WO2011149956A2 - Procédés de production de produits chimiques à partir de sous-produits de fermentation - Google Patents

Procédés de production de produits chimiques à partir de sous-produits de fermentation Download PDF

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WO2011149956A2
WO2011149956A2 PCT/US2011/037764 US2011037764W WO2011149956A2 WO 2011149956 A2 WO2011149956 A2 WO 2011149956A2 US 2011037764 W US2011037764 W US 2011037764W WO 2011149956 A2 WO2011149956 A2 WO 2011149956A2
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nrrl
byproducts
fermentation
biomass
rich stream
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PCT/US2011/037764
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English (en)
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WO2011149956A3 (fr
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Laura Angotti
Kevin Gray
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Qteros, Inc.
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Publication of WO2011149956A2 publication Critical patent/WO2011149956A2/fr
Publication of WO2011149956A3 publication Critical patent/WO2011149956A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/32—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from hydrolysates of wood or straw
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/06—Means for pre-treatment of biological substances by chemical means or hydrolysis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • WS whole stillage
  • WS comprises non-fermentable components of carbonaceous byproducts including undigested carbohydrates, oil, fiber, and protein.
  • TS thin stillage
  • WDG wet distillers grain
  • DG distillers grain
  • DDG dry distillers grains
  • TS is processed in a number of different ways.
  • TS can be concentrated and dried further to produce concentrated distillers solubles (CDS).
  • CDS is sold as animal feed supplement.
  • a fraction of TS is sent back to the head of the plant as make-up water for the fermentation process. This recycling stream of TS is commonly known as backset.
  • Some of the TS can also be sent to an evaporation process where the water is removed and the remaining dissolved and suspended solids are concentrated to what is commonly known as "syrup.”
  • CDS or syrup can be blended with the DG, DDG or WDG to form wet distillers grain with solubles (WDGS).
  • WDGS is usually dried to form dry distillers grains with solubles (DDGS) and sold as animal feed.
  • DDGS is sold as cattle feed; processes described herein can produce higher protein feed residuals, which can also be distributed, for example, in swine and poultry feed markets. In addition, processes described herein utilize these byproducts rich in carbonaceous material and thereby reduce waste.
  • a fermentation end product from two or more byproducts of biomass processing comprising: collecting two or more byproducts from a host plant and directing them to a consolidated bioprocessing process (CBP process); mixing the two or more byproducts with a mesophilic microorganism, wherein the mesophilic microorganism hydrolyzes and ferments lignocellulose or hemicellulose in the two or more byproducts; and, fermenting the two or more byproducts of biomass processing for a sufficient amount of time to allow the mesophilic microorganism to produce the fermentation end product from the two or more byproducts.
  • the biomass comprises plant matter, animal matter, or municipal waste.
  • the biomass comprises corn, wheat, rice, barley, soybeans, bamboo, cotton, crambe, jute, sorghum, high biomass sorghum, oats, tobacco, Miscanthus grass, switch grass, beans rape/canola, alfalfa, flax, sunflowers, safflowers, millet, rye, sugarcane, sugar beets, cocoa, tea, Brassica sp., cotton, coffee, sweet potatoes, flax, peanuts, clover, lettuce, tomatoes, cucurbits, cassava, potatoes, carrots, radishes, peas, lentils, cabbages, cauliflower, broccoli, Brussels sprouts, grapes, peppers, pineapples, apples, pears, peaches, apricots, walnuts, almonds, olives, avocadoes, bananas, coconuts, orchids, carnations, roses, castor beans, jatropha, peels, pits, fermentation waste, skins, straw, seeds, shells,
  • the biomass is corn.
  • the two or more byproducts comprise whole stillage (WS), thin stillage (TS), wet distillers grain (WDG), distillers grain (DG), concentrated distillers solubles (CDS), syrup or wet distillers grain with solubles (WDGS).
  • the two or more byproducts are carbonaceous byproducts substantially lacking starch.
  • the two or more byproducts comprise hemicelluloses or lignocellulose.
  • the two or more byproducts comprise C5 or C6 oligosaccharides.
  • the two or more byproducts are not pretreated. In one embodiment, at least one of the two or more byproducts are pretreated prior to the mixing step.
  • the pretreatment comprises acid hydrolysis, dilute acid hydrolysis, alkaline hydrolysis, autohydrolysis, or steam explosion. In one embodiment, the pretreatment comprises autohydrolysis and steam explosion. In one embodiment, the pretreatment comprises dilute acid hydrolysis.
  • One embodiment further comprises producing one or more fermentation byproducts from the fermentation of the two or more byproducts of biomass processing by the mesophilic microorganism. In one embodiment, the fermentation byproducts comprise WS, TS, WDG, DG, CDS, syrup, or WDGS. In one embodiment, the fermentation byproducts comprise higher protein distillers grains (HPDG). In one embodiment, the one or more fermentation byproducts are concentrated to produce an animal feed product. In one embodiment, the animal feed product is enriched in protein and substantially free of carbohydrates.
  • the animal feed product is treated to destroy any residual microorganisms.
  • the mesophilic microorganism is a Gram-positive bacterium.
  • the Gram-positive bacterium is a strain of Clostridium.
  • the strain is C. phytofermentans.
  • the strain is a Clostridium sp. Q.D.
  • the strain is a C.
  • the strain is a C.
  • the strain is genetically modified.
  • the fermentation end product is an alcohol.
  • the alcohol is ethanol.
  • Also disclosed herein are methods of producing a fermentation end product from two or more byproducts of biomass processing comprising: collecting two or more byproducts from a host plant and directing them to a consolidated bioprocessing process (CBP process); fractionating at least one of the two or more byproducts to form a fiber-rich stream, an oil-rich stream, and/or a protein-rich stream; mixing the fiber-rich stream and any unfractionated byproduct with a mesophilic microorganism, wherein the mesophilic microorganism hydrolyzes and ferments lignocellulose or hemicellulose in the fiber-rich stream and any unfractionated byproduct; and, fermenting the fiber-rich stream and any unfractionated byproduct of biomass processing for a sufficient amount of time to allow the mesophilic microorganism to produce the fermentation end product from the fiber-rich stream and any
  • the biomass comprises plant matter, animal matter, or municipal waste.
  • the biomass comprises corn, wheat, rice, barley, soybeans, bamboo, cotton, crambe, jute, sorghum, high biomass sorghum, oats, tobacco, Miscanthus grass, switch grass, beans rape/canola, alfalfa, flax, sunflowers, safflowers, millet, rye, sugarcane, sugar beets, cocoa, tea, Brassica sp., cotton, coffee, sweet potatoes, flax, peanuts, clover, lettuce, tomatoes, cucurbits, cassava, potatoes, carrots, radishes, peas, lentils, cabbages, cauliflower, broccoli, Brussels sprouts, grapes, peppers, pineapples, apples, pears, peaches, apricots, walnuts, almonds, olives, avocadoes, bananas, coconuts, orchids, carnations, roses, castor beans,
  • the biomass is corn.
  • the two or more byproducts comprise whole stillage (WS), thin stillage (TS), wet distillers grain (WDG), distillers grain (DG), concentrated distillers solubles (CDS), syrup or wet distillers grain with solubles (WDGS).
  • the two or more byproducts are carbonaceous byproducts substantially lacking starch.
  • the two or more byproducts comprise hemicelluloses or lignocellulose.
  • the fiber-rich stream comprises hemicelluloses or lignocellulose.
  • the two or more byproducts comprise C5 or C6 oligosaccharides.
  • the fractionating comprises centrifugation or filtering.
  • the fiber-rich stream is not pretreated. In one embodiment, the fiber-rich stream is pretreated. In one embodiment, the pretreatment comprises acid hydrolysis, dilute acid hydrolysis, alkaline hydrolysis, autohydrolysis, or steam explosion. In one embodiment, the pretreatment comprises autohydrolysis and steam explosion. In one embodiment, the pretreatment comprises dilute acid hydrolysis.
  • One embodiment further comprises producing one or more fermentation byproducts from the fermentation of the two or more byproducts of biomass processing by the mesophilic microorganism. In one embodiment, the fermentation byproducts comprise WS, TS, WDG, DG, CDS, syrup, or WDGS. In one embodiment, the fermentation byproducts comprise higher protein distillers grains (HPDG).
  • the one or more fermentation byproducts are concentrated to produce an animal feed product.
  • the protein-rich stream and/or the oil-rich stream are combined with the fermentation byproducts prior to the concentrating to produce an animal feed product.
  • the animal feed product is enriched in protein and substantially free of carbohydrates.
  • the animal feed product is treated to destroy any residual microorganisms.
  • the mesophilic microorganism is a Gram-positive bacterium.
  • the Gram-positive bacterium is a strain of Clostridium.
  • the strain is C. phytofermentans.
  • the strain is a Clostridium sp. Q.D.
  • the strain is a C phytofermentans American Type Culture Collection 700394T or a strain assigned the NRRL deposit accession number NRRL B-50361 , NRRL B-50362, NRRL B-50363, NRRL B-50364, NRRL B-50436, NRRL B-50437, NRRL B-50351 , NRRL B-50352, NRRL B-50353, NRRL B-50354, NRRL B-50355, NRRL B-50356, NRRL B-50357, NRRL B-50358, NRRL B-50359, or NRRL B-50498.
  • the strain is a C. phytofermentans ISDgT.
  • the strain is genetically modified.
  • the fermentation end product is an alcohol. In one embodiment, the alcohol is ethanol.
  • Also disclosed herein are methods of producing a fermentation end product from a fiber-rich stream comprising: collecting a fiber-rich stream from a host plant operating on a fractionated feedstock, wherein the fractionated feedstock forms a fiber-rich stream, a germ-rich stream and a starch-rich stream, and directing the fiber-rich stream to a consolidated bioprocessing process (CBP process); mixing the fiber-rich stream with a mesophilic microorganism, wherein the mesophilic microorganism hydrolyzes and ferments lignocellulose or hemicelluloses in the fiber-rich stream; and, fermenting the fiber-rich stream for a sufficient amount of time to allow the mesophilic microorganism to produce the fermentation end product from the fiber-rich stream.
  • CBP process consolidated bioprocessing process
  • the biomass comprises plant matter, animal matter, or municipal waste.
  • the biomass comprises corn, wheat, rice, barley, soybeans, bamboo, cotton, crambe, jute, sorghum, high biomass sorghum, oats, tobacco, Miscanthus grass, switch grass, beans rape/canola, alfalfa, flax, sunflowers, safflowers, millet, rye, sugarcane, sugar beets, cocoa, tea, Brassica sp., cotton, coffee, sweet potatoes, flax, peanuts, clover, lettuce, tomatoes, cucurbits, cassava, potatoes, carrots, radishes, peas, lentils, cabbages, cauliflower, broccoli, Brussels sprouts, grapes, peppers, pineapples, apples, pears, peaches, apricots, walnuts, almonds, olives, avocadoes, bananas, coconuts, orchids, carnations, roses, castor beans, jatropha, peels
  • the biomass is corn.
  • the fiber-rich stream comprises hemicelluloses or lignocellulose. In one embodiment, the fiber-rich stream is not pretreated. In one embodiment, the fiber-rich stream is pretreated. In one embodiment, the pretreatment comprises acid hydrolysis, dilute acid hydrolysis, alkaline hydrolysis, autohydrolysis, or steam explosion. In one embodiment, the pretreatment comprises autohydrolysis and steam explosion. In one embodiment, the pretreatment comprises dilute acid hydrolysis.
  • the germ-rich stream is mixed and fermented with the fiber-rich stream. In one embodiment, the germ-rich stream is pretreated to remove fats and/or oils.
  • One embodiment further comprises producing one or more fermentation byproducts from the fermentation of the fiber- rich stream by the mesophilic microorganism.
  • the fermentation byproducts comprise WS, TS, WDG, DG, CDS, syrup, or WDGS.
  • the fermentation byproducts comprise higher protein distillers grains (HPDG).
  • the one or more fermentation byproducts are concentrated to produce an animal feed product.
  • the animal feed product is enriched in protein and substantially free of carbohydrates.
  • the animal feed product is treated to destroy any residual microorganisms.
  • the mesophilic microorganism is a Gram-positive bacterium.
  • the Gram-positive bacterium is a strain of Clostridium.
  • the strain is C phytofermentans. In one embodiment, the strain is a Clostridium sp. Q.D. In one embodiment, the strain is a C phytofermentans American Type Culture Collection 700394T or a strain assigned the NRRL deposit accession number NRRL B-50361, NRRL B-50362, NRRL B-50363, NRRL B-50364, NRRL B-50436, NRRL B-50437, NRRL B-50351, NRRL B-50352, NRRL B-50353, NRRL B-50354, NRRL B-50355, NRRL B-50356, NRRL B-50357, NRRL B-50358, NRRL B-50359, or NRRL B-50498. In one embodiment, the strain is a C. phytofermentans ISDgT. In one embodiment, the strain is genetically modified. In one embodiment, the fermentation end product is an alcohol. In one embodiment, the alcohol is ethanol.
  • a CBP plant comprises: two or more byproducts from a host plant that processes biomass; a mesophilic microorganism, wherein the mesophilic microorganism hydrolyzes and ferments lignocellulose or hemicelluloses in the two or more byproducts from the host plant that processes biomass; and, a fermentation vessel.
  • the CBP plant further comprises a pretreatment reactor.
  • the CBP plant further comprises a beer well.
  • the CBP plant further comprises a distillation column.
  • the CBP plant further comprises a centrifuge.
  • the CBP plant further comprises dryers.
  • the system further produces an animal feed product from the two or more byproducts.
  • systems for the production of a fermentation end product and an animal feed product from the two or more byproducts of biomass processing comprising a CBP plant, wherein the CBP plant comprises: two or more byproducts from a host plant that processes biomass; a mesophilic microorganism, wherein the mesophilic microorganism hydrolyzes and ferments lignocellulose or hemicelluloses in the two or more byproducts from the host plant that processes biomass; a fermentation vessel; a beer well; a distillation column; a centrifuge; and, dryers.
  • the CBP plant further comprises a pretreatment reactor.
  • the biomass comprises plant matter, animal matter, or municipal waste.
  • the biomass comprises corn, wheat, rice, barley, soybeans, bamboo, cotton, crambe, jute, sorghum, high biomass sorghum, oats, tobacco, Miscanthus grass, switch grass, beans rape/canola, alfalfa, flax, sunflowers, safflowers, millet, rye, sugarcane, sugar beets, cocoa, tea, Brassica sp., cotton, coffee, sweet potatoes, flax, peanuts, clover, lettuce, tomatoes, cucurbits, cassava, potatoes, carrots, radishes, peas, lentils, cabbages, cauliflower, broccoli, Brussels sprouts, grapes, peppers, pineapples, apples, pears, peaches, apricots, walnuts, almonds, olives, avocadoes, bananas, coconuts, orchids
  • the biomass is corn.
  • the two or more byproducts comprise whole stillage (WS), thin stillage (TS), wet distillers grain (WDG), distillers grain (DG), concentrated distillers solubles (CDS), syrup or wet distillers grain with solubles (WDGS).
  • the two or more byproducts are carbonaceous byproducts substantially lacking starch.
  • the two or more byproducts comprise hemicelluloses or lignocellulose.
  • the two or more byproducts comprise C5 or C6 oligosaccharides.
  • the two or more byproducts are not pretreated. In some embodiments, at least one of the two or more byproducts are pretreated prior to the mixing step.
  • the pretreatment comprises acid hydrolysis, dilute acid hydrolysis, alkaline hydrolysis, autohydrolysis, or steam explosion. In some embodiments, the pretreatment comprises autohydrolysis and steam explosion. In some embodiments, the pretreatment comprises dilute acid hydrolysis.
  • the animal feed product is enriched in protein and substantially free of carbohydrates. In some embodiments, the animal feed product is treated to destroy any residual microorganisms.
