WO2012012590A2

WO2012012590A2 - Processes for producing fermentation products

Info

Publication number: WO2012012590A2
Application number: PCT/US2011/044770
Authority: WO
Inventors: Lars Saaby Pedersen; Donald L. Higgins; Hui Xu; Johan Mogensen; Hans Sejr Olsen; Jan Larsen
Original assignee: Novozymes A/S; Novozymes North America, Inc.
Priority date: 2010-07-23
Filing date: 2011-07-21
Publication date: 2012-01-26
Also published as: WO2012012590A3

Abstract

The invention relates to a process of producing a fermentation product from lignocellulose-containing material, comprising the steps of pretreating lignocellulose-containing material (step (a)); hydrolyzing pretreated lignocellulose-containing material (step (b)), and fermenting hydrolyzed pretreated lignocellulose-containing material (step (c)), wherein the initially fermented pretreated lignocellulose-containing material is subjected to further hydrolysis (step (d)) and further fermentation (step (e)), and optionally recovering the fermentation product (step (f)).

Description

PROCESSES FOR PRODUCING FERMENTATION PRODUCTS

TECHNICAL FIELD

The present invention relates to processes of producing fermentation products from lignocellulose-containing material.

BACKGROUND OF THE INVENTION

Lignocellulose-containing feed stock is available in abundance and can be used for producing renewable fuels such as ethanol. Producing fermentation products from lignocelluloses- containing material is known in the art and generally includes pre-treating, hydrolyzing, fermenting the material, and optionally recovering the fermentation products. The structure of lignocellulose is not directly accessible to enzymatic hydrolysis. Therefore, the lignocellulose-containing material is pre-treated in order to break the lignin seal and disrupt the crystalline structure of cellulose. Cellulolytic enzymes used during hydrolysis are strongly product inhibited by, e.g., glucose. Also, the enzymes may be inhibited by the fermentation product, such as ethanol.

There is still a need for providing improved and more cost efficient processes for producing fermentation products from lignocellulose-containing materials.

SUMMARY OF THE INVENTION

The present invention relates to improved processes for producing fermentation products, especially ethanol, from lignocellulose-containing materials.

In the first aspect the invention relates to a process for producing a fermentation product from lignocellulose-containing material, comprising the steps of pretreating lignocellulose- containing material (step (a)); hydrolyzing pretreated lignocellulose-containing material (step (b)), and fermenting hydrolyzed pretreated lignocellulose-containing material (step (c)), wherein the initially fermented pretreated lignocellulose-containing material is subjected to further hydrolysis (step (d)) and further fermentation (step (e)), and optionally recovering the fermentation product (step (f)).

By using a process of the invention the enzyme dosage may be reduced and/or higher cellulose conversion may be accomplished. This is illustrated in Example 1 .

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows a flow diagram of an embodiment of the invention including steps a-f.

Fig. 2 shows a flow diagram of an embodiment of the invention including steps a-h. Fig. 3 shows the glucose formation during a multi-stage (MS) hydrolysis and fermentation process of the invention using PCS as substrate compared to a standard (Std) hydrolysis process.

Fig. 4 shows glucose, ethanol, temperature and dry matter profiles for stepwise hydrolysis and fermentation.

DETAILED DESCRIPTION OF THE INVENTION

The process of the invention is a multi-stage hydrolysis and fermentation process. The invention comprises the combination of two or more separate hydrolysis steps with intermediate fermentation and optionally a fermentation product recovery step, such as an ethanol stripping or distillation step. The most efficient utilization of cellulolytic enzymes is obtained by using reaction conditions for the enzymes in a single pass hydrolysis process.

The inventors have found that subjecting already hydrolysed and fermented pretreated lignocellulose-containing material to further hydrolysis and fermentation results in a decrease in cellulolytic enzyme inhibition. This enables a decrease in the cellulolytic enzyme dosage and/or results in higher cellulose conversion, i.e., high amount of sugars, such as glucose, produced per amount of enzyme used. A significant part of the enzymes used for hydrolysis are still active after the initial hydrolysis step. Therefore, according to the invention no further enzymes need to be added after initial hydrolysis and initial fermentation and before further hydrolysis and further fermentation. However, it is also contemplated to add further cellulolytic enzymes or cellulolytic preparation during the further hydrolysis and/or further fermentation steps.

