WO2011072264A2 - Procédés et compositions pour traitement de la biomasse - Google Patents

Procédés et compositions pour traitement de la biomasse Download PDF

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Publication number
WO2011072264A2
WO2011072264A2 PCT/US2010/059962 US2010059962W WO2011072264A2 WO 2011072264 A2 WO2011072264 A2 WO 2011072264A2 US 2010059962 W US2010059962 W US 2010059962W WO 2011072264 A2 WO2011072264 A2 WO 2011072264A2
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Prior art keywords
acid
catalase
microorganism
biomass
composition
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PCT/US2010/059962
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English (en)
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WO2011072264A3 (fr
Inventor
Glenn Richards
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Qteros, Inc.
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Publication of WO2011072264A2 publication Critical patent/WO2011072264A2/fr
Publication of WO2011072264A3 publication Critical patent/WO2011072264A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Biomass is a renewable alternative to fossilized raw materials in production of fuels and other chemicals. Increase of bio fuels production has been sought to improve energy security and reduce green house gas emissions. Hydrocarbons are extracted directly from biomass or obtained by various conversion processes utilizing biomass and microorganisms. The conversion process may produce toxic by-products and inhibitors that often can negatively impact the ability of microorganisms to hydro lyze and/or ferment biomass to one or more end- products. Therefore, there remains a need to improve biomass conversion to fermentation end-pro duct(s) by neutralizing or removing toxic by-products from a fermentation mixture.
  • Methods and compositions described herein include a composition for production of fermentation end-products comprising: a carbonaceous biomass; a microorganism capable of directly processing said biomass; and at least one exogenous antioxidant agent. Methods are included for enhancing yield of or rate of fermentation end-product production, comprising: contacting a carbonaceous biomass with one or more microorganisms and at least one agent capable of antioxidant activity.
  • a composition for production of a fermentation end-product comprising: a carbonaceous biomass; a microorganism that processes the biomass to produce said fermentation end-product; and at least one exogenous antioxidant agent.
  • the microorganism ferments C5 and C6 carbohydrates of said carbonaceous biomass.
  • the microorganism is a bacterium.
  • t microorganism is a species of Clostridia.
  • the microorganism is a species of Clostridia.
  • Clostridium phytofermentans Clostridium sp. Q.D, Clostridium
  • the microorganism is genetically modified. In one embodiment, the microorganism is genetically modified to enhance activity of at least one antioxidant that is the same or different from said exogenous antioxidant. In one embodiment, the antioxidant is an enzyme capable of decomposing hydrogen peroxide. In one embodiment, the enzyme is catalase, superoxide dismutase or glutathione peroxidase. In one embodiment, the catalase is yeast catalase CTT1. In one embodiment, the catalase is yeast catalase CTA1.
  • the antioxidant is an amino acid, imidazole, peptide, carotinoid, carotene, chlorogenic acid, liponic acid, aurothioglucose, propylthiouracil, thiol, sulfoximine compound, chelator, a-hydroxy acid humic acid, lipoid acid, EDTA, EGTA, unsaturated fatty acid, folic acid, ubiquinone, ubiquinol, vitamin C (ascorbic acid), tocopherol, vitamin A, coniferyl benzoate, rutinic acid, uric acid, mannose, zinc compound, selenium compound, stilbene, melatonin, or peroxiredoxin.
  • the carbonaceous biomass comprises woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material,
  • hemicellulosic material carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, sorghum, bamboo, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, or algae.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers Dried Grains with Solubles
  • a composition for production of ethanol comprising: a carbonaceous biomass, a Clostridium microorganism that ferments C5 and C6 carbohydrates in said biomass, and a catalase.
  • the microorganism is Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12
  • the catalase is yeast catalase CTT1. In one embodiment, the catalase is yeast catalase CTA1. In one embodiment, the carbonaceous biomass comprises woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, sorghum, bamboo, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, or algae.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers
  • a method for producing a fermentation end- product comprising: contacting a carbonaceous biomass with one or more microorganisms that processes said carbonaceous biomass to produce said fermentation end-product and at least one agent capable of antioxidant activity.
  • the one or more microorganisms ferments C5 and C6 carbohydrates of said carbonaceous biomass.
  • the one or more microorganisms is a bacterium.
  • the one or more microorganisms is a species of Clostridia.
  • the one or more microorganisms is Clostridium phytofermentans, Clostridium sp.
  • the antioxidant is an enzyme capable of decomposing hydrogen peroxide.
  • the enzyme is catalase, superoxide dismutase or glutathione peroxidase.
  • the enzyme is catalase
  • the catalase is yeast catalase CTT1.
  • the catalase is yeast catalase CTA1.
  • the antioxidant is an amino acid, imidazole, peptide, carotinoid, carotene, chlorogenic acid, liponic acid, aurothioglucose, propylthiouracil, thiol, sulfoximine compound, chelator, a-hydroxy acid, humic acid, lipoid acid, EDTA, EGTA, unsaturated fatty acid, folic acid, ubiquinone, ubiquinol, vitamin C (ascorbic acid), tocopherol, vitamin A, coniferyl benzoate, rutinic acid, uric acid, mannose, zinc compound, selenium compound, stilbene, melatonin, or peroxiredoxin.
  • the contacting occurs in a bioreactor containing said biomass.
  • the fermentation end product is an alcohol.
  • the fermentation end product is butanol, ethanol, or propanol.
  • a method for producing ethanol comprising: contacting a carbonaceous biomass with a Clostridia microorganism that ferments C5 and C6 carbohydrates in said biomass and a catalase.
  • the microorganism is Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13 or mutants thereof
  • the catalase is yeast catalase CTT1.
  • the catalase is yeast catalase CTA1.
  • the carbonaceous biomass comprises woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, sorghum, bamboo, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, or algae.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers Dried Grains with Solubles
  • a kit for production of bio fuel from biomass comprising: a microorganism that processes said carbonaceous biomass to produce said fermentation end-product; and an antioxidant.
  • the microorganism ferments C5 and C6 carbohydrates of the carbonaceous biomass.
  • the microorganism is a bacterium.
  • the microorganism is a species of Clostridia.
  • the microorganism is Clostridium phytofermentans,
  • the antioxidant is an enzyme capable of decomposing hydrogen peroxide.
  • the antioxidant is a catalase, superoxide dismutase or glutathione peroxidase.
  • the antioxidant is a catalase.
  • the catalase is yeast catalase CTT1.
  • the catalase is yeast catalase CTA1.
  • the antioxidant is an amino acid, imidazole, peptide, carotinoid, carotene, chlorogenic acid, liponic acid, aurothioglucose, propylthiouracil, thiol, sulfoximine compound, chelator, a-hydroxy acid, humic acid, lipoid acid, EDTA, EGTA, unsaturated fatty acid, folic acid, ubiquinone, ubiquinol, vitamin C (ascorbic acid), tocopherol, vitamin A, coniferyl benzoate, rutinic acid, uric acid, mannose, zinc compound, selenium compound, stilbene, melatonin, or peroxiredoxin.
  • a composition for producing a fermentation end-product comprising: a carbonaceous biomass and a microorganism genetically modified to produce an antioxidant and wherein the microorganism processes said biomass to produce said fermentation end product.
  • the microorganism is capable of direct fermentation of C5 and C6 carbohydrates.
  • the microorganism is a bacterium.
  • the microorganism is a species of Clostridia.
  • the microorganism is Clostridium phytofermentans, Clostridium sp.
  • the microorganism is genetically modified to express a protein encoded by a heterologous polynucleotide. In one embodiment the microorganism is genetically modified to express a protein encoded by an additional copy of a endogenous polynucleotide. In one embodiment, the polynucleotide encodes a catalase, superoxide dismutase or glutathione peroxidase. In one embodiment, the antioxidant is a catalase. In one embodiment, the catalase is yeast catalase CTT1. In one embodiment, the catalase is yeast catalase CTA1.
  • the composition is further comprising an exogenous antioxidant.
  • the composition is further comprising an exogenous antioxidant, wherein said antioxidant is an amino acid, imidazole, peptide, carotinoid, carotene, chlorogenic acid, liponic acid, aurothioglucose, propylthiouracil, thiol, sulfoximine compound, chelator, ⁇ -hydroxy acid, humic acid, lipoid acid, EDTA, EGTA, unsaturated fatty acid, folic acid, ubiquinone, ubiquinol, vitamin C (ascorbic acid), tocopherol, vitamin A, coniferyl benzoate, rutinic acid, uric acid, mannose, zinc compound, selenium compound, stilbene, melatonin, or peroxiredoxin.
