WO2011088422A2

WO2011088422A2 - Biofuel production using biofilm in fermentation

Info

Publication number: WO2011088422A2
Application number: PCT/US2011/021433
Authority: WO
Inventors: William G. Latouf; Matthias Schmalisch
Original assignee: Qteros, Inc.
Priority date: 2010-01-15
Filing date: 2011-01-14
Publication date: 2011-07-21
Also published as: WO2011088422A3

Abstract

Compositions and methods for improving the efficiency of bio fuels and chemicals, e.g., ethanol, production is described. Increased yield was obtained by facilitating biofilm formation in a bioreactor coupled with a particular microbial species. Compositions and methods for increased processing of solid biomass in a bioreactor are also described.

Description

BIOFUEL PRODUCTION USING BIOFILM IN FERMENTATION

CLAIM OF PRIORITY

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application Serial No. 61/295,405, filed on January 15, 2010, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Fermentation of biomass to produce a bio fuel such as alcohol (e.g. methanol, ethanol, butanol, and propanol) can provide much needed solutions for the world energy problem. Lignocellulosic biomass has cellulose and hemicellulose as two major components.

Hydrolysis of these components results in both hexose (C6) as well as pentose (C5) sugars. Biomass conversion efficiency is highly dependent on the range of carbohydrates that can be utilized by the microorganism used in the biomass to fuel conversion process. In particular, an inability to utilize both hexose (e.g. cellobiose, glucose) and pentose (e.g. arabinose, xylose) sugars for conversion into ethanol can dramatically limit the total amount of bio fuel that can be generated from a given quantity of biomass. In addition, commonly used fermentation conditions can limit the productivity of bio catalysts that have specific physical requirements for enzymatic hydrolysis. Therefore, to obtain a high conversion efficiency of lignocellulosic biomass to ethanol (yield) it is important to be able to successfully hydro lyze polymers and ferment both hexose and pentose sugars into ethanol in an environment that maximizes product synthesis.

SUMMARY OF THE INVENTION

[0003] In one aspect of the invention, a system is provided comprising (a) one or more microorganisms that hydro lyzes and ferments hexose and pentose carbohydrates, wherein said one or more microorganisms forms a bio film; (b) a biomass comprising hexose and pentose carbohydrates; and (c) a bioreactor comprising an impeller. In one embodiment, the impeller is a helical impeller. In another embodiment, one of the one or more

microorganisms is a Clostridium microorganism. In another embodiment, the one or more microorganisms comprises Clostridium phytofermentans, Clostridium sp. Q.D., Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium

celerecrescens, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella

cellulolytica, Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum, or a variant thereof. In another embodiment, one or more microorganisms comprises Clostridium phytofermentans or a variant thereof. In another embodiment, the one or more microorganisms comprises Clostridium sp. Q.D or a variant thereof. In another embodiment, the microorganism is genetically modified. In another embodiment, the impeller has a revolution of about 10-300 rpm. In another embodiment, the impeller has a revolution of about 50-250 rpm. In another embodiment, the impeller has a revolution of about 100-200 rpm. In another embodiment, the impeller has a revolution of about 100-150 rpm. In another embodiment, the impeller has a revolution of about 150-200 rpm. In another embodiment, the Clostridium phytofermentans, Clostridium sp. Q.D., or a variant thereof is genetically modified. In another embodiment, the bioreactor is a stirred tank reactor, continuous stirred tank reactor, packed bed reactor, fluidized bed reactor, airlift reactor, upflow anaerobic sludge blanket reactor, or a expanded granular sludge reactor. In another embodiment, the impeller has a revolution of about 120 rpm. In another embodiment, the impeller has a revolution of about 175 rpm. In another embodiment, the biomass comprises organic matter. In another embodiment, the biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin.

[0004] In another aspect of the invention, a system is provided for comprising (a) a biomass comprising hexose and pentose carbohydrates; (b) one or more microorganisms comprising Clostridium phytofermentans, Clostridium sp. Q.D., or a variant thereof, wherein said one or more microorganisms forms a bio film on said biomass or a support; and (c) a bioreactor comprising a helical impeller that has a revolution rate of about 100-200 rpm. In one embodiment, the Clostridium phytofermentans, Clostridium sp. Q.D., or a variant thereof is genetically modified. In another embodiment, the bioreactor is a stirred tank reactor, continuous stirred tank reactor, packed bed reactor, fluidized bed reactor, airlift reactor, upflow anaerobic sludge blanket reactor, or a expanded granular sludge reactor. In one embodiment, the one or more microorganisms forms a bio film on said biomass. In another embodiment, the one or more microorganisms forms a bio film on said support. In one embodiment, the support comprises metal, composite or a polymer. In another embodiment, the impeller has a revolution of about 120 rpm. In another embodiment, the impeller has a revolution of about 175 rpm. In another embodiment, the biomass comprises organic matter. In a further embodiment, the organic matter is plant matter or animal matter. In another embodiment, the biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin.

[0005] In another aspect of the invention, a method of culturing a microorganism is provided for, comprising (a) contacting a biomass comprising hexose and pentose carbohydrates with one or more microorganisms that hydro lyzes and ferments hexose and pentose

carbohydrates., or a variant thereof; and (b) culturing said one or more microorganisms by agitating said biomass and said one or more microorganisms at a rate wherein said one or more microorganisms forms a bio film on the surface of said biomass or a support. In one embodiment, one of the one or more microorganisms is a Clostridium microorganism. In another embodiment, the one or more microorganisms comprises Clostridium

phytofermentans, Clostridium sp. Q.D., Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium

polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium

chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica,

Thermoanaerobacterium thermo saccharolyticum and Thermoanaerobacterium

saccharolyticum, or a variant thereof. In another embodiment, the one or more

microorganisms comprises Clostridium phytofermentans or a variant thereof. In another embodiment, the one or more microorganisms comprises Clostridium sp. Q.D or a variant thereof. In another embodiment, the microorganism is genetically modified. In another embodiment, the one or more microorganisms forms a bio film on said biomass. In another embodiment, the one or more microorganisms forms a bio film on the support. In a further embodiment, the support comprises metal, composite or a polymer. In another embodiment, the agitation produces a low sheer rate. In another embodiment, the agitation does not substantially disrupt the bio film. In another embodiment, the biomass is agitated by the revolution of an impeller. In a further embodiment, the impeller is a helical impeller. In a further embodiment, the microorganism is genetically modified. In a further embodiment, the impeller has a revolution of about 10-300 rpm. In a further embodiment, the impeller has a revolution of about 50-250 rpm. In a further embodiment, the impeller has a revolution of about 100-200 rpm. In a further embodiment, the impeller has a revolution of about 100-150 rpm. In another embodiment, the impeller has a revolution at a rate of about 120 rpm. In another embodiment, the impeller has a revolution at a rate of about 175 rpm. In another embodiment, the biomass comprises organic matter. In another embodiment, the organic matter is plant matter or animal matter. In another embodiment, the biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin. In a further embodiment, the culturing produces a fermentation end product. In a further embodiment, the fermentation end product is a bio fuel. In a further embodiment, the fermentation end product is an alcohol. In a further embodiment, the fermentation end product is ethanol, methanol, propanol or butanol. In a further embodiment, the fermentation end product is ethanol.

[0006] In another aspect of the invention, a method of culturing a microorganism is provided for, comprising (a) contacting biomass comprising hexose and pentose carbohydrates with one or more microorganisms comprising Clostridium phytofermentans, Clostridium sp. Q.D., or a variant thereof, wherein said one or more microorganisms forms a bio film on said biomass or a support; and (b) culturing said one or more microorganisms in a bioreactor by agitating said biomass with an impeller at a rate that said bio film remains substantially intact. In one embodiment, the Clostridium phytofermentans, Clostridium sp. Q.D., or a variant thereof is genetically modified. In another embodiment, the impeller has a revolution at a rate of about 120 rpm. In another embodiment, the impeller has a revolution at a rate of about 175 rpm. In another embodiment, the biomass comprises organic matter. In another embodiment, the organic matter is plant matter or animal matter. In another embodiment, the biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin. In another embodiment, the one or more microorganisms forms a bio film on said biomass. In another embodiment, the one or more microorganisms forms a bio film on said support. In a further embodiment, the support comprises metal, composite or a polymer. In a further embodiment, the culturing produces a fermentation end product. In a further embodiment, the fermentation end product is a bio fuel. In a further embodiment, the fermentation end product is an alcohol. In a further embodiment, the fermentation end product is ethanol, methanol, propanol or butanol. In a further embodiment, the fermentation end product is ethanol.

[0007] In another aspect of the invention, a method of producing fermentation end product is provided for, comprising (a) contacting biomass with a medium and one or more

microorganisms that hydro lyzes and ferments hexose and pentose carbohydrates that forms a bio film on said biomass or a support; (b) culturing said one or more microorganisms in said bioreactor with a low sheer force so that said bio film remains substantially intact; (c) producing a fermentation end product from said biomass by said one or more

microorganisms; and (d) separating said fermentation end product from said one or more microorganisms and said biomass. In one embodiment, one of the one or more

cellulolytica, Thermoanaerobacterium thermo saccharolyticum and Thermoanaerobacterium saccharolyticum, or a variant thereof. In another embodiment, the one or more microorganisms comprises Clostridium phytofermentans or a variant thereof. In another embodiment, the one or more microorganisms comprises Clostridium sp. Q.D or a variant thereof. In another embodiment, the one or more microorganisms is genetically modified. In another embodiment, the one or more microorganisms forms a bio film on said biomass. In another embodiment, the one or more microorganisms forms a bio film on the support. In a further embodiment, the support comprises metal, composite or a polymer. In another embodiment, the bio film is irreversibly immobilized on the biomass. In another embodiment, the separating is by centrifugation. In another embodiment, the method further comprises extracting said fermentation end product by distillation. In another embodiment, the culturing is facilitated by agitating the culture. In another embodiment, the culturing is facilitated by static fermentation. In a further embodiment, the agitating is due to the action of an impeller. In a further embodiment, the impeller is a helical impeller. In a further embodiment, thethe impeller has a revolution of about 10-300 rpm. In a further embodiment, the impeller has a revolution of about 50-250 rpm. In a further embodiment, the impeller has a revolution of about 100-200 rpm. In a further embodiment, the impeller has a revolution of about 100-150 rpm. In a further embodiment, the impeller has a revolution of about 150-200 rpm. In a further embodiment, the agitation is achieved by an impeller having a revolution at a rate of 120 rpm. In a further embodiment, the agitation is achieved by an impeller having a revolution at a rate of 175 rpm. In another embodiment, the biomass is 15 % (w/w) of total weight of the biomass, the medium and the one or more microorganisms. In another embodiment, the biomass is 20 % (w/w) of total weight of the biomass, the medium and the one or more microorganisms. In another embodiment, the biomass is 30 % (w/w) of total weight of the biomass, said medium and the one or more microorganisms. In another embodiment, the biomass comprises organic matter. In another embodiment, the organic matter is plant matter or animal matter. In another embodiment, the biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin. In another embodiment, the fermentation end product is a biofuel. In another embodiment, the fermentation end product is an alcohol. In another embodiment, the fermentation end product is ethanol, methanol, propanol, butanol, 1,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3- hydroxybutyrolactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, or a terpene. In another embodiment, the fermentation end product is ethanol. In some embodiments, a fermentation end product is produced by the methods described herein. In a further embodiment, the fermentation end product is biofuel. In a further embodiment, the fermentation end product is an alcohol. In a further embodiment, the fermentation end product is ethanol, methanol, propanol, butanol,l,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, or a terpene. In a further embodiment, the fermentation end product is ethanol.

[0008] In yet another aspect of the invention, a composition for the production of a fermentative end product is provided for, comprising (a) a biomass comprising hexose and pentose carbohydrates; and (b) one or more microorganisms that hydro lyzes and ferments hexose and pentose carbohydrates, wherein said one or more microorganisms forms a bio film. In one embodiment, one of the one or more microorganisms is a Clostridium microorganism. In another embodiment, the one or more microorganisms comprises Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum,

Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium

Thermoanaerobacterium thermo saccharolyticum Thermoanaerobacterium saccharolyticum, or a variant thereof. In another embodiment, the one or more microorganisms comprises Clostridium phytofermentans or a variant thereof. In another embodiment, the one or more microorganisms comprises Clostridium sp. Q.D or a variant thereof. In another embodiment, the biomass comprises organic matter. In a further embodiment, the organic matter is plant matter or animal matter. In another embodiment, the biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed

Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin. In another embodiment, the biomass is 15 % (w/w) of total weight of the composition. In another embodiment, the biomass is 20 % (w/w) of total weight of the composition. In another embodiment, the biomass is 30 % (w/w) of total weight of the composition. In another embodiment, the fermentation end product is a bio fuel. In another embodiment, fermentation end product is an alcohol. In another embodiment, the fermentation end product is ethanol, methanol, propanol, butanol, 1,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3 -hydro xybutyro lactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, or a terpene. In yet another

embodiment, the fermentation end product is ethanol.

INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0011] Figure 1 illustrates increased ethanol production by formation of bio film facilitated by gentle agitation of the culture;

[0012] Figure 2 illustrates increased ethanol production by formation of bio film facilitated by static fermentation;

[0013] Figure 3 illustrates a pathway map for cellulose hydrolysis and fermentation;

[0014] 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; [0015] Figure 5 illustrates a method for producing fermentation end products from biomass by using solvent extraction or separation methods;

[0016] Figure 6 illustrates a method for producing fermentation end products from biomass by charging biomass to a fermentation vessel;

[0017] Figure 7 illustrates pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either fermented separately or together;

[0018] Figure 8 illustrates a plasmid map for pIMPl;

[0019] Figure 9 illustrates a plasmid map for pIMCphy;

[0020] Figure 10 illustrates a plasmid map for pCphyP3510;

[0021] Figure 11 illustrates a plasmid map for pCphyP3510-1163.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The following description and examples illustrate embodiments of the present invention in detail. Those of skill in the art will recognize that there are numerous variations and modifications of this invention that are encompassed within its scope. Accordingly, the description of a preferred embodiment should not be deemed to limit the scope of the present invention.

Definitions

[0023] Unless characterized differently, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0024] The term "about" as used herein refers to a range that is 15% plus or minus from a stated numerical value within the context of the particular usage. For example, about 10 would include a range from 8.5 to 11.5. The terms "function" and "functional" as used herein refer to biological or enzymatic function.

[0025] The term "gene" as used herein, refers to a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5' and 3' untranslated sequences).

[0026] 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.

[0027] The term "isolated" as used herein, refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide", as used herein, 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. Alternatively, 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.

[0028] 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 microorganism. 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.

[0029] The terms "polynucleotide" or "nucleic acid" as used herein designates mR A, R A, cR A, rR A, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0030] As will be understood by those skilled in the art, 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.

[0031] The terms "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. In this regard, it is well understood in the art that 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.

[0032] The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants that encode these enzymes. Examples of naturally-occurring variants include allelic variants (same locus), homo logs (different locus), and orthologs (different

microorganism). 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.).

[0033] 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. In certain aspects, 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). For polynucleotide sequences, 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. Generally, 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%o, 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. In one embodiment 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. In another embodiment a variant polynucleotide sequence encodes a protein with greater activity compared to a protein encoded by the respective reference polynucleotide sequence.

[0034] In one embodiment a method is 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. Typically, 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). Biologically 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. Typically, 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, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 600 or more contiguous amino acids, including all integers in between, of a reference polypeptide sequence. In certain embodiments, a biologically active fragment comprises a conserved enzymatic sequence, domain, or motif, as described elsewhere herein and known in the art. Suitably, 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. Additional methods for genetic modification can be found in U.S. Patent Publication US20100086981A1, which is herein incorporated by reference in its entirety.

[0035] The term "exogenous" as used herein, 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. Examples of "exogenous" polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein or enzyme.

[0036] The term "endogenous" as used herein, refers to naturally-occurring polynucleotide sequences or polypeptides that can be found in a given wild-type cell or microorganism. For example, 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. In this regard, it is also noted that even though 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.

[0037] The term "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. Thus, 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.

[0038] The term "transformation" as used herein, 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.

[0039] The term "vector" as used herein, 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. Accordingly, 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. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Such a vector can comprise specific sequences that allow recombination into a particular, desired site of the host chromosome. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. A vector can be one which is operably functional in a bacterial cell, such as a cyanobacterial cell. The vector can include a reporter gene, such as a green fluorescent protein (GFP), which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.

[0040] The terms "wild-type" and "naturally-occurring" as used herein are used

interchangeably to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type gene or gene product {e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene.

[0041] The term "bio film" as used herein refers to an aggregate of microorganisms in which cells are stuck to each other and/or to a surface. A "bio film" includes a layer of cells where microbial cells attach to a support, flocculate or aggregate together as "granules." Bio film formation can be a natural process or induced process in which cells are attracted to an absorbent material and form a bio film. [0042] In one embodiment bio film formation can be employed as a way of increasing cell concentration in industrial bioreactors. For certain microbial strains, increased concentration of cells leads to increased production of target chemicals or fermentation end products.

