WO2010014817A2

WO2010014817A2 - Producing fermentation products

Info

Publication number: WO2010014817A2
Application number: PCT/US2009/052266
Authority: WO
Inventors: Jason Holmes; Randy Deinhammer; Zhengfang Kang; Chee-Leong Soong
Original assignee: Novozymes A/S
Priority date: 2008-07-30
Filing date: 2009-07-30
Publication date: 2010-02-04
Also published as: WO2010014817A3

Abstract

A process of fermenting plant material in a fermentation medium into a fermentation product using a fermenting organism, wherein one or more transketolase enzymes/ thiaminepyrophosphate, or mixtures thereof are added to the fermentation medium is described. The fermentation constituents increase ethanol yield and/or promote yeast quality or yeast proliferation.

Description

PRODUCING FERMENTATION PRODUCTS

BACKGROUND

1. Technical Field

The present disclosure relates to methods of fermenting plant derived material into desired fermentation products and to processes of producing a fermentation product from plant material using one or more fermenting organisms, compositions, transgenic plants, and modified fermenting organisms. The present disclosure also relates to improving yeast quality and/or yeast proliferation in fermentation processes. The present disclosure also relates to increasing alcohol production in fermenting organisms through the addition of transketolase enzyme and/or cofactor thiaminepyrophosphate to fermentations.

2. Background

A vast number of commercial products that are difficult to produce synthetically are today produced by fermenting organisms. Such products including alcohols (e.g., ethanol, methanol, butanol, 1 ,3-propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂), and more complex compounds, including, for example, antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones. Fermentation is also commonly used in the consumable alcohol (e.g., beer and wine), dairy (e.g., in the production of yogurt and cheese), leather, and tobacco industries.

A vast number of processes of producing fermentation products, such as ethanol, by fermentation of sugars provided by degradation of starch-containing and/or lignocellulose- containing material are known in the art.

However, production of fermentation products, such as ethanol, from such plant materials is problematic in that it is costly, and there is a continuous need to maximize yield. Further, some processes are deficient in that they tend to stress the fermenting organism such as yeast resulting in poor fermenting organism quality and/or fermenting organism quantity or proliferation. It is thus desirable to have effective, more problem free, processes that can increase fermenting organism quality/quantity and/or increase the yield of the fermentation product and thereby be applied towards improving the environment and/or production costs.

SUMMARY

The present disclosure relates to methods of fermenting plant derived material such as fermentable sugars into a fermentation product. The present disclosure provides methods of fermenting sugars derived from plant material into a fermentation product in a fermentation medium using a fermenting organism by adding one or more constituents including transketolase enzyme, thiaminepyrophosphate, or combinations thereof to the fermentation medium. Furthermore, the present disclosure relates to compositions comprising one or more transketolase enzymes, alone or in combination with thiaminepyrophosphate suitable for use in methods and fermentation processes in accordance with the present disclosure. Moreover, the disclosure relates to transgenic plants and modified fermenting organisms.

In accordance with the present disclosure the starting material (e.g., substrate for the fermenting organism in question) may be any plant material, especially plant derived material. The material may be treated and/or untreated. In embodiments, the starting material may be starch-containing material. In embodiments, the starting material may be lignocellulose- containing material.

A first aspect of the present disclosure relates to methods of fermenting sugars derived from plant material in a fermentation medium into a fermentation product using a fermenting organism, wherein one or more transketolase enzymes are present in the fermentation medium. In accordance with the present disclosure the concentration/dose level of transketolase enzymes is increased compared to when no transketolase enzymes are added before and/or during fermentation. In embodiments, one or more transketolase enzymes are added to the fermentation in an amount effective to increase the yield of the fermentation product. For example, alcohol production may be increased in alcohol fermenting organisms. In embodiments, one or more transketolase enzymes are added to the fermentation in an amount effective to improve fermenting organism (e.g. yeast) quality and/or proliferation.

A second aspect of the present disclosure relates to methods of fermenting sugars derived from plant material in a fermentation medium into a fermentation product using a fermenting organism, wherein one or more transketolase enzymes are present in the fermentation medium in combination with cofactor thiaminepyrophosphate. In accordance with the present disclosure, the concentration/dose level of one or more transketolase enzymes and cofactor thiaminepyrophosphate is increased compared to when no transketolase enzymes and cofactor thiaminepyrophosphate are added before and/or during fermentation. In embodiments, one or more transketolase enzymes in combination with cofactor thiaminepyrophosphate are added to the fermentation in an amount effective to increase the yield of the fermentation product. For example, alcohol production may be increased in alcohol fermenting organisms. In embodiments, one or more transketolase enzymes and thiaminepyrophosphate are added to the fermentation in an amount effective to improve fermenting organism (e.g. yeast) quality and/or proliferation.

A third aspect of the present disclosure relates to methods of fermenting sugars derived from plant material in a fermentation medium into a fermentation product using a fermenting organism, wherein cofactor thiaminepyrophosphate is present in or added to the fermentation medium. In accordance with the present disclosure, the concentration/dose level of cofactor thiaminepyrophosphate is increased compared to when no co-factor thiaminepyrophosphate is added before and/or during fermentation. In embodiments, cofactor thiaminepyrophosphate is added to the fermentation in an amount effective to increase the yield of the fermentation product. For example, alcohol production may be increased in alcohol fermenting organisms. In embodiments, thiaminepyrophosphate is added to the fermentation in an amount effective to improve fermenting organism (e.g. yeast) quality and/or proliferation.

A fourth aspect of the present disclosure relates to processes of producing a fermentation product from starch-containing material comprising the steps of: i) liquefying starch-containing material; ii) saccharifying the liquefied material; iii) fermenting with one or more fermenting organisms in accordance with a fermentation method of the present disclosure. In embodiments, one or more constituents comprising transketolase enzymes, co-factor thiaminepyrophosphate, and combinations thereof may be added before or during the fermenting step in an effective amount to increase the yield of the fermentation product or improve fermenting organism (e.g. yeast) characteristics including quality and/or proliferation.

A fifth aspect of the present disclosure relates to processes of producing a fermentation product from starch-containing material, comprising the steps of:

(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material,

(b) fermenting using a fermenting organism, wherein fermentation is carried out in accordance with a fermentation method of the present disclosure. It is to be understood that the starch-containing material is not subjected to liquefaction, such as a conventional liquefaction step. In embodiments, one or more constituents comprising one or more transketolase enzymes, co-factor thiaminepyrophosphate, and combinations thereof may be added before or during the fermenting step in an effective amount to increase the yield of the fermentation product or improve fermenting organism (e.g. yeast) characteristics including quality and/or proliferation.

A sixth aspect of the present disclosure relates to processes of producing a fermentation product from lignocellulose-containing material, comprising the steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolyzing the material;

(c) fermenting using a fermenting organism in accordance with a fermentation method of the present disclosure. In embodiments, one or more transketolase enzymes and/or co-factor thiaminepyrophosphate may be added before or during the fermenting step in an effective amount to increase the yield of the fermentation product. In embodiments, one or more constituents comprising transketolase enzymes, co-factor thiaminepyrophosphate, and combinations thereof may be added before or during the fermenting step in an effective amount to increase the yield of the fermentation product and/or improve fermenting organism (e.g. yeast) characteristics including quality and/or proliferation.

A seventh aspect of the present disclosure relates to a composition comprising one or more transketolase enzymes and/or thiaminepyrophosphate alone or in combination.

An eighth aspect of the present disclosure relates to the use of transketolase enzyme or compositions of the present disclosure in a fermentation method or process of the present disclosure. In embodiments, such compositions also include thiaminepyrophosphate.

A ninth aspect of the present disclosure relates to a transgenic plant material, wherein plant material has been transformed with a polynucleotide sequence encoding transketolase enzyme.

A tenth aspect of the present disclosure relates to modified fermenting organisms, wherein fermenting organisms have been transformed with a polynucleotide encoding a transketolase enzyme, wherein the fermenting organism is capable of expressing transketolase enzyme at fermentation conditions.

An aspect of the present disclosure relates to a method of fermenting sugars into a fermentation product in a fermentation medium using a fermenting organism comprising adding one or more constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof to the fermentation medium.

An aspect of the present disclosure relates to a method of fermenting sugars derived from plant material into a fermentation product in a fermentation medium using a fermenting organism comprising adding one or more constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof to the fermentation medium.

Additional aspects of the present disclosure are apparent from the detailed description below.

The term "e.g." refers generally to an abbreviation for the Latin phrase exempli gratia. As used herein, "e.g." refers to one or more non-limiting examples. The term is non-limiting in that the object that is exemplified is not limited in scope to the specific examples provided.

As used herein the terms "such as" is used to refer generally to one or more non-limiting examples. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 a is a chart showing ethanol produced after 24 hour fermentation of 10% TS acid- pretreated and unwashed PCS hydrolysate filtrate in accordance with example 1.

Fig. 1 b is a chart showing glucose after 24 hour fermentation of 10% TS acid-pretreated and unwashed PCS hydrolysate filtrate in accordance with example 1. Fig. 2 is a chart showing enhancement of fermentation rate and ethanol yield of thiamine pyrophosphate in one-step SSF process with AA1 and AMG A combination in accordance with example 2.

Fig. 3 is a chart showing that the addition of TPP increased the conversion of fructose in accordance with example 2.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It has now been found that the addition of one or more constituents such as transketolase enzymes and/or thiaminepyrophosphate to a fermentation medium effectively maximizes or increases the yield and rate at which fermenting organisms such as wild type and recombinant yeasts, bacteria, and fungi produce fermentation products such as alcohol (e.g. ethanol) or fuels from carbon-based substances. Further, it has been found that fermentation methods in accordance with the present disclosure may be carried out with increased solids content. This saves water in biomass fermentations while providing an increased carbon conversion to alcohol. The methods of the present disclosure are useful in obtaining or maintaining fermentation organism (e.g. yeast) quality or proliferation during fermentation. The present disclosure is also useful in obtaining higher alcohol (e.g. ethanol) yields in the conversion of corn or Zea mays.

Without being bound by any particular theory, it is believed that the addition of one or more transketolase enzymes and/or cofactor thiaminepyrophosphate each alone or in combination to a fermentation medium increases fermentation product production by enhancing or accelerating the metabolic pathways of the fermentation organism. It is believed that intracellular pathways that use these molecules may be rate limiting factors in reactions such as the conversion of fermentable sugars into alcohols such as ethanol.

Accordingly, the present disclosure provides, inter alia, methods of fermenting sugars derived from plant material into a fermentation product in a fermentation medium using a fermenting organism by adding one or more constituents including transketolase enzyme, thiaminepyrophosphate, or combinations thereof to the fermentation medium.

In embodiments, the methods in accordance with the present disclosure include one or more steps which alter the chemistry of the fermentation to make the fermentation more favorable, such as the addition of constituents such as thiamine pyrophosphate, magnesium, thiamine and/or adenosine triphosphate.

In embodiments, methods in accordance with the present disclosure add constituents to a fermentation medium in an effective amount to improve the quality of the fermenting organism. For example, constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof are added to a fermentation medium in an effective amount to promote yeast quality and quantity during fermentation. Accordingly, retention of yeast quantity during fermentation may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times higher than fermenting organism not contacted with the constituents in accordance with the present disclosure. Further, fermenting organism quality may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times better than a fermenting organism not contacted with the constituents in accordance with the present disclosure.

In embodiments, methods in accordance with the present disclosure add constituents to a fermentation medium in an effective amount to increase fermentation product yield. For example, in embodiments, constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof are added to a fermentation mediumin an effective amount to promote fermentation product yield. Accordingly, fermentation product yield may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times higher than a similar fermentation not contacted with the constituents in accordance with the present disclosure.

In embodiments, methods in accordance with the present disclosure add constituents to a fermentation medium in an effective amount to increase ethanol yield. For example, in embodiments, constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof are added to a fermentation medium in an effective amount to promote ethanol yield. Accordingly, ethanol yield may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times higher than a similar fermentation not contacted with the constituents in accordance with the present disclosure. Transketolase enzyme

In accordance with the present disclosure the term transketolase refers generally to enzymes classified in EC 2.2.1.1 (Transketolase). EC classes are based on recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). A description of EC classes can be found on the internet, e.g., see the website www.expasy.org/enzyme/. As used herein the term "TK" refers to transketolase enzyme as described herein. Transketolase enzymes are enzymes that may catalyze the following reaction: sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate = D-ribose 5-phosphate + D- xylulose 5-phosphate

In embodiments, the systematic name for transketolase enzymes suitable for use in accordance with the present disclosure is sedoheptulose-7-phosphate;D-glyceraldehyde-3- phosphate glycolaldehydetransferase.