Consequently, in the first aspect the invention relates to a process for producing a fermentation product from lignocellulose-containing material, comprising the steps of pretreating lignocellulose-containing material (step (a)); hydrolyzing pretreated lignocellulose-containing material (step (b)), and fermenting pretreated, hydrolyzed lignocellulose-containing material (step (c)), wherein the initially pretreated, fermented lignocellulose-containing material is subjected to further hydrolysis (step (d)) and/or further fermentation (step (e)), and optionally recovering the fermentation product (step (f)).

In another preferred embodiment the process of the invention comprises the steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolyzing the pretreated lignocellulose-containing material at a dry matter content of 10-40% (w/w) and at a temperature in the range from 25-70°C;

(c) fermenting using a fermenting organism;

(d) hydrolyzing at a temperature in the range from 25-70°C; (e) fermenting using a fermenting organism;

(f) optionally recovering the fermentation product.

Fig. 1 depicts a flow diagram of this embodiment of the invention.

(a) pre-treating lignocellulose-containing material;

(c) fermenting using a fermenting organism;

(i) recovering the fermentation product;

(ii) concentrating and/or separating the remaining fraction from step (c)(i) into solid and liquid fractions;

(d) hydrolyzing the concentrated and/or separated solid fraction from step (c)(ii) at a temperature in the range from 25-70°C;

(e) fermenting using a fermenting organism;

(f) optionally recovering the fermentation product.

The process of the invention may comprise a step (step (g)) wherein the recovered fermentation product in step (f) is transferred to the recovering step (c)(i). In another preferred embodiment the process includes a step (step (h)) wherein the liquid fraction from step (c)(ii) is recycled to step (b). While cellulolytic enzymes generally adsorb to lignocellulosic materials beta-glucosidase remains in solution. Beta-glucosidase can therefore be recycled to step (b) via step (h). Fig. 2 depicts a flow diagram of this embodiment of the invention.

According to the invention a high substrate dry matter content (e.g., above 10% (w/w) dry matter, such as between about 10-40% (w/w) dry matter, preferably between 15 and 30% (w/w) dry matter) can be used. This also minimizes the required equipment size and energy consumption for heating of the reaction mix. In other words, the present invention efficiently utilizes lignocellulose-containing materials and enables that hydrolysis can be carried out at a high dry matter content concentration (e.g., between 10-40% (w/w)) and can be done at reduced enzyme use compared to a corresponding process where only the initial hydrolysis and initial fermentation is done.

The initial hydrolysis step (step (b)) may run until the degree of hydrolysis is in the range from 25-70%. In a preferred embodiment the dry matter content during initial hydrolysis (step (b)) is from 15-30% (w/w). According to the invention the temperature during initial hydrolysis (step (b)) is from 45-60°C. The dry matter content during further hydrolysis (step (d)) is preferably between from 10-30% (w/w). The temperature during further hydrolysis (step (d)) is from 45-60°C. The initially hydrolyzed pretreated lignocellulose-containing material (step (b)) is cooled to a suitable temperature for the fermenting organism (step (c)), in particular a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C. The further pretreated, hydrolyzed lignocellulose-containing material (step (d)) is cooled to a suitable temperature for the fermenting organism (e.g., step (e)), in particular a temperature in the range from 20-40°C, preferably from 25-35°C, especially around 32°C. The initial hydrolysis (step (b)) and initial fermentation (step (c)) may according to the invention be carried out either separately or simultaneously. Simultaneous initial hydrolysis and fermentation (steps (b) and (c)) would according to the invention be carried out at a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C. The initial hydrolysis (step (b)) would be carried out at a temperature from 45-70°C when it is done separately from the initial fermentation step (step (c)). According to the invention further hydrolysis (step (d)) and further fermentation (step (e)) may be carried out either separately or simultaneously. Further hydrolysis and fermentation (steps (d) and (e)) may be carried out at a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C. Further hydrolysis (step (d)) may be carried out at a temperature from 45-70°C when it is done separately from further fermentation (step (e)).