  • Figure 1 illustrates an experimental design testing the effect of catalase on
  • Figure 2 illustrates the effect of lignin and catalase on the production of ethanol.
  • Figure 3 illustrates the effect of lignin and catalase on total residual sugars.
  • Figure 4 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 5 illustrates a method for producing fermentation end products from biomass by charging biomass to a fermentation vessel.
  • Figure 6 illustrates pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either fermented separately or together.
  • Figure 7 illustrates a plasmid map for pIMPl .
  • Figure 8 illustrates a plasmid map for pIMCphy.
  • Figure 9 illustrates a plasmid map for pCphyP3510.
  • Figure 10 illustrates a plasmid map for pCphyP3510-1163. DETAILED DESCRIPTION OF THE INVENTION
  • the methods and compositions described herein comprise contacting a biomass with one or more antioxidants to enhance the yield or production rate for at least one fermentation end-product.
  • the biomass is contacted with a least one antioxidant and one or more microorganisms that facilitate saccharificaiton and fermentation of the biomass to one or more fermentation end products.
  • technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
  • enzyme reactive conditions refers to an environmental condition (i.e., such factors as temperature, pH, 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.
  • the term "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 which 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, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • 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.
  • the term "increased” or “increasing” as used herein, refers to the ability of one or more recombinant microorganisms to produce a greater amount of a given product or molecule (e.g., commodity chemical, bio fuel, or intermediate product thereof) as compared to a control microorganism, such as an unmodified microorganism or a differently-modified
  • 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 microorganism.
  • 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
  • a regulatory sequence element can be positioned with respect to a gene to be placed under its control in the same position as the element is situated in its in its natural setting with respect to the native gene it controls.
  • Constant 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.,
  • inducible promoters typically do not allow significant or measurable levels of transcriptional activity.
  • polynucleotide or “nucleic acid” as used herein designates mR A, R A, cRNA, rRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleo tides 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. In one embodiment additional coding or non-coding sequences can, be present within a polynucleotide. In another embodiment
  • a polynucleotide can 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 encoding a polypeptide can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably.
  • the maximum length of a polynucleotide sequence which can be used to transform a microorganism is governed only by the nature of the recombinant protocol employed.
  • polynucleotide variant and “variant” and the like refer to polynucleotides that display substantial sequence identity with any of the reference polynucleotide sequences or genes described herein, and to polynucleotides that hybridize with any polynucleotide reference sequence described herein, or any polynucleotide coding sequence of any gene or protein referred to herein, under low stringency, medium stringency, high stringency, or very high stringency conditions that are defined hereinafter and known in the art. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms
  • 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), homo logs (different locus), and orthologs (different locus).
  • 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 microorganism that encodes one or more genes having a suitable enzymatic activity described herein (e.g., C--C ligase, diol dehyodrogenase, 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%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, or 99% or more sequence identity with the reference polynucleotide 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.
  • hybridizes under low stringency, hybridizes medium stringency, hybridizes high stringency, or hybridizes very high stringency conditions refers to conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used.
  • low stringency refers to conditions that include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C.
  • Low stringency conditions also can include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2X SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • 1 mM EDTA 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C
  • 2X SSC 0.1% SDS
  • BSA Bovine Serum Albumin
  • Medium stringency refers to conditions that include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C.
  • Medium stringency conditions also can include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2X SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 5%> SDS for washing at 60-65° C.
  • BSA Bovine Serum Albumin
  • 1 mM EDTA 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C
  • 2X SSC 0.1% SDS
  • BSA Bovine Serum Albumin
  • High stringency refers to conditions that include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C, and about 0.01 M to about 0.02 M salt for washing at 55° C.
  • High stringency conditions also can include 1% BSA, 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2X SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.
  • One embodiment of high stringency conditions includes hybridizing in 6X SSC at about 45° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65° C.
  • amino acids can be substituted for other amino acids in a protein sequence without appreciable loss of the desired activity (see Table 1 below). 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.
  • hydrophobicity and charge characteristics are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); 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.
  • 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
  • a polynucleotide comprises codons in its protein coding sequence that are optimized to increase the thermostability of an mRNA transcribed from the
  • polynucleotide In one embodiment this optimization does not change the amino acid sequence encoded by the polynucleotide.
  • a polynucleotide comprises codons in its protein coding sequence that are optimized to increase translation efficiency of an mRNA from the polynucleotide in a host cell. In one embodiment this optimization does not change the amino acid sequence encoded by the polynucleotide.
  • RNA codon table below (Table 1) shows the 64 codons and the amino acid for each.
  • the direction of the mRNA is 5 ' to 3 '.
  • ACU Thr/T
  • AAU Ar/N
  • AUC Ile/I Isoleucine ACC (Thr/T) AAC (Asn/N) AGC (Ser/S) Serine
  • GCU (Ala/A) GAU (Asp/D) GGU (Gly/G)
  • GUC Val/V
  • GCC Al/A
  • GAC Al/D
  • GGC Gly/G
  • GCA Al/A
  • GAA Glu/E
  • GGA Gly/G
  • the codon AUG both codes for methionine and serves as an initiation site: the first AUG in an mRNA's coding region is where translation into protein begins.
  • a method disclosed which uses variants of full-length polypeptides having any of the enzymatic activities described herein, truncated fragments of these full- length polypeptides, variants of truncated fragments, as well as their related biologically active fragments.
  • biologically active fragments of a polypeptide can participate in an interaction, for example, an intra-molecular or an inter-molecular interaction.
  • An inter- molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken).
  • Bioly active fragments of a polypeptide/enzyme an enzymatic activity described herein include peptides comprising amino acid sequences sufficiently similar to, or derived from, the amino acid sequences of a (putative) full-length reference polypeptide sequence.
  • biologically active fragments comprise a domain or motif with at least one enzymatic activity, and can include one or more (and in some cases all) of the various active domains.
  • a biologically active fragment of a an enzyme can be a polypeptide fragment which is, for example, 10, 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, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 600 or more contiguous amino acids, including all integers in between, of a reference polypeptide sequence.
  • a biologically active fragment comprises a conserved enzymatic sequence, domain, or motif, as described elsewhere herein and known in the art.
  • the biologically-active fragment has no less than about 1%, 10%, 25%, or 50%) of an activity of the wild-type polypeptide from which it is derived.
  • exogenous refers to a polynucleotide sequence or polypeptide that does not naturally occur in a given wild-type cell or microorganism, but is typically introduced into the cell by a molecular biological technique, i.e., engineering to produce a recombinant microorganism.
  • exogenous polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein or enzyme.
  • endogenous refers to naturally-occurring polynucleotide sequences or polypeptides that can be found in a given wild-type cell or microorganism.
  • certain naturally-occurring bacterial or yeast species do not typically contain a benzaldehyde lyase gene, and, therefore, do not comprise an "endogenous" polynucleotide sequence that encodes a benzaldehyde lyase.
  • a microorganism can comprise an endogenous copy of a given polynucleotide sequence or gene
  • the introduction of a plasmid or vector encoding that sequence such as to over-express or otherwise regulate the expression of the encoded protein, represents an "exogenous" copy of that gene or polynucleotide sequence.
  • Any of the pathways, genes, or enzymes described herein can utilize or rely on an "endogenous” sequence, or can be provided as one or more "exogenous" polynucleotide sequences, and/or can be used according to the endogenous sequences already contained within a given microorganism.
  • sequence identity for example, comprising a “sequence 50%> identical to,” as used herein, refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base ⁇ e.g., A, T, C, G, I) or the identical amino acid residue ⁇ e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base ⁇ e.g., A, T, C, G, I
  • the identical amino acid residue ⁇ e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys
  • 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.
  • 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 addition of additional copies of an endogenous gene into a microorganism.
  • 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.
  • 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.
  • a reporter gene such as a green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
  • 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.
  • Biomass can include, but is not limited to, plant matter, such as woody or non- woody plant matter, crop plants, aquatic or marine biomass, fruit-based biomass such as fruit waste, and vegetable-based biomass such as vegetable waste, and animal based biomass among others.
  • aquatic or marine biomass include, but are not limited to, kelp, other seaweed, algae, and marine microflora, microalgae, sea grass, salt marsh grasses such as Spartina sp. or Phragmites sp. and the like.