Transketolase enzyme suitable for use in accordance with the present disclosure may be transketolase enzyme alone, or in combination with one or more additional transketolase enzymes. Suitable combinations of transketolase enzymes for use in accordance with the present disclosure may include a combination of two of more transketolase enzymes from any origin. Non-limiting examples of the origin of suitable transketolase enzymes includes mammalian, plant or microbial origin. Non-limiting examples of suitable microbial origins for use in accordance with the present disclosure include bacterial, fungal and/or yeast origin. In embodiments the transketolase enzyme is of fungal origin, such as of yeast origin. In embodiments the transketolase enzyme is derived from a strain of Saccharomyces, such as a strain of Saccharomyces cervisae.

Other non-limiting suitable origins of transketolase enzymes suitable for use in accordance with the present disclosure include those from Acinetobacter sp. (strain ADP1 ), Actinobacillus pleuropneumoniae serotype 5b, Aeromonas hydrophila, Aeropyrum pernix, Aspergillus fumigatus, Aspergillus niger, Bacillus cereus, Bacillus clausii, Bacillus licheniformis, Bacillus subtilis, Bacillus thuringiensis, Candida albicans, Escherichia coli, Homo sapiens, Pichia stipitis, Zea mays, Zymomonas mobilis, and combinations thereof. A description of many other suitable transketolase enzymes for use in accordance with the present disclosure can be found on the internet, See e.g., www.brenda- enzymes.info/php/result_flat.php4?ecno=2.2.1.1. See BRENDA, a collection of enzyme data available on the internet maintained and developed at the Institute of Biochemistry and Bioinformatics at the Technical University of Braunschweig, Germany.

Commercially available transketolase enzymes include those from Saccharomyces cerevisae available from Sigma-Aldrich (product # 90197 or product # T6133). Another available transketolase includes one from E. CoIi available from Sigma-Aldrich (product #88804). Thiamine pyrophosphate

Transketolase (TK) uses thiamine pyrophosphate (TPP), also known as thiamin diphosphate (ThDP) as its cofactor for catalysis. As used herein, the term cofactor refers generally to any non-protein substances that help an enzyme carry out its catalytic action. Suitable thiamine pyrophosphate for use in accordance with the present disclosure may be synthesized by the enzyme thiamin pyrophosphokinase, which requires free thiamin, magnesium, and adenosine triphosphate. The general chemical structure includes:

Thiamine diphosphate As used herein the term "TPP" refers to thiamine pyrophosphate or thiaminepyrophosphate in accordance with the present disclosure. Fermenting Organism

The term "fermenting organism" refers to any organism, including bacterial and fungal organisms, including yeast and filamentous fungi, suitable for producing a desired fermentation product. Especially suitable fermenting organisms according to the present disclosure are able to ferment, e.g., convert, sugars, glucose, xylose, fructose and/or maltose, directly or indirectly into the desired fermentation product. Non-limiting examples of fermenting organisms include fungal organisms, such as yeast. In embodiments, yeast includes strains of the genus Saccharomyces, in particular a strain of Saccharomyces cerevisiae or Saccharomyces uvarum; a strain of Pichia, in particular Pichia stipitis or Pichia pastoris; a strain of the genus Candida, in particular a strain of Candida utilis, Candida arabinofermentans, Candida diddensii, or Candida boidinii. Other contemplated yeast includes strains of Hansenula, in particular Hansenula polymorpha or Hansenula anomala; strains of Kluyveromyces in particular Kluyveromyces marxianus or Kluyveromyces fagilis, and strains of Schizosaccharomyces, in particular Schizosaccharomyces pombe.

In embodiments, non-limiting examples of bacterial fermenting organisms include strains of Escherichia, in particular Escherichia coli, strains of Zymomonas in particular Zymomonas mobilis, strains of Zymobacter in particular Zymobactor palmae, strains of Klebsiella in particular Klebsiella oxytoca, strains of Leuconostoc in particular Leuconostoc mesenteroides, strains of Clostridium in particular Clostridium butyricum, strains of Enterobacter in particular Enterobacter aerogenes and strains of Thermoanaerobacter, in particular Thermoanaerobacter BG1 L1 (Appl. Micrbiol. Biotech. 77, 61-86) and Thermoanarobacter ethanolicus.

In an embodiment the fermenting organism is a C6 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.

In connection with especially fermentation of lignocellulose derived materials C5 sugar fermenting organisms are contemplated. Most C5 sugar fermenting organisms also ferment C6 sugars. Non-limiting examples of C5 sugar fermenting organisms include strains of Pichia, e.g., Pichia stipitis. C5 sugar fermenting bacteria are also known. Also some Saccharomyces cerevisae strains ferment C5 (and C6) sugars. Non-limiting examples are genetically modified strains of Saccharomyces spp that are capable of fermenting C5 sugars include the ones concerned in, e.g., Ho et al. (1998), Applied and Environmental Microbiology, p. 1852-1859 and Karhumaa et al. (2006), Microbial Cell Factories, 5:18.

In embodiments, the fermenting organism is Clostridium phytophermentans or any fermenting organism used for consolidated bio processing. For example, the fermenting organism may be a strain known as the Q Microbe suitable for consolidated bio processing. Additional information relating to consolidated bio processing fermenting organisms can be found on the internet at the webpage www.csrees.usda. gov/nea/plants/pdfs/Leschine_20080123.pdf.

In embodiments, the fermenting organism is bacterium such as Saccharophagus degradans.

In embodiments the fermenting organism is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per ml. of fermentation medium is in the range from 1 x 10⁵ to 1 x 10¹², preferably from 1x 10⁷ to 1 x 10¹⁰, especially about 5x10⁷.

Non-limiting examples of commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

According to the present disclosure the fermenting organism capable of producing a desired fermentation product from fermentable sugars, such as, e.g., glucose, fructose maltose, xylose and/or arabinose, may be grown under precise conditions at a particular growth rate. When the fermenting organism is introduced into/added to the fermentation medium the inoculated fermenting organism pass through a number of stages. Initially growth does not occur. This period is referred to as the "lag phase" and may be considered a period of adaptation. During the next phase referred to as the "exponential phase" the growth rate gradually increases. After a period of maximum growth the rate ceases and the fermenting organism enters "stationary phase". After a further period of time the fermenting organism enters the "death phase" where the number of viable cells declines.

In embodiments transketolase enzyme(s) is(are) added to the fermentation medium when the fermenting organism is in lag phase.

In embodiments transketolase enzyme(s) added to the fermentation medium when the fermenting organism is in exponential phase.

In embodiments transketolase enzyme(s) is(are) added to the fermentation medium when the fermenting organism is in stationary phase.

In embodiments thiaminepyrophosphate is added to the fermentation medium when the fermenting organism is in lag phase.

In embodiments thiaminepyrophosphate is added to the fermentation medium when the fermenting organism is in exponential phase.

In embodiments thiaminepyrophosphate is added to the fermentation medium when the fermenting organism is in stationary phase. In embodiments one or more transketolase enzyme(s) and thiaminepyrophosphate is(are) added to the fermentation medium in effective amounts when the fermenting organism is in lag phase, exponential phase, or stationary phase. Fermentation Products

The term "fermentation product" as used herein refers to a product produced by a method or process including fermenting using a fermenting organism. Non-limiting examples of fermentation products contemplated according to the present disclosure include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones. In embodiments the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, e.g., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Non-limiting examples of beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. In embodiments, fermentation processes used include alcohol fermentation processes. The fermentation product, such as ethanol, obtained according to the present disclosure, may be used as fuel. However, in the case of ethanol it may also be used as potable ethanol. Fermentation Medium

The term "fermentation medium" refers to the environment in which fermentation is carried out. The fermentation medium may include the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism(s), and may include the fermenting organism(s).

The fermentation medium may comprise nutrients and/or growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; vitamins; and minerals, or combinations thereof. Fermentation

The plant starting material used in fermenting methods or processes of the present disclosure may be starch-containing material and/or lignocellulose-containing material. The fermentation conditions are determined based on, e.g., the kind of plant material, the available fermentable sugars, the fermenting organism(s) and/or the desired fermentation product. One skilled in the art can easily determine suitable fermentation conditions. The fermentation may according to the present disclosure be carried out at conventionally used conditions. In embodiments, fermentation processes are anaerobic processes. The methods or processes of the present disclosure may be performed as a batch or as a continuous process. Fermentations of the present disclosure may be conducted in an ultrafiltration system where the retentate is held under recirculation in the presence of solids, water, and the fermenting organism, and where the permeate is the desired fermentation product containing liquid. Equally contemplated are methods/processes conducted in continuous membrane reactors with ultrafiltration membranes and where the retentate is held under recirculation in presence of solids, water, the fermenting organism(s) and where the permeate is the fermentation product containing liquid.

The methods of the present disclosure include the addition of one or more constituents to the fermentation medium during or before fermentation. The constituents include transketolase enzyme, thiaminepyrophosphate, or combinations thereof added to the fermentation medium and contacted with the fermenting organism. The amount and type of constituent added can be adjusted depending upon the characteristics of the fermentation. For example, the size of the fermentation, pH, or type of fermenting organism may affect the amount of constituent added. In embodiments, one or more constituents are added in an effective amount. As used herein, "effective amount" refers to an amount sufficient to induce a positive benefit to the fermentation process. The positive benefit can be fermentation medium related, or it may be more chemical in nature, or it may be a combination of the two. For example, constituents may be added to the fermentation medium in an amount effective to improve an undesirable condition, improve fermentation product yield such as by altering or contributing to a biochemical pathway, improving the fermenting organism, or combinations of these benefits. In embodiments, the positive benefit is achieved by contacting the fermentation medium and/or fermenting organism with one or more transketolase enzymes, thiaminepyrophosphate, or combinations thereof to enhance fermentation product levels and/or increase yeast quality and/or proliferation. In embodiments, the positive benefit is achieved by contacting the fermentation medium with one or more constituents to increase ethanol yield in the fermentation process. In embodiments, the positive benefit is achieved by contacting the fermentation medium with one or more constituents to increase fermentation product yield in the fermentation process. The positive benefit can be achieved by adding the one or more constituents to the fermentation medium before and/or during fermentation.

In embodiments where the constituent is one or more transketolase enzymes, the transketolase is present in the fermentation medium or added thereto in an amount of 0.001 to 1000 Units per ml. of fermentation medium. In some embodiments, transketolase is present in the fermentation medium or added thereto in an amount of 0.001 to 100 Units per ml. of fermentation medium. In some embodiments, transketolase is present in the fermentation medium or added thereto in an amount of 0.001 to 10 Units per ml. of fermentation medium. In some embodiments, transketolase is present in the fermentation medium or added thereto in an amount of 0.01 to 5 Units per ml. of fermentation medium.

In embodiments where the constituent is thiaminepyrophosphate, the thiaminepyrophosphate is present in the fermentation medium or added thereto in an amount of 0.001 to 1000 mmol/L of fermentation medium. In some embodiments, thiaminepyrophosphate is present in the fermentation medium or added thereto in an amount of 0.001 to 100 mmol/L of fermentation medium. In some embodiments, thiaminepyrophosphate is present in the fermentation medium or added thereto in an amount of 0.01 to 100 mmol/L of fermentation medium. In some embodiments, thiaminepyrophosphate is present in the fermentation medium or added thereto in an amount of 0.01 to 10 mmol/L of fermentation medium. In some embodiments, thiaminepyrophosphate is present in the fermentation medium or added thereto in an amount of 0.01 to 5 mmol/L of fermentation medium. In embodiments, the thiaminepyrophosphate is present and/or added in an amount of about 10 mmol/L of fermentation medium.

In embodiments, both transketolase enzyme and thiaminepyrophosphate are added to or present in the fermentation medium. Non-limiting examples include fermentation mediums where transketolase is present in the fermentation medium or added thereto in an amount of 0.001 to 1000 Units per mL of fermentation medium and thiaminepyrophosphate is present in the fermentation medium or added thereto in an amount of 0.001 to 1000 mmol/L of fermentation medium. In some embodiments, transketolase is present in the fermentation medium or added thereto in an amount of 0.001 to 10 Units per mL of fermentation medium and thiaminepyrophosphate is present in the fermentation medium or added thereto in an amount of 0.01 to 10 mmol/L of fermentation medium. One of skill in the art would recognize that suitable amounts of transketolase and thiaminepyrophosphate used in combination include all combinations of all amounts listed above of each constituent individually. Further, the examples below specify suitable amounts for mixtures of the constituents for use in accordance with the present disclosure.

It is envisioned that after fermentation the fermenting organism may be separated from the fermented slurry and recycled. Fermentation of Starch-Derived Sugars

Different kinds of fermenting organisms may be used for fermenting sugars derived from starch-containing material. In embodiments, fermentations are conventionally carried out using yeast, such as Saccharomyces cerevisae, as the fermenting organism. However, bacteria and filamentous fungi may also be used as fermenting organisms. Some bacteria have higher fermentation temperature optimum than, e.g., Saccharomyces cerevisae. Therefore, fermentations may in such cases be carried out at temperatures as high as 75°C, e.g., between 40-70⁰C, such as between 50-60⁰C. However, bacteria with a significantly lower temperature optimum down to around room temperature (around 20⁰C) are also known. Non-limiting examples of suitable fermenting organisms are described above.

