PRODUCTION OF ETHANOL FROM LIGNOCELLULOSIC BIOMASS

BACKGROUND OF THE INVENTION

Energy conversion, utilization and access underlie many of the great challenges of our time, including those associated with sustainability, environmental quality, security, and poverty. New applications of emerging technologies are required to respond to these challenges. Biotechnology, one of the most powerful of the emerging technologies, can give rise to important new energy conversion processes.

Plant biomass and derivatives thereof are a resource for the biological conversion of energy to forms useful to humanity. Among forms of plant biomass, lignocellulosic biomass (“biomass”) is particularly well-suited for energy applications because of its large-scale availability, low cost, and environmentally benign production. In particular, many energy production and utilization cycles based on cellulosic biomass have near-zero greenhouse gas emissions on a life-cycle basis. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful fuels. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol. In order to convert these fractions to ethanol, the cellulose and hemicellulose must initially be converted or hydrolyzed into monosaccharides; this hydrolysis has historically proven to be problematic.

Biologically mediated processes are promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations may occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that CBP does not involve a dedicated process step for cellulase and/or hemicellulase production.

CBP offers the potential for lower cost and higher efficiency than processes requiring dedicated cellulase production. The benefits result in part from avoided capital costs for substrate and other raw materials, and utilities associated with cellulase production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed cellulase systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic and xylanolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase and hemicellulase system enabling cellulose and hemicellulose utilization.

Many bacteria have the ability to ferment simple hexose sugars into a mixture of acidic and pH-neutral products via the process of glycolysis. The glycolytic pathway is ubiquitous and comprises a series of enzymatic steps whereby a six carbon glucose molecule is broken down, via multiple intermediates, into two molecules of the three-carbon compound pyruvate. From this point, however, the pyruvate can be metabolized via several different pathways, only one of which produces ethanol. The majority of facultative anaerobic bacteria do not produce high yields of ethanol from pyruvate under either aerobic or anaerobic conditions. Most facultative anaerobes metabolize pyruvate aerobically via pyruvate dehydrogenase (PDH) and the tricarboxylic acid cycle (TCA), ultimately producing CO₂, water, and ATP. Under anaerobic conditions, the main energy pathway for the metabolism of pyruvate is via the pyruvate-formate-lyase (PFL) pathway to give formate and acetyl-CoA. Acetyl-CoA is then converted to acetate, via phosphotransacetylase (PTA) and acetate kinase (ACK) with the co-production of ATP, or reduced to ethanol via acetalaldehyde dehydrogenase (AcDH) and alcohol dehydrogenase (ADH). In order to maintain a balance of reducing equivalents, excess NADH produced from glycolysis is re-oxidized to NAD⁺ by lactate dehydrogenase (LDH) during the reduction of pyruvate to lactate. NADH can also be re-oxidized by AcDH and ADH during the reduction of acetyl-CoA to ethanol, but this pathway typically plays a minor role in cells with a functional LDH. Theoretical yields of ethanol, therefore, are not achieved because most acetyl CoA is converted to acetate to regenerate ATP, and excess NADH produced during glycolysis is oxidized by LDH.

Metabolic engineering of microorganisms could result in the creation of a targeted knockout of the genes encoding for the production of enzymes, such as lactate dehydrogenase. In this case, “knock out” of the genes means partial, substantial, or complete deletion, silencing, inactivation, or down-regulation. If the conversion of pyruvate to lactate (the salt form of lactic acid) by the action of LDH were not available in the early stages of the glycolytic pathway, then the pyruvate could be more efficiently converted to acetyl CoA by the action of pyruvate dehydrogenase or pyruvate-ferredoxin oxidoreductase. If the further conversion of acetyl CoA to acetate (the salt form of acetic acid) by phosphotransacetylase and acetate kinase were also unavailable, e.g., if the genes encoding for the production of PTA and ACK were knocked out, then the acetyl CoA could be more efficiently converted to ethanol by AcDH and ADH. Accordingly, a genetically-modified strain of microorganism with such targeted gene knockouts, which would decrease or eliminate the production of organic acids, should have an increased ability to produce ethanol as a fermentation product.

