BIOFUEL PRODUCTION USING BIOFILM IN FERMENTATION

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application Nos. 61/424,013, filed December 16, 2010 and 61/500,070, filed June 22, 2011, which applications are incorporated herein by reference their entireties.

BACKGROUND OF THE INVENTION

[0002] Fermentation of biomass to produce a biofuel 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 can be 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 biofuel that can be generated from a given quantity of biomass. Therefore, to obtain a high conversion efficiency of lignocellulosic biomass to ethanol (yield) it can be important to be able to successfully ferment both hexose and pentose sugars into ethanol. In one aspect, conversion efficiency of biomass to ethanol is enhanced by the formation of a biofilm by the microorganism used in the biomass fermentation to fuel conversion process.

SUMMARY OF THE INVENTION

[0003] Disclosed herein are methods for producing and recovering one or more fermentation end- products, comprising: a. providing a fermenter comprising a slurry having a biomass and one or more microorganisms, wherein at least one of the microorganisms can hydro lyze and/or ferment the biomass to produce the fermentation end-products; b. sparging the slurry with a gas, wherein the gas provides low shear mixing of the slurry, and wherein a portion of at least one of the fermentation end-products evaporates into the gas; and, c. recovering the fermentation end-products from the gas. In one

embodiment, the sparging is accomplished by a gas distribution member in fluid contact with the slurry in the fermenter. In one embodiment, the recovering comprises directing the gas comprising the

fermentation end-products to a condenser to form a condensate comprising the fermentation end-products. In one embodiment, the directing is accomplished by a compressor. In one embodiment, the compressor is downstream of the fermenter and upstream of the condenser. In one embodiment, the compressor maintains a first pressure between the fermenter and the compressor that is below atmospheric pressure, and a second pressure between the compressor and the condenser that is at or above atmospheric pressure. In one embodiment, the condensate is directed to a distilling column. In one embodiment, all or a part of the gas leaving the condenser is recycled to the fermenter. In one embodiment, the gas leaving the condenser passes through a sterile filter prior to being recycled to the fermenter. In one embodiment, the gas leaving the condenser is directed to a gas separator prior to the sterile filter. In one embodiment, the gas passes through a scrubber prior to the gas separator. In one embodiment, the biomass comprises 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, pectin, or a combination thereof. In one embodiment, the gas comprises C02, CO, N2, H2, He, Ne, Ar, N02, deoxygenated air, and/or a combination thereof. In one embodiment, the biomass comprises cellulose, hemicellulose, lignocellulose, and/or a combination thereof. In one embodiment, the biomass comprises C5 sugars (pentose sugars), C6 sugars (hexose sugars), or a combination thereof. In one embodiment, at least one of the microorganisms forms a biofilm on the biomass. In one embodiment, the biofilm is irreversibly immobilized on the biomass. In one embodiment, the biofilm is reversibly immobilized on the biomass. In one embodiment, at least one of the microorganisms can hydrolyze and ferment hemicellulosic or lignocellulosic material. In one embodiment, the microorganisms comprise a bacteria, a yeast, a non-yeast fungus, or a combination thereof. In one embodiment, the microorganisms comprise Clostridium phytofermentans , 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, Thermoanaerobacterium

saccharolyticum, or a combination thereof. In one embodiment, at least one of the microorganisms is a Clostridium strain. In one embodiment, the Clostridium strain is Clostridium phytofermentans,

Clostridium sp Q.D, or a variant thereof. In one embodiment, Clostridium strain is a C. phytofermentans American Type Culture Collection 700394T or a strain assigned the NRRL deposit accession number NRRL B-50351, NRRL B-50361, NRRL B-50362, NRRL B-50363, NRRL B-50364, NRRL B-50436, NRRL B-50437, NRRL B-50498, NRRL B-50511, NRRL B-50512, NRRL B-50447, NRRL B-50448, NRRL B-50449, and NRRL B-50450. In one embodiment, at least one of the fermentation end-products has a boiling point below that of water. In one embodiment, the fermentation end-products comprise one or more alcohols. In one embodiment, the alcohols comprise ethanol, methanol, propanol, butanol, or a combination thereof. In one embodiment, the alcohols comprise ethanol. In one embodiment, the method is capable of producing at least about 20 g/L, 30 g/L, 40 g/L, 50g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or more of ethanol. In one embodiment, at least one of the microorganisms is genetically modified. In one embodiment, at least one of the fermentation end-products is maintained at a concentration in the fermenter that is below an inhibitory concentration for the microorganisms. [0004] Also disclosed herein are low shear rate methods for producing one or more fermentation end- products, comprising directing a gas through a slurry comprising biomass with a C. phytofermentans biofilm, wherein said gas extracts a portion of at least one of said fermentation end-product from said slurry, and isolating said fermentation end-products from said gas vapor.

[0005] Also disclosed herein are systems for producing and recovering one or more fermentation end- products, the system comprising: a. a fermenter configured to house a slurry, the slurry comprising: i. a biomass, and ii. one or more microorganisms, wherein at least one of the microorganisms can hydrolyze and/or ferment the biomass to produce the fermentation end-products; b. a gas distribution member in fluid contact with the slurry in the fermenter, wherein the gas distribution member sparges a gas through the slurry, wherein the gas causes low shear mixing of the slurry and wherein a portion of at least one of the fermentation end-products evaporates into the gas; and, c. a condenser, wherein the gas comprising the portion of the fermentation end-products is cooled such that a condensate is formed comprising the fermentation end-products. Some embodiments further comprise a compressor downstream of the fermenter and upstream of the condenser. In one embodiment, the compressor maintains a first pressure between the fermenter and the compressor that is below atmospheric pressure, and a second pressure between the compressor and the condenser that is at or above atmospheric pressure. Some embodiments further comprise a scrubber downstream of the condenser. Some embodiments further comprise a sterile filter downstream of the scrubber. Some embodiments further comprise a gas separator downstream of the scrubber and upstream of the sterile filter. In one embodiment, all or a part of the gas is recycled to the fermenter. In one embodiment, the gas comprises C02, CO, N2, H2, He, Ne, Ar, N02, deoxygenated air, and/or a combination thereof. Some embodiments further comprise a distillation column, wherein the condensate is directed from the condenser to the distillation column, and wherein the fermentation end- products are further purified. In one embodiment, the biomass comprises cellulose, lignocellulose, hemicellulose, or a combination thereof. In one embodiment, the biomass comprises 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, pectin, or a combination thereof. In one embodiment, the biomass comprises C5 sugars (pentose sugars), C6 sugars (hexose sugars), or a combination thereof. In one embodiment, at least one of the microorganisms forms a biofilm on the biomass. In one embodiment, the biofilm is irreversibly immobilized on the biomass. In one embodiment, the biofilm is reversibly immobilized on the biomass. In one embodiment, at least one of the microorganisms can hydrolyze and ferment hemicellulosic or lignocellulosic material. In one embodiment, at least one of the microorganisms can hydrolyze and/or ferment C5 sugars (pentose sugars) and C6 sugars (hexose sugars). In one embodiment, the microorganisms comprise a yeast, a bacteria, a non-yeast fungus, or a combination thereof. In one embodiment, at least one of the microorganisms is Clostridium phytofermentans, 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, or Thermoanaerobacterium saccharolyticum. In one embodiment, at least one of the microorganisms is a Clostridium strain. In one embodiment, the Clostridium strain is Clostridium phytofermentans, Clostridium sp Q.D, or a variant thereof. In one embodiment, the Clostridium strain is a C. phytofermentans American Type Culture Collection 700394T or a strain assigned the NRRL deposit accession number NRRL B-50351, NRRL B-50361, NRRL B-50362, NRRL B-50363, NRRL B-50364, NRRL B-50436, NRRL B-50437, NRRL B-50498, NRRL B-50511, NRRL B-50512, NRRL B-50447, NRRL B-50448, NRRL B-50449, and NRRL B-50450. In one embodiment, at least one of the microorganisms is genetically modified. In one embodiment, the biomass is 15 % (w/w) of total weight of the composition. In one embodiment, the biomass is 20 % (w/w) of total weight of the composition. In one embodiment, the biomass is 30 % (w/w) of total weight of the composition. In one embodiment, the fermentation end-products comprise one or more alcohols. In one embodiment, the alcohols comprise ethanol, methanol, propanol, butanol, or a combination thereof. In one embodiment, at least one of the fermentation end-products is ethanol. In one embodiment, the system is capable of producing at least about 20 g/L, 30 g/L, 40 g/L, 50g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or more of ethanol. In one embodiment, at least one of the fermentation end-products has a boiling point below that of water.

[0006] Also disclosed herein are methods for the continuous production of one or more fermentation end-products, comprising: a. forming a slurry having a biomass and one or more microorganisms, wherein at least one of the microorganisms can hydro lyze and/or ferment the biomass in a reactor to produce the fermentation end-products; b. directing a gas through the slurry, wherein the gas extracts a portion of at least one of the fermentation end-products from the slurry; c. separating the gas comprising the portion of the fermentation end-products from the slurry; and d. recovering the fermentation end- products from the gas, wherein the fermentation end-product has a boiling point below water. In one embodiment, the directing the gas through the slurry is accomplished by a gas distribution member in fluid contact with the slurry in the fermenter. In one embodiment, the recovering comprises directing the gas comprising the fermentation end-products to a condenser to form a condensate comprising the fermentation end-products. In one embodiment, the directing is accomplished by a compressor. In one embodiment, the compressor is downstream of the fermenter and upstream of the condenser. In one embodiment, the compressor maintains a first pressure between the fermenter and the compressor that is below atmospheric pressure, and a second pressure between the compressor and the condenser that is at or above atmospheric pressure. In one embodiment, the condensate is directed to a distilling column. In one embodiment, all or a part of the gas leaving the condenser is recycled to the fermenter. In one embodiment, the gas leaving the condenser passes through a sterile filter prior to being recycled to the fermenter. In one embodiment, the gas leaving the condenser is directed to a gas separator prior to the sterile filter. In one embodiment, the gas passes through a scrubber prior to the gas separator. In one embodiment, the biomass comprises 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, pectin, or a combination thereof. In one embodiment, the gas comprises C02, CO, N2, H2, He, Ne, Ar, N02, deoxygenated air, and/or a combination thereof. In one embodiment, the biomass comprises cellulose, hemicellulose, lignocellulose, and/or a combination thereof. In one embodiment, the biomass comprises C5 sugars (pentose sugars), C6 sugars (hexose sugars), or a combination thereof. In one embodiment, at least one of the microorganisms forms a biofilm on the biomass. In one embodiment, the biofilm is irreversibly immobilized on the biomass. In one embodiment, the biofilm is reversibly immobilized on the biomass. In one embodiment, at least one of the microorganisms can hydrolyze and ferment hemicellulosic or lignocellulosic material. In one embodiment, the microorganisms comprise a bacteria, a yeast, a non-yeast fungus, or a combination thereof. In one embodiment, the microorganisms comprise Clostridium phytofermentans, 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, Thermoanaerobacterium saccharolyticum, or a combination thereof. In one embodiment, at least one of the microorganisms is a Clostridium strain. In one embodiment, the Clostridium strain is Clostridium phytofermentans, Clostridium sp Q.D, or a variant thereof. In one embodiment, Clostridium strain is a C. phytofermentans American Type Culture Collection 700394T or a strain assigned the NRRL deposit accession number NRRL B-50351, NRRL B-50361, NRRL B-50362, NRRL B-50363, NRRL B-50364, NRRL B-50436, NRRL B-50437, NRRL B-50498, NRRL B-50511, NRRL B-50512, NRRL B-50447, NRRL B-50448, NRRL B-50449, and NRRL B-50450. In one embodiment, at least one of the fermentation end-products has a boiling point below that of water. In one embodiment, the fermentation end-products comprise one or more alcohols. In one embodiment, the alcohols comprise ethanol, methanol, propanol, butanol, or a combination thereof. In one embodiment, the alcohols comprise ethanol. In one embodiment, the method is capable of producing at least about 20 g/L, 30 g/L, 40 g/L, 50g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or more of ethanol. In one embodiment, at least one of the microorganisms is genetically modified. In one embodiment, at least one of the fermentation end-products is maintained at a concentration in the fermenter that is below an inhibitory concentration for the microorganisms. INCORPORATION BY REFERENCE

[0007] 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

[0008] 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:

[0009] Figure 1 illustrates a pathway map for cellulose hydrolysis and fermentation.

[0010] Figure 2 illustrates increased ethanol production for fermentation using a strain of Clostridium phytofermentans (Q.27); y-axis is yield in grams per liter (g/L) and x-axis is time in hours (h).

[0011] Figure 3 illustrates increased ethanol production by formation of biofilm facilitated by gentle agitation of the culture; y-axis is yield in grams per liter (g/L) and x-axis is time in hours (h).

[0012] Figure 4 illustrates ethanol production by formation of biofilm facilitated by static fermentation; y-axis is yield in grams per liter (g/L) and x-axis is time in hours (h).

[0013] Figure 5 (A&B) illustrates exemplary fermentation product recovery systems.

[0014] Figure 6 (A&B) depicts a graph showing ethanol condensate rate as a function of ethanol concentration (A) and condenser temperature (B).

[0015] Figure 7 depicts a graph showing the rate of ethanol removal and ethanol mass fraction as a function of pressure.