[0043] The terms "upstream" and "downstream" can refer to the disposition of a first process unit operation ("unit operation") with respect to the disposition of other unit operations, such as a second unit operation. The term "upstream" can refer to a unit operation that is disposed toward the beginning or start, or earlier in time (with respect to fluid flow) of a particular process. The term "downstream" can refer to a unit operation that is disposed at a later point along a particular process. For example, if a first unit operation is upstream from a second unit operation, fluid flows from the first unit operation to the second unit operation. As another example, if a second unit operation is downstream from a first unit operation, fluid flows from the first unit operation to the second unit operation.

Biomass

[0044] The term "biomass" comprises organic material derived from living organisms, including any member from the kingdoms: Monera, Protista, Fungi, Plantae, or Animalia. Organic material that comprises oligosaccharides (e.g., pentose saccaharides, hexose saccharides, or longer saccharides) is of particular use in the processes disclosed herein. Organic material includes organisms or material derived therefrom. Organic material includes cellulosic, hemicellulosic, and/or lignocellulosic material. In one embodiment biomass comprises genetically-modified organisms or parts of organisms, such as genetically- modified plant matter, algal matter, animal matter. In another embodiment biomass comprises non-genetically modified organisms or parts of organisms, such as non-genetically modified plant matter, algal matter, animal matter The term "feedstock" is also used to refer to biomass being used in a process, such as those described herein.

[0045] Plant matter comprises members of the kingdom Plantae, such as terrestrial plants and aquatic or marine plants. In one embodiment terrestrial plants comprise crop plants (such as fruit, vegetable or grain plants). In one embodiment aquatic or marine plants include, but are not limited to, sea grass, salt marsh grasses (such as Spartina sp. or Phragmites sp.) or the like. In one embodiment a crop plant comprises a plant that is cultivated or harvested for oral consumption, or for utilization in an industrial, pharmaceutical, or commercial process. In one embodiment, crop plants include but are not limited to corn, wheat, rice, barley, soybeans, bamboo, cotton, crambe, jute, sorghum, high biomass sorghum, oats, tobacco, grasses, (e.g., Miscanthus grass or switch grass), trees (softwoods and hardwoods) or tree leaves, beans rape/canola, alfalfa, flax, sunflowers, safflowers, millet, rye, sugarcane, sugar beets, cocoa, tea, Brassica sp., cotton, coffee, sweet potatoes, flax, peanuts, clover; lettuce, tomatoes, cucurbits, cassava, potatoes, carrots, radishes, peas, lentils, cabbages, cauliflower, broccoli, brussels sprouts, grapes, peppers, or pineapples; tree fruits or nuts such as citrus, apples, pears, peaches, apricots, walnuts, almonds, olives, avocadoes, bananas, or coconuts; flowers such as orchids, carnations and roses; nonvascular plants such as ferns; oil producing plants (such as castor beans, jatropha, or olives); or gymnosperms such as palms. Plant matter also comprises material derived from a member of the kingdom Plantae, such as woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, or

hemicellulosic material. Plant matter includes carbohydrates (such as pectin, starch, inulin, fructans, glucans, lignin, cellulose, or xylan). Plant matter also includes sugar alcohols, such as glycerol. In one embodiment plant matter comprises a corn product, {e.g. corn stover, corn cobs, corn grain, corn steep liquor, corn steep solids, or corn grind), stillage, bagasse, leaves, pomace, or material derived therefrom. In another embodiment plant matter comprises distillers grains, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with

Solubles (DDGS), peels, pits, fermentation waste, skins, straw, seeds, shells, beancake, sawdust, wood flour, wood pulp, paper pulp, paper pulp waste streams, rice or oat hulls, bagasse, grass clippings, lumber, or food leftovers. These materials can come from farms, forestry, industrial sources, households, etc. In another embodiment plant matter comprises an agricultural waste byproduct or side stream. In another embodiment plant matter comprises a source of pectin such as citrus fruit {e.g., orange, grapefruit, lemon, or limes), potato, tomato, grape, mango, gooseberry, carrot, sugar-beet, and apple, among others. In another embodiment plant matter comprises plant peel {e.g., citrus peels) and/or pomace {e.g., grape pomace). In one embodiment plant matter is characterized by the chemical species present, such as proteins, polysaccharides or oils. In one embodiment plant matter is from a genetically modified plant. In one embodiment a genetically-modified plant produces hydro lytic enzymes (such as a cellulase, hemicellulase, or pectinase etc.) at or near the end of its life cycles. In another embodiment a genetically-modified plant encompasses a mutated species or a species that can initiate the breakdown of cell wall components. In another embodiment plant matter is from a non-genetically modified plant.

[0046] Animal matter comprises material derived from a member of the kingdom Animaliae {e.g., bone meal, hair, heads, tails, beaks, eyes, feathers, entrails, skin, shells, scales, meat trimmings, hooves or feet) or animal excrement {e.g., manure). In one embodiment animal matter comprises animal carcasses, milk, meat, fat, animal processing waste, or animal waste (manure from cattle, poultry, and hogs).

[0047] Algal matter comprises material derived from a member of the kingdoms Monera {e.g. Cyanobacteria) or Protista {e.g. algae (such as green algae, red algae, glaucophytes, cyanobacteria,) or fungus-like members of Protista (such as slime molds, water molds, etc). Algal matter includes seaweed (such as kelp or red macroalgae), or marine microflora, including plankton.

[0048] Organic material comprises waste from farms, forestry, industrial sources, households or municipalities. In one embodiment organic material comprises sewage, garbage, food waste {e.g., restaurant waste), waste paper, toilet paper, yard clippings, or cardboard.

[0049] The term "carbonaceous biomass" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biological materials that can be converted into a bio fuel, chemical or other product. Carbonaceous biomass can comprise municipal waste (waste paper, recycled toilet papers, yard clippings, etc.), wood, plant material, plant matter, plant extract, bacterial matter {e.g. bacterial cellulose), distillers' grains, a natural or synthetic polymer, or a combination thereof.

[0050] In one embodiment, 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.).

[0051] The term "broth" as used herein has its ordinary meaning as known to those skilled in the art and can include the entire contents of the combination of soluble and insoluble matter, suspended matter, cells and medium, such as for example the entire contents of a

fermentation reaction can be referred to as a fermentation broth.

[0052] The term "productivity" as used herein has its ordinary meaning as known to those skilled in the art and can include the mass of a material of interest produced in a given time in a given volume. Units can be, for example, grams per liter-hour, or some other combination of mass, volume, and time. In fermentation, productivity is frequently used to characterize how fast a product can be made within a given fermentation volume. The volume can be referenced to the total volume of the fermentation vessel, the working volume of the fermentation vessel, or the actual volume of broth being fermented. The context of the phrase will indicate the meaning intended to one of skill in the art. Productivity {e.g. g/L/d) is different from "titer" {e.g. g/L) in that productivity includes a time term, and titer is analogous to concentration. [0053] The term "saccharification" as used herein has its ordinary meaning as known to those skilled in the art and can include conversion of plant polysaccharides to lower molecular weight species that can be used by the microorganism at hand. For some microorganisms, this would include conversion to monosaccharides, disaccharides, trisaccharides, and oligosaccharides of up to about seven monomer units, as well as similar sized chains of sugar derivatives and combinations of sugars and sugar derivatives. For some microorganisms, the allowable chain-length can be longer (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units or more) and for some microorganisms the allowable chain- length can be shorter (e.g. 1, 2, 3, 4, 5, 6, or 7 monomer units).

[0054] The term "external source" as it relates to a quantity of an enzyme or enzymes provided to a product or a process means that the quantity of the enzyme or enzymes is not produced by a microorganism in the product or process. An external source of an enzyme can include, but is not limited to an enzyme provided in purified form, cell extracts, culture medium or an enzyme obtained from a commercially available source.

[0055] The term "biocatalyst" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more enzymes and/or microorganisms, including solutions, suspensions, and mixtures of enzymes and microorganisms. In some contexts this word will refer to the possible use of either enzymes or microorganisms to serve a particular function, in other contexts the word will refer to the combined use of the two, and in other contexts the word will refer to only one of the two. The context of the phrase will indicate the meaning intended to one of skill in the art.

[0056] The terms "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:

C₆Hi₂0₆ -> 2 C₂H₅OH + 2 C0₂

and 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. For substrates comprising a mixture of different carbon sources such as found in biomass (xylan, xylose, glucose, cellobiose, arabinose cellulose, hemicellulose etc.), 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. In some cases, the theoretical maximum conversion efficiency is calculated based on an assumed saccharification yield. In one embodiment, given carbon source comprising lOg of cellulose, 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. In this embodiment, 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. In other cases, 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% for any carbohydrate carbon sources larger than a single monosaccharide subunit.

[0057] 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. The terms "fed-batch" or "fed-batch fermentation" as used herein has its ordinary meaning as known to those skilled in the art and can include a method of culturing microorganisms where nutrients, other medium components, or biocatalysts (including, for example, enzymes, fresh microorganisms, extracellular broth, etc.) are supplied to the fermentor during cultivation, but culture broth is not harvested from the fermentor until the end of the fermentation, although it can also include "self seeding" or "partial harvest" techniques where a portion of the fermentor volume is harvested and then fresh medium is added to the remaining broth in the fermentor, with at least a portion of the inoculum being the broth that was left in the fermentor. In some embodiments, 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.

[0058] 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.

[0059] The term "fermentable sugars" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more sugars and/or sugar derivatives that can be 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 can break down these polymers, such as by hydrolysis, prior to incorporating the broken down material. Exemplary sugars include but are not limited hexose (C6) and pentose (C5) polysaccharides, which comprise 6 and 5 carbon sugars, respectively.

Exemplary fermentable sugars include, but are not limited to glucose, xylose, arabinose, galactose, mannose, rhamnose, cellobiose, lactose, sucrose, maltose, and fructose.

[0060] The term "plant polysaccharide" as used herein has its ordinary meaning as known to those skilled in the art and can comprise one or more carbohydrate polymers of sugars and sugar derivatives as well as derivatives of sugar polymers and/or other polymeric materials that occur in plant matter. Exemplary plant polysaccharides include lignin, cellulose, starch, pectin, and hemicellulose. Others are chitin, sulfonated polysaccharides such as alginic acid, agarose, carrageenan, porphyran, furcelleran and funoran. Generally, the polysaccharide can have two or more sugar units or derivatives of sugar units. The sugar units and/or derivatives of sugar units can repeat in a regular pattern, or 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.

[0061] Examples of 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. Microorganisms

[0062] Microorganisms useful in compositions and methods of the invention include, but are not limited to bacteria, or yeast. Examples of bacteria include, but are not limited to, any bacterium found in the genus of Clostridium, such as C. acetobutylicum, C. aerotolerans, C. beijerinckii, C. bifermentans, C. botulinum, C. butyricum, C. cadaveris, C. chauvoei, C. clostridioforme, C. colicanis, C. difficile, C.fallax, C. formicaceticum, C. histolyticum, C. innocuum, C. ljungdahlii, C. laramie, C. lavalense, C. novyi, C. oedematiens, C.

paraputrificum, C. perfringens, C. phytofermentans (including NRRL B-50364 or NRRL B- 50351), C. piliforme, C. ramosum, C. scatologenes, C. septicum, C. sordellii, C. sporogenes, C. sp. Q.D (such as NRRL B-50361, NRRL B-50362, or NRRL B-50363), C. tertium, C. tetani, C. tyrobutyricum, or variants thereof (e.g. C. phytofermentans Q.12 or C.

phytofermentans Q.13).

[0063] Examples of yeast that can be utilized in co-culture methods of the invention include but are not limited to, species found in Cryptococcaceae, Sporobolomycetaceae with the genera Cryptococcus, Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon, Rhodotorula and Sporobolomyces and Bullera, the families Endo- and Saccharomycetaceae, with the genera Saccharomyces, Debaromyces, Lipomyces, Hansenula, Endomycopsis, Pichia, Hanseniaspora, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolitica, Emericella nidulans, Aspergillus nidulans, Deparymyces hansenii and Torulaspora hansenii.

[0064] In another embodiment a microorganism can be wild type, or a genetically modified strain. In one embodiment 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.

Pretreatment of Biomass

[0065] Described herein are also methods and compositions for pre-treating biomass prior to extraction of industrially useful end-products. In some embodiments, more complete saccharification of biomass and fermentation of the saccharification products results in higher fuel yields.

[0066] In some embodiments, Clostridium phytofermentans, Clostridium, sp. Q.D or a variant thereof is contacted with pretreated or non-pretreated feedstock containing cellulosic, hemicellulosic, and/or lignocellulosic material. Additional nutrients can be present or added to the biomass material to be processed by the microorganism including nitrogen-containing compounds such as amino acids, proteins, hydro lyzed proteins, ammonia, urea, nitrate, nitrite, soy, soy derivatives, casein, casein derivatives, milk powder, milk derivatives, whey, yeast extract, hydro lyze yeast, autolyzed yeast, corn steep liquor, corn steep solids, monosodium glutamate, and/or other fermentation nitrogen sources, vitamins, and/or mineral supplements. In some embodiments, one or more additional lower molecular weight carbon sources can be added or be present such as glucose, sucrose, maltose, corn syrup, lactic acid, etc. Such lower molecular weight carbon sources can serve multiple functions including providing an initial carbon source at the start of the fermentation period, help build cell count, control the carbon/nitrogen ratio, remove excess nitrogen, or some other function.

[0067] In some embodiments aerobic/anaerobic cycling is employed for the bioconversion of cellulosic/lignocellulosic material to fuels and chemicals. In some embodiments, the anaerobic microorganism can ferment biomass directly without the need of a pretreatment. In certain embodiments, 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. In some

embodiments 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. In some embodiments 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. In some embodiments 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. [0068] In another embodiment, 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. In other embodiments, pretreatment of biomass comprises pH controlled liquid hot water treatment. Examples of pH controlled liquid hot water treatments are disclosed in N. Mosier et ah, Bioresource Technology, (2005) 96, 1986, incorporated by reference herein in its entirety. In other embodiments, pretreatment of biomass comprises aqueous ammonia recycle process (ARP). Examples of aqueous ammonia recycle process are described in T. H. Kim and Y. Y. Lee, Bioresource Technology, (2005)96, 2007, incorporated by reference herein in its entirety.

[0069] In another embodiment, 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 hydro lyzate stream. In the above methods, 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. As a result, 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. In some embodiments, pretreatment results in removal of about 20%, 30%, 40%>, 50%>, 60%>, 70%> or more of the lignin component of the feedstock. In other embodiments, more than 40%>, 50%>, 60%>, 70%>, 80% or more of the hemicellulose component of the feedstock remains after pretreatment. In some embodiments, the microorganism {e.g., Clostridium phytofermentans, Clostridium, sp. Q.D or a variant thereof) 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.

[0070] In another embodiment, a two-step pretreatment is used to partially or entirely remove C5 polysaccharides and other components. After washing, the second step consists of an alkali treatment to remove lignin components. The pretreated biomass is then washed prior to saccharification and fermentation. One such pretreatment consists of a dilute acid treatment at room temperature or an elevated temperature, followed by a washing or neutralization step, and then an alkaline contact to remove lignin. For example, 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 microorganism such as Clostridium phytofermentans,

Clostridium, sp. Q.D or a variant thereof.

[0071] In another embodiment, pretreatment of biomass comprises ionic liquid

pretreatment. Biomass can be pretreated by incubation with an ionic liquid, followed by extraction with a wash solvent such as alcohol or water. The treated biomass can then be separated from the ionic liquid/wash- solvent solution by centrifugation or filtration, and sent to the saccharification reactor or vessel. Examples of ionic liquid pretreatment are disclosed in US publication No. 2008/0227162, incorporated herein by reference in its entirety.

[0072] 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. 5473061 to Bredereck, et al, U.S. Patent No. 6416621 to Karstens., U.S. Patent No. 6106888 to Dale, et al, U.S. Patent No. 6176176 to Dale, et al, PCT publication

WO2008/020901 to Dale, et al, Felix, A., et al, Anim. Prod. 51, 47-61 (1990)., Wais, A.C., Jr., et al, Journal of Animal Science, 35, No. 1,109-112 (1972), which are incorporated herein by reference in their entireties.

[0073] In some embodiments, after pretreatment by any of the above methods the feedstock contains cellulose, hemicellulose, soluble oligomers, simple sugars, lignins, volatiles and/or ash. The parameters of the pretreatment can be changed to vary the concentration of the components of the pretreated feedstock. For example, in some embodiments a pretreatment is chosen so that the concentration of hemicellulose and/or soluble oligomers is high and the concentration of lignins is low after pretreatment. Examples of parameters of the pretreatment include temperature, pressure, time, and pH.

[0074] In some embodiments, 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. phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13.