  • the term "crop plant” is intended to encompass any plant that is cultivated or harvested for the purpose of producing plant material that is sought after by man for either oral consumption, or for utilization in an industrial,
  • the invention may be applied to any of a variety of plants, including, but not limited to maize, wheat, rice, barley, soybean, cotton, sorghum, oats, tobacco, Miscanthus grass, switch grass, trees (softwoods and hardwoods), beans in general, rape/canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, cotton, coffee, sweet potato, flax, peanut, clover; vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, brussels sprouts, peppers, and pineapple; tree fruits such as citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, and coconut; and flowers such as orchids, carnations and roses, and nonvascular plants such as ferns, and gymnosperms such as palms.
  • Biomass can also include genetically-modified organisms, such as recombinant algae or plants that can produce hydro lytic enzymes (such as cellulases, hemicellulases, or pectinases etc.) or antioxidant enzymes at or near the end of their life cycles.
  • hydro lytic enzymes such as cellulases, hemicellulases, or pectinases etc.
  • antioxidant enzymes such as cellulases, hemicellulases, or pectinases etc.
  • Such biomass can encompass mutated species as well as those that initiate the breakdown of cell wall components.
  • carbonaceous biomass as used herein has its ordinary meaning as known to those skilled in the art and may include one or more biological material that can be converted into a biofuel, chemical or other product.
  • Carbonaceous biomass can comprise municipal waste, wood, plant material, plant matter, plant extract, distillers' grains, a natural or synthetic polymer, or a combination thereof.
  • Plant matter can include, but is not limited to, woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, stillage, leaves, switch grass, bamboo, sorghum, high biomass sorghum, and material derived from these. Plant matter can be derived from a genetically modified plant. Plant matter can be further described by reference to the chemical species present, such as proteins, polysaccharides and oils.
  • 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.).
  • fruit and/or vegetable biomass include, but are not limited to, any source of pectin such as plant peel and pomace including citrus, orange, grapefruit, potato, tomato, grape, mango, gooseberry, carrot, sugar-beet, and apple, among others.
  • plant matter is characterized by the chemical species present, such as proteins, polysaccharides and oils.
  • plant matter includes agricultural waste byproducts or side streams such as pomace, corn steep liquor, corn steep solids, distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), peels, citrus peels, pits, fermentation waste, straw, lumber, sewage, garbage or food leftovers.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers Dried Grains with Solubles
  • peels citrus peels, pits, fermentation waste, straw, lumber, sewage, garbage or food leftovers.
  • feedstock is frequently used to refer to biomass being used for a process, such as those described herein.
  • broth has its ordinary meaning as known to those skilled in the art and can include the entire contents of the combination of soluble and insoluble matter, suspended matter, cells and medium, such as for example the entire contents of a fermentation reaction can be referred to as a fermentation broth.
  • productivity has its ordinary meaning as known to those skilled in the art and can include the mass of a material of interest produced in a given time in a given volume. Units can be, for example, grams per liter-hour, or some other combination of mass, volume, and time. In fermentation, productivity is frequently used to characterize how fast a product can be made within a given fermentation volume. The volume can be referenced to the total volume of the fermentation vessel, the working volume of the fermentation vessel, or the actual volume of broth being fermented. The context of the phrase will indicate the meaning intended to one of skill in the art.
  • Productivity e.g. g/L/d
  • 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% (w ). Frequently, the conversion efficiency will be referenced to the theoretical maximum, for example, "80% of the theoretical maximum.” In the case of conversion of glucose to ethanol, this statement would indicate a conversion efficiency of 41% (wt.).
  • the context of the phrase will indicate the substrate and product intended to one of skill in the art.
  • the theoretical maximum conversion efficiency of the biomass to ethanol is an average of the maximum conversion efficiencies of the individual carbon source constituents weighted by the relative concentration of each carbon source.
  • the theoretical maximum conversion efficiency is calculated based on an assumed saccharification yield.
  • the theoretical maximum conversion efficiency can be calculated by assuming saccharification of the cellulose to the assimilable carbon source glucose of about 75% by weight.
  • lOg of cellulose can provide 7.5g of glucose which can provide a maximum theoretical conversion efficiency of about 7.5g*51% or 3.8g of ethanol.
  • the efficiency of the saccharification step can be calculated or determined, i.e., saccharification yield.
  • Saccharification yields can include between about 10-100%, about 20- 90%, about 30-80%, about 40-70% or about 50-60%, such as about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
  • the saccharification yield takes into account the amount of ethanol, and acidic products produced plus the amount of residual monomeric sugars detected in the media.
  • the ethanol figures resulting from media components are not adjusted in this experiment. These can account for up to 3 g/1 ethanol production or equivalent of up to 6g/l sugar as much as +/- 10%>- 15% saccharification yield (or saccharification efficiency). For this reason the saccharification yield % can be greater than 100% for some plots.
  • 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.
  • 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.
  • transfermentable sugars as used herein has its ordinary meaning as known to those skilled in the art and may include one or more sugars and/or sugar derivatives that can be utilized 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 may 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.
  • plant polysaccharide as used herein has its ordinary meaning as known to those skilled in the art and may 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 may repeat in a regular pattern, or otherwise.
  • 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.
  • Plant polysaccharide can be derived from genetically modified plants. Such plants can be transgenic to express a catalase.
  • polysaccharides, oligosaccharides, monosaccharides or other sugar components of biomass include, but are not limited to, alginate, agar, carrageenan, fucoidan, 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.
  • Antioxidants are molecules capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction in which electrons are transferred from a substance to an oxidizing agent. Such a process can occur in biological organisms. The transfer of electrons in oxidation reactions can lead to the production of free radicals and reactive oxygen species (ROS) in cells and tissues in the course of normal metabolic activities. Examples of the most reactive ROS and free radicals include the superoxide anion (0 2 ⁇ ), singlet oxygen, hydrogen peroxide (H 2 0 2 ), lipid peroxides, peroxinitrite, and hydroxyl radicals.
  • Hydrogen peroxide is generated metabolically in the endoplasmic reticulum, in metal-catalyzed oxidations in peroxisomes, in oxidative phosphorylation in mitochondria, and in the cytosolic oxidation of xanthine.
  • mechanical breakdown of biomass, swelling of lignocellulosic material, or fermentation process can result in release of hydrogen peroxide accumulated in biomass.
  • an antioxidant suitable for use in a composition or method described herein is an enzyme.
  • Antioxidative enzymes include but are not limited to superoxide dismutase (SOD), catalase or glutathione peroxidase (GSH-Px). These enzymes function in concert to remove ROS-related species and free radicals from the organism. SOD is present in oxygen-respiring organisms, such as plants. SOD dismutates (breaks down) superoxide anion to hydrogen peroxide. Hydrogen peroxide, itself is a highly reactive and oxidative molecule. In the presence of the appropriate electron acceptors (hydrogen donors), catalase catalyzes further reduction of hydrogen peroxide to water and oxygen. In the presence of reduced glutathione (GSH), GSH reductase also mediates reduction of hydrogen peroxide to water by a separate pathway.
  • SOD superoxide dismutase
  • GSH-Px glutathione peroxidase
  • byproducts are generated that may be harmful or inhibitory to microorganisms present in a fermentation culture or mixture.
  • harmful byproducts include, but are not limited to, formaldehyde, formic acid, and phenols.
  • enzymatic reaction such as the action of cellulases or xylanases, it is favorable to convert byproducts into other, less interfering substances.
  • catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules.
  • Catalase can also oxidize different toxins or inhibitors of enzymatic reactions, such as formaldehyde, formic acid, phenols, or other byproducts that are inhibitory to fermenting organisms. Catalase decomposes hydrogen peroxide according to the following reaction: H 2 0 2 + H 2 R ⁇ 2H 2 0 + R.
  • a catalase is a yeast catalase (e.g. CTT1, CTA1). In another embodiment a catalase is a Aspergillus niger catalase. In another embodiment a catalase is a Corynebacterium glytamicum catalase. In another embodiment a catalase is a Micrococcus lysodeikticus catalase.
  • antioxidants can be utilized in methods and compositions of the invention to enhance saccharification and fermentation of biomass to one or more desired end-products.