[0016] Figure 8 (A&B) depicts a graph showing ethanol concentration and rate of ethanol removal from the fermenter as a function of condenser pressure (A) and fermenter ethanol concentration (B).

[0017] Figure 9 illustrates a fermentation product recovery system comprising a scrubber.

[0018] Figure 10 illustrates a fermentation product recovery system comprising a scrubber and a gas separator.

DETAILED DESCRIPTION OF THE INVENTION

[0019] 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

[0020] Unless characterized differently, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0021] The term "about" as used herein with a numerical value means a range of values within 15% plus or minus from the recited numerical value within the context of the particular usage. For example, about 10 would mean 8.5 to 11.5.

[0022] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase "the medium can optionally contain glucose" means that the medium may or may not contain glucose as an ingredient and that the description includes both media containing glucose and media not containing glucose.

[0023] The term "enzyme reactive conditions" as used herein refers to environmental conditions (i.e., such factors as temperature, pH, or lack of inhibiting substances) which will permit the enzyme to function. Enzyme reactive conditions can be either in vitro, such as in a test tube, or in vivo, such as within a cell.

[0024] The terms "function" and "functional" and the like as used herein refer to a 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 that 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 that has been purified from the sequences that flank it in a naturally- occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. 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 terms "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, biofuel, 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 term "operably linked" as used herein means placing a gene under the regulatory control of a promoter, which then controls the transcription and optionally the translation of the gene. In one example, for the construction of promoter/structural gene combinations, the genetic sequence or promoter is positioned at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting; e.g., the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, a regulatory sequence element can be positioned with respect to a gene to be placed under its control in the same position as the element is situated in its natural setting with respect to the native gene it controls.

[0030] The term "constitutive promoter" refers to a polynucleotide sequence that induces transcription or is typically active, (i.e., promotes transcription), under most conditions, such as those that occur in a host cell. A constitutive promoter is generally active in a host cell through a variety of different environmental conditions.

[0031] The term "inducible promoter" refers to a polynucleotide sequence that induces transcription or is typically active only under certain conditions, such as in the presence of a specific transcription factor or transcription factor complex, a given molecule factor (e.g., IPTG), or a given environmental condition (e.g., CO ₂ concentration, nutrient levels, light, heat). In the absence of that condition, inducible promoters typically do not allow significant or measurable levels of transcriptional activity.

[0032] The terms "polynucleotide" or "nucleic acid" as used herein designates RNA, mRNA, cRNA, rRNA, DNA, or cDNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0033] 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.

[0034] Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. In one embodiment, additional coding or non-coding sequences can, but need not, be present within a polynucleotide, and a polynucleotide can, but need not, be linked to other molecules and/or support materials.

[0035] Polynucleotides can comprise a native sequence (i.e., an endogenous sequence) or can comprise a variant, or a biological functional equivalent of such a sequence. Polynucleotide variants can contain one or more base substitutions, additions, deletions and/or insertions, as further described below. In one embodiment a polynucleotide variant encodes a polypeptide with the same sequence as the native protein. In another embodiment a polynucleotide variant encodes a polypeptide with substantially similar enzymatic activity as the native protein. In another embodiment a polynucleotide variant encodes a protein with increased enzymatic activity relative to the native polypeptide. The effect on the enzymatic activity of the encoded polypeptide can generally be assessed as described herein.

[0036] A polynucleotide, can be combined with other DNA sequences, such as promoters,

polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably. In one embodiment, the maximum length of a polynucleotide sequence which can be used to transform a microorganism is governed only by the nature of the recombinant protocol employed.

[0037] 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.

[0038] 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), homologs (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 dehydrogenase, pectate lyase, alginate lyase, diol dehydratase, transporter, etc.).

[0039] 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%, 80%, 85%, 90% to 95% or more, and even about 97% or 98% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters. 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.

[0040] The genetic code is redundant in that it contains 64 different codons (triplet nucleotide sequence) but only codes for 22 standard amino acids and a stop signal. Due to the degeneracy of the genetic code, nucleotides within a protein-coding polynucleotide sequence can be substituted without altering the encoded amino acid sequence. These changes (e.g., substitutions, mutations, optimizations, etc.) are therefore "silent". It is thus contemplated that various changes can be made within a disclosed nucleic acid sequence without any loss of biological activity relating to either the polynucleotide sequence or the encoded peptide sequence.

[0041] In one embodiment, a polynucleotide comprises codons, within a coding sequence, that are optimized to increase the thermostability of an mRNA transcribed from the polynucleotide. In one embodiment, this optimization does not change the amino acid sequence encoded by the polynucleotide (i.e. they are "silent"). In another embodiment, a polynucleotide comprises codons, within a protein coding sequence, that are optimized to increase translation efficiency of an mRNA transcribed from the polynucleotide in a host cell. In one embodiment, this optimization is silent (does not change the amino acid sequence encoded by the polynucleotide).

[0042] It will be appreciated by one of skill in the art that amino acids can be substituted for other amino acids in a protein sequence without appreciable loss of the desired activity. It is thus contemplated that various changes can be made in the peptide sequences of the disclosed protein sequences, or their corresponding nucleic acid sequences without appreciable loss of the biological activity.

[0043] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0044] Amino acids have been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);

tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);

glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0045] It is known in the art that certain amino acids can be substituted by other amino acids having a similar hydropathic index or score and result in a protein with similar biological activity, i.e., still obtain a biologically-functional protein. In one embodiment, the substitution of amino acids whose hydropathic indices are within +/-0.2 is preferred, those within +/-0.1 are more preferred, and those within +/-0.5 are most preferred.

[0046] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (Hopp, which is herein incorporated by reference in its entirety) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0);

aspartate/glutamate (+3.0.+-.1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5);

leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).

[0047] It is understood that an amino acid can be substituted by another amino acid having a similar hydrophilicity score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein. In one embodiment the substitution of amino acids whose hydropathic indices are within +/-0.2 is preferred, those within +/-0.1 are more preferred, and those within. +/-.0.5 are most preferred.

[0048] As outlined above, amino acid substitutions can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take any of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Changes that are not expected to be advantageous can also be used if these resulting proteins have the same or improved characteristics, relative to the unmodified polypeptide from which they are engineered.

[0049] In one embodiment, a method is provided for that uses variants of full-length polypeptides having any of the enzymatic activities described herein, truncated fragments of these full-length polypeptides, variants of truncated fragments, as well as their related biologically active fragments. Typically, biologically active fragments of a polypeptide can participate in an interaction, for example, an intramolecular 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 an enzyme can be a polypeptide fragment that is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 600 or more contiguous amino acids, including all integers in between, of a reference polypeptide sequence. 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.

[0050] 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.

[0051] 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 of pathways, genes, or enzymes described herein can utilize or rely on an "endogenous" sequence, or can be provided as one or more "exogenous" polynucleotide sequences, and/or can be used according to the endogenous sequences already contained within a given microorganism

[0052] 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.

[0053] The terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.

Because two polynucleotides can each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more)

polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window can be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also can be made to the BLAST family of programs as disclosed, for example, by Altschul et ah, 1997, Nucl. Acids Res. 25:3389, which is herein incorporated by reference in its entirety. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et ah, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994- 1998, Chapter 15, which is herein incorporated by reference in its entirety.

[0054] 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 transfer of additional copies of an endogenous gene into a microorganism.

[0055] The term "recombinant" as used herein, refers to an microorganism that is genetically modified to comprise one or more heterologous or endogenous nucleic acid molecules, such as in a plasmid or vector. Such nucleic acid molecules can be comprised extra-chromosomally or integrated into the chromosome of an microorganism. The term "non-recombinant" means an microorganism is not genetically modified. For example, a recombinant microorganism can be modified to overexpress an endogenous gene encoding an enzyme through modification of promoter elements (e.g., replacing an endogenous promoter element with a constitutive or highly active promoter). Alternatively, a recombinant microorganism can be modified by introducing a heterologous nucleic acid molecule encoding a protein that is not otherwise expressed in the host microorganism.

[0056] 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.

[0057] The terms "inactivate" or "inactivating" as used herein for a gene, refer to a reduction in expression and/or activity of the gene. The terms "inactivate" or "inactivating" as used herein for a biological pathway, refer to a reduction in the activity of an enzyme in a the pathway. For example, inactivating an enzyme of the lactic acid pathway would lead to the production of less lactic acid.

[0058] 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.

[0059] 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 saccharides, 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, or animal matter. In another embodiment biomass comprises non-genetically modified organisms or parts of organisms, such as non-genetically modified plant matter, algal matter, or animal matter. The term "feedstock" is also used to refer to biomass being used in a process, such as those described herein.

[0060] 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 human or animal use, 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 includes, but is not limited to, products and/ or byproducts from a host plant operating on a fractionated product ( e.g., WDG, TS, WS, syrup, etc.). In another embodiment plant matter includes, but is not limited to, products from corn fractionation (e.g., germ, oil, fiber, fiber-enriched fraction, residues from fractionation and separation, etc.).ln 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 hydrolytic 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.

[0061] 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). [0062] 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.

[0063] 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.

[0064] 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 biofuel, chemical or other product. Carbonaceous biomass can comprise municipal waste (waste paper, recycled toilet papers, yard clippings, etc.), wood, plant material, plant matter, plant extract, bacterial matter {e.g., bacterial cellulose), distillers' grains, a natural or synthetic polymer, or a combination thereof.

[0065] 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.).

[0066] Examples of polysaccharides, oligosaccharides, monosaccharides or other sugar components of biomass include, but are not limited to, alginate, agar, carrageenan, fucoidan, floridean starch, pectin, gluronate, mannuronate, mannitol, lyxose, cellulose, hemicellulose, glycerol, xylitol, glucose, mannose, galactose, xylose, xylan, mannan, arabinan, arabinose, glucuronate, galacturonate (including di- and tri- galacturonates), rhamnose, and the like.

[0067] The term "carbonaceous byproducts" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biological materials that can be converted into a biofuel, chemical or other product. One exemplary source of carbonaceous material is plant matter. Plant matter can be, for example, woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, sugar cane, grasses, switchgrass, bamboo, algae, and material derived from these. Plant matter can also be residual spent solids from alcoholic or other fermentation from materials such as corn and which contain lignin, starch, cellulose, hemicellulose, and proteins. Plant matter can be further described by reference to the chemical species present, such as proteins, polysaccharides (such as chitin) and oils. Polysaccharides include polymers of various monosaccharides and derivatives of monosaccharides including glucose, fructose, lactose, galacturonic acid, rhamnose, etc. Plant matter also includes agricultural waste byproducts or side streams such as pomace, corn steep liquor, corn steep solids, corn stover, corn stillage, corn cobs, corn grain, bagasse, distillers grains, peels, pits, fermentation waste, wood chips, saw dust, wood flour, wood pulp, paper pulp, paper pulp waste steams straw, lumber, demolition waste, hybrid poplar, milo, sewage, seed cake, husks, rice hulls, leaves, grass clippings, food waste, restaurant waste, or cooking oil. These materials can come from farms, forestry, industrial sources, households, etc. Plant matter also includes maltose, corn syrup, syrup, Whole Stillage, Thin Stillage, Thick Stillage, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Grains (DG), Wet Distillers Grains (WDG), Wet Distillers Grains with Solubles (WDGS), or Distillers Dried Grains with Solubles (DDGS). Another non- limiting example of biomass is animal matter, including, for example milk, meat, fat, bone meal, animal processing waste, and animal waste.

"Feedstock" is frequently used to refer to biomass being used for a process, such as those described herein. Another example of carbonaceous material or biomass is sewage and/or municipal waste, much of which contains indigestible materials such as paper and other cellulosic, hemicellulosic and lignocellulosic material.

[0068] 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.

[0069] 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.

[0070] 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:

C ₆ H ₁₂ 0 ₆ -» 2C ₂ H ₅ OH + 2CO ₂

[0071] and the theoretical maximum conversion efficiency or yield is 51% (wt). Frequently, the conversion efficiency will be referenced to the theoretical maximum, for example, "80% of the theoretical maximum." In the case of conversion of glucose to ethanol, this statement would indicate a conversion efficiency of 41 > (wt.). The context of the phrase will indicate the substrate and product intended to one of skill in the art. 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 l Og 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, l Og 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.

[0072] The saccharification yield takes into account the amount of ethanol and acidic products produced plus the amount of residual monomeric sugars detected in the media. These can account for up to 3 g/L ethanol production or equivalent of up to 6 g/L sugar as much as +/- 10%>-15%> saccharification yield (or saccharification efficiency).

[0073] 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.

[0074] 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.

[0075] The terms "pretreatment" or "pretreated" as used herein refer to any mechanical, chemical, thermal, biochemical process or combination of these processes whether in a combined step or performed sequentially, that achieves disruption or expansion of a biomass so as to render the biomass more susceptible to attack by enzymes and/or microorganisms. In some embodiments, pretreatment can include removal or disruption of lignin so is to make the cellulose and hemicellulose polymers in the plant biomass more available to cellulolytic enzymes and/or microorganisms, for example, by treatment with acid or base. In some embodiments, pretreatment can include the use of a microorganism of one type to render plant polysaccharides more accessible to microorganisms of another type. In some embodiments, pretreatment can also include disruption or expansion of cellulosic and/or hemicellulosic material. Steam explosion, and ammonia fiber expansion (or explosion) (AFEX) are well known thermal/chemical techniques. Hydrolysis, including methods that utilize acids and/or enzymes can be used.