For ethanol production using yeast, the fermentation may in one embodiment go on for 24 to 96 hours, in particular for 35 to 60 hours. In an embodiment the fermentation is carried out at a temperature between 20 to 40⁰C, or 26 to 34°C, in particular embodiments around 30°C-32°C. In an embodiment the pH is from pH 3 to 6, or around pH 4 to 5. Other fermentation products may be fermented at temperatures known to the skilled person in the art to be suitable for the fermenting organism in question. For example, in some embodiments (such as where Kluyveromyces is selected as yeast) the fermentation is carried out at a temperature from 40 to 60⁰C, or around 45°C.

In embodiments, fermentation is carried out at a temperature about 2O⁰C to about 4O⁰C, and in some embodiments about 26⁰C to about 34⁰C, and in some embodiments about 3O⁰C.

In embodiments, fermentation is carried out at a temperature from about 4O⁰C to about 9O⁰C, or from about 6O⁰C to about 8O⁰C, or about 7O⁰C.

Fermentations are typically carried out at a pH in the range between 3 and 7, and in embodiments from pH 3.5 to 6, such as around pH 5. Fermentations are typically ongoing for 6-130 hours, and in embodiments 24-96 hours. Fermentation of Lignocellulose-Derived Sugars

Different kinds of fermenting organisms may be used for fermenting sugars derived from lignocellulose-containing materials. Fermentations are typically carried out by yeast, bacteria or filamentous fungi, including the fermenting organism mentioned above. If the aim is C6 fermentable sugars the conditions are usually similar to starch fermentations as described above. However, if the aim is to ferment C5 sugars (e.g., xylose) or a combination of C6 and C5 fermentable sugars the fermenting organism(s) and/or fermentation conditions may differ.

Bacteria fermentations may be carried out at higher temperatures, such as up to 75°C, e.g., between 40-70⁰C, such as between 50-60⁰C, than conventional yeast fermentations, which are typically carried out at temperatures from 20-40°C. However, bacteria fermentations at temperature as low as 20⁰C are also known. Fermentations are typically carried out at a pH in the range between 3 and 7, or from pH 3.5 to 6, such as around pH 5. Fermentations are typically ongoing for 6-130 hours, and in embodiments 24-96 hours. Recovery

Subsequent to fermentation the fermentation product may be separated from the fermentation medium. The fermentation medium may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively, the fermentation product may be recovered by stripping. Methods for recovery are well known in the art. Production of Fermentation Products from Starch-Containing Material

Processes for producing fermentation products from gelatinized starch-containing material are described below.

One aspect of the present disclosure relates to processes for producing a fermentation product, such as ethanol, from starch-containing material, which process includes a liquefaction step, and sequentially or simultaneously performed saccharification and fermentation steps.

Accordingly, the present disclosure relates to a process for producing a fermentation product from starch-containing material comprising the steps of: i) liquefying starch-containing material; ii) saccharifying the liquefied material; iii) fermenting using one or more fermenting organisms, wherein fermentation is carried out in accordance with a fermentation method of the present disclosure, e.g., in the presence of one or more transketolase enzymes, thiaminepyrophosphate or combinations thereof.

Saccharification step ii) and fermentation step iii) may be carried out either sequentially or simultaneously. The one or more transketolase enzymes or thiaminepyrophosphate may be added before and/or during the fermentation step iii) or before and/or during simultaneous saccharification and fermentation step.

The desired fermentation product, such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. Non-limiting examples of suitable starch- containing starting materials are described in the herein as starch-containing materials. Non- limiting examples of contemplated enzymes are described as enzymes herein. In embodiments, the liquefaction is carried out in the presence of an alpha-amylase, for example a bacterial alpha-amylase and/or acid fungal alpha-amylase. In embodiments, the fermenting organism is yeast, for example a strain of Saccharomyces cerevisiae. Non-limiting examples of suitable fermenting organisms are described as fermenting organisms herein.

In a particular embodiment, the process of the present disclosure further comprises, prior to the step i), the steps of: x) reducing the particle size of the starch-containing material, for example by milling; y) forming a slurry comprising the starch-containing material and water.

The aqueous slurry may contain from 10-55 wt.-% dry solids (DS), or 25-45 wt.-% dry solids (DS), or 30-40% dry solids (DS) of starch-containing material. The slurry is heated to above the gelatinization temperature and alpha-amylase, for example bacterial and/or acid fungal alpha-amylase may be added to initiate liquefaction (thinning). The slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to an alpha- amylase in step i) of the present disclosure.

Liquefaction may be carried out as a three-step hot slurry process. The slurry is heated to between 60-95⁰C, or 80-85⁰C, and alpha-amylase is added to initiate liquefaction (thinning). Then the slurry may be jet-cooked at a temperature between 95-140⁰C, or 105-125⁰C, for about 1-15 minutes, or for about 3-10 minutes, an in embodiments around about 5 minutes. The slurry is cooled to 60-95⁰C and more alpha-amylase is added to finalize hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at pH 4.5-6.5, or in embodiments at a pH from 5 to 6.

The saccharification step (ii) may be carried out using conditions well know in the art. For instance, a full saccharification step may last up to from about 24 to about 72 hours, however, it is also common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65°C, typically about 6O⁰C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation step (SSF process). Saccharification is typically carried out at temperatures from 20-75°C, or in embodiments from 40-70°C, typically around 6O⁰C, and at a pH between about 4 and 5, normally at about pH 4.5.

In embodiments of the present disclosure, and in processes of making ethanol, production includes simultaneous saccharification and fermentation (SSF), in which there is no holding stage for the saccharification. Here the fermenting organism(s), for example yeast, and enzyme(s), including one or more transketolase enzymes, thiaminepyrophosphate, or combinations thereof may be added together. SSF are typically carried out at temperatures from 20°C to 40°C, such as from 26°C to 34°C, or in embodiments around 32°C. According to the present disclosure the temperature may be adjusted up or down during fermentation.

In accordance with the present disclosure the fermentation step (iii) includes, without limitation, fermentation processes of the present disclosure used to produce fermentation products as described herein.

Processes for producing fermentation products from un-gelatinized starch-containing material are suitable for use in accordance with the present disclosure.

Accordingly another aspect of the present disclosure relates to processes for producing a fermentation product from starch-containing material without gelatinization (often referred to as "without cooking") of the starch-containing material. According to the present disclosure the desired fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material. In one embodiment a process of the present disclosure includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, or in the presence of alpha-amylase and/or carbohydrate-source generating enzyme(s) to produce sugars that can be fermented into the desired fermentation product by a suitable fermenting organism.

In embodiments the desired fermentation product, for example ethanol, is produced from un-gelatinized (e.g., uncooked), or milled, cereal grains, such as corn.

Accordingly, one aspect the present disclosure relates to processes of producing a fermentation product from starch-containing material comprising the steps of: (a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material,

(b) fermenting using a fermenting organism, wherein the fermentation is carried out in accordance with a fermentation process of the present disclosure, e.g., in the presence of one or more constituents such as transketolase enzymes, thiaminepyrophosphate, or combinations thereof.

In embodiments steps (a) and (b) are carried out simultaneously (e.g., one-step fermentation) or sequentially.

The fermentation product, for example ethanol, may optionally be recovered after fermentation, e.g., by distillation. Non-limiting examples of suitable starch-containing starting materials are further described below. Non-limiting examples of contemplated enzymes are further described below. Typically amylase(s), such as glucoamylase(s) and/or other carbohydrate-source generating enzymes and/or alpha-amylase(s), is(are) present during fermentation.

Non-limiting examples of glucoamylases and other carbohydrate-source generating enzymes can be found below and includes raw starch hydrolyzing glucoamylases.

Non-limiting examples of alpha-amylase(s) include acid alpha-amylases, for example acid fungal alpha-amylases.

Non-limiting examples of fermenting organisms include yeast, for example a strain of Saccharomyces cerevisiae. Other suitable non-limiting examples of fermenting organisms are described above as fermenting organisms.

The term "initial gelatinization temperature" means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50⁰C and 75°C; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In context of the present disclosure the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/Starke, Vol. 44 (12) pp. 461-466 (1992).

Before step (a) a slurry of starch-containing material, such as granular starch, having 10-55 wt.-% dry solids (DS), or 25-45 wt.-% dry solids, or 30-40% dry solids of starch- containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants. Because the process of the present disclosure is carried out below the initial gelatinization temperature and thus no significant viscosity increase takes place, high levels of stillage may be used if desired. In an embodiment the aqueous slurry contains from about 1 to about 70 vol.-%, or in embodiments 15-60% vol.-%, or in embodiments from about 30 to 50 vol.-% water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants, or combinations thereof, or the like.

The starch-containing material may be prepared by reducing the particle size, or by dry or wet milling, to 0.05 to 3.0 mm, or 0.1-0.5 mm. After being subjected to a method or process of the present disclosure at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or in embodiments at least 99% of the dry solids in the starch- containing material is converted into a soluble starch hydrolysate.

In a particular embodiment, the process of the present disclosure further comprises, prior to the step a), the steps of: x) reducing the particle size of the starch-containing material, for example by milling; y) forming a slurry comprising the starch-containing material and water.

A process of the present disclosure is conducted at a temperature below the initial gelatinization temperature, which means that the temperature at which step (a) is carried out typically lies in the range between 30-75°C, or in embodiments at a temperature of 45-6O⁰C.

In embodiments, steps (a) and (b) are carried out as a simultaneous saccharification and fermentation process. In such embodiments the process is typically carried at a temperature from 20⁰C to 40⁰C, for example from 26°C to 34°C, or in embodiments around 32°C.

In an embodiment, fermentation is carried out so that the sugar level, such as glucose level, is kept at a low level, such as below 6 wt.-%, such as below about 3 wt.-%, such as below about 2 wt.-%,such as below about 1 wt.-%., such as below about 0.5%, or below 0.25% wt.-%, such as below about 0.1 wt.-%. Such low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism. A skilled person in the art can easily determine which doses/quantities of enzyme and fermenting organism to use. The employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 wt.-%, such as below about 0.2 wt.-%.

The process of the present disclosure may be carried out at a pH from about 3 and 7, or in embodiments from pH 3.5 to 6, or more in embodiments from pH 4 to 5. Starch-Containing Materials

Any suitable starch-containing starting material, including granular starch (raw uncooked starch), may be used in accordance with the present disclosure. The starting material is generally selected based on the desired fermentation product. Non-limiting examples of starch- containing starting materials, suitable for use in methods or processes of the present disclosure, include tubers, roots, stems, whole grains, corns, cobs, wheat, barley, rye, millet, milo, sago, cassava, tapioca, sorghum, rice peas, beans, or sweet potatoes, yams, or mixtures thereof, or cereals. Also contemplated are waxy and non-waxy types of corn and barley.

The term "granular starch" means raw uncooked starch, e.g., starch in its natural form found in cereal, tubers or grains. Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to 50⁰C to 75°C the swelling may be reversible. However, with higher temperatures an irreversible swelling called "gelatinization" begins. Granular starch to be processed may be a highly refined starch quality, for example at least 90%, at least 95%, at least 97% or at least 99.5% pure or it may be a more crude starch-containing materials comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers. The raw material, such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure and allowing for further processing. In embodiments, two processes are suitable for use in accordance with the present disclosure: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in production of, e.g., syrups. Both dry and wet milling is well known in the art of starch processing and is equally contemplated for a process of the present disclosure. In embodiments the particle size is reduced to a size of 0.05 to 3.0 mm, or in embodiments 0.1- 0.5 mm, or so that at least 30%, or at least 50%, or at least 70%, or at least 90% of the starch- containing material fit through a sieve with a 0.05 to 3.0 mm screen, or in embodiments a 0.1- 0.5 mm screen.

Production of fermentation products from lignocellulose-containing material is suitable for use in accordance with the present disclosure.

The present disclosure relates to processes of producing fermentation products from lignocellulose-containing material. Conversion of lignocellulose-containing material into fermentation products, for example ethanol, has the advantages of the ready availability of large amounts of feedstock, including wood, agricultural residues, herbaceous crops, municipal solid wastes etc. Lignocellulose-containing materials typically primarily include cellulose, hemicellulose, and lignin and are often referred to as biomass.

The structure of lignocellulose is not directly accessible to enzymatic hydrolysis. Therefore, the lignocellulose-containing material has to be pre-treated, e.g., by acid hydrolysis under adequate conditions of pressure and temperature, in order to break the lignin seal and disrupt the crystalline structure of cellulose. This causes solubilization of the hemicellulose and cellulose fractions. The cellulose and hemicelluloses can then be hydrolyzed enzymatically, e.g., by cellulolytic and/or hemicellulolytic enzymes, to convert the carbohydrate polymers into fermentable sugars which may be fermented into desired fermentation products, for example ethanol. Optionally the fermentation product may be recovered, e.g., by distillation as also described above. Accordingly, one aspect of the present disclosure relates to processes of producing a fermentation product from lignocellulose-containing material, comprising the steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolyzing the material;

(c) fermenting with a fermenting organism in accordance with a fermentation method of the present disclosure, e.g., in the presence of one or more transketolase enzymes, thiaminepyrophosphate, or combinations thereof.