According to the invention further hydrolysis (step (d)) runs until the degree of hydrolysis is in the range from 80-100%. The fermentation product may be recovered by distillation (step (f)), in particular by vacuum distillation.

In one embodiment of the invention hemicellulose from the pretreated lignocellulose- containing material is removed before initial hydrolysis (step (b)). The cellulose content after hemicellulose removal is in a preferred embodiment at least 40% (w/w), preferably at least 50% (w/w), more preferably at least 60% (w/w), more preferably at least 70% (w/w), more preferably at least 80% (w/w), more preferably at least 90% (w/w) of insoluble solids (IS).

Initial hydrolysis (step (b)) is preferably carried out at a pH in the range from 4-6, preferably around 5. Initial hydrolysis is preferably carried out for 12-120 hours. Further hydrolysis (step (d)) is preferably carried out at a pH in the range from 4-6, preferably around 5. Further hydrolysis is preferably carried out for 24-120 hours.

Initial fermentation (step (c)) is preferably carried out for 24-120 hours. Further fermentation (step (e)) is preferably carried out for 24-120 hours.

The pretreated lignocellulose-containing material is initially hydrolyzed (step (b)) by subjecting the material to a cellulolytic preparation. Suitable examples will be described below in the "Enzymes"-section. In an embodiment of the invention initial hydrolysis (step (b)) and/or further hydrolysis (step (d)) are carried out applying free fall mixing technology as described in WO 2006/056838 (which is hereby incorporated by reference).

Pre-treatment

The lignocellulose-containing material is pretreated before initial hydrolysis and fermentation (step (a)). In practicing a process of the invention, any pretreatment process known in the art can be used. The lignocellulose-containing material may be chemically, mechanically, physically and/or biologically pre-treated (step (a)). The lignocellulose-containing material may in one embodiment be washed before being pre-treated. In another embodiment the lignocellulose- containing material may be unwashed before being pre-treated in step (a). The pretreated lignocellulose-containing material may or may not be washed prior to the enzymatic hydrolysis. In an embodiment the pretreated lignocelluloses-containing material is washed prior to hydrolysis, preferably after pretreatment and before hydrolysis.

The lignocellulose-containing material may be subjected to particle size reduction, pre- soaking, wetting, washing, or conditioning prior to pretreatment using methods known in the art. Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment. Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical C0₂, supercritical H₂0, ozone, and gamma irradiation pretreatments.

The lignocellulose-containing material can be pretreated before initial hydrolysis and/or initial fermentation. Pretreatment is preferably performed prior to the initial hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with initial hydrolysis, such as simultaneously with treatment of the lignocellulose-containing material with a cellulolytic preparation to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases the pretreatment step itself results in some conversion of the lignocellulose-containing material to fermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g., hemicellulase, accessible to enzymes. The lignocellulose-containing material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time. Steam pretreatment is preferably done at 140-230°C, more preferably 160-200°C, and most preferably 170-190°C, where the optimal temperature range depends on any addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1-15 minutes, more preferably 3-12 minutes, and most preferably 4-10 minutes, where the optimal residence time depends on temperature range and any addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that the lignocellulose-containing material is generally only moist during the pretreatment. The steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No. 2002/0164730). During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.

A catalyst such as H₂S0₄ or S0₂ (typically 0.3 to 3% w/w) is often added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), and organosolv pretreatments. In dilute acid pretreatment, the lignocellulose-containing material is mixed with dilute acid, typically H₂S0₄, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure. The dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter- current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Schell et al., 2004, Bioresource Technol. 91 : 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-1 15).