[0075] In some embodiments, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is about l%-99%, such as about 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% 1-99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10- 30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15- 30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-80%, 15-90% 15-99%, 20-10%, 20-20%, 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20-99%, 25-10%, 25-20%, 25- 30%, 25-40%, 25-50%, 25-60%, 25-70%, 25-80%, 25-90% 25-99%, 30-10%, 30-20%, 30- 30%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% 30-99%, 35-10%, 35-20%, 35- 30%, 35-40%, 35-50%, 35-60%, 35-70%, 35-80%, 35-90% 35-99%, 40-10%, 40-20%, 40- 30%, 40-40%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90% 40-99%, 45-10%, 45-20%, 45- 30%, 45-40%, 45-50%, 45-60%, 45-70%, 45-80%, 45-90% 45-99%, 50-10%, 50-20%, 50- 30%, 50-40%, 50-50%, 50-60%, 50-70%, 50-80%, 50-90% 50-99%, 55-10%, 55-20%, 55- 30%, 55-40%, 55-50%, 55-60%, 55-70%, 55-80%, 55-90% 55-99%, 60-10%, 60-20%, 60- 30%, 60-40%, 60-50%, 60-60%, 60-70%, 60-80%, 60-90% 60-99%, 65-10%, 65-20%, 65- 30%, 65-40%, 65-50%, 65-60%, 65-70%, 65-80%, 65-90% 65-99%, 70-10%, 70-20%, 70- 30%, 70-40%, 70-50%, 70-60%, 70-70%, 70-80%, 70-90% 70-99%, 75-10%, 75-20%, 75- 30%, 75-40%, 75-50%, 75-60%, 75-70%, 75-80%, 75-90% 75-99%, 80-10%, 80-20%, 80- 30%, 80-40%, 80-50%, 80-60%, 80-70%, 80-80%, 80-90% 80-99%, 85-10%, 85-20%, 85- 30%, 85-40%, 85-50%, 85-60%, 85-70%, 85-80%, 85-90% 85-99%, 90-10%, 90-20%, 90- 30%, 90-40%, 90-50%, 90-60%, 90-70%, 90-80%, 90-90% 90-99%, 95-10%, 95-20%, 95- 30%, 95-40%, 95-50%, 95-60%, 95-70%, 95-80%, 95-90% 95-99%30%, 20-40%, 20-50%, 30-40% or 30-50%. In some embodiments, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is 5% to 30%. In some embodiments, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is 10%> to 20%>.

[0076] In some embodiments, the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is about l%-99%, such as about 1- 10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% 1-99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10-30%, 10- 40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15-30%, 15- 40%, 15-50%, 15-60%, 15-70%, 15-80%, 15-90% 15-99%, 20-10%, 20-20%, 20-30%, 20- 40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20-99%, 25-10%, 25-20%, 25-30%, 25- 40%, 25-50%, 25-60%, 25-70%, 25-80%, 25-90% 25-99%, 30-10%, 30-20%, 30-30%, 30- 40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% 30-99%, 35-10%, 35-20%, 35-30%, 35- 40%, 35-50%, 35-60%, 35-70%, 35-80%, 35-90% 35-99%, 40-10%, 40-20%, 40-30%, 40- 40%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90% 40-99%, 45-10%, 45-20%, 45-30%, 45- 40%, 45-50%, 45-60%, 45-70%, 45-80%, 45-90% 45-99%, 50-10%, 50-20%, 50-30%, 50- 40%, 50-50%, 50-60%, 50-70%, 50-80%, 50-90% 50-99%, 55-10%, 55-20%, 55-30%, 55- 40%, 55-50%, 55-60%, 55-70%, 55-80%, 55-90% 55-99%, 60-10%, 60-20%, 60-30%, 60- 40%, 60-50%, 60-60%, 60-70%, 60-80%, 60-90% 60-99%, 65-10%, 65-20%, 65-30%, 65- 40%, 65-50%, 65-60%, 65-70%, 65-80%, 65-90% 65-99%, 70-10%, 70-20%, 70-30%, 70- 40%, 70-50%, 70-60%, 70-70%, 70-80%, 70-90% 70-99%, 75-10%, 75-20%, 75-30%, 75- 40%, 75-50%, 75-60%, 75-70%, 75-80%, 75-90% 75-99%, 80-10%, 80-20%, 80-30%, 80- 40%, 80-50%, 80-60%, 80-70%, 80-80%, 80-90% 80-99%, 85-10%, 85-20%, 85-30%, 85- 40%, 85-50%, 85-60%, 85-70%, 85-80%, 85-90% 85-99%, 90-10%, 90-20%, 90-30%, 90- 40%, 90-50%, 90-60%, 90-70%, 90-80%, 90-90% 90-99%, 95-10%, 95-20%, 95-30%, 95- 40%, 95-50%, 95-60%, 95-70%, 95-80%, 95-90% 95-99%. In some embodiments, the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is 5% to 40%. In some embodiments, the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is 10% to 30%. In some embodiments, the parameters of the pretreatment are changed such that

concentration of soluble oligomers in the pretreated feedstock is about l%-99%, such as about 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% 1-99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10- 30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15- 30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-80%, 15-90% 15-99%, 20-10%, 20-20%, 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20-99%, 25-10%, 25-20%, 25- 30%, 25-40%, 25-50%, 25-60%, 25-70%, 25-80%, 25-90% 25-99%, 30-10%, 30-20%, 30- 30%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% 30-99%, 35-10%, 35-20%, 35- 30%, 35-40%, 35-50%, 35-60%, 35-70%, 35-80%, 35-90% 35-99%, 40-10%, 40-20%, 40- 30%, 40-40%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90% 40-99%, 45-10%, 45-20%, 45- 30%, 45-40%, 45-50%, 45-60%, 45-70%, 45-80%, 45-90% 45-99%, 50-10%, 50-20%, 50- 30%, 50-40%, 50-50%, 50-60%, 50-70%, 50-80%, 50-90% 50-99%, 55-10%, 55-20%, 55- 30%, 55-40%, 55-50%, 55-60%, 55-70%, 55-80%, 55-90% 55-99%, 60-10%, 60-20%, 60- 30%, 60-40%, 60-50%, 60-60%, 60-70%, 60-80%, 60-90% 60-99%, 65-10%, 65-20%, 65- 30%, 65-40%, 65-50%, 65-60%, 65-70%, 65-80%, 65-90% 65-99%, 70-10%, 70-20%, 70- 30%, 70-40%, 70-50%, 70-60%, 70-70%, 70-80%, 70-90% 70-99%, 75-10%, 75-20%, 75- 30%, 75-40%, 75-50%, 75-60%, 75-70%, 75-80%, 75-90% 75-99%, 80-10%, 80-20%, 80- 30%, 80-40%, 80-50%, 80-60%, 80-70%, 80-80%, 80-90% 80-99%, 85-10%, 85-20%, 85- 30%, 85-40%, 85-50%, 85-60%, 85-70%, 85-80%, 85-90% 85-99%, 90-10%, 90-20%, 90- 30%, 90-40%, 90-50%, 90-60%, 90-70%, 90-80%, 90-90% 90-99%, 95-10%, 95-20%, 95- 30%, 95-40%, 95-50%, 95-60%, 95-70%, 95-80%, 95-90% 95-99%. In some embodiments, the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Examples of soluble oligomers include, but are not limited to, cellobiose and xylobiose. In some embodiments, the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is 30% to 90%. In some embodiments, the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is 45% to 80%. In some embodiments, 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.

[0077] In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is about l%-99%, such as about 1- 10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% 1-99%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90% 5-99%, 10-10%, 10-20%, 10-30%, 10- 40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90% 10-99%, 15-10%, 15-20%, 15-30%, 15- 40%, 15-50%, 15-60%, 15-70%, 15-80%, 15-90% 15-99%, 20-10%, 20-20%, 20-30%, 20- 40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90% 20-99%, 25-10%, 25-20%, 25-30%, 25- 40%, 25-50%, 25-60%, 25-70%, 25-80%, 25-90% 25-99%, 30-10%, 30-20%, 30-30%, 30- 40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% 30-99%, 35-10%, 35-20%, 35-30%, 35- 40%, 35-50%, 35-60%, 35-70%, 35-80%, 35-90% 35-99%, 40-10%, 40-20%, 40-30%, 40- 40%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90% 40-99%, 45-10%, 45-20%, 45-30%, 45- 40%, 45-50%, 45-60%, 45-70%, 45-80%, 45-90% 45-99%, 50-10%, 50-20%, 50-30%, 50- 40%, 50-50%, 50-60%, 50-70%, 50-80%, 50-90% 50-99%, 55-10%, 55-20%, 55-30%, 55- 40%, 55-50%, 55-60%, 55-70%, 55-80%, 55-90% 55-99%, 60-10%, 60-20%, 60-30%, 60- 40%, 60-50%, 60-60%, 60-70%, 60-80%, 60-90% 60-99%, 65-10%, 65-20%, 65-30%, 65- 40%, 65-50%, 65-60%, 65-70%, 65-80%, 65-90% 65-99%, 70-10%, 70-20%, 70-30%, 70- 40%, 70-50%, 70-60%, 70-70%, 70-80%, 70-90% 70-99%, 75-10%, 75-20%, 75-30%, 75- 40%, 75-50%, 75-60%, 75-70%, 75-80%, 75-90% 75-99%, 80-10%, 80-20%, 80-30%, 80- 40%, 80-50%, 80-60%, 80-70%, 80-80%, 80-90% 80-99%, 85-10%, 85-20%, 85-30%, 85- 40%, 85-50%, 85-60%, 85-70%, 85-80%, 85-90% 85-99%, 90-10%, 90-20%, 90-30%, 90- 40%, 90-50%, 90-60%, 90-70%, 90-80%, 90-90% 90-99%, 95-10%, 95-20%, 95-30%, 95- 40%, 95-50%, 95-60%, 95-70%, 95-80%, 95-90% 95-99%. In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is about 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 20%. In some embodiments, the parameters of the pretreatment are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 5%. Examples of simple sugars include, but are not limited to, C5 and C6 monomers and dimers.

[0078] In some embodiments, the parameters of the pretreatment are changed such that concentration of lignins in the pretreated feedstock is about 1%, 10%>, 15%, 20%>, 25%, 30%>, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. 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%. In some embodiments, the parameters of the pretreatment are changed such that the concentration of phenolics is minimized.

[0079] 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%. [0080] In some embodiments, 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 l% to 2%.

[0081] In some embodiments, 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%). In some embodiments, the parameters of the pretreatment are changed to obtain a high concentration of hemicellulose and a low concentration of lignins such that concentration of the components in the pretreated stock is optimal for fermentation with a microorganism such as a member of the genus Clostridium, for example Clostridium phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.12 Clostridium phytofermentans Q.13 or variants thereof.

[0082] Certain conditions of pretreatment can be modified prior to, or concurrently with, introduction of a fermentative microorganism into the feedstock. For example, pretreated feedstock can be cooled to a temperature which allows for growth of the microorganism(s). As another example, pH can be altered prior to, or concurrently with, addition of one or more microorganisms.

[0083] 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. In another embodiment, 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. In some embodiments, 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. When more than one pH modifiers are utilized, they 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. [0084] In some embodiments, 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. In some instances, 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. In some embodiments, 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.

[0085] In another embodiment, biomass can be pre-treated at an elevated temperature and/or pressure. In one embodiment biomass is pre treated at a temperature range of 20°C to 400°C. In another embodiment 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. In another embodiment, elevated

temperatures are provided by the use of steam, hot water, or hot gases. In one embodiment steam can be injected into a biomass containing vessel. In another embodiment 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.

[0086] In another embodiment, a biomass can be treated at an elevated pressure. In one embodiment biomass is pre treated at a pressure range of about lpsi to about 30psi. In another embodiment 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. In some embodiments, biomass can be treated with elevated pressures by the injection of steam into a biomass containing vessel. In other embodiments, the biomass can be treated to vacuum conditions prior or subsequent to alkaline or acid treatment or any other treatment methods provided herein.

[0087] In one embodiment 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. In one embodiment, the drying step can be performed under vacuum to increase the rate of evaporation of water or other solvents. Alternatively, or additionally, 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. [0088] In some embodiments of the present invention, 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. In some embodiments, 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. In one embodiment 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. In one embodiment 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.

[0089] In some embodiments, biomass (e.g. corn stover) is processed or pretreated prior to fermentation. In one embodiment 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. In some embodiments, 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. In one embodiment size separation can provide for enhanced yields. In some embodiments, separation of finely shredded biomass (e.g. particles smaller than about 8 mm in diameter, such as, 8, 7.9, 7.7, 7.5, 7.3, 7, 6.9, 6.7, 6.5, 6.3, 6, 5.9, 5.7, 5.5, 5.3, 5, 4.9, 4.7, 4.5, 4.3, 4, 3.9, 3.7, 3.5, 3.3, 3, 2.9, 2.7, 2.5, 2.3, 2, 1.9, 1.7, 1.5, 1.3,

1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mm) from larger particles allows the recycling of the larger particles back into the size reduction process, thereby increasing the final yield of processed biomass. In one embodiment, a fermentative mixture is provided 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. In some instances, pretreatment of the lignocellulosic feedstock comprises adding an alkaline substance which raises the pH to an alkaline level, for example NaOH. In some embodiments, NaOH is added at a concentration of about 0.5% to about 2% by weight of the feedstock. In other embodiments, pretreatment also comprises addition of a chelating agent. In some embodiments, 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, Clostridium phytofermentans Q.13 or variant thereof.

[0090] 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. In some instances, the five-carbon sugar is xylose, arabinose, or a combination thereof. In other instances, the six-carbon sugar is glucose, galactose, mannose, or a combination thereof. In some embodiments, the alkaline substance is NaOH. In some embodiments, NaOH is added at a concentration of about 0.5% to about 2% by weight of the feedstock. In some embodiments, 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 or variants thereof. In still other embodiments, the microorganism is genetically modified to enhance activity of one or more hydro lytic enzymes.

[0091] Further provided herein is 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. In some embodiments the alkaline substance is NaOH. In some embodiments, NaOH is added at a concentration of about 0.5% to about 2% by weight of the feedstock. In some embodiments, 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. In other embodiments, 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.

[0092] 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. In some instances, the sources of cellulosic material are corn stover, bagasse, switchgrass or poplar. In some embodiments the alkaline substance is NaOH. In some embodiments, NaOH is added at a concentration of about 0.5% to about 2% by weight of the feedstock. In some embodiments, 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 or variants thereof.

[0093] In some embodiments, a process for simultaneous saccharification and fermentation of cellulosic solids from biomass into biofuel or another end-product is provided. In one embodiment the process comprises treating the biomass in a closed container with a microorganism under conditions where the microorganism produces saccharolytic enzymes sufficient to substantially convert the biomass into oligomers, monosaccharides and disaccharides. In one embodiment the microorganism subsequently converts the oligomers, monosaccharides and disaccharides into ethanol and/or another biofuel or product.

[0094] In an another embodiment, a process for saccharification and fermentation comprises treating the biomass in a container with the microorganism, and adding one or more enzymes before, concurrent or after contacting the biomass with the microorganism, wherein the enzymes added aid in the breakdown or detoxification of carbohydrates or lignocellulosic material.

[0095] In one embodiment, the bioconversion process comprises a separate hydrolysis and fermentation (SHF) process. In an SHF embodiment, the enzymes can be used under their optimal conditions regardless of the fermentation conditions and the microorganism is only required to ferment released sugars. In this embodiment, hydrolysis enzymes are externally added.

[0096] In another embodiment, the bioconversion process comprises a saccharification and fermentation (SSF) process. In an SSF embodiment, hydrolysis and fermentation take place in the same reactor under the same conditions.

[0097] In another embodiment, the bioconversion process comprises a consolidated bioprocess (CBP). In essence, CBP is a variation of SSF in which the enzymes are produced by the microorganism that carries out the fermentation. In this embodiment, enzymes can be both externally added enzymes and enzymes produced by the fermentative microorganism. In this embodiment, biomass is partially hydro lyzed with externally added enzymes at their optimal condition, the slurry is then transferred to a separate tank in which the fermentative

microorganism (e.g. Clostridium phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13 or variants thereof.) converts the hydro lyzed sugar into the desired product (e.g. fuel or chemical) and completes the hydrolysis of the residual cellulose and hemicellulose.

[0098] In one embodiment, pretreated biomass is partially hydrolyzed by externally added enzymes to reduce the viscosity. Hydrolysis occurs at the optimal pH and temperature conditions (e.g. pH 5.5, 50°C for fungal cellulases). Hydrolysis time and enzyme loading can be adjusted such that conversion is limited to cellodextrins (soluble and insoluble) and hemicellulose oligomers. At the conclusion of the hydrolysis time, 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, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13 or variants thereof.) and media. The microorganism can then produce endogenous enzymes to complete the hydrolysis into fermentable sugars (soluble oligomers) and convert those sugars into ethanol and/or other products in a production tank. The production tank can then be operated under fermentation optimal conditions (e.g. pH 6.5, 35°C). In this way externally added enzyme is minimized due to operation under the enzyme's optimal conditions and due to a portion of the enzyme coming from the microorganism (e.g. Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13 or variants thereof.)

[0099] In some embodiments, exogenous enzymes added include a xylanase, a

hemicellulase, a glucanase or a glucosidase. In some embodiments, exogenous enzymes added do not include a xylanase, a hemicellulase, a glucanase or a glucosidase. In other embodiments, the amount of exogenous cellulase is greatly reduced, one-quarter or less of the amount normally added to a fermentation by a microorganism that cannot saccharify the biomass.

[00100] In one embodiment a second microorganism can be used to convert residual carbohydrates into a fermentation end-product. In one embodiment the second

microorganism is a yeast such as Saccharomyces cerevisiae; a Clostridia species such as C. thermocellum, C. acetobutylicum, or C. cellovorans; or Zymomonas mobilis.

[00101] In one embodiment, a process of producing a bio fuel or chemical product from a lignin-containing biomass is provided. In one embodiment the process 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 phytofermentans Q.13 or a Clostridium phytofermentans Q.12 or variants thereof.) under conditions wherein 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.

