Co-fermentation of mixed biomass feedstocks using thermophilic bacterial co-cultures

Abstract

A method for direct simultaneous co-fermentation of two or more insoluble biomass feedstocks using thermophilic bacterial co-cultures.

Description

PATENT Attorney Docket No. TBFI.P2003WO / 00609658 CO-FERMENTATION OF MIXED BIOMASS FEEDSTOCKS USING THERMOPHILIC BACTERIAL CO-CULTURES CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of priority to United States Provisional Patent Application No.63 / 535,916 filed on August 31, 2023, the content of which is incorporated herein by reference in its entirety. GOVERNMENT RIGHTS

[0002] This invention was made with government support provided by the US Department of Agriculture SBIR awards 2020-33610-32002 & 2021-33610-35655 and by the Center for Bioenergy Innovation, which is a US Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science. The government has certain rights in the invention. BACKGROUND I. Field of the Invention

[0003] The disclosure relates to direct simultaneous co-fermentation of two or more different plant-derived materials to produce ethanol or other products. This disclosure specifically relates to direct simultaneous co-fermentation of mixtures of insoluble biomass sources, such as corn stover and corn kernels, with a co-culture of thermophilic bacteria to produce ethanol. II. Description of the Related Art

[0004] New approaches are required to provide for the world's growing energy needs while preserving the environment. Plants are an abundant source of carbohydrates that can be converted into products such as liquid fuels. Biotechnology offers an economically attractive strategy for large-scale liquid fuel production, but this promise has not been fully realized. In particular, the ability to effectively utilize lignocellulosic biomass as a feedstock could unlock enormous reserves of renewable energy stored in plants. To date, no existing technologies are available for conversion of lignocellulosic materials to liquid fuels in a cost-competitive manner relative to fossil fuels. Technology to convert renewable lignocellulosic biomass into fuels, such as ethanol, and other products thus represents an important unmet societal need. Moreover, it would be highly desirable to have a cost-competitive method for the direct bioconversion ofAttorney Docket No. TBFI.P2003WO / 00609658 insoluble biomass feedstocks into ethanol via thermophilic microorganisms that can operate at the elevated temperatures preferred by industry.

[0005] According to the International Energy Agency, 29.5 billion gallons of bioethanol were produced in 2023, with the US and Brazil dominating production at 15.6 billion gallons and 8.3 billion gallons, respectively. To date, all commercial processes use yeast to ferment soluble sugars to ethanol. In the US, corn kernels are ground and the exposed starch is thermally and enzymatically depolymerized in pretreatment steps using alpha-amylase and glucoamylase to afford soluble glucose (corn dextrose) feedstock for ethanol production. In Brazil, sugarcane is crushed and extracted to provide soluble sucrose-rich cane juice or molasses feedstock for ethanol production. The yeast Saccharomyces cerevisiae is a mesophilic fungus that is used in >90% of industrial ethanol production processes, thus representing the commercial state-of-the- art. Yeast naturally ferments soluble sugars such as glucose, fructose, maltose, and sucrose, but is unable to depolymerize or directly ferment insoluble feedstocks, including starch, cellulose, and lignocellulosic biomass such as corn stover, corn fiber, corn cobs, and sugarcane bagasse. Since commercial ethanol processes using yeast are unable to directly utilize insoluble feedstocks, processing costs are higher than desired and a vast amount of lignocellulosic feedstock that could be converted to ethanol remains unexploited.

[0006] In a conventional dry grind ethanol process, pre-separated corn grain (kernels) is treated with heat and enzymes to yield corn dextrose for yeast fermentation that typically yields on average 2.8 gallons of ethanol per bushel of corn. A large volume of corn stover, the non- kernel lignocellulosic portion of the corn plant, is produced in this process - 1 ton of harvested corn yields approximately 1 dry ton of corn stover. Currently at least 90% of corn stover is simply left in the fields. It is estimated that at least 110 million dry tons of corn stover could be sustainably harvested in the US per year and could be converted into at least 6.8 billion gallons of ethanol if cost-competitive lignocellulosic technologies were available. While corn stover could be used to generate a large amount of ethanol, direct simultaneous conversion of this lignocellulosic feedstock together with corn kernels represents an even more efficient streamlined process for low-cost commercial ethanol production.

[0007] The present disclosure relates to a process to manufacture low-cost ethanol using thermophilic bacteria co-cultures that directly co-ferment mixtures of insoluble feedstocks.Attorney Docket No. TBFI.P2003WO / 00609658 This technology, referred to as Consolidated Bioprocessing (CBP), enables direct fermentation of mixtures of insoluble feedstocks to ethanol in higher yield relative to conventional ethanol technology. Also, for any given biomass feedstock, the CBP technology disclosed herein avoids up to five high-cost processing steps that are essential for existing biomass-to-ethanol conversion methods: 1) no separation of fermentable and nonfermentable biomass components is required (e.g., separation of corn kernels and corn stover), 2) no grinding and little or no milling of the biomass is necessary, 3) no thermochemical liquefaction pretreatment of biomass is needed, 4) no enzymatic pretreatment steps are required, which entails separate enzyme production and feedstock depolymerization steps to solubilize the biomass feedstock prior to fermentation, and 5) no or low-burden stillage processing steps are needed.

[0008] Thermophilic anaerobic bacteria have been shown to have many of the desired features for solubilizing and fermenting insoluble biomass feedstocks. Clostridium thermocellum (also referred to as Acetivibrio thermocellus), a bacterium found in compost piles, can solubilize and produce ethanol from corn stover and other insoluble materials containing predominantly lignocellulose. In particular, Clostridium thermocellum (C. thermocellum) is among the most efficient cellulose degraders isolated to date and is one of the most promising host organisms for application of CBP. The development of efficient and reliable genetic tools has allowed the engineering of this organism to increase ethanol yield and tolerance to high ethanol titers (See: Mazzoli, R., Olson, D.G., Clostridium thermocellum: A microbial platform for high-value chemical production from lignocellulose, Adv Appl Microbiol, 2020, 1-50).

[0009] The thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum (T. saccharolyticum) digests hemicellulose, ferments xylan, the main polymer in hemicellulose, and also utilizes all other major biomass sugars, including cellobiose, glucose, mannose, xylose, galactose, and arabinose. Organic fermentation products from wild-type strains of T. saccharolyticum strains include ethanol, acetic acid, and lactic acid. This organism has been engineered to produce ethanol at yields equivalent to yeast and can produce ethanol titers as high as 70 g / L from mixtures of soluble substrates cellobiose and maltodextrin (See: Herring, C.D., et al., Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood, Biotechnol Biofuels, 2016, 9:125. Doi: 10.1186 / s13068-016-0536-8). However, T.Attorney Docket No. TBFI.P2003WO / 00609658 saccharolyticum alone is not effective in utilizing cellulose and cannot solubilize lignocellulose in corn kernels, corn stover, or other insoluble lignocellulosic feedstocks.

[0010] Thermanaerobacterium thermosaccharolyticum (T. thermosaccharolyticum; formerly called Clostridium thermosaccharolyticum) is a thermophilic anaerobic bacterium originally isolated in spoilage from canned foods. This microbe utilizes substrates such as glucose, fructose, xylose, and xylan, and can depolymerize hemicellulose in lignocellulosic feedstocks and produces n-butanol, lactic acid, acetic acid, butyric acid, hydrogen, and ethanol (Vancanneyt, M. et al., Ethanol Production in Batch and Continuous Culture from Some Carbohydrates with Clostridium thermosaccharolyticum LMG 6564, System Appl Microbiol, 1990, 13, 382-387). T. thermosaccharolyticum has been widely studied in the context of conversion of cellulosic biomass to ethanol. T. thermosaccharolyticum is similar in many respects to T. saccharolyticum, it is unable to efficiently utilize cellulose, and it cannot solubilize lignocellulose in corn kernels or corn stover or other lignocellulosic feedstocks. T. thermosaccharolyticum has a higher optimum pH range for growth (up to pH 7.0), which can offer co-culture process advantages.