Several methods of pretreatment under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, lime pretreatment, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX). Lime pretreatment is performed with calcium carbonate, sodium hydroxide, or ammonia at low temperatures of 85-150°C and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959- 1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/1 10891 , WO 2006/1 10899, WO 2006/1 10900, and WO 2006/1 10901 disclose pretreatment methods using ammonia. Wet oxidation is a thermal pretreatment performed typically at 180-200°C for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151 ; Palonen et al., 2004, Appl. Biochem. Biotechnol. 1 17: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81 : 1669-1677). The pretreatment is performed at preferably 1-40% dry matter, more preferably 2-30% dry matter, and most preferably 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate. A modification of the wet oxidation pretreatment method, known as wet explosion (combination of wet oxidation and steam explosion), can handle dry matter up to 30%. In wet explosion, the oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032282). Ammonia fiber explosion (AFEX) involves treating lignocellulose-containing material with liquid or gaseous ammonia at moderate temperatures such as 90-100°C and high pressure such as 17-20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231 ; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121 :1 133-1 141 ; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018). AFEX pretreatment results in the depolymerization of cellulose and partial hydrolysis of hemicellulose. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200°C for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473- 481 ; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861 ; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121 :219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of the hemicellulose is removed.

Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. 105-108: 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as an acid treatment, and more preferably as a continuous dilute and/or mild acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride or mixtures thereof. Mild acid treatment is conducted in the pH range of preferably 1-5, more preferably 1-4, and most preferably 1-3. In one aspect, the acid concentration is in the range from preferably 0.01 to 20 wt. % acid, more preferably 0.05 to 10 wt. % acid, even more preferably 0.1 to 5 wt. % acid, and most preferably 0.2 to 2.0 wt. % acid. The acid is contacted with the cellulosic material and held at a temperature in the range of preferably 160-220°C, and more preferably 165-195°C, for periods ranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiber explosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. In preferred aspects, the lignocellulose-containing material is present during pretreatment in amounts preferably between 10-80 wt. %, more preferably between 20-70 wt. %, and most preferably between 30-60 wt. %, such as around 50 wt. %. The pretreated lignocellulose-containing material can be unwashed or washed using any method known in the art, e.g., washed with water.

Mechanical Pretreatment: The term "mechanical pretreatment" refers to various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).

Physical Pretreatment: The term "physical pretreatment" refers to any pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from cellulosic material. For example, physical pretreatment can involve irradiation (e.g., microwave irradiation), steaming/steam explosion, hydrothermolysis, and combinations thereof. Physical pretreatment can involve high pressure and/or high temperature (steam explosion). In one aspect, high pressure means pressure in the range of preferably about 300 to about 600 psi, more preferably about 350 to about 550 psi, and most preferably about 400 to about 500 psi, such as around 450 psi. In another aspect, high temperature means temperatures in the range of about 100 to about 300°C, preferably about 140 to about 235°C. In a preferred aspect, mechanical pretreatment is performed in a batch-process, steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: The cellulosic material can be pretreated both physically and chemically. For instance, the pretreatment step can involve dilute or mild acid treatment and high temperature and/or pressure treatment. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired. A mechanical pretreatment can also be included. Accordingly, in a preferred aspect, the lignocellulose-containing material is subjected to mechanical, chemical, mechanical or physical pretreatment, or any combination thereof to promote the separation and/or release of cellulose, hemicellulose and/or lignin. Biological Pretreatment: The term "biological pretreatment" refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the cellulosic material. Biological pretreatment 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 and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic 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 and Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331 ; and Vallander and Eriksson, 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng Biotechnol. 42: 63- 95).

Recovery: Subsequent to fermentation the fermentation product may optionally be separated from the fermentation medium in any suitable way. For instance, the medium may be distilled to extract the fermentation product or the fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively the fermentation product may be recovered by stripping. By stripping the fermentation product, such as ethanol, from the reaction blend under vacuum, the denaturing of the enzymes in the stripper is minimized. Recovery methods are well known in the art.

Distillation: The term "distillation" is used in context of the present invention in its tradition sense, i.e. , a process in which a mixture of two or more substances is separated into its component fractions based on differences in their volatilities in a boiling liquid mixture. For instance, ethanol is removed from fermented mash by taking advantage of its boiling point. The ethanol distillation temperature is in the range between 60-100°C, preferably 70-90°C, especially around the boiling point of ethanol which is 78.3°C when operated at normal pressure of 0.1 MPa. Like the stripping stage mentioned above, the distillation can also be carried out at reduced pressure, which lowers the boiling point of the water, ethanol and the blend of these. The pressure during vacuum distillation is controlled so that the boiling point of the bottom fraction, which contains the enzyme, is kept at a temperature that does not cause significant inactivation of the enzyme. Normally the boiling temperature is kept below 50°C. Solid-Liquid Separation: According to the invention solid-liquid separation can be achieved in many ways well-known to one skilled in the art. For instance, solid-liquid separation can be done using a screw press, centrifuge, decanter centrifuge, belt press, drum filter, hydrocyclone and/or filter press, or any kind of apparatus which can handle solids-liquid separation, including gravity- fed systems or apparatuses.