The one or more transketolase enzymes, thiaminepyrophosphate, or combinations thereof may be added before and/or during fermentation. Hydrolysis steps (b) and fermentation step (c) may be carried out sequentially or simultaneously. In embodiments the steps are carried out as SHF or HHF process steps which will be described further below.

In embodiments where pretreatment chemicals or processes stress the fermenting organism such as yeast, methods and composition of the present disclosure can improve the quality and quantity of the fermenting organism. SSF, HHF and SHF

Hydrolysis and fermentation can be carried out as a simultaneous hydrolysis and fermentation step (SSF). In general this means that combined/simultaneous hydrolysis and fermentation are carried out at conditions (e.g., temperature and/or pH) suitable, preferably optimal, for the fermenting organism(s) in question.

Hydrolysis and fermentation can also be carried out as hybrid hydrolysis and fermentation (HHF). HHF typically begins with a separate partial hydrolysis step and ends with a simultaneous hydrolysis and fermentation step. The separate partial hydrolysis step is an enzymatic cellulose saccharification step typically carried out at conditions (e.g., at higher temperatures) suitable, preferably optimal, for the hydrolyzing enzyme(s) in question. The subsequent simultaneous hydrolysis and fermentation step is typically carried out at conditions suitable for the fermenting organism(s) (often at lower temperatures than the separate hydrolysis step).

Hydrolysis and fermentation can also be carried out as separate hydrolysis and fermentation, where the hydrolysis is taken to completion before initiation of fermentation. This is often referred to as "SHF". Pre-treatment

The lignocellulose-containing material may according to the present disclosure be pre- treated before being hydrolyzed and fermented. In embodiments the pre-treated material is hydrolyzed, for example enzymatically, before and/or during fermentation. The goal of pretreatment is to separate and/or release cellulose, hemicellulose and/or lignin and this way improve the rate of enzymatic hydrolysis.

According to the present disclosure pre-treatment step (a) may be a conventional pre- treatment step known in the art. Pre-treatment may take place in aqueous slurry. The lignocellulose-containing material may during pre-treatment be present in an amount between 10- 80 wt. %, for example between 20-50 wt.-%. Chemical, Mechanical and/or Biological Pre-treatment

The lignocellulose-containing material may according to the present disclosure be chemically, mechanically and/or biologically pre-treated before hydrolysis and/or fermentation. Mechanical treatment (often referred to as physical pre-treatment) may be used alone or in combination with subsequent or simultaneous hydrolysis, especially enzymatic hydrolysis, to promote the separation and/or release of cellulose, hemicellulose and/or lignin.

In embodiments, the chemical, mechanical and/or biological pre-treatment is carried out prior to the hydrolysis and/or fermentation. Alternatively, the chemical, mechanical and/or biological pre-treatment is carried out simultaneously with hydrolysis, such as simultaneously with addition of one or more cellulolytic enzymes, or other enzyme activities mentioned below, to release fermentable sugars, such as glucose and/or maltose.

In an embodiment of the present disclosure the pre-treated lignocellulose-containing material is washed and/or detoxified before or after hydrolysis step (b). This may improve the fermentability of, e.g., dilute-acid hydrolyzed lignocellulose-containing material, such as corn stover. Detoxification may be carried out in any suitable way, e.g., by steam stripping, evaporation, ion exchange, resin or charcoal treatment of the liquid fraction or by washing the pre-treated material. Chemical Pre-treatment

According to the present disclosure "chemical pre-treatment" refers to any chemical treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin. Non-limiting examples of suitable chemical pre-treatment steps include treatment with; for example, dilute acid, lime, alkaline, organic solvent, ammonia, sulphur dioxide, carbon dioxide. Further, wet oxidation and pH-controlled hydrothermolysis are also contemplated chemical pre- treatments.

In embodiments, the chemical pre-treatment is acid treatment, for example, a continuous dilute and/or mild acid treatment, such as, treatment with sulfuric acid, or another organic acid, such as acetic acid, citric acid, tartaric acid, succinic acid, or mixtures thereof. Other acids may also be used. Mild acid treatment means in the context of the present disclosure that the treatment pH lies in the range from 1-5, for example from pH 1-3. In a specific embodiment the acid concentration is in the range from 0.1 to 2.0 wt % acid, for example sulphuric acid. The acid may be mixed or contacted with the material to be fermented according to the present disclosure and the mixture may be held at a temperature in the range of 160-220°C, for example 165-195⁰C, for periods ranging from minutes to seconds, e.g., 1-60 minutes, for example 2-30 minutes or 3-12 minutes. Addition of strong acids, such as sulphuric acid, may be applied to remove hemicellulose. This enhances the digestibility of cellulose.

Cellulose solvent treatment, also contemplated according to the present disclosure, has been shown to convert about 90% of cellulose to glucose. It has also been shown that enzymatic hydrolysis could be greatly enhanced when the lignocellulosic structure is disrupted. Alkaline H₂O₂, ozone, organosolv (uses Lewis acids, FeCI₃, (AI)₂SO₄ in aqueous alcohols), glycerol, dioxane, phenol, or ethylene glycol are among solvents known to disrupt cellulose structure and promote hydrolysis (Mosier et al. Bioresource Technology 96 (2005), p. 673-686).

Alkaline chemical pre-treatment with base, e.g., NaOH, Na₂CO₃ and/or ammonia or the like, is also within the scope of the present disclosure. Pre-treatment methods using ammonia are described in, e.g., WO 2006/110891 , WO 2006/11899, WO 2006/11900, WO 2006/110901 , which are hereby incorporated by reference in their entirety.

Wet oxidation techniques involve use of oxidizing agents, such as: sulphite based oxidizing agents or the like. Non-limiting examples of solvent pre-treatments include treatment with DMSO (Dimethyl Sulfoxide) or the like. Chemical pre-treatment is generally carried out for 1 to 60 minutes, such as from 5 to 30 minutes, but may be carried out for shorter or longer periods of time dependent on the material to be pre-treated.

Other non-limiting examples of suitable pre-treatment methods are described by Schell et al. (2003) Appl. Biochem and Biotechn. Vol. 105-108, p. 69-85, and Mosier et al. Bioresource Technology 96 (2005) 673-686, and US publication no. 2002/0164730, which references are hereby all incorporated by reference in their entirety. Mechanical Pre-treatment

As used in context of the present disclosure the term "mechanical pre-treatment" refers to any mechanical or physical pre-treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin from lignocellulose-containing material. Non-limiting examples of mechanical pre-treatment includes various types of milling, irradiation, steaming/steam explosion, and hydrothermolysis.

Mechanical pre-treatment includes comminution (mechanical reduction of the particle size). Comminution includes dry milling, wet milling and vibratory ball milling. Mechanical pre-treatment may involve high pressure and/or high temperature (steam explosion). In an embodiment of the present disclosure high pressure means pressure in the amount of 300 to 600 psi, for example 400 to 500 psi, or for example around 450 psi. In an embodiment of the present disclosure high temperature means temperatures in the amount of from about 100 to 300°C, for example from about 140 to 235⁰C. In embodiments, mechanical pre-treatment is a batch-process, steam gun hydrolyzer system which uses high pressure and high temperature as defined above. A Sunds Hydrolyzer (available from Sunds Defibrator AB (Sweden) may be used for this. Combined Chemical and Mechanical Pre-treatment

In embodiments of the present disclosure, both chemical and mechanical pre-treatments are carried out involving, for example, both dilute or mild acid pretreatment and high temperature and pressure treatment. The chemical and mechanical pretreatment may be carried out sequentially or simultaneously, as desired.

Accordingly, in embodiments, the lignocellulose-containing material is subjected to both chemical and mechanical pre-treatment to promote the separation and/or release of cellulose, hemicellulose and/or lignin.

In embodiments the pre-treatment is carried out as a dilute and/or mild acid steam explosion step. In embodiments, pre-treatment is carried out as an ammonia fiber explosion step (or AFEX pretreatment step). Biological Pre-treatment

As used in the present disclosure the term "biological pre-treatment" refers to any biological pre-treatment which promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the lignocellulose-containing material. Biological pre-treatment techniques can involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T. -A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh, P., and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241 ; Olsson, L., and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331 ; and Vallander, L., and Eriksson, K. -E. L., 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

In embodiments, biological pre-treatment involves applying lignin degrading enzymes to lignin or pretreated material. Non-limiting examples of suitable lignin degrading enzymes include one or more lignolytic enzymes, one or more oxidoreductases, and combinations thereof. Non-limiting examples of lignolytic enzymes include manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, and combinations thereof. Non-limiting examples of suitable pretreatment enzymes also include one ore more laccases, cellobiose dehydrogenases and combinations thereof. In embodiments, lignin peroxidase such as "ligninase", EC number 1.14.99, is suitable for use in accordance with the present disclosure.

In one embodiment, Ethazyme™ Pre available from Zymetis is suitable for use in pretreatment in accordance with the present disclosure. Hydrolysis

Before and/or during fermentation the pre-treated lignocellulose-containing material may be hydrolyzed in order to break the lignin seal and disrupt the crystalline structure of cellulose. In embodiments, hydrolysis is carried out enzymatically. According to the present disclosure the pre-treated lignocellulose-containing material to be fermented may be hydrolyzed by one or more hydrolases (class E. C. 3 according to Enzyme Nomenclature), for example one or more carbohydrases including cellulolytic enzymes and hemicellulolytic enzymes, or combinations thereof. Further, protease, alpha-amylase, glucoamylase and/or the like may also be present during hydrolysis and/or fermentation as the lignocellulose-containing material may include some, e.g., starchy and/or proteinaceous material.

The enzyme(s) used for hydrolysis may be capable of directly or indirectly converting carbohydrate polymers into fermentable sugars, for example glucose and/or maltose, which can be fermented into a desired fermentation product, such as ethanol.

In embodiments, the carbohydrase(s) has(have) cellulolytic and/or hemicellulolytic enzyme activity.

In embodiments, hydrolysis is carried out using a cellulolytic enzyme preparation further including one or more polypeptides having cellulolytic enhancing activity. In embodiments, the polypeptide(s) having cellulolytic enhancing activity is(are) of family GH61A origin. Non-limiting examples of cellulolytic enzyme preparations and polypeptides having cellulolytic enhancing activity suitable for use in accordance with the present disclosure are described herein as cellulolytic enzymes and cellulolytic enhancing polypeptides.

Non-limiting suitable enzymes are described herein. Hemicellulose polymers can be broken down by hemicellullolytic enzymes and/or acid hydrolysis to release its five and six carbon sugar components. The six carbon sugars (hexoses), such as glucose, galactose, arabinose, and mannose, can readily be fermented to fermentation products such as ethanol, acetone, butanol, glycerol, citric acid, fumaric acid etc. by suitable fermenting organisms including yeast.

In embodiments, yeast is a suitable fermenting organism for ethanol fermentation. For example, strains of Saccharomyces, or strains of the species Saccharomyces cerevisiae, or in embodiments strains which are resistant towards high levels of ethanol, e.g., up to, about 10, 12, 15 or 20 vol. % or more ethanol are suitable for use in accordance with the present disclosure.

Enzymatic hydrolysis may be carried out in a suitable aqueous environment under conditions which can readily be determined by one skilled in the art. In embodiments, hydrolysis is carried out at suitable, or optimal, conditions for the enzyme(s) in question. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art. Hydrolysis is carried out at a temperature between 25⁰C and 7O⁰C, for example between 40 and 6O⁰C, or, in embodiments around 5O⁰C. In embodiments, the step may be carried out at a pH in the range from 3-8, for example pH 4-6. Hydrolysis may typically be carried out for between 12 and 96 hours, for example 16 to 72 hours, or in embodiments between 24 and 48 hours.

Fermentation of lignocellulose derived material is carried out in accordance with a fermentation method of the present disclosure as described above. Liqnocellulose-Containinq Material (Biomass)

Any suitable lignocellulose-containing material is contemplated for use in accordance with the present disclosure. Lignocellulose-containing material may be any material containing lignocellulose. In embodiments, the lignocellulose-containing material contains at least 50 wt. - %, for example at least 70 wt.-%, or in embodiments at least 90 wt.-% lignocellulose. It is to be understood that the lignocellulose-containing material may also include other constituents for example cellulosic material, e.g., cellulose, hemicellulose and may also include constituents like sugars, for example fermentable sugars and/or un-fermentable sugars.

Ligno-cellulose-containing material is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. Lignocellulosic material can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues. In embodiments, lignocellulose-containing material may be in the form of plant cell wall material containing lignin, cellulose, and hemi-cellulose in a mixed matrix.