[0011] Herbinix hemicellulosilytica and other Herbinix species are thermophilic, anaerobic bacteria that have been isolated from anaerobic biogas reactors and digestors and have been reported to display potent hemicellulose-degrading activity and the ability to ferment a variety of pentose sugars (Beri, D. et al., Coculture with hemicellulose^fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings, Biotechnol Biofuels, 2021, 14:24. Doi: 10.1186 / s13068-020-01867-w; Beri, D. et al., Development of a thermophilic coculture for corn fiber conversion to ethanol, Nature Comm., 2020, 11:1937. Doi: 10.1038 / s41467-020-15704-z; Koeck, D.A., et al., Herbinix hemicellulosilytica gen. nov., sp. nov., a thermophilic cellulose-degrading bacterium isolated from a thermophilic biogas reactor, Int. J System Evol Microbiol, 2015, 65, 2365–2371. Doi: 10.1099 / ijs.0.000264).

[0012] No single organism has been reported to fully depolymerize lignocellulosic biomass feedstocks and utilize the derived carbohydrates for ethanol production with high yield. Specific combinations of thermophilic anaerobic bacteria have been found to work together as co- cultures, such as C. thermocellum and T. saccharolyticum, to more effectively degrade certain types of lignocellulosic biomass such as corn fiber (Beri, D. et al., Coculture withAttorney Docket No. TBFI.P2003WO / 00609658 hemicellulose^fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings, Biotechnol Biofuels, 2021, 14:24. Doi: 10.1186 / s13068- 020-01867-w; Beri, D. et al., Development of a thermophilic coculture for corn fiber conversion to ethanol, Nature Comm., 2020, 11:1937. Doi: 10.1038 / s41467-020-15704-z).

[0013] The invention disclosed herein involves a streamlined process using thermophilic bacterial co-cultures for simultaneously depolymerizing two or more insoluble biomass feedstocks and fermenting the solubilized carbohydrates to ethanol with significantly higher yields and economic benefits relative to existing industrial ethanol processes based on yeast. SUMMARY

[0014] The present disclosure overcomes some of the technical limitations with existing industrial ethanol production processes, as described above. The present disclosure relates to a process to manufacture low-cost ethanol using thermophilic bacterial co-cultures that directly and simultaneously co-ferment mixtures of insoluble biomass feedstocks. This technology, referred to as Consolidated Bioprocessing (CBP), enables direct fermentation of mixtures of insoluble feedstocks to ethanol in higher yield relative to conventional ethanol technology. Also, for any given biomass feedstock, the CBP technology disclosed herein avoids up to five high-cost processing steps that are essential for existing industry standard biomass-to-ethanol conversion methods: 1) no separation of fermentable and nonfermentable biomass components is required (e.g., corn kernels separated from corn stover), 2) no grinding and little or no milling of the biomass is required, 3) no thermochemical liquefaction pretreatment of biomass is needed, 4) no enzymatic pretreatment steps are necessary, which entails separate enzyme production and feedstock depolymerization steps to solubilize the biomass feedstock prior to fermentation, and 5) no or low- burden stillage processing steps are needed.

[0015] In one embodiment, a method for direct simultaneous co-fermentation of two or more insoluble biomass sources to produce ethanol is disclosed. In another embodiment, a method for direct simultaneous co-fermentation of two or more insoluble biomass sources using two or more thermophilic bacteria to produce ethanol is disclosed.

[0016] In one embodiment, it is disclosed that a tri-culture of C. thermocellum with T. saccharolyticum and T. thermosaccharolyticum or a bi-culture of C. thermocellum with T. saccharolyticum is capable of co-fermenting a mixture of two different insoluble biomassAttorney Docket No. TBFI.P2003WO / 00609658 feedstocks to produce ethanol. In another embodiment, it is disclosed that a tri-culture of C. thermocellum with T. saccharolyticum and T. thermosaccharolyticum or a bi-culture of C. thermocellum with T. saccharolyticum is capable of co-fermenting a mixture of corn kernels and corn stover to produce ethanol. In another embodiment, it is disclosed that a tri-culture of C. thermocellum with T. saccharolyticum and T. thermosaccharolyticum is capable of co-fermenting a mixture of rice and rice straw to produce ethanol. In another embodiment, it is disclosed that a tri-culture of C. thermocellum with T. saccharolyticum and T. thermosaccharolyticum is capable of co-fermenting a mixture of rice and rice hulls to produce ethanol. In another embodiment, it is disclosed that a tri-culture of C. thermocellum with T. saccharolyticum and T. thermosaccharolyticum is capable of co-fermenting a mixture of wheat grain and wheat straw to produce ethanol.

[0017] In another embodiment, at least one of the co-culture strains is genetically engineered to improve ethanol production and other properties. In another embodiment, C. thermocellum is genetically engineered to improve ethanol production and other properties. In another embodiment, T. saccharolyticum is genetically engineered to improve ethanol production and other properties. In another embodiment, T. thermosaccharolyticum is genetically engineered to improve ethanol production and other properties. In another embodiment, at least two of the co- culture strains are genetically engineered to improve ethanol production and other properties. In another embodiment, at least three of the co-culture strains are genetically engineered to improve ethanol production and other properties.

[0018] In one embodiment of the present disclosure, a 1:1 mixture of two different insoluble biomass feedstocks is added to the fermenter and converted to ethanol by a co-culture. In another embodiment of the present disclosure, a 1:1:1 mixture of three different insoluble biomass feedstocks is added to the fermenter and converted to ethanol by a co-culture. In another embodiment of the present disclosure, a 1:1:1:1 mixture of four different insoluble biomass feedstocks is added to the fermenter and converted to ethanol by a co-culture. In another embodiment of the present disclosure, a mixture of up to four different insoluble biomass feedstocks in unequal quantities is added to the fermenter and converted to ethanol by a co-culture.

[0019] In some embodiments, a method for producing ethanol by directly and simultaneously co-fermenting two or more insoluble biomass sources may include at least theAttorney Docket No. TBFI.P2003WO / 00609658 following steps: (a) mixing the two or more insoluble biomass sources in a vessel to form a biomass mixture, (b) mixing two or more thermophilic bacteria with the insoluble biomass mixture to form a fermentation mixture, (c) co-fermenting the two or more insoluble biomass sources in the presence of the two or more thermophilic bacteria, and (d) collecting ethanol from the vessel.

[0020] In some embodiments, the two or more thermophilic bacteria are used in step (b). In some embodiments, the two or more thermophilic bacteria used in step (b) are also anaerobic bacteria. In one aspect, no yeast is used in the disclosed process. In another aspect, the two or more thermophilic bacteria comprise at least one genetically engineered strain. In another aspect, the two or more thermophilic bacteria comprise at least two different genetically engineered strains. In another aspect, the two or more thermophilic bacteria comprise at least three different genetically engineered strains. In one aspect, the three different genetically engineered strains belong to different species. In another aspect, two of the three genetically engineered strains belong to same genus. In another aspect, the three different genetically engineered strains belong to two different genera and three different species.

