-aconitate decarboxylase or the like, to create pathways for new products from biomass.
  • modifications include modifying endogenous nucleic acid regulatory elements to increase expression of one or more enzymes ⁇ e.g., operably linking a gene encoding a target enzyme to a strong promoter), introducing into a microorganism additional copies of endogenous nucleic acid molecules to provide enhanced activity of an enzyme by increasing its production, and operably linking genes encoding one or more enzymes to an inducible promoter or a combination thereof.
  • a microorganism in another embodiment can be modified to enhance an activity of one or more hydrolytic enzymes (such as cellulase(s), hemicellulase(s), or pectinases etc.) or antioxidants (such as catalase), or other enzymes associated with cellulose processing.
  • hydrolytic enzymes such as cellulase(s), hemicellulase(s), or pectinases etc.
  • antioxidants such as catalase
  • various microorganisms of the invention can be modified to enhance activity of one or more cellulases, or enzymes associated with cellulose processing.
  • a hydrolytic enzyme is selected from the annotated genome of C.
  • the hydrolytic enzyme is an endoglucanase, chitinase, cellobiohydrolase or endo-processive cellulases (either on reducing or non-reducing end).
  • a microorganism such as C. phytofermentans
  • a microorganism can be modified to enhance production of one or more hydrolytic enzymes (such as cellulase(s), hemicellulase(s), or pectinases etc.) or antioxidants (such as catalase), or other enzymes associated with cellulose processing such as one disclosed in U.S. Patent Application Serial No. 12/510,994, which is herein incorporated by reference in its entirety.
  • one or more enzymes can be heterologous expressed in a host ⁇ e.g., a bacteria or yeast).
  • bacteria or yeast can be modified through recombinant technology (e.g., Brat ei al. Appl. Env. Microbio. 2009; 75(8):2304-2311, disclosing expression of xylose isomerase in S. cerevisiae and which is herein incorporated by reference in its entirety).
  • RM systems in bacteria serve as a defense mechanism against foreign nucleic acids.
  • bacterial RM systems are capable of attacking heterologous DNA through the use of enzymes such as DNA methyltransferase (MTase) and restriction endonuclease (REase).
  • MTase DNA methyltransferase
  • REase restriction endonuclease
  • bacterial MTases methylate DNA, creating a "self signal
  • REases restriction enzyme that enymatically cleave DNA that is not methylated, "foreign” DNA.
  • a vector comprising a heterologous DNA sequence is methylated prior to transformation into a Clostridium species.
  • methylation can be accomplished by the phi3TI methyltransferase.
  • plasmid DNA can be transformed into DI- ⁇ E. coli harboring vector pDHKM (Zhao, et al. Appl. Environ. Microbiol. 69: 2831-41 (2003)) carrying an active copy of the phi3TI methyltransferase gene.
  • the invention provides for a DNA sequence comprising genetic material from a first microorganism, wherein the DNA sequence comprises restriction enzyme sites that are not recognized by a second microorganism.
  • the DNA sequence encodes for a gene, or genetically modified variant of the gene, from a Clostridium species, for example C. sp. Q.D.
  • the DNA sequence encodes for an expression product that is a protein, or fragment thereof, from a Clostridium species, for example C. sp. Q.D.
  • the first microorganism is a Clostridium species and the second microorganism is bacteria or yeast, e.g. E. coli.
  • the host microorganism can further comprise an additional heterologous DNA segment, the expression product of which is a protein involved in the transport of mono- and/or oligosaccharides into the recombinant host.
  • additional genes from the glycolytic pathway can be incorporated into the host. In such ways, an enhanced rate of ethanol production can be achieved.
  • promoters can be used to drive expression of the heterologous genes in a recombinant host microorganism.
  • Promoter elements can be selected and mobilized in a vector (e.g., pIMPCphy).
  • a transcription regulatory sequence is operably linked to gene(s) of interest (e.g., in a expression construct).
  • the promoter can be any array of DNA sequences that interact specifically with cellular transcription factors to regulate transcription of the downstream gene. The selection of a particular promoter depends on what cell type is to be used to express the protein of interest.
  • a transcription regulatory sequences can be derived from the host microorganism.
  • constitutive or inducible promoters are selected for use in a host cell.
  • constitutive and inducible promoters which are known and that can be engineered to function in the host cell.
  • FIG. 24 A map of the plasmid pIMPCphy is shown in FIG. 24, and the DNA sequence of this plasmid is provided as SEQ ID NO: 2.
  • the vector pIMPCphy was constructed as a shuttle vector for C. phytofermentans and is further described in U.S. Patent Application Publication US20100086981 , which is herein incorporated by reference in its entirety. It has an Ampicillin-resistance cassette and an Origin of Replication (ori) for selection and replication in E.coli. It contains a Gram-positive origin of replication that allows the replication of the plasmid in C. phytofermentans. In order to select for the presence of the plasmid, the pIMPCphy carries an erythromycin resistance gene under the control of the C. phytofermentans promoter of the gene Cphyl 029. This plasmid can be transferred to C. phytofermentans by
  • pIMPCphy is an effective replicative vector system for all microorganisms, including all gram + and gram " bacteria, and fungi (including yeasts).
  • a microorganism can be modified to enhance an activity of one or more cellulases, or enzymes associated with cellulose processing.
  • the classification of cellulases is usually based on grouping enzymes together that forms a family with similar or identical activity, but not necessary the same substrate specificity.
  • One of these classifications is the CAZy system (CAZy stands for Carbohydrate- Active enZymes), for example, where there are 115 different Glycoside Hydrolases (GH) listed, named GH1 to GH155.
  • GH Glycoside Hydrolases
  • Each of the different protein families usually has a corresponding enzyme activity.
  • This database includes both cellulose and hemicellulase active enzymes.
  • the entire annotated genome of C phytofermentans is available on the worldwideweb at www.ncbi.nlm.nih.gov/sites/entrez.
  • cellulase enzymes whose function can be enhanced for expression endogenously or for expression heterologously in a microorganism include one or more of the genes disclosed in Table 2.
  • mutagenic agents for example, nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) or the like, to increase the mutation frequency above that of spontaneous mutagenesis.
  • a mutagenic agent for example, nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) or the like.
  • Techniques for inducing mutagenesis include, but are not limited to, exposure of the bacteria to a mutagenic agent, such as x-rays or chemical mutagenic agents. More sophisticated procedures involve isolating the gene of interest and making a change in the desired location, then reinserting the gene into bacterial cells. This is site-directed mutagenesis.
  • Directed evolution is usually performed as three steps which can be repeated more than once. First, the gene encoding a protein of interest is mutated and/or recombined at random to create a large library of gene variants. The library is then screened or selected for the presence of mutants or variants that show the desired property. Screens enable the identification and isolation of high-performing mutants by hand; selections automatically eliminate all non functional mutants. Then the variants identified in the selection or screen are replicated , enabling DNA sequencing to determine what mutations occurred. Directed evolution can be carried out in vivo or in vitro. See, for example, Otten, L.G.; Quax, W.J. (2005). Biomolecular Engineering 22 (1 -3): 1 -9; Yuan, L., et al. (2005) Microbiol. Mol. Biol. Rev. 69 (3): 373-392.
  • methods and compositions of the invention comprise genetically modifying a microorganism to enhance enzyme activity of one or more enzymes, including but not limited to a metabolic intermediate.
  • modifications include modifying endogenous nucleic acid regulatory elements to increase expression of one or more enzymes (e.g. , operably linking a gene encoding a target enzyme to a strong promoter), introducing into a microorganism additional copies of nucleic acid molecules to provide enhanced activity of an enzyme, operably linking genes encoding one or more enzymes to an inducible promoter or a combination thereof.
  • microorganisms of the invention can be modified to enhance activity of one or more enzymes to produce novel chemicals.
  • a microorganism other than Clostridium species can be modified to express and/or overexpress any of these enzymes.
  • other bacteria or yeast can be modified through conventional recombinant technology to express enzymes.
  • the host can further comprise an additional heterologous DNA segment, the expression product of which is a protein involved in the transport of mono- and/or oligosaccharides into the recombinant host.
  • additional genes from the glycolytic pathway can be incorporated into the host. In such ways, an enhanced rate of chemical production can be achieved.
  • a microorganism can be obtained without the use of recombinant DNA techniques that exhibit desirable properties such as increased productivity, increased yield, or increased titer.
  • mutagenesis, or random mutagenesis can be performed by chemical means or by irradiation of the microorganism.
  • the population of mutagenized microorganisms can then be screened for beneficial mutations that exhibit one or more desirable properties. Screening can be performed by growing the mutagenized microorganisms on substrates that comprise carbon sources that will be utilized during the generation of end-products by fermentation. Screening can also include measuring the production of end-products during growth of the microorganism, or measuring the digestion or assimilation of the carbon source(s).
  • the isolates so obtained can further be transformed with recombinant polynucleotides or used in combination with any of the methods and compositions provided herein to further enhance biofuel production.
  • production of a fermentation end-product comprises: a carbonaceous biomass, a microorganism that is capable of direct hydrolysis and fermentation of the biomass to a fermentation end-product disclosed herein.
  • a product for production of a biofuel comprises: a carbonaceous biomass, a microorganism that is capable of hydrolysis and fermentation of the biomass, wherein the microorganism is modified to provide enhanced production of a fermentation end-product disclosed herein.
  • a product for production of fermentation end-products comprises: (a) a fermentation vessel comprising a carbonaceous biomass; (b) and a modified microorganism that is capable of hydrolysis and fermentation of the biomass; wherein the fermentation vessel is adapted to provide suitable conditions for fermentation of one or more carbohydrates into fermentation end- products.
  • a microorganism utilized in products or processes of the invention can be one that is capable of hydrolysis and fermentation of C5 and C6 carbohydrates (such as lignocellulose or hemicelluloses). In one embodiment, such a capability is achieved through modifying the microorganism to express one or more genes encoding proteins associated with C5 and C6
  • Microorganisms useful in compositions and methods of the invention include but are not limited to bacteria, yeast or fungi that can hydrolyze and ferment feedstock or biomass. In some embodiments, two or more different microorganisms can be utilized during saccharification and/or fermentation processes to produce an end-product. Microorganisms utilized in methods and compositions described herein can be recombinant.
  • a microorganism utilized in compositions or methods of the invention is a strain of Clostridia.
  • the microorganism is C. sp. Q.D, or genetically modified variant thereof.
  • Organisms of the invention can be modified to comprise one or more heterologous or exogenous polynucleotides that enhance enzyme function.
  • enzymatic function is increased for one or more cellulase enzymes.
  • a microorganism used in products and processes of the invention can be capable of uptake of one or more complex carbohydrates from biomass (e.g., biomass comprises a higher concentration of oligomeric carbohydrates relative to monomeric carbohydrates).
  • biomass e.g., biomass comprises a higher concentration of oligomeric carbohydrates relative to monomeric carbohydrates.
  • one or more enzymes are utilized in products and processes of the invention, which are added externally (e.g., enzymes provided in purified form, cell extracts, culture medium or commercially available source).