In embodiments, the lignocellulose-containing material is corn fiber, corn cobs, pine wood, wood chips, poplar, wheat straw, switchgrass, bagasse, paper and pulp processing waste.

Other non-limiting examples of lignocellulose-containing material for use in accordance with the present disclosure include corn stover, corn fiber, hardwood, such as poplar and birch, softwood, cereal straw, such as, wheat straw, switchgrass, municipal solid waste (MSW), industrial organic waste, office paper, or mixtures thereof.

In embodiments, the material is corn stover, corn fiber or combinations thereof. Enzymes

Even if not specifically mentioned in context of a method or process of the present disclosure, it is to be understood that enzyme(s) is(are) used in an effective amount. For example, an effective amount of enzyme may refer to an amount of one or more enzyme(s) in accordance with the present disclosure sufficient to induce a particular positive benefit to processes in accordance with the present disclosure. The positive benefit can be activity- related for example activity towards a substrate. Alpha-Amylase

In accordance with the present disclosure any alpha-amylase may be used, such as of fungal, bacterial or plant origin. For example the alpha-amylase may be an acid alpha-amylase, e.g., acid fungal alpha-amylase or acid bacterial alpha-amylase. The term "acid alpha-amylase" means an alpha-amylase (E. C. 3.2.1.1 ) which added in an effective amount has activity optimum at a pH in the range of 3 to 7, or in embodiments from 3.5 to 6, or in embodiments from 4-5. Bacterial Alpha-Amylase

In embodiments, suitable bacterial alpha-amylase for use in accordance with the present disclosure include those derived from the genus Bacillus.

In embodiments, the Bacillus alpha-amylase is derived from a strain of Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus stearothermophilus, but may also be derived from other Bacillus sp. Non-limiting examples of contemplated alpha- amylases include the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase SEQ ID NO: 5 in WO 99/19467 and the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 (all sequences hereby incorporated by reference in their entirety). In embodiments, the alpha- amylase may be an enzyme having a degree of identity of at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99% to any of the sequences shown in SEQ ID NOS: 1 , 2 or 3, respectively, in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid, especially one described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby incorporated by reference in their entirety). Specifically contemplated alpha-amylase variants are disclosed in US patent Nos. 6,093,562, 6,297,038 or US patent no. 6,187,576 (hereby incorporated by reference in their entirety) and include Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants having a deletion of one or two amino acid in positions R179 to G182, or a double deletion disclosed in WO 1996/023873 - see e.g., page 20, lines 1-10 (hereby incorporated by reference in its entirety), for example corresponding to delta(181-182) compared to the wild-type BSG alpha- amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids R179 and G180 using SEQ ID NO:3 in WO 99/19467 for numbering (which reference is hereby incorporated by reference in its entirety). Other non-limiting examples include Bacillus alpha-amylases, for example Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta(181-182) and further includes a N193F substitution (also denoted 1181^* + G182^* + N193F) compared to the wild-type BSG alpha- amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467. Bacterial Hybrid Alpha-Amylase

Bacterial hybrid alpha-amylase are suitable for use in accordance with the present disclosure. For example, a hybrid alpha-amylase specifically contemplated comprises 445 C- terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), with one or more, especially all, of the following substitution:

G48A+T49I+G107A+H156Y+A181T+N190F+I201 F+A209V+Q264S (using the Bacillus licheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Other non-limiting examples include variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha-amylase backbones): H154Y, A181T, N 190F, A209V and Q264S and/or deletion of two residues between positions 176 and 179, preferably deletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO 99/19467).

In an embodiment the bacterial alpha-amylase is dosed in an amount of 0.0005-5 KNU per g DS, or 0.001-1 KNU per g DS, or in embodiments around 0.050 KNU per g DS. Fungal Alpha-Amylase

Fungal alpha-amylases are suitable for use as enzymes in accordance with the present disclosure. Non-limiting examples include alpha-amylases derived from a strain of the genus Aspergillus, such as, Aspergillus oryzae, Aspergillus niger and Aspergillis kawachii alpha-amylases.

In embodiments, acidic fungal alpha-amylase includes a Fungamyl-like alpha-amylase which is derived from a strain of Aspergillus oryzae. According to the present disclosure, the term "Fungamyl-like alpha-amylase" indicates an alpha-amylase which exhibits a high identity, e.g. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.

Another non-limiting example of an acid alpha-amylase derived from a strain Aspergillus niger. In embodiments the acid fungal alpha-amylase is the one from Aspergillus niger disclosed as "AMYA_ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in WO 89/01969 (Example 3 - incorporated by reference in its entirety). In embodiments, a commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark) is suitable for use in accordance with the present disclosure.

Other non-limiting examples include contemplated wild-type alpha-amylases include those derived from a strain of the genera Rhizomucor and Meripilus, or a strain of Rhizomucor pusillus (See WO 2004/055178 incorporated by reference in its entirety) or Meripilus giganteus. In embodiments the alpha-amylase is derived from Aspergillus kawachii and disclosed by Kaneko et al. J. Ferment. Bioeng 81 :292-298(1996) "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachir, and further as EMBL: #AB008370.

In embodiments, the fungal alpha-amylase may also be a wild-type enzyme including a starch-binding domain (SBD) and an alpha-amylase catalytic domain (e.g., none-hybrid), or a variant thereof. In embodiments the wild-type alpha-amylase is derived from a strain of Aspergillus kawachii. Fungal Hybrid Alpha-Amylase

Fungal hybrid alpha-amylase enzymes are suitable for use in accordance with the present disclosure. In embodiments, the fungal acid alpha-amylase is a hybrid alpha-amylase. Non-limiting examples of fungal hybrid alpha-amylases for use in accordance with the present disclosure include the hybrid alpha-amylases disclosed in WO 2005/003311 or U.S. Patent Publication no. 2005/0054071 (Novozymes) or US patent application No. 60/638,614 (Novozymes) which is hereby incorporated by reference in its entirety. A hybrid alpha-amylase may include an alpha-amylase catalytic domain (CD) and a carbohydrate-binding domain/module (CBM), such as a starch binding domain, and optional a linker.

Non-limiting examples of contemplated hybrid alpha-amylases include those disclosed in Table 1 to 5 of the examples in US patent application no. 60/638,614, including Fungamyl variant with catalytic domain JA1 18 and Athelia rolfsii SBD (SEQ ID NO:100 in US 60/638,614), Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO:101 in US 60/638,614), Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO:20, SEQ ID NO:72 and SEQ ID NO:96 in US application no. 11/316,535) or as V039 in Table 5 in WO 2006/069290, and Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO:102 in US 60/638,614). Other non-limiting examples of hybrid alpha-amylases are any of those listed in Tables 3, 4, 5, and 6 in Example 4 in US application No. 1 1/316,535 and WO 2006/069290 (hereby incorporated by reference in their entirety).

Other non-limiting examples of contemplated hybrid alpha-amylases include those disclosed in U.S. Patent Publication no. 2005/0054071 , including those disclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and starch binding domain.

In embodiments, alpha-amylases include those which exhibit a high identity to any of above mention alpha-amylases, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequences. An acid alpha-amylases may according to the present disclosure be added in an amount of 0.001 to 10 AFAU/g DS, or in embodiments from 0.01 to 5 AFAU/g DS, or 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, or in embodiments 0.01 to 1 FAU-F/g DS. Commercial Alpha-Amylase Products

Commercial alpha-amylase enzymes are suitable for use in accordance with the present disclosure. Non-limiting examples of commercial compositions comprising alpha-amylase include MYCOLASE™ from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X, LIQUOZYME™ SC and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™ L-40,000, DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™ DELTA AA, and GC358™ (Genencor lnt.),FUELZYME™ (from Verenium Corp. USA), and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark). Carbohydrate-Source Generating Enzyme

As used herein the term "carbohydrate-source generating enzyme" includes glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators) and also pullulanase and alpha-glucosidase. A carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy- source by the fermenting organism(s) in question, for instance, when used in a process of the present disclosure for producing a fermentation product, for example ethanol. The generated carbohydrate may be converted directly or indirectly to the desired fermentation product, for example ethanol. According to the present disclosure a mixture of carbohydrate-source generating enzymes may be used. Especially contemplated blends are mixtures comprising at least a glucoamylase and an alpha-amylase, for example an acid amylase, or an acid fungal alpha-amylase. The ratio between glucoamylase activity (AGU) and acid fungal alpha- amylase activity (FAU-F) (e.g., AGU per FAU-F) may in embodiments of the present disclosure be in an amount of 0.1 and 100 AGU/FAU-F, or in embodiments 2 and 50 AGU/FAU-F, such as in an amount of 10-40 AGU/FAU-F, especially when doing one-step fermentation (Raw Starch Hydrolysis - RSH), e.g., when saccharification in step (a) and fermentation in step (b) are carried out simultaneously (e.g. without a liquefaction step).

In a conventional starch-to-ethanol process (e.g., including a liquefaction step (a)) the ratio may be as defined in EP 140,410-B1 , especially when saccharification in step ii) and fermentation in step iii) are carried out simultaneously. Glucoamylase

Glucoamylase enzymes are suitable for use in accordance with the present disclosure. Non-limiting examples include a glucoamylase derived from any suitable source, e.g., derived from a microorganism or a plant. Non-limiting examples of glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1 102), or variants thereof, such as those disclosed in WO 92/00381 , WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921 , Aspergillus oryzae glucoamylase (Agric. Biol. Chem. (1991 ), 55 (4), p. 941-949), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J. 301 , 275-281 ); disulphide bonds, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al. (1997), Protein Eng. 10, 1199-1204.

Other non-limiting examples of glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see US patent no. 4,727,026 and (Nagasaka,Y. et al. (1998) "Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (US patent no. 4,587,215).

Non-limiting examples of bacterial glucoamylases include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831 ) and Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289; or Peniophora rufomarginata disclosed in PCT/US2007/066618; or a mixture thereof. Also hybrid glucoamylase may be suitable for use in accordance with the present disclosure. Non-limiting examples include the hybrid glucoamylases disclosed in WO 2005/045018 and the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference in their entirety).

In embodiments, glucoamylases suitable for use in accordance with the present disclosure include those which exhibit a high identity to any of above mention glucoamylases, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzymes sequences mentioned above.

Non-limiting examples of commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME ULTRA™, and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480™ and GC147™ (from Genencor Int., USA): AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from Genencor Int.).

In embodiments, glucoamylases may be added in an amount of 0.0001-20 AGU/g DS, or in embodiments 0.001-10 AGU/g DS, or 0.01-5 AGU/g DS, for example 0.1-2 AGU/g DS. Beta-amylase

Beta-amylase enzymes are suitable for use in accordance with the present disclosure. A beta-amylase (E. C 3.2.1.2) is the name traditionally given to exo-acting maltogenic amylases, which catalyze the hydrolysis of 1 ,4-alpha-glucosidic linkages in amylose, amylopectin and related glucose polymers. Maltose units are successively removed from the non-reducing chain ends in a step-wise manner until the molecule is degraded or, in the case of amylopectin, until a branch point is reached. The maltose released has the beta anomeric configuration, hence the name beta-amylase.

Beta-amylases have been isolated from various plants and microorganisms (W. M. Fogarty and CT. Kelly, Progress in Industrial Microbiology, vol. 15, pp. 1 12-115, 1979). These beta-amylases are characterized by having optimum temperatures in the range from 40⁰C to 65°C and optimum pH in the range from 4.5 to 7. Non-limiting examples of beta-amylase suitable for use in accordance with the present disclosure include the commercially available beta-amylase from barley is NOVOZYM™ WBA from Novozymes A/S, Denmark and SPEZYME™ BBA 1500 from Genencor Int., USA. Maltogenic Amylase

Maltogenic amylase is an enzyme suitable for use in accordance with the present disclosure. A "maltogenic alpha-amylase" (glucan 1 ,4-alpha-maltohydrolase, E. C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. Non-limiting examples of maltogenic amylase includes those from Bacillus stearothermophilus strain NCIB 1 1837 which is commercially available from Novozymes A/S. Additional examples of maltogenic alpha-amylases include those described in US Patent nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference in their entity.

In embodiments, maltogenic amylase may be added in an amount of 0.05-5 mg total protein/gram DS or in embodiments in an amount of 0.05- 5 MANU/g DS. Cellulolvtic Activity

The term "cellulolytic activity" as used herein refers to enzymes having cellobiohydrolase activity. For non-limiting examples see enzyme classification (EC 3.2.1.91 ), as well as cellobiohydrolase I and cellobiohydrolase II, endo-glucanase activity (EC 3.2.1.4) and beta- glucosidase activity (EC 3.2.1.21).