[0076] Other thermal, chemical, biochemical, enzymatic techniques can also be used.

[0077] The term "xylano lytic" as used herein refers to any substance capable of breaking down xylan. The term "cellulolytic" as used herein refers to any substance capable of breaking down cellulose.

[0078] 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 microorganisms, including solutions, suspensions, and mixtures of enzymes and microorganisms. In some contexts this word will refer to the possible use of either enzymes or microorganisms to serve a particular function, in other contexts the word will refer to the combined use of the two, and in other contexts the word will refer to only one of the two. The context of the phrase will indicate the meaning intended to one of skill in the art. For example, the term

"Clostridium biocatalyst" as used herein indicates one or more Clostridium strains {e.g., C.

phytofermentans , Clostridium Sp. Q.D, C. phytofermentans Q8, Clostridium sp. Q.D-5, Clostridium sp. Q.D-7, Clostridium phytofermentans Q.7D, Clostridium phytofermentans Q.13, Clostridium

phytofermentans Q.27, derivatives of the same, etc.) including any and all genetically modified or wild- type variations thereof. In one embodiment, a Clostridium biocatalyst can simultaneously hydrolyze and ferment lignocellulosic biomass. In one embodiment, a Clostridium biocatalyst can hydrolyze and ferment hexose (C6) and pentose (C5) polysaccharides {e.g., carbohydrates).

[0079] Generally, compositions and methods are provided for enzyme conditioning of feedstock or biomass to allow saccharification and fermentation to one or more industrially useful fermentation end- products.

[0080] The term "biofilm" as used herein refers to an aggregate of microorganisms in which cells are stuck to each other and/or to a surface. A "biofilm" includes a layer of cells where microbial cells attach to a support, flocculate or aggregate together as "granules." Biofilm formation can be a natural process or induced process in which cells are attracted to an absorbent material and form a biofilm.

[0081] Biofilm formation has been 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.

[0082] The term "fuel" or "biofuel" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more compounds suitable as liquid fuels, gaseous fuels, reagents, chemical feedstocks and includes, but is not limited to, hydrocarbons, 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.).

[0083] 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, chemical additives, processing aids, food additives, organic acids {e.g., acetic, lactic, formic, citric acid etc.), derivatives of organic acids such as esters {e.g., wax esters, glycerides, etc.) and other functional compounds including, but not limited to, 1, 2- propanediol, 1, 3 -propanediol, lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid, enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases and can be present as a pure compound, a mixture, or an impure or diluted form.

[0084] 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.

[0085] 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. 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.

[0086] 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.

[0087] 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 fermentable sugars include, but are not limited to glucose, xylose, arabinose, galactose, mannose, rhamnose, cellobiose, lactose, sucrose, maltose, and fructose.

[0088] 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 utilized 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 monomer units).

[0089] 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. [0090] The term "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.

[0091] The term "sugar compounds" as used herein has its ordinary meaning as known to those skilled in the art and can include monosaccharide sugars, including but not limited to hexoses and pentoses; sugar alcohols; sugar acids; sugar amines; compounds containing two or more of these linked together directly or indirectly through covalent or ionic bonds; and mixtures thereof. Included within this description are disaccharides; trisaccharides; oligosaccharides; polysaccharides; and sugar chains, branched and/or linear, of any length.

[0092] 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.

Pretreatment of Biomass

[0093] 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.

[0094] In some embodiments, a Clostridium species, for example 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, hydrolyzed proteins, ammonia, urea, nitrate, nitrite, soy, soy derivatives, casein, casein derivatives, milk powder, milk derivatives, whey, yeast extract, hydrolyze yeast, autolyzed yeast, corn steep liquor, corn steep solids, monosodium glutamate, and/or other fermentation nitrogen sources, vitamins, and/or mineral supplements. 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.

[0095] 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 some embodiments, the anaerobic microorganism can hydrolyze and ferment a biomass 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 disclosure 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 O.Olg 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 O.Olg of alkaline (e.g., caustic), 0.02g, 0.03g, 0.04g, 0.05g, 0.075g, O. lg, 0.2g, 0.3g, 0.4g, 0.5g, 0.75g, lg, 2g, or about 3g of alkaline (e.g., caustic) for every gram of biomass to be treated.

[0096] In another embodiment, pretreatment of biomass comprises dilute acid hydrolysis. Examples 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, incorporated by reference herein in its entirety.

[0097] In some embodiments, pretreatment of biomass comprises autohydro lysis (i.e., hot water treatment). In one embodiment, a hot water treatment can be performed between about 100°C and 200°C, for example, between about 100°C and 110°C, 100°C and 120°C, 100°C and 130°C, 100°C and 140°C, 100°C and 150°C, 100°C and 160°C, 100°C and 170°C, 100°C and 180°C, 100°C and 190°C, 100°C and 200°C, 110°C and 120°C, 110°C and 130°C, 110°C and 140°C, 110°C and 150°C, 110°C and 160°C, 110°C and l70°C, 110°C and 180°C, 110°C and 190°C, 110°C and 200°C, 120°C and 130°C, 120°C and 140°C, 120°C and 150°C, 120°C and 160°C, 120°C and 170°C, 120°C and 180°C, 120°C and 190°C, 120°C and 200°C, 130°C and 140°C, 130°C and 150°C, 130°C and 160°C, 130°C and 170°C, 130°C and 180°C, 130°C and 190°C, 130°C and 200°C, 140°C and 150°C, 140°C and 160°C, 140°C and 170°C, 140°C and 180°C, 140°C and 190°C, 140°C and 200°C, 150°C and 160°C, 150°C and 170°C, 150°C and 180°C, 150°C and 190°C, 150°C and 200°C, 160°C and 170°C, 160°C and 180°C, 160°C and 190°C, 160°C and 200°C, 170°C and 180°C, 170°C and 190°C, 170°C and 200°C, 180°C and 190°C, 180°C and 200°C, or 190°C and 200°C. The autohydrolysis temperature can be about 100°C, 101 °C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, 1 1 1 °C, 1 12°C, 1 13°C, 1 14°C, 1 15°C, 1 16°C, 1 17°C, 118°C, 1 19°C, 120°C, 121 °C, 122°C, 123°C, 124°C, 125°C, 126°C, 127°C, 128°C, 129°C, 130°C, 131 °C, 132°C, 133°C, 134°C, 135°C, 136°C, 137°C, 138°C, 139°C, 140°C, 141 °C, 142°C, 143°C, 144°C, 145°C, 146°C, 147°C, 148°C, 149°C, 150°C, 151 °C, 152°C, 153°C, 154°C, 155°C, 156°C, 157°C, 158°C, 159°C, 160°C, 161 °C, 162°C, 163°C, 164°C, 165°C, 166°C, 167°C, 168°C, 169°C, 170°C, 171 °C, 172°C, 173°C, 174°C, 175°C, 176°C, 177°C, 178°C, 179°C, 180°C, 181 °C, 182°C, 183°C, 184°C, 185°C, 186°C, 187°C, 188°C, 189°C, 190°C, 191 °C, 192°C, 193°C, 194°C, 195°C, 196°C, 197°C, 198°C, 199°C or 200°C. In some embodiments, the duration of autohydrolysis pretreatment is between about lmin and 60 min, for example, about 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 1 1 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, or 60 min. In some embodiments, the duration of

autohydrolysis treatment is between about 1 hour and 24 hours, for example, about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0098] In some embodiments, pretreatment can also include disruption or expansion of cellulosic and/or hemicellulosic material. In one embodiment, pretreatment comprises steam explosion, ammonia fiber expansion (or explosion) (AFEX) or another thermal/chemical pretreatment technique. In another embodiment, biomass can be pretreated by liquid hot water treatment (e.g., autohydrolysis) followed by steam explosion.

[0099] 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 hydrolysate 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 carbohydrates (C5 and C6) in the alkali-based pretreatment methods, while glucose is the major product in the hydrolysate from the low and neutral pH methods. The enzymatic digestibility of the residual biomass is somewhat better for the high-pH methods due to the removal of lignin that can interfere with the accessibility of cellulase enzyme to cellulose. 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%o, 50%), 60%o, 70%), 80%o 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.

[00100] 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, malic acid, or oxalic acid) or inorganic (such as nitric, hydrochloric, or sulfuric acid), followed by washing and an alkaline treatment in 0.5 to 2.0%> NaOH. This type of pretreatment can result in a higher percentage of oligomeric to monomeric saccharides, which can be preferentially fermented by a microorganism such as Clostridium phytofermentans, Clostridium, sp. Q.D or a variant thereof.

[00101] 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.

[00102] 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.

[00103] 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.

[00104] 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 microorganism such as a Clostridium biocatalyst such as C. phytofermentans , Clostridium sp. Q.D, Clostridium phytofermentans Q.27, Clostridium phytofermentans Q.13, or a variant thereof.

[00105] 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%.

[00106] 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%.

[00107] 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.

[00108] 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 monomers and dimers.

[00109] 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.

[00110] 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%.

[00111] 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 1% to 2%.

[00112] 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.27, Clostridium phytofermentans Q.13 or variants thereof.

[00113] Certain conditions of pretreatment can be modified prior to, or concurrently with, introduction of a fermentation 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.

[00114] 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, each of which is hereby incorporated by reference in its entirety. [00115] 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.

[00116] 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.

[00117] 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.

[00118] 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.

[00119] In some embodiments, 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 described herein 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 0.005 microns 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.

[00120] 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 fermentation 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.27, Clostridium phytofermentans Q.13 or variant thereof.

[00121] The present disclosure also provides a fermentation 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.27 or Clostridium phytofermentans Q.13 or variants thereof. In still other embodiments, the microorganism is genetically modified to enhance activity of one or more hydrolytic enzymes.

[00122] Further provided herein is a fermentation 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.27, Clostridium phytofermentans Q.13, or variants thereof. In other embodiments, the microorganism is genetically modified to express or increase expression of an enzyme capable of hydro lyzing the oligosaccharide, a transporter capable of transporting the oligosaccharide, or a combination thereof.

[00123] Another aspect of the present disclosure provides a fermentation mixture comprising a cellulosic feedstock comprising cellulosic material from one or more sources, wherein the feedstock is pre-treated with a substance that increases the pH to an alkaline level, at a temperature of about 80°C to about 120°C; and a microorganism capable of fermenting the cellulosic material from at least two different sources to produce a fermentation end-product at substantially a same yield coefficient. 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.27 or Clostridium phytofermentans Q.13 or variants thereof.

[00124] 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.

[00125] 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.

[00126] 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.

[00127] 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.

[00128] 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 hydrolyzed with externally added enzymes at their optimal condition, the slurry is then transferred to a separate tank in which the fermentative microorganism such as a Clostridium biocatalyst (e.g.,

Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27 or Clostridium phytofermentans Q.13 or variants thereof) converts the hydrolyzed sugar into the desired product (e.g., fuel or chemical) and completes the hydrolysis of the residual cellulose and hemicellulose.

[00129] 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 such as a Clostridium biocatalyst (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27 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 such as a Clostridium biocatalyst (e.g., Clostridium phytofermentans, Clostridium sp. Q.D, Clostridium phytofermentans Q.27or Clostridium phytofermentans Q.13 or variants thereof).

[00130] 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.

[00131] 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.

[00132] In one embodiment, a process of producing a biofuel 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 biocatalyst (such as Clostridium phytofermentans , a Clostridium sp. Q.D, a Clostridium

phytofermentans Q.13 or a Clostridium phytofermentans Q.27 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 biofuel 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 biofuel.

[00133] 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 -l,4-linkage. The degree of polymerization ranges from 100 to 20,000. Adjacent cellulose molecules are coupled by extensive hydrogen bonds and van der Waals forces, resulting in a parallel alignment. The parallel sheet-like structure renders cellulose very stable.

[00134] 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 materials: 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 lignocellulosic biomass have been published as U.S. Pat. Nos. 4,304,649, 5,366,558, 5,411,603, and 5,705,369.

Biomass Processing

[00135] 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units or more) and or shorter {e.g., 1, 2, 3, 4, 5, 6 monomer units). As used herein, "directly processing" means that a microorganism is capable of both hydrolyzing biomass and fermenting without the need for conditioning the biomass, such as subjecting the biomass to chemical, heat, enzymatic treatment or combinations thereof.

[00136] 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. In some embodiments, fed batch and or staged feeding techniques can be utilized to manage, for example, the carbohydrate balance of the fermentation medium and/or the growth rate of the microorganism or the group of microorganisms.

[00137] Organisms disclosed herein can be incorporated into methods and compositions 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 hydro lyzing and fermenting hexose (C6) and pentose (C5) polysaccharides (e.g., carbohydrates). In addition, C. phytofermentans is capable of acting directly on lignocellulosic biomass without any pretreatment. Other examples of microorganisms that can hydrolyze and ferment hexose (C6) and pentose (C5) polysaccharides include Clostridium sp. Q.D, or variants of Clostridium phytofermentans (e.g., mutagenized or recombinant), such as Clostridium Q.8, Clostridium Q.27, or Clostridium phytofermentans Q.13. Additionally, these organisms can produce hemicellulases, pectinases, xylansases, or chitinases.

[00138] In one embodiment, modified microorganisms are provided which ferment hexose and pentose polysaccharides which are part of a biomass. In some embodiments, a Clostridium hydrolyzes and ferment hexose and pentose polysaccharides which are part of a biomass. In a further embodiment, C. phytofermentans or variants thereof hydrolyze and ferment hexose and pentose polysaccharides which are part of a biomass. In some embodiments, the biomass comprises lignocellulose. In some embodiments, the biomass comprises hemicellulose.