  • Enzyme activity can also be enhanced by modifying conditions in a reaction vessel, including but not limited to time, pH of a culture medium, temperature, concentration of nutrients and/or catalyst, or a combination thereof.
  • a reaction vessel can also be configured to separate one or more desired end- products.
  • Products or processes of the invention provide for hydrolysis of biomass resulting in a greater concentration of cellobiose relative to monomeric carbohydrates.
  • monomeric carbohydrates can comprise xylose and arabinose.
  • batch fermentation with a microorganism of the invention and of a mixture of hexose and pentose saccharides using processes of the present invention provides uptake rates of about 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 2, 3, 4, 5, or about 6 g/L/h or more of hexose (e.g. glucose, cellulose, cellobiose etc.), and about 0.1, 0.2, 0.4, 0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or about 6 g/L/h or more of pentose (xylose, xylan, hemicellulose etc.).
  • hexose e.g. glucose, cellulose, cellobiose etc.
  • pentose xylose, xylan, hemicellulose etc.
  • Clostridium sp. Q.D. or variants thereof are capable of hydrolysis and fermentation of C5 and C6 sugars.
  • Biofuel plant and process of producing biofuel are Biofuel plant and process of producing biofuel
  • a fuel plant that includes a hydrolysis unit configured to hydrolyze a biomass material comprising a high molecular weight carbohydrate, and a fermentor configured to house a medium and one or more species of microorganisms.
  • the microorganism is Clostridium sp. Q.D.
  • a fuel or chemical end-product that includes combining a microorganism (such ⁇ Clostridium sp. Q.D or a similar species of Clostridium that hydro lyzes and ferments C5/C6 carbohydrates) and a lignocellulosic material (and/or other biomass material) in a medium, and fermenting the lignocellulosic material under conditions and for a time sufficient to produce a fermentation end-product, ⁇ e.g., ethanol, propanol, methane, or hydrogen).
  • a microorganism such ⁇ Clostridium sp. Q.D or a similar species of Clostridium that hydro lyzes and ferments C5/C6 carbohydrates
  • a lignocellulosic material and/or other biomass material
  • a process is provided for producing a fermentation end-product from biomass using acid hydrolysis pretreatment. In some embodiments, a process is provided for producing a fermentation end-product from biomass using enzymatic hydrolysis pretreatment. In another embodiment a process is provided for producing a fermentation end-product from biomass using biomass that has not been enzymatically pretreated. In another embodiment a process is provided for producing a fermentation end-product from biomass using biomass that has not been chemically or enzymatically pretreated, but is optionally steam treated.
  • Ethanologenic genes have been integrated chromosomally in E. coli B; see Ohta et al. (1991) Appl. Environ. Microbiol. 57:893-900. In general, this is accomplished by purification of a DNA fragment containing (1) the desired genes upstream from an antibiotic resistance gene and (2) a fragment of homologous DNA from the target microorganism. This DNA can be ligated to form circles without replicons and used for transformation.
  • the gene of interest can be introduced in a heterologous host such as E. coli, and short, random fragments can be isolated and ligated in
  • a fermentation end-product ⁇ e.g., ethanol) from biomass is produced on a large scale utilizing a microorganism, such as Clostridium sp. Q.D or variants thereof.
  • a biomass that includes high molecular weight carbohydrates is hydrolyzed to lower molecular weight carbohydrates, which are then fermented using a microorganism to produce ethanol.
  • the biomass is fermented without chemical and/or enzymatic pretreatment.
  • hydrolysis can be accomplished using acids, e.g., Bronsted acids ⁇ e.g., sulfuric or hydrochloric acid), bases, e.g., sodium hydroxide, hydrothermal processes, steam explosion, ammonia fiber explosion processes ("AFEX”), lime processes, enzymes, or combination of these.
  • Acids e.g., Bronsted acids ⁇ e.g., sulfuric or hydrochloric acid
  • bases e.g., sodium hydroxide
  • hydrothermal processes e.g., sodium hydroxide
  • steam explosion e.g., sodium hydroxide
  • AFEX ammonia fiber explosion processes
  • lime processes e.g., lime processes, enzymes, or combination of these.
  • Hydrogen, and other products of the fermentation can be captured and purified if desired, or disposed of, e.g., by burning.
  • the hydrogen gas can be flared, or used as an energy source in the process, e.g., to drive a steam boiler, e.g
  • Hydrolysis and/or steam treatment of the biomass can,increase porosity and/or surface area of the biomass, often leaving the cellulosic materials more exposed to the microorganismal cells, which can increase fermentation rate and yield.
  • removal of lignin can provide a combustible fuel for driving a boiler, and can also increase porosity and/or surface area of the biomass, often increasing fermentation rate and yield.
  • the initial concentration of the carbohydrates in the medium is greater than 20 mM, e.g., greater than 30 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, or even greater than 500 mM.
  • the invention features a fuel plant that comprises a hydrolysis unit configured to hydrolyze a biomass material that includes a high molecular weight carbohydrate; a fermentor configured to house a medium with a C5/C6 hydrolyzing and fermenting microorganism ⁇ e.g., Clostridium sp. Q.D or variants thereof); and one or more product recovery system(s) to isolate a fermentation end- product or end- products and associated by-products and co-products.
  • the invention features methods of making a fermentation end-product or end- products that include combining a C5/C6 hydrolyzing and fermenting microorganism (e.g., Clostridium sp.
  • a carbonaceous biomass in a medium, and fermenting the biomass material under conditions and for a time sufficient to produce a fermentation end-products (e.g. ethanol, propanol, hydrogen, lignin, terpenoids, and the like).
  • a fermentation end-product e.g. ethanol, propanol, hydrogen, lignin, terpenoids, and the like.
  • the fermentation end-product is a biofuel or chemical product.
  • the invention features one or more fermentation end-products made by any of the processes described herein.
  • one or more fermentation end-products can be produced from biomass on a large scale utilizing a C5/C6 hydrolyzing and fermenting microorganism (e.g., Clostridium sp. Q.D or variants thereof).
  • the process can comprise a milling of the carbonaceous material, via wet or dry milling, to reduce the material in size and increase the surface to volume ratio (physical modification).
  • the treatment includes treatment of a biomass with acid.
  • the acid is dilute.
  • the acid treatment is carried out at elevated temperatures of between about 85 and 140°C.
  • the method further comprises the recovery of the acid treated biomass solids, for example by use of a sieve.
  • the sieve comprises openings of approximately 150-250 microns in diameter.
  • the method further comprises washing the acid treated biomass with water or other solvents.
  • the method further comprises neutralizing the acid with alkali.
  • the method further comprises drying the acid treated biomass. In some embodiments, the drying step is carried out at elevated temperatures between about 15-45°C.
  • the liquid portion of the separated material is further treated to remove toxic materials.
  • the liquid portion is separated from the solid and then fermented separately.
  • a slurry of solids and liquids are formed from acid treatment and then fermented together.
  • FIG. 15 illustrates an example of a method for producing a fermentation end-product from biomass by first treating biomass with an acid at elevated temperature and pressure in a hydrolysis unit.
  • the biomass can first be heated by addition of hot water or steam.
  • the biomass can be acidified by bubbling gaseous sulfur dioxide through the biomass that is suspended in water, or by adding a strong acid, e.g., sulfuric, hydrochloric, or nitric acid with or without preheating/presteaming/water addition.
  • a weak organic acid can also be used.
  • the pH is maintained at a low level, e.g., below about 5.
  • the temperature and pressure can be elevated after acid addition.
  • a metal salt such as ferrous sulfate, ferric sulfate, ferric chloride, aluminum sulfate, aluminum chloride, magnesium sulfate, or mixtures of these can be added to aid in the hydrolysis of the biomass.
  • the acid-impregnated biomass is fed into the hydrolysis section of the pretreatment unit.
  • Steam is injected into the hydrolysis portion of the pretreatment unit to directly contact and heat the biomass to the desired temperature.
  • the temperature of the biomass after steam addition is, e.g., between about 130° C and 220° C.
  • the hydrolysate is then discharged into the flash tank portion of the pretreatment unit, and is held in the tank for a period of time to further hydrolyze the biomass, e.g., into oligosaccharides and monomeric sugars. Steam explosion can also be used to further break down biomass. Alternatively, the biomass can be subject to discharge through a pressure lock for any high-pressure pretreatment process. Hydrolysate is then discharged from the pretreatment reactor, with or without the addition of water, e.g., at solids concentrations between about 15% and 60%.
  • the biomass after pretreatment, can be dewatered and/or washed with a quantity of water, e.g. by squeezing or by centrifugation, or by filtration using, e.g. a countercurrent extractor, wash press, filter press, pressure filter, a screw conveyor extractor, or a vacuum belt extractor to remove acidified fluid.
  • the acidified fluid with or without further treatment, e.g. addition of alkali (e.g. lime) and or ammonia (e.g. ammonium phosphate), can be re-used, e.g., in the acidification portion of the pretreatment unit, or added to the fermentation, or collected for other use/treatment.
  • Products can be derived from treatment of the acidified fluid, e.g., gypsum or ammonium phosphate.
  • Enzymes or a mixture of enzymes can be added during pretreatment to assist, e.g. endoglucanases, exoglucanases, cellobiohydrolases (CBH), beta-glucosidases, glycoside hydrolases,
  • glycosyltransferases lyases, and esterases active against components of cellulose, hemicelluloses, pectin, and starch, in the hydrolysis of high molecular weight components.
  • the fermentor is fed with hydrolyzed biomass; any liquid fraction from biomass pretreatment; an active seed culture of Clostridium sp. Q.D or a mutagenized or genetically- modified variant thereof, optionally a co-fermenting microorganism (e.g., yeast or E. coli) and, as needed, nutrients to promote growth of the Clostridium cells or other microorganisms.
  • a co-fermenting microorganism e.g., yeast or E. coli
  • the pretreated biomass or liquid fraction can be split into multiple fermentors, each containing a different strain of Clostridium sp. Q.D, or a mutagenized or genetically-modified variant thereof and/or other microorganisms; with each fermentor operating under specific physical conditions.
  • Fermentation is allowed to proceed for a period of time, e.g., between about 15 and 150 hours, while maintaining a temperature of, e.g., between about 25° C and 50° C.
  • Gas produced during the fermentation is swept from fermentor and is discharged, collected, or flared with or without additional processing, e.g. hydrogen gas can be collected and used as a power source or purified as a co-product.
  • the contents of the fermentor are transferred to product recovery.
  • Products are extracted, e.g., ethanol is recovered through distillation and rectification.
  • Methods and compositions described herein can include extracting or separating fermentation end-products, such as ethanol, from biomass. Depending on the product formed, different methods and processes of recovery can be provided.
  • a method for extraction of lactic acid from a fermentation broth uses freezing and thawing of the broth followed by centrifugation, filtration, and evaporation.
  • Other methods that can be utilized are membrane filtration, resin adsorption, and crystallization. (See, e.g., Huh, et al. 2006 Process Biochemistry).
  • the process can take advantage of preferential partitioning of the product into one phase or the other. In some cases the product might be carried in the aqueous phase rather than the solvent phase.
  • the pH is manipulated to produce more or less acid from the salt synthesized from the microorganism. The acid phase is then extracted by vaporization, distillation, or other methods. (See FIG. 16.)
  • a system for production of fermentation end-products comprises:
  • a fermentation vessel comprising a carbonaceous biomass; (b) and a microorganism that is capable of hydrolysis and fermentation of the biomass; wherein the fermentation vessel is adapted to provide suitable conditions for fermentation of one or more carbohydrates into fermentation end-products.