The separated liquid can be recycled in accordance with the process of the invention (e.g., step (h)).

Fermentation Products: The present invention may be used for producing any liquid fermentation product. Preferred fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone) etc.

In a preferred embodiment the fermentation product is an alcohol, especially ethanol. The fermentation product, such as ethanol, obtained according to the invention, may preferably be used as fuel alcohol/ethanol. However, in the case of ethanol it may also be used as potable ethanol.

Fermenting Organisms: 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 invention are able to ferment, i.e., convert, sugars, glucose, xylose, fructose and/or maltose, directly or indirectly into the desired fermentation product. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred 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.

Preferred 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(s) is(are) C6 sugar fermenting organisms, such as of 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. Examples of C5 sugar fermenting organisms include strains of Pichia, such as of the species Pichia stipitis. C5 sugar fermenting bacteria are also known. Also some Saccharomyces cerevisae strains ferment C5 (and C6) sugars. 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 one embodiment 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 10⁵ to 10¹², preferably from 10⁷ to 10¹⁰, especially about 5x10⁷.

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), FERMIOL (available from DSM Specialties), and a modified yeast from Royal Nedalco, NL.

Liqnocellulose-Containinq Material: The term "Ngnocellulose-containing material" means material containing a significant content of cellulose, hemicellulose, and lignin. Lignocellulose- containing material is often referred to as "biomass".

The lignocellulose-containing material may be any material containing lignocellulose. The lignocellulose-containing material preferably contains at least 30% (w/w), preferably at least 50% (w/w), more preferably at least 70% (w/w), even more preferably at least 90% (w/w) lignocellulose. It is to be understood that lignocellulose-containing material may also comprise other constituents such as proteinaceous material, starchy material, sugars, such as fermentable sugars and/or un- fermentable sugars.

Lignocellulose-containing material is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. Lignocellulose-containing 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. It is to be understood that lignocellulose-containing material may be in the form of plant cell wall material containing lignin, cellulose and hemicellulose in a mixed matrix. In a preferred embodiment the lignocellulose-containing material is cereal straw such as wheat straw, corn stover, corn cobs, corn fiber, rice straw, hard wood or soft wood, such as, e.g., pine spruce, birch, eucalyptus and poplar wood or wood chips, bagasse, paper and pulp processing waste and saw mill waste.

Other examples include, switch grass, Miscanthus, rice hulls, municipal solid waste (MSW), industrial organic waste, recycled paper, office paper and card board, or mixtures thereof.

In a preferred embodiment the lignocellulose-containing material is wheat straw. In another preferred embodiment the lignocellulose- corn stover or corn cobs. In another preferred embodiment, the lignocellulose-containing material is corn fiber. In another preferred embodiment, the lignocellulose-containing material is switch grass. In another preferred embodiment, the lignocellulose-containing material is bagasse.

Enzymes

Even if not specifically mentioned in context of a process of the invention, it is to be understood that the enzyme(s) (as well as other compounds) are used in an effective amount.

Hydrolyzing Enzymes: According to the invention hydrolyzing enzymes include especially cellulolytic enzymes, hemicellulolytic enzymes, including those listed below. The cellulolytic enzymes typically used for hydrolyzing lignocellulose-containing material adsorb to the material. The only exception is the beta-glucosidase that remains in solution.

Cellulolytic Enzymes and Cellulolytic Preparation: The term "cellulolytic enzymes" as used herein are understood as comprising enzymes having cellobiohydrolase activity (EC 3.2.1.91 ), e.g., cellobiohydrolase I and cellobiohydrolase II, as well as endo-glucanase activity (EC 3.2.1.4) and beta-glucosidase activity (EC 3.2.1.21 ).