  • the mesophilic microorganism is a Gram-positive bacterium. In some embodiments, the Gram-positive bacterium is a strain of Clostridium. In some embodiments, the strain is C. phytofermentans. In some embodiments, the strain is a Clostridium sp. Q.D.
  • the strain is a C phytofermentans American Type Culture Collection 700394T or a strain assigned the NRRL deposit accession number NRRL B- 50361, NRRL B-50362, NRRL B-50363, NRRL B-50364, NRRL B-50436, NRRL B-50437, NRRL B- 50351, NRRL B-50352, NRRL B-50353, NRRL B-50354, NRRL B-50355, NRRL B-50356, NRRL B- 50357, NRRL B-50358, NRRL B-50359, or NRRL B-50498.
  • the strain is a C. phytofermentans ISDgT.
  • the strain is genetically modified.
  • the fermentation end product is an alcohol.
  • the alcohol is ethanol.
  • a CBP plant comprising: a fiber storage system; a fiber-rich stream from a host plant that processes biomass, wherein the host plant is operating on a fractionated feedstock, wherein the fractionated feedstock forms the fiber-rich stream, a germ-rich stream and a starch-rich stream; a mesophilic microorganism, wherein the mesophilic microorganism hydrolyzes and ferments lignocellulose or hemicelluloses in the fiber-rich stream; and, a fermentation vessel.
  • the CBP plant further comprises a pretreatment reactor.
  • the CBP plant further comprises a beer well. In one embodiment, the CBP plant further comprises a distillation column. In one embodiment, the CBP plant further comprises a centrifuge. In one embodiment, the CBP plant further comprises dryers. In one embodiment, the system further produces an animal feed product from the two or more byproducts.
  • the CBP plant comprises: a fiber storage system; a fiber-rich stream from a host plant that processes biomass, wherein the host plant is operating on a fractionated feedstock, wherein the fractionated feedstock forms the fiber-rich stream, a germ-rich stream and a starch-rich stream
  • the CBP plant further comprises a pretreatment reactor.
  • the biomass comprises plant matter, animal matter, or municipal waste.
  • the biomass comprises corn, wheat, rice, barley, soybeans, bamboo, cotton, crambe, jute, sorghum, high biomass sorghum, oats, tobacco, Miscanthus grass, switch grass, beans rape/canola, alfalfa, flax, sunflowers, safflowers, millet, rye, sugarcane, sugar beets, cocoa, tea, Brassica sp., cotton, coffee, sweet potatoes, flax, peanuts, clover, lettuce, tomatoes, cucurbits, cassava, potatoes, carrots, radishes, peas, lentils, cabbages, cauliflower, broccoli, Brussels sprouts, grapes, peppers, pineapples, apples, pears, peaches, apricots, walnuts, almonds, olives, avocadoes, bananas, coconuts, orchids
  • the biomass is corn.
  • the fiber-rich stream comprises hemicelluloses or lignocellulose.
  • the fiber-rich stream is not pretreated.
  • the fiber-rich stream is pretreated.
  • the pretreatment comprises acid hydrolysis, dilute acid hydrolysis, alkaline hydrolysis, autohydrolysis, or steam explosion. In some embodiments, the pretreatment comprises autohydrolysis and steam explosion. In some embodiments, the pretreatment comprises dilute acid hydrolysis.
  • the germ-rich stream is mixed with the fiber-rich stream. In some embodiments, the germ-rich stream is pretreated to remove fats and/or oils
  • the animal feed product is enriched in protein and substantially free of carbohydrates. In some embodiments, the animal feed product is treated to destroy any residual microorganisms.
  • the mesophilic microorganism is a Gram-positive bacterium. In some embodiments, the Gram-positive bacterium is a strain of Clostridium.
  • the strain is C. phytofermentans. In some embodiments, the strain is a Clostridium sp. Q.D. In some embodiments, the strain is a C phytofermentans American Type Culture Collection 700394T or a strain assigned the NRRL deposit accession number NRRL B- 50361, NRRL B-50362, NRRL B-50363, NRRL B-50364, NRRL B-50436, NRRL B-50437, NRRL B- 50351, NRRL B-50352, NRRL B-50353, NRRL B-50354, NRRL B-50355, NRRL B-50356, NRRL B- 50357, NRRL B-50358, NRRL B-50359, or NRRL B-50498. In some embodiments, the strain is a C. phytofermentans ISDgT. In some embodiments, the strain is genetically modified. In some
  • the fermentation end product is an alcohol.
  • the alcohol is ethanol.
  • Disclosed herein are methods of producing sugars from two or more byproducts of biomass processing comprising: collecting two or more byproducts from a host plant and directing them to a consolidated bioprocessing process (CBP process); mixing the two or more byproducts with a mesophilic microorganism, wherein the mesophilic microorganism hydrolyzes and ferments lignocellulose or hemicellulose in the two or more byproducts; and, fermenting the two or more byproducts of biomass processing for a sufficient amount of time to allow the mesophilic microorganism to produce the fermentation end product from the two or more byproducts.
  • CBP process consolidated bioprocessing process
  • compositions for the production of a fermentation end product comprising: TS; WDG; syrup; and, a Clostridium strain that can hydrolyze and ferment hemicelluloses or hgnocellulose in the TS, WDG, or syrup.
  • compositions for the production of a fermentation end product comprising: TS; WDG; syrup; and, a C. phytofermentans that can hydrolyze and ferment hemicelluloses or hgnocellulose in the TS, WDG, or syrup.
  • compositions for the production of a fermentation end product comprising: TS; WDG; syrup; and, Clostridium sp. Q.D., wherein the Clostridium sp. Q.D. can hydrolyze and ferment hemicelluloses or hgnocellulose in the TS, WDG, or syrup.
  • Disclosed herein are methods of producing an animal feed product enriched in grain-based protein substantially free of carbohydrates comprising: collecting one or more byproducts from a host plant; mixing the one or more byproducts with a mesophilic microorganism, wherein the mesophilic organism can hydrolyze and ferment hemicelluloses or hgnocellulose; producing ethanol from the one or more byproducts; and, concentrating leftover of the one or more byproducts.
  • Also disclosed herein are method of producing an animal feed product enriched in corn-based protein substantially free of carbohydrates comprising: collecting one or more byproducts from a corn processing plant; mixing the one or more byproducts with a mesophilic microorganism, wherein the mesophilic organism can hydrolyze and ferment hemicelluloses or hgnocellulose; producing ethanol from the one or more byproducts; and, concentrating leftover of the one or more byproducts.
  • Also disclosed herein are methods of producing ethanol from one or more byproducts of grain processing comprising collecting the one or more byproducts from a grain processing plant; mixing the one or more byproducts with a mesophilic microorganism, wherein the mesophilic microorganism can hydrolyze and ferment hgnocellulose and hemicellulose; and, producing the ethanol from the one or more byproducts.
  • Also disclosed herein are methods of reducing animal feed production cost in a dry milling process comprising: directing one or more feed streams to a consolidated bioprocessing process; mixing one or more byproducts obtained from the one or more feed streams with a mesophilic microorganism, wherein the mesophilic organism can hydrolyze and ferment hemicelluloses or hgnocellulose; and, producing animal feed from the one or more byproducts.
  • Also disclosed herein are methods of processing grain comprising: contacting grain to produce byproducts comprising WS, TS, WDG, and/or syrup; directing the byproducts to a consolidated bioprocessing process; contacting the directed byproducts with a mesophilic microorganism, wherein the mesophilic organism can hydrolyze and ferment hemicelluloses or hgnocellulose; and, producing ethanol and animal feed product from the directed byproducts.
  • Also disclosed herein are methods of processing corn comprising: contacting corn to produce byproducts comprising WS, TS, WDG, and/or syrup; directing the byproducts to a consolidated bioprocessing process; contacting the directed byproducts with a mesophilic
  • the mesophilic organism can hydrolyze and ferment hemicelluloses or lignocellulose; and, producing ethanol and an animal feed product from the directed byproducts.
  • the mesophilic microorganism is a Gram-positive bacterium.
  • the Gram-positive bacterium is a strain of Clostridium.
  • the Clostridium can hydrolyze and ferment hemicellulose.
  • the Clostridium can hydrolyze and ferment lignocellulose.
  • the strain is C. phytofermentans.
  • the strain is a Clostridium sp. Q.D.
  • the strain is a C.
  • the strain is a C. phytofermentans ISDgT. In some embodiments, the strain is genetically modified.
  • FIG. 1 is a flow diagram showing a consolidated bioprocessing (CBP) process attached to a corn-milling process.
  • CBP consolidated bioprocessing
  • Figure 2 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 3 illustrates a method for producing fermentation end products from biomass by using solvent extraction or separation methods.
  • Figure 4 illustrates a method for producing fermentation end products from biomass by charging biomass to a fermentation vessel.
  • Figure 5 A-C illustrates pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either fermented separately or together.
  • Figure 6 illustrates the plasmid pQInt
  • Figure 7 illustrates the plasmid pQIntl .
  • Figure 8 illustrates the plasmid pQInt2.
  • Figure 9 A-D illustrates corn byproduct processing options: A. direct feed; B. back-end fractionation; C. post-treatment processing; and, D. front-end fractionation.
  • Figure 10 illustrates a CBP process attached to a host plant wherein whole distillers grains (WDG) is utilitzed with pretreatment.
  • WDG whole distillers grains
  • Figure 11 illustrates a CPB process attached to a host plant wherein the host plant feedstock is fractionated prior to processing.
  • Figure 12 illustrates optional process flows for a CBP process attached to a host plant wherein the host factory feedstock is fractionated prior to processing.
  • Figure 13 illustrates a compositional analysis of a batch of WDG.
  • Figure 14 illustrates carbohydrate hydrolysis during pretreatment of WDG.
  • Figure 15 illustrates ethanol yields from a simultaneous saccharification and fermentation at 100 mL scale of pretreated WDG at 10% solids.
  • Figure 16 illustrates ethanol yields from simultaneous saccharification and fermentation at 1 L scale of pretreated WDG at 5% and 10% solids.
  • Figure 17 summarizes cellulosic ethanol yields from simultaneous saccharification and fermentation of autohydrolyzed WDG.
  • Figure 18 A-B illustrates compositional analysis of a batch of WDG.
  • Figure 19 illustrates ethanol yield from fermentation at 1L scale of pretreated WDG at 6% solids.
  • Figure 20 summarizes the final composition and ethanol yields from simultaneous
  • CBP processes utilizing carbonaceous byproducts of biomass processing plants, which produce sugars, ethanol or other fermentation end products from the carbonaceous byproducts.
  • CBP processes described herein comprise a module that can be readily adapted and attached to any known biomass processing plant. As an additional module, it collects byproducts of biomass processing plants and boosts production of sugars, ethanol or other fermentation products by utilizing carbonaceous material in the collected material.
  • a CBP process can be implemented as a stand-alone processing plant in which byproducts from multiple biomass plants are processed.
  • microorganisms useful for CBP processes are microorganisms useful for CBP processes.
  • a microorganism disclosed herein produces sugars, ethanol or fermentation products from carbonaceous byproducts fed into the CBP processes.
  • CBP processes for example, the total amount of ethanol production is increased while producing a high protein animal feed.
  • CBP processes described herein have other benefits including, but not limited to: decreased waste; lower production cost; and simplified steps for producing animal feeds or other valuable chemical products.
  • Raw plant material useful in described processes includes, but is not limited to, oats, wheat, barley, rice, sugar cane, energy cane, sugar beets, sorghum (milo), cassava, soft or hard woods, bagasse, stover, algae, Camelina sp., Jatropha sp., peel, seed cake, seed, sugar beet, wood chip, or any combination thereof.
  • a feed stream can include any material that is directed to a CBP process.
  • a feed stream can be any material directed from a host plant to a CBP process.
  • Various feed streams containing carbonaceous byproducts can be utilized in a CBP process.
  • a feed stream includes, but is not limited to, byproducts such as whole stillage (WS), thin stillage, (TS), wet distiller's grain (WDG), distillers grains (DG), syrup, condensed distillers grains with solubles (CDS) and wet distillers grains with solubles (WDGS).
  • a feed stream can be pretreated.
  • a feed stream is not pretreated.
  • a feed stream includes, but is not limited to, products from a fractionation process (e.g. , germ, oil, fiber, fiber-enriched fraction, residues from fractionation and separation, etc. ).
  • a fractionation process produces a fiber-enriched fraction (e.g. , a cellulosic fraction), a carbohydrate- enriched fraction (e.g. , a starch fraction), and a protein and oil-enriched fraction (e.g. , a germ fraction).
  • a feed stream includes, but is not limited to, products and/ or byproducts from a host plant operating on a fractionated product (e.g. , WDG, TS, WS, syrup, etc. ).
  • a feed stream can be sawdust from sawmilling process.
  • a CBP process comprises contacting streams of carbonaceous byproducts with a microorganism capable of producing sugar molecules from the byproducts.
  • the sugar molecules are fed into yeast that converts sugar molecules to ethanol.
  • the microorganism is a bacterium.
  • the microorganism is a mesophilic bacterium
  • ethanol is produced directly from a microorganism growing on carbonaceous byproducts.
  • alcohol other than ethanol is produced directly from a microorganism growing on carbonaceous byproducts.
  • a fermentation product is produced directly from a microorganism growing on carbonaceous byproducts.
  • biofuel is an end product of a CBP process.
  • the term "about” in relation to a reference numerical value includes a range of values plus or minus 15% from that value. For example the amount “about 10" includes amounts from 8.5 to 1 1.5.
  • “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • 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.
  • 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.
  • a host cell that comprises a recombinant vector 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 polynucleotide refers to a polynucleotide that has been purified from the sequences that flank it in a naturally- occurring state, e.g. , a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • an "isolated peptide” or an “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 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.
  • 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, -1 1 °C, -12°C, -13°C, -14°C, or -15°C.
  • polynucleotide or “nucleic acid” as used herein designates RNA, mRNA, cRNA, rRNA, DNA, or cDNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides 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 sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments 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, 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 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.).
  • 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 genetic code is redundant in that it contains 64 different codons (triplet nucleotide sequence) but only codes for 22 standard amino acids and a stop signal. Due to the degeneracy of the genetic code, nucleotides within a protein-coding polynucleotide sequence can be substituted without altering the encoded amino acid sequence. These changes ⁇ e.g. substitutions, mutations, optimizations, etc.) are therefore "silent". It is thus contemplated that various changes can be made within a disclosed nucleic acid sequence without any loss of biological activity relating to either the polynucleotide sequence or the encoded peptide sequence.
  • a polynucleotide comprises codons, within a 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 (i.e. they are "silent"). In another embodiment, a polynucleotide comprises codons, within a protein coding sequence, that are optimized to increase translation efficiency of an mRNA transcribed from the polynucleotide in a host cell. In one embodiment, this optimization is silent (does not change the amino acid sequence encoded by the polynucleotide).
  • 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 (+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);
  • glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • 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 that are not expected to be advantageous can also be used if these resulting proteins have the same or improved
  • a method for that 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 an enzyme can be a polypeptide fragment that is, for example, 10, 1 1 , 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, 1 10, 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.
  • 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 al
  • FASTA Altschul et al
  • TFASTA TFASTA
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al , "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15, which is herein incorporated by reference in its entirety.
  • 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 transfer of additional copies of an endogenous gene into a microorganism.
  • the term "recombinant” as used herein, refers to an organism that is 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 extra-chromosomally 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 oil (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 keto
  • reaction 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.
  • organic acids e.g. acetic, lactic, formic, citric acid etc.
  • esters e.g. wax esters, glycerides, etc.
  • end-products include, but are not limited to, alcohols (e.g. ethanol, butanol, methanol, 1 , 2-propanediol, 1 , 3 -propanediol, etc.), acids (e.g.
  • lactic acid formic acid, acetic acid, succinic acid, pyruvic acid, etc.
  • enzymes e.g.cellulases, polysaccharases, lipases, proteases, ligninases, hemicellulases, etc.