  • the microorganism is genetically modified. In another embodiment the microorganism is not genetically modified.
  • FIG. 17 depicts a method for producing chemicals from biomass by charging biomass to a fermentation vessel.
  • the biomass can be allowed to soak for a period of time, with or without addition of heat, water, enzymes, or acid/alkali.
  • the pressure in the processing vessel can be maintained at or above atmospheric pressure. Acid or alkali can be added at the end of the pretreatment period for neutralization.
  • an active seed culture of a C5/C6 hydrolyzing and fermenting microorganism e.g., Clostridium sp. Q.D or variant thereof
  • a co-fermenting microorganism e.g., yeast or E.
  • a C5/C6 hydrolyzing and fermenting microorganism ⁇ e.g. , Clostridium sp. Q.D or variant thereof) can be used alone or
  • microorganisms e.g. yeasts, fungi, or other bacteria.
  • different methods can be used within a single plant to produce different end-products.
  • the invention features a fuel plant that includes a hydrolysis unit configured to hydrolyze a biomass material that includes a high molecular weight carbohydrate, a fermentor configured to house a medium and contains a C5/C6 hydrolyzing and fermenting microorganism ⁇ e.g., Clostridium sp. Q.D or mutagenized or genetically-modified cells thereof).
  • a hydrolysis unit configured to hydrolyze a biomass material that includes a high molecular weight carbohydrate
  • a fermentor configured to house a medium and contains a C5/C6 hydrolyzing and fermenting microorganism ⁇ e.g., Clostridium sp. Q.D or mutagenized or genetically-modified cells thereof).
  • the invention features a chemical production plant that includes a hydrolysis unit configured to hydrolyze a biomass material that includes a high molecular weight carbohydrate, a fermentor configured to house a medium and contains a C5/C6 hydrolyzing and fermenting microorganism (e.g., Clostridiu sp. Q.D or mutagenized or genetically-modified cells thereof).
  • a hydrolysis unit configured to hydrolyze a biomass material that includes a high molecular weight carbohydrate
  • a fermentor configured to house a medium and contains a C5/C6 hydrolyzing and fermenting microorganism (e.g., Clostridiu sp. Q.D or mutagenized or genetically-modified cells thereof).
  • the invention features methods of making a chemical(s) or fuel(s) that include combining a C5/C6 hydrolyzing and fermenting microorganism (e.g., Clostridium sp. Q.D or mutagenized or genetically-modified cells thereof ), and a lignocellulosic material (and/or other biomass material) in a medium, and fermenting the lignocellulosic material under conditions and for a time sufficient to produce a chemical(s) or fuel(s), e.g., ethanol, propanol and/or hydrogen or another chemical compound.
  • a chemical(s) or fuel(s) e.g., ethanol, propanol and/or hydrogen or another chemical compound.
  • the present invention provides a process for producing ethanol and hydrogen from biomass using acid hydrolysis pretreatment. In some embodiments, the present invention provides a process for producing ethanol and hydrogen from biomass using enzymatic hydrolysis pretreatment. Other embodiments provide a process for producing ethanol and hydrogen from biomass using biomass that has not been enzymatically pretreated. Still other embodiments disclose a process for producing ethanol and hydrogen from biomass using biomass that has not been chemically or enzymatically pretreated, but is optionally steam treated.
  • FIG. 18 discloses pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either, fermented separately or together.
  • FIG. 18A depicts a process (e.g., acid pretreatment) that produces a solids phase and a liquid phase which are then fermented separately.
  • FIG. 18B depicts a similar pretreatment that produces a solids phase and liquids phase.
  • the liquids phase is separated from the solids and elements that are toxic to the fermenting microorganism are removed prior to fermentation.
  • the two phases are recombined and cofermented together. This is a more cost-effective process than fermenting the phases separately.
  • the third process (FIG. 18C) is the least costly.
  • the pretreatment results in a slurry of liquids or solids that are then cofermented. There is little loss of saccharides component and minimal equipment required.
  • Clostridium sp. Q.D includes all strains, mutants and recombinants, and can in some embodiments be defined based on the phenotypic and genotypic characteristics of the cultured strain as described infra. Aspects described herein generally include systems, methods, and compositions for producing fuels, such as ethanol, and/or other useful organic products involving, for example,
  • Clostridium sp. Q.D and/or any other strain of the species including those which can be derived from Clostridium sp. Q.D, including genetically modified strains, or strains separately isolated.
  • Some exemplary species can be defined using standard taxonomic considerations (Stackebrandt and Goebel, International Journal of Systematic Bacteriology, 44:846-9, 1994): Strains with 16S rRNA sequence homology values of 97% and higher as compared to the type Clostridium sp. Q.D, and strains with DNA re-association values of at least about 70% can be considered Clostridium sp. Q.D.
  • strains with DNA re-association values of at least about 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% can be considered Clostridium sp. Q.D.
  • Analyses of assembled contigs from the sequence of Clostridium sp. Q.D indicate the presence of large numbers of genes and genetic loci that are likely to be involved in mechanisms and pathways for plant polysaccharide fermentation, giving rise to the unusual fermentation properties of this microorganism which can be found in all or nearly all strains of the species Clostridium sp. Q.D and can be natural isolates, or genetically modified strains.
  • an isolated Clostridium bacterium disclosed herein can comprise a 16S rRNA nucleotide sequence that is encoded in part by a nucleotide sequence with greater than at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97.1 , 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6,
  • Clostridium sp. Q.D genome was sequenced using the short-read (50mers) sequencing technology. These 20672042 short reads were then assembled by velvet, and produced 727 contigs (or nodes) that are longer than 2000 bases. The 727 contigs are collectively disclosed as SEQ ID NO: 1.
  • Velvet is a de novo genomic assembler, designed for short read sequencing technologies, developed at the European Bioinformatics Institue (EMBL-EBI). The velvet algorithm was published in the journal article "Velvet: algorithms for de novo short read assembly using de Bruijn graphs. D.R. Zerbino and E. Birney, Genome Research 18:821 -829", which is hereby incorporated by reference.
  • compositions of the invention comprise genes encoded by any of the nodes listed in Table 3 : [00274] Table 3
  • GH 9 Cellulose 1 ,4-beta-cellobiosidase 38713 to 40617*
  • an isolated Clostridium bacterium disclosed herein can comprise a 16S rRNA nucleotide sequence that is encoded in part by a nucleotide sequence with greater than at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97.1 , 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% identity to SEQ ID NO: 13.
  • an isolated Clostridium bacterium disclosed herein can comprise one or more nucleotide sequences comprising at a significant level of identity to SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.
  • a significant amount can be at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97.1 , 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98.0, 98.1 , 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% identity to a region of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40 ,41 , or 42.
  • a nucleotide sequence in this context, can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more nucleotides in length.
  • An isolated Clostridium bacterium disclosed herein e.g. Clostridium sp.
  • Q.D or variants thereof can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotide sequences with significant identity to a region of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40 ,41 , or 42.
  • a nucleotide sequence can encode a protein or protein fragment.
  • an isolated Clostridium bacterium disclosed herein can comprise one or more nucleotide sequences comprising at a significant level of identity to SEQ ID NO: 1.
  • a significant amount can be at least 80, 81, 82, 83,
  • a nucleotide sequence in this context, can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more nucleotides in length.
  • An isolated Clostridium bacterium disclosed herein can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotide sequences with significant identity to a region of SEQ ID NO: 1.
  • a nucleotide sequence can encode a protein or protein fragment.
  • an isolated Clostridium bacterium disclosed herein can comprise a 16S rRNA nucleotide sequence that is encoded in part by a nucleotide sequence with greater than at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97.1 , 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% identity to SEQ ID NO: 13 and 1 , 2, 3, 4, 5, 6, 7,
  • an isolated Clostridium bacterium disclosed herein can comprise a 16S rRNA nucleotide sequence that is encoded in part by a nucleotide sequence with greater than at least 80, 81, 82, 83, 84,
  • a nucleotide sequence in any embodiment, can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more nucleotides in length.
  • a nucleotide sequence can encode a protein or protein fragment.
  • An isolated Clostridium bacterium disclosed herein can be genetically modified.
  • an isolated Clostridium bacterium e.g. Clostridium sp.
  • Q.D or variants thereof can be genetically modified to express one or more heterologous genes.
  • an isolated Clostridium bacterium disclosed herein can be genecially modified to inhibit the expression of one or more endogenous genes.
  • an isolated Clostridium bacterium disclosed herein can be genetically modified to enhance the activity of one or more endogenous enzymes.
  • the microorganism Clostridium sp. Q.D, provides useful advantages for the conversion of biomass to ethanol and other products.
  • One advantage of this microorganism is its ability to produce enzymes capable of hydrolyzing polysaccharides and higher molecular weight saccharides to lower molecular weight saccharides, such as oligosaccharides, disaccharides, and monosaccharides.
  • Clostridium sp. Q.D can produce a wide spectrum of hydrolytic enzymes, which can facilitate fermenting of various biomass materials, including cellulosic, hemicellulosic, lignocellulosic materials; pectins; starches; wood; paper; agricultural products; forest waste; tree waste; tree bark; leaves; grasses; sawgrass; woody plant matter; non-woody plant matter; carbohydrates; pectin; starch; inulin; fructans; glucans; corn; sugar cane; grasses; bamboo, algae, and material derived from these materials.
  • various biomass materials including cellulosic, hemicellulosic, lignocellulosic materials; pectins; starches; wood; paper; agricultural products; forest waste; tree waste; tree bark; leaves; grasses; sawgrass; woody plant matter; non-woody plant matter; carbohydrates; pectin; starch; inulin; fructans; glucans; corn; sugar cane;
  • the organism can usually produce these enzymes as needed, frequently without excessive production of unnecessary hydrolytic enzymes, or in one embodiment, one or more enzymes is added to further improve the organism's production capability.
  • This ability to produce a very wide range of hydrolytic enzymes gives Clostridium sp. Q.D and the associated technology distinct advantages in biomass fermentation, especially those fermentations not utilizing simple sugars as the feedstock.
  • Various fermentation conditions can enhance the activities of the organism, resulting in higher yields, higher productivity, greater product selectivity, and/or greater conversion efficiency.
  • fermentation conditions include fed batch operation and fed batch operation with cell augmentation; addition of complex nitrogen sources such as corn steep powder or yeast extract; addition of specific amino acids including proline, glycine, isoleucine, and/or histidine; addition of a complex material containing one or more of these amino acids; addition of other nutrients or other compounds such as phytate, proteases enzymes, or polysaccharase enzymes.
  • fermentation conditions can include supplementation of a medium with an organic nitrogen source.
  • fermentation conditions can include supplementation of a medium with an inorganic nitrogen source.
  • the addition of one material provides supplements that fit into more than one category, such as providing amino acids and phytate.