[0021] In some embodiments, the disclosed process further includes a step of sterilizing the fermentation mixture, wherein the sterilization step is performed after step (a) but before step (b). In other embodiments, the disclosed process further includes a step of sterilizing the biomass mixture in one vessel and then transferring it aseptically to another vessel containing the thermophilic bacterial co-culture. In one aspect, the collecting step (d) comprises a distillation process to obtain the ethanol from the fermentation broth. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a flow chart describing one embodiment of the disclosed methods involving co-fermentation of corn kernels and corn stover using tri-cultures of Strains X, Y, and Z, as defined in Table 1.

[0023] FIG. 2 graphically depicts the ethanol yield in grams produced using tri- cultures by (A) co-fermentation of corn kernels and corn stover, and (B) fermentation of corn kernels, in a 1 L fermentation vessel. Example (C) shows the ethanol yield expected by fermenting corn kernels using yeast, which does not utilize lignocellulose in the kernels. Tri-cultures comprise Strains X, Y, and Z, as defined in Table 1.Attorney Docket No. TBFI.P2003WO / 00609658 DETAILED DESCRIPTION

[0024] The novel features of the subject matter described herein are set forth specifically in the appended claims. A better understanding of the features and benefits of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the claimed embodiments are utilized. To facilitate a full understanding of the disclosure set forth herein, a number of terms are defined below. Terminology

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of terms and definitions set forth below shall control.

[0026] The embodiment(s) described, and references in the specification to "one embodiment", "an embodiment", "an example embodiment", etc., indicate that the embodiment(s) described can include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is understood that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0027] The description of "a" or "an" or “the” item herein may refer to a single item or multiple items. It is understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of' and / or "consisting essentially of' are also provided. Thus, for example, reference to "a polynucleotide"Attorney Docket No. TBFI.P2003WO / 00609658 includes a plurality of such polynucleotides and reference to "the microorganism" includes reference to one or more microorganisms, and so forth.

[0028] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0029] As used herein, the term “aerobic” when used in reference to a culture or growth condition means that free oxygen (O2) is available in the culture or growth condition. This includes when the dissolved oxygen in the liquid medium is more than 50% of saturation.

[0030] "Aerobic metabolism" refers to a biochemical process in which oxygen is used as a terminal electron acceptor to make energy, typically in the form of ATP, from carbohy- drates. Aerobic metabolism typically occurs, for example, via glycolysis and the TCA cycle, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen.

[0031] As used herein, the term "anaerobic" refers to an organism, biochemical reaction or process that is active or occurs under conditions of an absence of oxygen.

[0032] "Anaerobic conditions" are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor.

[0033] “Anaerobic metabolism" refers to a biochemical process in which oxygen is not the final acceptor of electrons generated. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, andAttorney Docket No. TBFI.P2003WO / 00609658 substrate level phosphorylation, in which no exogenous electron acceptor is used and products of an intermediate oxidation state are generated via a "fermentative pathway."

[0034] The terms “ferment,” “fermentation,” or “fermentation process” as used herein refer to a metabolic process whereby microorganisms, such as yeast and bacteria, utilize enzymatic pathways to convert a feedstock, for example organic molecules such as carbohydrates, into a range of products such as ethanol, lactic acid, hydrogen, and carbon dioxide. Anaerobic fermentation processes occur in the absence of oxygen and produce by-products such as ethanol and / or hydrogen as a means for the organism to re-oxidize NADH to NAD+ required for glycolysis and ATP generation to proceed. To produce ethanol, a fermentation process converts one glucose molecule into two ethanol molecules and two carbon dioxide (CO2) molecules and acetaldehyde is the final electron acceptor. The terms "fermenting" and "fermentation" are intended herein to include the enzymatic process (e.g., cellular or acellular, e.g., a lysate or purified polypeptide mixture) by which ethanol is produced from a carbohydrate, in particular, as a product of fermentation.

[0035] The terms “co-fermentation” or “co-ferment” refer to a process whereby one or more microorganisms simultaneously ferments two or more different types of feedstock in the same fermenter to a product or products of interest. For example, a mixed feedstock such as corn kernels (starch-rich) and corn stover (fiber-rich) can be co-fermented to ethanol. Alternatively, a mixed feedstock such as rice grain and rice straw or wheat grain and wheat straw may be used.

[0036] The term “direct co-fermentation” as used herein refers to a fermentation process involving a co-culture of microorganisms that is able to directly depolymerize and ferment two or more different insoluble biomass feedstocks with no or minimal milling and without prior grinding or thermochemical / enzymatic pretreatment of the biomass.

[0037] The term “simultaneous co-fermentation” as used herein refers to a fermentation process involving a co-culture of microorganisms that is able to concurrently depolymerize and ferment two or more different insoluble biomass feedstocks added to the same reaction vessel.

[0038] In "fermentative pathways", the amount of NAD(P)H generated by glycolysis is balanced by the consumption of the same amount of NAD(P)H in subsequent steps. For example, in one of the fermentative pathways of certain yeast strains, NAD(P)H generated throughAttorney Docket No. TBFI.P2003WO / 00609658 glycolysis donates its electrons to acetaldehyde, yielding ethanol. Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain.

[0039] As used herein, the term "flux" is the rate of flow of molecules through a metabolic pathway, akin to the flow of material in a process.

[0040] As used herein, the term "end-product" refers to a chemical compound that is not or cannot be used by a cell, and so is excreted or allowed to diffuse into the extracellular environment. Common examples of end-products from anaerobic fermentation include, but are not limited to, ethanol, acetic acid, formic acid, lactic acid, hydrogen and carbon dioxide.

[0041] As used herein, a "pathway" is a group of biochemical reactions that together can convert one compound into another compound in a multi-step process. A product of the first step in a pathway may be a substrate for the second step, and a product of the second step may be a substrate for the third, and so on. Pathways of the present invention include, but are not limited to, the lactate production pathway, the ethanol production pathway, and the acetate production pathway.

[0042] A "plasmid" or "vector" refers to an extrachromosomal element often carrying one or more genes and is usually in the form of a circular double-stranded DNA molecule. Plasmids and vectors may also contain additional genetic elements such as autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences. They may also be linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source. Plasmids and vectors may be constructed by known techniques in which a number of nucleotide sequences have been joined or recombined into a unique construction. Plasmids and vectors generally also include a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence. Generally, the plasmids of the present invention are stable and self-replicating.

[0043] The term "recombination" or "recombinant" refers to the physical exchange of DNA between two identical (homologous), or nearly identical, DNA molecules. Recombination is used for targeted gene deletion to modify the sequence of a gene. The term "recombinant microorganism" and "recombinant host cell" are used interchangeably herein and refer to microorganisms that have been genetically modified to express or over-express endogenousAttorney Docket No. TBFI.P2003WO / 00609658 polynucleotides, or to express heterologous polynucleotides, such as those included in a vector, or which have a modification in expression of an endogenous gene. By "modification" it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modification.

[0044] As used herein, the term "ethanol pathway" refers to the canonical pathway of ethanol production from pyruvate generated by glycolysis. Components of the ethanol pathway consist of all substrates, cofactors, byproducts, intermediates, end products, and enzymes in the pathway.

[0045] As used herein, the term "glycolysis" or "glycolytic pathway" refers to the canonical pathway of basic metabolism in which a sugar such as glucose is broken down into more oxidized products, generating energy and / or compounds required for cell growth. The pathway consists of all substrates, cofactors, byproducts, end-products, and enzymes in the pathway.