In order to be efficient, the digestion of cellulose may require several types of enzymes acting cooperatively. At least three categories of cellulolytic enzymes are often needed to convert cellulose into glucose: endoglucanases (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 are the key enzymes for the degradation of native crystalline cellulose. The term "cellobiohydrolase I" is defined herein as a cellulose 1 ,4-beta-cellobiosidase (also referred to as Exo-glucanase, Exo-cellobiohydrolase or 1 ,4-beta-cellobiohydrolase) activity, as defined in the enzyme class EC 3.2.1 .91 , which catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose and cellotetraose, by the release of cellobiose from the non-reducing ends of the chains. The definition of the term "cellobiohydrolase II activity" is identical, except that cellobiohydrolase II attacks from the reducing ends of the chains.

The cellulolytic enzymes may comprise a carbohydrate-binding module (CBM) which enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme. A CBM is defined as contiguous amino acid sequence within a carbohydrate-active enzyme with a discreet fold having carbohydrate-binding activity. For further information of CBMs see the CAZy internet server (Supra) or Tomme et al. (1995) in Enzymatic Degradation of Insoluble Polysaccharides (Saddler and Penner, eds.), Cellulose- binding domains: classification and properties, pp. 142-163, American Chemical Society, Washington.

The cellulolytic enzymes may be in the form of a cellulolytic preparation comprising enzymes of fungal origin, such as from a strain of the genus Trichoderma, preferably a strain of Trichoderma reesei; a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense (see, e.g., US publication no. 2007/0238155 from Dyadic Inc, USA).

In a preferred embodiment the cellulolytic preparation contains one or more of the following activities: hemicellulolytic activity, cellulolytic enzyme enhancing activity, cellulolytic activity, including beta-glucosidase activity, endoglucanase activity, cellobiohydrolase I and/or II activity, or xylose-isomerase activity.

In a preferred embodiment the cellulolytic enzymes may be a cellulolytic preparation as defined WO 2008/151079, which is hereby incorporated by reference.

In an embodiment the cellulolytic preparation comprising a polypeptide having cellulolytic enhancing activity, such as the GH61 polypeptides disclosed in WO 2005/074647, WO 2008/148131 , WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868, WO 2010/065830.

In a preferred embodiment the cellulolytic preparation comprising a polypeptide having cellulolytic enhancing activity of fungal origin, such as filamentous fungus origin, preferably from a strain of the genus Thermoascus, more preferably a strain of Thermoascus aurantiacus, especially the ones disclosed in WO 2005/074656 (GH61A) or WO 2010/065830 (GH61 B), or a combination thereof.

The cellulolytic preparation may further comprise a beta-glucosidase, such as a beta- glucosidase of fungal origin, such as filamentous fungi origin, derived from a strain of the genus Trichoderma, Aspergillus or Penicillium, including the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637 (Novozymes), the beta-glucosidase derived from Aspergillus fumigatus disclosed as SEQ ID NO: 2 in WO 2005/047499 (Novozymes) or the Penicillium brasillianum beta-glucosidase disclosed in WO 2007/019442 (Novozymes).

In an embodiment the cellulolytic preparation may also comprise a CBH II, preferably of fungal origin, such as filamentous fungi origin, preferably a strain of the genus Thielavia, preferably a strain of Thielavia terrestris cellobiohydrolase II (CEL6A), e.g., disclosed in WO 2006/074435, or a strain of the genus Myceliophthora, preferably a strain of Myceliophthora thermophila (CEL6A) CBHII, e.g., disclosed in WO 2009/042871 (Novozymes).

In another preferred embodiment the cellulolytic preparation may also comprise cellulolytic enzymes, preferably one derived from a strain of Trichoderma, preferably a strain of Trichoderma reesei; or a strain of Humicola, preferably a strain of Humicola insolens and/or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense.

In an embodiment the cellulolytic preparation comprises a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; a cellobiohydrolase, such as Thielavia terrestris cellobiohydrolase II (CEL6A), a beta-glucosidase (e.g., the fusion protein disclosed in WO 2008/057634) and cellulolytic enzymes, e.g., derived from Trichoderma reesei.