  • the Clostridium sp. Q.D organism is used to hydrolyze various higher saccharides (higher molecular weight) present in biomass to lower saccharides (lower molecular weight), such as in preparation for fermentation to produce ethanol, hydrogen, or other chemicals such as organic acids including formic acid, acetic acid, and lactic acid.
  • Another advantage of Clostridium sp. Q.D is its ability to hydrolyze polysaccharides and higher saccharides that contain hexose sugar units or that contain pentose sugar units, and that contain both, into lower saccharides and in some cases monosaccharides.
  • These enzymes and/or the hydrolysate can be used in fermentations to produce various products including fuels, and other chemicals.
  • Clostridium sp. Q.D Another advantage of Clostridium sp. Q.D is its ability to produce ethanol, hydrogen, and other fuels or compounds such as organic acids including acetic acid, formic acid, and lactic acid from lower sugars (lower molecular weight) such as monosaccharides. Another advantage of Clostridium sp. Q.D is its ability to perform the combined steps of hydrolyzing a higher molecular weight biomass containing sugars and/or higher saccharides or polysaccharides to lower sugars and fermenting these lower sugars into desirable products including ethanol, hydrogen, and other compounds such as organic acids including formic acid, acetic acid, and lactic acid.
  • Clostridium sp. Q.D Another advantage of Clostridium sp. Q.D is its ability to grow under conditions that include elevated ethanol concentration, high sugar concentration, low sugar concentration, utilize insoluble carbon sources, and/or operate under anaerobic conditions. These characteristics, in various combinations, can be used to achieve operation with long fermentation cycles and can be used in combination with batch fermentations, fed batch fermentations, self-seeding/partial harvest fermentations, and recycle of cells from the final fermentation as inoculum.
  • the enzymes of the method are produced by Clostridium sp. Q.D, including a range of hydrolytic enzymes suitable for the biomass materials used in the fermentation methods.
  • Clostridium sp. Q.D is grown under conditions appropriate to induce and/or promote production of the enzymes needed for the saccharification of the polysaccharide present.
  • the production of these enzymes can occur in a separate vessel, such as a seed fermentation vessel or other fermentation vessel, or in the production fermentation vessel where ethanol production occurs.
  • the enzymes are produced in a separate vessel, they can, for example, be transferred to the production fermentation vessel along with the cells, or as a relatively cell free solution liquid containing the intercellular medium with the enzymes.
  • the enzymes When the enzymes are produced in a separate vessel, they can also be dried and/or purified prior to adding them to the production fermentation vessel.
  • the conditions appropriate for production of the enzymes are frequently managed by growing the cells in a medium that includes the biomass that the cells will be expected to hydrolyze in subsequent fermentation steps. Additional medium components, such as salt supplements, growth factors, and cofactors including, but not limited to phytate, amino acids, and peptides can also assist in the production of the enzymes utilized by the microorganism in the production of the desired products.
  • a method of fermenting a carbonaceous material comprising contacting the carbonaceous material with an effective, fermenting amount of an isolated Gram- positive bacterium, wherein the bacterium is an anaerobic, obligate mesophile that produces colonies that are beige or orange pigmented, wherein the bacterium can use polysaccharides as a sole carbon source and can reduce acetaldehyde into ethanol, whereby contacting the carbonaceous material with the bacterium ferments the carbonaceous material.
  • an "effective amount" is within the knowledge of one skilled in the art.
  • the bacterium comprises a 16S rRNA nucleic acid identified in FIG. 2. In another aspect, the bacterium comprises a 16S rRNA nucleic acid that is greater than about 98% similar to the nucleic acid identified in FIG. 2. In yet another aspect, the bacterium comprises a 16S rRNA nucleic acid that is greater than about 99% similar to the nucleic acid identified in FIG. 2. In one aspect, the bacterium comprises a 16S rRNA nucleic acid identified as in FIG. 2.
  • the contacting step of the disclosed method can occur at a pH of from about 5.0 to about 7.5. In one aspect, the contacting step occurs at a pH of from about 6.0 to about 6.5.
  • the method disclosed can be carried out at a temperature from about 30° C. to about 40° C. In another aspect, the disclosed method can be carried out at a temperature from about 35° C. to about 37° C.
  • a method of growing an isolated Gram-positive bacterium designated Q.D and deposited under NRRL Accession No. B-50361.
  • the bacterium designated Q.D and deposited under NRRL Accession No. B-50362, NRRL Accession No. B-50363, or designated C. phytofermentans and deposited under NRRL Accession No. B-50364.
  • the bacterium is an anaerobic, obligate mesophile that produces colonies that are beige-pigmented, wherein the bacterium can use cellulose as a sole carbon source and can oxidize acetaldehyde into ethanol, comprising culturing the bacterium at a temperature and on a medium effective to promote growth of the bacterium.
  • the bacterium can grow at a temperature from about 30° C. to about 40° C. In one aspect, the bacterium can grow at a temperature from about 35° C. to about 37° C. Further, the bacterium can grow on medium wherein the pH is from about 5.0 to about 7.5. In one aspect, the pH of the medium can be from about 6.0 to about 6.5.
  • Media are currently known that are effective in promoting growth of the disclosed bacterium.
  • methods are provided for the recovery of the fermentation end products, such as an alcohol ⁇ e.g. ethanol, propanol, methanol, butanol, etc.) another biofuel or chemical product.
  • broth will be harvested at some point during of the fermentation, and fermentation end product or products will be recovered.
  • the broth with ethanol to be recovered will include both ethanol and impurities.
  • the impurities include materials such as water, cell bodies, cellular debris, excess carbon substrate, excess nitrogen substrate, other remaining nutrients, non-ethanol metabolites, and other medium components or digested medium components.
  • the broth can be heated and/or reacted with various reagents, resulting in additional impurities in the broth.
  • the processing steps to recover ethanol frequently includes several separation steps, including, for example, distillation of a high concentration ethanol material from a less pure ethanol-containing material.
  • the high concentration ethanol material can be further concentrated to achieve very high concentration ethanol, such as 98% or 99% or 99.5% (wt.) or even higher.
  • Other separation steps, such as filtration, centrifugation, extraction, adsorption, etc. can also be a part of some recovery processes for ethanol as a product or biofuel, or other biofuels or chemical products.
  • a process can be scaled to produce commercially useful biofuels.
  • Clostridium sp. Q.D is used to produce an alcohol, e.g., ethanol, butanol, propanol, methanol, or a fuel such as hydrocarbons hydrogen, methane, and hydroxy compounds.
  • Clostridium sp. Q.D is used to produce a carbonyl compound such as an aldehyde or ketone ⁇ e.g. acetone, formaldehyde, 1 -propanal, etc.), an organic acid, a derivative of an organic acid such as an ester ⁇ e.g.
  • wax ester 1 , 2-propanediol, 1 , 3 -propanediol, lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid, or an enzyme such as a cellulase, polysaccharase, lipases, protease, ligninase, and hemicellulase.
  • a fed-batch fermentation for production of fermentation end product is described.
  • a fed-batch fermentation for production of ethanol is described.
  • Fed-batch culture is a kind of microbial process in which medium components, such as carbon substrate, nitrogen substrate, vitamins, minerals, growth factors, cofactors, etc. or biocatalysts
  • Clostridium sp. Q.D can be used in long or short fermentation cycles, it is particularly well-suited for long fermentation cycles and for use in fermentations with partial harvest, self-seeding, and broth recycle operations due to the anaerobic conditions of the fermentation, the presence of alcohol, the very fast growth rate of the organism compared to other Clostridia, and, in one embodiment, the use of a solid carbon substrate, whether or not resulting in low sugar concentrations in the broth.
  • a fermentation to produce ethanol is performed by culturing a strain of Clostridium sp. Q.D in a medium having a high concentration of one or more carbon sources, and/or augmenting the culture with addition of fresh cells of Clostridium sp. Q.D during the course of the fermentation.
  • the resulting production of ethanol can be up to 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, and in some cases up to 10-fold and higher in volumetric productivity than a batch process and achieve a carbon conversion efficiency approaching the theoretical maximum.
  • the theoretical maximum can vary with the substrate and product.
  • the generally accepted maximum efficiency for conversion of glucose to ethanol is 0.51 g ethanol/g glucose.
  • Clostridium sp. Q.D can produce about 40-100% of a theoretical maximum yield of ethanol.
  • Clostridium sp. Q.D can produce up to about 40% of the theoretical maximum yield of ethanol.
  • Clostridium sp. Q.D can produce up to about 50%> of the theoretical maximum yield of ethanol.
  • Clostridium sp. Q.D can produce about 70%) of the theoretical maximum yield of ethanol.
  • Clostridium sp. Q.D can produce about 90%> of the theoretical maximum yield of ethanol.
  • Clostridium sp. Q.D can produce about 95% of the theoretical maximum yield of ethanol. In another embodiment, Clostridium sp. Q.D can produce about 95% of the theoretical maximum yield of ethanol. In another embodiment, Clostridium sp. Q.D can produce about 99% of the theoretical maximum yield of ethanol. In another embodiment, Clostridium sp. Q.D can produce about 100% of the theoretical maximum yield of ethanol. In one embodiment Clostridium sp.
  • Q.D can produce up to about 1 %>, 2 %>, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 31 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, 50 %, 51 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %, 58 %, 59 %, 60 %,
  • Clostridium sp. Q.D cells used for the seed inoculum or for cell augmentation can be prepared or treated in ways that relate to their ability to produce enzymes useful for hydrolyzing the components of the production medium.
  • the cells can produce useful enzymes after they are transferred to the production medium or production fermentor.
  • Clostridium sp. Q.D cells can have already produced useful enzymes prior to transfer to the production medium or the production fermentor.
  • Clostridium sp. Q.D cells can be ready to produce useful enzymes once transferred to the production medium or the production fermentor, or Clostridium sp. Q.D cells can have some combination of these enzyme production characteristics.
  • the seed can be grown initially in a medium containing a simple sugar source, such as corn syrup, and then transitioned to the production medium carbon source prior to transfer to the production medium.
  • a simple sugar source such as corn syrup
  • the seed is grown on a combination of simple sugars and production medium carbon source prior to transfer to the production medium.
  • the seed is grown on the production medium carbon source from the start.
  • the seed is grown on one production medium carbon source and then transitioned to another production medium carbon source prior to transfer to the production medium.
  • the seed is grown on a combination of production medium carbon sources prior to transfer to the production medium.
  • the seed is grown on a carbon source that favors production of hydrolytic enzymes with activity toward the components of the production medium.
  • a fermentation to produce ethanol is performed by culturing a strain of Clostridium sp. Q.D microorganism and adding fresh medium components and fresh Clostridium sp. Q.D cells while the cells in the fermentor are growing.
  • Medium components such as carbon, nitrogen, and combinations of these, can be added as disclosed herein, as well as other nutrients, including vitamins, factors, cofactors, enzymes, minerals, salts, and such, sufficient to maintain an effective level of these nutrients in the medium.
  • the medium and Clostridium sp. Q.D can be added simultaneously, or one at a time.
  • Q.D cells can be added when hydrolytic enzyme activity decreases, especially when the activity of those hydrolytic enzymes that are more sensitive to the presence of alcohol decreases.
  • a nitrogen feed or a combination of nitrogen and carbon feed and/or other medium components can be fed, prolonging the enzymatic production or other activity of the cells.