[0046] As used herein, the terms "biomass" or “plant biomass” refer to non-fossilized renewable materials that are derived from plants and includes any plant-derived matter (woody or non-woody) that can be renewably grown and is available on a sustainable basis. The terms “biomass” or “plant biomass” can include any type of plant-derived biomass known in the art or described herein. Plant biomass may include, but is not limited to, purposefully grown agricultural materials such as corn, sugar beets, and sorghum, agricultural crop wastes, and agricultural components such as corn kernels, corn cobs, corn stover, corn processing byproducts such as corn bran or corn fiber, wheat grain, wheat straw, rice, rice straw, and the like, grass crops, such as sugar cane, switch grass, alfalfa, winter rye, and the like. Plant biomass may further include, but is not limited to, woody energy crops, wood wastes and residues such as trees, softwood, forest thinnings, barky wastes, sawdust, paper and pulp industry residues or waste streams, wood fiber, and the like. In urban areas, plant biomass may include yard waste, such as grass clippings, leaves, tree clippings, brush, etc., vegetable processing waste, as well as recycled cardboard and paper products. Biomass also includes plant-derived starch and hexose and pentose sugars.

[0047] The terms “lignocellulose,” "lignocellulosic material," "lignocellulosic substrate," “lignocellulosic biomass,” and "cellulosic biomass" as used herein can be usedAttorney Docket No. TBFI.P2003WO / 00609658 interchangeably and mean any type of plant-derived fibrous material comprising cellulose, hemicellulose, and lignin, or combinations thereof. As used herein, “lignocellulose” is a term used to refer to any polymeric plant-based materials that are the major components of plant cell walls and is distinguished from other homogeneous polysaccharides such as starch or cellulose. Lignocellulosic biomass includes but is not limited to woody biomass, such as hardwood (e.g., poplar, oak, walnut, ash, maple, birch), softwood (e.g., evergreens; fir, cedar, redwood), recycled wood pulp fiber, paper mill waste streams, paper mill sludge, forestry residues and / or forestry wastes, wood wastes and residues such as trees, forest thinnings, barky wastes, sawdust, waste- water-treatment sludge, municipal solid waste, grasses including forage grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and / or agricultural residues, such as but not limited to rice straw, rice hulls, barley straw, cereal straw, wheat straw, canola straw, oat straw, oat hulls, soybean hulls and stover, corn fiber, corn stover, corn cobs, corn fiber and corn stover from wet and dry mill corn ethanol plants, and sugar beet- or sugar cane-processing residues, such as bagasse, or any combination thereof. Lignocellulosic material may comprise one species of fiber; alternatively, lignocellulosic material may comprise a mixture of fibers that originate from different lignocellulosic materials.

[0048] The terms “hemicellulose,” "hemicellulosics," "hemicellulosic portions," and "hemicellulosic fractions" mean the non-lignin, non-cellulose elements of lignocellulosic mate rial, such as but not limited to hemicellulose (i.e., comprising xyloglucan, xylan, glucuronoxylan, arabinoxylan, mannan, glucomannan, and galactoglucomannan), pectins (e.g., homogalacturonans, rhanmogalacturonan I and II, and xylogalacturonan), and proteoglycans (e.g., arabinogalactan-protein, extensin, and praline-rich proteins).

[0049] A “pentose sugar” is a simple sugar, or monosaccharide, with five carbon atoms and the general chemical formula C^H^^O^. Examples of pentose sugars are ribose, xylose, and arabinose.

[0050] A “hexose sugar” is a simple sugar, or monosaccharide, with six carbon atoms and the general chemical formula C6H12O6. Examples of hexose sugars are glucose, mannose, and galactose.Attorney Docket No. TBFI.P2003WO / 00609658

[0051] A "pentose sugar utilizing enzyme" can be any enzyme involved in pentose sugar digestion, metabolism and / or hydrolysis.

[0052] The terms "feedstock,” “biomass feedstock,” or “biomass source” are used interchangeably herein and refer to a raw material or mixture of raw materials supplied to a microorganism, a co-culture of microorganisms, or a fermentation process to facilitate growth and from which other products can be made. For example, a carbon source, such as biomass or the carbon compounds derived from biomass, such as glucose, cellobiose, or sucrose, are feedstocks for a microorganism that produces a product such as ethanol in a fermentation process. A feedstock also can contain nutrients other than a carbon source.

[0053] The terms “insoluble feedstock,” “insoluble substrate,” “insoluble biomass feedstock,” or “insoluble biomass source” as used herein to refer to polymerized feedstocks comprised of carbohydrate units whereby the polymeric material used as input for a fermentation process is not soluble in water or other aqueous fermentation media. Examples of insoluble feedstocks include, but are not limited by starch, cellulose, hemicellulose, fiber, and lignocellulosic biomass.

[0054] The terms “soluble feedstock,” “soluble substrate,” “soluble sugars,” or “soluble carbohydrates” as used herein to refer to monosaccharides or disaccharides, or depolymerized feedstocks containing a small number of carbohydrate units (typically less than 20 sugars) whereby the material used as input for a fermentation process is soluble in water or other aqueous fermentation media. Examples of soluble feedstocks include, but are not limited to, glucose, sucrose, fructose, maltose, cellobiose, and cellotriose.

[0055] The terms “fermentable feedstock,” “fermentable biomass,” and “fermentable biomass components” as used herein refer to biomass or biomass components that can be converted to carbohydrates that are fermentable under a given set of conditions. For example, in commercial ethanol processes based on yeast fermentations, the starch in corn kernels (grain) is a fermentable biomass component, whereas the lignocellulosic corn stover is non-fermentable.

[0056] The terms “nonfermentable feedstock,” “nonfermentable biomass,” and “nonfermentable biomass components” as used herein refer to biomass or biomass components that can’t be converted to carbohydrates that are fermentable under a given set of conditions. For example, in commercial ethanol processes based on yeast fermentations, the lignocellulosic cornAttorney Docket No. TBFI.P2003WO / 00609658 stover is nonfermentable, whereas lignocellulosic corn stover is fermentable when thermophilic bacterial co-cultures are implemented.

[0057] The term "yield" is defined as the amount of product obtained per unit weight of raw material and may be expressed as grams of product per gram of substrate (g / g) or grams of product per bushel of biomass. Yield may be expressed as a percentage of the theoretical yield. "Theoretical yield" is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to ethanol is 0.51 g EtOH per 1 g glucose. As such, a yield of 4.8 g ethanol from 10 g of glucose would be expressed as 94% of theoretical or 94% theoretical yield.

[0058] The term "titer" is defined as the strength of a solution or the concentration of a substance in solution. For example, the titer of a product in a fermentation broth is described as grams of product in solution per liter of fermentation broth (g / L) or as g / kg broth.

[0059] The terms “microbial,” “microbial organism” and “microorganism” as used herein refer to any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the terms encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The terms also include cells of any species that can be cultured for the production of a biochemical (e.g., ethanol).

[0060] "Bacteria" or "eubacteria" refer to a domain of prokaryotic organisms. Bacteria include Gram-positive (Gram+) bacteria and Gram-negative (Gram-) bacteria. In some embodiments of the invention, the host cell is a prokaryotic microorganism. In some embodiments, the host cell is a bacterium. In some embodiments, the host cell is able to digest and ferment cellulose. In some embodiments, the host cell is able to digest and ferment lignocellulose. In some embodiments, the host cell is a thermophilic bacterium. In some embodiments, the host cell is a thermophilic anaerobic bacterium. In some embodiments, the microorganism is from the genus Clostridium. In some embodiments, the microorganism is from the genus Clostridium (or Acetivibrio) and the species thermocellum (or thermocellus). In some embodiments, the microorganism is from the genus Thermoanaerobacterium. In some embodiments, theAttorney Docket No. TBFI.P2003WO / 00609658 microorganism is from the genus Thermoanaerobacterium and the species saccharolyticum or thermosaccharolyticum.