Co-Culture Methods and Compositions

[00139] Methods can also include co-culture with a microorganism that naturally produces or is genetically modified to produce one or more enzymes, such as hydrolytic enzymes (such as cellulase(s), hemicellulase(s), or pectinases etc.) or antioxidants (such as catalase, superoxide dismutase or glutathione peroxidase). A culture medium containing such a microorganism can be contacted with biomass (e.g., in a bioreactor) prior to, concurrent with, or subsequent to contact with a second microorganism. 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. phytofermentans or Clostridium sp. Q.D. Mixtures of microorganisms can be provided as solid mixtures (e.g., freeze-dried mixtures), or as liquid dispersions of the microorganisms, and grown in co-culture with a second microorganism Co-culture methods capable of use are known, such as those disclosed in U.S. Patent Application Publication No. 20070178569, which is hereby incorporated by reference in its entirety.

Fermentation end-product

[00140] 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. [00141] 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 -hydroxybutyro lactone, glycerol, sorbitol, xylitol/arabitol, butanediol, butanol, isopentenyl diphosphate, methane, methanol, ethane, ethene, ethanol, n-propane, 1-propene, 1-propanol, propanal, acetone, propionate, n-butane, 1-butene, 1 -butanol, butanal, butanoate, isobutanal, isobutanol, 2-methylbutanal, 2-methylbutanol, 3-methylbutanal, 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, 1- phenyl-2-butanol, 4-phenyl-2-butanol, l-phenyl-2-butanone, 4-phenyl-2-butanone, l-phenyl-2,3- butandiol, 1 -phenyl-3-hydroxy-2-butanone, 4-phenyl-3-hydroxy-2-butanone, 1 -phenyl-2,3-butanedione, n-pentane, ethylphenol, ethenylphenol, 2-(4-hydroxyphenyl)ethanol, 4-hydroxyphenylacetaldehyde, 1- (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, l-(4- hydroxyphenyl)-2-butanone, 4-(4-hydroxyphenyl)-2-butanone, 1 -(4-hydroxyphenyl)-2,3-butandiol, 1 -(4- hydroxyphenyl)-3-hydroxy-2-butanone, 4-(4-hydroxyphenyl)-3-hydroxy-2-butanone, l-(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- 1 -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-hydroxy-2-pentanone, 4-methyl-2,3- pentanedione, 1-phenylpentane, 1 -phenyl- 1 -pentene, l-phenyl-2-pentene, l-phenyl-3-pentene, 1-phenyl- 2-pentanol, l-phenyl-3-pentanol, l-phenyl-2-pentanone, l-phenyl-3-pentanone, l-phenyl-2,3- pentanediol, l-phenyl-2-hydroxy-3-pentanone, l-phenyl-3-hydroxy-2-pentanone, l-phenyl-2,3- pentanedione, 4-methyl- 1 -phenylpentane, 4-methyl- 1 -phenyl- 1 -pentene, 4-methyl- 1 -phenyl-2-pentene, 4-methyl- 1 -phenyl-3-pentene, 4-methyl- 1 -phenyl-3-pentanol, 4-methyl- 1 -phenyl-2-pentanol, 4-methyl- 1 -phenyl-3-pentanone, 4-methyl- 1 -phenyl-2-pentanone, 4-methyl- 1 -phenyl-2,3-pentanediol, 4-methyl- 1 - phenyl-2,3-pentanedione, 4-methyl-l -phenyl-3-hydroxy-2-pentanone, 4-methyl-l-phenyl-2-hydroxy-3- pentanone, 1 -(4-hydroxyphenyl) pentane, 1 -(4-hydroxyphenyl)- 1 -pentene, l-(4-hydroxyphenyl)-2- pentene, l-(4-hydroxyphenyl)-3-pentene, l-(4-hydroxyphenyl)-2-pentanol, l-(4-hydroxyphenyl)-3- pentanol, l-(4-hydroxyphenyl)-2-pentanone, l-(4-hydroxyphenyl)-3-pentanone, 1 -(4-hydroxyphenyl)- 2,3-pentanediol, 1 -(4-hydroxyphenyl)-2-hydroxy-3-pentanone, 1 -(4-hydroxyphenyl)-3-hydroxy-2- pentanone, l-(4-hydroxyphenyl)-2,3-pentanedione, 4-methyl- 1 -(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-l-(4-hydroxyphenyl)-3-pentanol, 4-methyl- l-(4-hydroxyphenyl)-2-pentanol, 4- methyl- 1 -(4-hydroxyphenyl)-3-pentanone, 4-methyl- 1 -(4-hydroxyphenyl)-2-pentanone, 4-methyl- 1 -(4- hydroxyphenyl)-2,3-pentanediol, 4-methyl-l-(4-hydroxyphenyl)-2,3-pentanedione, 4-methyl- 1 -(4- hydroxyphenyl)-3-hydroxy-2-pentanone, 4-methyl- l-(4-hydroxyphenyl)-2-hydroxy-3-pentanone, 1- indole-3-pentane, l-(indole-3)-l-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, 1 -(indole-3)-2-hydroxy-3-pentanone, 1 -(indole-3)-3-hydroxy-2-pentanone, 1 -(indole-3)-2,3- pentanedione, 4-methyl-l-(indole-3-)pentane, 4-methyl-l-(indole-3)-2-pentene, 4-methyl-l-(indole-3)-3- pentene, 4-methyl-l-(indole-3)-l-pentene, 4-methyl-2-(indole-3)-3-pentanol, 4-methyl- l-(indole-3)-2- pentanol, 4-methyl-l -(indole-3)-3-pentanone, 4-methyl-l-(indole-3)-2-pentanone, 4-methyl- l-(indole-3)-

2.3- pentanediol, 4-methyl-l-(indole-3)-2,3-pentanedione, 4-methyl-l-(indole-3)-3-hydroxy-2-pentanone, 4-methyl-l-(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-hydroxy-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- hydroxy-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-l-phenylhexane, 4-methyl- 1 -phenylhexane, 5-methyl-l - phenyl- 1-hexene, 5-methyl-l-phenyl-2-hexene, 5-methyl-l-phenyl-3-hexene, 4-methyl- 1 -phenyl- 1- hexene, 4-methyl- 1 -phenyl-2-hexene, 4-methyl- 1 -phenyl-3 -hex ene, 5-methyl-l -phenyl-2-hexanol, 5- methyl- 1 -phenyl-3 -hexanol, 4-methyl- 1 -phenyl-2-hexanol, 4-methyl- 1 -phenyl-3 -hexanol, 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-p henyl- 3-hydroxy-2-hexanone, 5-methyl-l-phenyl-2-hydroxy-3-hexanone, 4-methyl- 1 -phenyl-3 -hydroxy-2- hexanone, 4-methyl-l-phenyl-2-hydroxy-3-hexanone, 5-methyl-l-phenyl-2,3-hexanedione, 4-methyl-l- phenyl-2,3-hexanedione, 4-methyl- 1 -(4-hydroxyphenyl)hexane, 5-methyl- 1 -(4-hydroxyphenyl)- 1 - hex ene, 5-methyl- 1 -(4-hydroxyphenyl)-2-hexene, 5-methyl- 1 -(4-hydroxyphenyl)-3-hexene, 4-methyl- 1 - (4-hydroxyphenyl)- 1 -hex ene, 4-methyl- 1 -(4-hydroxyphenyl)-2-hexene, 4-methyl- 1 -(4-hydroxyphenyl)- 3-hexene, 5-methyl- l-(4-hydroxyphenyl)-2-hexanol, 5-methyl-l-(4-hydroxyphenyl)-3-hexanol, 4- methyl- 1 -(4-hydroxyphenyl)-2-hexanol, 4-methyl- 1 -(4-hydroxyphenyl)-3-hexanol, 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-l-(4-hydroxyphenyl)-2,3-hexanediol, 5-methyl- 1 -(4- hydroxyphenyl)-3-hydroxy-2-hexanone, 5-methyl-l-(4-hydroxyphenyl)-2-hydroxy-3-hexanone, 4- methyl-l-(4-hydroxyphenyl)-3-hydroxy-2-hexanone, 4-methyl- 1 -(4-hydroxyp henyl)-2-hydroxy-3- hexanone, 5-methyl-l-(4-hydroxyphenyl)-2,3-hexanedione, 4-methyl-l-(4-hydroxyphenyl)-2,3- hexanedione, 4-methyl- 1 -(indole-3 -)hexane, 5-methyl- 1 -(indole-3)- 1 -hexene, 5-methyl- 1 -(indole-3)-2- hexene, 5-methyl-l -(indole-3)-3-hexene, 4-methyl- 1 -(indole-3)- 1 -hexene, 4-methyl- l-(indole-3)-2- hexene, 4-methyl- l -(indole-3)-3 -hexene, 5-methyl- l-(indole-3)-2-hexanol, 5-methyl-l -(indole-3)-3- hexanol, 4-methyl- l-(indole-3)-2-hexanol, 4-methyl- l-(indole-3)-3-hexanol, 5-methyl- l-(indole-3)-2- hexanone, 5-methyl-l-(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-l-(indole-3)-2,3-hexanediol, 5-methyl-l - (indole-3)-3-hydroxy-2-hexanone, 5-methyl-l-(indole-3)-2-hydroxy-3-hexanone, 4-methyl- l-(indole-3)- 3-hydroxy-2-hexanone, 4-methyl-l-(indole-3)-2-hydroxy-3-hexanone, 5-methyl-l -(indole-3)-2,3- hexanedione, 4-methyl-l-(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-hydroxy-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-hydroxy-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-hydroxy-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, 1-dodecanol, ddodecanal, dodecanoate, 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, cyclop entanone, cyclopentanol, (S)-2-aceto lactate, (R)-2,3-Dihydroxy-isovalerate, 2-oxoisovalerate, isobutyryl-CoA, isobutyrate, isobutyraldehyde, 5- amino pentaldehyde, 1 , 10-diaminodecane, l,10-diamino-5-decene, l,10-diamino-5-hydroxydecane, 1 , 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, 1 ,4-diphenyl-2,3-butanediol, 1 ,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- 1 -butene, 1 -(indole-3)-4-phenyl-2-butene, 1 -(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-hydroxy-2-butanone, 4-hydroxyphenylacetoaldehyde, 1 ,4-di(4- hydroxyphenyl)butane, 1 ,4-di(4-hydroxyphenyl)- 1 -butene, 1 ,4-di(4-hydroxyphenyl)-2-butene, 1 ,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, 1 -(4- hydroxyphenyl)-4-(indole-3)-2,3-butanediol, l-(4-hydroxyphenyl-4-(indole-3)-3-hydroxy-2-butanone, indole-3-acetoaldehyde, 1 ,4-di(indole-3-)butane, 1 ,4-di(indole-3)-l -butene, 1 ,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, 1 ,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, succini acid, malic acid, isoprenoids, polyisoprenes (including rubber), and terpenes. Additional fermentation end products, and methods of production thereof, can be found in U.S. Patent Application US12/969,582, which is herein incorporated by reference in its entirety.

Microorganism

[00142] Microorganisms useful in these compositions and methods 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 cellulolyticum, C. chauvoei, C clostridioforme, C. colicanis, C difficile, C fallax, C. formicaceticum, C. histolyticum, C innocuum, C. Ijungdahlii, C. laramie, C. lavalense, C. novyi, C oedematiens, C. paraputrificum, C. perfringens, C. phytofermentans (including NRRL B-50364 or NRRL B-50351), C. piliforme, C. ramosum, C 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.27 (NRRL B-50498) or C. phytofermentans Q.13) and Clostridium biocatalysts.

[00143] Examples of yeast that can be utilized in co-culture methods described herein include but are not limited to, species found in Cryptococcaceae, Sporobolomycetaceae with the genera Cryptococcus , Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon, Rhodotorula and Sporobolomyces and Bullera, the families Endo- and Saccharomycetaceae, with the genera

Saccharomyces , Debaromyces, Lipomyces, Hansenula, Endomycopsis , Pichia, Hanseniaspora,

Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolitica, Emericella nidulans, Aspergillus nidulans, Deparymyces hansenii and Torulaspora hansenii.

[00144] 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.

[00145] Anaerobic digestion usually takes place in one of three temperature ranges: psychrophilic (less than 20°C), mesophilic (between 20°C and 40°C), and thermophilic (between 40°C and 70°C). In one embodiment, digestion is performed by a psychrophilic microorganism. In another embodiment, digestion is performed by a thermophilic microorganism. In another embodiment, digestion is performed by a mesophilic microorganism. In one embodiment, the microorganism is a naturally mesophilic microorganism. In another embodiment, the microorganism is genetically modified to be a mesophilic microorganism. In one embodiment, carbonaceous byproducts are fermented at a temperature optimal for mesophilic digestion rather than thermophilic digestion. Toxicity from residues of pretreatment is lower and the costs of raising temperatures for fermentation are also reduced. In one embodiment, a mesophilic microorganism digests carbonaceous byproducts obtained from one or more feed streams. In one embodiment, a digester is operated between about 35 - 37°C. In another embodiment, a digester is operated between about 20 - 25°C. In another embodiment, a digester is operated between about 22 - 27°C. In another embodiment, a digester is operated between about 24 - 29°C. In another embodiment, a digester is operated between about 26 - 30°C. In another embodiment, a digester is operated between about 28 - 32°C. In another embodiment, a digester is operated between about 30 - 35°C. In another embodiment, a digester is operated between about 32 - 37°C. In another embodiment, a digester is operated between about 34 - 39°C. In another embodiment, a digester is operated between about 36 - 40°C. In another embodiment, a digester is operated between about 37- 42°C. In another embodiment, a digester is operated at about 32°C. In another embodiment, a digester is operated at about 37°C. In another embodiment, a digester is operated at about 30°C. In another embodiment, a digester is operated at about 28°C.