  • the cells can be added with sufficient carbon and nitrogen to prolong the enzymatic production or other activity of the cells sufficiently until the next addition of fresh cells.
  • fresh Clostridium sp. Q.D cells can be added when both the nitrogen level and carbon level present in the fermentor increase.
  • Q.D cells can be added when the viable cell count decreases, especially when the nitrogen level is relatively stable or increasing.
  • fresh cells can be added when a significant portion of the viable cells are in the process of sporulation, or have sporulated.
  • Q.D cells can be when the portion of cells in the process of sporulation or have sporulated is about 2% to about 100%, about 10% to about 75%, about 20% to about 50%, or about 25% to about 30% of the cells are in the process of sporulation or have sporulated.
  • Clostridium sp. Q.D-5 or Clostridium sp. Q.D-7 cells or another sporulation mutant can be used in fermentation to continue the production of fermentation end products without the cells entering a resting state. In this instance, cells will continue to grow and divide, or just continue to metabolize without reproduction.
  • a fermentation to produce ethanol is performed by culturing a strain of Clostridium sp. Q.D microorganism and adding recycled Clostridium sp. Q.D cells while the cells in the fermentor are cell expansion stage ⁇ e.g. seed stage) and/or the final fermentation stage of a
  • the results described herein indicate that the recycled cells have a tolerance of higher ethanol concentrations and the ability to grow in such an environment. Thus, such a tolerance and ability can be useful for situations such as the cell expansion stage ⁇ e.g. seed stage) and the final fermentation stage of a fermentation where these concentrations of ethanol are present, including ethanol production fermentations, or for the production of other products in the presence of these concentrations of ethanol.
  • particular medium components can have beneficial effects on the performance of the fermentation, such as increasing the titer of desired products, or increasing the rate that the desired products are produced.
  • Specific compounds can be supplied as a specific, pure ingredient, such as a particular amino acid, or it can be supplied as a component of a more complex ingredient, such as using a microbial, plant or animal product as a medium ingredient to provide a particular amino acid, promoter, cofactor, or other beneficial compound.
  • the particular compound supplied in the medium ingredient can be combined with other compounds by the organism resulting in a fermentation-beneficial compound.
  • a medium ingredient provides a specific amino acid which the organism uses to make an enzyme beneficial to the fermentation.
  • Other examples can include medium components that are used to generate growth or product promoters, etc. In such cases, it can be possible to obtain a fermentation- beneficial result by supplementing the enzyme, promoter, growth factor, etc. or by adding the precursor. In some situations, the specific mechanism whereby the medium component benefits the fermentation is not known, only that a beneficial result is achieved.
  • beneficial fermentation results can be achieved by adding yeast extract.
  • the addition of the yeast extract can result in increased ethanol titer in batch fermentation, improved productivity and reduced production of side products such as organic acids.
  • beneficial results with yeast extract can be achieved at usage levels of about 0.5 to about 50 g/L, about 5 to about 30 g/L, or about 10 to about 30 g/L.
  • the yeast extract is used at level about 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L, 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6 g/L, 1.7 g/L, 1.8 g/L, 1.9 g/L, 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, 3.1 g/L, 3.2 g/L, 3.3 g/L, 3.4 g/L, 3.5 g/L, 3.6 g/L, 3.7 g/L, 3.8 g/L, 3.9 g/L, 4 g//L
  • the yeast extract can also be fed throughout the course of the entire fermentation or a portion of the fermentation, continuously or delivered at intervals.
  • usage levels include maintaining a nitrogen concentration of about 0.05 g/L to about 3g/L (as nitrogen), where at least a portion of the nitrogen is supplied from corn steep powder; or about 0.3g/L to 1.3g/L; or 0.4 g/L to about 0.9 g/L.
  • the nitrogen concentration is about 0.05 g/L, 0.06 g/L, 0.07 g/L, 0.08 g/L, 0.09 g/L, 0.1 g/L, 0.1 1 g/L, 0.12 g/L, 0.13 g/L, 0.14 g/L, 0.15 g/L, 0.16 g/L, 0.17 g/L, 0.18 g/L, 0.19 g/L, 0.2 g/L, 0.21 g/L, 0.22 g/L, 0.23 g/L, 0.24 g/L, 0.25 g/L, 0.26 g/L, 0.27 g/L, 0.28 g/L, 0.29 g/L, 0.3 g/L, 0.31 g/L, 0.32 g/L, 0.33 g/L, 0.34 g/L, 0.35 g/L, 0.36 g/L, 0.37 g/L, 0.38 g/L, 0.39 g/L,
  • beneficial fermentation results can be achieved by adding corn steep powder to the fermentation.
  • the addition of the corn steep powder can result in increased ethanol titer in batch fermentation, improved productivity and reduced production of side products such as organic acids.
  • beneficial results with corn steep powder can be achieved at usage levels of about 3 to about 20 g/L, about 5 to about 15 g/L, or about 8 to about 12 g/L.
  • beneficial results with steep powder can be achieved at a level of about 3 g/L, 3.1 g/L, 3.2 g/L, 3.3 g/L, 3.4 g/L, 3.5 g/L, 3.6 g/L, 3.7 g/L, 3.8 g/L, 3.9 g/L, 4 g/L, 4.1 g/L, 4.2 g/L, 4.3 g/L, 4.4 g/L, 4.5 g/L, 4.6 g/L, 4.7 g/L, 4.8 g/L, 4.9 g/L, 5 g/L, 5.1 g/L, 5.2 g/L, 5.3 g/L, 5.4 g/L, 5.5 g/L, 5.6 g/L, 5.7 g/L, 5.8 g/L, 5.9 g/L, 6 g/L, 6.1 g/L, 6.2 g/L, 6.3 g/L, 6.4 g/L
  • corn steep powder can also be fed throughout the course of the entire fermentation or a portion of the fermentation, continuously or delivered at intervals.
  • usage levels include maintaining a nitrogen concentration of about 0.05 g/L to about 3g/L (as nitrogen), where at least a portion of the nitrogen is supplied from corn steep powder; about 0.3g/L to 1.3g/L; or about 0.4 g/L to about 0.9 g/L.
  • the nitrogen level is about 0.05 g/L, 0.06 g/L, 0.07 g/L, 0.08 g/L, 0.09 g/L, 0.1 g/L, 0.1 1 g/L, 0.12 g/L, 0.13 g/L, 0.14 g/L, 0.15 g/L, 0.16 g/L, 0.17 g/L, 0.18 g/L, 0.19 g/L, 0.2 g/L, 0.21 g/L, 0.22 g/L, 0.23 g/L, 0.24 g/L, 0.25 g/L, 0.26 g/L, 0.27 g/L, 0.28 g/L, 0.29 g/L, 0.3 g/L, 0.31 g/L, 0.32 g/L, 0.33 g/L, 0.34 g/L, 0.35 g/L, 0.36 g/L, 0.37 g/L, 0.38 g/L, 0.39 g/L,
  • corn steep liquor or corn steep solids.
  • the usage rate would be approximately the same as for corn steep solids on a solids basis.
  • the corn steep powder (or solids or liquor) is added in relation to the amount of carbon substrate that is present or that will be added.
  • beneficial amounts of corn steep powder (or liquor or solids) can include about 1 : 1 to about 1 :6 g/g carbon, about 1 : 1 to about 1 :5 g/g carbon, or about 1 :2 to about 1 :4 g/g carbon.
  • ratios as high as about 1.5: 1 g/g carbon or about 3 : 1 g/g carbon or as low as about 1 : 8 g/g carbon or about 1 : 10 g/g carbon are used.
  • the ratio is 2: 1 g/g carbon, 1.9: 1 g/g carbon, 1.8: 1 g/g carbon, 1.7:1 g/g carbon, 1.6: 1 g/g carbon, 1.5: 1 g/g carbon, 1.4: 1 g/g carbon, 1.3 : 1 g/g carbon, 1.2: 1 g/g carbon, 1.1 : 1 g/g carbon, 1 : 1 g/g carbon, 1 : 1.1 g/g carbon, 1 : 1.2 g/g carbon, 1 : 1.3 g/g carbon, 1 : 1.4 g/g carbon, 1 : 1.5 g/g carbon, 1 : 1.6 g/g carbon, 1 : 1.7 g/g carbon, 1 : 1.8 g
  • beneficial fermentation results can be achieved by adding corn steep powder in combination with yeast extract to the fermentation.
  • Beneficial results with corn steep powder in combination with yeast extract can be achieved at corn steep powder usage levels of about 3 to about 20 g/L, about 5 to about 15 g/L, or about 8 to about 12 g/L and yeast extract usage levels of about 3 to 50 g/L, about 5 to about 30 g/L, or about 10 to about 30 g/L.
  • the corn steep powder and yeast extract can also be fed throughout the course of the entire fermentation or a portion of the fermentation, continuously or delivered at intervals.
  • the beneficial compounds from corn steep powder and/or yeast extract such as glycine, histidine, isoleucine, proline, or phytate as well as combinations of these compounds can be added to the medium or broth to obtain a beneficial effect.
  • Various embodiments offer benefits relating to improving the titer and/or productivity of alcohol production by Clostridium sp. Q.D by culturing the organism in a medium comprising one or more compounds comprising particular fatty acid moieties and/or culturing the organism under conditions of controlled pH.
  • Production of high levels of alcohol requires both the ability for the organism to thrive generally in the presence of elevated alcohol levels and the ability to continue to produce alcohol without undue inhibition or suppression by the alcohol and/or other components present.
  • different metabolic pathways will be implicated for each of these.
  • pathways related to cell growth generally include those related to protein production, membrane production as well as the production of all of the cellular subsystems necessary for the cell to survive.
  • Pathways related to alcohol production will frequently be more specific, such as those pathways related to the metabolism of sugars leading to production of alcohol and the enzymes that are necessary for the production of alcohol and intermediates.
  • the pathway for one alcohol e.g., ethanol, can share some similar enzymes, etc., but will also have enzymes and substrates unique to that pathway.
  • alcohol intolerance or alcohol-induced toxicity can be related to permeabilization of the cell membrane by elevated levels of alcohol, leading to leakage of intracellular enzymes and nutrients.
  • alcohol tolerance and the ability to produce high alcohol titers is related to the ability of intracellular enzymes to withstand denaturing by the alcohol present, e.g., within the cell, whether due to production by the cell itself or from transport across the cell membrane.
  • a more robust membrane will allow a higher alcohol gradient to be present across the membrane, thus allowing the cells to grow and/or continue to produce alcohol at higher external alcohol concentrations.
  • Clostridium sp. Q.D is fermented with a substrate at about pH 5-8.5
  • a Clostridium sp. Q.D is fermented at pH of about 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5.
  • methods of producing alcohol comprising culturing
  • Clostridium sp. Q.D in a medium under conditions of controlled pH.
  • a culture of Clostridium sp. Q.D can be grown at an acidic pH are provided herein.
  • the medium that the culture is grown in can include a carbon source such as agricultural crops, crop residues, trees, wood chips, sawdust, paper, cardboard, or other materials containing cellulose, hemicellulosic, lignocellulose, pectin, polyglucose, polyfructose, and/or hydrolyzed forms of these (collectively, "Feedstock").
  • Additional nutrients can be present including sulfur- and nitrogen-containing compounds such as amino acids, proteins, hydrolyzed proteins, ammonia, urea, nitrate, nitrite, soy, soy derivatives, casein, casein derivatives, milk powder, milk derivatives, whey, yeast extract, hydrolyze yeast, autolyzed yeast, corn steep liquor, corn steep solids, monosodium glutamate, and/or other fermentation nitrogen sources, vitamins, cofactors and/or mineral supplements.
  • the Feedstock can be pretreated or not, such as described in U.S. Provisional Patent Application No. 61/032048, filed February 27, 2008 or U.S. Provisional Application No. 61/158,581 , filed on March 9, 2009, which are herein incorporated by reference in their entireties. .
  • the procedures and techniques for growing the organism to produce a fuel or other desirable chemical such as is described in incorporated U.S. Patent Application No.
  • the pH of the medium is controlled at less than about pH 7.2 for at least a portion of the fermentation.
  • the pH is controlled within a range of about pH 3.0 to about 7.1 or about pH 4.5 to about 7.1, or about pH 5.0 to about 6.3, or about pH 5.5 to about 6.3, or about pH 6.0 to about 6.5, or about pH 5.5 to about 6.9 or about pH 6.2 to about 6.7.
  • the pH can be controlled by the addition of a pH modifier.
  • a pH modifier is an acid, a base, a buffer, or a material that reacts with other materials present to serve to raise of lower the pH.
  • more than one pH modifier can be used, such as more than one acid, more than one base, one or more acid with one or more bases, one or more acids with one or more buffers, one or more bases with one or more buffers, or one or more acids with one or more bases with one or more buffers.
  • more than one pH modifiers are utilized, they can be added at the same time or at different times.
  • one or more acids and one or more bases can be combined, resulting in a buffer.
  • media components such as a carbon source or a nitrogen source can also serve as a pH modifier; suitable media components include those with high or low pH or those with buffering capacity.
  • Exemplary media components include acid- or base-hydrolyzed plant polysaccharides having with residual acid or base, AFEX treated plant material with residual ammonia, lactic acid, corn steep solids or liquor.