[0061] The terms “culture” or “microbial culture” refer to a process for multiplying microorganisms in a controlled setting by allowing them to reproduce, grow, and proliferate in a predetermined culture medium. The culture medium can be liquid, gelled (solid), or semi- solid. Aseptic techniques are used to prevent contamination, and the microorganisms can be grown in liquid broth or solid nutrient agar culture media.

[0062] The term “co-culture”, “microbial co-culture”, “mixed culture”, or “consortium,” may be used interchangeably and refers to a biological system where two or more different microorganisms or strains of microorganisms are grown together in the same environment, such as a shake flask, a bioreactor, or fermenter. Co-cultures comprising two different microorganisms can be referred to as bi-cultures. Co-cultures comprising three different microorganisms can be referred to as tri-cultures.

[0063] The terms “engineered,” or “genetically engineered,” as used here in reference to any peptide, polypeptide, protein, nucleic acid, polynucleotide, or microorganism described herein refer to a sequence of amino acids or nucleic acids having at least one alteration (e.g., substitution, insertion, deletion) at an amino acid residue or nucleic acid base as compared to a parent sequence or as compared to a parent or wild-type organism, in the case of a microorganism. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a modified variant of a wild-type sequence or homolog thereof. Such an engineered or variant sequence of amino acids or nucleic acids is not naturally occurring. An engineered microorganism can have its natural DNA sequence altered through chromosomal insertion, deletion, or mutation or through the introduction of a replicating plasmid bearing genes of interest that alter the cellular properties of the microorganism.

[0064] The term “amino acid” refers to naturally occurring and non-naturally occurring alpha-amino acids, as well as alpha-amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring alpha-amino acids. Naturally encoded amino acids are the 22 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid. glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine). Amino acidAttorney Docket No. TBFI.P2003WO / 00609658 analogs or derivatives refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and a side chain R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms “non-natural amino acid” or “non-proteinogenic amino acid” or “unnatural amino acid” or “non-canonical” refer to alpha-amino acids that contain different side chains (different R groups) relative to those that appear in the twenty-two common or naturally occurring amino acids listed above. In addition, these terms also can refer to amino acids that are described as having D-stereochemistry, rather than L-stereochemistry of natural amino acids, despite the fact that some amino acids do occur in the D-stereochemical form in nature (e.g., D- alanine and D-serine).

[0065] The terms “isolated,” “isolate,” and “isolating,” or grammatical equivalent thereof, when used in reference to a microbial organism, nucleic acid, protein, polypeptide, or peptide, refer to a microbial organism, nucleic acid, protein, polypeptide, or peptide that is substantially free of at least one component relative to the referenced microbial organism, nucleic acid, protein, polypeptide, or peptide is found in nature or in its current environment. The term includes a microbial organism, nucleic acid, protein, polypeptide, or peptide that is removed from some or all components as it is found in its natural environment. Therefore, an isolated microbial organism, nucleic acid, protein, polypeptide, or peptide is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments (e.g., laboratories). Specific examples of isolated microbial organism, nucleic acid, protein, polypeptide, or peptide include a partially pure microbial organism, nucleic acid, protein, polypeptide, or peptide, a substantially pure microbial organism, nucleic acid, protein, polypeptide, or peptide, a microbial organism cultured in a medium that is non-naturally occurring, a protein, polypeptide, or peptide purified from other components and substances present their natural environment, including other proteins, polypeptides, or peptides, or an isolated nucleic acid that is substantially separated from other genome DNA sequences as well asAttorney Docket No. TBFI.P2003WO / 00609658 proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. As another example, an isolated nucleic acid can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A substantially pure molecule can include isolated forms of the molecule.

[0066] The terms “non-naturally occurring,” “non-natural,” “unnatural” and “non- native” as used herein refer to a material, substance, molecule, cell, nucleic acid, oligonucleotide, nucleotide, enzyme, protein, polypeptide, peptide, or amino acid that is not known to exist or is not found in Nature or that has been structurally modified and / or synthesized by humans. Such terms when used in reference to a microbial organism, cell extract, or nucleic acid of the disclosure mean that the microbial organism, cell extract, or nucleic acid has at least one genetic alteration not normally found in a naturally occurring strain or a naturally occurring nucleic acid of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, introduction of expressible oligonucleotides or nucleic acids encoding polypeptides, nucleic acid additions, substitutions, or deletions and / or other functional disruption of the microbial organism’s genetic material. Such alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic coding regions and functional fragments thereof, used for heterologous, homologous or both heterologous and homologous expression of polypeptides. Additional alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic non-coding and / or regulatory regions in which the modifications alter expression of a gene or operon. Such terms when used in reference to a protein, polypeptide, or peptide are used to refer to a protein, polypeptide, or peptide having amino acids that are introduced into the amino acid sequence of the protein, polypeptide, or peptide to modify the properties of the polypeptide.

[0067] The term “physiological conditions” as used herein refers to the conditions of the external or internal milieu that occurs in nature for a healthy or normal functioning organism or cell system. Physiological conditions include a number of parameters, including temperature, pressure, pH, glucose concentration, oxygen concentration, gravity, and electromagnetism, all of which will depend upon the healthy or normal physiological conditions of the organism or cell system.Attorney Docket No. TBFI.P2003WO / 00609658

[0068] The term “wild-type” refers to organisms, cells, genes, biosynthetic gene clusters, enzymes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature (in the wild). Consolidated Bioprocessing (CBP) for Co-fermentation of Mixtures of Insoluble Biomass Feedstocks

[0069] The present disclosure relates to methods for simultaneous direct co- fermentation of two or more insoluble biomass feedstocks by co-cultures of thermophilic microorganisms to produce ethanol or other products. The CBP method disclosed herein comprises: 1) mixing two or more insoluble biomass feedstocks such as a first insoluble biomass (e.g., corn stover) and a second insoluble biomass (e.g., corn kernels) in a fermentation vessel and sterilizing by autoclaving or steam treatment; 2) Adding media components and adjusting temperature and pH of the mixture; 3) adding two or more thermophilic anaerobic bacterial strains to the vessel as a co-culture; 4) co-fermenting the two or more insoluble biomass feedstocks ; 5) separating the fermentation broth from the remaining cells and insoluble materials; and 6) recovering ethanol from the fermentation broth by distillation. The methods described herein may be conducted in either batch or continuous modes. The two or more thermophilic anaerobic bacteria can be engineered for high ethanol production. In one embodiment, the methods of the present disclosure can enable co-fermentation of an easily fermented feedstock (e.g., corn kernels) together with a difficult-to-ferment lignocellulosic feedstock (e.g., corn stover) to allow for reaching commercially viable performance more rapidly. In another embodiment, the sterilizing by autoclaving or steam treatment in step (1) above can be applied to the first and second biomass independently before the two biomass materials are mixed.