[00102] Of various molecules typically found in 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 β- 1 ,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.

[00103] 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 (between the chains of linear cellulosic polymer) 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. Of course, with respect to utilization of any of the methods disclosed herein, conditioning of a biomass can be concurrent to contact with a microorganism that is capable of saccharification and fermentation. In addition, other examples describing the pretreatment of hgnocellulosic biomass have been published as U.S. Pat. Nos. 4,304,649, 5,366,558, 5,41 1,603, and 5,705,369.

Biomass Processing

[00104] Described herein are compositions and methods allowing saccharification and fermentation to one or more industrially useful fermentation end-products. Saccharification includes conversion of long-chain sugar polymers, such as cellulose, to monosaccharides, disaccharides, trisaccharides, and oligosaccharides of up to about seven monomer units, as well as similar sized chains of sugar derivatives and combinations of sugars and sugar derivatives. The chain-length for saccharides can be longer (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units or more) and or shorter (e.g. 1, 2, 3, 4, 5, 6 monomer units). As used herein, "directly processing" means that a microorganism is capable of both hydro lyzing biomass and fermenting without the need for conditioning the biomass, such as subjecting the biomass to chemical, heat, enzymatic treatment or combinations thereof. [00105] Methods and compositions described herein contemplate utilizing fermentation process for extracting industrially useful fermentation end-products from biomass. The term "fermentation" as used herein has its ordinary meaning as known to those skilled in the art and can include culturing of a microorganism or group of microorganisms in or on a suitable medium for the microorganisms. The microorganisms can be aerobes, anaerobes, facultative anaerobes, heterotrophs, autotrophs, photoautotrophs, photoheterotrophs, chemoautotrophs, and/or chemoheterotrophs. The cellular activity, including cell growth can be growing aerobic, microaerophilic, or anaerobic. The cells can be in any phase of growth, including lag (or conduction), exponential, transition, stationary, death, dormant, vegetative, sporulating, etc.

[00106] 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. One example of such a microorganism is Clostridium phytofermentans ("C. phytofermentans^'"), which can simultaneously hydrolyze and ferment lignocellulosic biomass. Furthermore, C. phytofermentans is capable of fermenting hexose (C6) and pentose (C5) polysaccharides. In addition, C. phytofermentans is capable of acting directly on lignocellulosic biomass without any pretreatment. Other examples of microorganisms that can ferment hexose (C6) and pentose (C5 ) polysaccharides include Clostridium sp. Q.D, or mutagen ized variants of

Clostridium phytofermentans, such as Clostridium Q.12, or Clostridium phytofermentans Q.13. Additionally, these organisms produce hernicellulases, pectinases, xylansases, and chitinases. Co-Culture Methods and Compositions

[00107] Methods of the invention can also included co-culture with an microorganism that naturally produces or is genetically modified to produce one or more enzymes, such as hydro lytic enzymes (such as cellulase(s), hemicellulase(s), or pectinases etc.) or antioxidants (such as catalase, superoxide dismutase or glutathione peroxidase). A culture medium containing such a microorganism can be contacted with biomass (e.g., in a bioreactor) prior to, concurrent with, or subsequent to contact with a second microorganism. In one embodiment a first microorganism produces saccharifying enzyme while a second microorganism ferments C5 and C6 sugars. In one embodiment, the first microorganism is C. phytopfermentans or C. sp. Q.D. Mixtures of microorganisms can be provided as solid mixtures (e.g., freeze-dried mixtures), or as liquid dispersions of the microorganisms, and grown in co-culture with a second microorganism. Co-culture methods capable of use with the present invention are known, such as those disclosed in U.S. Pat. Application No.

20070178569. Fermentation end-product

[00108] The term "fuel" or "bio fuel" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more compounds suitable as liquid fuels, gaseous fuels, biodiesel fuels (long-chain alkyl (methyl, propyl or ethyl) esters), heating oils (hydrocarbons in the 14-20 carbon range), reagents, chemical feedstocks and includes, but is not limited to, hydrocarbons (both light and heavy), hydrogen, methane, hydroxy compounds such as alcohols (e.g. ethanol, butanol, propanol, methanol, etc.), and carbonyl compounds such as aldehydes and ketones (e.g. acetone, formaldehyde, 1-propanal, etc.).

[00109] The term "fermentation end-product" or "end-product" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biofuels,or chemicals,(such as additives, processing aids, food additives, organic acids (e.g. acetic, lactic, formic, citric acid etc.), derivatives of organic acids such as esters (e.g. wax esters, glycerides, etc.) or other compounds). These end-products include, but are not limited to, an alcohol (such as ethanol, butanol, methanol, 1 , 2-propanediol, or 1, 3-propanediol), an acid (such as lactic acid, formic acid, acetic acid, succinic acid, or pyruvic acid), enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases and can be present as a pure compound, a mixture, or an impure or diluted form. In one embodiment a fermentation end-product is made using a process or microorganism disclosed herein. In another embodiment production of a fermentation end-product is enhanced through saccharification and fermentation using enzyme-enhancing products or processes.

[00110] In one embodiment a fermentation end-product is a 1 ,4 diacid (succinic, fumaric and malic), 2,5 furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol/arabitol, butanediol, butanol, isopentenyl diphosphate, methane, methanol, ethane, ethene, ethanol, n-propane, 1-propene, 1 -propanol, propanal, acetone, propionate, n-butane, 1-butene, 1 -butanol, butanal, butanoate, isobutanal, isobutanol, 2-methylbutanal, 2- methylbutanol, 3-methylbutanal, 3-methylbutanol, 2-butene, 2-butanol, 2-butanone, 2,3- butanediol, 3-hydroxy-2-butanone, 2,3-butanedione, ethylbenzene, ethenylbenzene, 2- phenylethanol, phenylacetaldehyde, 1-phenylbutane, 4-phenyl- 1-butene, 4-phenyl-2-butene,

1- phenyl-2-butene, l-phenyl-2-butanol, 4-phenyl-2-butanol, l-phenyl-2-butanone, 4-phenyl-

2- butanone, l-phenyl-2,3-butandiol, l-phenyl-3 -hydro xy-2-butanone, 4-phenyl-3 -hydro xy-2- butanone, l-phenyl-2,3-butanedione, n-pentane, ethylphenol, ethenylphenol, 2-(4- hydroxyphenyl)ethanol, 4-hydroxyphenylacetaldehyde, l-(4-hydroxyphenyl) butane, 4-(4- hydroxyphenyl)- 1-butene, 4-(4-hydroxyphenyl)-2-butene, l-(4-hydroxyphenyl)- 1-butene, 1- (4-hydroxyphenyl)-2-butanol, 4-(4-hydroxyphenyl)-2-butanol, 1 -(4-hydroxyphenyl)-2- butanone, 4-(4-hydroxyphenyl)-2-butanone, l-(4-hydroxyphenyl)-2,3-butandiol, l-(4- hydroxyphenyl)-3 -hydro xy-2-butanone, 4-(4-hydroxyphenyl)-3 -hydro xy-2-butanone, 1 -(4- hydroxyphenyl)-2,3-butanonedione, indolylethane, indolylethene, 2-(indole-3-)ethanol, n- pentane, 1 -pentene, 1-pentanol, pentanal, pentanoate, 2-pentene, 2-pentanol, 3-pentanol, 2- pentanone, 3-pentanone, 4-methylpentanal, 4-methylpentanol, 2,3-pentanediol, 2-hydroxy-3- pentanone, 3-hydroxy-2-pentanone, 2,3-pentanedione, 2-methylpentane, 4-methyl-l -pentene, 4-methyl-2-pentene, 4-methyl-3 -pentene, 4-methyl-2-pentanol, 2-methyl-3-pentanol, 4- methyl-2-pentanone, 2-methyl-3 -pentanone, 4-methyl-2,3-pentanediol, 4-methyl-2-hydroxy- 3-pentanone, 4-methyl-3 -hydro xy-2-pentanone, 4-methyl-2,3-pentanedione, 1-phenylpentane, 1 -phenyl- 1 -pentene, 1 -phenyl-2-pentene, 1 -phenyl-3 -pentene, 1 -phenyl-2-pentano 1, 1 -phenyl- 3-pentanol, l-phenyl-2-pentanone, 1 -phenyl-3 -pentanone, l-phenyl-2,3-pentanediol, 1- phenyl-2-hydroxy-3 -pentanone, 1 -phenyl-3 -hydroxy-2-pentanone, 1 -phenyl-2,3- pentanedione, 4-methyl- 1-phenylpentane, 4-methyl-l -phenyl- 1 -pentene, 4-methyl- 1-phenyl- 2-pentene, 4-methyl- 1 -phenyl-3 -pentene, 4-methyl- 1 -phenyl-3 -pentano 1, 4-methyl- 1 -phenyl- 2-pentanol, 4-methyl-l -phenyl-3 -pentanone, 4-methyl- l-phenyl-2-pentanone, 4-methyl- 1- phenyl-2,3-pentanediol, 4-methyl- 1 -phenyl-2,3-pentanedione, 4-methyl- 1 -phenyl-3 -hydro xy- 2-pentanone, 4-methyl- l-phenyl-2-hydroxy-3 -pentanone, l-(4-hydroxyphenyl) pentane, l-(4- hydroxyphenyl)- 1 -pentene, 1 -(4-hydroxyphenyl)-2-pentene, 1 -(4-hydroxyphenyl)-3 -pentene, 1 -(4-hydroxyphenyl)-2-pentano 1, 1 -(4-hydroxyphenyl)-3 -pentano 1, 1 -(4-hydroxyphenyl)-2- pentanone, l-(4-hydroxyphenyl)-3 -pentanone, l-(4-hydroxyphenyl)-2,3-pentanediol, l-(4- hydroxyphenyl)-2-hydroxy-3 -pentanone, 1 -(4-hydroxyphenyl)-3 -hydro xy-2-pentanone, 1 -(4- hydroxyphenyl)-2,3-pentanedione, 4-methyl- l-(4-hydroxyphenyl) pentane, 4-methyl-l -(4- hydroxyphenyl)-2-pentene, 4-methyl- 1 -(4-hydroxyphenyl)-3 -pentene, 4-methyl- 1 -(4- hydroxyphenyl)- 1 -pentene, 4-methyl- 1 -(4-hydroxyphenyl)-3 -pentano 1, 4-methyl- 1 -(4- hydroxyphenyl)-2-pentano 1, 4-methyl- 1 -(4-hydroxyphenyl)-3 -pentanone, 4-methyl- 1 -(4- hydroxyphenyl)-2-pentanone, 4-methyl- 1 -(4-hydroxyphenyl)-2,3-pentanediol, 4-methyl- 1 -(4- hydroxyphenyl)-2,3-pentanedione, 4-methyl- 1 -(4-hydroxyphenyl)-3-hydroxy-2-pentanone, 4- methyl- 1 -(4-hydroxyphenyl)-2-hydroxy-3-pentanone, 1 -indole-3 -pentane, 1 -(indole-3)- 1 - pentene, l-(indole-3)-2-pentene, l-(indole-3)-3-pentene, l-(indole-3)-2-pentanol, l-(indole- 3)-3-pentanol, l-(indole-3)-2-pentanone, l-(indole-3)-3-pentanone, l-(indole-3)-2,3- pentanediol, l-(indole-3)-2-hydroxy-3-pentanone, l-(indole-3)-3-hydroxy-2-pentanone, 1- (indole-3)-2,3-pentanedione, 4-methyl- 1 -(indole-3 -)pentane, 4-methyl- 1 -(indole-3)-2- pentene, 4-methyl- l-(indole-3)-3-pentene, 4-methyl-l -(indole-3)- 1 -pentene, 4-methyl-2- (indole-3)-3-pentanol, 4-methyl- 1 -(indole-3)-2-pentanol, 4-methyl- 1 -(indole-3)-3-pentanone, 4-methyl- 1 -(indole-3)-2-pentanone, 4-methyl- 1 -(indole-3)-2,3-pentanediol, 4-methyl- 1 - (indole-3)-2,3-pentanedione, 4-methyl- 1 -(indole-3)-3-hydroxy-2-pentanone, 4-methyl- 1 - (indole-3)-2-hydroxy-3-pentanone, n-hexane, 1-hexene, 1-hexanol, hexanal, hexanoate, 2- hexene, 3-hexene, 2-hexanol, 3-hexanol, 2-hexanone, 3-hexanone, 2,3-hexanediol, 2,3- hexanedione, 3,4-hexanediol, 3,4-hexanedione, 2-hydroxy-3-hexanone, 3-hydroxy-2- hexanone, 3-hydroxy-4-hexanone, 4-hydroxy-3-hexanone, 2-methylhexane, 3-methylhexane, 2-methyl-2-hexene, 2-methyl-3-hexene, 5-methyl- 1-hexene, 5-methyl-2-hexene, 4-methyl- 1- hexene, 4-methyl-2-hexene, 3-methyl-3-hexene, 3-methyl-2-hexene, 3 -methyl- 1-hexene, 2- methyl-3-hexanol, 5-methyl-2-hexanol, 5 -methyl- 3-hexanol, 2-methyl-3-hexanone, 5-methyl- 2-hexanone, 5-methyl-3-hexanone, 2-methyl-3,4-hexanediol, 2-methyl-3,4-hexanedione, 5- methyl-2,3-hexanediol, 5-methyl-2,3-hexanedione, 4-methyl-2,3-hexanediol, 4-methyl-2,3- hexanedione, 2-methyl-3 -hydro xy-4-hexanone, 2-methyl-4-hydroxy-3-hexanone, 5-methyl-2- hydroxy-3-hexanone, 5-methyl-3-hydroxy-2-hexanone, 4-methyl-2-hydroxy-3-hexanone, 4- methyl-3 -hydro xy-2-hexanone, 2,5-dimethylhexane, 2,5-dimethyl-2-hexene, 2,5-dimethyl-3- hexene, 2,5-dimethyl-3-hexanol, 2,5-dimethyl-3-hexanone, 2,5-dimethyl-3,4-hexanediol, 2,5- dimethyl-3,4-hexanedione, 2,5-dimethyl-3-hydroxy-4-hexanone, 5 -methyl- 1-phenylhexane, 4-methyl- 1 -phenylhexane, 5 -methyl- 1 -phenyl- 1 -hexene, 5 -methyl- 1 -phenyl-2-hexene, 5 - methyl- l-phenyl-3-hexene, 4-methyl- 1 -phenyl- 1-hexene, 4-methyl- l-phenyl-2-hexene, 4- methyl- 1 -phenyl-3 -hexene, 5 -methyl- 1 -phenyl-2-hexano 1, 5 -methyl- 1 -phenyl-3 -hexano 1, 4- methyl- 1 -phenyl-2-hexano 1, 4-methyl- 1 -phenyl-3 -hexano 1, 5 -methyl- 1 -phenyl-2-hexanone, 5 -methyl- 1 -phenyl-3 -hexanone, 4-methyl- 1 -phenyl-2-hexanone, 4-methyl- 1 -phenyl-3 - hexanone, 5-methyl- l-phenyl-2, 3 -hexanediol, 4-methyl- l-phenyl-2,3-hexanediol, 5-methyl- 1 -phenyl-3 -hydro xy-2-hexanone, 5 -methyl- 1 -phenyl-2-hydroxy-3 -hexanone, 4-methyl- 1 - phenyl-3 -hydro xy-2-hexanone, 4-methyl- 1 -phenyl-2-hydroxy-3 -hexanone, 5 -methyl- 1 - phenyl-2,3-hexanedione, 4-methyl- 1 -phenyl-2,3-hexanedione, 4-methyl- 1 -(4- hydroxyphenyl)hexane, 5 -methyl- 1 -(4-hydroxyphenyl)- 1 -hexene, 5 -methyl- 1 -(4- hydroxyphenyl)-2-hexene, 5 -methyl- 1 -(4-hydroxyphenyl)-3 -hexene, 4-methyl- 1 -(4- hydroxyphenyl)- 1 -hexene, 4-methyl- 1 -(4-hydroxyphenyl)-2-hexene, 4-methyl- 1 -(4- hydroxyphenyl)-3 -hexene, 5 -methyl- 1 -(4-hydroxyphenyl)-2-hexano 1, 5 -methyl- 1 -(4- hydroxyphenyl)-3 -hexano 1, 4-methyl- 1 -(4-hydroxyphenyl)-2-hexano 1, 4-methyl- 1 -(4- hydroxyphenyl)-3 -hexano 1, 5 -methyl- 1 -(4-hydroxyphenyl)-2-hexanone, 5 -methyl- 1 -(4- hydroxyphenyl)-3 -hexanone, 4-methyl- 1 -(4-hydroxyphenyl)-2-hexanone, 4-methyl- 1 -(4- hydroxyphenyl)-3 -hexanone, 5-methyl- 1 -(4-hydroxyphenyl)-2,3-hexanediol, 4-methyl- 1 -(4- hydro xyphenyl)-2,3-hexanediol, 5-methyl- 1 -(4-hydroxyphenyl)-3-hydroxy-2-hexanone, 5- methyl- 1 -(4-hydroxyphenyl)-2-hydroxy-3 -hexanone, 4-methyl- 1 -(4-hydroxyphenyl)-3 - hydro xy-2-hexanone, 4-methyl- 1 -(4-hydroxyphenyl)-2-hydroxy-3 -hexanone, 5 -methyl- 1 -(4- hydroxyphenyl)-2,3-hexanedione, 4-methyl- 1 -(4-hydroxyphenyl)-2,3-hexanedione, 4-methyl-