  • the pH modifier can be added as a part of the medium components prior to inoculation with Clostridium sp. Q.D. In one embodiment, the pH modifier can also be added after inoculation with the Clostridium sp. Q.D. In one embodiment, sufficient buffer capacity can be added to the seed fermentation by way of various pH modifiers and/or other medium components and/or metabolites to provide adequate pH control during the final fermentation stage. In other cases, pH modifier can be added only to the final fermentation stage. In still other cases, pH modifier can be added to both the seed stage and the final stage. In one embodiment, the pH is monitored throughout the fermentation and is adjusted in response to changes in the fermentation.
  • the pH modifier is added whenever the pH of the fermentation changes by a pH value of about 0.005, 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5 or more at any stage of the fermentation.
  • the pH modifier is added whenever the alcohol content of the fermentation is about 0.5 g/L, 1.0 g/L, 2.0 g/L, or 5.0 g/L or more.
  • different types of pH modifiers can be utilized at different stages or points in the fermentation, such as a buffer being used at the seed stage, and base and/or acid added in the final fermenter, or an acid being used at one time and a base at another time.
  • a constant pH can be utilized throughout the fermentation.
  • the timing and/or amount of pH reduction can be related to the growth conditions of the cells, such as in relation to the cell count, the alcohol produced, the alcohol present, or the rate of alcohol production.
  • the pH reduction can be made in relation to physical or chemical properties of the fermentation, such as viscosity, medium composition, gas production, off gas composition, etc.
  • Non-limiting examples of suitable buffers include salts of phosphoric acid, including monobasic, dibasic, and tribasic salts, mixtures of these salts and mixtures with the acid; salts of citric acid, including the various basic forms, mixtures and mixtures with the acid; and salts of carbonate.
  • Suitable acids and bases that can be used as pH modifiers include any liquid or gaseous acid or base that is compatible with the organism. Examples include ammonia, ammonium hydroxide, sulfuric acid, lactic acid, citric acid, phosphoric acid, sodium hydroxide, and HC1.
  • the selection of the acid or base can be influenced by the compatibility of the acid or base with equipment being used for fermentation.
  • both an acid addition, to lower pH or consume base, and a base addition, to raise pH or consume acid can be used in the same fermentation.
  • the timing and amount of pH modifier to add can be determined from a measurement of the pH of the contents of the fermentor, such as by grab sample or by a submerged pH probe, or it can be determined based on other parameters such as the time into the fermentation, gas generation, viscosity, alcohol production, titration, etc. In one embodiment, a combination of these techniques can be used.
  • the pH of the fermentation is initiated at a neutral pH and then is reduced to an acidic pH when the production of alcohol is detected.
  • the pH of the fermentation is initiated at an acidic pH and is maintained at an acidic pH until the fermentation reaches a stationary phase of growth.
  • a combination of adding a fatty acid comprising compound to the medium and fermenting at reduced pH can be used.
  • addition of a fatty acid such as a free fatty acid fulfills both techniques: adding a fatty acid compound and lowering the pH of the fermentation.
  • different compounds can be added to accomplish each technique. For example, a vegetable oil can be added to the medium to supply the fatty acid and then a mineral acid or an organic acid can be added during the fermentation to reduce the pH to a suitable level, as described above.
  • the methods and techniques described herein for each type of operation separately can be used together.
  • the operation at low pH and the presence of the fatty acid comprising compounds will be at the same time.
  • the presence of fatty acid comprising compounds will precede operation at low pH, and in one embodiment, operation at low pH will precede the addition of fatty acid comprising compounds.
  • the operation at low pH and the presence of the fatty acid will be prior to inoculation with Clostridium sp. Q.D.
  • the operation at low pH will be prior to inoculation with Clostridium sp. Q.D and the presence of the fatty acid will occur after or during to inoculation with Clostridium sp. Q.D.
  • the presence of the fatty acid will be prior to inoculation with Clostridium sp. Q.D and the operation at low pH will occur after or during to inoculation with Clostridium sp. Q.D. In one embodiment, the operation at low pH and the presence of the fatty acid will be after inoculation with Clostridium sp. Q.D. In one embodiment, the operation at low pH and the presence of the fatty acid will be at other stages of fermentation.
  • compositions and methods to produce a fuel such as one or more alcohols, e.g., ethanol, by the creation and use of a genetically modified Clostridium sp. Q.D are provided.
  • regulating fermentative biochemical pathways, expression of saccharolytic enzymes, or increasing tolerance of environmental conditions during fermentation of Clostridium sp. Q.D is provided.
  • Clostridium sp. Q.D is transformed with heterologous polynucleotides encoding one or more genes for the pathway, enzyme, or protein of interest.
  • Clostridium sp. Q.D is transformed to produce multiple copies of one or more genes for the pathway, enzyme, or protein of interest.
  • Clostridium sp. Q.D is transformed with heterologous polynucleotides encoding one or more genes encoding enzymes for the hydrolysis and/or fermentation of a hexose, wherein said genes are expressed at sufficient levels to confer upon the Clostridium sp. Q.D transformant the ability to produce ethanol at increased concentrations, productivity levels or yields compared to Clostridium sp. Q.D that is not transformed. In such ways, an enhanced rate of ethanol production can be achieved,
  • Clostridium sp. Q.D is transformed with heterologous polynucleotides encoding one or more genes encoding saccharolytic enzymes for the saccharification of a
  • the saccharolytic DNA can be native to the host, although more often the DNA will be foreign, and heterologous.
  • Advantageous saccharolytic genes include cellulolytic, xylanolytic, and starch-degrading enzymes such as cellulases, xylanases, and amylases.
  • the saccharolytic enzymes can be at least partially secreted by the host, or it can be accumulated substantially intracellularly for subsequent release.
  • intracellularly-accumulated enzymes which are thermostable can be released when desired by heat-induced lysis. Combinations of enzymes can be encoded by the heterologous DNA, some of which are secreted, and some of which are accumulated. Examples of a host expressing a saccharolytic enzyme can be found in U.S. Pat.
  • the host can further comprise an additional heterologous DNA segment, the expression product of which is a protein involved in the transport of mono- and/or oligosaccharides into the recombinant host.
  • additional genes from the glycolytic pathway can be incorporated into the host to redirect the bioenergetics of the ethanolic production pathways. In such ways, an enhanced rate of ethanol production can be achieved.
  • modifications can be made in transcriptional regulators, genes for the formation of organic acids, carbohydrate transporter genes, sporulation genes, genes that influence the formation/regenerate of enzymatic cofactors, genes that influence ethanol tolerance, genes that influence salt tolerance, genes that influence growth rate, genes that influence oxygen tolerance, genes that influence catabolite repression, genes that influence hydrogen production, genes that influence resistance to heavy metals, genes that influence resistance to acids or genes that influence resistance to aldehydes.
  • transcriptional regulators genes for the formation of organic acids, carbohydrate transporter genes, sporulation genes, genes that influence the formation/regenerate of enzymatic cofactors, genes that influence ethanol tolerance, genes that influence salt tolerance, genes that influence growth rate, genes that influence oxygen tolerance, genes that influence catabolite repression, genes that influence hydrogen production, genes that influence resistance to heavy metals, genes that influence resistance to acids or genes that influence resistance to aldehydes.
  • Ethanologenic genes have been integrated chromosomally in E. coli B; see Ohta et al. (1991) Appl. Environ. Microbiol. 57:893-900. In general, this is accomplished by purification of a DNA fragment containing (1) the desired genes upstream from an antibiotic resistance gene and (2) a fragment of homologous DNA from the target organism. This DNA can be ligated to form circles without replicons and used for transformation. Thus, the gene of interest can be introduced in a heterologous host such as E. coli, and short, random fragments can be isolated and ligated in
  • Strain Q.D was generated in a screening of anaerobic bacteria for increased cellulase expression in the presence of cellobiose. Cultures were plated onto QM plates with maltose and Azo- Carboxymethyl-cellulose (Azo-CMC). QM medium composition is shown in Table 4. Strains were then tested for their ability to produce ethanol from complex cellulosic material like corn stover. One of the strains derived from these screenings was named "Q.D” (hereinafter Clostridium sp. Q.D).
  • the seed propagation media was prepared according to Table 4 above. Base media, salts and substrates were degassed with nitrogen prior to autoclave sterilization. Following sterilization, 94 ml of base media was combined with 1ml of 100X salts and 5mls of 20X substrate to achieve a final concentrations. All additions were prepared anaerobically and aseptically.
  • Clostridium sp. Q.D forms moist, shiny, beige, opaque, irregular or undulate colonies.
  • the cells are entire, small, short rods, diplo or chains, motile, and form subterminal endospores.
  • FIG. 3 illustrates: (A) a picture of Clostridium sp. Q.D cells taken at logarithmic phase; (B) pictures of Clostridium sp. Q.D cells in stationary phase showing endospores; and (C) a photographic enlargement of Clostridium sp. Q.D cells in stationary phase, showing that spores are formed subterminally.
  • Clostridium sp. Q.D is able to grow on yeast extract as a sole carbon source and is able to utilize a wide variety of sugars, including cellobiose, glucose, fructose and rhamnose. Azo-CMC plate assays indicates that Clostridium sp. Q.D possesses cellulolytic capability as well as xylanolytic activity and it grows well on pretreated corn stover. Its major fermentation end products of biomass are ethanol and acetic acid in nearly identical quantities. [00333] Clostridium sp. Q.D was tested for growth at 35° C, 39° C and 42° C and in a pH range of 6.0 to 7.5. It grew best at 35° C; growth also occurred at 39° C, albeit slower and cultures did not reach the same cell density compared with those at 35°C. No growth was observed at 42° C.
  • Clostridium sp. Q.D Growth of Clostridium sp. Q.D was tested on Cellobiose, Starch, Lactose, Xylose, Rhamnose, Fucose, Glucose, Xylan, Maltose as well as on corn stover and Avicel. Clostridium sp. Q.D grows slowly, but to high density on yeast extract.
  • Example 4 Growth of Clostridium sp. Q.D compared to strains of Clostridium phytofermentans
  • Clostridium sp. Q.D demonstrated endoglucanase activity in the presence of specific carbon substrates, maltose and glucose.
  • WTQ C. phytofermentans
  • Q.8 and Q.I Clostridium sp. Q.D grows faster, and produces a larger colony and greater zone of clearing (sugar metabolism) than C. phytofermentans (FIG. 6).
  • Higher levels of glucose FIGG. 7 produced a color change in the colony to a more orange hue.
  • Example 5 Induction of cellulase activity on CMC agar
  • Clostridium sp. Q.D cultures were spotted on QM plates with 1% of the monosaccharides depicted in FIG. 8 and grown anaerobically at 35°C until the colony was between 5- 10mm in size, wherein an overlay of 0.3% CMC agar was poured and the plates incubated for lh at 35°C. The plates were stained with Congo red and destained with 1.0M NaCl to visualize activity of the cellulases expressed and secreted during growth.
  • the 16S rRNA gene sequence from Clostridium sp. Q.D was used to search against 99097 isolated bacterial 16S rRNA sequences from Ribosomal Database Project Release 10.
  • the BLAST result showed that Clostridium sp. Q.D only shared 90%> similarity to Clostridium phytofermentans, but was closer to Clostridium algidixylanolyticum starin SPL73 (99%>), Clostridium sp. U201 (99%>), and swine fecal bacterium strain RF3G-Cel2 (99%>). From the blast result, 100 species with at least 90%> similarity were selected.