[00146] In one aspect, a microorganism is genetically modified to robustly metabolize carbonaceous byproducts.

[00147] In another aspect a microorganism is genetically modified to robustly metabolize carbonaceous byproducts at a mesophilic optimal temperature. In another aspect, the genetic modification comprises genetically engineering a naturally mesophilic microorganism. In one embodiment, a mesophilic microorganism is a bacterium In one embodiment, a mesophilic microorganism is a species of

Clostridium. In one embodiment the species of Clostridium is Clostridium phytofermentans or

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

[00148] In one embodiment, a microorganism is an isolated Gram-positive bacterium, wherein the bacterium is an obligate anaerobic, mesophilic, cellulolytic organism. In another embodiment, a microorganism is an isolated Gram-positive bacterium that produces colonies that are beige pigmented, wherein the bacterium can use polysaccharides as a sole carbon source and can oxidize glucose into one or more fermentation end-products, such as ethanol or one or more organic acids.

[00149] In one embodiment, a microorganism is Clostridium sp. Q.D, having the NRRL patent deposit designation NRRL B-50361. Clostridium sp. Q.D consists of motile rods that form terminal spores. These Gram-positive rods were isolated from a 0.3% Maltose, 5% Azo-CM-Cellulose, QM plate comprising a mutated pool of Clostridium phytofermentans and were cultured in liquid QM media. Endoglucases activity was noted on the plate after four days' incubation at 35° C. The Clostridium sp. Q.D bacterium was distinguishable from Clostridium phytofermentans in that Q.D is a faster-growing colony of larger size having a larger clearing zone in the presence of glucose and 5% Azo-CM-Cellulose plates. It also displays a color modification in the presence of higher concentrations of glucose (2-3% glucose), changing to an orange color. Percent identity values for Q.D bacterium compared with representative members of the Clostridium sp. ranged from 99.8%> with Clostridium xylanolyticum to 99.7%) with Clostridium algidixylanolyticum. Also C. xylanolyticum has terminal endospores whereas C. algidixylanolyticum has subterminal endospores.

[00150] In another embodiment, a microorganism is an obligate anaerobic mesophile that can ferment carbonaceous byproducts into ethanol, organic acids or another fermentation end-product. In another embodiment, the mesophile degrades cellulose and/or xylose into ethanol and acetic or lactic acid.

[00151] In one embodiment, a mesophilic microorganism is C. phytofermentans, which includes American Type Culture Collection 700394T. In one embodiment, a C. phytofermentans carries the phenotypic and genotypic characteristics of a cultured strain, ISDgT (Warnick et al., International Journal of Systematic and Evolutionary Microbiology, 52:1155-60, 2002). In another embodiment, a C. phytofermentans is a strain derived from ISDgT or another species of Clostridium phytofermentans . In one embodiment, the derivation is genetic modification or mutagenesis. In another embodiment, the derivation is isolation from nature.

[00152] Some exemplary species useful for processes described herein are defined by standard taxonomic considerations (Stackebrandt and Goebel, International Journal of Systematic Bacteriology, 44:846-9, 1994): Strains with 16S rRNA sequence homology values of 97% and higher as compared to the type strain (ISDgT), and strains with DNA re-association values of at least about 70% can be considered Clostridium phytofermentans. Considerable evidence exists to indicate that many microbes which have 70% or greater DNA re-association values also have at least 96%> DNA sequence identity and share phenotypic traits defining a species. Analyses of the genome sequence of Clostridium phytofermentans strain ISDgT indicate the presence of large numbers of genes and genetic loci that are likely to be involved in mechanisms and pathways for plant polysaccharide fermentation, giving rise to the unusual fermentation properties of this microbe which can be found in all or nearly all strains of the species Clostridium phytofermentans.

[00153] C. phytofermentans and Clostridium sp. Q.D provide useful advantages for the conversion of carbonaceous byproducts to ethanol and other products. In one embodiment, the C. phytofermentans employed in a CBP process produce enzymes capable of hydrolyzing polysaccharides and higher saccharides to lower molecular weight saccharides, oligosaccharides, disaccharides, and

monosaccharides. In another embodiment, C. phytofermentans or Clostridium sp. Q.D employed in processes described herein produces a wide spectrum of hydrolytic enzymes capable of facilitating fermentation of various biomass materials, including cellulosic, hemicellulosic, hgnocellulosic materials; pectins; starches; wood; paper; agricultural products; forest waste; tree waste; tree bark; leaves; grasses; sawgrass; woody plant matter; non-woody plant matter; algae; carbohydrates; pectin; starch; inulin; fructans; glucans; corn; sugar cane; energy cane; milo, grasses; bamboo, and byproduct material derived from these materials. The organism can usually produce these enzymes as needed, frequently without excessive production of unnecessary hydrolytic enzymes. In some embodiments, one or more enzymes are added to further improve the organism's production capability.

[00154] Various fermentation conditions can enhance the activities of the organism, resulting in higher yields, higher productivity, greater product selectivity, and/or greater conversion efficiency. In some embodiments, fermentation conditions can include fed batch operation and fed batch operation with cell augmentation; addition of complex nitrogen sources such as corn steep powder or yeast extract; addition of specific amino acids including proline, glycine, isoleucine, and/or histidine; addition of a complex material containing one or more of these amino acids; addition of other nutrients or other compounds such as phytate, proteases enzymes, or polysaccharase enzymes. In some embodiments, the addition of one material provides supplements that fit into more than one category, such as providing amino acids and phytate.

[00155] In some embodiments, C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze various higher saccharides present in biomass to lower saccharides, such as in preparation for fermentation to produce ethanol, hydrogen, or other chemicals such as organic acids including formic acid, acetic acid, and lactic acid. In another embodiment, C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze polysaccharides and higher saccharides such as hexose saccharides. In another embodiment, C.

phytofermentans is used to hydrolyze polysaccharides and higher saccharides such as pentose saccharides. In another embodiment, C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze polysaccharides and higher saccharides that contain both hexose and pentose sugar units. In another embodiment, C. phytofermentans or Clostridium sp. Q.D is used to hydrolyze polysaccharides and higher saccharides into lower saccharides or monosaccharides. In another embodiment, hydrolysate from C. phytofermentans or Clostridium sp. Q.D treatment is used in a fermentation process to produce one or more fermentation end-products such as a biofuels. In another embodiment, C. phytofermentans or Clostridium sp. Q.D is used to produce ethanol, hydrogen, or compounds such as organic acids including acetic acid, formic acid, and lactic acid from a lower sugar such as monosaccharide or a disaccharide. In another embodiment, C. phytofermentans or Clostridium sp. Q.D is used to perform the combined steps of hydrolyzing a higher molecular weight biomass containing sugars and/or higher saccharides or polysaccharides to lower sugars and fermenting these lower sugars into desirable products including ethanol, hydrogen, and other compounds such as organic acids including formic acid, acetic acid, and lactic acid, or other fermentation end products.

[00156] In one embodiment, C. phytofermentans, Clostridium sp. Q.D, or any variant thereof is used in a CBP process to grow under conditions that include elevated ethanol concentration, high sugar concentration, or low sugar concentration. In another embodiment, C. phytofermentans, Clostridium sp. Q.D, or any variant thereof is used in a process described herein to utilize insoluble carbon sources and/or operate under anaerobic conditions. In another embodiment, C. phytofermentans or Clostridium sp. Q.D is used in a process described herein to achieve operation with long fermentation cycles. In another embodiment, the microbe is used in combination with batch fermentations, fed batch

fermentations, or self-seeding/partial harvest fermentations. In another embodiment, the microbe is recycled from the final fermentation as inoculum.

[00157] In one embodiment, a mesophilic bacterium useful for processes described herein is a species of Clostridium. In another embodiment, a mesophilic bacterium is a species of Bacillus. Examples of Bacillus species useful for processes described herein include, but are not limited to, Bacillus subtilis, Bacillus alvei, Bacillus amylolyticus, Bacillus azotofixans, Bacillus glucanolyticus, Bacillus larvae, Bacillus lautus, Bacillus lentimorbus, Bacillus macerans, Bacillus macquariensis, Bacillus pabuli, Bacillus polymyxa, Bacillus popilliae, Bacillus psychrosaccharolyticus , Bacillus pulvifaciens , Bacillus thiaminolyticus, Bacillus avlidus, Bacillus alcalophilus, Bacillus amyloiquefaciens, Bacillus atrophaeus, Bacillus carotarum, Bacillus firmus, Bacillus flexus, Bacillus laterosporus , Bacillus megaterium, Bacillusmycoides, Bacillus niacini, Bacillus pantothenticus, Bacillus pumilus, Bacillus simplex, Bacillus thuringiensis , Bacillus sphaericus, Bacillus anthracis, Bacillus azotoformans , Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus lentus, Bacillus lichemformis, Bacillus megaterium, Bacillus natto, Bacillus stearothermophilus, Bacillus halodurans, and Bacillus pallidus. In another embodiment, a mesophilic bacterium is a species of Oceanobacillus iheyensis. In another embodiment, a mesophilic bacterium is a species of Lactococcus lactis, Bifidobacterium LAFTB94, Lactobaccillus acidophilus, Lactobaccillus acidophilus LAFTI L10, Lactobaccillus casei, Lactobaccillus casei LAFTI L26, Bifidobacterium animalis subsp. Bifidobacterium lactis, Bifidobacterium lactis BB-12, Bifidobacterium lactis HN019, Bifidobacterium breve, Bifidobacterium breve Yakult, Bifidobacterium infantis Bifidobacterium, Bifidobacterium infantis 35624, Bifidobacterium longum, Bifidobacterium longum BB536, Bifidobacterium bifidum BB012, E. coli M-17, E. coli Nissle 1917, Baccillus coagulans, and Streptococcus thermophilus, Lactobaccillus acidophilus DDS-1, Lactobaccillus acidophilus LA-5, Lactobaccillus acidophilus NCFM, Lactobaccillus acidophilus NCFM, Lactobaccillus acidophilus CD 1285, Lactobaccillus casei 431, Lactobaccillus casei F19, Lactobaccillus casei Shirota, Lactobaccillus paracasei, Lactobaccillus paracasei Stl 1, Lactobaccillus johnsonii, Lactobaccillus johnsonii Lai, Lactobaccillus lactis, Lactobaccillus lactis L1A, Lactobaccillus plantarum, Lactobaccillus plantarum 299v, Lactobaccillus reuteri, Lactobaccillus reuteri ATTC 55730, Lactobaccillus rhamnosus,

Lactobaccillus rhamnosus ATCC 53013, Lactobaccillus rhamnosus LB21, Lactobaccillus rhamnosus GR-1, Lactobaccillus rhamnosus RC-14, Lactobaccillus reuteri RC014, Lactobaccillus rhamnosus R011, Lactobaccillus helveticus, and Lactobaccillus helveticus R0052.

Modification to Alter Enzyme Activity

[00158] In various embodiments, one or more modification of conditions for hydrolysis and/or fermentation is implemented to enhance end-product production. Examples of such modifications include genetic modification to enhance enzyme activity in a microorganism that already comprises genes for encoding one or more target enzymes, introducing one or more heterogeneous nucleic acid molecules into a host microorganism to express and enhance activity of an enzyme not otherwise expressed in the host, genetic modifications to disrupt the expression of one or more metabolic pathway genes to direct, modifying physical and chemical conditions to enhance enzyme function (e.g., modifying and/or maintaining a certain temperature, pH, nutrient concentration, temporal), or a combination of one or more such modifications. Other embodiments include overexpression of an endogenous nucleic acid molecule into the host microorganism to express and enhance activity of an enzyme already expressed in the host or to express activity of an enzyme in the host when the enzyme would not normally be expressed in the naturally- occurring host microorganism.

Genetic modification of microorganisms

[00159] For the fermentation described herein, any microbes capable of forming a biofilm can be used. In one embodiment two or more genetically distinct microorganisms can be used to form a biofilm. In another embodiment a naturally occurring microorganism can be introduced to a broth containing biomass to form a biofilm with genetically modified microorganisms. In another embodiment a two or more genetically modified microorganisms can be used to form a biofilm.

[00160] Clostridium phytofermentans isolates can be obtained without the use of recombinant DNA techniques that exhibit desirable properties such as increased productivity, increased yield, or increased titer. For example, mutagenesis, or random mutagenesis can be performed by chemical means or by irradiation of the microorganism. The population of mutagenized microorganisms can then be screened for beneficial mutations that exhibit one or more desirable properties. Screening can be performed by growing the mutagenized microorganisms on substrates that comprise carbon sources that will be utilized during the generation of end-products by fermentation. Screening can also include measuring the production of end-products during growth of the microorganism, or measuring the digestion or assimilation of the carbon source(s). The isolates so obtained can further be transformed with

recombinant polynucleotides or used in combination with any of the methods and compositions provided herein to further enhance biofuel production.