1- (indole-3-)hexane, 5-methyl- l-(indole-3)-l-hexene, 5-methyl- l-(indole-3)-2-hexene, 5- methyl- 1 -(indole-3)-3-hexene, 4-methyl- 1 -(indole-3)- 1 -hexene, 4-methyl- 1 -(indole-3)-2- hexene, 4-methyl- l-(indole-3)-3-hexene, 5-methyl- l-(indole-3)-2-hexanol, 5-methyl- 1- (indole-3)-3-hexanol, 4-methyl- l-(indole-3)-2-hexanol, 4-methyl- l-(indole-3)-3-hexanol, 5- methyl- 1 -(indole-3)-2-hexanone, 5-methyl- 1 -(indole-3)-3-hexanone, 4-methyl- 1 -(indole-3)-

2- hexanone, 4-methyl- l-(indole-3)-3-hexanone, 5-methyl- l-(indole-3)-2,3-hexanediol, 4- methyl- 1 -(indole-3)-2,3-hexanediol, 5-methyl- 1 -(indole-3)-3-hydroxy-2-hexanone, 5-methyl- 1 -(indole-3)-2-hydroxy-3-hexanone, 4-methyl- 1 -(indole-3)-3-hydroxy-2-hexanone, 4-methyl- 1 -(indole-3)-2-hydroxy-3-hexanone, 5-methyl- 1 -(indole-3)-2,3-hexanedione, 4-methyl- 1 - (indole-3)-2,3-hexanedione, n-heptane, 1-heptene, 1-heptanol, heptanal, heptanoate, 2- heptene, 3-heptene, 2-heptanol, 3-heptanol, 4-heptanol, 2-heptanone, 3-heptanone, 4- heptanone, 2,3-heptanediol, 2,3-heptanedione, 3,4-heptanediol, 3,4-heptanedione, 2-hydroxy-

3- heptanone, 3-hydroxy-2-heptanone, 3-hydroxy-4-heptanone, 4-hydroxy-3 -heptanone, 2- methylheptane, 3-methylheptane, 6-methyl-2-heptene, 6-methyl-3-heptene, 2-methyl-3- heptene, 2-methyl-2-heptene, 5-methyl-2-heptene, 5-methyl-3-heptene, 3-methyl-3-heptene, 2-methyl-3-heptanol, 2-methyl-4-heptanol, 6-methyl-3-heptanol, 5-methyl-3-heptanol, 3- methyl-4-heptanol, 2-methyl-3 -heptanone, 2-methyl-4-heptanone, 6-methyl-3 -heptanone, 5- methyl-3 -heptanone, 3-methyl-4-heptanone, 2-methyl-3,4-heptanediol, 2-methyl-3,4- heptanedione, 6-methyl-3,4-heptanediol, 6-methyl-3,4-heptanedione, 5-methyl-3,4- heptanediol, 5-methyl-3,4-heptanedione, 2-methyl-3-hydroxy-4-heptanone, 2-methyl-4- hydroxy-3 -heptanone, 6-methyl-3 -hydro xy-4-heptanone, 6-methyl-4-hydroxy-3 -heptanone, 5-methyl-3-hydroxy-4-heptanone, 5 -methyl-4-hydroxy-3 -heptanone, 2,6-dimethylheptane, 2,5-dimethylheptane, 2,6-dimethyl-2-heptene, 2,6-dimethyl-3-heptene, 2,5-dimethyl-2- heptene, 2,5-dimethyl-3-heptene, 3,6-dimethyl-3-heptene, 2,6-dimethyl-3-heptanol, 2,6- dimethyl-4-heptanol, 2,5-dimethyl-3-heptanol, 2,5-dimethyl-4-heptanol, 2,6-dimethyl-3,4- heptanediol, 2,6-dimethyl-3,4-heptanedione, 2,5-dimethyl-3,4-heptanediol, 2,5-dimethyl-3,4- heptanedione, 2,6-dimethyl-3-hydroxy-4-heptanone, 2,6-dimethyl-4-hydroxy-3-heptanone, 2,5-dimethyl-3-hydroxy-4-heptanone, 2,5-dimethyl-4-hydroxy-3-heptanone, n-octane, 1- octene, 2-octene, 1-octanol, octanal, octanoate, 3-octene, 4-octene, 4-octanol, 4-octanone, 4,5-octanediol, 4,5-octanedione, 4-hydroxy-5-octanone, 2-methyloctane, 2-methyl-3-octene, 2- methyl-4-octene, 7-methyl-3-octene, 3-methyl-3-octene, 3-methyl-4-octene, 6-methyl-3- octene, 2-methyl-4-octanol, 7-methyl-4-octanol, 3-methyl-4-octanol, 6-methyl-4-octanol, 2- methyl-4-octanone, 7-methyl-4-octanone, 3-methyl-4-octanone, 6-methyl-4-octanone, 2- methyl-4,5-octanediol, 2-methyl-4,5-octanedione, 3-methyl-4,5-octanediol, 3-methyl-4,5- octanedione, 2-methyl-4-hydroxy-5-octanone, 2-methyl-5-hydroxy-4-octanone, 3-methyl-4- hydroxy-5-octanone, 3-methyl-5-hydroxy-4-octanone, 2,7-dimethyloctane, 2,7-dimethyl-3- octene, 2,7-dimethyl-4-octene, 2,7-dimethyl-4-octanol, 2,7-dimethyl-4-octanone, 2,7- dimethyl-4,5-octanediol, 2,7-dimethyl-4,5-octanedione, 2,7-dimethyl-4-hydroxy-5-octanone, 2,6-dimethyloctane, 2,6-dimethyl-3-octene, 2,6-dimethyl-4-octene, 3,7-dimethyl-3-octene, 2,6-dimethyl-4-octanol, 3,7-dimethyl-4-octanol, 2,6-dimethyl-4-octanone, 3,7-dimethyl-4- octanone, 2,6-dimethyl-4,5-octanediol, 2,6-dimethyl-4,5-octanedione, 2,6-dimethyl-4- hydroxy-5-octanone, 2,6-dimethyl-5-hydroxy-4-octanone, 3,6-dimethyloctane, 3,6-dimethyl-

3- octene, 3,6-dimethyl-4-octene, 3,6-dimethyl-4-octanol, 3,6-dimethyl-4-octanone, 3,6- dimethyl-4,5-octanediol, 3,6-dimethyl-4,5-octanedione, 3,6-dimethyl-4-hydroxy-5-octanone, n-nonane, 1-nonene, 1-nonanol, nonanal, nonanoate, 2-methylnonane, 2-methyl-4-nonene, 2- methyl-5-nonene, 8-methyl-4-nonene, 2-methyl-5-nonanol, 8-methyl-4-nonanol, 2-methyl-5- nonanone, 8-methyl-4-nonanone, 8-methyl-4,5-nonanediol, 8-methyl-4,5-nonanedione, 8- methyl-4-hydroxy-5-nonanone, 8-methyl-5-hydroxy-4-nonanone, 2,8-dimethylnonane, 2,8- dimethyl-3-nonene, 2,8-dimethyl-4-nonene, 2,8-dimethyl-5-nonene, 2,8-dimethyl-4-nonanol, 2,8-dimethyl-5-nonanol, 2,8-dimethyl-4-nonanone, 2,8-dimethyl-5-nonanone, 2,8-dimethyl- 4,5-nonanediol, 2,8-dimethyl-4,5-nonanedione, 2,8-dimethyl-4-hydroxy-5-nonanone, 2,8- dimethyl-5 -hydro xy-4-nonanone, 2,7-dimethylnonane, 3,8-dimethyl-3-nonene, 3,8-dimethyl-

4- nonene, 3,8-dimethyl-5-nonene, 3,8-dimethyl-4-nonanol, 3,8-dimethyl-5-nonanol, 3,8- dimethyl-4-nonanone, 3,8-dimethyl-5-nonanone, 3,8-dimethyl-4,5-nonanediol, 3,8-dimethyl- 4,5 -nonanedione, 3 , 8-dimethyl-4-hydroxy-5 -nonanone, 3 , 8-dimethyl-5 -hydro xy-4-nonanone, n-decane, 1-decene, 1-decanol, decanoate, 2,9-dimethyldecane, 2,9-dimethyl-3-decene, 2,9- dimethyl-4-decene, 2,9-dimethyl-5-decanol, 2,9-dimethyl-5-decanone, 2,9-dimethyl-5,6- decanediol, 2,9-dimethyl-6-hydroxy-5-decanone, 2,9-dimethyl-5,6-decanedionen-undecane, 1-undecene, 1-undecanol, undecanal. undecanoate, n-dodecane, 1-dodecene, 1-dodecanol, dodecanal, dodecanoate, n-dodecane, 1-decadecene, n-tridecane, 1-tridecene, 1-tridecanol, tridecanal, tridecanoate, n-tetradecane, 1-tetradecene, 1-tetradecanol, tetradecanal, tetradecanoate, n-pentadecane, 1-pentadecene, 1-pentadecanol, pentadecanal, pentadecanoate, n-hexadecane, 1-hexadecene, 1-hexadecanol, hexadecanal, hexadecanoate, n-heptadecane, 1- heptadecene, 1-heptadecanol, heptadecanal, heptadecanoate, n-octadecane, 1-octadecene, 1- octadecanol, octadecanal, octadecanoate, n-nonadecane, 1-nonadecene, 1-nonadecanol, nonadecanal, nonadecanoate, eicosane, 1-eicosene, 1-eicosanol, eicosanal, eicosanoate, 3- hydroxy propanal, 1,3-propanediol, 4-hydroxybutanal, 1,4-butanediol, 3-hydroxy-2-butanone, 2,3-butandiol, 1,5-pentane diol, homocitrate, homoisocitorate, b-hydroxy adipate, glutarate, glutarsemialdehyde, glutaraldehyde, 2-hydroxy-l-cyclopentanone, 1,2-cyclopentanediol, cyclopentanone, cyclopentanol, (S)-2-acetolactate, (R)-2,3-Dihydroxy-isovalerate, 2- oxoiso valerate, isobutyryl-CoA, isobutyrate, isobutyraldehyde, 5 -amino pentaldehyde, 1,10- diaminodecane, l,10-diamino-5-decene, l,10-diamino-5-hydroxydecane, l,10-diamino-5- decanone, 1 , 10-diamino-5,6-decanediol, 1 , 10-diamino-6-hydroxy-5-decanone,

phenylacetoaldehyde, 1 ,4-diphenylbutane, 1,4-diphenyl-l-butene, 1 ,4-diphenyl-2-butene, 1,4- diphenyl-2-butanol, 1 ,4-diphenyl-2-butanone, l,4-diphenyl-2,3-butanediol, l,4-diphenyl-3- hydroxy-2-butanone, 1 -(4-hydeoxyphenyl)-4-phenylbutane, 1 -(4-hydeoxyphenyl)-4-phenyl- 1 -butene, 1 -(4-hydeoxyphenyl)-4-phenyl-2-butene, 1 -(4-hydeoxyphenyl)-4-phenyl-2-butanol, 1 -(4-hydeoxyphenyl)-4-phenyl-2-butanone, 1 -(4-hydeoxyphenyl)-4-phenyl-2,3-butanediol, 1 - (4-hydeoxyphenyl)-4-phenyl-3-hydroxy-2-butanone, 1 -(indole-3)-4-phenylbutane, 1 -(indole- 3)-4-phenyl-l -butene, l-(indole-3)-4-phenyl-2-butene, l-(indole-3)-4-phenyl-2-butanol, 1- (indole-3)-4-phenyl-2-butanone, 1 -(indole-3)-4-phenyl-2,3-butanediol, 1 -(indole-3)-4- phenyl-3 -hydro xy-2-butanone, 4-hydroxyphenylacetoaldehyde, 1 ,4-di(4- hydroxyphenyl)butane, 1 ,4-di(4-hydroxyphenyl)- 1 -butene, 1 ,4-di(4-hydroxyphenyl)-2- butene, l,4-di(4-hydroxyphenyl)-2-butanol, l,4-di(4-hydroxyphenyl)-2-butanone, l,4-di(4- hydroxyphenyl)-2,3-butanediol, 1 ,4-di(4-hydroxyphenyl)-3-hydroxy-2-butanone, 1 -(4- hydroxyphenyl)-4-(indole-3-)butane, 1 -(4-hydroxyphenyl)-4-(indole-3)- 1 -butene, 1 -di(4- hydroxyphenyl)-4-(indole-3)-2-butene, 1 -(4-hydroxyphenyl)-4-(indole-3)-2-butanol, 1 -(4- hydroxyphenyl)-4-(indole-3)-2-butanone, l-(4-hydroxyphenyl)-4-(indole-3)-2,3-butanediol, 1 -(4-hydroxyphenyl-4-(indole-3)-3-hydroxy-2-butanone, indole-3-acetoaldehyde, 1 ,4- di(indole-3-)butane, l,4-di(indole-3)-l -butene, l,4-di(indole-3)-2-butene, l,4-di(indole-3)-2- butanol, l,4-di(indole-3)-2-butanone, l,4-di(indole-3)-2,3-butanediol, l,4-di(indole-3)-3- hydroxy-2-butanone, succinate semialdehyde, hexane-l,8-dicarboxylic acid, 3-hexene-l,8- dicarboxylic acid, 3-hydroxy-hexane-l,8-dicarboxylic acid, 3-hexanone-l,8-dicarboxylic acid, 3,4-hexanediol-l,8-dicarboxylic acid, 4-hydroxy-3-hexanone-l,8-dicarboxylic acid, fucoidan, iodine, chlorophyll, carotenoid, calcium, magnesium, iron, sodium, potassium, phosphate, lactic acid, acetic acid, formic acid, an isoprenoid or terpene. Additional fermentation end products, and methods of production thereof, can be found in U.S. Patent Application US 12/969,582, which is herein incorporated by reference in its entirety. Biofuel plant and process of producing biofuel

[00111] In one aspect, provided herein is a fuel plant that includes a hydrolysis unit configured to hydro lyze a biomass material comprising a high molecular weight

carbohydrate, and a fermentor configured to house a medium and one or more species of microorganisms. In one embodiment 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.

[00112] In another aspect, provided herein are methods of making a fuel or chemical end- product that includes combining a microorganism (such as Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13 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).

[00113] In some embodiments, a process is provided for producing a fermentation end- product from biomass using acid hydrolysis pretreatment. In some embodiments, a process is provided for producing a fermentation end-product from biomass using enzymatic hydrolysis pretreatment. In another embodiment a process is provided for producing a fermentation end- product from biomass using biomass that has not been enzymatically pretreated. In another embodiment a process is provided for producing a fermentation end-product from biomass using biomass that has not been chemically or enzymatically pretreated, but is optionally steam treated.

[00114] In another aspect, provided herein are end-products made by any of the processes described herein. Those skilled in the art will appreciate that a number of genetic

modifications can be made to the methods exemplified herein. For example, a variety of promoters can be utilized to drive expression of the heterologous genes in a recombinant microorganism (such as Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12 or Clostridium phytofermentans Q.13). The skilled artisan, having the benefit of the instant disclosure, will be able to readily choose and utilize any one of the various promoters available for this purpose. Similarly, skilled artisans, as a matter of routine preference, can utilize a higher copy number plasmid. In another embodiment, 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 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. Thus, the gene of interest can be introduced in a heterologous host such as E. coli, and short, random fragments can be isolated and ligated in Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytqfermentans Q.12, Clostridium phytofermentans Q.13, or genetically-modified mutants thereof, to promote homologous recombination.

Large Scale Fermentation End-Product Production from Biomass

[00115] In one aspect 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, phytqfermentans Q.12 Clostridium phytofermentans Q.13 or variants thereof. In one embodiment, a biomass that includes high molecular weight carbohydrates is hydrolyzed to lower molecular weight carbohydrates, which are then fermented using a microorganism to produce ethanol. In another embodiment, the biomass is fermented without chemical and/or enzymatic pretreatment. In one embodiment, hydrolysis can be accomplished using acids, e.g., Bronsted acids (e.g., sulfuric or hydrochloric acid), bases, e.g., sodium hydroxide, hydrothermal processes, steam explosion, ammonia fiber explosion processes ("AFEX"), lime processes, enzymes, or combination of these. Hydrogen, and other products of the fermentation can be captured and purified if desired, or disposed of, e.g., by burning. For example, 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,increase porosity and/or surface area of the biomass, often leaving the cellulosic materials more exposed to the microorganismal cells, which can increase fermentation rate and yield. In another embodiment removal of lignin can provide a combustible fuel for driving a boiler, and can also increase porosity and/or surface area of the biomass, often increasing

fermentation rate and yield. In some embodiments, 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.

[00116] In one aspect, the invention features a fuel plant that comprises a hydrolysis unit configured to hydro lyze a biomass material that includes a high molecular weight

carbohydrate; 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, Clostridium phytofermentans Q.13, or variants thereof); and one or more product recovery system(s) to isolate a fermentation end- product or end- products and associated by-products and co -pro ducts.

[00117] In another aspect, the invention features methods of making a fermentation end- product or end- products that include combining a C5/C6 hydro lyzing microorganism (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.V2,

Clostridium phytofermentans Q.13, or variants thereof) and a carbonaceous bio mass 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). In one embodiment the fermentation end-product is a bio fuel or chemical product.