  • suitable antioxidants include, but are not limited to, amino acids (for example glycine, histidine, tyrosine, tryptophane) and derivatives thereof; imidazoles (for example urocanic acid) and derivatives thereof; peptides, such as D,L-carnosine, D-carnosine, L- carnosine and derivatives thereof (for example anserine); carotinoids; carotenes (for example a-carotene, ⁇ -carotene, lycopene) and derivatives thereof; chlorogenic acid and derivatives thereof; liponic acid and derivatives thereof (for example dihydroliponic acid);
  • amino acids for example glycine, histidine, tyrosine, tryptophane
  • imidazoles for example urocanic acid
  • peptides such as D,L-carnosine, D-
  • aurothioglucose propylthiouracil and other thiols (for example thioredoxine, glutathione, cysteine, cystine, cystamine and glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, ⁇ -linoleyl, cholesteryl and glyceryl esters thereof) and their salts; dilaurylthiodipropionate; distearylthiodipropionate; thiodipropionic acid and derivatives thereof (esters, ethers, peptides, lipids, nucleotides, nucleosides and salts);sulfoximine compounds (for example butionine sulfoximines, homocysteine sulfoximine, butionine sulfones, penta-, hexa-and hepta-thi
  • an antioxidant that is a component of a composition or used in a method described herein is an exogenous antioxidant.
  • an antioxidant that is a component of a composition or used in a method described herein is produced by a microorganism present in the composition or used in the method.
  • the microorganism is genetically modified to produce the antioxidant.
  • the microorganism is genetically modified to produce more of the antioxidant.
  • antioxidants can be utilized in methods and compositions of the invention to enhance saccharification and fermentation of biomass to one or more desired end-products.
  • commercially available antioxidants can be utilized in methods and compositions of the invention to enhance production of a fermentation end product, such as ethanol by a microorganism described herein, such as a Clostridium.
  • catalases can include, but are not limited to, catalase from: CATAZYME® (Novozymes), Aspergillus niger (EMD Chemical group and Sigma- Aldrich), bovine liver (EMD Chemical group), human erythrocytes (EMD Chemical group and Sigma-Aldrich), bison liver (Sigma- Aldrich), Corynebacterium glytamicum (Sigma- Aldrich), and Micrococcus lysodeikticus (Sigma-Aldrich).
  • SOD's can include, but are not limited to, SOD from: E. coli.
  • GSH-Px's can include, but are not limited to, GSH-Px from: bovine erythrocytes (Sigma-Aldrich) or human erythrocytes (Sigma- Aldrich).
  • One or more antioxidants can be added to a bioreactor or fermentation vessel in amounts of, or expressed by one or more microorganisms in a bioreactor or fermentation vessel in amounts of, about 0.1 mg/liter, about 0.2 mg/liter, about 0.3 mg/liter, about 0.4 mg/liter, about 0.5 mg/liter, about 0.6 mg/liter, about 0.7 mg/liter, about 0.8 mg/liter, about 0.9 mg/liter, about 1 mg/liter, about 2 mg/liter, about 3 mg/liter, about 4 mg/liter, about 5 mg/liter, about 6 mg/liter, about 7 mg/liter, about 8 mg/liter, about 9 mg/liter, about 10 mg/liter, about 20 mg/liter, about 30 mg/liter, about 40 mg/liter, about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, about 100 mg/liter, about 200 mg/liter, about 300 mg/liter, about 400 mg/liter, about 500 mg/liter, about 600 mg/liter
  • a carbonaceous biomass, a microorganism and a mixture of one or more antioxidants are combined in a bioreactor to produce a fermentation end-product.
  • the mixture comprises two or more antioxidants (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 antioxidants).
  • the microorganism can ferment hexose and pentose carbohydrates of the biomass.
  • the microorganism is a Clostridium.
  • the microorganism is Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.13. Clostridium phytofermentans Q.12 or a similar C5/C6
  • the fermentation end-product is produced in a higher amount in the presence of the mixture of one or more antioxidants than in the absence of the mixture of one or more antioxidants.
  • the fermentation end-product is an alcohol.
  • the fermentation end product is ethanol.
  • the antioxidant mixture can comprise catalase in any of the catalase concentrations described herein as one part of an antioxidant mixture.
  • one part of antioxidant can be added to at least one part of hydro genated biomass.
  • One part of antioxidant can be added to parts of hydro genated biomass which include, but are not limited to: 10, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, 10,000, 20,000, 40,000, 60,000, 80,000, 100,000, 500,000, and 1,000,000.
  • one volume of catalase can be added to a volume of hydro genated biomass.
  • One volume of catalase can be added to volumes (L) of hydro genated biomass which include, but are not limited to: 5, 50, 250, 300, 500, 700, 900, 1500, 3000, 5000, 7000, 9000, 15,000, 30,000, 50,000, 70,000, 90,000, 150,000,
  • Microorganisms useful in compositions and methods of the invention include, but are not limited to bacteria, or yeast.
  • bacteria include, but are not limited to, any bacterium found in the genus of Clostridium, such as C. acetobutylicum, C. aerotolerans, C. beijerinckii, C. bifermentans, C. botulinum, C. butyricum, C. cadaveris, C. chauvoei, C.
  • yeast that can provide a source for an antioxidant protein or can be utilized in co-culture methods of the invention include but are not limited to, species found in Cryptococcaceae, Sporobolomycetaceae with the genera Cryptococcus, Torulopsis,
  • Pityrosporum Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon, Rhodotorula and Sporobolomyces and Bullera, the families Endo- and Saccharomycetaceae, with the genera Saccharomyces, Debaromyces, Lipomyces, Hansenula, Endomycopsis, Pichia, Hanseniaspora, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolitica, Emericella nidulans, Aspergillus nidulans, Deparymyces hansenii and Torulaspora hansenii.
  • a microorganism in another embodiment can be wild type, or a genetically modified strain.
  • a microorganism can be genetically modified to express one or more polypeptides capable of neutralizing a toxic by-product or inhibitor, which can result in enhanced end-product production in yield and/or rate of production. Examples of
  • 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.
  • two or more different microorganisms may be utilized during saccharification and/or fermentation processes to produce an end-product.
  • a bacterium e.g. Clostridia
  • a yeast e.g., a yeast and be cultured with a carbonaceous biomass and one or more antioxidants to produce a fermentation end product, such as ethanol.
  • Microorganisms utilized in compositions or methods of the invention include
  • Clostridia One such Clostridia is C. phytofermentans, C. sp. Q.D or mutants thereof, such as C. phytofermentans Q.8, C. phytofermentans Q.12, or C. phytofermentans Q.13.
  • Microorganisms of the invention can be modified to comprise one or more heterologous polynucleotides that encode an expansin or swollenin, catalase, cellulase, hemicellulase or xylanse enzymes.
  • a microorganism that utilized in products and processes of the invention can be capable of uptake of one or more complex carbohydrates from biomass ⁇ e.g., biomass comprises a higher concentration of oligomeric carbohydrates relative to monomeric carbohydrates).
  • the NRRL has assigned the following NRRL deposit accession numbers to strains: Clostridium sp. Q.D (NRRL B-50361), Clostridium sp. Q.D-5 (NRRL B- 50362), Clostridium sp. Q.D-7 (NRRL B-50363), Clostridium phytofermentans Q.7D (NRRL B-50364), all of which were deposited on April 9, 2010.
  • the NRRL has assigned the following NRRL deposit accession numbers to strains: Clostridium phytofermentans Q.8 (NRRL B- 50351), deposited on March 9, 2010; Clostridium phytofermentans Q.12 (NRRL B-50436), and Clostridium phytofermentans Q.13 (NRRL B-50437), deposited on November 3, 2010.
  • the depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
  • the deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject matter disclosed herein in derogation of patent rights granted by governmental action.
  • 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.
  • 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 0.0 lg 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 0.0 lg of alkaline (e.g. caustic), 0.02g, 0.03g, 0.04g, 0.05g, 0.075g, O. lg, 0.2g, 0.3g, 0.4g, 0.5g, 0.75g, lg, 2g, or about 3g of alkaline (e.g. caustic) for every gram of biomass to be treated.
  • pretreatment of biomass comprises dilute acid hydrolysis.
  • Example of dilute acid hydrolysis treatment are disclosed in T. A. Lloyd and C. E Wyman, Bioresource Technology, (2005) 96, 1967), incorporated by reference herein in its entirety.
  • 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.
  • the above-mentioned methods have two steps: a pretreatment step that leads to a wash stream, and an enzymatic hydrolysis step of pretreated-biomass that produces a hydrolyzate stream.
  • the pH at which the pretreatment step is carried out increases progressively from dilute acid hydrolysis to hot water pretreatment to alkaline reagent based methods (AFEX, ARP, and lime pretreatments).
  • Dilute acid and hot water treatment methods solubilize mostly hemicellulose, whereas methods employing alkaline reagents remove most lignin during the pretreatment step.