[00161] In another aspect, the present disclosure provides compositions and methods to produce a fuel such as one or more alcohols, e.g., ethanol, by the creation and use of a genetically modified Clostridium phytofermentans. This disclosure contemplates, in particular, regulating fermentative biochemical pathways, expression of saccharolytic enzymes, or increasing tolerance of environmental conditions during fermentation of a Clostridium phytofermentans. In one embodiment, the Clostridium

phytofermentans is transformed with polynucleotides encoding one or more genes for the pathway, enzyme, or protein of interest. In another embodiment, the Clostridium phytofermentans is transformed to produce multiple copies of one or more genes for the pathway, enzyme, or protein of interest. In some cases, the polynucleotide transformed into the Clostridium phytofermentans is heterologous. In other cases, the polynucleotide is derived from Clostridium phytofermentans. In one embodiment, the

Clostridium phytofermentans is transformed with heterologous polynucleotides encoding one or more genes encoding enzymes for the fermentation of a hexose, wherein the genes are expressed at sufficient levels to confer upon the Clostridium phytofermentans transformant the ability to produce ethanol at increased concentrations, productivity levels or yields compared to a Clostridium phytofermentans that is not transformed. In another embodiment, the Clostridium phytofermentans is transformed with heterologous polynucleotides encoding one or more genes encoding enzymes for the fermentation of a pentose, wherein the genes are expressed at sufficient levels to confer upon the Clostridium

phytofermentans transformant the ability to produce ethanol at increased concentrations, productivity levels or yields compared to a Clostridium phytofermentans that is not transformed. In still other embodiments, the Clostridium phytofermentans is transformed with a combination of enzymes for fermentation of hexose and pentose saccharides. In such ways, an enhanced rate of ethanol production can be achieved.

[00162] In one embodiment, the Clostridium phytofermentans is transformed with heterologous polynucleotides encoding one or more genes encoding saccharolytic enzymes for the saccharification of a polysaccharide, wherein the genes are expressed at sufficient levels to confer upon the Clostridium phytofermentans transformant the ability to saccharify a polysaccharide to mono-, di- or oligosaccharides at increased concentrations, rates of saccharification or yields of mono-, di- or oligosaccharides compared to a Clostridium phytofermentans that is not transformed. The production of a saccharolytic enzyme by the host, and the subsequent release of that saccharolytic enzyme into the medium, reduces the amount of commercial enzyme necessary to degrade biomass or polysaccharides into fermentable monosaccharides and oligosaccharides. The saccharolytic DNA can be native to the host, although more often the DNA will be foreign, i.e., heterologous. Advantageous saccharolytic genes include cellulolytic, xylano lytic, and starch- degrading enzymes such as cellulases, xylanases, glucanases, glucosidases, and amylases. The saccharolytic enzymes can be at least partially secreted by the host, or it can be accumulated substantially intracellularly for subsequent release. Advantageously, intracellularly-accumulated enzymes which are thermostable can be released when desired by heat-induced lysis. Combinations of enzymes can be encoded by the heterologous DNA, some of which are secreted, and some of which are accumulated.

[00163] Other modifications can be made to enhance the ethanol production of the recombinant bacteria. For example, the host can further comprise an additional heterologous DNA segment, the expression product of which is a protein involved in the transport of mono- and/or oligosaccharides into the recombinant host. 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.

[00164] In order to improve the production of fermentation end-products such as biofuels (e.g., ethanol), modifications can be made in transcriptional regulators, genes for the formation of organic acids, carbohydrate transporter genes, sporulation genes, genes that influence the formation/regenerate of enzymatic cofactors, genes that influence ethanol tolerance, genes that influence salt tolerance, genes that influence growth rate, genes that influence oxygen tolerance, genes that influence catabolite repression, genes that influence hydrogen production, genes that influence resistance to heavy metals, genes that influence resistance to acids or genes that influence resistance to aldehydes.

[00165] Those skilled in the art will appreciate that a number of 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 the recombinant Clostridium phytofermentans host. 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 to promote homologous recombination.

[00166] Various microorganisms can be modified to enhance activity of one or more cellulases, or enzymes associated with cellulose processing {e.g., FIG. 1). The classification of cellulases is usually based on grouping enzymes together that forms a family with similar or identical activity, but not necessary the same substrate specificity. One of these classifications is the CAZY system (CAZY stands for Carbohydrate- Active enZymes), for example, where there are 115 different Glycoside Hydrolases (GH) listed, named GH1 to GH155. Each of the different protein families usually has a corresponding enzyme activity. This database includes both cellulose and hemicellulase active enzymes. Furthermore, the entire annotated genome of C. phytofermentans is available on the worldwideweb at

www. ncbi.nlm. nih. gov/ sites/ entrez.

[00167] Attachment to cellulosic substrate, or any biomass, can be facilitated by genetic modification. Polypeptides contemplated to be utilized for facilitating attachment process include, but not limited to, DUF291 of Cphy3367. DUF291 has two, tandem C-terminal domains. The function of these domains is unknown. But these domains are found in other C. phy. genes, such as Cphy2128 (GH26), Cphy3202 (GH5) and Cphy3368 (GH48). Other domains homologous to DUF291are found in the scaffoldin proteins of various Clostridia species, such as C. cellulolyticum (CipC), C. cellulovorans (CbpA) and C. josui (CipA). Homologous domains are found in the hydrolases as well, e.g., CelY and CelZ from C.

stercorarium. The CbpA DUF291 domains are thought to be capable of binding both cellulose and the cell wall. Thus, it has been proposed as an anchor protein attaching the cell wall to cellulose. The outcome of anchoring is facilitation of biofilm formation, resulting in increased cellulose degradation. Thus, the DUF291 domains of Cphy3367 can also play a role in attaching Cphy3367 to the cell surface.

Biofilms

[00168] Industrial fermentations are generally performed in vessels outfitting to control process parameters such as pH, oxygen levels, nutrient availability, and temperature control. [00169] Batch additions of pH control chemicals, nutrients or gasses, as well as temperature control, generally utilize agitation or mixing and cultures are 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.

[00170] When fermenting insoluble carbohydrate sources such as biomass (e.g., cellulosic, hemi- cellulosic, starch based), microbes 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. This allows them to reduce the diffusion of sugars away from their own cellular transport machinery and effectively increase the local concentration. In one embodiment, a microorganism that reduces diffusion of sugars can increase growth or productivity by optimizing sugar uptake rates and minimizing the energy needed to degrade the substrate. In one embodiment a microorganism that does not form a biofilm produces more enzymes to achieve acceptable soluble sugar levels to support its growth.

[00171] In one embodiment a microorganism that forms a biofilm has an advantage over a

microorganism that does not because its enzyme kinetics is optimized by immediate contact with the substrate. In one embodiment a bioreactor is designed for continuous and/or vigorous mixing. In another embodiment a bioreactor comprising a microorganism that forms a biofilm employs static fermentation or agitation that avoids disrupting the biofilm. In one embodiment the agitation is low sheer agitation.

[00172] 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 microbe cultures, 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).

[00173] 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; Clostridium phytofermentans Q.27 (NRRL B-50498), deposited on April 28, 2011; and Q.32 (NRRL B-50511), Q.33 (NRRL B-50512), Q.8 I2 C10 (NRRL B-50447), Q.8 I2 C11 (NRRL B-50448), Q.8 I2 C12 (NRRL B-50449), and Q.8 I2 H9 (NRRL B-50450). 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.

[00174] In addition to naturally formed biofilms (via flocculation or aggregation), biofilms can be formed on a supporting material. Supporting material can be porous, non-porous, biodegradable, non-degradable, digestible by the microbe growing on the supporting material, or an absorbent material attracting microbes. Microbes 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 microbes are attracted to the supporting material. The attraction can occur, for example, via chemotaxis. There can be no attraction, but microbes can begin to attach to the supporting material, without being bound by theory, for example, via Brownian motion.

[00175] In one embodiment a fermentation end product is produced with a microorganism that forms a biofilm. Biofilms can be formed of a single microbial species. Biofilms 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 thermosaccharolyticum and Thermoanaerobacterium saccharolyticum. [00176] Biofilms contemplated or described herein can be used in various types of bioreactors. Examples of bioreactor designs compatible with biofilms 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). Various configurations of these reactor designs are compatible and useful for biofilms described herein.

[00177] Biofilms contemplated or described herein can be used -with various types of impellers.

Examples of impellers include, but are not limited to, impellers creating radial flow, such as Rushton impeller, or impellers creating vertical flow. In one embodiment, the impeller is a helical impeller.

[00178] Utilizing methods and compositions described herein, a bioreactor can process a high percentage of solid biomass. Examples of the source of solid biomass include, but 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). In one embodiment, the solid biomass is about 20% of total weight of mixture comprising water (or medium) and C. phytofermentans inoculum. 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

[00179] Various embodiments of the present disclosure offer benefits relating to improving the titer and/or productivity of fermentation end-product production by microorganisms, such as Clostridium phytofermentans, Clostridium algidixylanolyticum or Clostridium xylanolyticum, by culturing the microorganism in a medium comprising one or more compounds comprising hexose and/or pentose sugars. In some embodiments, the process comprises conversion of the starting material to a biofuel, such as one or more alcohols. In one embodiment, methods disclosed herein can comprise contacting one or more substrates comprising both hexose (e.g., glucose, cellobiose) and pentose (e.g., xylose, arabinose) saccharides with C. phytofermentans to produce ethanol.

[00180] In some embodiments, batch fermentation with Clostridium phytofermentans of a mixture of hexose and pentose saccharides using the methods disclosed herein provides uptake rates of about 0.1, 0.2, 0.4, 0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or about 6 g/L/h or more of hexose (e.g., glucose, cellulose, cellobiose etc.), and about 0.1, 0.2, 0.4, 0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or about 6 g/L/h or more of pentose (xylose, xylan, hemicellulose etc.). 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 disclosure 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 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 disclosed herein can be due to the simultaneous fermentation of hexose and pentose saccharides.

[00181] Several factors can influence production of high levels of alcohol from biomass: the ability for the microorganism to thrive generally in the presence of elevated alcohol levels; the ability 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.

[00182] In some embodiments, a strain of Clostridium phytofermentans is able to attain an ethanol concentration of at least about 15 g/L after about 36 - 48 hours of batch fermentation, with carbon substrate remaining in the broth. In one embodiment, lowering the fermentation pH to about 6.5 and/or adding unsaturated fatty acids results 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 in the broth following a 48 to 72 to 96 - hours or longer fermentation. In yet another embodiment, the productivity of the microorganism is higher (to about 10 g/L-d) when the ethanol titer is 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 may 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 embodiments, the simultaneous fermentation of hexose and pentose carbohydrate substrates is in combination with fermentation at reduced pH and/or with the addition of fatty acids to further increase productivity, and/or yield.

[00183] 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. 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.

[00184] Forms of agitations useful for avoiding disruption of the biofilm 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 biofilm is contained; and agitating with impeller blade having a unique shape or blade having unique angle to provide low sheer agitation.

[00185] 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

[00186] A static fermentation is achieved, for example, by flocculating microorganisms with or without flocculent, depending on the microbe'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

[00187] 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 microbes 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 microenvironment between the biomass and the cell wall of microbe 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.

[00188] Molecules diffusing out of the barrier can also be further processed by exogenously adding enzymes to a culture containing biofilm. Any enzymes capable of breaking down carbohydrates are useful for exogenously added to a culture containing biofilm. Examples of enzymes include, but are not limited to, cellulases, hemicellulases, beta-galactosidases, glycosyl hydrolase family 9 enzymes (GH9) such as ABX43720 of Cphy, endoglucanases, cellobiohydrolases, chitinases and endo-processive cellulases.

Recovery of Ethanol or other fermentation end-products

[00189] In some cases, to achieve significant titers of fermentation end-products, a fermentation vessel can include a significant amount of carbohydrate content derived from a biomass {e.g., pretreated hydrolyzed lignocellulosic feedstock slurry). For example, about 150 grams per liter to 350 grams per liter of insoluble solids can be present in a fermentation broth/slurry. At such high concentrations, kinematic and static viscosities can become substantially high so as to cause difficulty with mixing via standard rotating agitation pitched blade impellers. Such high viscosities can decrease the lifetime of mixing equipment, leading to increases in manufacturing costs and considerable downtime. In addition, fermentations utilizing Clostridium phytofermentans and similar microorganisms typically require relatively low shear so as to maintain microorganism, biofilm, and enzymes expressed from the microorganism in contact with the biomass. Accordingly, standard mixing equipment might not be desirable in certain cases.

[00190] In one aspect, methods are provided for achieving low shear mixing and/or mixing under high viscosity conditions. In one embodiment, a method comprises utilizing gas bubbles rising due to buoyancy forces (Archimedes principle) and bubble encapsulation due to surface tension. As the bubbles rise due to buoyancy, they provide a motive force to cause a fermentation broth/slurry in a fermentation vessel to rise with the bubbles, and an array of such bubbles can cause a significant amount of low shear mixing to occur and material to be turned over inside the fermentation vessel, evenly distributing solids and minimizing chemical concentration, temperature, and/or pH gradients.