[0070] This disclosure relates to the direct simultaneous co-fermentation of mixtures of two or more insoluble biomass sources. In some embodiments, the two or more insoluble biomass sources are from the same plant species. Examples of the same plant species may be corn, wheat, rice, or other species. In other embodiments, the two or more insoluble biomass sources are from different plant species. In some embodiments, the two or more insoluble biomass sources comprise a first insoluble biomass and a second insoluble biomass, and the first insoluble biomass and the second insoluble biomass have different starch and fiber contents. In some embodiments, the two or more insoluble biomass sources comprise a first insoluble biomass containingAttorney Docket No. TBFI.P2003WO / 00609658 predominantly fibrous lignocellulose and a second insoluble biomass containing predominantly a homogeneous polysaccharide such as starch or cellulose. In one aspect, the first insoluble biomass has a fiber content that is at least 50% higher, or at least 60%, or at least 70% or at least 80% higher than the fiber content of the second insoluble biomass. In another aspect, the second insoluble biomass has a starch content that is at least 50%, or at least 60%, or at least 70% or at least 80%higher than the starch content of the first insoluble biomass. In some embodiments, one insoluble biomass material contains predominantly (for example, more than 60%, or more than 70%, or more than 80%) fibrous lignocellulose and the other insoluble biomass material contains predominantly (for example, more than 60%, or more than 70%, or more than 80%) starch. In another embodiment, each of the two biomass sources, namely, the first biomass and the second biomass, constitutes at least 20%, 30, 40%, or 50% by weight of the total biomass.

[0071] In one aspect, the first insoluble biomass source is corn stover. In another aspect, the second insoluble biomass source is corn kernel.

[0072] In some embodiments, the corn stover and corn kernel used in step (1) above are separated before mixing. In other embodiments, the corn stover and corn kernel used in step (1) above are introduced directly from a whole corn plant without separating the corn kernel from the corn stover.

[0073] In some embodiments the two or more insoluble biomass feedstocks are added together into the fermentation vessel in step (1) without prior grinding, milling, or pretreatment. In some embodiments the two or more insoluble biomass feedstocks are added together into the fermentation vessel in step (1) after grinding or milling.

[0074] In some embodiments, the first biomass is a fiber-enriched derivative of corn kernels, such as corn bran, corn fiber, dried distillers grains with solubles (DDGS), wet cake (wet distillers grains), or whole stillage.

[0075] In some embodiments, the weight ratio between the first insoluble biomass source and the second insoluble biomass source used in step (1) is between 2:1 and 1:2, or between 1.2:1 and 1:1.2, or about 1.2:1, or about 1:1, or about 1:1.2.

[0076] In some embodiments, the two or more thermophilic bacteria are used in step (3). In some embodiments, the two or more thermophilic bacteria used in step (3) are also anaerobicAttorney Docket No. TBFI.P2003WO / 00609658 bacteria. In some embodiments, the two or more thermophilic bacteria used in step (3) are comprised of at least one of C. thermocellum, T. saccharolyticum, T. thermosaccharolyticum, or Herbinix spp. In one aspect, no yeast is used in the disclosed process. In another aspect, the two or more thermophilic bacteria comprise at least one genetically engineered strain. In another aspect, the two or more thermophilic bacteria comprise at least two different genetically engineered strains. In another aspect, the two or more thermophilic bacteria comprise at least three different genetically engineered strains. In one aspect, the three different genetically engineered strains belong to different species. In another aspect, two of the three genetically engineered strains belong to the same genus. In another aspect, the three different genetically engineered strains belong to two different genera and three different species.

[0077] In some embodiments, the three genetically engineered strains (Strains X, Y, and Z) belong to these three species: C. thermocellum, T. saccharolyticum, T. thermosaccharolyticum, respectively. In certain embodiments, these three strains have been genetically engineered and comprise the following genotypes: 1. Strain X. C. thermocellum strain LL1043 (pta(í) L-ldh(í); Reference: DA Argyros et al.2011. High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microbiol. 77(23):8288-94.) 2. Strain Y. T. saccharolyticum strain M1442 (pta / ack(í) L-ldh(í) Tsac_0795(í) urease(+) metE(+) EPSoperon(í); Reference: CD Herring CD et al.2016. Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood. Biotechnol Biofuels.16;9:125.) 3. Strain Z. T. thermosaccharolyticum strain TC2 (pta / ack(í) L-ldh(í) thiolase(-) hpt(-); unpublished)

[0078] In some embodiments, wild-type or genetically engineered strains of the genus Herbinix are included in the co-culture of step (3) above.

[0079] In some embodiments, the disclosed process is conducted in either batch or continuous mode.

[0080] In some embodiments, the ethanol yield as measured by the amount (in grams, liters, or gallons) of ethanol produced from a certain amount of biomass (in grams, tons, or bushels)Attorney Docket No. TBFI.P2003WO / 00609658 resulting from the disclosed process is higher than the ethanol yield resulting from fermentation of the same substrates with yeast using conventional technology. In some embodiments, the ethanol titer as measured by the total amount of ethanol in grams per liter of total liquid resulting from the disclosed process is higher than the ethanol titer resulting from fermentation of either the first insoluble biomass or the second insoluble biomass alone. In some embodiments, the ethanol titer achieved according to the disclosed process is 30-70 g / L, 40-60 g / L or 35-45 g / L.

[0081] In some embodiments, the final ethanol concentration of the co-fermentation is higher than the maximum ethanol concentration achievable with only the predominantly starch- or cellulose-rich substrates, even at higher initial concentrations of that individual substrate.

[0082] In some embodiments, fermentation residue from corn stover and kernels can be used as soil amendment or used to make fuel pellets or animal feed.

[0083] One embodiment of the present disclosure relates to methods for co-fermenting corn kernels and corn stover using engineered thermophilic bacteria. FIG.1 shows a representative flow chart of the method of the present disclosure involving co-fermentation of corn kernels and corn stover using a co-culture comprised of three different thermophilic anaerobic bacteria. In one aspect, a 1:1 mixture of corn kernels:corn stover allows for significant production of cellulosic ethanol. In some embodiments, the disclosed methods help achieve an ethanol titer of at least 30 g / L, or at least 35 g / L, or at least 37 g / L, or about 37 g / L. In another aspect, the weight ratio of corn kernels:corn stover is between 2:1 and 1:2, or between 1.2:1 and 1:1.2, or about 1.2:1, or about 1:1, or about 1:1.2. In another aspect, use of bi-cultures or tri-cultures of certain thermophilic bacteria would allow for access to, depolymerization of, and fermentation of the corn fiber cellulose, hemicellulose, and recalcitrant starch in corn kernels. Thus, the ethanol yield from corn kernels (corn starch and corn fiber) is significantly higher than that estimated based on the industry standard of 2.85 gallons of ethanol per bushel of corn produced in a commercial yeast fermentation process. In another embodiment, use of bi-cultures or tri-cultures of certain thermophilic bacteria allow at least 16% higher ethanol yield from corn kernels relative to industry standard yeast fermentation processes.

[0084] Various embodiments of the inventions of the present disclosure can be further illustrated by the following Items:Attorney Docket No. TBFI.P2003WO / 00609658

[0085] Item 1. A method for producing ethanol by simultaneously co-fermenting two or more insoluble biomass sources, comprising: a) mixing the two or more insoluble biomass sources to form an insoluble biomass mixture, b) mixing the insoluble biomass mixture of step (a) with two or more thermophilic bacteria to form a fermentation mixture, c) simultaneously co-fermenting the two or more insoluble biomass sources in the presence of the two or more thermophilic bacteria, and d) collecting ethanol produced in step (c).