At least three categories of enzymes are important for converting cellulose into fermentable sugars: endo-glucanases (EC 3.2.1.4) that cut the cellulose chains at random; cellobiohydrolases (EC 3.2.1.91) which cleave cellobiosyl units from the cellulose chain ends and beta-glucosidases (EC 3.2.1.21 ) that convert cellobiose and soluble cellodextrins into glucose. Among these three categories of enzymes involved in the biodegradation of cellulose, cellobiohydrolases seems to be the key enzymes for degrading native crystalline cellulose. The cellulolytic activity may, in embodiments, be in the form of a preparation of enzymes of fungal origin, such as from a strain of the genus Trichoderma, or a strain of Trichoderma reesei; a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosporium, or a strain of Chrysosporium lucknowense (see e.g., US publication # 2007/0238155 from Dyadic Inc, USA).

In embodiments, the cellulolytic enzyme preparation contains one or more of the following activities: cellulase, hemicellulase, cellulolytic enzyme enhancing activity, beta-glucosidase activity, endoglucanase, cellubiohydrolase, xylose-isomerase, or a combination thereof.

In embodiments, cellulolytic enzyme preparation is a composition concerned in co-pending application U.S. Application No. 60/941 ,251 , which is hereby incorporated by reference in its entirety. In embodiments, the cellulolytic enzyme preparation comprises a polypeptide having cellulolytic enhancing activity, for example a family GH61A polypeptide, or the one disclosed in WO 2005/074656 (Novozymes). The cellulolytic enzyme preparation may further comprise a beta-glucosidase, such as a beta-glucosidase derived from a strain of the genus Trichoderma, Aspergillus or Penicillium, including the fusion protein having beta-glucosidase activity disclosed in co-pending Application U.S. 60/832,511 (PCT/US2007/074038) (Novozymes). In embodiments, the cellulolytic enzyme preparation may also include a CBH Il enzyme, for example Thielavia terrestris cellobiohydrolase Il (CEL6A). In embodiments the cellulolytic enzyme preparation may also include cellulolytic enzymes, for example those derived from Trichoderma reesei, Humicola insolens and/or Chrysosporium lucknowense.

In embodiments, the cellulolytic enzyme preparation may also comprise a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; a beta- glucosidase (fusion protein disclosed in US 60/832,511 or PCT/US2007/074038) and cellulolytic enzymes derived from Trichoderma reesei.

In embodiments, the cellulolytic enzyme preparation may include a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; a beta-glucosidase (fusion protein disclosed in US 60/832,51 1 or PCT/US2007/074038); a CBH Il enzyme from Thielavia terrestris (CEL6A; and cellulolytic enzymes derived from Trichoderma reesei.

In embodiments, the cellulolytic enzyme composition is the commercially available product CELLUCLAST™ 1.5L, CELLUZYME™ (from Novozymes A/S, Denmark) or ACCELERASE™ 1000 (from Genencor Inc. USA).

The cellulolytic activity may be dosed in the amount of from 0.1-100 FPU per gram total solids (TS), or in embodiments 0.5-50 FPU per gram TS, or 1-20 FPU per gram TS. Endoglucanase (EG)

Endoglucanse is suitable enzyme for use in accordance with the present disclosure. As used herein the term "endoglucanase" refers to an endo-1 ,4-(1 ,3;1 ,4)-beta-D-glucan 4- glucanohydrolase (E. C. No. 3.2.1.4). Such enzymes catalyze endo-hydrolysis of 1 ,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta- D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity may be determined using carboxymethyl cellulose (CMC) hydrolysis according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268.

Non-limiting examples of endoglucanases include those derived from a strain of the genus Trichoderma, for example a strain of Trichoderma reesei; a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosporium, for example a strain of Chrysospoήum lucknowense. Cellobiohvdrolase (CBH)

Cellobiohydrolase enzymes are suitable for use in accordance with the present disclosure. As used herein the term "cellobiohydrolase" means a 1 ,4-beta-D-glucan cellobiohydrolase (E. C. 3.2.1.91 ). Such enzymes are able to catalyzes the hydrolysis of 1 ,4- beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain.

Non-limiting examples of cellobiohydroloses are mentioned above including CBH I and CBH Il from Trichoderma reseei; Humicola insolens and CBH Il from Thielavia terrestris cellobiohydrolase (CELL6A).

Cellobiohydrolase activity may be determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279 and by van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288. The Lever et al. method is suitable for assessing hydrolysis of cellulose in corn stover and the method of van Tilbeurgh et al. is suitable for determining the cellobiohydrolase activity on a fluorescent disaccharide derivative. Beta-glucosidase

Beta-glucosidase enzymes are suitable for use in accordance with the present disclosure especially during hydrolysis. As used herein term "beta-glucosidase" refers to a beta-D- glucoside glucohydrolase (E. C. 3.2.1.21 ). Such enzymes are typically suited to catalyze the hydrolysis of terminal non-reducing beta- D-g Iu cose residues with the release of beta-D- glucose. For purposes of the present disclosure, beta-glucosidase activity is determined according to the basic procedure described by Venturi et al., 2002, J. Basic Microbiol. 42: 55- 66, except different conditions were employed as described herein. One unit of beta- glucosidase activity is defined as 1.0 μmole of p-nitrophenol produced per minute at 50⁰C, pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate, 0.01% TWEEN® 20.

In embodiments beta-glucosidase in accordance with the present disclosure is of fungal origin, for example a strain of the genus Trichoderma, Aspergillus or Penicillium. Non-limiting examples of beta-glucosidase includes those derived from Trichoderma reesei, such as the beta-glucosidase encoded by the bgl1 gene (see Fig. 1 of EP 562003), or beta-glucosidase derived from Aspergillus oryzae (recombinantly produced in Aspergillus oryzae according to

WO 02/095014), Aspergillus fumigatus (recombinantly produced in Aspergillus oryzae according to Example 22 of WO 02/095014) or Aspergillus niger (1981 , J. Appl. VoI 3, pp 157-

163).

Hemicellulolvtic enzymes

Hemicellulolytic enzymes are suitable for use in accordance with the present disclosure. For example, the pre-treated lignocellulose-containing material may further be subjected to one or more hemicellulolytic enzymes, e.g., one or more hemicellulases.

Hemicellulose can be broken down by hemicellulases and/or acid hydrolysis to release its five and six carbon sugar components.

In an embodiment of the present disclosure the lignocellulose derived material may be treated with one or more hemicellulases.

Any hemicellulase suitable for use in hydrolyzing hemicellulose, for example into xylose, may be used. Non-limiting examples of hemicellulases include xylanases, arabinofuranosidases, acetyl xylan esterase, feruloyl esterase, glucuronidases, endo-galactanase, mannases, endo or exo arabinases, exo-galactanses, and mixtures of two or more thereof. In embodiments, the hemicellulase for use in the present disclosure is an exo-acting hemicellulase. In embodiments, the hemicellulase is an exo-acting hemicellulase which has the ability to hydrolyze hemicellulose under acidic conditions of below pH 7, or in embodiments pH 3-7. A non-limiting example of hemicellulase suitable for use in the present disclosure includes VISCOZYME™ (available from Novozymes A/S, Denmark).

In an embodiment the hemicellulase is a xylanase. In an embodiment the xylanase may be of microbial origin, such as of fungal origin (e.g., Trichoderma, Meripilus, Humicola, Aspergillus, Fusarium) or from a bacterium (e.g., Bacillus). In embodiments the xylanase is derived from a filamentous fungus, for example derived from a strain of Aspergillus, such as Aspergillus aculeatus; or a strain of Humicola, for example Humicola lanuginosa. The xylanase may be an endo-1 ,4-beta-xylanase, or an endo-1 ,4-beta-xylanase of GH10 or GH11. Non-limiting examples of commercial xylanases include SHEARZYME™ and BIOFEED WHEAT™ from Novozymes A/S, Denmark.

In embodiments, hemicellulase is added in an amount effective to hydrolyze hemicellulose, for example, in amounts from about 0.001 to 0.5 wt.-% of total solids (TS), or in embodiments in an amount of from about 0.05 to 0.5 wt.-% of TS.

Xylanases may be added in amounts of 0.001-1.0 g/kg DM (dry matter) substrate, or in the amounts of 0.005-0.5 g/kg DM substrate, or in embodiments from 0.05-0.10 g/kg DM substrate. Cellulolvtic Enhancing Activity As used herein the term "cellulolytic enhancing activity" refers to a biological activity that enhances the hydrolysis of a lignocellulose derived material by proteins having cellulolytic activity. For purposes of the present disclosure, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or in the increase of the total of cellobiose and glucose from the hydrolysis of a lignocellulose derived material, e.g., pre-treated lignocellulose- containing material by cellulolytic protein under the following conditions: 1-50 mg of total protein/g of cellulose in PCS (pre-treated corn stover), wherein total protein is comprised of 80- 99.5% w/w cellulolytic protein/g of cellulose in PCS and 0.5-20% w/w protein of cellulolytic enhancing activity for 1-7 day at 50⁰C compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS).

The polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a lignocellulose derived material catalyzed by proteins having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis by at least 0.1- fold, or at least 0.2-fold, or at least 0.3-fold, or at least 0.4-fold, or at least 0.5-fold, or at least 1- fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 100-fold.

In embodiments the hydrolysis and/or fermentation is carried out in the presence of a cellulolytic enzyme in combination with a polypeptide having enhancing activity. In embodiments, the polypeptide having enhancing activity is a family GH61A polypeptide. WO 2005/074647 discloses isolated polypeptides having cellulolytic enhancing activity and polynucleotides thereof from Thielavia terrestris. WO 2005/074656 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Thermoascus aurantiacus. U.S. Published Application Serial No. 2007/0077630 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Trichoderma reesei. Proteases

Protease enzymes are suitable for use in accordance with the present disclosure. For example, a protease may be added during hydrolysis in step ii), fermentation in step iii) or simultaneous hydrolysis and fermentation. The protease may be any protease. In embodiments the protease is an acid protease of microbial origin, for example of fungal or bacterial origin. In embodiments, an acid fungal protease is suitable for use in accordance with the present disclosure, but also other proteases can be used.

Non-limiting examples of suitable proteases include microbial proteases, for example fungal and bacterial proteases. In embodiments, proteases are acidic proteases, e.g., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7. Non-limited examples of acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand Torulopsis. Additional non-limiting examples include proteases derived from Aspergillus niger (see, e.g., Koaze et al., (1964), Agr. Biol. Chem. Japan, 28, 216), Aspergillus saitoi (see, e.g., Yoshida, (1954) J. Agr. Chem. Soc. Japan, 28, 66), Aspergillus awamori (Hayashida et al., (1977) Agric. Biol. Chem., 42(5), 927-933, Aspergillus aculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepA protease; and acidic proteases from Mucor pusillus or Mucor miehei.

Additional non-limiting examples of proteases include neutral or alkaline proteases, for example a protease derived from a strain of Bacillus. A particular protease contemplated for the present disclosure is protease derived from Bacillus amyloliquefaciens and has the sequence obtainable at Swissprot as Accession No. P06832. Also contemplated are the proteases having at least 90% identity to amino acid sequence obtainable at Swissprot as Accession No. P06832 such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.

Non-limiting examples of proteases also include the proteases having at least 90% identity to amino acid sequence disclosed as SEQ.ID.NO:1 in the WO 2003/048353 such as at 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.

Non-limiting examples of proteases also include papain-like proteases such as proteases within E.C. 3.4.22.^* (cysteine protease), such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricain).

In embodiments the protease is a protease preparation derived from a strain of Aspergillus, for example Aspergillus oryzae. In another embodiment the protease is derived from a strain of Rhizomucor, for example Rhizomucor mehei. In another embodiment the protease is a protease preparation, for example a mixture of a proteolytic preparation derived from a strain of Aspergillus, {e.g. Aspergillus oryzae) and a protease derived from a strain of Rhizomucor, for example Rhizomucor mehei.

Other suitable proteases include aspartic acid proteases for example those described in, Hand-book of Proteolytic Enzymes, Edited by A.J. Barrett, N. D. Rawlings and J. F. Woessner, Aca-demic Press, San Diego, 1998, Chapter 270). Non-limiting examples of aspartic acid protease include, e.g., those disclosed in R. M. Berka et al. Gene, 96, 313 (1990)); (R.M. Berka et al. Gene, 125, 195-198 (1993)); and Gomi et al. Biosci. Biotech. Biochem. 57, 1095-1 100 (1993), which are hereby incorporated by reference in their entirety.

Non-limiting examples of commercially available protease products include ALCALASE®, ESPERASE™, FLAVO U RZYME ™, PROMIX™, NEUTRASE®, RENNILASE®, NOVOZYM™ FM 2.0L, and NOVOZYM™ 50006 (available from Novozymes A/S, Denmark) and GC106™ and SPEZYME™ FAN from Genencor Int., Inc., USA. Additional enzymes include FERMGEN™ and GC 212 from Genencor.