  • End- products 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, alcohols (e.g. ethanol, butanol, methanol, 1, 2-propanediol, 1 , 3 -propanediol), acids (e.g. lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid), and enzymes (e.g. cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases) and can be present as a pure compound, a mixture, or an impure or diluted form.
  • alcohols e.g. ethanol, butanol, methanol, 1, 2-propanediol, 1 , 3 -propanediol
  • acids e.g. lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid
  • 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, 1 1, 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, or animal matter.
  • biomass comprises non-genetically modified organisms or parts of organisms, such as non-genetically modified plant matter, algal matter, or 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 human or animal use, 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., cotton, coffee, sweet potatoes, flax, peanuts, clover; lettuce, tomatoes, cucurbits, cassava, potatoes, carrots, radishes, peas, lentils, cabbages, cauliflower, broccoli, Brussels sprouts, grapes, peppers, or pineapples; tree fruits or nuts such as citrus, apples, pears, peaches, apricots, walnuts, almonds, olives, avocadoes, bananas, or coconuts; flowers such as orchids, carnations and roses; nonvascular plants such as ferns; oil producing plants (such as castor beans, jatropha, or olives); or gymnosperms such as palms.
  • 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
  • plant matter includes, but is not limited to, products and/ or byproducts from a host plant operating on a fractionated product (e.g. , WDG, TS, WS, syrup, etc. ).
  • plant matter includes, but is not limited to, products from corn fractionation (e.g. , germ, oil, fiber, fiber- enriched fraction, residues from fractionation and separation, 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.
  • hydrolytic enzymes such as a cellulase, hemicellulase, or pectinase etc.
  • 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.).
  • 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.
  • carbonaceous byproducts 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.
  • One exemplary source of carbonaceous material is plant matter.
  • Plant matter can be, for example, woody plant matter, non-woody plant matter, cellulosic material, hgnocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, sugar cane, grasses, switchgrass, bamboo, algae, and material derived from these.
  • Plant matter can also be residual spent solids from alcoholic or other fermentation from materials such as corn and which contain lignin, starch, cellulose, hemicellulose, and proteins. Plant matter can be further described by reference to the chemical species present, such as proteins, polysaccharides (such as chitin) and oils.
  • Polysaccharides include polymers of various monosaccharides and derivatives of monosaccharides including glucose, fructose, lactose, galacturonic acid, rhamnose, etc.
  • Plant matter also includes agricultural waste byproducts or side streams such as pomace, corn steep liquor, corn steep solids, corn stover, corn stillage, corn cobs, corn grain, bagasse, distillers grains, peels, pits, fermentation waste, wood chips, saw dust, wood flour, wood pulp, paper pulp, paper pulp waste steams straw, lumber, demolition waste, hybrid poplar, milo, sewage, seed cake, husks, rice hulls, leaves, grass clippings, food waste, restaurant waste, or cooking oil.
  • These materials can come from farms, forestry, industrial sources, households, etc.
  • Plant matter also includes maltose, corn syrup, syrup, Whole Stillage, Thin Stillage, Thick Stillage, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Grains (DG), Wet Distillers Grains (WDG), Wet Distillers Grains with Solubles (WDGS), or Distillers Dried Grains with Solubles (DDGS).
  • Another non- limiting example of biomass is animal matter, including, for example milk, meat, fat, bone meal, animal processing waste, and animal waste. "Feedstock” is frequently used to refer to biomass being used for a process, such as those described herein.
  • Another example of carbonaceous material or biomass is sewage and/or municipal waste, much of which contains indigestible materials such as paper and other cellulosic, hemicellulosic and Hgnocellulosic material.
  • indigestible materials such as paper and other cellulosic, hemicellulosic and Hgnocellulosic material.
  • the term "broth” as used herein 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
  • titer e.g. g/L
  • 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. These can account for up to 3 g/L ethanol production or equivalent of up to 6 g/L sugar as much as +/- 10%>-15%>
  • fed-batch or fed-batch fermentation
  • the terms "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.
  • 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.
  • 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 is 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.
  • xylanolytic 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. In some contexts 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. For example, the term “Clostridium biocatalyst” as used herein indicates one or more Clostridium strains ⁇ e.g. , C.
  • a Clostridium biocatalyst can simultaneously hydrolyze and ferment lignocellulosic biomass.
  • a Clostridium biocatalyst can hydrolyze and ferment hexose (C6) and pentose (C5) polysaccharides ⁇ e.g. carbohydrates).
  • 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 Clostridum phytofermentans, 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.
  • 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.
  • pretreatment of biomass comprises dilute acid hydrolysis. Examples 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.
  • pretreatment of biomass comprises autohydrolysis (i.e. , hot water treatment).
  • a hot water treatment can be performed between about 100°C and 200°C, for example, between about 100°C and 1 10°C, 100°C and 120°C, 100°C and 130°C, 100°C and 140°C, 100°C and 150°C, 100°C and 160°C, 100°C and 170°C, 100°C and 180°C, 100°C and 190°C, 100°C and 200°C, 1 10°C and 120°C, 1 10°C and 130°C, 1 10°C and 140°C, 1 10°C and 150°C, 1 10°C and 160°C, 1 10°C and 170°C, 1 10°C and 180°C, 1 10°C and 190°C, 1 10°C and 200°C, 120°C and 130°C, 120°C and 140°C, 120°C and 150°C, 120°C and 160°C, 120°C and 150°C, 120°C
  • the autohydrolysis temperature can be about 100°C, 101 °C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 1 10°C, 1 11 °C, 1 12°C, 1 13°C, 1 14°C, 115°C, 1 16°C, 1 17°C, 1 18°C, 119°C, 120°C, 121 °C, 122°C, 123°C, 124°C, 125°C, 126°C, 127°C, 128°C, 129°C, 130°C, 131 °C, 132°C, 133°C, 134°C, 135°C, 136°C, 137°C, 138°C, 139°C, 140°C, 141 °C, 142°C, 143°C, 144°C, 145°C, 146°C, 147°C, 148°C,
  • the duration of autohydrolysis pretreatment is between about lmin and 60 min, for example, about 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, or 60 min.
  • the duration of autohydrolysis treatment is between about 1 hour and 24 hours, for example, about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.
  • pretreatment can also include disruption or expansion of cellulosic and/or hemicellulosic material.
  • pretreatment comprises steam explosion, ammonia fiber expansion (or explosion) (AFEX) or another thermal/chemical pretreatment technique.
  • biomass can be pretreated by liquid hot water treatment (i.e. , autohydrolysis) followed by steam explosion.
  • 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 hydrolysate 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, A P, 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.
  • 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 phytofermentans, 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 such as a Clostridium biocatalyst such as C. phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or a variant thereof.
  • a microorganism such as a Clostridium biocatalyst such as C. phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, 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%,
  • 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%
  • 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%).
  • the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 20%.
  • 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%.
  • the parameters of the pretreatment are changed such that the
  • 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%).
  • hemicellulose e.g. , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher
  • 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 phytofermentans , Clostridium sp. Q.D, Clostridium
  • 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, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more times.
  • a pH modifier can be added.
  • 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, each of which is hereby incorporated by reference in its entirety.
  • 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.
  • biomass in some embodiments, can be pre-treated at an elevated temperature and/or pressure. In one embodiment 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 pretreatment), or before the drying step.
  • the solids recovery step provided by the methods described herein 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
  • biomass is processed or pretreated prior to
  • 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 phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13 or 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 phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27 or Clostridium
  • 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 phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27, Clostridium
  • the microorganism is genetically modified to express or increase expression of an enzyme capable of hydrolyzing 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 that increases the pH to an alkaline level, at a temperature of 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 phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.27 or Clostridium
  • 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
  • SHF fermentation 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.
  • SSF saccharification and fermentation
  • hydrolysis and fermentation take place in the same reactor under the same conditions.
  • 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 such as a Clostridium biocatalyst (e.g. Clostridium phytofermentans, Clostridium sp.
  • Clostridium biocatalyst e.g. Clostridium phytofermentans, Clostridium sp.
  • Clostridium phytofermentans Q.27 or Clostridium phytofermentans Q.13 or variants thereof converts the hydro lyzed sugar into the desired product (e.g. fuel or chemical) and completes the hydrolysis of the residual cellulose and hemicellulose.
  • 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 such as a Clostridium biocatalyst (e.g. Clostridium phytofermentans, Clostridium sp.
  • a microorganism such as a Clostridium biocatalyst (e.g. Clostridium phytofermentans, Clostridium sp.
  • 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 such as a Clostridium biocatalyst (e.g. Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27or Clostridium phytofermentans Q.13 or variants thereof).
  • a Clostridium biocatalyst e.g. Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27or Clostridium phytofermentans Q.13 or variants 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.
  • a second microorganism can be used to convert residual carbohydrates into a fermentation end-product.
  • the second microorganism is a yeast such as
  • Saccharomyces cerevisiae a Clostridia species such as C. thermocellum, C. acetobutylicum, or C. cellovorans; or Zymomonas mobilis.
  • 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. 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9); 3) treating the biomass in a closed container with a Clostridium biocatalyst (such as Clostridium phytofermentans , a Clostridium sp. Q.D, a Clostridium
  • 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.
  • 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.
  • fed batch and or staged feeding techniques can be utilized to manage, for example, the carbohydrate balance of the fermentation medium and/or the growth rate of the microorganism or the group of microorganisms.
  • Organisms disclosed herein can be incorporated into methods and compositions so as to enhance fermentation end-product yield and/or rate of production.
  • Clostridium phytofermentans Clostridium phytofermentans
  • C. phytofermentans Clostridium phytofermentans
  • C. phytofermentans is capable of hydrolyzing and fermenting hexose (C6) and pentose (C5) polysaccharides (e.g. carbohydrates).
  • C. phytofermentans is capable of acting directly on lignocellulosic biomass without any pretreatment.
  • Other examples of microorganisms that can hydrolyze and ferment hexose (C6) and pentose (C5) polysaccharides include Clostridium sp. Q.D, or variants of Clostridium phvtofennentans (e.g.
  • Clostridium Q.8 Clostridium Q.27
  • Clostridium phytofermentans Q.13 Clostridium phytofermentans
  • these organisms can produce hemicellulases, pectinases, xylansases, or chitinases.
  • 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.
  • C. phytofermentans or variants thereof hydrolyze and ferment hexose and pentose polysaccharides which are part of a biomass.
  • the biomass comprises lignocellulose.
  • the biomass comprises hemicellulose.
  • Methods can also include co-culture with a 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 is C. phytofermentans or Clostridium 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 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
  • reaction end-product 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 or chemicals
  • additives 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).
  • 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
  • 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
  • a byproduct is anything produced in a process that is not the primary product.
  • a fermentation process produces a primary fermentation end product (e.g., ethanol) and a byproduct called whole stillage (WS), which is the mixture of liquids and solids that remains after the primary fermentation end product is removed.
  • WS can be fractionated ( e.g., by centrifugation and/ or filtration) to produce thin stillage (TS) and wet distillers grains (WDG).
  • TS comprises the liquid fraction of WS, including any soluble molecules
  • WDG comprises the solids fraction of WS, including undigested carbohydrates, oil, fiber (e.g., hemicelluloses or lignocellulose), and protein.
  • TS and WS are byproducts of the fermentation process.
  • TS can be concentrated ( e.g., by an evaporation/distillation process) to produce concentrated distillers solubles (CDS), which is also termed syrup.
  • WDG can be processed ( e.g., by evaporation/ drying) to produce dry distillers grains (DDG).
  • DDG dry distillers grains
  • WDG or DDG and CDS/syrup can be combined to form wet distillers grain with solubles (WDGS).
  • WDGS can be processed ( e.g., by evaporation/drying) to produce dry distillers grains with solubles (DDGS).
  • Each of these materials are byproducts of the original fermentation process. Any of these byproducts can be fed to a consolidated bioprocessing process (CBP process).
  • the CBP process utilizes the hydrolysable and/ or fermentable portion of the byproducts (e.g., carbohydrates and fiber) to produce a desired product (e.g. , sugars, ethanol or other fermentation end product).
  • the CBP process produces its own byproducts as well, for example, higher protein distillers grains (HPDG).
  • a CBP process is adapted and attached to a host biomass processing plant.
  • a CBP process is adapted and attached to a corn milling process.
  • the corn milling process is a dry milling process.
  • the corn milling process is a wet milling process.
  • a byproduct from a corn milling process is sent to a CBP process.
  • a byproduct includes, but is not limited to, carbonaceous byproducts such as WS, TS, DG, WDG, CDS, syrup or WDGS.
  • a single byproduct is directed to a CBP process.
  • more than one byproducts are directed to a CBP process.
  • carbonaceous byproducts fed to CBP process comprise WDG. In another embodiment, carbonaceous byproducts fed to CBP process comprise WDGS. In another embodiment, carbonaceous byproducts fed to CBP process comprise WS. In another embodiment, carbonaceous byproducts fed to CBP process comprise syrup. In another embodiment, carbonaceous byproducts fed to CBP process comprise TS. In another embodiment, carbonaceous byproducts fed to CBP process comprise CDS. In another embodiment, carbonaceous byproducts fed to CBP process comprise material from DG.
  • combined carbonaceous byproducts comprise material from two or more byproducts.
  • Combined carbonaceous byproducts for CBP process comprise any two, three or four byproducts such as WS, TS, DG, WDG, CDS, syrup or WDGS.
  • carbonaceous byproducts for CBP process comprise WS, WDG and syrup.
  • carbonaceous byproducts fed to CBP process can vary throughout the year. Without being limiting, for example, winter wheat can first be fermented to a carbonaceous byproduct and the resulting byproduct fed to CBP process and, subsequently, in the same plant, corn cobs can be fermented to a carbonaceous byproduct in the fall and the byproduct fed to the CBP process.
  • amount of WS diverted to CBP process is up to about 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100%.
  • combined carbonaceous byproducts comprise about 15% of WS, about 35%) of syrup, and about 35% of WDG.
  • the percentage of syrup is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% of the combined carbonaceous byproducts.
  • the percentage of WDG is about 5%, 10%, 15%, 20%, 25%, 30%, 35%), 40%), 45%), 50%), 55% or 60% of the combined carbonaceous byproducts.
  • the percentage of WS is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% of the combined carbonaceous byproducts.
  • total carbohydrate content can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the combined carbonaceous byproducts.
  • the total carbohydrate content can be between 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 100%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 100%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 100%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 100%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80%, 40% to 90%, 40% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 100%, 60% to 70%, 60% to 80%, 60% to 90%, 60% to 100%, 70% to 80%, 70% to 90%, 70% to 100%, 80% to 90%, 80% to 100%, or 90% to 100% of
  • 100% of WS is committed to CBP process and centrifugation or drying steps are no longer utilized to handle byproducts.
  • 100% of WS from one batch of biomass processing plant is mixed with syrup obtained from another batch of biomass processing plant and then provided for CBP process.
  • 100% of WS from one batch of biomass processing plant is mixed with WDG obtained from another batch of biomass processing plant and then provided for CBP process.
  • 100% of WS from one batch of biomass processing plant is mixed with DG obtained from another batch of biomass processing plant and then provided for CBP process.
  • the byproducts can come from multiple sources, e.g., beet pulp and wood waste.
  • the amount of available stillage for producing DDGS is inversely proportional to the amount of WS committed to the CBP process. In one embodiment, about 50% of WS is committed to CBP process and the rest is committed to producing DDGS. In one embodiment, the amount of WS committed to CBP process is adjusted based on demand for ethanol, animal feed, or a fermentation product. In one embodiment the demand is the predicted market demand of one ethanol, animal feed, or a fermentation product. In one embodiment, the amount of WS committed to CBP process is increased when ethanol production rate is below the level of predicted market demand. In another embodiment, the amount of WS committed to CBP process is decreased when ethanol production rate is above the level of predicted market demand.