Ideally, desirable characteristics of different microorganisms could be utilized simultaneously by fermenting lignocellulosic biomass with co-cultures of the microorganisms. However, the optimal conditions for fermentation of lignocellulosic biomass vary greatly from species to species. Under the most favorable conditions, monocultures of bacteria can replicate very quickly and efficiently produce the desired fermentation product. However, due to evolutionary pressure, when a co-culture of microorganisms is present, the species that can grow the fastest often dominates. Many variables influence the success of bacterial fermentation of lignocellulosic biomass, including but not limited to: temperature, pH, growth medium, and pre-treatment protocol. Identifying the small window of conditions suitable for co-culturing at least two microorganisms, while the organisms simultaneously ferment lignocellulosic biomass, presents a significant challenge.

SUMMARY OF THE INVENTION

In certain embodiments, the first microorganism is a cellulolytic microorganism. In certain embodiments, the second microorganism is a xylanolytic microorganism. In certain embodiments, the first microorganism is a cellulolytic microorganism; and the second microorganism is a xylanolytic microorganism. In certain embodiments, the first microorganism is a native cellulolytic microorganism. In certain embodiments, the second microorganism is a genetically engineered xylanolytic microorganism. In certain embodiments, the first microorganism is a native cellulolytic microorganism; and the second microorganism is a genetically engineered xylanolytic microorganism. In certain embodiments, the first microorganism is native Clostridium thermocellum. In certain embodiments, the second microorganism is a genetically engineered Thermoanaerobacterium saccharolyticum. In certain embodiments, the first microorganism is native Clostridium thermocellum; and the second microorganism is a genetically engineered Thermoanaerobacterium saccharolyticum.

In certain embodiments, the first microorganism is a xylanolytic microorganism. In certain embodiments, the second microorganism is a cellulolytic microorganism. In certain embodiments, the first microorganism is a xylanolytic microorganism; and the second microorganism is a cellulolytic microorganism. In certain embodiments, the first microorganism is a native xylanolytic microorganism. In certain embodiments, the second microorganism is a genetically engineered cellulolytic microorganism. In certain embodiments, the first microorganism is a native xylanolytic microorganism; and the second microorganism is a genetically engineered cellulolytic microorganism. In certain embodiments, the first microorganism is native Thermoanaerobacterium saccharolyticum. In certain embodiments, the second microorganism is a genetically engineered Clostridium thermocellum. In certain embodiments, the first microorganism is native Thermoanaerobacterium saccharolyticum; and the second microorganism is a genetically engineered Clostridium thermocellum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the fermentation product profiles of: (a) a co-culture of C. thermocellum with engineered T. saccharolyticum adapted to pH 7 and 60° C. (represented by squares) on 5% mixed hardwoods in serum bottles; and (b) C. thermocellum (represented by diamonds) monoculture on 5% mixed hardwoods in serum bottles.

FIG. 2 depicts a total product profile comparison of: (a) a co-culture of C. thermocellum with engineered T. saccharolyticum adapted to pH 7 and 60° C. on 2% unwashed mixed hardwoods (represented by X); (b) a co-culture of C. thermocellum with engineered T. saccharolyticum adapted to pH 7 and 60° C. on 2% washed mixed hardwoods (represented by squares); (c) C. thermocellum monoculture on 2% unwashed mixed hardwoods (represented by triangles); and (d) C. thermocellum monoculture on 2% washed mixed hardwoods (represented by diamonds).

FIG. 3 depicts an ethanol yield comparison of: (a) a co-culture of C. thermocellum with engineered T. saccharolyticum adapted to pH 7 and 60° C. on 2% unwashed mixed hardwoods (represented by X); (b) a co-culture of C. thermocellum with engineered T. saccharolyticum adapted to pH 7 and 60° C. on 2% washed mixed hardwoods (represented by squares); (c) C. thermocellum monoculture on 2% unwashed mixed hardwoods (represented by triangles); and (d) C. thermocellum monoculture on 2% washed mixed hardwoods (represented by diamonds).