  • the wash stream from the pretreatment step in the former methods contains mostly hemicellulose-based sugars, whereas this stream has mostly lignin for the high-pH methods.
  • the subsequent enzymatic hydrolysis of the residual feedstock leads to mixed sugars (C5 and C6) in the alkali-based pretreatment methods, while glucose is the major product in the hydro lysate 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., C. phytofermentans
  • 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.
  • 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, 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 organism such as C. phytofermentans or C. sp. Q.D.
  • 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 bio mass can then be separated from the ionic liquid/wash- solvent solution by centrifugation or filtration, and sent to the
  • 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. U.S. Patent No. 5171592 to Holtzapple, et al, et al., U.S. Patent No. 5939544 to Karstens, et al., U.S. Patent No.
  • 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.
  • a pretreatment is chosen so that the concentration of hemicellulose and/or soluble oligomers is high and the concentration of lignins is low after pretreatment.
  • 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 microbe such as C.
  • the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is 1%, 5%, 10%, 12%>, 13%>, 14%, 15%, 16%, 17%, 19%, 20%, 30%, 40% or 50%. 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 1%>, 5%>, 10%>, 12%>, 13%>, 14%>, 15%, 16%, 17%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40% or 50%.
  • 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 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 1%, 5%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 19%, 20%, 30%, 40% or 50%. In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 20%. In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 5%. Examples of simple sugars include, but are not limited to, C5 and C6 monomers and dimers.
  • the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is 1%, 5%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 19%, 20%, 30%, 40% or 50%. In some embodiments, the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is 0% to 20%. In some embodiments, the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is 0% to 5%. In some embodiments, 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 concentration of phenolics is minimized. [00113] 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 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 1% to 2%.
  • the parameters of the pretreatment are changed such that concentration of accessible cellulose is 10% to 20 %, the concentration of hemicellulose is 10% to 30%, the concentration of soluble oligomers is 45% to 80%, the concentration of simple sugars is 0% to 5%, and the concentration of lignins is 0% to 5% and the concentration of furfural and low molecular weight lignins in the pretreated feedstock is less than 1% to 2%.
  • the parameters of the pretreatment are changed to obtain a high concentration of hemicellulose (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher) and a low concentration of lignins (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%).
  • the parameters of the pretreatment are changed to obtain a high concentration of hemicellulose and a low concentration of lignins such that
  • concentration of the components in the pretreated stock is optimal for fermentation with a microbe such as C. phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13, or other mutagenized species of Clostridium.
  • a microbe such as C. phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13, or other mutagenized species of 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more times.
  • a pH modifier can be added. For example, an acid, a buffer, or a material that reacts with other materials present can be added to modulate the pH of the feedstock.
  • more than one pH modifier can be used, such as one or more bases, one or more bases with one or more buffers, one or more acids, one or more acids with one or more buffers, or one or more buffers.
  • more than one pH modifiers can be added at the same time or at different times.
  • Other non- limiting exemplary methods for neutralizing feedstocks treated with alkaline substances have been described, for example in U.S. Patent Nos. 4,048,341 ; 4, 182,780; and 5,693,296.
  • one or more acids can be combined, resulting in a buffer.
  • Suitable acids and buffers that can be used as pH modifiers include any liquid or gaseous acid that is compatible with the microorganism. Non-limiting examples include peroxyacetic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, and hydrochloric acid.
  • the pH can be lowered to neutral pH or acidic pH, for example a pH of 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, or lower.
  • the pH is lowered and/or maintained within a range of about pH 4.5 to about 7.1, or about 4.5 to about 6.9, or about pH 5.0 to about 6.3, or about pH 5.5 to about 6.3, or about pH 6.0 to about 6.5, or about pH 5.5 to about 6.9 or about pH 6.2 to about 6.7.
  • biomass can be pre-treated at an elevated temperature and/or pressure.
  • biomass is pre treated at a temperature range of 20°C to 400°C.
  • biomass is pretreated at a temperature of about 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 80°C, 90°C, 100°C, 120°C, 150°C, 200°C, 250°C, 300°C, 350°C, 400°C or higher.
  • elevated temperatures are provided by the use of steam, hot water, or hot gases.
  • steam can be injected into a biomass containing vessel.
  • the steam, hot water, or hot gas can be injected into a vessel jacket such that it heats, but does not directly contact the biomass.
  • a biomass can be treated at an elevated pressure.
  • biomass is pre treated at a pressure range of about lpsi to about 30psi.
  • biomass is pre treated at a pressure or about lpsi, 2psi, 3psi, 4psi, 5psi, 6psi, 7psi, 8psi, 9psi, lOpsi, 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 of the present invention includes the use of a sieve, filter, screen, or a membrane for separating the liquid and solids fractions.
  • a suitable sieve pore diameter size ranges from about 0.001 microns to 8mm, such as about 0.005microns to 3mm or about 0.01 microns to 1mm.
  • a sieve pore size has a pore diameter of about O.Olmicrons, 0.02 microns, 0.05 microns, 0.1 microns, 0.5 microns, 1 micron, 2 microns, 4 microns, 5 microns, 10 microns, 20 microns, 25 microns, 50 microns, 75 microns, 100 microns, 125 microns, 150 microns, 200 microns, 250 microns, 300 microns, 400 microns, 500 microns, 750 microns, 1mm or more.
  • biomass e.g. corn stover
  • a method of pre-treatment includes but is not limited to, biomass particle size reduction, such as for example shredding, milling, chipping, crushing, grinding, or pulverizing.
  • biomass particle size reduction can include size separation methods such as sieving, or other suitable methods known in the art to separate materials based on size.
  • size separation can provide for enhanced yields.
  • separation of finely shredded biomass e.g.
  • 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.12 or Clostridium phytofermentans Q.13.
  • 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 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.12 or Clostridium phytofermentans Q.13.
  • the microorganism is genetically modified to enhance activity of one or more hydro lytic 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.12 or Clostridium phytofermentans Q.13.
  • Clostridium phytofermentans for example Clostridium phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13.
  • the microorganism is genetically modified to express or increase expression of an enzyme capable of hydro lyzing said 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 said feedstock is pre-treated with a substance which increases the pH to an alkaline level, at a temperature of from about 80°C to about 120°C; and a microorganism capable of fermenting said 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.12 or Clostridium phytofermentans Q.13.
  • Clostridium phytofermentans for example Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13.
  • a process for simultaneous saccharification and fermentation of cellulosic solids from biomass into bio fuel or another end-product comprises treating the biomass in a closed container with a
  • microorganism and one or more antioxidants (e.g., catalase) under conditions where the microorganism produces saccharo lytic enzymes sufficient to substantially convert the biomass into oligomers, monosaccharides and disaccharides.
  • the organism subsequently converts the oligomers, monosaccharides and disaccharides into ethanol and/or another bio fuel or product.
  • a process for saccharification and fermentation comprises treating the biomass in a container with the microorganism, one or more antioxidants (e.g. , catalase) 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.
  • one or more antioxidants e.g. , catalase
  • the bioconversion process comprises a separate hydrolysis and fermentation (SHF) process.
  • SHF hydrolysis and fermentation
  • the enzymes can be used under their optimal conditions regardless of the fermentation conditions and the organism is only required to ferment released sugars.
  • hydrolysis enzymes are externally added.
  • one or more antioxidants e.g. , catalase
  • 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.
  • one or more antioxidants e.g., catalase
  • exogenous hydrolysis enzymes can be externally added.
  • the byconversion process comprises a consolidated bioprocess (CBP).
  • CBP is a variation of SSF in which the enzymes are produced by the organism that carries out the fermentation.
  • enzymes can be both externally added enzymes and enzymes produced by the fermentative microbe.
  • bio mass is partially hydrolyzed with externally added enzymes at their optimal condition, the slurry is then transferred to a separate tank in which the fermentative microbe (e.g. Clostridium phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13) converts the hydrolyzed sugar into the desired product (e.g. fuel or chemical) and completes the hydrolysis of the residual cellulose and hemicellulose.
  • the desired product e.g. fuel or chemical
  • one or more antioxidants e.g., catalase
  • pretreated biomass is partially hydrolyzed by externally added enzymes to reduce the viscosity.
  • Bydro lysis occurs at the optimal pH and temperature conditions (e.g. pH 5.5, 50 C for fungal ceilulases). Hydroisysis 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. For example, the resultant mixture can be pumped over time (fed batch) into a reactor containing a microorganism (e.g. Clostridium phytofermentans, Clostridium sp. Q.D.