  • the amount of WS committed to CBP process is decreased when production rate for animal feed is below the level of predicted market demand for animal feed. In another embodiment, the amount of WS committed to CBP process is increased when production rate for animal feed is above the level of predicted market demand for animal feed.
  • the byproducts of a CBP process e.g. , high-protein distillers grains
  • CBP microbes e.g. , C. phytofermentans biocatalysts.
  • processed byproducts of a CBP process are utilized as live stock feed (e.g. , cattle feed, swine feed, poultry feed, etc.).
  • byproducts e.g. , WS, WDG, TS, Syrup, etc.
  • a CBP process Fig. 1&9A
  • byproducts are pretreated prior to use in a CBP process (Fig. 10)
  • the amount, and identity, of byproducts committed to a CBP process is dependent upon one or more front-end fractionation processes (Fig. 9D, 11, & 12).
  • a feedstock of a host plant is fractionated to produce fractionated products (e.g., a fiber-rich stream, which can comprise hemicelluloses and lignocelluloses; a germ-rich stream, which can comprise protein and oil; and a starch-rich stream, which can comprise soluble carbohydrates).
  • the fiber-rich fraction is dedicated to the CBP process.
  • the germ-rich stream is dedicated to a CBP process.
  • the germ-rich stream is pretreated prior to use in a CBP process.
  • the germ-rich stream is pretreated to remove fats and/or oils.
  • byproducts e.g. , WDG, TS, WS, syrup, etc.
  • byproducts e.g. , WDG, TS, WS, syrup, etc.
  • WDG, TS, WS, syrup, etc. from the host plant operating on the starch-rich stream are committed to the CBP process.
  • byproducts e.g. , WDG, TS, WS, syrup, etc.
  • any combination of front-end fractionated products and/ or byproducts from a host factory can be committed to a CBP process.
  • one or more byproducts from a host plant are subjected to one or more back-end fractionation processes prior to CBP processing ( Fig. 9B).
  • fractionation involves a filtration centrifuge, a decanter centrifuge, a pressure screen, or a paddle screen. Exemplary processes are disclosed in PCT/US2009/045163, which is hereby incorporated by reference in its entirety.
  • a byproduct is fractionated into a protein-rich and/ or oil-rich stream and a fiber-rich stream.
  • the fiber -rich stream is fed into a CBP process.
  • the fiber -rich stream from a front end or back end fractionation process contains a high percentage of C5 carbohydrates.
  • C5 carbohydrates make up between about 5% and 50% of the total fiber content, for example between 5% and 10%, 5% and 15%, 5% and 20%, 5% and 25%, 5% and 30%, 5% and 35%, 5% and 40%, 5% and 45%, 5% and 50%, 10% and 15 %, 10% and 20%, 10% and 25 %, 10% and 30%, 10% and 35 %, 10% and 40%, 10% and 45 %, 10% and 50%, 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, 15% and 45%, 15% and 50%, 20% and 25%, 20% and 30%, 20% and 35%, 20% and 40%, 20% and 45%, 20% and 50%, 25 % and 30%, 25 % and 35 %, 25% and 40%, 25% and 45 %, 25% and 50%, 30% and 35%, 30% and 40%, 30% and 45%, 30% and 50%, 35% and 40%, 35% and 45%, 35% and 50%, 40% and 45%, 40% and 50%, or 45% and 50%.
  • C5 carbohydrates make up about 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%, 3 1 %, 32%, 33 %, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43 %, 44%, 45%, 46%, 47%, 48%, 49%, or 50 % of the total fiber content of a fiber-rich stream.
  • a total yield of ethanol or other fermentation end-product per dried weight of plant material is proportional to the amount of feed streams committed to CBP process.
  • the plant material is corn.
  • the plant material comprises oats, wheat, barley, rice, sugar cane, sugar beets, sorghum (milo), cassava, soft or hard woods, bagasse, stover, algae, peel, seed cake, seeds, sugar beets, or wood chips.
  • the plant material consists essentially of oats, wheat, barley, rice, sugar cane, sugar beets, sorghum (milo), cassava, soft or hard woods, bagasse, stover, algae, peel, seed cake, seeds, sugar beets, or wood chips.
  • a CBP process increases the yield of ethanol or another chemical product by about 1 -50%. In another embodiment, a CBP process increases the yield of ethanol or another chemical product by about 5-20%. In another embodiment, a CBP process increases the yield of ethanol or another chemical product by about 10-30%. In another embodiment, a CBP process increases the yield of ethanol or another chemical product by about 20-40%.
  • a CBP process increases the yield of ethanol or another chemical product by greater than 20%. In another embodiment, the total yield is increased by diverting high percentage of feed streams to a CBP process. In some embodiments, a CBP process increases ethanol or another chemical product by 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%, or 50%.
  • the total yield of combined CBP and fermentation process is about 2-fold higher than the yield of fermentation process alone. In another embodiment, the total yield of combined CBP and fermentation process is about 3- fold higher than the yield of fermentation process alone. In another embodiment, the total yield of combined CBP and fermentation process is about 4-fold higher than the yield of fermentation process alone. In another embodiment, the total yield of combined CBP and fermentation process is about 5- fold higher than the yield of fermentation process alone. In another embodiment, the total yield of combined CBP and fermentation process is up to about 6-fold higher than the yield of a fermentation process alone.
  • Microorganisms useful in these compositions and methods 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. bifermentans, C. botulinum, C. butyricum, C. cadaveris, C. chauvoei, C. clostridioforme, C. colicanis, C. difficile, C. fallax, C. formicaceticum, C. histolyticum, C. innocuum, C. Ijungdahlii, C. laramie, C.
  • yeast that can be utilized in co-culture methods described herein 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 lipoUtica, 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.
  • Anaerobic digestion usually takes place in one of three temperature ranges: psychrophilic (less than 20°C), mesophilic (between 20°C and 40°C), and thermophilic (between 40°C and 70°C).
  • digestion is performed by a psychrophilic microorganism.
  • digestion is performed by a thermophilic microorganism.
  • digestion is performed by a mesophilic microorganism.
  • the microorganism is a naturally mesophilic microorganism.
  • the microorganism is genetically modified to be a mesophilic microorganism.
  • carbonaceous byproducts are fermented at a temperature optimal for mesophilic digestion rather than thermophilic digestion. Toxicity from residues of pretreatment is lower and the costs of raising temperatures for fermentation are also reduced.
  • a mesophilic microorganism digests carbonaceous byproducts obtained from one or more feed streams.
  • a digester is operated between about 35 - 37°C.
  • a digester is operated between about 20 - 25°C.
  • a digester is operated between about 22 - 27°C.
  • a digester is operated between about 24 - 29°C.
  • a digester is operated between about 26 - 30°C.
  • a digester is operated between about 28 - 32°C. In another embodiment, a digester is operated between about 30 - 35°C. In another embodiment, a digester is operated between about 32 - 37°C. In another embodiment, a digester is operated between about 34 - 39°C. In another embodiment, a digester is operated between about 36 - 40°C. In another embodiment, a digester is operated between about 37- 42°C. In another embodiment, a digester is operated at about 32°C. In another embodiment, a digester is operated at about 37°C. In another embodiment, a digester is operated at about 30°C. In another embodiment, a digester is operated at about 28°C.
  • a microorganism is genetically modified to robustly metabolize carbonaceous byproducts.
  • a microorganism is genetically modified to robustly metabolize carbonaceous byproducts at a mesophilic optimal temperature.
  • the genetic modification comprises genetically engineering a naturally mesophilic microorganism
  • a mesophilic microorganism is a bacterium.
  • a mesophilic microorganism is a species of Clostridium.
  • the species of Clostridium is
  • a microorganism is an isolated Gram-positive bacterium, wherein the bacterium is an obligate anaerobic, mesophilic, cellulolytic organism.
  • a microorganism is an isolated Gram-positive bacterium 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.
  • a microorganism is Clostridium sp. Q.D, having the NRRL patent deposit designation NRRL B-50361.
  • Clostridium sp. Q.D consists of motile rods that form terminal spores. These Gram-positive rods were isolated from a 0.3% Maltose, 5% Azo-CM-Cellulose, QM plate comprising a mutated pool of Clostridium phytofermentans and were cultured in liquid QM media. Endoglucases activity was noted on the plate after four days' incubation at 35° C. The Clostridium sp.
  • Q.D bacterium was distinguishable from Clostridium phytofermentans in that Q.D is a faster-growing colony of larger size having a larger clearing zone in the presence of glucose and 5% Azo-CM- Cellulose plates. It also displays a color modification in the presence of higher concentrations of glucose (2-3% glucose), changing to an orange color.
  • 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. Also C. xylanolyticum has terminal endospores whereas C. algidixylanolyticum has subterminal endospores.
  • a microorganism is an obligate anaerobic mesophile that can ferment carbonaceous byproducts into ethanol, organic acids or another fermentation product.
  • the mesophile degrades cellulose and/or xylose into ethanol and acetic or lactic acid.
  • a mesophilic microorganism is C. phytofermentans, which includes American Type Culture Collection 700394T.
  • a C. phytofermentans carries the phenotypic and genotypic characteristics of a cultured strain, ISDgT (Warnick et al., International Journal of Systematic and Evolutionary Microbiology, 52: 1 155-60, 2002).
  • a C. phytofermentans is a strain derived from ISDgT or another species of Clostridium phytofermentans.
  • the derivation is genetic modification or mutagenesis.
  • the derivation is isolation from nature.
  • Some exemplary species useful for CBP process are defined by 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 strain (ISDgT), and strains with DNA re-association values of at least about 70% can be considered Clostridium phytofermentans. Considerable evidence exists to indicate that many microbes which have 70%) or greater DNA re-association values also have at least 96%> DNA sequence identity and share phenotypic traits defining a species.
  • C. phytofermentans and Clostridium sp. Q.D provide useful advantages for the conversion of carbonaceous byproducts to ethanol and other products.
  • the C. phytofermentans employed in a CBP process produce enzymes capable of hydrolyzing polysaccharides and higher saccharides to lower molecular weight saccharides, oligosaccharides, disaccharides, and
  • C. phytofermentans or Clostridium sp. Q.D employed in a CBP process produces a wide spectrum of hydro lytic enzymes capable of facilitating fermentation of various biomass materials, including cellulosic, hemicellulosic, lignocellulosic materials; pectins; starches; wood; paper; agricultural products; forest waste; tree waste; tree bark; leaves; grasses;
  • 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 can 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.
  • addition of one material provides supplements that fit into more than one category, such as providing amino acids and phytate.
  • C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze various higher saccharides present in biomass to lower saccharides, 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.
  • C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze polysaccharides and higher saccharides such as hexose saccharides.
  • C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze polysaccharides and higher saccharides such as hexose saccharides.
  • phytofermentans is used to hydrolyze polysaccharides and higher saccharides such as pentose saccharides.
  • C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze polysaccharides and higher saccharides that contain both hexose and pentose sugar units.
  • C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze polysaccharides and higher saccharides into lower saccharides or monosaccharides.
  • hydrolysate from C. phytofermentans or Clostridium sp. Q.D treatment is used in a fermentation process to produce one or more fermentation products such as a biofuels.
  • C phytofermentans or Clostridium sp. Q.D is used to produce ethanol, hydrogen, or compounds such as organic acids including acetic acid, formic acid, and lactic acid from a lower sugar such as monosaccharide or a disaccharide.
  • C. phytofermentans or Clostridium sp. Q.D is used 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, or other fermentation end products.
  • C. phytofermentans, Clostridium sp. Q.D, or any variant thereof is used in a CBP process to grow under conditions that include elevated ethanol concentration, high sugar concentration, or low sugar concentration.
  • C. phytofermentans, Clostridium sp. Q.D, or any variant thereof is used in a CBP process to utilize insoluble carbon sources and/or operate under anaerobic conditions.
  • C. phytofermentans or Clostridium sp. Q.D is used in CBP process to achieve operation with long fermentation cycles.
  • the microbe is used in combination with batch fermentations, fed batch fermentations, or self- seeding/partial harvest fermentations.
  • a mesophilic bacterium useful for processes described herein is a species of Clostridium. In another embodiment, a mesophilic bacterium is a species of Bacillus.
  • Bacillus species useful for processes described herein include, but are not limited to, Bacillus subtilis, Bacillus alvei, Bacillus amylolyticus, Bacillus azotofixans, Bacillus glucanolyticus, Bacillus larvae, Bacillus lautus, Bacillus lentimorbus, Bacillus macerans, Bacillus macquariensis, Bacillus pabuli, Bacillus polymyxa, Bacillus popilliae, Bacillus psychrosaccharolyticus, Bacillus pulvifaciens, Bacillus thiaminolyticus, Bacillus avlidus, Bacillus alcalophilus, Bacillus amyloiquefaciens, Bacillus atrophaeus, Bacillus carotarum, Bacillus firmus, Bacillus flexus, Bacillus laterosporus, Bacillus megaterium, Bacillusmycoides, Bacillus niacini, Bacillus pantoth
  • LAFT B94 Lactobacillus acidophilus, Lactobacillus acidophilus LAFTI L10, Lactobacillus casei, Lactobacillus casei LAFTI L26, Bifidobacterium animalis subsp.
  • Lactobacillus acidophilus DDS-1 Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus CD 1285, Lactobacillus casei 431, Lactobacillus casei F19, Lactobacillus casei Shirota, Lactobacillus paracasei, Lactobacillus paracasei Stl 1, Lactobacillus johnsonii, Lactobacillus johnsonii Lai, Lactobacillus lactis, Lactobacillus lactis LI A, Lactobacillus plantarum, Lactobacillus plantarum 299v, Lactobacillus reuteri, Lactobacillus reuteri ATTC 55730, Lactobacillus rhamnosus, Lactobacillus rhamnosus ATCC 53013, Lactobacillus
  • a CBP process described herein is a module that can be readily adapted and attached to any conventional biomass processing plant.
  • a CBP process is attached to a conventional dry milling process upon which various feed streams are drawn to the CBP process for increased productions of ethanol and other fermentation products.
  • a CBP process is attached to a sawmill process upon which sawdust and any soft or hard wood material are drawn to the CBP process for further processing of carbonaceous material therein.
  • a CBP process is attached to wet milling process to increase productions of ethanol and other fermentation products.
  • a CBP process By attaching a CBP process to a known biomass processing plant, byproducts such as undigested carbohydrates, lipid, or proteins are collected and fed into the CBP process in which the byproducts are further processed to produce sugars, ethanol, or other fermentation products.
  • a CBP process reduces waste by directing substantial amounts of byproducts to the CBP process.
  • a CBP process obviates the need for drying or concentrating TS, CDS, or WDG by feeding these byproducts directly to CBP process.
  • residual material is collected at the end of a CBP process and re-fed into a CBP process to achieve complete utilization of any residual carbonaceous material therein.
  • residual material is optionally concentrated, then packaged as an animal food or a food supplement.
  • food or a food supplement is a non- human animal food or a food supplement (e.g. , cattle feed, swine feed, poultry feed, fish feed, sheep feed, etc.).
  • the food or a food supplement is a human food or a food supplement.
  • protein content is separated from the residual material and packaged for commercial application, such as an animal food or a food supplement.
  • lipid content is separated from the residual material and packaged for commercial application.
  • vitamin content is separated from the residual material and packaged for commercial application.
  • a mesophilic organism is utilized to digest carbonaceous material in the plant material fed into a CBP process.
  • a mesophilic organism produces sugars.
  • a mesophilic organism produces ethanol or hydrogen.
  • a mesophilic organism produces one or more fermentation products.
  • a mesophilic microorganism produces acetic acid or lactic acid or both by contacting carbonaceous byproducts from single or combined feed stream (such as 2,3,4,5,6,7,8,9, 10 or more streams).
  • a single byproduct is fed into a CBP process.
  • the byproduct is WS and a mesophilic microorganism produces sugars, ethanol, or fermentation products by contacting WS and digesting carbonaceous material therein.