[00118] In another aspect, the invention features one or more fermentation end-products made by any of the processes described herein. In one embodiment one or more fermentation end-products can be produced from biomass on a large scale utilizing a C5/C6 hydrolyzing microorganism (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13, or variants thereof). In one embodiment depending on the type of biomass and its physical manifestation, 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).

[00119] In some embodiments, the treatment includes treatment of a biomass with acid. In some embodiments, the acid is dilute. In some embodiments, the acid treatment is carried out at elevated temperatures of between about 85 and 140°C. In some embodiments, the method further comprises the recovery of the acid treated biomass solids, for example by use of a sieve. In some embodiments, the sieve comprises openings of approximately 150-250 microns in diameter. In some embodiments, the method further comprises washing the acid treated biomass with water or other solvents. In some embodiments, the method further comprises neutralizing the acid with alkali. In some embodiments, the method further comprises drying the acid treated biomass. In some embodiments, the drying step is carried out at elevated temperatures between about 15-45°C. In some embodiments, the liquid portion of the separated material is further treated to remove toxic materials. In some embodiments, the liquid portion is separated from the solid and then fermented separately. In some embodiments, a slurry of solids and liquids are formed from acid treatment and then fermented together. [00120] Fig. 4 illustrates an example of a method for producing a fermentation end-product from biomass by first treating biomass with an acid at elevated temperature and pressure in a hydrolysis unit. The biomass can first be heated by addition of hot water or steam. The biomass can be acidified by bubbling gaseous sulfur dioxide through the biomass that is suspended in water, or by adding a strong acid, e.g., sulfuric, hydrochloric, or nitric acid with or without preheating/presteaming/water addition. During the acidification, the pH is maintained at a low level, e.g., below about 5. The temperature and pressure can be elevated after acid addition. In addition to the acid already in the acidification unit, optionally, 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 hydro lysate 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 hydro lyze 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%.

[00121] In some embodiments, after pretreatment, 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.

[00122] In one embodiment 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 phytofermentans Q.13, mutagenized or genetically-modified cells thereof, optionally a co-fermenting

microorganism(e.g., yeast or E. coli) and, as needed, nutrients to promote growth of the Clostridium cells or other microorganisms. In another embodiment the pretreated biomass or liquid fraction can be split into multiple fermentors, each containing a different strain of Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.12,

Clostridium phytofermentans Q.13 mutagenized or genet ica 1 ly-mod ified cells thereof and/or other microorganisms; with each fermentor operating under specific physical conditions. Fermentation is allowed to proceed for a period of time, e.g., between about 15 and 150 hours, while maintaining a temperature of, e.g., between about 25° C and 50° C. Gas produced during the fermentation is swept from fermentor and is discharged, collected, or flared with or without additional processing, e.g. hydrogen gas can be collected and used as a power source or purified as a co-product.

[00123] After fermentation, the contents of the fermentor are transferred to product recovery. Products are extracted, e.g., ethanol is recovered through distillation and rectification.

Methods and compositions described herein can include extracting or separating fermentation end-products, such as ethanol, from biomass. Depending on the product formed, different methods and processes of recovery can be provided.

[00124] In one embodiment, a method for extraction of lactic acid from a fermentation broth uses freezing and thawing of the broth followed by centrifugation, filtration, and evaporation. (Omar, et al. 2009 African J. Biotech. 8:5807-5813) Other methods that can be utilized are membrane filtration, resin adsorption, and crystallization. (See, e.g., Huh, et al. 2006 Process Biochemistry).

[00125] In another embodiment for solvent extraction of a variety of organic acids (such as ethyl lactate, ethyl acetate, formic, butyric, lactic, acetic, succinic), the process can take advantage of preferential partitioning of the product into one phase or the other. In some cases the product might be carried in the aqueous phase rather than the solvent phase. In other embodiments, the pH is manipulated to produce more or less acid from the salt synthesized from the microorganism. The acid phase is then extracted by vaporization, distillation, or other methods. See Fig. 5.

[00126] In yet a further embodiment, a system for production of fermentation end-products comprises: (a) a fermentation vessel comprising a carbonaceous biomass; (b) and a microorganism that is capable of direct hydrolysis and fermentation of the biomass; wherein the fermentation vessel is adapted to provide suitable conditions for fermentation of one or more carbohydrates into fermentation end-products. In one embodiment the microorganism is genetically modified. In another embodiment the microorganism is not genetically modified. Chemical Production From Biomass

[00127] Fig. 6 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. At the end of the pretreatment period, or at the same time as pretreatment begins, an active seed culture of a C5/C6 hydrolyzing microorganism (e.g., Clostridium phytofermentans, Clostridium sp. Q.D. Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13 or variant thereof) and, if desired, a co-fermenting microorganism, e.g., yeast or E. coli, and, if required, nutrients to promote growth of 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 are added. 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 microorganisms (e.g. yeasts, fungi, or other bacteria). In some embodiments different methods can be used within a single plant to produce different end-products.

[00128] In another aspect, the invention features a fuel plant that includes a hydrolysis unit configured to hydro lyze a biomass material that includes a high molecular weight

carbohydrate, 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).

[00129] In another aspect, the invention features a chemical production plant that includes a hydrolysis unit configured to hydrolyze a biomass material that includes a high molecular weight carbohydrate, a fermentor configured to house a medium and contains a C5/C6 hydrolyzing microorganism (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q. 1 2, Clostridium phytofermentans Q.13, or mutagenized or genetically-modified cells thereof).

[00130] In another aspect, the invention features methods of making a chemical(s) or fuel(s) that include combining a C5/C6 hydro lyzing microorganism {e.g., Clostridium

phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q. 1 2. or Clostridium phytofermentans Q.13), and a lignocellulosic material (and/or other biomass material) in a medium, and fermenting the lignocellulosic material under conditions and for a time sufficient to produce a chemical(s) or fuel(s), e.g., ethanol, propanol and/or hydrogen or another chemical compound.

[00131] In some embodiments, 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.

[00132] Fig. 7 discloses pretreatments that produce hexose or pentose saccharides or oligomers that are then unprocessed or processed further and either, fermented separately or together. Figure 7A depicts a process {e.g., acid pretreatment) that produces a solids phase and a liquid phase which are then fermented separately. Figure 7B 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. At initiation of 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 7C) 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.

Modification to Enhance Enzyme Activity

[00133] In one embodiment one or more modifications of hydrolysis and/or fermentation conditions can be implemented to enhance end-product production. Examples of such modifications include genetic modification to enhance enzyme activity in a microorganism that already comprises genes for encoding one or more target enzymes, introducing one or more heterogeneous nucleic acid molecules into a host microorganism to express and enhance activity of an enzyme not otherwise expressed in the host, modifying physical and chemical conditions to enhance enzyme function (e.g., modifying and/or maintaining a certain temperature, pH, nutrient concentration, temporal), or a combination of one or more such modifications.

Genetic Modification

[00134] In one embodiment, a microorganism can be genetically modified to enhance enzyme activity of one or more enzymes, including but not limited to hydrolytic enzymes (such as cellulase(s), hemicellulase(s), or pectinase(s) etc.). In one embodiment a method is used to genetically modify a microorganism (such as a Clostridium species) that is disclosed in US 20100086981 or PCT/US2010/40494, which are herein incorporated by reference in their entirety. In another embodiment, an enzyme can be selected from the annotated genome of C. phytofermentans, another bacterial species, such as B. subtilis, E. coli, various

Clostridium species, or yeasts such as S. cerevisiae for utilization in products and processes described herein. Examples include enzymes such as L-butanediol dehydrogenase, acetoin reductase, 3-hydroxyacyl-CoA dehydrogenase, cis-aconitate decarboxylase or the like, to create pathways for new products from biomass.

[00135] Examples of such 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.

[00136] In another embodiment a microorganism can be modified to enhance an activity of one or more hydrolytic enzymes (such as cellulase(s), hemicellulase(s), or pectinases etc.) or antioxidants (such as catalase), or other enzymes associated with cellulose processing. For example, in the case of cellulases, various microorganisms of the invention can be modified to enhance activity of one or more cellulases, or enzymes associated with cellulose processing (e.g., FIG. 3).

[00137] In one embodiment a hydrolytic enzyme is selected from the annotated genome of C. phytofermentans for utilization in a product or process disclosed herein. In another embodiment the hydrolytic enzyme is an endoglucanase, chitinase, cellobiohydrolase or endo-processive cellulases (either on reducing or non-reducing end).

[00138] In one embodiment a microorganism, such as C. phytofermentans, can be modified to enhance production of one or more hydrolases. In another embodiment one or more enzymes can be heterologous expressed in a host (e.g., a bacteria or yeast). For heterologous expression 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).

[00139] In another embodiment other modifications can be made to enhance end-product (e.g., ethanol) production in a recombinant microorganism. For example, the host

microorganism can further comprise an additional heterologous DNA segment, the expression product of which is a protein involved in the transport of mono- and/or oligosaccharides into the recombinant host. Likewise, additional genes from the glycolytic pathway can be incorporated into the host. In such ways, an enhanced rate of ethanol production can be achieved.

[00140] A variety of promoters (e.g., constitutive promoters, inducible promoters) can be used to drive expression of the heterologous genes in a recombinant host microorganism.

[00141] Promoter elements can be selected and mobilized in a vector (e.g., pIMPCphy). For example, 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. In one embodiment a transcription regulatory sequences can be derived from the host microorganism. In various embodiments, 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.

[00142] A map of the plasmid pIMPCphy is shown in Figure 9, and the DNA sequence of this plasmid is provided as SEQ ID NO: l.

[00143] SEQ ID NO: 1 :

gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaa ttgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaaagctttggctaacacacacgccattccaaccaat agttttctcggcataaagccatgctctgacgcttaaatgcactaatgccttaaaaaaacattaaagtctaacacactagacttatttacttcgt aattaagtcgttaaaccgtgtgctctacgaccaaaagtataaaacctttaagaactttcttttttcttgtaaaaaaagaaactagataaatctc tcatatcttttattcaataatcgcatcagattgcagtataaatttaacgatcactcatcatgttcatatttatcagagctccttatattttatttcgat ttatttgttatttatttaacatttttctattgacctcatcttttctatgtgttattcttttgttaattgtttacaaataatctacgatacatagaaggagg aaaaactagtatactagtatgaacgagaaaaatataaaacacagtcaaaactttattacttcaaaacataatatagataaaataatgacaa atataagattaaatgaacatgataatatctttgaaatcggctcaggaaaagggcattttacccttgaattagtacagaggtgtaatttcgtaa ctgccattgaaatagaccataaattatgcaaaactacagaaaataaacttgttgatcacgataatttccaagttttaaacaaggatatattgc agtttaaatttcctaaaaaccaatcctataaaatatttggtaatataccttataacataagtacggatataatacgcaaaattgtttttgatagta tagctgatgagatttatttaatcgtggaatacgggtttgctaaaagattattaaatacaaaacgctcattggcattatttttaatggcagaagtt gatatttctatattaagtatggttccaagagaatattttcatcctaaacctaaagtgaatagctcacttatcagattaaatagaaaaaaatcaa gaatatcacacaaagataaacagaagtataattatttcgttatgaaatgggttaacaaagaatacaagaaaatatttacaaaaaatcaattt aacaattccttaaaacatgcaggaattgacgatttaaacaatattagctttgaacaattcttatctcttttcaatagctataaattatttaataagt aagttaagggatgcataaactgcatcccttaacttgtttttcgtgtacctattttttgtgaatcgatccggccagcctcgcagagcaggattc ccgttgagcaccgccaggtgcgaataagggacagtgaagaaggaacacccgctcgcgggtgggcctacttcacctatcctgcccgg atcgattatgtcttttgcgcattcacttcttttctatataaatatgagcgaagcgaataagcgtcggaaaagcagcaaaaagtttcctttttgc tgttggagcatgggggttcagggggtgcagtatctgacgtcaatgccgagcgaaagcgagccgaagggtagcatttacgttagataa ccccctgatatgctccgacgctttatatagaaaagaagattcaactaggtaaaatcttaatataggttgagatgataaggtttataaggaat

agtgagaaaaagatgaaagaaagatatggaacagtctataaaggctctcagaggctcatagacgaagaaagtggagaagtcataga ggtagacaagttataccgtaaacaaacgtctggtaacttcgtaaaggcatatatagtgcaattaataagtatgttagatatgattggcgga aaaaaacttaaaatcgttaactatatcctagataatgtccacttaagtaacaatacaatgatagctacaacaagagaaatagcaaaagcta caggaacaagtctacaaacagtaataacaacacttaaaatcttagaagaaggaaatattataaaaagaaaaactggagtattaatgttaa accctgaactactaatgagaggcgacgaccaaaaacaaaaatacctcttactcgaatttgggaactttgagcaagaggcaaatgaaat agattgacctcccaataacaccacgtagttattgggaggtcaatctatgaaatgcgattaagcttagcttggctgcaggtcgacggatcc ccgggaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctt tcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatg cggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccag ccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctcc gggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaat gtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaa atatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcc cttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcac gagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaa agttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggtt gagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataaca ctgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcctt gatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcg caaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttct gcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggc cagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggat ctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatca aaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatc aagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggcc accacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtct taccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgg agcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggaca ggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtc gggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcct ttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtg agctgataccgctcgccgcagccgaacgccgagcgcagcgagtcagtgagcgaggaagcggaaga

[00144] 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. phytofermentans by electroporation or by transconjugation with an E.coli strain that has a mobilizing plasmid, for example pRK2030. A plasmid map of pIMPCphy is depicted in Fig. 9. pIMPCphy is an effective replicative vector system for all microorganisms, including all gram⁺ and gram^" bacteria, and fungi (including yeasts). A further discussion of promoters, regulation of gene expression products, and additional genetic modifications can be found in U.S. Patent Application Publication US

20100086981A1, which is herein incorporated by reference in its entirety.

[00145] For the fermentation described herein, any microorganisms capable of forming a bio film can be used. In one embodiment, two or more genetically distinct microorganisms can be used to form a bio film. In another embodiment a naturally occurring microorganism can be introduced to a broth containing biomass to form a bio film with genetically modified microorganisms. In another embodiment, two or more genetically modified microorganisms can be used to form a bio film. Biofilms

[00146] In one embodiment fermentation and or hydrolysis is in a vessel designed to control process parameters such as pH, oxygen levels, nutrient availability, and temperature control. Batch additions of pH control chemicals, nutrients or gasses, as well as temperature control, generally utilize agitation or mixing and cultures can be kept homogenous with respect to these parameters by continual agitation or mixing with internal stirrers. In small scale "shake flask" experiments this is accomplished by agitation on a rotating platform.

[00147] In one embodiment, a microorganism of the invention attaches to or form films on the surface of insoluble carbohydrate sources. When fermenting insoluble carbohydrate sources such as biomass (e.g., lignocellulosic, cellulosic, hemi-cellulosic, or starch based), microorganisms secrete enzymes to degrade the insoluble food source to soluble

carbohydrates (sugars) capable of transport inside the cell. To control the availability of food from the process (i.e. to prevent their cellular machinery from feeding other cells), a microorganism can attach to or form films on the surface of these insoluble substrates. Such a process permits a reduction in the diffusion of sugars away from the cellular transport machinery of a microorganism and effectively increases the local concentration of the food source. In another embodiment, a microorganism reduces diffusion of sugars such that it increases growth or productivity by optimizing sugar uptake rates and minimizing the energy needed to degrade the substrate. In one embodiment, a microorganism that ferments biomass in a bio film demonstrates a greater yield of product, such as a bio fuel, compared to fermentation in a planktonic state. In another embodiment, a microorganism that does not form a bio film produces more enzymes to achieve acceptable soluble sugar levels to support its growth.

[00148] In one embodiment a microorganism that forms a bio film has an advantage over a microorganism that does not because its enzyme kinetics are optimized by immediate contact with the substrate. A microorganism that forms a bio film has increased local concentration of substrate to be converted into a fermentation end-product, and therefore can increase production of a fermentation end product, such as ethanol. In one embodiment a bioreactor is designed for continuous and/or vigorous mixing. In another embodiment a bioreactor comprising a microorganism that forms a bio film employs static fermentation or agitation that avoids disrupting the bio film. In one embodiment the agitation is low sheer agitation.

[00149] Compositions and methods disclosed herein include static or minimally agitated cultures. In one embodiment various compositions of substrate concentrations disclosed herein surpass the concentration limit conventionally observed in conventional stirred tank reactors (STR)-operation. Conditions disclosed herein can improve fermentation and growth rates as well as insoluble biomass substrate conversion efficiency for cultures of

microorganisms, e.g., Clostridia species froml6s RNA Group 14A (Clostridia species classification according to 16S rRNA gene phylogeny). In one embodiment, the Clostridia species is C. phytofermentans (Cphy), Clostridium sp. Q.D, Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13 or variant thereof.

[00150] The isolated strains disclosed herein have been deposited in the Agricultural Research Service culture Collection (NRRL), an International Depositary Authority, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A. in accordance with and under the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures plus five years after the last request for a sample from the deposit. The strains were tested by the NRRL and determined to be viable. 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.