[00191] In one embodiment, gas bubbles are directed to a fermentation vessel during formation (or production) of one or more fermentation end-products. In another embodiment, gas bubbles are directed to a fermentation vessel with the aid of a gas distribution member in fluid communication with the fermentation vessel. In another embodiment, gas bubbles are directed through a fermentation vessel with the aid of a gas distribution member. The gas distribution member can be a blower or sparger. In another embodiment, gas bubbles are injected into the bottom portion of a fermentation vessel. In another embodiment, to inject the bubbles into the bottom of the fermentation vessel with a slurry present, a blower discharge is connected to a sparger with holes on the bottom side to release the bubbles into the slurry. The blower suction is connected to the head space, and a balancing valve is utilized on the blower discharge to control gas pressures and volumes as the fermentation results in additional gas production.

[00192] In one embodiment, mixing of a slurry having one or more microorganisms and biomass is facilitated by a gas or vapor comprising 0 ₂ , CO ₂ , CO, N ₂ , H ₂ , He, Ne, Ar, NO ₂ , or a combination thereof. In another embodiment, mixing of the slurry is facilitated by de-oxygenated air. In another embodiment, mixing of a slurry having one or more microorganisms and biomass is facilitated by CO ₂ . The pressure and flow rate of the gas or vapor can be selected to aid in producing one or more fermentation end- products at a desirable rate of production. For example, the flow rate of the gas or vapor (e.g., CO ₂ ) into a fermentation vessel can be selected to affect a desirable rate of production of ethanol. In some embodiments, slurry mixing can be provided by an impeller in a fermentation vessel and gas bubbles provided to the fermentation vessel. The rotation speed of the impeller can be selected to aid in providing low shear mixing.

[00193] In one embodiment, a method for promoting mixing is to utilize both gas bubbles and a pitched blade agitator or impeller rotating at relatively low speeds. Due to the possible dewatering of the slurry from differential pressure forces being stronger than the capillary surface tension of the liquid, and the subsequent "stove pipe" effect of bubbles rising through a column of water in the slurry and a subsequent lack of mixing, low speed agitation can be required to eliminate such "stove pipes" and enable the bubbles to continue to provide a motive force for mixing.

[00194] In one embodiment, in addition to causing mixing, gas bubbles can allow for fermentation end- products (e.g., alcohols, e.g., methanol, ethanol, propanol, butanol, etc.) to diffuse across the surface of the bubble into the gas space, and evaporate into the gas. When the bubbles reach the surface, the gases are released, along with the diffused and evaporated fermentation end-products, into the head space. With the blower taking suction on the head space, the gases can then routed through a condenser (or cooler), which can cool the gases and condense the fermentation end-products. The gases can be routed to either a balancing line through a balancing valve or back to the sparger in the bottom of fermentation vessel, and the fermentation end-products can be drained by gravity to a liquid discharge lien from the condenser. The liquid effluent can be provided to a separation system located downstream from the fermentation system. Such separation system can include a distillation column, for example. This can result in stripping a significant amount of one or more fermentation end-products out of the fermentation broth. Stripping fermentation end-products from the fermenter can allow higher amounts of the fermentation end-products to be produced. Increased production of fermentation end-products can be due to maintaining a concentration of the fermentation end-products in the fermenter at a concentration that is below an inhibitory concentration.

[00195] In one embodiment, a system is provided for separating one or more fermentation end-products from a broth comprising the fermentation end-products, the broth disposed in a fermentation vessel ("fermenter"). The fermentation end-products can be recycled to the fermenter. The system can comprise one or more unit operations for recovering the fermentation end-products. In one embodiment, the system can include the fermentation vessel (or reactor) and one or more of a separation vessel, such as a distillation column, absorption column, a heat exchanger (e.g., condenser), a pump and a compressor. In another embodiment, the system can include a fermentation vessel, one or more condensers and one or more compressors. The condenser can be configured to convert a vapor or vapor-containing mixture into a liquid or liquid-containing mixture. The compressor can be configured to raise a pressure of a compressible fluid from a first pressure to a second pressure, the second pressure being larger than the first pressure. In another embodiment, in addition to various unit operations, the system can include valves, such as throttle valves, and a control system to regulate the flow rate of fermentation products and by-products.

[00196] In one embodiment, a stripping gas is provided to a fermenter to aid in the removal of one or more fermentation end-products from the fermenter. A mixture of stripping gas and fermentation gas can be directed to a condenser, which condenses at least a portion of the fermentation end-products. The stripping gas can be recycled to the fermenter. In some embodiments, at least one fermentation end- product has a boiling point lower than water.

[00197] In one embodiment, at some point during of the fermentation, broth can be harvested and the final desired fermentation end-product or products will be recovered. The broth with the fermentation end- products to be recovered can include both the fermentation end-products and impurities. The impurities can 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, which can result in additional impurities in the broth.

[00198] In one embodiment, 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.

[00199] With reference to FIG. 5A, an exemplary fermentation end-product recovery system (also "recovery system" herein) is shown. The recovery system includes a first heat exchanger downstream from a fermentation vessel ("fermenter", as illustrated), a compressor downstream from the first condenser, and a second heat exchanger downstream from the compressor. The exemplary recovery system further includes a sterile filter between the second condenser and the fermenter. In the illustrated embodiment, the first and second heat exchangers are condensers. In other embodiments, the first and second heat exchangers can facilitate heat exchange between two or more fluids or gasses without producing a phase change in the fluids or gasses.

[00200] With continued reference to FIG. 5A, a fermentation vessel ("fermenter") is provided having a broth comprising one or more fermentation end-products. The fermenter can comprise a headspace filled with a gas located above the broth. The gas in the headspace can comprise a portion of the fermentation end-products in a vapor phase. The gas in the headspace can comprise water vapor. Gas from the top of the fermenter can be directed through the first heat exchanger (or condenser), which lowers the temperature of the gas and can condense some water and the fermentation end-products (e.g., one or more alcohols, e.g., ethanol, methanol, butanol, propanol, etc.) into a liquid phase, after which the liquid phase can be removed from the gas stream. Following the first heat exchanger, the gas can be directed through a compressor, e.g., in order to increase the pressure of the gas. The compressed gas can subsequently be directed through the second heat exchanger (or condenser). In one embodiment, the second heat exchanger is optional. In another embodiment, the location of the condensers can be selected so as to strike a balance between proximity to the fermenter and the losses associated with transporting the gas to the condensers. In one embodiment, some of the gas leaving the second condenser can be purged. This purge rate could be determined by the pressure that will be maintained in the fermenter.

[00201] Next, after the compressor (and second condenser) the gas can be directed through a sterile filter and subsequently recycled to the fermenter. The sterile filter can be configured to maintain the sterility of the fermenter. In one embodiment, sterile feed, water, pH regulating agents, antifoam agents and surfactants can be supplied to the fermenter as the fermentation proceeds. The broth can be drawn from the bottom of the fermenter, either in batch mode or continuous mode. In one embodiment, the broth can be drawn from the fermenter under sterile conditions.

[00202] In one embodiment, the fermenter can include one tank (or vessel) or several tanks in series, parallel, or a combination of series and parallel arrangements.

[00203] Mixing in the fermenter can accomplished by recycling at least a portion of the gas generated in the fermenter from the top of the fermenter to a sparge ring at the bottom of the fermenter. In one embodiment, the sparge ring is designed (or configured) to provide mixing in the fermenter. The superficial velocity of the gas in the fermenter can be sufficient to supply mixing energy to the fermenter. In one embodiment, as the gas is recycled through the fermenter, a portion of one or more fermentation end-products (e.g., one or more alcohols, e.g., methanol, ethanol, propanol, butanol, etc.) and other substances, such as water, can evaporate into the gas from which the fermentation end-products can be recovered. In one embodiment, the fermentation end-products include any fermentation end-products provided herein. In another embodiment, the fermentation end-products comprise one or more alcohols. In one embodiment, the alcohols comprise methanol, ethanol, propanol, butanol, or a combination thereof.

[00204] In one embodiment, for a specific superficial velocity of gas, the volumetric flow rate of the gas per volume of fermentation broth depends upon the height of the liquid in the fermenter. This gas volumetric flow rate can be referred to as volume of gas per minute per volume of fermentation broth ("wm"). For bubble columns, the wm can be between about 0.4 and 9. For production fermenters having heights between about 10 and 20 meters, the wm can be between about 0.5 to 2. In one embodiment, a fermenter is provided having a wm value of at least about 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20.

[00205] In one embodiment, the stripping capability of the recycle gas depends proportionally on the wm. The rate of ethanol removal (and water) by the stripping gas can be directly proportional to the wm; for example, if the wm is 3 for a specific fermenter, the rate of ethanol removal can be three times that of a fermenter wherein the wm is 1. [00206] With reference to FIG. 5A, in one embodiment, gas is recycled through the fermenter. Gas or vapor from a top portion of the fermenter passes through the first heat exchanger, which lowers the temperature of the gas and condenses some water and ethanol, which is removed from the gas stream.

[00207] In another aspect of the invention, a recovery system is provided that is operated at pressures lower than atmospheric pressure. The recovery system can include a compressor, which can be used to create a vacuum and provide mixing.

[00208] With reference to FIG. 5B, a recovery system is illustrated having a fermenter, a compressor downstream from the fermenter, and a heat exchanger downstream from the compressor. The heat exchanger of the illustrated embodiment is a condenser. The system further includes a flowmeter downstream from the condenser and between the condenser and a sterile filter.

[00209] With continued reference to FIG. 5B, the compressor can be designed to provide a pressure drop larger than necessary to overcome one or more pressure drops in the lines at the desired gas flow rates, and also to overcome the static head in the fermenter. The compressor can also be designed to provide a gas flow rate sufficient for mixing. In one embodiment, gas comprising one or more fermentation end- products (e.g., one or more alcohols, e.g., methanol, ethanol, propanol, butanol, etc.) is directed from the fermenter to the compressor, which is located downstream from the fermenter. The compressor can raise the pressure of the gas comprising the fermentation end-products from a first pressure to a second pressure. The gas comprising the fermentation end-products at the second pressure can be subsequently directed to the condenser downstream from the compressor. At least a portion of the fermentation end- products can condense to a liquid phase in the condenser. The portion of the fermentation product in liquid form (condensate) can then be recovered. The condensate can be directed to further processing, such as to a distillation column for further separation.

[00210] In one embodiment, the compressor raises the pressure of the gas comprising the fermentation end-products from below atmospheric pressure to a pressure at or above atmospheric pressure. The gas (or vapor) discharge from the compressor can be at a pressure that is at or above atmospheric pressure. In one embodiment, carbon dioxide and other gases are generated in the fermenter (during fermentation); such gases can be released to the atmosphere to maintain the pressure of the system constant or nearly constant. Pressure in the fermenter can be used to release gas from the fermenter (through "Off Gas Out", as illustrated).

[00211] In one embodiment, the absolute pressure at an inlet of the compressor (e.g., from the fermenter to the compressor) is between about 0 bar and 1 bar; for example, about 0 bar, 0.1 bar, 0.2 bar, 0.3 bar, 0.4 bar, 0.5 bar, 0.6 bar, 0.7 bar, 0.8 bar, 0.9 bar, 1 bar, or more, or any intervening fraction. In another embodiment, the absolute pressure at an outlet of the compressor (e.g., from the compressor to the condenser) is between about 1 bar and 4 bar; for example about 1 bar, 1.1 bar, 1.2 bar, 1.3 bar, 1.4 bar, 1.5 bar, 1.6 bar, 1.7 bar, 1.8 bar, 1.9 bar, 2 bar, 2.1 bar, 2.2 bar, 2.3 bar, 2.4 bar, 2.5 bar, 2.6 bar, 2.7 bar, 2.8 bar, 2.9 bar, 3 bar, 3.1 bar, 3.2 bar, 3.3 bar, 3.4 bar, 3.5 bar, 3.6 bar, 3.7 bar, 3.8 bar, 3.9 bar, 4 bar, or more, or any intervening fraction. [00212] With continued reference to FIG. 5B, the gas flow rate through the fermenter can be measured by a flow meter. The flow meter can control a valve on the inlet gas flow line to the fermenter. The valve can be adjusted by a system configured to maintain the pressure of the recovery system to within certain limits, dependent upon the density and height of the column of liquid contained in the fermentation vessel. The minimum suction pressure going to the compressor can be close to an absolute vacuum (0 psia) and the maximum pressure can be based upon the limitations of the blower and the density and height of the column of liquid (net positive head pressure at the bottom of the vessel).

[00213] In one embodiment, the formation of one or more fermentation end-products (e.g., one or more alcohols, e.g., methanol, ethanol, propanol, butanol, etc.) in a fermenter is accompanied by the formation of carbon dioxide. Gas, comprising the carbon dioxide produced during fermentation and any external or recycled sources, can bubble through the fermenter. The gas bubbles can strip one or more of the fermentation end-products and water from the fermenter as the fermentation end-products and water evaporate into the bubbles. This stripping occurs at the temperature and pressure of the fermenter. The temperature of the fermenter can be selected so as to provide optimum processing conditions. The gas can then be directed to the first heat exchanger (or condenser), where the temperature of the gas is lowered, causing some of the gaseous fermentation product and water to condense. This condensed product can be removed from the gas phase.