In an embodiment the cellulolytic preparation comprises a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; a beta-glucosidase (e.g., the fusion protein disclosed in WO 2008/057637) and cellulolytic enzymes, e.g., derived from Trichoderma reesei.

In an embodiment the cellullolytic preparation is a blend from Trichoderma reesei, Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity disclosed in WO 2005/074656, Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499, Aspergillus aculeatus xylanase disclosed as Xyl II in WO 1994/021785.

In an embodiment the cellulolytic enzyme composition is the commercially available product CELLUCLAST™ 1.5L, CELLUZYME™, CELLIC™ CTEC2 (from Novozymes A/S, Denmark) or ACCELERASE™ 1000, ACCELERASE™ 1500, ACCELERASE™ DUET (from Genencor Inc. USA).

The cellulolytic enzymes or cellulolytic preparation may used for hydrolyzing the pre-treated lignocellulose-containing material and may preferably be dosed from 0.1-100 FPU per g cellulose, preferably 0.5-50 FPU per g cellulose, especially 1-20 FPU per g cellulose. In another embodiment at least 0.1 mg cellulolytic enzyme or cellulolytic preparation per g cellulose is used, preferably at least 3 mg cellulolytic enzyme or cellulolytic preparation per g cellulose, such as between 5 and 10 mg cellulolytic enzyme or cellulolytic preparation per g cellulose.

Endoqlucanase (EG): The term "endoglucanase" means an endo-1 ,4-(1 ,3;1 ,4)-beta-D-glucan 4- glucanohydrolase (E.C. No. 3.2.1.4), which catalyzes 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.

In a preferred embodiment endoglucanases may be derived from a strain of the genus Trichoderma, preferably a strain of Trichoderma reesei; a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense.

Cellobiohydrolase (CBH): The term "cellobiohydrolase" means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 ), which 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.

Examples of cellobiohydroloses are mentioned above including CBH I and CBH II from Trichoderma reseei; Humicola insolens and CBH II from Thielavia terrestris cellobiohydrolase (CEL6A).

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: One or more beta-glucosidases may be present during hydrolysis.

The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21 ), which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. For purposes of the present invention, 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 μη-iole of p-nitrophenol produced per minute at 50°C, pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate, 0.01 % TWEEN® 20.

In a preferred embodiment the beta-glucosidase is of fungal origin, such as a strain of the genus Trichoderma, Aspergillus or Penicillium. In a preferred embodiment the beta- glucosidase is a derived from Trichoderma reesei, such as the beta-glucosidase encoded by the bgll gene (see Fig. 1 of EP 562003). In another preferred embodiments the beta-glucosidase may be derived from Aspergillus oryzae (recombinantly produced in Aspergillus oryzae according to WO 02/095014), Aspergillus fumigatus disclosed in WO 2005/047499 (e.g., recombinantly produced in Aspergillus oryzae according to Example 22 of WO 02/095014) or Aspergillus niger (1981 , J. Appl. 3: 157-163), Penicillium brasillianum disclosed in WO 2007/019442 or the beta-glucosidase fusion protein disclosed in WO 2008/057637 (Novozymes Inc, USA) comprising Humicola insolens endoglucanase V core domain fused to Aspergillus oryzae beta-glucosidase.

Hemicellulolvtic Enzymes: According to the invention the pre-treated lignocellulose-containing material may 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 invention the lignocellulose derived material may be treated with one or more hemicellulases.

Any hemicellulase suitable for use in hydrolyzing hemicellulose, preferably into xylose, may be used. Preferred 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. Preferably, the hemicellulase for use in the present invention is an endo-acting hemicellulase, and more preferably, the hemicellulase is an endo-acting hemicellulase which has the ability to hydrolyze hemicellulose under acidic conditions of below pH 7, preferably pH 3-7.

An example of hemicellulase suitable for use in the present invention includes VISCOZYME™ (available from Novozymes A/S, Denmark).