  • a microorganism e.g. Clostridium phytofermentans, Clostridium sp. Q.D.
  • Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13 and media.
  • the microorganism can then produce endogenous enzymes to complete the hydrolysis into fermentable sugars (soluble oligomers) and convert those sugars into ethanoi and/or other products in a production tank.
  • the production tank can then be operated under fermentatio 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 C. phytofermentans.
  • One or more antioxidants e.g., catalase
  • exogenous enzymes added include a xylanase, a
  • exogenous enzymes added do not include a xylanase, a hemicellulase, a glucanase or a glucosidase.
  • 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 Saccharomyces cerevisiae; a Clostridia species such as C. thermocellum, C. acetobutylicum, and C. cellovorans; or Zymomonas mobilis.
  • a process of producing a bio fuel 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 microorganism, (such as Clostridium phytofermentans , a Clostridium sp. Q.D.
  • a Clostridium microorganism such as Clostridium phytofermentans , a Clostridium sp. Q.D.
  • Clostridium phytofermentans Q.13 or a Clostridium phytofermentans Q.12 a Clostridium phytofermentans Q.13 or a Clostridium phytofermentans Q.12
  • one or more antioxidants e.g., catalase
  • the Clostridium microorganism optionally with the addition of one or more hydrolytic enzymes to the container, substantially converts the treated biomass into oligomers, monosaccharides and disaccharides, and/or bio fuel or other fermentation end-product; and 4) optionally, introducing a culture of a second microorganism wherein the second microorganism is capable of substantially converting the oligomers, monosaccharides and disaccharides into bio fuel.
  • Methods and compositions described herein can include extracting or separating fermentation end-products, such as ethanol, from biomass.
  • 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 P-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 an organism 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, 5,411,603, and 5,705,369.
  • 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 may be longer (e.g. 8, 9, 10, 11, 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 an organism is capable of both hydrolyzing biomass and fermenting without the need for conditioning the biomass, such as subjecting the biomass to chemical, heat, enzymatic treatment or combinations thereof.
  • microorganisms can be aerobes, anaerobes, facultative anaerobes, heterotrophs, autotrophs, photoautotrophs, photoheterotrophs, chemoautotrophs, and/or chemoheterotrophs.
  • the cellular activity, including cell growth can be growing aerobic, microaerophilic, or anaerobic.
  • the cells can be in any phase of growth, including lag (or conduction), exponential, transition, stationary, death, dormant, vegetative, sporulating, etc.
  • Organisms disclosed herein can be incorporated into methods and compositions of the inventon so as to enhance fermentation end-product yield and/or rate of production.
  • Clostridium phytofermentans (“C. phytofermentans ' "), which can simultaneously hydrolyze and ferment lignocellulose biomass.
  • C. phytofermentans ' Clostridium phytofermentans
  • phytofermentans is capable of fermenting hexose (C6) and pentose (C5) polysaccharides.
  • C. phytofermentans is capable of acting directly on lignocellulosic biomass without any pretreatment.
  • Other examples include Clostridium sp. Q.D, or mutagen izc species of Clostridium phytofermentans, such as Clostridium Q.12, Clostridium phytofermentans Q. 1 . or a genetically-modified species of C. phytofermentans.
  • a genetically modified organism of the invention can be further modified to heterologously express one or more catalases, or further modified to enhance expression of one or more endogenous cellulases in addition to incorporation of nucleic acid allowing expression of an antioxidant, thereby further enhancing the hydrolysis of biomass.
  • Products of the invention include a product for production of a biofuel comprising: a carbonaceous biomass, a microorganism that is capable of direct hydrolysis and fermentation of said biomass, wherein said microorganism is modified to provide enhanced activity of one or more cellulases and an antioxidant.
  • the methods and compositions described herein comprise fermenting biomass with an antioxidant.
  • Any of the microorganisms described herein can be used for fermentation.
  • a strain for fermentation is Clostridium phytofermentans.
  • the biomass includes, but is not limited to, plant material, carbonaceous biomass or any material containing cellulosic, hemicellulosic, and/or lignocellulosic material. Examples of such biomass can be pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, bamboo and algae.
  • the antioxidant is a catalase. Any of the antioxidants described herein can be used in the fermentation process.
  • An antioxidant can be directly added to a bioreactor containing biomass and microorganisms capable of fermenting the biomass.
  • An antioxidant-producing microorganism can be added to a bioreactor containing biomass and microorganisms capable of fermenting the biomass.
  • An antioxidant-producing organism useful for the present invention includes, but is not limited to, yeast.
  • yeast For example, S. cerevisiae or Aspergillus niger can be included in the fermentation.
  • Methods of the invention can also included co-culture with an organism that naturally produces or is genetically modified to produce one or more antioxidants.
  • a culture medium containing such an organism can be contacted with biomass (e.g., in a bioreactor) prior to, concurrent with, or subsequent to contact with a second organism.
  • biomass e.g., in a bioreactor
  • a first organism produces an antioxidant while a second organism saccharifies and ferments C5 and C6 sugars.
  • the organism producing an antioxidant can also ferment biomass to produce a fermentation end-product (e.g., a yeast strain disclosed herein can produce catalase in co-culture with Clostridium phytofermentans, Clostridium sp. Q.D,
  • Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13 Mixtures of microorganisms can be provided as solid mixtures (e.g., freeze-dried mixtures), or as liquid dispersions of the microorganisms, and grown in co-culture with a second microorganism.
  • Co- culture methods capable of use with the present invention are known, such as those disclosed in U.S. Pat. Application No. 20070178569.
  • Production of various fermentation end-products can be made or enhanced by methods and compositions described herein and include but are not limited to to 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, 3-methylbutanol, 2-butene, 2-butanol, 2- butanone, 2,3-butanediol, 3-hydroxy-2-butanone, 2,3-butanedione, ethylbenzene,
  • a fuel plant that includes a hydrolysis unit configured to hydro lyze a bio mass material comprising a high molecular weight carbohydrate, and a fermentor configured to house a medium and one or more species of microorganisms and one or more antioxidants (e.g., catalase).
  • a hydrolysis unit configured to hydro lyze a bio mass material comprising a high molecular weight carbohydrate
  • a fermentor configured to house a medium and one or more species of microorganisms and one or more antioxidants (e.g., catalase).
  • the microorganism is
  • Clostridium phytofermentans In another embodiment, the microorganism is Clostridium sp. Q.D. In another embodiment, the microorganism is Clostridium phytofermentans Q.12 In another embodiment, the microorganism is Clostridium phytofermentans Q.13.
  • a fuel or chemical end product that includes combining a microorganism (such as Clostridium phytofermentans cells, Clostridium sp. Q.D, Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.D or a similar C5/C6 Clostridium species) 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 is a fuel.
  • a process is provided for producing a fermentation end- product (such as ethanol or hydrogen) from biomass using acid hydrolysis pretreatment.
  • a process is provided for producing a fermentation end-product (such as ethanol or hydrogen) from biomass using enzymatic hydrolysis pretreatment.
  • a process is provided for producing a fermentation end-product (such as ethanol or hydrogen) from biomass using biomass that has not been enzymatically pretreated.
  • a process is provided for producing a fermentation end-product (such as ethanol or hydrogen) from biomass using biomass that has not been chemically or
  • constructs can be prepared for chromosomal integration of the desired genes. Chromosomal integration of foreign genes can offer several advantages over plasmid-based constructions, the latter having certain limitations for commercial processes. 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
  • This DNA can be ligated to form circles without replicons and used for
  • 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.12 or Clostridium phytofennentans Q.13 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.12 or Clostridium phytofennentans Q.13.
  • a microorganism such as C. phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofennentans Q.13.
  • hydrolysis can be accomplished using acids, e.g., Bronsted acids (e.g., sulfuric or hydroch
  • Hydrothermal processes steam explosion, ammonia fiber explosion processes ("AFEX"), 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., by burning.
  • Hydrolysis and/or steam treatment of the biomass can, e.g., increase porosity and/or surface area of the biomass, often leaving the cellulosic materials more exposed to the microbial cells, which can increase fermentation rate and yield.
  • Removal of lignin can, e.g., provide a combustible fuel for driving a boiler, and can also, e.g., increase porosity and/or surface area of the biomass, often increasing fermentation rate and yield.
  • the initial concentration of the carbohydrates in the medium is greater than 20 mM, e.g., greater than 30 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, or even greater than 500 mM.