[0086] Item 2. The method of Item 1, wherein the two or more insoluble biomass sources comprise a first insoluble biomass and a second insoluble biomass, said first insoluble biomass and said second insoluble biomass having different starch and fiber contents, wherein the first insoluble biomass has a fiber content that is at least 50% higher than the fiber content of the second insoluble biomass.

[0087] Item 3. The method of Items 1 and 2, wherein the second insoluble biomass has a starch content that is at least 50% higher than the starch content of the first insoluble biomass.

[0088] Item 4. The method of any preceding Items, wherein the two or more insoluble biomass sources are from the same plant source.

[0089] Item 5. The method of any preceding Items, wherein the same plant source is selected from the group consisting of corn, wheat, and rice.

[0090] Item 6. The method of any preceding Items, wherein the first insoluble biomass source is corn stover, and the second insoluble biomass source is corn kernel.

[0091] Item 7. The method of any preceding Items, wherein the first insoluble biomass source is a fiber-enriched derivative of corn kernels and the second insoluble biomass source is corn kernel.

[0092] Item 8. The method of any preceding Items, wherein the corn stover and corn kernel used in step (a) are directly from a whole corn plant without separating the corn kernel from the corn stover first.Attorney Docket No. TBFI.P2003WO / 00609658

[0093] Item 9. The method of any preceding Items, wherein the weight ratio between the first insoluble biomass source and the second insoluble biomass source in step (a) is about 1:1.

[0094] Item 10. The method of any preceding Items, wherein the two or more thermophilic bacteria comprise two or more different genetically engineered strains, wherein the two or more different genetically engineered strains belong to different species.

[0095] Item 11. The method of any preceding Items, wherein two or more genetically engineered strains belong to same genus.

[0096] Item 12. The method of any preceding Items, wherein the two or more thermophilic bacteria comprise three genetically engineered strains that belong to three different species, the three different species being C. thermocellum, T. saccharolyticum, T. thermosaccharolyticum, respectively.

[0097] Item 13. The method of any preceding Items, further comprising sterilizing the fermentation mixture, wherein the sterilization step is performed after step (a) but before step (b).

[0098] Item 14. The method of any preceding Items, further comprising sterilizing the fermentation mixture, wherein the sterilization step is performed before step (a).

[0099] Item 15. The method of any preceding Items, wherein the collecting step (d) comprises a distillation process.

[0100] Item 16. The method of any preceding Items, wherein the method is conducted in either batch or continuous mode.

[0101] Item 17. The method of any preceding Items, wherein the two or more insoluble biomass sources comprise a first insoluble biomass and a second insoluble biomass, said first insoluble biomass having more than 60% fiber, and said second insoluble biomass having more than 60% starch.

[0102] Item 18. The method of any preceding Items, where the two or more insoluble biomass sources are from different genomically-defined plant species.Attorney Docket No. TBFI.P2003WO / 00609658

[0103] Item 19. The method of any preceding Items, where the two or more insoluble biomass sources are subjected to a grinding or milling step prior to mixing in a vessel to form an insoluble biomass mixture in step (a).

[0104] Item 20. The method of any preceding Items, wherein the corn stover and corn kernel from the corn plant are separated into corn kernel and corn stover before being mixed in step (a).

[0105] It will be readily apparent to those skilled in the art that the systems and methods described herein may be modified and substitutions may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. EXAMPLES Example 1 Co-fermentation of corn stover and corn kernels Preparation of pre-grown inocula

[0106] For Clostridium thermocellum (Strain X), the inoculum was prepared by growing cells at pH 7 and 55 degrees Celsius for approximately 24 hours using 20 g / L Avicel feedstock (a powdered cellulose) with additional growth nutrients (CC8 medium). The cells and broth (OD § 1) were then transferred into a sterile, nitrogen-flushed serum bottle, sealed with a butyl rubber stopper and crimp cap, then frozen at -80 degrees Celsius. The frozen inoculum bottles, prepared in advance, were defrosted for approximately 2 hours prior to use. Cells from the defrosted bottle were removed with a 50 ml syringe, then transferred into a 2 L fermenter (1 L working volume) containing sterile solid substrates and CC8 growth nutrients, previously flushed with nitrogen to remove oxygen.

[0107] For T. saccharolyticum (Strain Y) and T. thermosaccharolyticum (Strain Z), previously frozen aliquots of cells were defrosted in an anaerobic glove bag, then 50 microliters were transferred into 5 ml of CTFUD medium at pH 7, in a 14 ml snap cap tube. The tube was incubated at 55 degrees Celsius in the glove bag for about 16 hours. Then approximately 1 mL was used to inoculate between 10 and 50 mL of CTFUD medium in snap-cap or screw-cap tubes,Attorney Docket No. TBFI.P2003WO / 00609658 which were incubated in the glove bag at 55 degrees C for 3-6 additional hours, or until the optical density was approximately 1.0. The designated amount of each inoculum was then transferred via syringe into the same bioreactor in which C. thermocellum was previously inoculated. Mixed biomass co-fermentations using co-cultures

[0108] To determine the optimum conditions to utilize corn stover and corn kernels to make ethanol, corn stover and / or corn kernels were mixed in a fermentation vessel at a concentration or ratio as shown in Table 1. The mixture was sterilized by autoclaving or steam treatment without being subject to any other pretreatment. The pH of the mixture was then adjusted to 6.6. Temperature was then adjusted to 55 degrees Celsius, a temperature suitable for a co-culture of C. thermocellum, T. saccharolyticum and / or T. thermosaccharolyticum to grow. Pre-grown inocula of two strains (Strains X and Y described above) or three strains (Strains X, Y, and Z described above) were next added to the 2 L vessel at 5% (50 mL of Strain X inoculum), 1% (10 mL of Strain Y inoculum) and 1% (10 mL of Strain Z inoculum) of the total fermentation working volume (1 L). The mixture of biomass and microorganisms was allowed to ferment in the vessel for up to 7 days. The fermentation broth and the solids were then separated and recovered by centrifugation and the ethanol concentration in the broth was measured by HPLC. The solid residue was dried and the sugar content was analyzed by complete acid hydrolysis followed by HPLC (Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D.2008. Determination of structural carbohydrates and lignin in biomass. US Technical Report NREL / TP-510-42618.).

[0109] Experimental results are shown in Table 1 and demonstrate higher ethanol production in the co-fermentation process relative to that expected from the industry standard process based on yeast.Attorney Docket No. TBFI.P2003WO / 00609658

[0110] Table 1. Experimental results using the methods of the present disclosure for co-fermenting corn stover and corn kernels, compared to separate fermentation of the same substrates.*Engineered strains described above: X=C. thermocellum; Y=T. saccharolyticum; Z=T. thermosaccharolyticum ** Predicted ethanol concentrations were calculated based on the accepted industry standard yield of 2.85 gallons ethanol per bushel corn kernels at 15.5% moisture.

[0111] The results in Table 1 show that the final ethanol concentration using engineered thermophilic bacterial co-cultures was higher than that predicted if the same material was fermented using yeast and conventional technology for corn fermentation. Such optimized, state-of-the-art industrial technology typically yields ca.2.85 gallons of ethanol per bushel of corn. In the case of corn kernels alone (Row 1), the ethanol concentration using thermophilic co-cultures was 16% higher than the maximum ethanol titer predicted if yeast was used. The final ethanol concentration in rows 4-7 with co-cultures and mixed corn kernels and stover is much higher than could be expected from yeast, because yeast cannot ferment lignocellulose.