FIG. 4 depicts growth curves as a function of time for: (a) unadapted T. saccharolyticum (MO 355) at 60° C. and pH 7 (represented by diamonds); (b) pH-adapted T. saccharolyticum (MO 521) at 60° C. and pH 7 (represented by squares); (c) pH-adapted T. saccharolyticum (MO 699) at 60° C. and pH 7 (represented by triangles); (d) pH-adapted T. saccharolyticum (MO 694) at 60° C. and pH 7 (represented by X); and (e) pH-adapted T. saccharolyticum (MO 728) at 60° C. and pH 7 (represented by asterisks).

FIG. 5 depicts ethanol yields obtained with T. saccharolyticum alone, C. thermocellum LDH KO 1313 alone, and C. thermocellum strains 27405 and LDH KO 1313, respectively, co-cultured with T. saccharolyticum.

FIG. 6 depicts total product yields obtained with T. saccharolyticum alone, C. thermocellum LDH KO 1313 alone, and C. thermocellum strains 27405 and LDH KO 1313, respectively, co-cultured with T. saccharolyticum.

FIG. 7 depicts variation of ethanol, acetate and total yield on 20 g/L Avicel of the residue that remains after yeast fermentation.

FIG. 8 depicts the fermentation profile of 80 g/L Avicel in a co-culture at 55° C. and pH 6.

FIG. 9 depicts the fermentation profile for a co-culture fermentation on 160 g/L Avicel.

FIG. 10 depicts ethanol concentration and yields at various Avicel concentrations.

FIG. 11 depicts the total product yield from unwashed MS 149 (milled and unmilled) at 20 g/L solids.

FIG. 12 depicts the theoretical ethanol yield from unwashed MS 149 (milled and unmilled) at 20 g/L solids.

FIG. 13 depicts the product distribution from unwashed MS 149 (milled and unmilled) at 20 g/L solids.

FIG. 14 depicts ethanol concentrations from 2-7.5% unwashed solids at times from 236 h to 400 h.

FIG. 15 depicts ethanol concentrations from 2-7.5% washed solids at times from 236 h to 400 h.

FIG. 16 depicts ethanol yields from 2-7.5% washed solids at times from 236 h to 400 h.

FIG. 17 depicts the final product concentrations from 5% MS 419 using a monoculture and co-culture.

FIG. 18 depicts product distribution from paper sludge at 100 g/L solids.

FIG. 19 depicts the product concentrations from 14.9% unwashed mixed hardwoods and 3% paper sludge in a co-culture.

FIG. 20 depicts results from a stable, mutualistic consortium co-culture intentionally contaminated with Geobacillus thermoglucosidiasus.

FIG. 21 depicts the product concentrations from mono- and co-cultures of C. thermocellum and T. thermosaccharolyticum.

FIG. 22 depicts product concentrations and yields on the transfer of co-cultures on Avicel, xylan, and xylose.

FIG. 23 depicts a comparison of the production of ethanol and exopolysaccharides (EPS) from a monoculture and co-culture.

DETAILED DESCRIPTION OF THE INVENTION
Overview

Aspects of the present invention relate to a process by which the efficiency and cost of ethanol production from cellulosic biomass-containing materials can be reduced by using a novel consolidated bioprocessing (CBP) methodology. In particular, the present invention provides numerous methods for increasing the efficiency of ethanol production from biomass by microorganisms.

One aspect of the invention relates to a method for the conversion of lignocellulosic biomass into ethanol utilizing co-cultures of at least two microorganisms. By exploiting certain desirable characteristics from each organism in the co-culture, unexpectedly high levels of ethanol are produced in comparison to the levels of ethanol produced in monocultures of the individual microorganisms. For example, because substrates often contain cellulose and xylan (hemi-cellulose) components, a microorganism capable of utilizing cellulose is combined with a microorganism capable of utilizing xylan in certain embodiments of the invention. In this respect, the efforts of the microorganisms are orthogonal, but complementary. Processes utilizing co-cultures, therefore, offer significant benefits over standard monoculture-based processes.

By virtue of a novel integration of processing steps, commonly known as consolidated bioprocessing, aspects of the present invention provide for more efficient production of ethanol from cellulosic-biomass-containing raw materials. One of the leading economic challenges in converting biomass to ethanol is the cost of additional enzymes that are typically added to the broth. One aspect of the present inventions provides for a process in which no external enzymes are added, thus making the process extremely cost-effective. Additionally, the incorporation of genetically-modified thermophilic or mesophilic microorganisms in the processing of said materials allows for fermentation steps to be conducted at higher temperatures, thereby improving process economics. For example, reaction kinetics are typically a function of temperature, so higher temperatures are generally associated with increases in the overall rate of production. Additionally, higher temperatures facilitate the removal of volatile products from the broth, and reduces the need for cooling of the substrate after pretreatment (a preceding step that is typically conducted at an elevated temperature).