[00151] In addition to naturally formed bio films (via flocculation or aggregation), bio films can be formed on a supporting material. Supporting material can be porous, non-porous, biodegradable, non-degradable, digestible by the microorganism growing on the supporting material, or an absorbent material attracting microorganisms. The supporting material can be virtually of any shape including, but not limited to, pipe, rod, bead, slat, tube, screen, honeycomb, sphere, web, or a shape with latticework. Microorganisms can attach to or be immobilized on the surface of supporting material. The attachment can be reversible. The attachment or immobilization can be a microbial process autonomously occurring in the presence of supporting material. An example of autonomous process is secretion of extracellular polymeric substances that binds firmly to the surface of supporting material. The attachment or immobilization can be an artificially induced process in which microorganisms are attracted to the supporting material. The attraction can occur, for example, via

chemotaxis. In one embodiment a microorganism is not attracted to a support. In one embodiment a microorganism attaches to the supporting material by Brownian motion, motion of the microorganisms resulting from asymmetry in the kinetic impacts of molecules that make up the liquid surrounding the microorganisms. The supporting material can be coated with a homing material that is used to cultivate the microorganisms on the substrate. The homing material can comprise at least one chemoattractant and/or carbon compound. The carbon compound can be selected from the group comprising, but not limited to, glucose, fructose, glycerol, mannitol, asparagines, casein, adonitol, 1-arabinose, cellobiose, dextrose, dulcitol, d-galactose, inositol, inulin, lactose, levulose, maltose, d-mannitol, d-mannose, melibiose, raffinose, rhamnose, sucrose, salicin, d-sorbintol, trihalose and d-xylose or any combination thereof. In one embodiment, baffles are present to assist flow of fluid in the correct flow pattern around the supporting material.

[00152] In one embodiment a fermentative end product is produced with a microorganism that forms a bio film. Bio films can be formed of a single microbial species. Bio films can be formed of a heterogeneous mixture of two or more microbial species. Examples of species include, but not limited to, Clostridium phytofermentans, Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum,

Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium sp. Q.D.,

Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica, Thermoanaerobacterium thermos accharolyticum and Thermoanaerobacterium saccharolyticum or variants thereof.

[00153] Bio films contemplated or described herein can be used in various types of bioreactors. Examples of bioreactor designs compatible with bio films contemplated or described herein include, but not limited to, stirred tank reactors (STRs), continuous stirred tank reactions (CSTRs), packed bed reactors (PBRs), fluidized bed reactors (FBRs), airlift reactors (ARs), upflow anaerobic sludge blanket reactors (UASBRs), and expanded granular sludge reactors (EGSBRs). Manufactures of such bioreactors can include Sartorius Stedim North American, Inc. (Bohemia, NY), B. Braun Biotech, Inc. (Allentown, PA), Lambda Laboratory Instruments (Zurich, Switzerland) or New Brunswick Scientific (Edison, NJ). Various configurations of these reactor designs are compatible and useful for bio films described herein.

[00154] Bio films contemplated or described herein can be used -with various types of impellers supplied by manufacturers such as Lightnin (Rochester, NY), Proquip (Macedonia, OH), Chemineer (Dayton, OH), Lotus (Nokomis, FL), or Dynamic (Richmond BD, Canada). Examples of impellers include, but are not limited to, impellers creating radial flow (e.g. a Rushton impeller, fabricated marine impeller, pitch blade turbine impeller, retreat blade impeller, sawtooth disperse blade impeller, or impellers creating vertical flow). In one embodiment, the impeller is a helical impeller. Commercially available impellers components include plastic, TEFLON, KYNAR, polypropylene, polyethylene, mixing impellers, along with stirring propellers, and a mixing tank (White Mountain Process, Pembroke, MA).

[00155] In one embodiment, a bioreactor can process a high percentage of solid biomass. Examples of the source of solid biomass include, but are not limited to, sawdust, wood flour, wood pulp, paper pulp, paper pulp waste streams, grasses, such as, switchgrass, biomass plants and crops, such as, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, corn grind, distillers grains, and pectin. Examples of solid material that can be processed in a mixture include, but not limited to, about 15 % (w/w), about 16 % (w/w), about 17 % (w/w), about 18 % (w/w), about 19 % (w/w), about 20 % (w/w), about 21 % (w/w), about 22 % (w/w), about 23 % (w/w), about 24 % (w/w), about 25 % (w/w), about 26 % (w/w), about 27 % (w/w), about 28 % (w/w), about 29 % (w/w), about 30 % (w/w), about 31 % (w/w), about 32 % (w/w), about 33 %(w/w), about 34 % (w/w), about 35 % (w/w), about 36 % (w/w), about 37 % (w/w), about 38 % (w/w), about 39 % (w/w), about 40 % (w/w), about 41 % (w/w), about 42 % (w/w), about 43 %(w/w), about 44 % (w/w), about 45 % (w/w), about 46 % (w/w), about 47 % (w/w), about 48 % (w/w), about 49 % (w/w), about 50 % (w/w), and about 51 % (w/w). As used herein, "w/w" corresponds to percent of the mixture, which is equal to the (mass of the solid biomass material / mass of the mixture) x 100. In one embodiment, the solid biomass is about 20% of total weight of mixture comprising water (or medium) and inoculums of C. phytofermentans microorganism, such as C. phytofermentans (Cphy), Clostridium sp. Q.D, Clostridium phytofennentans Q.12. Clostridium phytofennentans Q.13 or variant thereof. In another embodiment, the solid biomass is about 30% of total weight of mixture comprising water (or medium) and C.

phytofermentans inoculum. The solid biomass can be mixed in a liquid media containing C. phytofermentans inoculum without any processing. Alternatively, the solid biomass can be processed by pre-treatment methods disclosed herein.

Gentle agitation or static fermentation

Gentle agitation

[00156] Various embodiments of the invention offer benefits relating to improving the titer and/or productivity of fermentation end-product production by microorganisms by culturing the microorganism in a medium comprising one or more compounds

comprising hexose and/or pentose sugars. Exemplary microorganisms include but are not limited Clostridia (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofennentans Q. 12, Clostridium phytofermentans Q.13, Clostridium algidixylanolyticum or Clostridium xylanolyticum). In some embodiments, the process comprises conversion of the starting material to a fermentation end product. Examplary fermentation end- products include but are not limited to one or more bio fuels and chemicals. Bio fuels can comprise one or more alcohols {e.g. ethanol) or other chemicals {e.g. aspartic acid, aspartate, glutamic acid, glutamate, malic acid or malate). In one embodiment, methods of the invention comprise contacting one or more substrates comprising both hexose {e.g. glucose, cellobiose) and pentose {e.g. xylose, arabinose) saccharides with C.

phytofermentans, Clostridium sp. Q.D, Clostridium phytofennentans Q.12, Clostridium phytofennentans Q.13, Clostridium algidixylanolyticum or Clostridium xylanolyticum to produce ethanol. In one embodiment, methods of the invention comprise contacting one or more substrates comprising both hexose {e.g. glucose, cellobiose) and pentose {e.g.

xylose, arabinose) saccharides with C. phytofermentans, Clostridium sp. Q.D, Clostridium phytofennentans Q.12, Clostridium phytofermentans Q.13, Clostridium algidixylanolyticum or Clostridium xylanolyticum to produce aspartic acid, aspartate, glutamic acid,

glutamate, malic acid or malate.

[00157] In some embodiments batch fermentation with a microorganism and a mixture of hexose and pentose saccharides using the methods of the present invention provides uptake rates of about 0.1, 0.2, 0.4, 0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or about 6 g/L/h or more of hexose (e.g. glucose, cellulose, cellobiose etc.), and about 0.1, 0.2, 0.4, 0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or about 6 g/L/h or more of pentose (xylose, xylan, hemicellulose etc.). In some

embodiments, the microorganism is a Clostridium species. Exemplary Clostridia include but are not limited to C. phytofermentans, Clostridium, sp. Q.D. Clostridium phytofermentans Q.12, Clostridium phytofermentans Q.13, Clostridium algidixylanolyticum or Clostridium

xylanolyticum, or variant thereof. The uptake rates for hexose can range between about 0.1- 0.5, 0.2-0.6, 0.3-0.7, 0.4-0.8, 0.5-1, 0.6-2, 0.7-3, 0.8-4, 1-5, 2-6, 3-7, or about 4-8 g/L/h. The uptake rates for pentose can range between about 0.1-0.5, 0.2-0.6, 0.3-0.7, 0.4-0.8, 0.5-1, 0.6- 2, 0.7-3, 0.8-4, 1-5, 2-6, 3-7, or about 4-8 g/L/h. The present invention also provides methods for production of about 15 g/L, 20g/L, 25g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 100 g/L or more ethanol in 40 hours by the fermentation of biomass. The ethanol produced by the present invention can range between about 13-17 g/L, 14-18 g/L, 18- 21 g/L, 19-24 g/L, 23-27 g/L, 24-29 g/L, 28-31 g/L, 29-33 g/L, 31-36 g/L, 33-37 g/L, 34-39 g/L, 36-41 g/L, 37-42 g/L, 38-43 g/L, 41-44 g/L, 42-47 g/L, 46-51 g/L, 48-52 g/L, 55-65 g/L, 58-61 g/L, 65-75 g/L, 68-72 g/L, 75-85g/L, 78-82g/L, 95-105 g/L, or 98-101 g/L. In some cases, the ethanol productivities provided by the methods of the present invention are due to the simultaneous fermentation of hexose and pentose saccharides.

[00158] In some embodiments several factors can influence production of high levels of alcohol (e.g., ethanol) from biomass: the ability of the microorganism to thrive generally in the presence of elevated alcohol levels; the ability of the microorganism to continue to produce alcohol without undue inhibition or suppression by the alcohol and/or other components present; and the ability to efficiently convert the multitude of different hexose and pentose carbon sources found in a biomass feedstock.

[00159] It has been observed in other work with Clostridium phytofermentans that ethanol concentration attains a plateau of about 15 g/L after about 36 - 48 hours of batch

fermentation, with carbon substrate remaining in the broth. Lowering the fermentation pH to about 6.5 and/or adding unsaturated fatty acids resulted in a significant increase in the amount of ethanol produced by the microorganism, with between about 20 g/L to about 30, 40, 50g/L or more of ethanol observed in the broth following a 48 to 72 to 96— hour or longer fermentation. In addition, it has also been observed that the productivity of the microorganism was higher (to about 10 g/L-d) when the ethanol titer was low and lower (to about 2 g/L-d) when the ethanol concentration was higher. Fermentation at reduced pH and/or with the addition of fatty acids can result in about a three to five to 10 fold or higher increase in the ethanol production rate. In some embodiments of the present invention, simultaneous fermentation of both hexose and pentose saccharides can also enable increases in ethanol productivity and/or yield. In some cases, the simultaneous fermentation of hexose and pentose carbohydrate substrates can be utilized in combination with fermentation at reduced pH and/or with the addition of fatty acids to further increase productivity, and/or yield.

[00160] Described herein is static or gentle agitation in which a mixture is either culture in a static condition or mixed in various low-mixing rates. For example, in a bioreactor equipped with a radial impeller, mixing rate can be, measured by the rotation of the impeller, about 0 rpm, about 10 rpm, about 20 rpm, about 30 rpm, about 40 rpm, about 50 rpm, about 60 rpm, about 70 rpm, about 80 rpm, about 90 rpm, about 100 rpm, about 110 rpm, about 120 rpm, about 130 rpm, about 140 rpm, about 150 rpm, about 160 rpm, about 170 rpm, about 180 rpm, about 190 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm. In one embodiment, the mixing rate is 175 rpm. In another embodiment, the bioreactor is mixed with a helical impeller at the rate of 120 rpm. In one embodiment the mixing rate can range between about 0-510 rpm, such as 0-200 rpm, 50-400 rpm 100-300 rpm, 100-200rpm 150- 200 rpm, or 150-300rpm. In another embodiment the mixing rate can range between about 0- 5, 5-15, 9-11, 12-18, 15-25, 19-21, 25-35, 29-31, 35-45, 39-41, 45-55, 49-51, 55-65, 59-61, 65-75, 68-72, 75-85, 78-82, 85-95, 88-92, 95-105, 98-102, 105-115, 108-112, 115-125, 118- 121, 125-135, 128-132, 135-145, 138-142, 145-155, 148-152, 155-165, 158-162, 165-175, 168-172, 169-173, 170-174, 171-176, 172-177, 174-187, 175-185, 178-182, 185-195, 188- 192, 195-205, 198-202, 285-310, 295-305, 298-302, 385-410, 395-405, 398-402, 485-510, 495-505, or 498-502 rpm.

[00161] Forms of agitations useful for avoiding disruption of the bio film include, but not limited to agitating using pulsating liquid flow; intermediately stirring liquid; rolling, vibrating, moving back and forth, or tilting the housing in which liquid and bio film is contained; and agitating with impeller blade having a unique shape or blade having unique angle to provide low sheer agitation.

[00162] Structural elements useful for providing gentle agitation include, but not limited to, impellers providing circular flow, pumps providing pulsating or vertical flow (pneumatic action or peristaltic action, for example), orbital shakers, rollers, tumblers, rockers, stirrers, and any equipment providing movement to liquid in a container. In one embodiment, the structural element providing gentle agitation is an impeller. In another embodiment, the structural element providing gentle agitation is a helical impeller.

Static fermentation

[00163] A static fermentation is achieved, for example, by flocculating microorganisms with or without flocculent, depending on the microorganism's ability to flocculate without adding exogenous flocculent, and fermenting biomass with flocculate. A static fermentation can also be achieved without disturbing the culture after the microbial inoculum is introduced to a medium containing biomass. Static fermentation can be performed in a fermenting chamber lacking any moving parts, such as a sedimentation chamber, allowing a mixture of an inoculum and biomass to sit for a period of time without being disturbed. For static fermentation, an inoculum and biomass can be layered in a manner that a layer of inoculum is sandwiched between layers of biomass. Alternatively, an inoculum prepared from an exponentially growing microbial culture can be expanded to a larger volume of culture and the larger volume of culture can be layered between biomass layers. Static fermentation can last up to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 hours. The time for static fermentation can be determined by continuously monitoring the rate of end-product production, or intermittently monitoring the rate of production. In one embodiment, a static fermentation lasts up to 200 hours with the fermentation progress being monitored every 50 hours.

Fermentation and formation of biofilm in the presence of exogenous enzyme

[00164] The fermentation methods disclosed herein are compatible with fermenting methods utilizing exogenous enzymes facilitating the release or digestion of biomass. A biofilm formed on the surface of biomass creates a barrier in which digestive enzymes secreted by the microorganisms on to the biomass do not diffuse out of areas in close proximity of the biomass. The barrier also entraps substrate, or byproduct molecules digested from the biomass. Thus, the biofilm creates a micro environment between the biomass and the cell wall of microorganism in which high concentration of enzyme-substrate reaction can occur. This highly concentrated activity results in higher breakdown rates of biomass than breaking down the biomass without forming a biofilm.

[00165] In one embodiment one or more exogenous enzymes are added to a culture comprising a biofilm. In one embodiment the one or more exogenous enzymes includes, but is not limited to a cellulase, B-glucosidase, hemicellulase, pectinase, xylanase, beta- galactosidase, glycosyl hydrolase family 9 enzymes (GH9)( such as ABX43720 of Cphy), endoglucanase, cellobiohydrolase, chitinase or a endo-processive cellulase. Enzymes exogenously added to a culture containing bio film can be from commercial or noncommercial sources. Examples of commercially available enzymes include (all available from Novozymes): NS50013 cellulase, NS50010 B-glucosidase, NS50012 hemicellulase, NS50030 xylinase, NS22002 glucanse/zylanase, as well as cocktail mixtures of these enzymes.

[00166] In specific embodiments an exogenously added enzyme is added to a culture containing bio film during pretreatment, wherein the enzyme comprises cellulase, B- glucosidase, hemicellulase, xylinase, glucanse/zylanase, or a cocktail mixture of any of these enzymes. In another further specific embodiment, exogenous enzyme is added during the fermentation step.

Recovery of ethanol or other fermentation end-products

[00167] At some point during of the fermentation, broth will be harvested and the final desired product or products will be recovered. The broth with ethanol to be recovered will include both ethanol and impurities. The impurities include materials such as water, cell bodies, cellular debris, excess carbon substrate, excess nitrogen substrate, other remaining nutrients, non-ethanol metabolites, and other medium components or digested medium components. During the course of processing the broth, the broth can be heated and/or reacted with various reagents, resulting in additional impurities in the broth.

[00168] In the case of recovery of ethanol, the processing steps frequently includes several separation steps, including, for example, distillation of a high concentration ethanol material from a less pure ethanol-containing material and in some cases the high concentration ethanol material can be further concentrated to achieve very high concentration ethanol, such as 98% or 99% or 99.5% (wt.) or even higher. Other separation steps, such as filtration, centrifugation, extraction, adsorption, etc. can also be a part of some recovery processes.

EXAMPLES

Example 1. Increased ethanol production using C. phytofermentans in a biofilm-forming gentle agitation culture.

[00169] A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 g/L (10% w/v), were inoculated with exponential phase C. phytofermentans {Cphy) at 2% (v/v). The culture was incubated at 35°C with continuous agitation at 175 rpm, pH was adjusted and samples were taken daily. Total ethanol yield, acid by-product production and residual sugar production over time was graphed (FIG. 1). The following calculation was performed to calculate conversion rate: assuming 100 g/L biomass has about 80% carbohydrate, each culture has a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] = 80). Also assumed that complete fermentation of 80 g/L carbohydrate would result in about 40.8 g/L product. Agitation at 175 rpm resulted in an average yield of 17.9 g/L plus 4.07 g/L total acid resulted ([17.9+4.07]/40.8) x 100 = 54 percent total conversion with 17.1 g/L total sugar unfermented (potential 8.7 g/L ethanol additional).