[0112] The expected final concentration of ethanol from the present disclosure can be calculated from the data obtained when corn stover and corn kernels were fermented separately, shown in rows 1 and 2 of Table 1. It is predicted that 27.3 g / L ethanol would be produced in a fermentation of 40 g / L kernels + 40 g / L stover, based on those values (18.4 + 17.8 / 2). The result shown in row 5 of Table 1 is consistent with that calculated prediction when the tri-culture is used. RIN Credit Calculation

[0113] The present disclosure produces ethanol from two different feedstocks. In example 1, those feedstocks are: 1) corn kernels, which are predominantly starch; and 2) corn stover, which is predominantly fiber (Table 2).Attorney Docket No. TBFI.P2003WO / 00609658

[0114] Table 2. Carbohydrate breakdown of corn kernels, corn stover, and a mixture thereof.

[0115] The value of ethanol made from cellulosic feedstocks is higher than the value of ethanol made from sugar or starch feedstocks due to government incentives under the Renewable Fuel Standard, which is implemented with Renewable Identification Numbers (RIN) to identify the source of ethanol. D3 RINs designate ethanol produced from cellulose. Corn kernels contain a small amount of lignocellulose, approximately 10%. Conventional yeast-based processes used commercially for ethanol production are unable to access, degrade, and ferment lignocellulose. Co-fermenting corn kernels and corn stover with thermophilic bacterial co-cultures avoids this problem, and allows a straightforward calculation of what percent of the final product is cellulosic: A = B – (C * D * E) where A = cellulosic ethanol (gallons), B = total ethanol (gallons), C = mass of feedstock (tons), D = expected ethanol yield from corn kernels fermented by yeast (gallons / ton), E = fraction of feedstock that is corn kernels.

[0116] The benefits of co-fermentation of corn kernel and corn stover relative to the industry standard yeast fermentation process are illustrated in FIG.2. In this example, 50 g / L corn stover + 50 g / L corn kernels were simultaneously and directly co-fermented using the triculture of Strains X, Y, and Z, producing 33 g / L ethanol, as per Row 6 of Table 1. The expected ethanol concentration if those feedstocks were fermented with yeast is 20 g / L. Therefore, 13 g / L of the total 33 g / L ethanol can be considered cellulosic, for the purpose of assigning RIN values. Based on separate corn stover and corn kernel fermentations (rows 1 and 2 of Table 1), we expect 11 g / L of the 33 g / L total to come from the corn stover, and 23 g / L to come from the corn kernels. A portion of the 23 g / L from corn kernels, approximately 10%, comes from the small fraction of lignocellulose that occurs in corn kernels.Attorney Docket No. TBFI.P2003WO / 00609658 Example 2 Using wheat or rice as alternative source of biomass

[0117] Analogous experiments were conducted using (1) wheat grain + wheat straw, (2) rice grain + rice straw, or (3) rice grain + rice hulls using the same three co-culture strains (Strains X, Y, and Z) as in Example 1. Table 3 below shows the ethanol concentration after fermenting each individual component, and when they are combined, with the tri-culture of Strains X, Y, and Z, as defined in Table 1. This example shows three additional insoluble biomass feedstock mixtures were successfully co-fermented to produce ethanol concentrations that are higher when two biomass sources are combined relative to separate fermentations. While the efficiency of these co-fermentation reactions may be improved further to ensure all of the biomass sugars are converted and the maximum amount of ethanol is produced, these examples demonstrate the economic benefits of the CBP co-fermentation process that produces higher final ethanol titers from mixtures of less expensive feedstocks and far greater RIN credits relative to the industry standard yeast-based ethanol process.

[0118] This approach of showing how much ethanol comes from each substrate is an aspect of the invention. With government incentives (D3 RIN credits for cellulosic ethanol), there is a higher value for ethanol produced from lignocellulosic substrates compared to those from grains. Clarifying how much ethanol came from each biomass source is an advantage of this co- fermentation technology.

[0119] Table 3. Ethanol production using the methods of the present disclosure for co-fermenting a range of mixed insoluble biomass feedstocks compared with fermentation of individual feedstocks.Attorney Docket No. TBFI.P2003WO / 00609658

[0120] The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application or listed below are hereby expressly incorporated by reference in their entirety for any purpose into the present disclosure. The disclosure may employ, unless otherwise indicated, conventional techniques of microbiology, molecular biology and cell biology, which are well known in the art.

[0121] The disclosed methods may be modified without departing from the scope hereof. It should be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

Claims

Attorney Docket No. TBFI.P2003WO / 00609658 CLAIMS What is claimed is:

1. A method for producing ethanol by simultaneously co-fermenting two or more insoluble biomass sources, comprising: a) mixing the two or more insoluble biomass sources to form an insoluble biomass mixture, b) mixing the insoluble biomass mixture of step (a) with two or more thermophilic bacteria to form a fermentation mixture, c) simultaneously co-fermenting the two or more insoluble biomass sources in the presence of the two or more thermophilic bacteria, and d) collecting ethanol produced in step (c).

2. The method of claim 1, wherein the two or more insoluble biomass sources comprise a first insoluble biomass and a second insoluble biomass, said first insoluble biomass and said second insoluble biomass having different starch and fiber contents, wherein the first insoluble biomass has a fiber content that is at least 50% higher than the fiber content of the second insoluble biomass.

3. The method of claim 2, wherein the second insoluble biomass has a starch content that is at least 50% higher than the starch content of the first insoluble biomass.

4. The method of claim 3, wherein the two or more insoluble biomass sources are from the same plant source.

5. The method of claim 4, wherein the same plant source is selected from the group consisting of corn, wheat, and rice.

6. The method of claim 5, wherein the first insoluble biomass source is corn stover, and the second insoluble biomass source is corn kernel.

7. The method of claim 5, wherein the first insoluble biomass source is a fiber-enriched derivative of corn kernels and the second insoluble biomass source is corn kernel.

8. The method of claim 6, wherein the corn stover and corn kernel used in step (a) are directly from a whole corn plant without separating the corn kernel from the corn stover first.Attorney Docket No. TBFI.P2003WO / 00609658 9. The method of claim 6, wherein the weight ratio between the first insoluble biomass source and the second insoluble biomass source in step (a) is about 1:

1.

10. The method of claim 1, wherein the two or more thermophilic bacteria comprise two or more different genetically engineered strains, wherein the two or more different genetically engineered strains belong to different species.

11. The method of claim 10, wherein two or more genetically engineered strains belong to same genus.

12. The method of claim 10, wherein the two or more thermophilic bacteria comprise three genetically engineered strains that belong to three different species, the three different species being C. thermocellum, T. saccharolyticum, T. thermosaccharolyticum, respectively.

13. The method of claim 1, further comprising sterilizing the fermentation mixture, wherein the sterilization step is performed after step (a) but before step (b).

14. The method of claim 1, further comprising sterilizing the fermentation mixture, wherein the sterilization step is performed before step (a).

15. The method of claim 1, wherein the collecting step (d) comprises a distillation process.

16. The method of claim 1, wherein the method is conducted in either batch or continuous mode.

17. The method of claim 1, wherein the two or more insoluble biomass sources comprise a first insoluble biomass and a second insoluble biomass, said first insoluble biomass having more than 60% fiber, and said second insoluble biomass having more than 60% starch.

18. The method of claim 1 where the two or more insoluble biomass sources are from different genomically-defined plant species.

19. The method of claim 1 where the two or more insoluble biomass sources are subjected to a grinding or milling step prior to mixing in a vessel to form an insoluble biomass mixture in step (a).

20. The method of claim 6, wherein the corn stover and corn kernel from the corn plant are separated into corn kernel and corn stover before being mixed in step (a).

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