  • FIG. 2 shows the BLAST results of the Clostridium sp. Q.D. 16S rRNA sequence (Query) compared to Clostridium sp. U201 gene for 16S rRNA, Clostridium algidixylanolyticum 16S rRNA, and Clostridium sp. Kas401-4 gene for 16S rRNA, as well as calculated percentage similarities for 16S rRNA gene sequences for Clostridium sp. nov. and these three other species of Clostridium. On this basis and the characteristics described supra that distinguish it from other known species of microorganisms, Clostridium sp. Q.D. was determined to be a new cellulolytic species of the genus Clostridium.
  • Example 8 Sporulation knock-out in Clostridium sp. Q.D and Clostridium phytofermentans.
  • a plasmid was constructed to inactivate the spoIID (Cphy3479) gene of C. phytofermentans and closely related species by integration of an antibiotic marker inside the ORF.
  • the plasmid incorporates an erythromycin resistance gene flanked by the upstream part and downstream sequences of the gene Cphy3479.
  • the knock-out strains produced using this plasmid demonstrate increased product formation and enhanced yield.
  • the plasmid was constructed based on the pGEM-T-Easy vector from Promega (Promega Corp., 2800 Woods Hollow Rd., Madison, WI 5371 1).
  • the plasmid (FIG. 9) contains an erythromycin resistance gene under the control of the Clostridium phytofermentans promoter S12 (ribosomal protein). It is flanked by two 500 bp region upstream and downstream of Cphy3479.
  • the plasmid also contains an oriT, which allows transfer to Clostridum phytofermentans by
  • FIGS. 10 and 11 The sequences necessary for generating the knockout by homologous recombination are shown in FIGS. 10 and 11. They were cloned into the pGEM-T easy plasmid to generate the plasmid pGEMspo2DS12EmJoriT. Specifically, the sequences are:
  • phytofermentans (genome position 291176-291317) (SEQ ID NO:5);
  • phytofermentans Cphy3479, (bases 1058-1584 of the gene) (SEQ ID NO:9).
  • Example 10 Electroporation Conditions for Clostridium sp. Q.D
  • Example 11 Electroporation
  • the plasmid was transformed into E.coli, purified and then transformed into Clostridium sp. Q.D by electroporation. All procedures were conducted anaerobically except centrifugation wherein the centrifuge tubes were sealed from the atmosphere.
  • the metal contacts of the electroporation cuvette were cleaned with a Kimwipe or other adsorbent material to ensure no trace of moisture was present.
  • Electroporation was conducted using a Gene Pulser XcellTM apparatus (BioRad, Inc.) at 1500-2500 V, 25 ⁇ , and 600 ohms.
  • the ideal time constant was in the interval of 0.8 ms to 1.8 ms.
  • the erythromycin resistant colonies were screened for double homologous recombination events obtained from the electroporation for sporulation-deficient mutants. Three recombinants were selected: two produced from Clostridium sp. Q.D ⁇ Clostridium sp. Q.D-5 and Clostridium sp. Q.D-7) and one from Clostridium phytofermentans (Clostridium phytofermentans Q.7D).
  • Clostridium sp. Q.D-5, Clostridium sp. Q.D-7, and Closteridium phytofermentans Q.7D were compared to Clostridium sp. Q.D on cellobiose as a carbon source (FIG. 13).
  • Closteridium phytofermentans Q.7D is labeled "Q.I”. Growth of the sporulation mutants was as good or better than that of the non-recombinant strain as measured by cell density.
  • Fermentation profiles were measured over five days using 5% corn stover as a biomass substrate (FIG. 14). Both recombinant strains of Clostridium sp. Q.D fermented corn stover well, hydrolyzing the polysaccharide substrate at a rate almost as fast as the parent strain. Slightly more acid (acetic acid) was produced in the knockout strains.
  • Fermentation conditions of varying pH were examined with Clostridium sp. Q.D using cellobiose (50 g/L initia i) as substrate. The pH was maintained at 6.5 for 48 hrs (FIG. 19), or 5.5 (FIG. 20), and without adjustment (FIG. 21). The systems were examined as to the effect on growth and fermentation performance. Fermentations were performed in BM (low -2.5 g/L yeast extract (YE)) with cellobiose, OD660 was monitored throughout, and final time points were analyzed for product.
  • BM low -2.5 g/L yeast extract (YE)
  • C. sp. Q.D demonstrated direct fermentation and use of organic nitrogen at pH 5.5 without pH maintenance, as the overall product yield in g/L exceeded the carbohydrate consumed.
  • Example 15 Synergistic Characteristics of C. sp. Q.D
  • Clostridium sp. Q.D is a Clostridium microorganism capable of fermentation of pentose and hexose polysaccharides.
  • the saccharification yield from NaOH pretreated corn stover solids was observed after contact with Clostridium sp. Q.D, either alone or with the addition of a cellulase mixture that produces a high amount of oligosaccharides.
  • Example 16 Propagation and Fermentation Media for C. phytofermentans and other mesophilic Clostridium species
  • Seed propagation media (QM1) recipe [00374] Seed propagation media (QM1) recipe:
  • Seed propagation media (BM) recipe :
  • Seed propagation medium was prepared according to the recipe above. Base media, salts and substrates were degassed with nitrogen prior to autoclave sterilization. Following sterilization, 87 ml of base media was combined with 10ml of lOx Substrate stock and 1ml each of 100X salts solution, lOOx amino acids. All additions were prepared anaerobically and aseptically.
  • Fermentation media (FM media)
  • Base media (g/L) was prepared with: 50g/L NaOH pretreated corn stover, yeast extract 10 g/L, corn steep powder 5 g/L, K 2 HP0 4 3 g/1, KH 2 P0 4 1.6 g/L, TriSodium citrate2H 2 0 2 2 g/L, Citric acid3 ⁇ 40 1.2 g/L, (NH 4 ) 2 S0 4 0.5 g/L, NaCl 1 g/L, Cysteine.HCl 1.0 g/L, dissolved in deionized water to achieve final volume, adjusted to pH to 6.5, degassed with nitrogen and autoclaved 121°C for 30 min.
  • the fermentation media was prepared according to the protocol above. Components of the Base media were degassed with nitrogen prior to sterilization. A 100X salts stock was prepared and sterilized separately. After sterilization base media was supplemented with a 1% v/v dose of 100X salts to achieve a final concentration. All additions were prepared anaerobically and aseptically.
  • Example 17 Microorganism Modification and Vector Construction
  • FIG. 27 A general illustration of an integrating replicative plasmid, pQInt, is shown in FIG. 27.
  • Identified elements include a Multi-cloning site (MCS) with a LacZ-a reporter for use in E. coli; a gram-positive replication origin; the homologous integration sequence; an antibiotic-resistance cassette; the ColEl gram-negative replication origin and the traJ origin for conjugal transfer.
  • MCS Multi-cloning site
  • Several unique restriction sites are indicated but are not meant to be limiting on any embodiment. The arrangement of the elements can be modified.
  • FIGS. 28 and 29 Another embodiment, depicted in FIGS. 28 and 29, is a map of the plasmids pQIntl and pQInt2. These plasmids contain gram-negative (ColEl) and gram-positive (repA/Orf2) replication origins; the bi-functional aad9 spectinomycin-resistance gene; traJ origin for conjugal transfer; LacZ- a/MCS and the 1606-1607 region of chromosomal homology. Since the 1606-1607 region of homology is cloned into a single Ascl site, it can be obtained in two different orientations in a single cloning step.
  • Plasmid pQInt2 is identical to pQIntl except the orientation of the homology region is reversed.
  • These plasmids consist of five key elements. 1) A gram-negative origin of replication for propagation of the plasmid in E. coli or other gram-negative host(s). 2) A gram-positive replication origin for propagation of the plasmid in gram-positive organisms. In C. phytofermentans, this origin allows for suitable levels of replication prior to integration. 3) A selectable marker; typically a gene encoding antibiotic resistance. 4) An integration sequence; a sequence of DNA at least 400 base pairs in length and identical to a locus in the host chromosome. This represents the preferred site of integration. 5) A multi-cloning site ("MCS") with or without a heterologous gene expression cassette cloned. An additional element for conjugal transfer of plasmid DNA is an optional element described in certain embodiments.
  • the plasmid is digested with suitable restriction enzyme(s) to ligate a heterologous gene expression cassette ("insert") into the MCS.
  • Ligation products are transformed into a suitable cloning host, typically E. coli.
  • Antibiotic resistant transformants are screened to verify the presence of the desired insert.
  • the plasmid is then transformed into C. phytofermentans or other suitable expression host strain.
  • Transformants are selected based on resistance to the appropriate antibiotic. Resistant colonies are propagated in the presence of antibiotic to allow for homologous recombination integration of the plasmid. Integration is verified by a "junction PCR" protocol. This protocol uses either a preparation of host chromosomal DNA or a sample of transformed cells.
  • the junction PCR utilizes one primer that hybridizes to the plasmid backbone flanking the MCS and a second primer that hybridizes to the chromosome flanking the site of integration.
  • the primers must be designed so they are unique. That is, the plasmid primer cannot hybridize to chromosomal sequences and the chromosomal primer cannot hybridize to the plasmid.
  • the ability to amplify a PCR product demonstrates integration at the correct site (see FIGS. 27-29).
  • Standard gene expression systems use autonomously replicating plasmids ("episomes” or “episomal plasmids”). Such plasmids are not suitable for use in C. phytofermentans, C. sp. Q.D. and most other Clostridia due to segregational instability.
  • the use of homologous sequences to allow for integration of a replicative gene expression in C. phytofermentans is not usual for transformation.
  • true suicide integration which utilizes non-replicating plasmids.
  • true suicide integration the only way to obtain an antibiotic resistant transformant is to have the plasmid integrate immediately after transformation. This is a low probability event. Replication from the gram-positive origin after transformation results in a greater number of transformed cells which makes the integration event statistically more likely.
  • the integrated plasmid is stable indefinitely.
  • the transformed strain can be indefinitely propagated without loss of plasmid DNA.
  • the transformant can be evaluated for heterologous gene expression under any suitable conditions. Stability of the integrated DNA can be ensured by continuous culture in the presence of the appropriate antibiotic. It is also possible to remove the antibiotic if so desired.
  • Plasmids suitable for use in Clostridium phytofermentans were constructed using pQInt with the promoter from the C. phytofermentans pyruvate ferredoxin oxidase reductase gene Cphy_3558 and the C phytofermentans cellulase gene Cphy_3202.
  • the sequence of this vector (pMTL82351-P3558- 3202) inserted DNA (SEQ ID NO: 10) is as follows:
  • promoters from C. phytofermentans were chosen for vector use that show high expression of their corresponding genes in all growth stages as well as on different substrates.
  • a promoter element is selected by selecting key genes that would necessarily be involved in constitutive pathways ⁇ e.g., ribosomal genes, or for ethanol production, alcohol dehydrogenase genes). Examples of promoters from such genes include but are not limited to:
  • Cphy l 029 iron-containing alcohol dehydrogenase
  • Cphy_3510 Ig domain-containing protein
  • Cphy_3925 bifunctional acetaldehyde-CoA/alcohol dehydrogenase
  • One or more genes disclosed (see Table 2), which can include each gene's own ribosome binding sites, were amplified via PCR and subsequently digested with the appropriate enzymes as described previously under Cloning of Promoter. Resulting plasmids were also treated with the corresponding restriction enzymes and the amplified genes are mobilized into plasmids through standard ligation. E.coli were transformed with the plasmids and correct inserts were verified from transformants selected on selection plates.
  • E. coli DH5a along with the helper plasmid pRK2030, were transformed with the different plasmids discussed above. E.coli colonies with both of the foregoing plasmids were selected on LB plates with 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin after growing overnight at 37°C. Single colonies were obtained after re-streaking on selective plates at 37°C. Growth media for E.coli ⁇ e.g. LB or LB supplemented with 1% glucose and 1% cellobiose) was inoculated with a single colony and either grown aerobically at 37°C or anaerobically at 35°C overnight. Fresh growth media was inoculated 1 :100 with the overnight culture and grown until mid log phase. A C. phytofermentans strain was also grown in the same media until mid log.
  • the bacteria mixture was either spread directly onto plates or first grown on liquid media for 6h to 18h and then plated.
  • the plates contain 10 ⁇ g/ml erythromycin as selective agent for C. phytofermentans and 10 ⁇ g/ml Trimethoprim, 150 ⁇ g/ml