  • the byproduct is TS and a mesophilic microorganism produces sugars, ethanol, hydrogen or fermentation products by contacting TS and digesting carbonaceous material therein.
  • the byproduct is CDS and a mesophilic microorganism produces sugars, ethanol, hydrogen or fermentation products by contacting CDS and digesting carbonaceous material therein.
  • the byproduct is WDG and a mesophilic microorganism produces sugars, ethanol, hydrogen or fermentation products by contacting WDG and digesting carbonaceous material therein.
  • the byproduct is DG and a mesophilic microorganism produces sugars, ethanol, hydrogen or fermentation products by contacting DG and digesting carbonaceous material therein.
  • the byproduct is syrup and a mesophilic microorganism produces sugars, ethanol, hydrogen or fermentation products by contacting syrup and digesting carbonaceous material therein.
  • the byproduct is WDGS and a mesophilic microorganism produces sugars, ethanol, hydrogen or fermentation products by contacting WDGS and digesting carbonaceous material therein.
  • two or more byproducts are fed into a CBP process, producing a combined carbonaceous byproduct.
  • a mesophilic microorganism produces sugars, ethanol, hydrogen or fermentation products by contacting the combined carbonaceous byproducts.
  • the combined carbonaceous byproducts comprise any two, three or four byproducts selected from the group consisting of WS, TS, DG, WDG, CDS, syrup or WDGS.
  • one or more byproducts are fed into a CBP process without pretreatment. In some embodiments, one or more byproducts are fed into a CBP process following pretreatment. In one embodiment, one or more byproducts are acid pretreated. In one embodiment, one or more byproducts are pretreated with dilute acid. In one embodiment, one or more byproducts are alkaline pretreated. In one embodiment, one or more byproducts are hot water treated. In one embodiment, one or more byproducts are pretreated by steam explosion. In one embodiment, one or more byproducts are subjected to two or more pretreatment processes (e.g., hot water treatment followed by steam explosion). In some embodiments, one or more pretreated and one or more non-pretreated byproducts are combined in a CBP process.
  • the amount, and identity, of byproducts committed to a CBP process is dependent upon one or more front-end fractionation processes (Fig. 9D, 11, & 12).
  • a feedstock of a host plant is fractionated to produce fractionated products (e.g., a fiber-rich stream, which can comprise hemicelluloses and lignocelluloses; a germ-rich stream, which can comprise protein and oil; and a starch-rich stream, which can comprises soluble carbohydrates).
  • the fiber-rich fraction is dedicated to the CBP process.
  • the germ-rich stream is dedicated to a CBP process.
  • the germ-rich stream is pretreated prior to use in a CBP process.
  • the germ-rich stream is pretreated to remove fats and/or oils.
  • byproducts e.g., WDG, TS, WS, syrup, etc.
  • WDG, TS, WS, syrup, etc. from the host plant operating on the starch-rich stream are committed to the CBP process.
  • byproducts e.g., WDG, TS, WS, syrup, etc.
  • any combination of front-end fractionated products and/ or byproducts from a host factory can be committed to a CBP process.
  • a CBP process comprises treating carbonaceous byproducts with a microorganism capable of saccharifying C5/C6 saccharides.
  • a CBP process comprises treating the carbonaceous byproducts with Clostridium phytofermentans or another
  • Clostridium species such as Clostridium sp. Q.D under conditions wherein the Clostridium
  • a CBP process comprises treating the carbonaceous byproducts with a microorganism capable of saccharifying C5/C6 saccharides and adding one or more enzymes to aid in the breakdown or detoxification of carbohydrates or lignocellulosic material.
  • a CBP process comprises treating the carbonaceous byproducts in a container with a Clostridium phytofermentans or another similar C5/C6 Clostridium species and adding one or more enzymes to aid in the breakdown or detoxification of carbohydrates or lignocellulosic material.
  • a CBP process comprises, after fermentation with a first microorganism (such as Clostridium phytofermentans or Clostridium sp. Q.D), contacting carbonaceous byproducts and with a second microorganism where the second organism is capable of substantially converting the monosaccharides and disaccharides into a desired fermentation products, such as a fuel ⁇ e.g. ethanol or butanol).
  • a first microorganism such as Clostridium phytofermentans or Clostridium sp. Q.D
  • a second microorganism where the second organism is capable of substantially converting the monosaccharides and disaccharides into a desired fermentation products, such as a fuel ⁇ e.g. ethanol or butanol.
  • the second microorganisms is a fungi. In another embodiment the second microorganism is a yeast. In another embodiment the second microorganism is Saccharomyces bayanus , Saccharomyces boulardii , Saccharomyces bulderi , Saccharomyces cariocanus ,
  • Saccharomyces cariocus Saccharomyces cerevisiae , Saccharomyces chevalieri , Saccharomyces dairenensis , Saccharomyces ellipsoideus , Saccharomyces martiniae , Saccharomyces monacensis , Saccharomyces norbensis , Saccharomyces paradoxus , Saccharomyces pastorianus , Saccharomyces spencerorum , Saccharomyces turicensis , Saccharomyces unisporus , Saccharomyces uvarum , Saccharomyces zonatus.
  • the second microorganism is Saccharomyces or Candidia.
  • the second microorganism is a Clostridia species such as C.
  • thermocellum C. acetobutylicum, and C. cellovorans, or Zymomonas mobilis.
  • CBP processes described herein can increase the value of an animal feed product produced from fermentation byproducts by at least a factor of 1.1, 1.2. 1.3, 1.4, 1.5, 1.6, 1.7,
  • processes described herein cuts the volume to process these byproducts by about 50%.
  • a digestion is performed at a temperature of about 30 °C to about 45 °C and at a pH of about 6 to 7. In another embodiment, a digestion is performed at about 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C.
  • a digestion is performed at about pH 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 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,
  • adapting a biomass processing plant with a CBP process to ferment byproducts produces ethanol about 10 g/1 to 20 g/1 in 2-6 days or less.
  • adapting a biomass processing plant with a CBP process produces ethanol about 10 g/1, 1 1 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, 25g/L, 26 g/L, 27 g/L, 28 g/L, 29 g/L, 30 g/L , 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g
  • a microorganism that produces a fermentative product tolerates the presence of high alcohol (e.g. ethanol or butanol) concentrations.
  • Clostridium phytofermentans or Clostridium sp. Q.D tolerates the presence of high alcohol (e.g. ethanol or butanol) concentrations.
  • a microorganism described herein grows in alcohol (e.g. ethanol or butanol) concentrations up to about 15% v/v.
  • a microorganism described herein grows in alcohol (e.g.
  • ethanol or butanol concentrations of up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, or 15% v/v.
  • functioning in high alcohol concentrations includes the ability to continue to produce alcohol without undue inhibition or suppression by alcohol and/or other components present; the ability to efficiently convert hexose and pentose carbon sources in a carbonaceous byproducts feedstock to a fermentation product such as an alcohol.
  • ethanol concentration can attain a plateau of about 15 g/L after about 36 - 48 hours of batch fermentation, with carbon substrate remaining in the medium.
  • the fermentation pH is lowered to about 6.5.
  • unsaturated fatty acids are added to the fermentation medium to significantly increase the amount of ethanol produced by the organism.
  • a combination of reduced pH and addition of unsaturated fatty acids increases ethanol production to about 20 g/L L or more in the medium following a 48 - 120 hrs or longer fermentation.
  • the productivity of a microorganism is higher (about 10 g/L-d) when the ethanol titer is low (about 2 g/L-d).
  • fermentation at reduced pH and/or with the addition of a lipid (e.g. , fatty acids) can result in about a two to ten fold (such as a 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 1 Ox increase) or higher increase in the ethanol production rate as compared to the unadjusted fermentation medium.
  • both hexose and pentose saccharides are simultaneously fermented in a CBP process to increase ethanol productivity and/or yield.
  • the simultaneous fermentation of hexose and pentose carbohydrate substrates is utilized in combination with
  • a lipid e.g. , fatty acids
  • a lipid is a fat or oil, including without limitation the glyceride esters of fatty acids along with associated phosphatides, sterols, alcohols, hydrocarbons, ketones, and related compounds.
  • a lipid is a phospholipid.
  • a fatty acid is an aliphatic or aromatic monocarboxylic acid.
  • a fatty acid is an unsaturated fatty acid.
  • an unsaturated fatty acid is a fatty acid with 1 to 3 double bonds.
  • a "highly unsaturated fatty acid” is a fatty acid with 4 or more double bonds.
  • an unsaturated fatty acid is an omega-3 highly unsaturated fatty acid, such as eicosapentaenoic acid, docosapentaenoic acid, alpha linolenic acid, docosahexaenoic acid, and conjugates thereof.
  • a fatty acid is a saturated fatty acid.
  • a fatty acid is a vegetable oil, such as partially hydrogenated, include palm oil, cottonseed oil, corn oil, peanut oil, palm kernel oil, babassu oil, sunflower oil, safflower oil, or mixtures thereof.
  • a composition comprising a fatty acid further comprises a wax, such as beeswax, petroleum wax, rice bran wax, castor wax, microcrystalline wax, or mixtures thereof.
  • carbonaceous byproducts are pre-treated with a surfactant prior to fermentation with a microorganism.
  • carbonaceous byproducts are contacted with a surfactant during fermentation with a microorganism.
  • the surfactant is a Tween series of surfactant (e.g., Tween 20 or Tween 80) or a Triton series of surfactant (e.g. Triton X- 100).
  • the surfactant is polysorbate 60, polysorbate 80, propylene glycol, sodium dioctylsulfoesuccmate, sodium lauryl sulfate, lactylic esters of fatty acids, polyglycerol esters of fatty acids, or mixtures thereof.
  • carbonaceous byproducts are pre-treated with a surfactant and a lipid prior to fermentation with a microorganism.
  • carbonaceous byproducts are contacted with a surfactant and a lipid during fermentation with a microorganism.
  • a process for producing a biofuel or other chemical from a lignin-containing carbonaceous byproducts. The process comprises: 1) contacting the lignin-containing carbonaceous byproducts with an aqueous acid solution at a concentration sufficient to hydrolyze at least a portion of the lignin-containing carbonaceous byproducts; 2) neutralizing the treated
  • Q.D optionally with the addition of one or more enzymes to the container, substantially converts the treated carbonaceous byproducts into monosaccharides and disaccharides, and/or biofuel or other fermentation product; and 4) optionally, introducing a culture of a second microorganism wherein the second organism is capable of substantially converting the monosaccharides and disaccharides into a fermentation product, such as a biofuel.
  • CBP process utilizes a Clostridium biocatalyst, which can
  • one or more modification of conditions for hydrolysis and/or fermentation is implemented to enhance end-product production.
  • 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, genetic modifications to disrupt the expression of one or more metabolic pathway genes to direct, 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.
  • inventions 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.), decarboxylases (e.g. pyruvate decarboxylase), dehydrogenases (e.g. alcohol dehydrogenase), and synthetases (e.g. Acetyl CoA synthetase).
  • hydrolytic enzymes such as cellulase(s), hemicellulase(s), or pectinase(s) etc.
  • decarboxylases e.g. pyruvate decarboxylase
  • dehydrogenases e.g. alcohol dehydrogenase
  • synthetases e.g. Acetyl CoA synthetase
  • a method is used to genetically modify a microorganism (such as a Clostridium species) that is disclosed in US 20100086981 or PCT/US2010/40494, which are herein incorporated by reference in their entirety.
  • a microorganism such as a Clostridium species
  • 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.
  • 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.
  • 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 e.g., operably linking genes encoding one or more enzymes to an inducible promoter or a combination thereof.
  • a variety of promoters can be used to drive expression of the heterologous genes in a recombinant host microorganism.
  • 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.
  • SEQ ID NO: 61 contains the sequence of this vector (pMTL82351-P3558-3202) inserted DNA.
  • Promoters typically used in recombinant technology such as E. coli lac and trp operons, the tac promoter, the bacteriophage pL promoter, bacteriophage T7 and SP6 promoters, beta-actin promoter, insulin promoter, baculo viral polyhedrin and plO promoter, can be used to initiate transcription.
  • a constitutive promoter can be used including, but not limited to the int promoter of bacteriophage lamda, the bla promoter of the beta- lactamase gene sequence of pBR322, hydA or thlA in Clostridium, S. coelicolor hrdB, or whiE, the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, Staphylococcal constitutive promoter blaZ and the like.
  • an inducible promoter can be used that regulates the expression of downstream gene in a controlled manner, such as under a specific condition of a cell culture.
  • inducible prokaryotic promoters include, but are not limited to, the major right and left promoters of bacteriophage, the trp, reca, lacZ, AraC and gal promoters of E. coli, the alpha-amylase (Ulmanen Ett at., J. Bacteriol. 162: 176-182, 1985, which is herein incorporated by reference in its entirety) and the sigma-28-specific promoters of B.
  • subtilis (Gilman et ah , Gene sequence 32: 1 1 -20 (1984) , which is herein incorporated by reference in its entirety), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982) , which is herein incorporated by reference in its entirety), Streptomyces promoters (Ward et at., Mol. Gen. Genet. 203 :468-478, 1986, which is herein incorporated by reference in its entirety), and the like. Exemplary prokaryotic promoters are reviewed by Glick (J. Ind. Microtiot.
  • a promoter that is constitutively active under certain culture conditions can be inactive in other conditions.
  • the promoter of the hydA gene from Clostridium acetobutylicum wherein expression is known to be regulated by the environmental pH.
  • temperature-regulated promoters are also known and can be used.
  • a pH-regulated or temperature-regulated promoter can be used with an expression constructs to initiate transcription.
  • Other pH-regulatable promoters are known, such as PI 70 functioning in lactic acid bacteria, as disclosed in US Patent Application No. 20020137140, which is herein incorporated by reference in its entirety.
  • promoters can be used; e.g. , the original promoter of the gene, promoters of antibiotic resistance genes such as for instance kanamycin resistant gene of Tn5, ampicillin resistant gene of pBR322, and promoters of lambda phage and any promoters which can be functional in the host cell.
  • promoters of antibiotic resistance genes such as for instance kanamycin resistant gene of Tn5, ampicillin resistant gene of pBR322, and promoters of lambda phage and any promoters which can be functional in the host cell.
  • other regulatory elements such as for instance a Shine-Dalgarno (SD) sequence (e.g.
  • promoters examples include those disclosed in the following patent documents: US20040171824, US 6410317, WO 2005/024019 , which are herein incorporated by reference in their entirety.
  • Several promoter- operator systems such as lac, (D. V. Goeddel et al , "Expression in Escherichia coli of Chemically Synthesized Genes for Human Insulin", Proc. Nat. Acad. Sci. U.S.A., 76: 106-1 10 (1979) , which is herein incorporated by reference in its entirety); trp (J. D. Windass et al.
  • Repressors are protein molecules that bind specifically to particular operators.
  • the lac repressor molecule binds to the operator of the lac promoter-operator system, while the cro repressor binds to the operator of the lambda pR promoter.
  • Other combinations of repressor and operator are known in the art. See, e.g., J. D. Watson et al, Molecular Biology Of The Gene, p. 373 (4th ed. 1987), which is herein incorporated by reference in its entirety.
  • the structure formed by the repressor and operator blocks the productive interaction of the associated promoter with RNA polymerase, thereby preventing transcription.
  • Other molecules termed inducers, bind to repressors, thereby preventing the repressor from binding to its operator.
  • inducers bind to repressors, thereby preventing the repressor from binding to its operator.
  • Analogous promoter-operator systems and inducers are known in other microorganisms.
  • yeast the GALI O and GALl promoters are repressed by extracellular glucose, and activated by addition of galactose, an inducer.
  • Protein GAL80 is a repressor for the system, and GAL4 is a transcriptional activator. Binding of GAL80 to galactose prevents GAL80 from binding GALA Then, GAL4 can bind to an upstream activation sequence (UAS) activating transcription. See Y.