  • the invention features a fuel plant that includes a hydrolysis unit configured to hydro lyze a biomass material that includes a high molecular weight
  • a fermentor configured to house a medium with a C5/C6 hydrolyzing
  • microorganism e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium
  • phytofermentans Q.12 or Clostridium phytofermentans Q.13 and one or more antioxidants (e.g., catalase); and one or more product recovery system(s) to isolate a fermentation end- product or end- products and associated by-products and co-products.
  • antioxidants e.g., catalase
  • product recovery system(s) to isolate a fermentation end- product or end- products and associated by-products and co-products.
  • the invention features methods of making a fermentation end- product or end- products that include combining a C5/C6 hydrolyzing microorganism (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13 one or more antioxidants (e.g., catalase) 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).
  • a fermentation end-product is a bio fuel or chemical product.
  • the invention features one or more end-products made by any of the processes described herein.
  • one or more fermentation end-products can be produced from biomass on a large scale utilizing a C5/C6 hydrolyzing microorganism (e.g.,
  • 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.
  • 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. 4 illustrates an example of a method for producing chemical products 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. Hydro lysate 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 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.12, Clostridium phytofennentans Q.13, or mutagen ized. or genetically-modified cells thereof, if desired a co-fermenting microbe, e.g., yeast or E. coli; one or more antioxidants (e.g., catalase); and, as needed, nutrients to promote growth of
  • Clostridium phytofermentans or other microbes can be split into multiple fermentors, each containing a different strain of Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12,
  • antioxidants e.g., catalase
  • 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 -pro duct.
  • the contents of the fermentor are transferred to product recovery. Products are extracted, e.g., ethanol is recovered through distilled and rectification.
  • Fig. 5 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 microorganism e.g., Clostridium phytofermentans, Clostridium sp. Q.D.
  • Clostridium phytofermentans Q.13, or mutagen izc or genetically-modified cells thereof are added.
  • One or more antioxidants e.g., catalase
  • Fermentation is allowed to proceed as described above. After fermentation, the contents of the fermentor are transferred to product recovery as described above. Any combination of the chemical production methods and/or features can be utilized to make a hybrid production method. In any of the methods described herein, products can be removed, added, or combined at any step.
  • a C5/C6 hydrolyzing microorganism e.g., Clostridium phytofermentans, Clostridium sp. Q.D,
  • Clostridium phytofermentans Q.12, or Clostridium phytofermentans Q.13 can be used alone or synergistically in combination with one or more other microbes ⁇ e.g. yeasts, fungi, or other bacteria). More than one or more antioxidants ⁇ e.g., catalase). In some embodiments different methods can be used within a single plant to produce different end-products.
  • the invention features a fuel plant that includes a hydrolysis unit configured to hydro lyze a biomass material that includes a high molecular weight
  • a fermentor configured to house a medium and contains a C5/C6 hydrolyzing microorganism (e.g., Clostridium phytofermentans, Clostridium, sp. Q.D, Clostridium
  • phytofermentans Q.12 Clostridium phytofermentans Q.13, or mutagenized or genetically- modified cells thereof
  • one or more antioxidants ⁇ e.g., catalase
  • the invention features methods of making a fuel or fuels that include combining a C5/C6 hydrolyzing microorganism ⁇ e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12, or Clostridium phytqfermentans Q.13), one or more antioxidants (e.g., catalase) 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 fuel or fuels, e.g., ethanol, propanol and/or hydrogen or another chemical compound.
  • a fuel or fuels e.g., ethanol, propanol and/or hydrogen or another chemical compound.
  • the present invention provides a process for producing ethanol and hydrogen from biomass using acid hydrolysis pretreatment. In some embodiments, the present invention provides a process for producing ethanol and hydrogen from biomass using enzymatic hydrolysis pretreatment. Other embodiments provide a process for producing ethanol and hydrogen from biomass using biomass that has not been enzymatically pretreated. Still other embodiments disclose a process for producing ethanol and hydrogen from biomass using biomass that has not been chemically or enzymatically pretreated, but is optionally steam treated.
  • Fig. 6 discloses pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either, fermented separately or together.
  • Figure 6A depicts a process (e.g., acid pretreatment) that produces a solids phase and a liquid phase which are then fermented separately.
  • Figure 6B 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 ( Figure 6C) 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.
  • one or more modifications hydrolysis and/or fermentation conditions can be 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, 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.
  • a microorganism can be genetically modified to enhance enzyme activity of one or more enzymes, including but not limited to hydro lytic enzymes (such as cellulase(s), hemicellulase(s), or pectinases etc.) and antioxidative enzymes (such as superoxide dismutase (SOD), catalase(s) and glutathione peroxidase (GSH-Px) etc.).
  • hydro lytic enzymes such as cellulase(s), hemicellulase(s), or pectinases etc.
  • antioxidative enzymes such as superoxide dismutase (SOD), catalase(s) and glutathione peroxidase (GSH-Px) etc.
  • 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 microorganism can be modified to enhance an activity of one or more cellulases, or enzymes associated with cellulose processing.
  • the classification of cellulases is usually based on grouping enzymes together that forms a family with similar or identical activity, but not necessary the same substrate specificity.
  • One of these classifications is the CAZy system (CAZy stands for Carbohydrate- Active enZymes), for example, where there are 115 different Glycoside Hydrolases (GH) listed, named GH1 to GH155.
  • GH Glycoside Hydrolases
  • Each of the different protein families usually has a corresponding enzyme activity.
  • This database includes both cellulose and hemicellulase active enzymes.
  • the entire annotated genome of C. phytofermentans is available on the worldwideweb at www.ncbi.nlm.nih.gov/sites/entrez.
  • cellulase enzymes whose function can be enhanced for expression endogenously or for expression heterologously in a microorganism include one or more of the genes disclosed in Table 2.
  • the Glycosyl hydrolase family 9 O-Glycosyl hydrolases are a widespread group of enzymes that hydro lyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety.
  • a classification system for glycosyl hydrolases based on sequence similarity, has led to the definition of 85 different families PUBMED:7624375, PUBMED:8535779, PUBMED:. This classification is available on the CAZy (CArbohydrate-Active EnZymes) web site PUBMED. Because the fold of proteins is better conserved than their sequences, some of the families can be grouped in 'clans'.
  • the Glycoside hydrolase family 9 comprises enzymes with several known activities, such as endoglucanase and cellobiohydrolase. In C. phytofermentans, a GH9 cellulase is ABX43720 (Table 2).
  • Cellulase enzyme activity can be enhanced in a microorganism.
  • a cellulase disclosed in Table 2 is enhanced in a microorganism.
  • a hydrolytic enzyme is selected from the annotated genome of C. phytofermentans for utilization in a product or process disclosed herein.
  • 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
  • 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 et al. Appl. Env. Microbio. 2009; 75(8):2304-2311, disclosing expression of xylose isomerase in S. cerevisiae and which is herein incorporated by reference in its entirety).
  • other modifications can be made to enhance end-product (e.g., ethanol) production in a recombinant microorganism.
  • the host e.g., ethanol
  • 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 variety of promoters can be used to drive expression of the heterologous genes in a recombinant host microorganism.
  • Promoter elements can be selected and mobilized in a vector (e.g., pIMPCphy).
  • a transcription regulatory sequence is operably linked to gene(s) of interest (e.g., in a expression construct).
  • the promoter can be any array of DNA sequences that interact specifically with cellular transcription factors to regulate transcription of the downstream gene. The selection of a particular promoter depends on what cell type is to be used to express the protein of interest.
  • a transcription regulatory sequences can be derived from the host microorganism.
  • constitutive or inducible promoters are selected for use in a host cell. Depending on the host cell, there are potentially hundreds of constitutive and inducible promoters which are known and that can be engineered to function in the host cell.
  • a map of the plasmid pIMPCphy is shown in Figure 8, and the DNA sequence of this plasmid is provided as SEQ ID NO: l .
  • SEQ ID NO: 1 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 Cphyl029. This plasmid can be transferred to C.
  • pIMPCphy is an effective replicative vector system for all microbes, including all gram + and gram " bacteria, and fungi (including yeasts).
  • 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 al, Gene sequence 32: 11-20 (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 constitutive ly 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 transcrription.
  • 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.
  • 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.
  • regulatory elements such as for instance a Shine-Dalgarno (SD) sequence (e.g., AGGAGG and so on including natural and synthetic sequences operable in a host cell) and a transcriptional terminator (inverted repeat structure including any natural and synthetic sequence) which are operable in a host cell (into which a coding sequence is introduced to provide a recombinant cell) can be used with the above described promoters.