In embodiments, protease may be present in an amount of 0.0001-1 mg enzyme protein per g DS, or in some embodiments an amount of 0.001 to 0.1 mg enzyme protein per g DS. Alternatively, the protease may be present in an amount of 0.0001 to 1 LAPU/g DS, or in embodiments in an amount of 0.001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/g DS, or 0.001 to 0.1 mAU-RH/g DS. Non-limiting Commercial Enzymes

In embodiments, granular starch hydrolyzing enzymes are suitable for use as enzymes in accordance with the present disclosure. For example, alpha-amylase and/or glucoamylase may be blending for the processing of uncooked starch. Commercially available blends include STARGEN ™ 001 and STARGEN ™ 002 available from Genencor.

In embodiments, enzymes suitable for use in accordance with the present disclosure include enzymes for starch liquefaction such as SPEZYME® ALPHA, SPEZYME® FRED L, SPEZYME® HPA and SPEZYME® XTRA brand enzymes from Genencor.

In embodiments, enzymes suitable for use in accordance with the present disclosure include enzymes such as FERMENZYME® C and FERMENZYME® L-400 brand enzymes available from Genencor.

Other commercially available enzymes for use in accordance with the present disclosure include enzymes such as DISTILLASE® L-400, DISTILLASE® L-500, DISTILLASE® VHP, G- ZYME®480 ETHANOL, OPTIMASE TBG, OPTIMASH VR, OPTIMASH XL and OPTIMASH BG brand enzymes available from Genencor.

Additional enzymes for use in accordance with the present disclosure include commercially available enzyme products. One non-limiting example of enzyme product suitable for use herein includes Maxaliq™ ONE available from Genencor. Composition

The present disclosure further relates to a composition including one or more transketolase enzymes, thiamine pyrophosphate, or combinations thereof. Non-limiting examples of suitable transketolase enzymes are described above. Non-limiting examples of suitable thiaminepyrophosphate is described above.

In embodiments, compositions in accordance with the present disclosure comprise one or more carbohydrases, such as alpha-amylases. In embodiments the alpha-amylase is an acid alpha-amylase or a fungal alpha-amylase, for example an acid fungal alpha-amylase.

In embodiments, compositions in accordance with the present disclosure comprise one or more carbohydrate-source generating enzymes. Non-limiting examples of suitable carbohydrate-source generating enzymes include glucoamylases, beta-amylases, maltogenic amylases, pullulanases, alpha-glucosidases, or a mixture thereof. In embodiments, compositions in accordance with the present disclosure comprise enzymes selected from the group consisting of cellulolytic enzymes, for example cellulases, and/or hemicellulolytic enzymes, such as hemicellulases.

In embodiments, compositions in accordance with the present disclosure comprise one or more transketolase enzymes and/or thiamine pyrophosphate and further one or more fermenting organisms, for example yeast and/or bacteria. Non-limiting examples of fermenting organisms are described above.

In embodiments, compositions for use in accordance with the present disclosure contain one or more constituents such as one or more transketolase enzymes, thiamine pyrophosphate, or combinations thereof in an effective amount to improve yeast quality. For example, in embodiments, formulations in accordance with the present disclosure include constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof in an effective amount to promote fermenting organism (e.g. yeast) quality and quantity during fermentation. Moreover, retention of fermentation organism during fermentation may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times higher than a fermenting organism not contacted with the constituents in accordance with the present disclosure.

The particular amount of constituent in a composition of the present disclosure generally depends on the purpose for which the constituent is to be applied. For example, the amount of constituent can vary depending upon the type of fermenting organism used in fermentation, amount of fermenting organism in fermentation, the time the constituent is applied during fermentation, and/or type of composition (e.g. solid or liquid). In embodiments, one or more constituents are added to the composition of the present disclosure such that the constituent is present in an amount of 0.01%-20% by weight of the total composition. In embodiments, one or more constituents are present in an amount of about 0.5 to 10% by weight of the total composition. In embodiments, one or more constituents are present in an amount of about 0.2 to 0.4% by weight of the total composition. In embodiments, thiaminepyrophosphate is present in an amount of about 0.001 to 10% by weight of the total composition.

In embodiments, thiaminepyrophosphate is present in an amount of about 0.002125 to 0.425% by weight of the total composition.

In embodiments, transketolase enzyme is present in an amount that results in about 5 Units to 50 Units per kg of dry solids present in the fermentation. Use

In this aspect the present disclosure relates to the use of one or more transketolase enzymes and/or thiamine pyrophosphate in a fermentation process. In embodiments, one or more transketolase enzymes and thiamine pyrophosphate are used for improving the fermentation product yield. In an embodiment one or more transketolase enzymes and thiamine pyrophosphate are used for increasing growth and quality of the fermenting organism(s).

In embodiments, one or more transketolase enzymes are used for improving the fermentation product yield. In an embodiment one or more transketolase enzymes are used for increasing growth and quality of the fermenting organism(s).

In embodiments, thiamine pyrophosphate is used for improving the fermentation product yield. In an embodiment thiamine pyrophosphate is used for increasing growth and quality of the fermenting organism(s). Transgenic Plant Material

Another aspect of the present disclosure relates to transgenic plant material transformed with one or more transketolase enzyme genes.

In one embodiment the present disclosure relates to a transgenic plant, plant part, or plant cell which has been transformed with a polynucleotide sequence encoding a transketolase enzyme so as to express and produce the enzyme. The enzyme may be recovered from the plant or plant part, but in context of the present disclosure the plant or plant part containing the recombinant transketolase enzyme may be used in one or more of the methods or processes of the present disclosure concerned and described above.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Non-limiting examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and corn.

Non-limiting examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.

Non-limiting examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilization of the present disclosure are also considered plant parts, e.g., embryos, endosperms, aleurone and seeds coats.

Also included within the scope of the present disclosure are the progeny of such plants, plant parts, and plant cells.

Also included are plants generated with total vegetative growth for the reduction, and/or prevention of, transgene escape. Such plants may be used to increase biomass production in a plant. (See U.S. Patent Application No: 1 1/056,948 entitled Development of controlled total vegetative growth for prevention of transgene escape from genetically modified plants and for enhancing biomass production (herein incorporated by reference in its entirety).

Non-limiting examples of suitable transgenic plant material for use in accordance with the present disclosure also includes any plant(s) modified for the purposes of adding commercially desirable, agronomically important or end-product traits to the plant. Such traits include, but are not limited to, herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, fungal, nematode), stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress and oxidative stress, increased yield, food or feed content and value, physical appearance, male sterility, drydown, standability, prolificacy, starch quantity and quality, oil quantity and quality, protein quality and quantity, amino acid composition, and the like. See for example U.S. Patent Application No. 09/757,089 entitled Maize chloroplast aldolase promoter compositions and methods for use thereof (herein incorporated by reference in its entirety).

The transgenic plant or plant cell expressing a transketolase enzyme may be constructed in accordance with methods well known in the art. In short, the plant or plant cell is constructed by incorporating one or more expression constructs encoding the transketolase enzyme into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell. Examples of plant transformation constructs are known in the art (see for example, Sambrook et al., 1989; Gelvin et al., 1990).

The expression construct is conveniently a nucleic acid construct which comprises a polynucleotide encoding transketolase enzyme operably linked with appropriate regulatory sequences required for expression of the polynucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the enzyme is desired to be expressed. For instance, the expression of the gene encoding a transketolase enzyme may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1 , and the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21 : 285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang et al., 1991 , Plant Cell 3: 1 155-1165). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant MoI. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152: 708- 711 ), a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941 ), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573- 588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higher expression of a transketolase enzyme in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the polynucleotide sequence encoding a transketolase enzyme. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including /Agrobacter/t/m-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-meώated gene transfer is one method for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) and can also be used for transforming monocots, although other transformation methods may be used for these plants. Another method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281 ; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21 : 415-428. Suitable transformation methods for use in accordance with the present disclosure include those described in U.S. Patent No. 6,395,966 entitled Methods and compositions for the increase of yield in plants (herein incorporated by reference in its entirety) and U.S. Patent Application No. 09/757,089.

Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well-known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co- transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase. The production and characterization of stably transformed plants is further described in U.S. Patent Application No. 09/757,089.

In embodiments, it is envisioned that one could employ techniques for the site-specific integration or excision of transformation constructs prepared in accordance with the present disclosure. An advantage of site-specific integration or excision is described in U.S. Patent Application No. 09/757,089.

In embodiments, in addition to direct transformation of a particular plant genotype with a construct prepared according to the present disclosure, transgenic plants may be made by crossing a plant having a construct of the present disclosure to a second plant lacking the construct. For example, a construct encoding transketolase enzyme or a portion thereof can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the present disclosure not only encompasses a plant directly regenerated from cells which have been transformed in accordance with the present disclosure, but also the progeny of such plants. As used herein the term "progeny" denotes the offspring of any generation of a parent plant prepared in accordance with the present disclosure, wherein the progeny includes a DNA construct prepared in accordance with the present disclosure, or a portion of the a DNA construct prepared in accordance with the present disclosure. "Crossing" a plant to provide a plant line having one or more added transgenes relative to a starting plant line, as disclosed herein, is defined as the techniques that result in a transgene of the present disclosure being introduced into a plant line by crossing a starting line with a donor plant line that comprises a transgene of the present disclosure. Non-limiting examples of such steps are further articulated in U.S. Patent Application No. 09/757,089 incorporated herein by reference in its entirety.

As used herein, introgression of a DNA element into a plant genotype is defined as the result of the process of backcross conversion. A plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly a plant genotype lacking the desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.

In embodiments, a method for producing transketolase enzyme in a plant would comprise: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding an transketolase enzyme under conditions conducive for production of the enzyme. The transgenic plant in accordance with the present disclosure is capable of expressing one or more transketolase enzyme in increased amounts compared to corresponding unmodified plant material. Modified Fermenting Organism

Another aspect of the present disclosure relates to a modified fermenting organism transformed with a polynucleotide encoding a transketolase enzyme, wherein the fermenting organism is capable of expressing transketolase enzyme at fermentation conditions.

In embodiments the fermentation conditions are as defined according to the present disclosure. In embodiments, the fermenting organism is a microbial organism, such as yeast or filamentous fungus, or a bacterium. Non-limiting examples of other fermenting organisms are described above.

A fermenting organism may be transformed with a transketolase enzyme encoding genes using techniques well know in the art.

The following non-limiting examples further illustrate methods in accordance with this disclosure.

MATERIALS & METHODS Materials:

Transketolase from Saccharomyces cerevisae was obtained Sigma-Aldrich (product # 90197 or product # T6133).

Glucoamylase (AMG A): Glucoamylase derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289 and available from Novozymes A/S.

Alpha-Amylase A: Hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S).

Cellulolvtic preparation A: Cellulolytic composition comprising a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; a beta-glucosidase (fusion protein disclosed in US 60/832,511 ); and cellulolytic enzymes preparation derived from Trichoderma reesei. Cellulolytic preparation A is disclosed in co-pending US application No. 60/941 ,251. Yeast: RED STAR™ available from Red Star/Lesaffre, USA Methods: Identity The relatedness between two amino acid sequences or between two polynucleotide sequences is described by the parameter "identity".

For purposes of the present disclosure, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=1 , gap penalty=3, windows=5, and diagonals=5.

For purposes of the present disclosure, the degree of identity between two polynucleotide sequences is determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=3, gap penalty=3, and windows=20. SIGMA Enzymatic Assay for Transketolase

1 Unit (U) corresponds to the amount of enzyme which will produce 1 μmol of glyceraldehyde-3-phosphate from xylulose-5-phosphate per minute at pH 7.7 and 25°C, in the presence of ribose-5-phosphate, thiamine pyrophosphate and Mg²⁺.

Glucoamylase activity

Glucoamylase activity may be measured in Glucoamylase Units (AGU). Glucoamylase activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37°C, pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

A folder (EB-SM-0131.02/01 ) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference. Alpha-amylase activity (KNU)

The alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (e.g., at 37°C +/- 0.05; 0.0003 M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference. Acid Alpha-Amylase Activity

When used according to the present disclosure the activity of an acid alpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units) or FAU-F. Acid alpha-amylase activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 AFAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.

Acid alpha-amylase, an endo-alpha-amylase (1 ,4-alpha-D-glucan-glucanohydrolase, E. C. 3.2.1.1 ) hydrolyzes alpha-1 ,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths. The intensity of color formed with iodine is directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.

ALPHA - AMYLASE

STARCH + IODINE 40° , pH 2,5 DEXTRINS + OLIGOSACCHARIDES λ = 590 nm blue/violet t = 23 sec. decoloration Standard conditions/reaction conditions:

Substrate: Soluble starch, approx. 0.17 g/L

Buffer: Citrate, approx. 0.03 M

Iodine (12): 0.03 g/L

CaCI₂: 1.85 mM pH: 2.50 ± 0.05

Incubation temperature: 40⁰C

Reaction time: 23 seconds

Wavelength: 590nm

Enzyme concentration: 0.025 AFAU/mL

Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference. Determination of FAU-F

FAU-F Rjngal Alpha-Amylase LJnits (Fungamyl) is measured relative to an enzyme standard of a declared strength.