  • UAS upstream activation sequence
  • Mata2 is a temperature-regulated promoter system in yeast. A repressor protein, operator and promoter sites have been identified in this system. A. Z. Sledziewski et al , "Construction Of Temperature-Regulated Yeast Promoters Using The Mata2 Repression System", Bio/Technology, 6:41 1 -16 (1988), which is herein incorporated by reference in its entirety.
  • CUP1 promoter Another example of a repressor system in yeast is the CUP1 promoter, which can be induced by Cu 2 ions.
  • the CUP1 promoter is regulated by a metallothionine protein. J. A. Gorman et al ,
  • 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.
  • the DNA sequence of the plasmid pIMPCphy is provided as SEQ ID NO: 1.
  • 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).
  • 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 C. phytofermentans .
  • 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.
  • a DNA sequence comprising genetic material from a first microorganism is provided, 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 C. phytofermentans.
  • the DNA sequence encodes for an expression product that is a protein, or fragment thereof, from C. phytofermentans.
  • the first microorganism is a Clostridium species and the second microorganism is bacteria or yeast, e.g. E. coli.
  • a mesophilic microorganism is modified to disrupt the expression of one or more metabolic pathway genes ⁇ e.g. lactate dehydrogenase).
  • the organism can be a naturally- occurring mesophilic organism or a mutated or recombinant organism.
  • wild-type refers to any of these organisms with metabolic pathway gene activity that is normal for that organism
  • a non "wild- type” knockout is the wild-type organism that has been modified to reduce or eliminate activity of a metabolic pathway gene, e.g. lactate dehydrogenase activity compared to the wild-type activity level of that enzyme.
  • the nucleic acid sequence for a gene of interest ⁇ e.g. lactate dehydrogenase
  • a target gene ⁇ e.g. lactate dehydrogenase
  • the lactate dehydrogenase gene is inactivated by the integration of a plasmid that achieves natural homologous recombination or integration between the plasmid and the microorganism's chromosome.
  • Chromosomal integrants can be selected for on the basis of their resistance to an antibacterial agent (for example, kanamycin).
  • the integration into the lactate dehydrogenase gene can occur by a single cross-over recombination event or by a double (or more) cross-over recombination event.
  • an effective form is an expression vector.
  • the DNA construct is a plasmid or vector.
  • the plasmid comprises the nucleic acid sequence of SEQ ID NO: 2.
  • the plasmid comprises a nucleic acid with 70-99.9% similarity to the sequence of SEQ ID NO: 2.
  • the plasmid comprises a nucleic acid with 70% similarity to the sequence of SEQ ID NO: 2.
  • the plasmid comprises a nucleic acid with 75% similarity to the sequence of SEQ ID NO: 2.
  • the plasmid comprises a nucleic acid with 80% similarity to the sequence of SEQ ID NO: 2.
  • the plasmid comprises a nucleic acid with 85%) similarity to the sequence of SEQ ID NO: 2. In another embodiment, the plasmid comprises a nucleic acid with 90%> similarity to the sequence of SEQ ID NO:2. In another embodiment, the plasmid comprises a nucleic acid with 95% similarity to the sequence of SEQ ID NO: 2. In another
  • the plasmid comprises a nucleic acid with 99% similarity to the sequence of SEQ ID NO: 2.
  • the DNA construct can only replicate in the host microorganism through recombination with the genome of the host microorganism.
  • the pMA-0923071 plasmid lacks a gram positive origin of replication, and contains chloramphenicol acetyltransferase (catP) and kanamycin acetyltransferase sites, conferring
  • FIG. 6 A general illustration of an integrating replicative plasmid, pQInt, is shown in Fig. 6.
  • 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.
  • FIG. 7 and Fig. 8 Another embodiment, depicted in Fig. 7 and Fig. 8, 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.
  • a gram-negative origin of replication for propagation of the plasmid in E. coli or other gram-negative host(s).
  • 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.
  • a selectable marker typically a gene encoding antibiotic resistance.
  • 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.
  • An additional element for conjugal transfer of plasmid DNA is an optional element described in certain embodiments.
  • the DNA constructs in these embodiments can also incorporate a suitable reporter gene as an indicator of successful transformation.
  • the reporter gene is an antibiotic resistance gene, such as a kanamycin, ampicillin or chloramphenicol resistance gene.
  • the DNA constructs can also incorporate multiple reporter genes, as appropriate.
  • microorganisms described herein can be cultured under conventional culture conditions, depending on the mesophilic microorganism chosen.
  • the choice of substrates, temperature, pH and other growth conditions can be selected based on known culture requirements, for example see WO01/49865 and WO01/85966, the content of each being incorporated herein by reference in their entirety.
  • compositions and methods are provided to produce a fermentation product such as one or more alcohols, e.g., ethanol or other fermentation products, by the creation and use of a genetically modified microorganism.
  • the genetically modified microorganism is Clostridium phytofermentans.
  • a genetic modification is to a nucleic acid sequence that regulates or encodes a protein related to a fermentative biochemical pathway, expression of saccharolytic enzymes, or increasing tolerance of environmental conditions during fermentation.
  • the genetic modification is to a nucleic acid sequence in a microorganism.
  • the microorganism is transformed with polynucleotides encoding one or more genes for the pathway, enzyme, or protein of interest. In another embodiment, the microorganism is transformed to produce multiple copies of one or more genes for the pathway, enzyme, or protein of interest. In some embodiments, the polynucleotide transformed into the microorganism is heterologous. In other embodiments, the polynucleotide is derived from the microorganism.
  • the microorganism is transformed with heterologous polynucleotides encoding one or more genes encoding enzymes for the fermentation of a hexose, wherein said genes are expressed at sufficient levels to confer upon said microorganism transformant the ability to produce ethanol at increased concentrations, productivity levels or yields compared to a microorganism that is not transformed.
  • the microorganism is transformed with heterologous polynucleotides encoding one or more genes encoding enzymes for the fermentation of a pentose, wherein said genes are expressed at sufficient levels to confer upon said microorganism transformant the ability to produce ethanol or other products at increased concentrations, productivity levels or yields compared to a microorganism that is not transformed.
  • the microorganism is transformed with a combination of enzymes for fermentation of hexose and pentose saccharides. In some embodiments, an enhanced rate of product production can be achieved.
  • the microorganism is transformed with heterologous polynucleotides encoding one or more genes encoding saccharolytic enzymes for the saccharification of a polysaccharide, wherein said genes are expressed at sufficient levels to confer upon the transformed microorganism an ability to saccharify a polysaccharide to mono-, di- or oligosaccharides at increased concentrations, rates of saccharification or yields of mono-, di- or oligosaccharides compared to a microorganism that is not transformed.
  • the genetic modification is to a nucleic acid sequence of a Clostridium phytofermentans or Clostridium sp. Q.D.
  • the Clostridium phytofermentans or Clostridium sp. Q.D is transformed with polynucleotides encoding one or more genes for the pathway, enzyme, or protein of interest.
  • the Clostridium phytofermentans or Clostridium sp. Q.D is transformed to produce multiple copies of one or more genes for the pathway, enzyme, or protein of interest.
  • the polynucleotide transformed into the Clostridium phytofermentans is heterologous.
  • the polynucleotide is derived from
  • Q.D is transformed with heterologous polynucleotides encoding one or more genes encoding enzymes for the fermentation of a pentose, wherein said genes are expressed at sufficient levels to confer upon said Clostridium phytofermentans or Clostridium sp.
  • Q.D transformant the ability to produce ethanol or other products at increased concentrations, productivity levels or yields compared to a Clostridium phytofermentans or Clostridium sp. Q.D that is not transformed.
  • the Clostridium phytofermentans or Clostridium sp. Q.D is transformed with a combination of enzymes for fermentation of hexose and pentose saccharides. In some embodiments, an enhanced rate of product production can be achieved.
  • the Clostridium phytofermentans or Clostridium sp. Q.D is transformed with heterologous polynucleotides encoding one or more genes encoding saccharolytic enzymes for the saccharification of a polysaccharide, wherein said genes are expressed at sufficient levels to confer upon said Clostridium phytofermentans or Clostridium sp.
  • Q.D transformant the ability to saccharify a polysaccharide to mono-, di- or oligosaccharides at increased concentrations, rates of saccharification or yields of mono-, di- or oligosaccharides compared to a Clostridium phytofermentans or Clostridium sp. Q.D that is not transformed.
  • saccharolytic DNA can be native to the host, although more often the DNA will be foreign, i.e., heterologous.
  • Advantageous saccharolytic genes include cellulolytic, xylanolytic, and starch-degrading enzymes such as cellulases, xylanases, glucanases, glucosidases, and amylases.
  • the saccharolytic enzymes can be at least partially secreted by the host, or it can be accumulated substantially intracellularly for subsequent release. Combinations of enzymes can be encoded by the heterologous DNA, some of which are secreted, and some of which are accumulated.
  • 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.
  • transcriptional regulators genes for the formation of organic acids or other chemical products, 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.
  • 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 Clostridium phytofermentans or Clostridium sp. Q.D to promote homologous recombination.
  • 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.
  • 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.
  • a microorganism 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 described herein 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):2304-231 1 , disclosing expression of xylose isomerase in S. cerevisiae and which is herein incorporated by reference in its entirety).
  • 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 1 15 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
  • 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 1.
  • a mesophilic microorganism is modified to disrupt the expression of one or more lactic acid synthesis pathway genes. Inactivating the lactate dehydrogenase gene helps prevent the breakdown of pyruvate into lactate, and therefore promotes, under appropriate conditions, the breakdown of pyruvate into ethanol using pyruvate decarboxylase and alcohol dehydrogenase.
  • one or more naturally- occurring lactate dehydrogenase genes are disrupted by a deletion within or of the gene.
  • lactate dehydrogenase is reduced or eliminated by a chemically-induced or naturally-occurring mutation.
  • a mesophilic microorganism is modified to disrupt the expression of one or more lactate dehydrogenase pathway genes. In one embodiment, a mesophilic microorganism is modified to disrupt the expression of one or more lactate dehydrogenase genes.
  • the nucleic acid sequence for a lactate dehydrogenase can be used to target the lactate dehydrogenase gene to inactivate the gene through different mechanisms.
  • a lactate dehydrogenase gene is inactivated by the insertion of a transposon, or by the deletion of the gene sequence or a portion of the gene sequence.
  • the lactate dehydrogenase gene is inactivated by the integration of a plasmid that achieves natural homologous recombination or integration between the plasmid and the microorganism's chromosome. Chromosomal integrants can be selected for on the basis of their resistance to an antibacterial agent (for example, kanamycin).
  • the integration into the lactate dehydrogenase gene can occur by a single cross-over recombination event or by a double (or more) cross-over recombination event.
  • a recombinant organism wherein the organism lacks expression of LDH or demonstrates reduced synthesis of lactate is useful for the biofuel processes disclosed herein.
  • the recombinant microorganism used for the biofuel processes is C. phytofermentans demonstrating little or no expression of LDH.
  • a recombinant microorganism used for the biofuel processes is C. phytofermentans showing lactic acid synthesis of 100- 90%, 90- 80%, 80-70%, 70-60%, 60-50%, 50-40%, 40- 30%, 30-20%, 20%-10% , or lower, compared to the wild-type organism.
  • a recombinant microorganism used for the generation of a fermentation end-product is a C5/C6 hydrolyzing and fermenting microorganism ⁇ e.g. , Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or genetically-modified cells thereof) lacking LDH activity.
  • the microorganism is capable of enhanced production of biofuel(s) or chemical(s) as compared to a wild-type microorganism.
  • a microorganism engineered to knockout or reduce naturally- occurring lactate dehydrogenase is useful for producing ethanol and other chemical products, fermentive end products and/or bio fuels at a higher yield than that of natural, wild-type microorganism
  • a genetically modified microorganism such as a Clostridium species expressing reduced yields of lactic acid produces ethanol at a rate measurably faster than a corresponding wild-type microorganism, such as a Clostridium species that does not incorporate LDH knockout DNA construct.
  • a genetically modified microorganism comprises one or more heterologous genes in addition to an LDH knockout DNA construct.
  • the heterologous gene is a cellulase, a xylanase, a hemicellulase, an endoglucanase, an exoglucanase, a cellobiohydrolase (CBH), a beta-glycosidase, a glycoside hydrolase, a glycosyltransferase, a lysase, an esterase, a chitinase, or a pectinase.
  • CBH cellobiohydrolase
  • the genetically modified microorganism that is further transformed is a Clostridium strain.
  • the Clostridium strain is C. phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or genetically-modified cells thereof.
  • the heterologous gene is an acetic acid or formic acid knockout DNA construct.
  • the acetic acid knockout DNA construct comprises all or part of: a phosphotransacetylase (PTA) gene, such as Cphy_1326, an acetyl kinase gene, such as Cphy_1327, and/or a pyruvate formate lyase gene such as Cphy l 174. (See Table 2.)
  • PTA phosphotransacetylase
  • Cphy_1326 Cphy_1326
  • Cphy_1327 acetyl kinase gene
  • a pyruvate formate lyase gene such as Cphy l 174.
  • the genetically modified microorganism that is further transformed is a Clostridium strain.
  • the Clostridium strain is C. phytofermentans, Clostridium, sp. Q.D, Clostridium
  • phytofermentans Q.8 Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or genetically-modified cells thereof.
  • Glycolysis is the metabolic pathway that converts glucose, ⁇ nOe, into pyruvate
  • Glucose enters the glycolysis pathway by conversion to glucose-6-phosphate. Early in this pathway, the hexose, fructose-6-bisphosphate, is split into two triose sugars, dihydroxyacetone phosphate, a ketone, and glyceraldehyde 3-phosphate, an aldehyde, thus two molecules of pyruvate are generated for each glucose molecule that is metabolized.
  • Anaerobic organisms lack a respiratory chain. They must reoxidize NADH produced in glycolysis through some other reaction, because NAD + is needed for the glyceraldehyde-3 -phosphate dehydrogenases reaction. Usually NADH is reoxidized as pyruvate is converted to a more reduced compound. For example, lactate dehydrogenase catalyzes the reduction of the keto group in pyruvate to a hydroxyl, yielding lactate, as NADH is oxidized to NAD + . In C. phytofermentans or Q.D, very little lactate dehydrogenase is synthesized however.
  • 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.
  • a redirection of glycolytic or solventogenic pathways can be used to alter the yield of end products such as ethanol or used to reduce ethanol inhibition.
  • a heterologous alcohol dehydrogenase for example, the adhB enzyme from Zymomonas mobilis
  • a microorganism for example a Clostridium species (e.g. Clostridum
  • overexpression of an alcohol dehydrogenase tolerant to high ethanol titers can boost the ethanol production to 50, 55, 60, 65, 70, and even 75 g/L, thus generating higher overall yields.
  • a microorganism can be modified to enhance an activity of one or more decarboxylases ⁇ e.g. pyruvate decarboxylase), dehydrogenases ⁇ e.g. alcohol dehydrogenase), synthetases ⁇ e.g. Acetyl CoA synthetase) or other enzymes associated with glycolic processing).
  • decarboxylases e.g. pyruvate decarboxylase
  • dehydrogenases e.g. alcohol dehydrogenase
  • synthetases e.g. Acetyl CoA synthetase
  • incorporation of a pyruvate decarboxylase into an organism such as C. phytofermentans or Q.D can redirect most of the conversion of pyruvate from glycolysis directly into acetaldehyde and subsequently to ethanol, reducing substantially the amount of acetic acid synthesized to practically nothing.
  • the oxidized NAD + can enter back into glycolysis.
  • no acetic acid is synthesized and the small amount of Acetyl-CoA produced is utilized in essential pathways, such as fatty acid synthesis.
  • acetyl-CoA synthetase is overexpressed to recycle the acetic acid synthesized so that additional ATP is generated and there is no buildup of acetic acid product.
  • one or more genes found in Table 3 are heterologously expressed in a microorganism, for example a Clostridium species ⁇ e.g. Clostridum phytofermentans, Clostridium sp. Q.D or a variant thereof).