Operating CBP processes at thermophilic temperatures offers several important benefits over conventional mesophilic fermentation temperatures of 30-37° C. In particular, costs associated with having a process step dedicated to cellulase production are eliminated for CBP. Costs associated with fermentor cooling and heat-exchange before and after fermentation are also expected to be reduced for CBP. Moreover, processes featuring thermophilic biocatalysts may be less susceptible to microbial contamination as compared to processes featuring conventional mesophilic biocatalysts. Additionally, combining strains of complementary microorganisms that are good at utilizing cellulose and xylan, respectively, and producing ethanol as the major product renders the processes more economical than current industrial processes because, for example, a greater proportion of the biomass is converted to ethanol, and added enzymes are not needed.

In one embodiment, the present invention provides for a method of converting to ethanol hardwoods pretreated by autohydrolysis via fermentation with a co-culture of a cellulolytic and xylanolytic microorganisms, without the use of exogenous enzymes.

DEFINITIONS

The term “expression” is intended to include the expression of a gene at least at the level of mRNA production.

The term “expression product” is intended to include the resultant product, e.g., a polypeptide, of an expressed gene.

The term “increased expression” is intended to include an alteration in gene expression at least at the level of increased mRNA production and, preferably, at the level of polypeptide expression. The term “increased production” is intended to include an increase in the amount of a polypeptide expressed, in the level of the enzymatic activity of the polypeptide, or a combination thereof.

The terms “activity,” “activities,” “enzymatic activity,” and “enzymatic activities” are used interchangeably and are intended to include any functional activity normally attributed to a selected polypeptide when produced under favorable conditions. Typically, the activity of a selected polypeptide encompasses the total enzymatic activity associated with the produced polypeptide. The polypeptide produced by a host cell and having enzymatic activity may be located in the intracellular space of the cell, cell-associated, secreted into the extracellular milieu, or a combination thereof. Techniques for determining total activity as compared to secreted activity are described herein and are known in the art.

The term “xylanolytic activity” is intended to include the ability to hydrolyze glycosidic linkages in oligopentoses and polypentoses.

The term “cellulolytic activity” is intended to include the ability to hydrolyze partially, substantially or completely cellulose or any of its constituents. Cellulolytic activity may also include the ability to depolymerize or debranch cellulose and hemicellulose.

The term “xylanolytic activity” is intended to include the ability to hydrolyze glycosidic linkages in oligopentoses and polypentoses.

As used herein, the term “lactate dehydrogenase” or “LDH” is intended to include the enzyme capable of converting pyruvate into lactate. It is understood that LDH can also catalyze the oxidation of hydroxybutyrate.

As used herein the term “alcohol dehydrogenase” or “ADH” is intended to include the enzyme capable of converting acetaldehyde into an alcohol, advantageously, ethanol.

The term “pyruvate decarboxylase activity” is intended to include the ability of a polypeptide to enzymatically convert pyruvate into acetaldehyde (e.g., “pyruvate decarboxylase” or “PDC”). Typically, the activity of a selected polypeptide encompasses the total enzymatic activity associated with the produced polypeptide, comprising, e.g., the superior substrate affinity of the enzyme, thermostability, stability at different pHs, or a combination of these attributes.

The term “ethanologenic” is intended to include the ability of a microorganism to produce ethanol from a carbohydrate as a fermentation product. The term is intended to include, but is not limited to, naturally occurring ethanologenic organisms, ethanologenic organisms with naturally occurring or induced mutations, and ethanologenic organisms which have been genetically modified.

The terms “fermenting” and “fermentation” are intended 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.

By “thermophilic” is meant an organism that thrives at a temperature of about 45° C. or higher.

By “mesophilic” is meant an organism that thrives at a temperature of about 20° C.-45° C.