A folder (EB-SM-0216.02) describing this standard method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference. Measurement of Cellulase Activity Using Filter Paper Assay (FPU assay) 1. Source of Method 1.1 The method is disclosed in a document entitled "Measurement of Cellulase Activities" by Adney, B. and Baker, J. 1996. Laboratory Analytical Procedure, LAP-006, National Renewable Energy Laboratory (NREL). It is based on the IUPAC method for measuring cellulase activity (Ghose, T.K., Measurement of Cellulse Activities, Pure & Appl. Chem. 59, pp. 257-268, 1987. 2. Procedure

2.1 The method is carried out as described by Adney and Baker, 1996, supra, except for the use of a 96 well plates to read the absorbance values after color development, as described below.

2.2 Enzyme Assay Tubes:

2.2.1 A rolled filter paper strip (#1 Whatman; 1 X 6 cm; 50 mg) is added to the bottom of a test tube (13 X 100 mm).

2.2.2 To the tube is added 1.0 mL of 0.05 M Na-citrate buffer (pH 4.80).

2.2.3 The tubes containing filter paper and buffer are incubated 5 min. at 50° C (± 0.1° C) in a circulating water bath.

2.2.4 Following incubation, 0.5 mL of enzyme dilution in citrate buffer is added to the tube. Enzyme dilutions are designed to produce values slightly above and below the target value of 2.0 mg glucose.

2.2.5 The tube contents are mixed by gently vortexing for 3 seconds.

2.2.6 After vortexing, the tubes are incubated for 60 mins. at 50° C (± 0.1° C) in a circulating water bath.

2.2.7 Immediately following the 60 min. incubation, the tubes are removed from the water bath, and 3.0 mL of DNS reagent is added to each tube to stop the reaction. The tubes are vortexed 3 seconds to mix.

2.3 Blank and Controls

2.3.1 A reagent blank is prepared by adding 1.5 mL of citrate buffer to a test tube.

2.3.2 A substrate control is prepared by placing a rolled filter paper strip into the bottom of a test tube, and adding 1.5 mL of citrate buffer.

2.3.3 Enzyme controls are prepared for each enzyme dilution by mixing 1.0 mL of citrate buffer with 0.5 mL of the appropriate enzyme dilution.

2.3.4 The reagent blank, substrate control, and enzyme controls are assayed in the same manner as the enzyme assay tubes, and done along with them.

2.4 Glucose Standards

2.4.1 A 100 mL stock solution of glucose (10.0 mg/mL) is prepared, and 5 mL aliquots are frozen. Prior to use, aliquots are thawed and vortexed to mix.

2.4.2 Dilutions of the stock solution are made in citrate buffer as follows: G1 = 1.0 mL stock + 0.5 mL buffer = 6.7 mg/mL = 3.3 mg/0.5 mL

G2 = 0.75 mL stock + 0.75 mL buffer = 5.0 mg/mL = 2.5 mg/0.5 mL G3 = 0.5 ml. stock + 1.O mL buffer = 3.3 mg/mL = 1.7 mg/0.5 mL G4 = 0.2 ml. stock + 0.8 ml. buffer = 2.0 mg/mL = 1.0 mg/0.5 mL

2.4.3 Glucose standard tubes are prepared by adding 0.5 mL of each dilution to 1.0 mL of citrate buffer.

2.4.4 The glucose standard tubes are assayed in the same manner as the enzyme assay tubes, and done along with them.

2.5 Color Development

2.5.1 Following the 60 min. incubation and addition of DNS, the tubes are all boiled together for 5 mins. in a water bath.

2.5.2 After boiling, they are immediately cooled in an ice/water bath.

2.5.3 When cool, the tubes are briefly vortexed, and the pulp is allowed to settle. Then each tube is diluted by adding 50 microL from the tube to 200 microL of ddH2O in a 96-well plate. Each well is mixed, and the absorbance is read at 540 nm.

2.6 Calculations (examples are given in the NREL document)

2.6.1 A glucose standard curve is prepared by graphing glucose concentration (mg/0.5 mL) for the four standards (G1-G4) vs. A₅₄₀. This is fitted using a linear regression (Prism Software), and the equation for the line is used to determine the glucose produced for each of the enzyme assay tubes.

2.6.2 A plot of glucose produced (mg/0.5 mL) vs. total enzyme dilution is prepared, with the Y- axis (enzyme dilution) being on a log scale.

2.6.3 A line is drawn between the enzyme dilution that produced just above 2.0 mg glucose and the dilution that produced just below that. From this line, it is determined the enzyme dilution that would have produced exactly 2.0 mg of glucose.

2.6.4 The Filter Paper Units/mL (FPU/mL) are calculated as follows: FPU/mL = 0.37/ enzyme dilution producing 2.0 mg glucose

Protease Assay method - AU(RH)

The proteolytic activity may be determined with denatured hemoglobin as substrate. In the Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA). The amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan. One Anson Unit (AU-RH) is defined as the amount of enzyme which under standard conditions (e.g. 25°C, pH 5.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.

The AU(RH) method is described in EAL-SM-0350 and is available from Novozymes A/S Denmark on request. Protease assay method (LAPU)

1 Leucine Amino Peptidase Unit (LAPU) is the amount of enzyme which decomposes 1 microM substrate per minute at the following conditions: 26 mM of L-leucine-p-nitroanilide as substrate, 0.1 M Tris buffer (pH 8.0), 37⁰C, 10 minutes reaction time.

LAPU is described in EB-SM-0298.02/01 available from Novozymes A/S Denmark on request. Determination of Maltogenic Amylase activity (MANU)

One MANU (Maltogenic Amylase jsjovo JJ.nit) may be defined as the amount of enzyme required to release one micro mole of maltose per minute at a concentration of 10 mg of maltotriose (Sigma M 8378) substrate per ml of 0.1 M citrate buffer, pH 5.0 at 37⁰C for 30 minutes. Example 1

A test was conducted to examine the effect of thiamine pyrophosphate (TPP) and transketolase (TK) in the fermentation of pretreated corn stover (PCS) hydrolysates to produce ethanol.

All treatments were evaluated via mini-scale fermentations. NREL dilute acid steam exploded corn stover (PCS) was diluted with water and adjusted to pH 5.0 with NH₄OH. Penicillin and citrate buffer and YP (yeast extract and peptone) medium were also added prior to the hydrolysis. The total solids (TS) level was 20%. The sample was hydrolyzed for 72 hours at 50⁰C with Cellulolytic preparation A. Following the hydrolysis step, the sample was sterile- filtered to remove the solids and the filtrate was used for fermentation. Fermentation was carried on in 20 ml mini vials at 30⁰C. Each vial contained 2.5 ml PCS hydrolysates, 1.95 ml YPDX (yeast extract, peptone, glucose and xylose) medium and certain amount of water to make the final total working volume as 5 ml. Each vial was dosed with the appropriate amount of TPP/TK based on the dosage shown in Table 1 below, followed by inoculation of 0.25 ml over-night Red Star yeast propagate. After inoculation, the flasks were incubated in the 30°C shaker at 150 rpm. All tests were conducted in triplicate. Samples were taken during the fermentation and at the end of fermentation to measure the ethanol, glucose, xylose, acetic acid and glycerol levels by HPLC. The HPLC preparation consisted of stopping the reaction by addition of 40% H₂SO₄ (1% v/v addition), centrifuging, and filtering through a 0.20 micrometer filter. Samples were stored at 4°C until analysis. Agilent™ 1100 HPLC system coupled with Rl detector was used. The separation column was aminex HPX-87H ion exclusion column (300mm x 7.8mm) from BioRad™.

Table 1

The results indicate that the addition of the optimal dosage of TPP or combination of TPP and TK in the fermentation of PCS hydrolysates increased ethanol yield. These results are shown in Figure 1a and Figure. 1 b.

Example 2

Tests were conducted to determine the effect of thiamine pyrophosphate (TPP) toward combination of α-amylase (AA1 ) and glucoamylase (AMG A) in one-step simultaneous and fermentation (SSF) process.

All treatments were evaluated via mini-scale fermentations. 410 g of ground yellow dent corn (with an average particle size around 0.5 mm) was added to 590 g tap water. This mixture was supplemented with 3.0 ml 1g/L penicillin and 1g of urea. The pH of this slurry was adjusted to 4.5 with 40% H₂SO₄. Dry solid (DS) level was determined to be about 35 wt. %. Approximately 5 g of this slurry was added to 20 ml vials. Each vial was dosed with the appropriate amount of enzyme dosage shown in table 2 below followed by addition of 200 micro liters yeast propagate/5 g slurry. Actual enzyme dosages were based on the exact weight of corn slurry in each vial. Vials were incubated at 32°C. Nine replicate fermentations of each treatment were run. Three replicates were selected for 24 hours, 48 hours and 70 hours time point analysis. Vials were vortexed at 24, 48 and 70 hours and analyzed by HPLC. The HPLC preparation consisted of stopping the reaction by addition of 50 micro liters of 40% H₂SO₄, centrifuging, and filtering through a 0.45 micrometer filter. Samples were stored at 4°C until analysis. Agilent™ 1 100 HPLC system coupled with Rl detector was used to determine ethanol and oligosaccharides concentration. The separation column was aminex HPX-87H ion exclusion column (300mm x 7.8mm) from BioRad™. The treatments shown in Table 2 below: Table 2

The results are shown in Table 3: Table 3

Ethanol yield over time was measured for thiamine pyrophosphate at different concentrations. These results are also shown in Figure 2 and Figure 3.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of various aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure, including definitions will be controlling.

Claims

WHAT IS CLAIMED IS:

1. A method of fermenting sugars derived from plant material into a fermentation product in a fermentation medium using a fermenting organism comprising adding one or more constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof to the fermentation medium.

2. A method in accordance with claim 1 , wherein the one or more constituents are added to the fermentation medium before or during fermentation.

3. A method in accordance with claims 1 , wherein the transketolase enzyme is present or added in an amount of 0.001 to 1000 Units per ml. of fermentation medium.

4. A method in accordance with claims 1 , wherein the thiaminepyrophosphate is present or added in an amount of 0.001 to 1000 mmol/L of fermentation medium.

5. A method in accordance with claims 4, wherein the thiaminepyrophosphate is present or added in an amount of about 10 mmol/L of fermentation medium.

6. A process of producing a fermentation product from starch-containing material comprising the steps of: i) liquefying starch-containing material; ii) saccharifying the liquefied material; iii) fermenting with one or more fermenting organisms, wherein one or more constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof are added to the fermentation medium.

7. A process in accordance with claim 6, wherein steps ii) and iii) are carried out simultaneously or sequentially.

8. A process in accordance with claim 6 or 7, further comprising, prior to the step i), the steps of: x) reducing the particle size of starch-containing material; preferably by milling; and y) forming a slurry comprising the starch-containing material and water.

9. A process of producing a fermentation product from starch-containing material, comprising the steps of: (a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material,

(b) fermenting using a fermenting organism, wherein one or more constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof are added to the fermentation medium.

10. A process in accordance with claim 9, wherein steps ii) and iii) are carried out simultaneously or sequentially.

11. A process in accordance with claim 9 or 10, further comprising, prior to the step a), the steps of: x) reducing the particle size of starch-containing material; preferably by milling; and y) forming a slurry comprising the starch-containing material and water.

12. A process in accordance with any of claims 6-11 , wherein the starch-containing material comprises tubers, roots, stems, whole grains, corn, cobs, wheat, barley, rye, millet, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, potatoes, yams, cereals, or mixtures thereof.

13. A process of producing a fermentation product from lignocellulose-containing material, comprising the steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolysing the material;

(c) fermenting with a fermenting organism, wherein one or more constituents comprising transketolase enzyme, thiaminepyrophosphate, or combinations thereof are added to the fermentation medium.

14. A process in accordance with claim 13, wherein the lignocellulose-containing material originates from materials selected from the group consisting of: corn stover, corn cobs, corn fiber, hardwoods, softwoods, cereal straw, switchgrass, bagasse, rice hulls, municipal solid waste, industrial organic waste, office paper, or mixtures thereof.

15. A method or process in accordance with any of claims 1-14, wherein the fermenting organism is a yeast, preferably a strain of the genus Saccharomyces, especially Saccharomyces cerevisiea, or a strain of Pichia, preferably a strain of Pichia stipitis or Pichia pastoris.

16. A method or process in accordance with any of claims 1-15, wherein the fermentation product is an alcohol, preferably ethanol or butanol.

17. A method or process in accordance with any of claims 1-16, wherein the fermentation product is recovered after fermentation.

18. Transgenic plant material transformed with a polynucleotide sequence encoding transketolase or progeny of such transgenic plant material capable of expressing transketolase.

19. A modified fermenting organism transformed with a polynucleotide encoding a transketolase, wherein the fermenting organism is capable of expressing transketolase at fermentation conditions.

20. The modified fermentation organism of claim 19, wherein the fermenting organism is a microbial organism such as yeast or filamentous fungus, or a bacterium.