  • a Clostridium species ⁇ e.g. Clostridum phytofermentans, Clostridium sp. Q.D or a variant thereof.
  • Zymomonas mobilis pyruvate decarboxylase (pdc) is expressed in a microorganism.
  • Z. mobilis alcohol dehydrogenase II (adhB) is expressed in a microorganism.
  • both pdc and adhB from Z. mobilis are expressed in a microorganism.
  • the microorganism is a Clostridium species ⁇ e.g.
  • acetyl- CoA synthetase (acs) from Escherichia coli is heterologously expressed in a microorganism with or without the expression of pdc and/or adhB from Z. mobilus.
  • a recombinant organism disclosed herein can be further genetically modified to reduce or eliminate the expression of lactate dehydrogenase (ldh).
  • a genetically modified microorganism ⁇ e.g. a Clostridium bacterium, e.g. Clostridum phytofermentans, Clostridium sp. Q.D or a variant thereof) expressing a gene from a glycolytic or solventogenic pathway ⁇ e.g. a gene from Table 3, e.g. pyruvate decarboxylase) produces an increased yield of a fermentation end-product ⁇ e.g. an alcohol, e.g. ethanol) as compared to a control strain.
  • a Clostridium bacterium e.g. Clostridum phytofermentans, Clostridium sp. Q.D or a variant thereof
  • a gene from a glycolytic or solventogenic pathway e.g. a gene from Table 3, e.g. pyruvate decarboxylase
  • the increase in production can be 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, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 g/L, or more.
  • This increase can be, for example, at least a 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%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%
  • An increase in yield from a genetically modified microorganism can be 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 or more times the yield of a non-genetically modified microorganism.
  • a species of C. phytofermentans expressing a heterologous pdc gene from Z. mobilis produces 8-10 g/L more ethanol than a control strain under conditions detailed in Example 5.
  • host cells e.g., microorganisms
  • a single transformed cell can contain exogenous nucleic acids encoding an entire glycolytic or solventogenic pathway.
  • a pathway can include genes encoding a pyruvate decarboxylase, a heterologous alcohol dehydrogenase, and/or a synthetase.
  • Such cells transformed with entire pathways and/or enzymes extracted from them can ferment certain components of biomass more efficiently than the naturally- occurring organism.
  • Constructs can contain multiple copies of the same gene, and/or multiple genes encoding the same enzyme from different organisms, and/or multiple genes with mutations in one or more parts of the coding sequences.
  • Other constructs can contain plasmids to disrupt the activity of certain enzymes, such as lactate dehydrogenase (See, for example, U.S. application Serial No. 12/729, 037, which is herein incorporated by reference in its entirety).
  • the nucleic acid sequences encoding the genes can be similar or identical to the endogenous gene.
  • the gene inserted into the microbe's genome does not have an endogenous counterpart. There can be a percent similarity of 70% or more in comparing the base pairs of the sequences. Examples of genes that can be used in the methods described supra are shown in Table 3 (supra) and Table 4.
  • Bacillus subtilis GenBank AF027868.1 expansin (yoaJ) (12919..13617)
  • Bacillus subtilis GenBank AAB84448.1 expansin (yoaJ) Polypeptide sequence
  • Bacillus subtilis GenBank EU585783.1 beta-galactosidase (lacA) Polynucleotide sequence
  • Escherichia coli GenBank AERR01000023.1 toxin
  • KNase mazF 132931..133266
  • more effective biomass fermentation pathways can be created by transforming host cells with multiple copies of enzymes of a pathway and then combining the cells producing the individual enzymes. This approach allows for the combination of enzymes to more particularly match the biomass of interest by altering the relative ratios of the multiple-transformed strains. In one embodiment two times as many cells expressing the first enzyme of a pathway can be added to a mix where the first step of the reaction pathway is a limiting step of the overall reaction pathway.
  • a biofuel plant or process disclosed herein is useful for producing biofuel with a microorganism engineered to knockout or reduce naturally- occurring lactate
  • LDH knockout dehydrogenase
  • An LDH knockout is useful for increasing yields of ethanol or other biofuels, or other chemical products from the hydrolysis of biomass in comparison to other mesophilic fermenting microorganisms.
  • a mesophilic LDH knockout can be used for reducing the amount of lactic acid in the yield of ethanol or other biofuels or fermentive end products.
  • an LDH knockout construct can be expressed in a microorganism that does not express pyruvate carboxylase.
  • an LDH knockout construct can be expressed in a microorganism that does not produce ethanol as a primary product of its metabolic process.
  • a microorganism that does not produce ethanol as a primary product can be a naturally occurring, or a genetically modified microorganism.
  • the microorganism in a microorganism producing ethanol, lactic acid and acetic acid, the microorganism can be engineered to produce undetectable amount of lactic acid and acetic acid.
  • the microorganism can further be engineered to express an acetic acid knockout and/or a formic acid knockout.
  • increased fermentive yield activity is obtained by transforming a microorganism with an LDH knockout construct.
  • the microorganism is selected from the group of Clostridia.
  • the microorganism is a strain selected from C. phytofermentans .
  • a microorganism comprises a heterologous alcohol dehydrogenase gene and a pyruvate decarboxylase gene.
  • the pyruvated decarboxylase gene can be endogenous or heterologous.
  • the expression of the heterologous genes results in the production of enzymes which redirect the metabolism to yield ethanol as a primary fermentation product.
  • the heterologous genes can be obtained from microorganisms that typically undergo anaerobic fermentation, including Zymomonas species, including Zymomonas mobilis.
  • the wild-type microorganism is mesophilic or thermophilic.
  • the microorganism is a Clostridium species.
  • the Clostridium species is C. phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or genetically-modified cells thereof.
  • the microorganism is cellulolytic.
  • the microorganism is xylanolytic.
  • the microorganism is gram negative or gram positive.
  • the microorganism is anaerobic.
  • Microorganisms selected for modification are said to be "wild-type" and are useful in the fermentation of carbonaceous biomass.
  • the microorganisms can be mutants or strains of Clostridium sp. and are mesophilic, anaerobic, and C5/C6 saccharifying microorganisms.
  • the microorganisms can be isolated from environmental samples expected to contain mesophiles. Isolated wild-type microorganisms will have the ability to produce ethanol but, unmodified, lactate is likely to be a fermentation product.
  • the isolates are also selected for their ability to grow on hexose and/or pentose sugars, and oligomers thereof, at mesophilic (10°C to 40°C) temperatures.
  • the microorganism described herein has characteristics that permit it to be used in a fermentation process.
  • the microorganism should be stable to at least 6% ethanol and should have the ability to utilize C3, C5 and C6 sugars (or their oligomers) as a substrate, including cellobiose and starch.
  • the microorganism can saccharify C5 and C6
  • the microorganism produces ethanol in a yield of at least 50g/l over a 5-8 day fermentation.
  • the microorganism is a spore-former. In another embodiment, the microorganism does not sporulate.
  • the success of the fermentation process does not depend necessarily on the ability of the microorganism to sporulate, although in certain circumstances it can be preferable to have a sporulator, e.g. when it is desirable to use the microorganism as an animal feed-stock at the end of the fermentation process. This is due to the ability of sporulators to provide a good immune stimulation when used as an animal feed-stock. Spore-forming microorganisms also have the ability to settle out during fermentation, and therefore can be isolated without the need for centrifugation.
  • the microorganisms can be used in an animal feed-stock without the need for complicated or expensive separation procedures.
  • 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 described herein 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 these embodiments include but are not limited to bacteria, yeast or fungi that can hydrolyze and ferment feedstock or biomass.
  • 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 described herein is a strain of Clostridia.
  • the microorganism is Clostridium phytofermentans, C. sp. Q.D, or genetically modified variant thereof.
  • Organisms described herein 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 described herein 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).
  • one or more enzymes are utilized in products and processes in these embodiments, 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 described in these embodiments 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 described herein and of a mixture of hexose and pentose saccharides using methods and processes disclosed herein 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.).
  • pentose xylose, xylan, hemicellulose etc.
  • phytofermentans Clostridium sp. Q.D. or variants thereof are capable of hydrolysis and fermentation of C5 and C6 sugars.
  • the wild-type strain of C. phytofermentans and eight lactate dehydrogenase derivative strains (LDH knockout strains) were deposited in the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL)(International Depositary Authority), National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A.
  • phytofermentans Q8 (NRRL B-50351), C. phytofermentans 1117-1( NRRL B-50352), C. phytofermentans 1117-2 (NRRL B-50353), C. phytofermentans 1117-3 (NRRL B-50354), C.
  • phytofermentans 1117-4 (NRRL B-50355), C. phytofermentans 1232-1 (NRRL B-50356), C.
  • phytofermentans 1232-4 (NRRL B-50357), C. phytofermentans 1232-5 (NRRL B-50358), and C. phytofermentans 1232-6 (NRRL B-50359).
  • 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 a Clostridium biocatalyst (e.g., C. phytofermentans, Clostridium Sp. Q.D, C. phytofermentans Q8, Clostridium sp. Q.D-5, Clostridium sp. Q.D-7, Clostridium phytofermentans Q.7D, Clostridium phytofermentans Q.13, Clostridium phytofermentans Q.27, etc.).
  • a Clostridium biocatalyst e.g., C. phytofermentans, Clostridium Sp. Q.D, C. phytofermentans Q8, Clostridium sp. Q.D-5, Clostridium sp. Q.D-7, Clostridium phytofermentans Q.
  • the microorganism is Clostridium phytofermentans. In another embodiment, the microorganism is Clostridium sp. Q.D. In another embodiment, the microorganism is Clostridium phytofermentans Q.8. In another embodiment, the microorganism is Clostridium phytofermentans Q.27. In another embodiment, the microorganism is Clostridium phytofermentans Q.13.
  • a fuel or chemical end-product that includes combining a microorganism such as Clostridium a biocatalyst (such as Clostridium
  • phytofermentans Clostridium sp. Q.D, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13 or a similar species of Clostridium that hydrolyzes 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 fermentation end-product ⁇ e.g., ethanol, propanol, methane, or hydrogen
  • a process for producing a fermentation end-product from biomass that is pretreated.
  • the biomass is pretreated by acid hydrolysis.
  • the biomass is pretreated by hot water treatment.
  • the biomass is pretreated by alkaline pretreatment.
  • the biomass is pretreated by steam explosion.
  • a process is provided for producing a fermentation end-product from biomass using enzymatic hydrolysis pretreatment.
  • a process is provided for producing a fermentation end-product from biomass using biomass that has not been enzymatically pretreated.
  • 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.
  • fermentation end-products made by any of the processes described herein.
  • Those skilled in the art will appreciate that a number of genetic modifications can be made to the methods exemplified herein.
  • a variety of promoters can be utilized to drive expression of the heterologous genes in a recombinant microorganism (such as Clostridium
  • coli B see Ohta et al. (1991) Appl. Environ. Microbiol. 57:893-900.
  • 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 Clostridium phytofermentans, Clostridium, sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or variants thereof, to promote homologous recombination.
  • a fermentation end-product ⁇ e.g., ethanol) from biomass is produced on a large scale utilizing a microorganism, such as C. phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13 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.
  • these embodiments feature 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 phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofennentans Q. I 3, 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.
  • 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 phytofermentans, Clostridium sp. Q.D
  • these embodiments feature methods of making a fermentation end- product or end- products that include combining a C5/C6 hydrolyzing and fermenting microorganism such as a
  • Clostridium biocatalyst ⁇ e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or variants thereof) and 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).
  • the fermentation end-product is a biofuel or chemical product.
  • these embodiments feature 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 hydro lyzing and fermenting
  • 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. 2 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 strong acid e.g. , sulfuric, hydrochloric, or nitric acid with or without preheating/presteaming/water addition.
  • 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 phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, a mutagenized or genetically-modified variant thereof, optionally a co-fermenting microorganism ⁇ . ⁇ . , yeast or E. coli) and, as needed, nutrients to promote growth of the Clostridium cells or other microorganisms.
  • the pretreated biomass or liquid fraction can be split into multiple fermentors, each containing a different strain of Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, 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. 3).
  • a system for production of fermentation end-products comprises: (a) 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. 4 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 such as a Clostridium biocatalyst (e.g., Clostridium phytofermentans, Clostridium sp.
  • a co-fermenting microorganism e.g., yeast or E. coli
  • nutrients to promote growth of a C5/C6 hydrolyzing and fermenting microorganism e.g., Clostridium phytofermentans, Clostridium
  • a C5/C6 hydrolyzing and fermenting microorganism e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, or Clostridium phytofermentans Q.13
  • Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, or Clostridium phytofermentans Q.13 can be used alone or synergistically in combination with one or more other microorganisms (e.g. yeasts, fungi, or other bacteria).
  • different methods can be used within a single plant to produce different end-products.
  • these embodiments feature 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 phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, 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 phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phyto
  • 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 such as a Clostridium biocatalyst (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or mutagenized or genetically- modified cells thereof).
  • a Clostridium biocatalyst e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.8, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or mutagenized or genetically- modified cells thereof.
  • these embodiments feature methods of making a chemical(s) or fuel(s) that include combining a C5/C6 hydrolyzing and fermenting microorganism such as a Clostridium biocatalyst (e.g., Clostridium phytofermentans, Clostridium sp.
  • a Clostridium biocatalyst e.g., Clostridium phytofermentans, Clostridium sp.
  • a chemical(s) or fuel(s) e.g., ethanol, propanol and/or hydrogen or another chemical compound.
  • a process is provided for producing ethanol and hydrogen from biomass using acid hydrolysis pretreatment. In some embodiments, a process is provided 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
  • 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. 5 discloses pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either, fermented separately or together.
  • Fig. 5A depicts a process (e.g., acid pretreatment) that produces a solids phase and a liquid phase which are then fermented separately.
  • Fig. 5B 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. 5C) 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.
  • EXAMPLE 1 Increased ethanol production from a mixture of carbonaceous byproducts via CBP process.
  • the CBP Plant collects a feed stream of stillage from the plant distillation column bottoms. Stillage direct from the distillation column, at 15% solids, is below the approximately 22% solids required to support a 50 g/L cellulosic ethanol fermentation. The required 22% solids concentration that is fed to the fermentor is derived by balancing three streams: plant stillage without additional processing (at approximately 15% solids); plant WDG from the portion of the stillage centrifuged (at approximately 35% solids); and syrup from the plant evaporation system (approximately 35% solids) (Fig. 1).
  • the combined and CBP Plant TS is evaporated and the syrup is added to the CBP feed.
  • the combined WS, WDG, and syrup at 50% stillage diversion results in a feed stream to CBP that is 22% solids, of which approximately 50% are fermentable by a Clostridium biocatalyst after addition of media and seed microbe. This CBP feed stream is pumpable.
  • Feed is pumped from the Host Plant centrifuge and evaporators to the CBP, where it is fed into the fermentors, along with the seed culture (5% of final fermentor volume) and media (10% of final fermentor volume). CBP fermentation takes place over a period of several days, and carbohydrates is converted to ethanol and C02.
  • Product from the CBP fermentors is fed to a beer well and from there to a distillation system specific to the fermentation process with a Clostridium biocatalyst. Because the volume of CBP beer fermentation is relative to plant volume (an additional 40-50% liquid volume). To contain different fermentation byproducts, a new CBP Plant distillation system is installed. Rectification, drying, and product handling systems are maintained separately for the CBP plant.
  • This CBP Plant design locates a cellulosic ethanol plant using a Clostridium biocatalyst adjacent to an existing corn ethanol plant [i.e., Host Plant). While a 100 MGY (million gallons per year) corn ethanol plant was used for design purposes, all results are expected to scale to 90-120+ MGY corn plants with essentially equivalent economics. However, smaller Host Plant sizes will have significantly lower returns due to economies of scale.
  • the CBP process entails the pretreatment and subsequent fermentation of the existing WDG stream to produce both ethanol and a distiller's grain residue.