Example 2. Increased ethanol production using C. phytofermentans in a biofilm-forming static fermentation.

[00170] A standard fermentation medium, containing required nwtrient sources and a biomass carbon source at 100 g/L (10% w/v), were inoculated with exponential phase Cphy at 2% (v/v). The culture was incubated at 35°C without agitation (i.e., static fermentation), except for homo genizat ion during pH adjustments or sampling. Total ethanol yield, acid byproduct production and residual sugar production over time was graphed (FIG. 2). The following calculation was performed to calculate conversion rate: assuming 100 g/L biomass has about 80% carbohydrate, each culture has a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] =80). Also assumed that complete fermentation of 80 g/L

carbohydrate would result in about 40.8 g/L product. Static fermentation resulted in an average yield of 30.3 g/L plus 6.8 g/L total acid resulted ([30.3+6.08]/40.8) x 100 = 90 percent total conversion with 8.1 g/L total sugar unfermented (potential 4 g/L ethanol additional). Some additional ethanol can result from residual carbohydrates in nutritional supplements.

Example 3. Increased aspartic acid production using C. phytofermentans in a biofilm- forming gentle agitation culture.

[00171] A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 g/L (10% w/v), is inoculated with exponential phase Cphy at 2% (v/v). The culture is incubated at 35°C with continuous agitation at 175 rpm, the pH is adjusted and samples taken daily. Total chemical fermentation end-product yield, acid byproduct production and residual sugar production over time is graphed. The following calculation is performed to calculate conversion rate: assuming 100 g/L biomass has about 80% carbohydrate, each culture will have a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] = 80). Agitation at 175rpm is applied that leads to production of the

fermentation end product of aspartic acid or aspartate.

Example 4. Increased glutamic acid production using C. phytofermentans in a biofilm- forming gentle agitation culture.

[00172] A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 g/L (10% w/v), is inoculated with exponential phase Cphy at 2% (v/v). The culture is incubated at 35°C with continuous agitation at 175 rpm, the pH is adjusted and samples taken daily. Total chemical fermentation end-product yield, acid byproduct production and residual sugar production over time is graphed. The following calculation is performed to calculate conversion rate: assuming 100 g/L biomass has about 80% carbohydrate, each culture will have a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] = 80). Agitation at 175rpm is applied that leads to production of the

fermentation end product of glutamic acid or glutamate.

Example 5. Increased malic acid production using C. phytofermentans in a biofilm- forming gentle agitation culture.

[00173] A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 g/L (10%> w/v), is inoculated with exponential phase Cphy at 2%> (v/v). The culture is incubated at 35°C with continuous agitation at 175 rpm, the pH is adjusted and samples taken daily. Total chemical fermentation end-product yield, acid byproduct production and residual sugar production over time is graphed. The following calculation is performed to calculate conversion rate: assuming 100 g/L biomass has about 80% carbohydrate, each culture will have a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] = 80). Agitation at 175rpm is applied that leads to production of the

fermentation end product of malic acid or malate.

Example 6. Increased aspartic acid production using C. phytofermentans in a biofilm- forming static fermentation.

[00174] A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 g/L (10%> w/v), is inoculated with exponential phase Cphy at 2% (v/v). The culture is incubated at 35°C without agitation (i.e., static fermentation), except for homogenization during pH adjustments or sampling. Total ethanol yield, acid by-product production and residual sugar production over time is graphed. The following calculation is performed to calculate conversion rate: assuming 100 g/L biomass has about 80%

carbohydrate, each culture has a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] =80). Static fermentation is applied in order to arrive at the fermentation end product of aspartic acid or aspartate.

Example 7. Increased glutamic acid production using C. phytofermentans in a biofilm- forming static fermentation.

[00175] A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 g/L (10%> w/v), is inoculated with exponential phase Cphy at 2% (v/v). The culture is incubated at 35°C without agitation (i.e., static fermentation), except for homogenization during pH adjustments or sampling. Total ethanol yield, acid by-product production and residual sugar production over time is graphed. The following calculation is performed to calculate conversion rate: assuming 100 g/L biomass has about 80%

carbohydrate, each culture has a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] =80). Static fermentation is applied in order to arrive at the fermentation end product of glutamic acid or glutamate.

Example 8. Increased malic acid production using C. phytofermentans in a biofilm- forming static fermentation.

[00176] A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 A standard fermentation medium, containing required nutrient sources and a biomass carbon source at 100 g/L (10% w/v), is inoculated with exponential phase Cphy at 2% (v/v). The culture is incubated at 35°C without agitation (i.e., static fermentation), except for homogenization during pH adjustments or sampling. Total ethanol yield, acid by-product production and residual sugar production over time is graphed. The following calculation is performed to calculate conversion rate: assuming 100 g/L biomass has about 80% carbohydrate, each culture has a potential 80 g/L total carbohydrate for conversion ([100 x 0.8] =80). Static fermentation is applied in order to arrive at the fermentation end product of malic acid or malate.

[00177] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A system comprising:

a. one or more microorganisms that hydro lyzes and ferments hexose and pentose carbohydrates, wherein said one or more microorganisms forms a bio film; b. a biomass comprising hexose and pentose carbohydrates; and

c. a bioreactor comprising an impeller.

2. The system of claim 1 wherein said impeller is a helical impeller.

3. The system of claim 1 wherein one of said one or more microorganisms is a

Clostridium microorganism.

4. The system of claim 1 wherein said one or more microorganisms comprises

Clostridium phytofermentans, Clostridium sp. Q.D., Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium

stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens,

Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica,

Thermoanaerobacterium thermo saccharolyticum and Thermoanaerobacterium saccharolyticum, or a variant thereof.

5. The system of claim 1 wherein said one or more microorganisms comprises

Clostridium phytofermentans or a variant thereof.

6. The system of claim 1 wherein said one or more microorganisms comprises

Clostridium sp. Q.D or a variant thereof.

7. The system of claim 1 wherein said microorganism is genetically modified.

8. The system of claim 1 wherein said impeller has a revolution of about 10-300 rpm.

9. The system of claim 1 wherein said impeller has a revolution of about 50-250 rpm.

10. The system of claim 1 wherein said impeller has a revolution of about 100-200 rpm.

11. The system of claim 1 wherein said impeller has a revolution of about 100-150 rpm.

12. The system of claim 1 wherein said impeller has a revolution of about 150-200 rpm.

13. A system comprising :

a. a biomass comprising hexose and pentose carbohydrates;

b. one or more microorganisms comprising Clostridium phytofermentans,

Clostridium sp. Q.D., or a variant thereof, wherein said one or more microorganisms forms a bio film on said biomass or a support; and

c. a bioreactor comprising a helical impeller that has a revolution rate of about 100-200 rpm.

14. The system of claim 13, wherein said Clostridium phytofermentans, Clostridium sp.

Q.D., or a variant thereof is genetically modified.

15. The system of claims 1 or 13, wherein said bioreactor is a stirred tank reactor, continuous stirred tank reactor, packed bed reactor, fluidized bed reactor, airlift reactor, upflow anaerobic sludge blanket reactor, or a expanded granular sludge reactor.

16. The system of claim 13, wherein said one or more microorganisms forms a bio film on said biomass.

17. The system of claim 13, wherein said one or more microorganisms forms a bio film on said support.

18. The system of claim 13, wherein said support comprises metal, composite or a polymer.

19. The system of claims 1 or 13, wherein said impeller has a revolution of about 120 rpm.

20. The system of claims 1 or 13, wherein said impeller has a revolution of about 175 rpm.

21. The system of claims 1 or 13, wherein said biomass comprises organic matter.

22. The system of claim 21 wherein said organic matter is plant matter or animal matter

23. The system of claims 1 or 13, wherein said biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin.

24. A method of culturing a microorganism comprising:

a. contacting a biomass comprising hexose and pentose carbohydrates with one or more microorganisms that hydro lyzes and ferments hexose and pentose carbohydrates., or a variant thereof; and

b. culturing said one or more microorganisms by agitating said biomass and said one or more microorganisms at a rate wherein said one or more microorganisms forms a bio film on the surface of said biomass or a support.

25. The method of claim 24, wherein one of said one or more microorganisms is a

Clostridium microorganism.

26. The method of claim 24, wherein said one or more microorganisms comprises

27. The method of claim 24, wherein said one or more microorganisms comprises

Clostridium phytofermentans or a variant thereof.

28. The method of claim 24, wherein said one or more microorganisms comprises

Clostridium sp. Q.D or a variant thereof.

29. The method of claim 24, wherein said microorganism is genetically modified.

30. The method of claim 24, wherein said one or more microorganisms forms a bio film on said biomass.

31. The method of claim 24, wherein said one or more microorganisms forms a bio film on said support.

32. The method of claim 31 , wherein said support comprises metal, composite or a polymer.

33. The method of claim 24, wherein said agitation produces a low sheer rate.

34. The method of claim 24, wherein said agitation does not substantially disrupt the bio film.

35. The method of claim 24, wherein said biomass is agitated by the revolution of an impeller.

36. The method of claim 35, wherein said impeller is a helical impeller.

37. The method of claim 35, wherein said microorganism is genetically modified.

38. The method of claim 35, wherein said impeller has a revolution of about 10-300 rpm.

39. The method of claim 35, wherein said impeller has a revolution of about 50-250 rpm.

40. The method of claim 35, wherein said impeller has a revolution of about 100-200 rpm.

41. The method of claim 35, wherein said impeller has a revolution of about 100-150 rpm.

42. The method of claim 35, wherein said impeller has a revolution of about 150-200 rpm.

43. A method of culturing a microorganism comprising:

a. contacting biomass comprising hexose and pentose carbohydrates with one or more microorganisms comprising Clostridium phytofermentans, Clostridium sp. Q.D., or a variant thereof, wherein said one or more microorganisms forms a bio film on said biomass or a support; and

b. culturing said one or more microorganisms in a bioreactor by agitating said biomass with an impeller at a rate that said bio film remains substantially intact.

44. The method of claim 43, wherein said Clostridium phytofermentans, Clostridium sp.

Q.D., or a variant thereof is genetically modified.

45. The method of claims 24 or 43, wherein said impeller has a revolution at a rate of about 120 rpm.

46. The method of claims 24 or 43, wherein said impeller has a revolution at a rate of about 175 rpm.

47. The method of claims 24 or 43, wherein said biomass comprises organic matter.

48. The method of claims 24 or 43, wherein said organic matter is plant matter or animal matter

49. The method of claims 24 or 43, wherein said biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin.

50. The method of claim 43, wherein said one or more microorganisms forms a bio film on said biomass.

51. The method of claim 43, wherein said one or more microorganisms forms a bio film on said support.

52. The method of claim 51 , wherein said support comprises metal, composite or a polymer.

53. The method of claims 24 or 43, wherein said culturing produces a fermentation end product.

54. The method of claims 24 or 43, wherein said fermentation end product is a biofuel.

55. The method of claims 24 or 43, wherein said fermentation end product is an alcohol.

56. The method of claims 24 or 43, wherein said fermentation end product is ethanol, methanol, propanol or butanol.

57. The method of claims 24 or 43, wherein said fermentation end product is ethanol.

58. A method of producing fermentation end product comprising:

a. contacting biomass with a medium and one or more microorganisms that hydro lyzes and ferments hexose and pentose carbohydrates that forms a bio film on said biomass or a support;

b. culturing said one or more microorganisms in said bioreactor with a low sheer force so that said bio film remains substantially intact;

c. producing a fermentation end product from said biomass by said one or more microorganisms; and

d. separating said fermentation end product from said one or more

microorganisms and said biomass.

59. The method of claim 58, wherein one of said one or more microorganisms is a

Clostridium microorganism.

60. The method of claim 58, wherein said one or more microorganisms comprises

Clostridium phytofermentans, Clostridium sp. Q.D. , Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium

61. The method of claim 58, wherein said one or more microorganisms comprises

Clostridium phytofermentans or a variant thereof.

62. The method of claim 58, wherein said one or more microorganisms comprises

Clostridium sp. Q.D or a variant thereof.

63. The method of claim 58, wherein said one or more microorganisms is genetically modified.

64. The method of claim 58, wherein said one or more microorganisms forms a bio film on said biomass.

65. The method of claim 58, wherein said one or more microorganisms forms a bio film on said support.

66. The method of claim 65, wherein said support comprises metal, composite or a polymer.

67. The method of claim 58, wherein said bio film is irreversibly immobilized on said biomass.

68. The method of claim 58, wherein said separating is by centrifugation.

69. The method of claim 58, further comprising extracting said fermentation end product by distillation.

70. The method of claim 58, wherein said culturing is facilitated by agitating said culture.

71. The method of claim 58, wherein said culturing is facilitated by static fermentation.

72. The method of claim 70, wherein said agitating is due to the action of an impeller.

73. The method of claim 72, wherein said impeller is a helical impeller.

74. The method of claim 70, wherein said impeller has a revolution of about 10-300 rpm.

75. The method of claim 70, wherein said impeller has a revolution of about 50-250 rpm.

76. The method of claim 70, wherein said impeller has a revolution of about 100-200 rpm.

77. The method of claim 70, wherein said impeller has a revolution of about 100-150 rpm.

78. The method of claim 70, wherein said impeller has a revolution of about 150-200 rpm.

79. The method of claim 70, wherein said agitation is achieved by an impeller having a revolution at a rate of 120 rpm.

80. The method of claim 70, wherein said agitation is achieved by an impeller having a revolution at a rate of 175 rpm.

81. The method of claim 58, wherein said biomass is 15 % (w/w) of total weight of said biomass, said medium and said one or more microorganisms.

82. The method of claim 58, wherein said biomass is 20 % (w/w) of total weight of said biomass, said medium and said one or more microorganisms.

83. The method of claim 58, wherein said biomass is 30 % (w/w) of total weight of said biomass, said medium and said one or more microorganisms.

84. The method of claim 58, wherein said biomass comprises organic matter.

85. The method of claim 58, wherein said organic matter is plant matter or animal matter

86. The method of claim 58, wherein said biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin.

87. The method of claim 58, wherein said fermentation end product is a biofuel.

88. The method of claim 58, wherein said fermentation end product is an alcohol.

89. The method of claim 58, wherein said fermentation end product is ethanol, methanol, propanol, butanol, 1,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3 -hydro xybutyro lactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, or a terpene.

90. The method of claim 58, wherein said fermentation end product is ethanol.

91. A fermentation end product produced by the method of claims 58 to 86.

92. The fermentation end product of claim 91, wherein said fermentation end product is biofuel.

93. The fermentation end product of claim 91, wherein said fermentation end product is an alcohol.

94. The fermentation end product of claim 91, wherein said fermentation end product is ethanol, methanol, propanol, butanol, 1,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, or a terpene.

95. The fermentation end product of claim 91, wherein said fermentation end product is ethanol.

96. A composition for the production of a fermentative end product comprising

a. a biomass comprising hexose and pentose carbohydrates; and

b. one or more microorganisms that hydro lyzes and ferments hexose and pentose carbohydrates, wherein said one or more microorganisms forms a bio film.

97. The composition of claim 96, wherein one of said one or more microorganisms is a Clostridium microorganism.

98. The composition of claim 96, wherein said one or more microorganisms comprises Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium

Thermoanaerobacterium thermo saccharolyticum Thermoanaerobacterium saccharolyticum, or a variant thereof.

99. The composition of claim 96, wherein said one or more microorganisms comprises Clostridium phytofermentans or a variant thereof.

100. The composition of claim 96, wherein said one or more microorganisms comprises Clostridium sp. Q.D or a variant thereof.

101. The composition of claim 96, wherein said biomass comprises organic matter

102. The composition of claim 101, wherein said organic matter is plant matter or animal matter

103. The composition of claim 101, wherein said biomass comprises sawdust, wood flour, wood pulp, paper pulp, grass, switchgrass, crambe, algae, rice hulls, bagasse, jute, leaves, grass clippings, corn stover, corn cobs, corn grain, distillers grains, citrus peels, food waste, distillers grains, Distillers Dried Solubles, Distillers Dried Grains, Condensed Distillers Solubles, Distillers Wet Grains, Distillers Dried Grains with Solubles, or pectin.

104. The composition of claim 96, wherein said biomass is 15 % (w/w) of total weight of said composition.

105. The composition of claim 96, wherein said biomass is 20 % (w/w) of total weight of said composition.

106. The composition of claim 101, wherein said biomass is 30 % (w/w) of total weight of said composition.

107. The composition of claim 96, wherein said fermentation end product is a bio fuel.

108. The composition of claim 96, wherein fermentation end product is an alcohol.

109. The composition of claim 96, wherein fermentation end product is ethanol, methanol, propanol, butanol, 1,4 diacid (succinic, fumaric or malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, aspartate, glucaric acid, glutamic acid, glutamate, malate, itaconic acid, levulinic acid, 3 -hydro xybutyro lactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, an isoprenoid, or a terpene.

110. The composition of claim 96, wherein said fermentation end product is ethanol.