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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne une nouvelle bactérie Clostridium Gram positif. La bactérie est un mésophile anaérobie, cellulolytique et xylanolytique qui produit des colonies pigmentées de beige. Elle peut utiliser la biomasse carbonée en tant que source de carbone et peut fermenter les sucres en éthanol, biocarburants et autres produits chimiques. L'invention concerne également des souches recombinantes de plusieurs espèces Clostridium dans lesquelles la sporulation est interrompue. L'invention concerne en outre des procédés d'utilisation de la bactérie et des souches pour dégrader la matière organique et pour une utilisation dans des processus industriels.
PCT/US2011/033832 2010-04-22 2011-04-25 Nouvelle bactérie pour la production de produits chimiques et ses recombinants WO2011133984A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32705110P 2010-04-22 2010-04-22
US61/327,051 2010-04-22

Publications (2)

Publication Number Publication Date
WO2011133984A2 true WO2011133984A2 (fr) 2011-10-27
WO2011133984A3 WO2011133984A3 (fr) 2012-05-10

Family

ID=44834851

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2011/033688 WO2011133952A2 (fr) 2010-04-22 2011-04-22 Nouvelle bactérie pour la production de produits chimiques et bactéries recombinées obtenues à partir de celle-ci
PCT/US2011/033832 WO2011133984A2 (fr) 2010-04-22 2011-04-25 Nouvelle bactérie pour la production de produits chimiques et ses recombinants

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2011/033688 WO2011133952A2 (fr) 2010-04-22 2011-04-22 Nouvelle bactérie pour la production de produits chimiques et bactéries recombinées obtenues à partir de celle-ci

Country Status (1)

Country Link
WO (2) WO2011133952A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITNO20120002A1 (it) * 2012-03-20 2013-09-21 Novamont Spa Processo per la produzione di composti organici a partire da specie vegetali
WO2021163584A1 (fr) * 2020-02-12 2021-08-19 The Broad Institute, Inc. Moyen de diagnostic crispr-cas utilisable sur place et méthodes d'utilisation de celui-ci
US11453907B2 (en) 2020-03-23 2022-09-27 The Broad Institute, Inc. Crispr effector system based coronavirus diagnostics

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 09 December 2003 Database accession no. AY353957 *
LDESVAUX, M. ET AL.: 'Cellulose catabolism by Clostirdium cellulolyticum growing in batch culture on defined medium.' APP. ENVIRON. MICROBIOL. vol. 66, no. 6, 2000, pages 2461 - 2470 *
PARSHINA S. N. ET AL.: 'Soehngenia saccarolytica gen. nov., sp. nov. and Clostridium amygdalinum sp. nov., two novel anaerobic, benzaldehyde- converting bacteria.' INT. J. SYST. EVOL. MICROBIOL. vol. 53, 2003, pages 1791 - 1799 *
SCOTCHER, M. C. ET AL.: 'SpolIE regulates sporulation but does not directly affect solventogenesis in clostridium acetobutylicum ATCC 824.' J. BACTERIOL. vol. 187, no. 6, 2005, pages 1930 - 1936 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources
US11840500B2 (en) 2016-02-19 2023-12-12 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources

Also Published As

Publication number Publication date
WO2011133984A3 (fr) 2012-05-10
WO2011133952A2 (fr) 2011-10-27
WO2011133952A3 (fr) 2012-05-03

Similar Documents

Publication Publication Date Title
AU2007210012B2 (en) Systems and methods for producing biofuels and related materials
WO2011149956A2 (fr) Procédés de production de produits chimiques à partir de sous-produits de fermentation
US20100268000A1 (en) Compositions and Methods for Fermentation of Biomass
WO2011008564A2 (fr) Compositions et procédés pour la saccharification améliorée d'une biomasse
US20110183382A1 (en) Methods and compositions for producing chemical products from c. phytofermentans
US20120107888A1 (en) Modulation of fermentation products through vitamin supplementation
US20110269201A1 (en) Redirected bioenergetics in recombinant cellulolytic clostridium microorganisms
WO2011116358A2 (fr) Microorganismes comprenant un gène inactivé de lactate déshydrogénase (ldh) destinés à la production chimique
JP2012519500A (ja) クロストリジウム（Ｃｌｏｓｔｒｉｄｉｕｍ）種からの発酵最終産物の生産
US20120064592A1 (en) Biocatalysts synthesizing deregulated cellulases
WO2011088422A2 (fr) Production de biocarburant en utilisant un biofilm en fermentation
WO2013148415A1 (fr) Fermentation renforcée à partir de produits de prétraitement
WO2014026154A1 (fr) Prétraitement amélioré de biomasse
WO2012083244A2 (fr) Production de biocarburant utilisant un biofilm dans un processus de fermentation
WO2012068310A2 (fr) Compositions et procédés pour la saccharification améliorée de biomasse dérivée de plantes génétiquement modifiées
WO2013155496A1 (fr) Procédés et systèmes pour la saccharification d'une biomasse
WO2011106576A2 (fr) Procédés et compositions pour activité enzymatique améliorée dans micro-organismes en fermentation
CA2780974C (fr) Compositions enzymatiques a multiples cellulases pour hydrolyse de biomasse cellulosique
WO2011133984A2 (fr) Nouvelle bactérie pour la production de produits chimiques et ses recombinants
WO2011072264A2 (fr) Procédés et compositions pour traitement de la biomasse
WO2012068537A2 (fr) Nouveaux biocatalyseurs et amorces pour la production de produits chimiques
WO2012103385A2 (fr) Biocatalyseurs synthétisant des cellulases dérégulées
Tukur ADAPTIVE LABORATORY EVOLUTION STRATEGY FOR IMPROVEMENT OF GROWTH AND SUBSTRATE UTILIZATION OF BACILLUS SUBTILIS IN HAZELNUT SHELLS HYDROLYSATE
WO2011028623A2 (fr) Organismes modifiés utilisés pour une meilleure saccharification de la biomasse
GB2468558A (en) Fermentation process comprising microorganism and external source of enzymes such as cellulase

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: 11772856

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: 11772856

Country of ref document: EP

Kind code of ref document: A2