  • the DDGs associated with this process are expected to have a higher protein content than the initial yeast-based stream due to the conversion of the carbohydrate content to cellulosic ethanol during the fermentation with a Clostridium biocatalyst. While this high protein distiller's grain product is expected to have an enhanced value due to the increased protein content, a smaller quantity of DDGs will be produced than that from the Host Plant alone.
  • the CBP Plant is designed to minimize physical and operational impact on the Host Plant and maintains complete biological independence [i.e., at no time does a stream that contacts the Clostridium biocatalyst come into contact with a stream that goes into the Host Plant fermenters).
  • FIG. 10 A block flow diagram for a CBP Plant using a WDG feedstock, and its connection to the Host Plant, is shown in Fig. 10.
  • CBP Processes” indicates a process boundary between the Host Plant and the CBP Plant.
  • the large broken line outlines those processes on the left within the Host Plant that remain unchanged.
  • the CBP Plant will collect a feed stream of wet distillers grains (WDGs) from the Host Plant centrifuges.
  • WDGs wet distillers grains
  • the CBP Plant will also collect syrup from the Host Plant evaporation train. There is no mixing of process streams from the CBP Plant with those from the Host Plant.
  • the solids concentration of the WDG feed stream to the fermenter will be below the approximately 30% solids content required to achieve an economical ethanol titer. [00344] Consequently, a filter for water removal may be required following pretreatment in order to provide the needed solids concentration, and a concentration system added for the liquid portion to concentrate solubilized sugars for addition to production fermentation without diluting overall sugar concentration.
  • the fermentation system is designed such that one production fermenter will be processed per shift. Assuming a 50 g/L ethanol titer, the production fermenters will be approximately 275,000 gallons each.
  • Feed material is expected to be transferred to the production fermenters including pretreated WDG, Host Plant evaporator syrup, and concentrated pretreatment liquor, where it will be mixed with the Clostridium biocatalyst seed culture (approx. 5% of final fermenter volume) and media (10% of final fermenter volume).
  • Production fermentation will take place over a period of approximately 100 hours, and the monomer and oligimer C5 and C6 will be converted to ethanol and CO 2 .
  • the Clostridium biocatalyst's endogenous enzymes will continue to release sugars during fermentation.
  • the output from the production fermenters will be fed to a beer well and from there to a distillation system specific to the CBP Plant. Because the incremental volume of beer produced by the CBP Plant is expected to be relatively large (e.g., an additional 40-50%> liquid volume compared to the Host Plant), and it will contain different fermentation by-products, it has been assumed that a new distillation system will be required for the CBP Plant.
  • FIG. 9B An optional back-end fractionation process is illustrated in Fig. 9B.
  • Fig. 9C illustrates a process flow diagram including an optional post-treatment process.
  • An exemplary post-treatment process was developed by Biodynamics (West Des Moines, IA, USA). This process passes thin stillage from a Host Plant through a fungal digestion process that converts the input material into a high-protein feed product. Projections indicate that the resulting feed product will have a value of approximately $250/ton, which has already been certified as safe for feed use.
  • the CBP Plant is designed to minimize impact on the Host Plant, while utilizing existing equipment wherever it provides a process advantage
  • the design locates a cellulosic ethanol plant using a Clostridium biocatalyst (i.e., CBP Plant) adjacent to an existing corn ethanol plant (i.e., Host Plant). While a 110 MGY corn ethanol plant was used for base design purposes, all results are expected to scale to 90-120+ MGY corn plants with essentially equivalent economics. However, smaller Host Plant sizes will have less compelling economics due to economies of scale.
  • CBP Plant Clostridium biocatalyst
  • FIG. 11 A block flow diagram for a CBP Plant using a fiber feedstock is shown in Fig. 11.
  • the fiber fraction is expected to be conveyed to the CBP Plant as a clean stream with a uniform and relatively small particle size. This feedstock is suitable for direct introduction into the pretreatment reactor.
  • Pretreatment for the fiber fraction includes acid digestion at low acid levels. Because of the nature of the feed stream, pretreatment conditions are less harsh than those typically employed for a less processed feed or one with a higher lignin content, such as bagasse or corn stover.
  • the material exiting the pretreatment reactor can require additional treatment.
  • Expected operations can include solid- liquid separation and concentration of liquids to enable higher solids loadings to the production fermenters, or detoxification of pretreatment liquor to remove or neutralize inhibitors generated by pretreatment.
  • Clostridium biocatalyst produces its own hydrolytic enzymes, one of the major advantages of Clostridium species, the commercial process can be enhanced by the use of exogenous hydrolytic enzyme applied in a prehydrolysis step. This releases monomer and oligomer sugars to enable exponential growth of the Clostridium biocatalyst as production fermentation is initiated.
  • Pretreated feed material is transferred to the production fermenters, where it is mixed with the Clostridium biocatalyst culture (approx. 10% of final fermenter volume) and media (10% of final fermenter volume).
  • Production fermentation takes place over a period of approximately 100 hours, and the monomer and oligomer C5 and C6 are converted to ethanol and CO 2 .
  • the Clostridium biocatalysts' endogenous enzymes continue to release sugars during fermentation.
  • the output from the production fermenters is fed to a beer well and from there to a distillation system specific to the CBP Plant.
  • a distillation system specific to the CBP Plant For the one plant option, it is possible that the CBP Plant beer is distilled in the existing Host Plant system; however, concerns about cross-contamination can prevent this. Therefore, it has been assumed that a new distillation system is required for the CBP Plant.
  • Separate rectification, drying, and product handling systems are also assumed for the CBP plant.
  • the boost in production resulting from the combined facilities can be handled by the existing Host Plant systems, and the concern over cross-contamination can be, at a minimum, significantly less after distillation. The need to maintain product segregation can outweigh the benefits associated with additional integration.
  • this material is disposed of as a solid waste. However, this material has potential as either a source of energy for the plant, or as a feed or other product.
  • the corn fiber feed is collected from the fractionation process equipment and used as a feedstock to the CBP Plant. This requires no process integration and no shared equipment.
  • the CBP plant will maintain its own cooling system, distillation and product handling systems, and steam generation plant.
  • EXAMPLE 4 Pretreatment of Feedstock at Bench Scale
  • the feedstock can be impregnated with acid by soaking the feedstock in a solution containing a predetermined acid concentration at 40 to 60 °C, and recirculating the soaking fluids through the feedstock utilizing a filter-basket for the feedstock, submerged in a vat that has a suction drawn to a centrifugal pump at the bottom and a spray nozzle discharge at the top to spray the impregnation fluid over the feedstock.
  • the basket is partially or completely submerged, and the spray nozzle distributes the fluid over the top of the basket, with flow from the top of the vat to the bottom.
  • the feedstock is then dewatered with a hydraulic press and filter basket, pressing out the impregnation fluid along with some few extractive compounds and loss of suspended solids. The dewatering is effectively to about 40% solids content.
  • the feedstock is then fed into a steam gun in 1 ⁇ 2 kg quantities. Steam is introduced at sufficient pressure to reach saturation temperatures of 160 °C for about 20 minutes. During this steam introduction, the feedstock is fluidized to maintain good contact of all portions of the feedstock with the steam.
  • the feedstock is then discharged through a shear device (die or orifice) utilizing a rapidly opening poppet valve to cause a size reduction of the feedstock particles through steam "exploding" from the particles.
  • a shear device die or orifice
  • EXAMPLE 5 Pretreatment and shake flask fermentations:
  • Pretreatment of high fiber distillers grain were conducted at 100ml operating volumes in a Milestone ETHOS EZ microwave digestion system in sealed Teflon vessels.
  • the components of the WDG prior to pretreatment can be found in Fig. 13.
  • the desired pretreatment temperature was provided through microwave radiation and a standardized ramp time of 5 minutes.
  • Biomass loadings for pretreatments were standardized at 20% w/w (20 grams total solids per 100 grams of reaction mixture) and treatments carried out over 5, 12.5, 20, 22.5 or 40 minutes under auto hydrolysis, dilute sulfuric acid, and caustic sodium hydroxide conditions.
  • Deionized water was added to the biomass in the pretreatment vessels to achieve a biomass suspension of 20% solids for autohydrolysis conditions.
  • Fig. 14 illustrates carbohydrate hydrolysis after the various pretreatments.
  • Pretreated biomass samples were transferred to 100ml anaerobic shake flasks and diluted with fermentation medium to a final concentration of 10% total solids. Fermentation medium was added to produce a final concentration of 2.5g/L Bacto yeast extract, 2 g/L Ammonium sulfate, 10 mg/L Riboflavin, 30 mg/L Nicotinic acid, 10 mg/L Pyridoxine, 10 mg/L Cyanocobalamine, 10 mg/L Pantethine, 10 mg/L thiamine, 0.3 mg/L Folinic acid, 1 g/L cysteine hydrochloride, 0.25 g/L histidine, 1.6 g/L potassium dihydrogen phosphate, 3.0 g/L dipotassium phosphate, 0.1 mg/L Trisodium
  • Fig. 15 displays the ethanol yields by pretreatment condition. The highest cellulosic ethanol yields were derived from WDG pretreated by autohydrolysis for 20 minutes at 160°C for one source of WDG.
  • the fermentations conducted at 1L scale used media containing final concentrations of: 20g/L Dried brewer's yeast (DBY), 10 mg/L Riboflavin, 30 mg/L Nicotinic acid, 10 mg/L Pyridoxine, 10 mg/L Cyanocobalamine, 10 mg/L Pantethine, 10 mg/L thiamine, 0.3 mg/L Folinic acid, 1 g/L cysteine hydrochloride, 1.6 g/L potassium dihydrogen phosphate, 1 mg/L Trisodium Citrate-2H 2 0, 5 mg/L CaCL 2 -2H 2 0, 60 mg/L MgSO 4 -7H- 2 0, 4 mg/L FeS0 4 -7H 2 0, 2 mg/CoS0 4 -H 2 0, 2 mg/1 ZnS0 4 -H 2 0, 2 mg/L NiCl 2 , 5 mg/L MnS0 4 -H 2 0, 0.4 mg/L CuS0 4 -5H 2 0, 0.4 mg/L K
  • FIG. 18 A-B Another sample of WDG having a higher protein content (Fig. 18 A-B) was pretreated with dilute acid and processed at a 6% solids fermentation.
  • Fig. 19 shows the ethanol yield over 120 hours and the final composition and higher actual ethanol yield (compared to theoretical yield) is shown in Fig. 20.

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Abstract

L'invention concerne des procédés dans lesquels des sous-produits carbonés issus d'installations de production d'éthanol à base de maïs ou de transformation de grains sont utilisés comme charges d'alimentation dans un nouveau procédé de biotraitement consolidé (CBP). Les procédés CBP de l'invention viennent en complément à l'étape initiale de fermentation du processus de broyage à sec. Dans les procédés CBP, les micro-organismes décrits ici produisent soit des sucres, soit de l'éthanol, soit encore d'autres produits chimiques directement à partir de diverses charges d'alimentation introduites dans le dispositif CBP. L'emploi de dispositifs CBP permet d'augmenter la quantité totale du produit obtenu. Les procédés CBP de l'invention présentent en outre d'autres avantages: réduction des déchets, coût de production inférieur, et valorisation accrue des aliments pour animaux ou autres produits chimiques de valeur.
PCT/US2011/037764 2010-05-24 2011-05-24 Procédés de production de produits chimiques à partir de sous-produits de fermentation WO2011149956A2 (fr)

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US11800882B2 (en) 2011-12-02 2023-10-31 The South Dakota Board Of Regents, As Governing Board For South Dakota State University Microbial-based process for high quality protein concentrate
US9359557B2 (en) 2012-05-17 2016-06-07 Shell Oil Company Process for producing volatile organic compounds from biomass material
WO2013173560A1 (fr) * 2012-05-17 2013-11-21 Shell Oil Company Produits pour l'alimentation animale et leurs procédés de fabrication
US9624436B2 (en) 2012-05-17 2017-04-18 Shell Oil Company Methods and systems for processing biomass material
CN104271730A (zh) * 2012-05-17 2015-01-07 国际壳牌研究有限公司 动物饲料产品及其制造方法
US8946491B2 (en) 2012-05-17 2015-02-03 Shell Oil Company Process for producing volatile organic compounds from biomass material
US9242915B2 (en) 2012-05-17 2016-01-26 Shell Oil Company Process for producing volatile organic compounds from biomass material
US20150223517A1 (en) * 2012-07-13 2015-08-13 Kind Consumer Limited Process for using tobacco
WO2014072580A1 (fr) * 2012-11-07 2014-05-15 Maa- Ja Elintarviketalouden Tutkimuskeskus Bioraffinerie durable
US10376837B2 (en) 2013-03-14 2019-08-13 The University Of Wyoming Research Corporation Conversion of carbon dioxide utilizing chemoautotrophic microorganisms systems and methods
US10557155B2 (en) 2013-03-14 2020-02-11 The University Of Wyoming Research Corporation Methods and systems for biological coal-to-biofuels and bioproducts
CN103193834B (zh) * 2013-04-28 2016-08-03 山东福田药业有限公司 一种d-木糖和l-阿拉伯糖的提取方法
CN103193834A (zh) * 2013-04-28 2013-07-10 山东福田药业有限公司 一种d-木糖和l-阿拉伯糖的提取方法
WO2015063549A1 (fr) * 2013-10-29 2015-05-07 Indian Oil Corporation Limited Procédé de préparation de sucres fermentables à partir de ressources de biomasse
US9394209B2 (en) 2014-12-05 2016-07-19 NutriChem Marketing, Inc. Alternative method for the manufacture of granulated nutrients
CZ306795B6 (cs) * 2015-02-16 2017-07-12 Vysoká škola chemicko-technologická v Praze Úplné kultivační médium pro mikrobiální produkci etanolu nebo butanolu, způsoby jeho výroby a způsoby výroby etanolu a butanolu
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
JP7476177B2 (ja) 2018-05-21 2024-04-30 ジュペン バイオ （エイチケイ） リミテッド 細菌細胞由来のタンパク産物の収量を増加させるための系および方法
JP7408645B2 (ja) 2018-05-21 2024-01-05 ジュペン バイオ （エイチケイ） リミテッド 細菌発酵プロセスからタンパク質富化栄養素サプリメントを得るためのシステム
JP2021527442A (ja) * 2018-05-21 2021-10-14 ジュペン バイオ （エイチケイ） リミテッド 細菌発酵プロセスからタンパク質富化栄養素サプリメントを得るためのシステム
JP2021527445A (ja) * 2018-05-21 2021-10-14 ジュペン バイオ （エイチケイ） リミテッド 細菌細胞由来のタンパク産物の収量を増加させるための系および方法
CN108977401A (zh) * 2018-08-17 2018-12-11 中国科学院青岛生物能源与过程研究所 采用木质纤维素培养微藻的方法
CN108977401B (zh) * 2018-08-17 2021-09-28 中国科学院青岛生物能源与过程研究所 采用木质纤维素培养微藻的方法
WO2020058325A1 (fr) * 2018-09-19 2020-03-26 Fermentationexperts A/S Procédé de régulation d'un procédé de fermentation
CN109735454B (zh) * 2019-02-22 2022-03-25 天津农学院 一种用于真姬菇菌种的木屑保藏培养基及其保藏方法
CN109735454A (zh) * 2019-02-22 2019-05-10 天津农学院 一种用于真姬菇菌种的木屑保藏培养基及其保藏方法
CN112586701A (zh) * 2020-12-14 2021-04-02 江西长通实业有限公司 一种微生态果蔬酵素及其制备方法
DE102022206546A1 (de) 2022-06-28 2023-12-28 Freie Universität Berlin, Körperschaft des öffentlichen Rechts Biologisches Verfahren zum Aufschluss von lignocellulosehaltiger Biomasse und Umsetzung von lignocellulosehaltiger Biomasse zu niedrigmolekularen Verbindungen

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