  • SD Shine-Dalgarno
  • 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.
  • 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.
  • 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.
  • the suppression of protein expression by repressor molecules can be reversed by reducing the concentration of repressor (depression) or by neutralizing the repressor with an inducer.
  • yeast the GAL 10 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 GAL4. Then, GAL4 can bind to an upstream activation sequence (UAS) activating transcription.
  • 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, 6:411-16 (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, "Regulation Of The Yeast Metallothionine Gene", Gene, 48: 13-22 (1986), which is herein incorporated by reference in its entirety.
  • plasmid can be used to obtain a desired expression level of one or more catalases.
  • Constructs can be prepared for chromosomal integration of the desired genes. Chromosomal integration of foreign genes can offer several advantages over plasmid-based constructions. Ethanologenic genes have been integrated chromosomally in E. coli B; see Ohta et al. (1991) Appl. Environ. Microbiol. 57:893-900, which is herein incorporated by reference in its entirety. In general, this is accomplished by purification of a DNA fragment containing (1) the desired genes upstream from an antibiotic resistance gene and (2) a fragment of homologous DNA from the target microorganism.
  • This DNA can be ligated to form circles without replicons and used for transformation.
  • the gene of interest can be introduced in a heterologous host such as E. coli, and short, random fragments can be isolated and operably linked to target genes ⁇ e.g., genes encoding cellulase enzymes) 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 used 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 bio fuel production.
  • host cells e.g., microorganisms
  • a single transformed cell can contain exogenous nucleic acids encoding an entire bio degradation pathway.
  • a pathway can include a polynucleotide encoding an exo-P-glucanase, and endo- ⁇ - glucanase, and an endoxylanase.
  • Such cells transformed with entire pathways and/or enzymes extracted from them can saccharify certain components of biomass more rapidly than the naturally-occurring organism. Constructs can contain multiple copies of the same
  • polynucleotide and/or multiple polynucleotides encoding the same enzyme from different organisms, and/or multiple polynucleotides with mutations in one or more parts of the coding sequences.
  • the polynucleotides can be similar or identical to the endogenous gene. There can be a percent similarity of 70% (e.g. 70, 75, 80, 85, 90, or 95%) or more in comparing the base pairs of the sequences.
  • more effective biomass degradation pathways can be created by transforming host cells with multiple copies of polynucleotides encoding enzymes of the 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.
  • 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.
  • biomass-degrading enzymes are made by transforming host cells (e.g., microbial cells such as bacteria, especially Clostridial cells, algae, and fungi) and/or organisms comprising host cells with polynucleotides encoding one or more different biomass degrading enzymes (e.g., cellulolytic enzymes, hemicellulo lytic enzymes, xylanases, lignases and cellulases) or antioxidative enzymes (e.g. superoxide dismutase (SOD), catalase or glutathione peroxidase (GSH-Px)).
  • a single enzyme can be produced.
  • a cellulase which breaks down pretreated cellulose fragments into cellodextrins or double glucose molecules (cellobiose) or a cellulase which splits cellobiose into glucose can be produced.
  • multiple copies of an enzyme can be transformed into an organism to overcome a rate-limiting step of a reaction pathway.
  • a Clostridum microrganism is genetically modified to express a yeast catalase protein.
  • the protein is CTTl or CTAl .
  • a Clostridum microrganism is genetically modified to express a yeast catalase protein.
  • the protein expressed is SEQ ID NOS: 3 5, 7, 9, or variants thereof.
  • the protein expressed is encoded by polynucleodies, or variants thereof, of SEQ ID NOS: 2, 4, 6, or 8.
  • Candida albicans SC5314 TRFSTVGGELGSADTARDPRGFATKFYTEEGNLDLVY NTPVF hypothetical protein (CTA1) FIRDPSKFPHFIHTQKRNPETHLKDANMFWDYLTSNEESIHQVM
  • glycolate oxidase GO
  • CTTl S. cerevisiae catalase T
  • 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.
  • EXAMPLE 1 Production of ethanol and residual sugar from biomass.
  • Catalase and lignin were mixed according to the design illustrated in Figure 1.
  • the catalase may be any of the catalases described herein.
  • triplicates were prepared for each combination of catalase and lignin.
  • Letters a, b, and c indicates triplicates of the same condition.
  • Numerical numbers 1 through 9 indicates the number assigned to each set of triplicates.
  • the concentrations of lignin and catalase are indicated.
  • the fermentation condition was as follows: Pretreated corn stover (5% w/w, 50g/L), plus Clostridia culture media, plus Clostridia culture salts, at 35°C. Growth conditions were anaerobic (Oxygen maintained at 0 to less than 1 ppm), at 35°C.
  • the growth medium QM contained (per liter): 10.6g K2HP04 , 1.92g KH2P04 , 4.6g (NH4)2S04 , 3g Na3C6H507 , 6g Bacto yeast extract , lg Cysteine » HCl adjusted to pH 7.5 with NaOH.
  • Fermentation medium FM contained (per liter): 20g Bacto yeast extract, 1.5g Corn Steep Powder, 1.36g KH2P04, 2g Na3C6H507 , 1.2g C6H807 » H20, 0.5g (NH4)2S04, Ig NaCI, lg Cysteine'HCl, 11.45g TES (Tris EthaneSulfonic acid) adjusted to pH 8.0 with NaOH.
  • Composition of lOOx Salts solution each salt is added in order (allowing each to dissolve prior to next addition) to 1000 g ddH20 and mixed) in Table 4.
  • Total volume should be 100 mL.
  • Corn stover was pretreated at 121 °C for 1 hour in 1% NaOH, thoroughly washed.
  • Cellulase enzymes utilized for fermentation were cellulase complex, ⁇ -glucosidase, an enzyme complex known as NS50012 (Novozyme), Xylanase and hemicellulase.
  • NS50012 Novozyme
  • Clostria culture media was prepared by day one and inoculation took place on day two. Fermentation lasted for 7 days, starting from the morning of day two the morning of 7th day. Sampling took place at 0, 6, 24, 48, 72, 96, 120, 144, 168 hours. H was adjusted each time accordingly.
  • kits for production of bio fuel from biomass comprises a microorganism that is capable of direct fermentation of pentose and hexose carbohydrates and an antioxidant, wherein the
  • kits can also include an organism that is capable of hydrolysis and fermentation of lignocellulosic biomass and an amount of one or more antioxidant (e.g., catalase).
  • a kit can also include two microorganisms whereby one microorganism is capable of producing one or more antioxidant and secreting the same into a culture medium. Examples of such microorganisms and antioxidants are disclosed throughout herein.

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Abstract

La présente invention concerne des procédés et des compositions permettant le traitement amélioré de la biomasse. La production de biocarburant est améliorée par l'utilisation d'un antioxydant, tel qu'une catalase, durant la fermentation de la biomasse.
PCT/US2010/059962 2009-12-10 2010-12-10 Procédés et compositions pour traitement de la biomasse WO2011072264A2 (fr)

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WO2013063584A1 (fr) * 2011-10-27 2013-05-02 Utah State University Procédés pour produire de l'acétone, du butanol et de l'éthanol
WO2014105405A1 (fr) * 2012-12-12 2014-07-03 Flsmidth A/S Antioxydants pour utilisation dans des systèmes d'extraction par solvant
CN108893517A (zh) * 2018-07-19 2018-11-27 威海利达生物科技有限公司 一种红法夫酵母发酵生产虾青素的发酵培养基及方法
CN109563526A (zh) * 2016-06-20 2019-04-02 巴斯夫欧洲公司 包括干磨和在发酵的醪中添加链烷磺酸的由玉米制备乙醇的方法
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013063584A1 (fr) * 2011-10-27 2013-05-02 Utah State University Procédés pour produire de l'acétone, du butanol et de l'éthanol
WO2014105405A1 (fr) * 2012-12-12 2014-07-03 Flsmidth A/S Antioxydants pour utilisation dans des systèmes d'extraction par solvant
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
CN109563526A (zh) * 2016-06-20 2019-04-02 巴斯夫欧洲公司 包括干磨和在发酵的醪中添加链烷磺酸的由玉米制备乙醇的方法
CN109563526B (zh) * 2016-06-20 2023-02-28 巴斯夫欧洲公司 包括干磨和在发酵的醪中添加链烷磺酸的由玉米制备乙醇的方法
CN108893517A (zh) * 2018-07-19 2018-11-27 威海利达生物科技有限公司 一种红法夫酵母发酵生产虾青素的发酵培养基及方法

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