The term “organic acid” is art-recognized. The term “lactic acid” refers to the organic acid 2-hydroxypropionic acid in either the free acid or salt form. The salt form of lactic acid is referred to as “lactate” regardless of the neutralizing agent, i.e., calcium carbonate or ammonium hydroxide. The term “acetic acid” refers to the organic acid methanecarboxylic acid, also known as ethanoic acid, in either free acid or salt form. The salt form of acetic acid is referred to as “acetate.”

The terms “lignocellulosic material,” “lignocellulosic substrate,” and “cellulosic biomass” mean any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and/or agricultural residues, forestry residues and/or forestry wastes, paper-production sludge and/or waste paper sludge, waste-water-treatment sludge, municipal solid waste, corn fiber from wet and dry mill corn ethanol plants, and sugar-processing residues.

The term “co-culture” means a mixture of at least two microorganisms that have been reproduced in predetermined culture media under controlled laboratory conditions, either together or separately.

Exemplary Methods

Aspects of the present invention relate to methods useful in the production of ethanol from lignocellulosic biomass substrates.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the first microorganism is a xylanolytic microorganism. In certain embodiments, the second microorganism is a cellulolytic microorganism. In certain embodiments, the first microorganism is a xylanolytic microorganism; and the second microorganism is a cellulolytic microorganism. In certain embodiments, the first microorganism is a native xylanolytic microorganism. In certain embodiments, the second microorganism is a genetically engineered cellulolytic microorganism. In certain embodiments, the first microorganism is a native xylanolytic microorganism; and the second microorganism is a genetically engineered cellulolytic microorganism. In certain embodiments, wherein the first microorganism is native Thermoanaerobacterium saccharolyticum. In certain embodiments, the second microorganism is a genetically engineered Clostridium thermocellum. In certain embodiments, wherein the first microorganism is native Thermoanaerobacterium saccharolyticum; and the second microorganism is a genetically engineered Clostridium thermocellum.

In certain embodiments, the invention relates to a method for converting lignocellulosic biomass to ethanol, comprising the step of contacting the lignocellulosic biomass with a mixture for a period of time at an initial temperature and an initial pH, thereby producing an amount of ethanol; wherein the mixture comprises at least two microorganisms; and at least one of the microorganisms comprises at least one genetic modification. In certain embodiments, the invention relates to a method utilizing one or more genetically-modified thermophilic or mesophilic microorganisms comprising a gene or a particular polynucleotide sequence that has been partially, substantially, or completely deleted, silenced, inactivated, or down-regulated, which gene or polynucleotide sequence encodes for an enzyme that confers upon the microorganism the ability to produce organic acids as fermentation products; thereby increasing the ability of the microorganism to produce ethanol as a fermentation product.

In certain embodiments, the invention relates to a method for converting lignocellulosic biomass to ethanol, comprising the step of contacting the lignocellulosic biomass with a mixture for a period of time at an initial temperature and an initial pH, thereby producing an amount of ethanol; wherein the mixture comprises at least two microorganisms; and at least one of the microorganisms comprises at least one genetic modification. In certain embodiments, the invention relates to a method utilizing one or more genetically-modified thermophilic or mesophilic microorganisms, wherein (a) a first native gene has been partially, substantially, or completely deleted, silenced, inactivated, or down-regulated, which first native gene encodes a first native enzyme involved in the metabolic production of an organic acid or a salt thereof, and (b) a first non-native gene has been inserted, which first non-native gene encodes a first non-native enzyme involved in the metabolic production of ethanol; thereby increasing the ability of said thermophilic or mesophilic microorganism to produce ethanol as a fermentation product.