[00214] Another exemplary fermentation end-product recovery system is shown in FIG. 9. The recovery system includes a compressor downstream from a fermentation vessel ("fermenter", as illustrated), a heat exchanger downstream from the compressor, and a scrubber downstream from the heat exchanger. The recovery system further includes a sterile filter between the scrubber and the fermenter. In the illustrated embodiment, the heat exchanger is a condenser. The fermentation vessel can be an agitated vessel with associated control systems, such as those provided by DCI, Appache Stainless, or Feldmeier. The scrubber can be a wet scrubber, such as that provided by Mikropul, Eisenmann, or Monroe

Environmental. The filter can be a Donaldson Ultrapure™ R-EG/R-TF or Kaeser Series FST filters. The heat exchanger/condenser can be a tube and shell heat exchanger or a plate and frame heat exchanger, such as those provided by Tranter, ITT Standard, or Paul Mueller Co.

[00215] With continued reference to FIG. 9, a fermentation vessel ("fermenter") is provided having a broth or slurry comprising a biomass and one or more microorganisms that can hydrolyze and/or ferment the biomass to produce one or more fermentation end-products. In one embodiment, the biomass comprises cellulose, hemicellulose, lignocellulose, or a combination thereof. In another embodiment, the biomass comprises C5 (pentose) and/or C6 (hexose) sugars. The fermentation end-products can comprise one or more alcohols, such as methanol, ethanol, propanol, or butanol. The fermentation end-products can be included in a gas over the broth or in liquid form in the broth.

[00216] In some embodiments, integrating scrubbing with the gas mixing (i) facilitates the operation of the scrubber more efficiently at higher pressures, and (ii) permits greater efficiency in the extraction of one or more fermentation end-products (e.g., one or more alcohols, e.g., methanol, ethanol, propanol, butanol, etc.) from the fermenter by a mixing gas. [00217] With reference to FIG. 10, another exemplary fermentation end-product recovery system is shown. The recovery system includes a compressor downstream from a fermentation vessel ("fermenter", as illustrated), a heat exchanger downstream from the compressor, a scrubber downstream from the heat exchanger, and a gas separator downstream of the scrubber. The recovery system further includes a sterile filter between the gas separator and the fermenter. In the illustrated embodiment, the heat exchanger is a condenser.

[00218] With continued reference to FIG. 10, a fermentation vessel ("fermenter") is provided having a broth that can be harvested to form a fermentation end-product. The fermentation product can be included in a gas over the broth or in liquid form in the broth.

[00219] In one embodiment, the control of the gas components of the fermentation product recovery system improves fermentation rate and yield. For example, controlled gas components include, without limitation, carbon dioxide, carbon monoxide, nitrogen, hydrogen, methane, ethane, and/or fluorocarbons. In further embodiments, redox potential, pH, the solubility of the product, and/or Gibbs Free Energy equilibria are be altered in addition to controlling gas components gases.

[00220] In some embodiments, gas stripping and recycling steps described herein enhance the production of one or more fermentation end-products from a mixture comprising a biomass, microorganism, and gas. In further embodiments, the gas comprising the fermentation end-product in the form of a vapor is separated from the mixture and the fermentation end-product is separated from the vapor.

EXAMPLES

Example 1. Increased ethanol production using C. phytofermentans.

[00221] Standard fermentation medium containing hexose (C6) and pentose (C5) sugars (xylose 24 g/L, total glucose 21 g/L, total cellobiose 20.6 g/L, and arabinose 2.6 g/L) was inoculated with 10% or 20% inoculums of C. phy strain Q.27). The culture was incubated at 35°C with continuous agitation at 175 rpm, pH was adjusted and samples taken over the course of 100 hours. Total ethanol yield production over time is graphed (FIG. 2). The maximum ethanol title recorded was 39.5 g/L, which was recorded for the sample containing 20%> inoculum. An increase in inoculums amount was correlated with an increase in ethanol production rate and titer.

Example 2. Increased ethanol production using C. phytofermentans in a biofilm-forming gentle agitation culture.

[00222] 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. phy. at 2%> (v/v). The culture was incubated at 35°C with continuous agitation at 175 rpm, pH was adjusted and samples taken daily. Total ethanol yield, acid by-product production and residual sugar production over time is graphed (FIG. 3). 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). 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 ethanol 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 3. Increased ethanol production using C. phytofermentans in a biofilm-forming static fermentation.

[00223] 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 Cphy at 2% (v/v). The culture was 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 (FIG. 4). 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). 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 ethanol 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 4. Ethanol stripping with gas recycling.

[00224] Calculations were performed to assess the stripping of a fermentation end-product (ethanol) from a fermenter. The process considered in these calculations involved a system having a fermenter and a condenser, such as the system of FIG. 5A. The calculations assumed that gas (primarily carbon dioxide) bubbled through the fermenter and stripped (or removed) the fermentation end-product and water from the fermenter. This stripping occurred at the temperature and pressure of the fermenter. The gas then proceeded to the condenser where the temperature of the gas was lowered, causing some of the gaseous fermentation end-product and water to condense. This condensed fermentation end-product and water mixture was removed from the gas phase as a condensate.

[00225] The calculations further assumed an equilibrium condition between the liquid and gas phases of the fermentation end-product and water in the fermenter and the condenser. The liquid in the fermenter was assumed to be composed of water, fermentation end-product, and lactic acid. The low lactic acid concentration had little effect on the equilibrium.

[00226] Mass balances and phase equilibria calculations yielded the amount of fermentation end-product and the composition of the liquid which was condensed. The van Laar equations were used to calculate the activity coefficients for the phase equilibria in both the fermenter and the condenser. The Antione equation was used to calculate vapor pressures. A mathematical solver function {e.g., Solver routine on Excel) was used to obtain the solution of non linear simultaneous equations. Three different equation sequences were formulated before a formulation that converged with the solver function was obtained.

[00227] Three variables were changed in the calculations: the pressure of the gas, the fermentation end- product concentration in the fermenter, and the temperature of the condenser. The pressure in the fermenter had little effect on the performance of the system.

[00228] The effect of the fermentation end-product concentration is shown in FIG. 6A. The rate at which fermentation end-product is removed from the fermentation by the recycling gas is shown on the left-hand axis in FIG. 6A. With increasing fermentation end-product concentration in the fermentation broth, the fermentation end-product removed by the condensate also increases. For example, at a broth ethanol concentration of about 60 grams per liter, the ethanol removal rate is about 0.8 g/hr per liter of fermentation broth. This is with a gas flow rate of about 1 wm (volume of gas per minute per volume of fermentation broth). If the gas flow rate is increased to about 3 wm, the ethanol removal rate from the fermenter would be about 2.4 g/hr per liter fermentation broth. This is above the production rate of Clostridium phytofermentans . The calculations show that it would be possible to maintain the ethanol concentration in fermenter below 60 g/1 while the fermentation is producing ethanol. At 60 g/1 ethanol concentration in the fermenter, the expected concentration of the condensate leaving the condenser will be about 30 weight percent ethanol. Thus the stripping gas has increased the ethanol concentration from about 6 weight percent to 30 weight percent.

[00229] The stripping gas is also removing water from the fermenter. According to the calculations, at 1 wm, the gas is removing water at the rate of about 1.86 g/hr per liter of fermentation broth. If the gas flow rate is increased to 3 wm, the water removal rate will be about 5.6 g/hr per liter of fermentation broth. At this rate, the water removed from the fermenter will be 134 g per liter of fermentation broth in 24 hours. This rate of evaporation may require that sterile water be added to the fermentation as it proceeds.

[00230] The calculations also show that the temperature maintained in the condenser can affect the performance of the system. The results of calculations varying the condenser temperature are shown in FIG. 6B.

[00231] The rate of ethanol removal from the fermenter decreases greatly as the condenser temperature ranges from 0°C to 20°C. In addition, the concentration of ethanol in the condensate decreases with increasing condenser temperature. This suggests that the condenser temperature can be maintained as low as possible.

[00232] While the plot of FIG. 6B shows the lowest temperature to be 0°C, it is possible to operate the system below this temperature. Such a condenser would use glycol or salt solution as the cooling liquid. The 30% by weight ethanol solution can have a freezing point below this temperature.

[00233] Such gas mixing and ethanol stripping process is particularly useful for fermentation processes in which Clostridium phytofermentans (or other microbe) cannot tolerate high ethanol concentrations and requires mixing of high viscosity broths. However, it can also apply to the traditional yeast ethanol fermentations using sugar or corn starch.

Example 5. Pressure effects on ethanol stripping with gas recycle.

[00234] A model system having a fermenter, a condenser and a compressor downstream from the condenser was simulated to determine the influence of pressure on the condensate rate and mass fraction, as well as the concentration of carbon dioxide in the fermenter. A plot generated from the simulation is shown in FIG. 6, which shows that the rate of ethanol removal ("Ethanol Rate") and the ethanol mass fraction ("Ethanol Mass Fraction") remain nearly constant with fermenter and condenser pressure, and increasing CO ₂ concentration (or partial pressure).

[00235] As can be seen in the graph of FIG. 7, pressure can have little effect on the rate of ethanol removal in the condenser and on the ethanol concentration in the condensate from the condenser.

However, pressure can have a significant effect on the concentration of carbon dioxide in the

fermentation broth. This is calculated as if the fermenter is pure water. Any other ionic components in the broth will change the concentrations of the ions of carbonic acid. These calculations suggest that operating a fermenter at different pressures can improve the fermentation process.

Example 6. Pressure effects on ethanol stripping with gas recycle.

[00236] A model system having a fermenter, a compressor and a condenser downstream from the condenser (see FIG. 5B) was simulated to calculate the influence condenser pressure on the ethanol mass fraction in the condensate and the rate of ethanol removal from the fermenter. For such calculations, the following parameters were used: the fermenter temperature was 35°C, the lactate concentration was 5 g/1, the fermenter pressure was 800 mmHg, the gas flow rate through the fermenter was 1 wm, and the condenser temperature was 5°C. The results of calculations performed using this model are shown in FIGs. 8A and 8B. The higher pressure after the condenser permits a greater amount of the ethanol and water to be removed from the stream at the same condenser temperature.

[00237] The first variable investigated was the condenser pressure. The calculations were performed up to a pressure of 2400 mmHg, which would correspond to a fermenter with a liquid level of about 20 meters. This pressure also corresponds to approximately the pressure output of a variable frequency drive (VFD) single stage screw compressor.

[00238] The plot of FIG. 8A indicates that increasing pressure in the condenser increases the ethanol concentration in the condensate out of the condenser, as well as the rate of ethanol removal from the fermenter. For example, increasing the pressure in the condenser from 800 to 2400 mmHg (absolute) gave a 64% increase in the ethanol removal rate as well as a 42%> increase in the ethanol concentration. The heat removal requirement for the condenser after the compressor can be significantly greater.

[00239] For a system having a condenser before (or upstream from) the compressor, the concentration of ethanol in the fermenter had a significant effect on the condensate ethanol concentration and the ethanol removal rate. FIG. 8B shows a plot of ethanol mass fraction in the condensate and the rate of ethanol removal from the fermenter for a system having a condenser after the fermenter and before the compressor. As shown by the plot, the fermenter ethanol concentration had a significant effect on the condensate ethanol concentration and on the ethanol removal rate. For example, at 120 g/1 ethanol concentration in the fermenter, the condensate ethanol concentration was above 50%>, and the liquid could go directly to the rectifying section of the column or directly to a membrane separation unit. Additionally, the removal rate could maintain the ethanol concentration at a lower level, thereby increasing the productivity of the fermenter. The plot of FIG. 8B indicates that the ethanol concentration in the fermenter can be maintained or lowered by adjusting the gas flow rate.

Example 7. Gas mixing and ethanol stripping

[00240] As described in Example 4, gas mixing by recycling the gas generated from a biofilm

fermentation system using a C. phy. strain has the advantage of stripping ethanol from the fermenter, thereby reducing the ethanol toxicity for the fermentation, and resulting in enhanced ethanol yield. Such a gas mixing system and process can additionally improve the scrubbing of the exhaust gas from the fermenter and thereby further increase the yield of ethanol recovery.

[00241] The addition of a scrubber unit to such an ethanol recovery system further removes ethanol from the fermenter, thereby reducing the ethanol toxicity and consequently enhancing ethanol yield. For example, a model system having a scrubber, a fermenter, and a compressor downstream of a condenser (see FIG. 9) can be used for enhancing ethanol yield. A scrubber unit, such as a scrubbing column, works at a higher pressure than typical for ethanol plants. A gas mixing system as described herein provides for the pressure requirements of a scrubbing column, as a compressor and higher pressure levels are available in such a system.

Example 8. Controlled gas atmosphere in anaerobic fermentation.

[00242] A system described herein, e.g., that of Example 4, for recycling gas generated by biofilm fermentation using a C. phy. strain which results enhanced ethanol yield. In another example, the gas products of the fermentation can be controlled to improve fermentation rate and yield. Methods of controlling gas concentrations to enhance generation of ethanol as a fermentation end-product is described in U.S. Patent Publication 2010/0120106 which is herein incorporated by reference in its entirety.

[00243] The addition of a gas separator to the ethanol recovery system described in Example 7 further enhances ethanol yield. For example, a model system having a scrubber, a gas separator, a fermenter, and a compressor downstream of a condenser (see FIG. 10) can be used for enhancing ethanol yield. Gases that can be controlled include without limitation, carbon dioxide, carbon monoxide, nitrogen, hydrogen, methane, ethane, and fluorocarbons. Additionally, redox potential, pH, solubility, and Gibbs free energy equilibria can be altered with these gases.

[00244] 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.