In certain embodiments, the invention relates to a method for converting lignocellulosic biomass to ethanol, comprising the step of contacting the lignocellulosic biomass with a mixture for a period of time at an initial temperature and an initial pH, thereby producing an amount of ethanol; wherein the mixture comprises at least two microorganisms; and at least one of the microorganisms comprises at least one genetic modification. In certain embodiments, the invention relates to a method utilizing one or more genetically-modified thermophilic or mesophilic microorganisms, wherein (a) a first native gene has been partially, substantially, or completely deleted, silenced, inactivated, or down-regulated, which first native gene encodes a first native enzyme involved in the metabolic production of an organic acid or a salt thereof, and (b) a first non-native gene has been inserted, which first non-native gene encodes a first non-native enzyme involved in the hydrolysis of a polysaccharide; thereby increasing the ability of said thermophilic or mesophilic microorganism to produce ethanol as a fermentation product.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the amount of ethanol produced is at least about 60% of the theoretical yield based on the amount of lignocellulosic biomass metabolized.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the amount of ethanol produced is at least about 70% of the theoretical yield based on the amount of lignocellulosic biomass metabolized.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the amount of ethanol produced is at least about 80% of the theoretical yield based on the amount of lignocellulosic biomass metabolized.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the amount of ethanol produced is at least about 90% of the theoretical yield based on the amount of lignocellulosic biomass metabolized.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the period of time is about 10 hours to about 300 hours.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the period of time is about 50 hours to about 200 hours.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the period of time is about 80 hours to about 160 hours.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the period of time is about 80 hours (h), about 85 h, about 90 h, about 95 h, about 100 h, about 105 h, about 110 h, about 115 h, about 120 h, about 125 h, about 130 h, about 135 h, about 140 h, about 145 h, about 150 h, about 155 h, or about 160 h.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the period of time is about 120 hours.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial temperature is about 30° C. to about 75° C.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial temperature is about 45° C. to about 75° C.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial temperature is about 55° C. to about 65° C.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial temperature is about 60° C.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial pH is between about 5 and about 9.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial pH is between about 6 and about 8.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial pH is about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial pH is about 6, about 6.5, about 7, about 7.5, or about 8.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the initial pH is about 7 or about 7.5.

In one embodiment, the invention relates to a method for converting lignocellulosic biomass to ethanol, comprising the step of contacting the lignocellulosic biomass with a mixture for a period of time at an initial temperature and an initial pH, thereby producing an amount of ethanol; wherein the mixture comprises a first microorganism and a second microorganism; the first microorganism is selected from the group consisting of Clostridium thermocellum, Clostridium cellulolyticum, Thermoanaerobacterium saccharolyticum, Clostridium stercorarium, Clostridium stercorarium II, Caldiscellulosiruptor kristjanssonii, and Clostridium phytofermentans; and the second microorganism is selected from the group consisting of Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobium brockii, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter ethanolicus, Thermoanaerobacter brocki, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium phytofermentans, Clostridium straminosolvens, Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, Caldicellulosiruptor acetigenus, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus, and Anaerocellum thermophilum.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the first microorganism is Clostridium thermocellum; and the second microorganism is Thermoanaerobacterium saccharolyticum.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the first microorganism or the second microorganism comprises at least one genetic modification.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the second microorganism comprises at least one genetic modification.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein the first microorganism comprises at least one genetic modification.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein at least one of the microorganisms is a genetically-modified thermophilic or mesophilic microorganism comprising a gene or a particular polynucleotide sequence that has been partially, substantially, or completely deleted, silenced, inactivated, or down-regulated, which gene or polynucleotide sequence encodes for an enzyme that confers upon the microorganism the ability to produce organic acids as fermentation products; thereby increasing the ability of the microorganism to produce ethanol as a fermentation product.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein at least one of the microorganisms is a genetically-modified thermophilic or mesophilic microorganism comprising (a) a first native gene that has been partially, substantially, or completely deleted, silenced, inactivated, or down-regulated, which first native gene encodes a first native enzyme involved in the metabolic production of an organic acid or a salt thereof, and (b) a first non-native gene that has been inserted, which first non-native gene encodes a first non-native enzyme involved in the metabolic production of ethanol; thereby increasing the ability of said thermophilic or mesophilic microorganism to produce ethanol as a fermentation product.

In certain embodiments, the invention relates to any one of the above-mentioned methods, wherein at least one of the microorganisms is a genetically-modified thermophilic or mesophilic microorganism comprising (a) a first native gene that has been partially, substantially, or completely deleted, silenced, inactivated, or down-regulated, which first native gene encodes a first native enzyme involved in the metabolic production of an organic acid or a salt thereof, and (b) a first non-native gene that has been inserted, which first non-native gene encodes a first non-native enzyme involved in the hydrolysis of a polysaccharide; thereby increasing the ability of said thermophilic or mesophilic microorganism to produce ethanol as a fermentation product.