In an embodiment the hemicellulase is a xylanase. In an embodiment the xylanase may preferably be of microbial origin, such as of fungal origin (e.g., Trichoderma, Meripilus, Humicola, Aspergillus, Fusarium) or from a bacterium (e.g., Bacillus). In a preferred embodiment the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeatus; or a strain of Humicola, preferably Humicola lanuginosa. The xylanase may preferably be an endo-1 ,4-beta-xylanase, more preferably an endo-1 ,4-beta-xylanase of GH10 or GH1 1. Examples of commercial xylanases include SHEARZYME™ and BIOFEED WHEAT™ from Novozymes A S, Denmark.

The hemicellulase may be added in an amount effective to hydrolyze hemicellulose, such as, in amounts from about 0.001 to 0.5% (w/w), more preferably from about 0.05 to 0.5% (w/w) of hemicellulose.

Cellulolytic Enhancing Activity: The term "cellulolytic enhancing activity" is defined herein as a biological activity that enhances the hydrolysis of a lignocellulose derived material by proteins having cellulolytic activity. For purposes of the present invention, 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 50°C 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 preferably at least 0.1 -fold, more at least 0.2-fold, more preferably at least 0.3-fold, more preferably at least 0.4-fold, more preferably at least 0.5-fold, more preferably at least 1-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 10-fold, more preferably at least 20-fold, even more preferably at least 30-fold, most preferably at least 50-fold, and even most preferably at least 100-fold.

MATERIALS & METHODS

Materials:

Cellic™ Ctec2: Cellulolytic preparation from Trichoderma reesei available from Novozymes A/S, Denmark). Yeast: RED STAR™ available from Red Star/Lesaffre, USA

Pre-treated corn stover used in Example 1 is dilute acid-catalyzed steam explosion corn stover (29.5 wt. % DS - batch 1752-91 ) obtained from NREL (National Renewable Research Laboratory, USA).

Reactor used in Example 1 : Jar tester (250 g capacity) Methods:

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 lUPAC method for measuring cellulase activity (Ghose, 1987, Measurement of Cellulase Activities, Pure & Appl. C em. 59: 257-268.

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 + 1.0 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 ddH20 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

EXAMPLES

Example 1

Washed pretreated corn stover (PCS) (20% TS) was dosed with CELLIC™ CTEC2 at 6 mg protein/g cellulose (around 12 FPU/g cellulose) and incubated at 50°C, pH 5, with agitation for 3 days. The batch was split into 2 smaller reactors. One reactor was returned to 50°C for an additional 4 days. The second reactor was dosed with RED STAR™ yeast and incubated at 32°C for 24 hours before returning it to 50°C for an additional 3 days.

Table 1. PCS cellulose conversion after standard and multi-stage processes.

Fig. 3 shows the test results.

Example 2

Material:

Biomass: Single-step autohydrolysis pretreated wheat straw produced by Inbicon (Inbicon A/S, DK), 25% dry matter content and pH of 4.7

Yeast: THERMOSACC® Dry Yeast, available from Lallemand Ethanol Technology, CAN

Enzymes: Celluclast 1.5 L and Novozym188. Enzyme preparations available from Novozymes A/S, Denmark.

An amount of 108 kg biomass was enzymatically hydrolyzed and fermented in the following sequence. Both the hydrolysis and the fermentation were carried out in a free fall mixer (WO 2006/056838). The enzyme dosages were 2.5 I Celluclast 1.5L (7.21 FPU/g biomass dry matter) and 0.5 I Novozym188.

The biomass was first hydrolyzed for 18 hours at 55°C. Then the temperature was lowered to 32°C and 250 g of yeast was added. After 121 hours of simultaneous hydrolysis and fermentation the temperature was elevated to 50°C again. This inactivates the yeast and the enzymatic hydrolysis was continued for 46 hours. Total time for the sequence was 183 hours.

The cellulose and ethanol concentration, as well as temperature profile and dry matter content in the reactions slurry was plotted versus process time in figure 4.

During the first part of the hydrolysis the glucose content increased as the hydrolysis proceed. After lowering of the temperature and addition of yeast the glucose was consumed by the yeast and ethanol formed. Ethanol formation continued until the temperature was raised to 55°C again, which killed or inactivated the yeast. After increasing the temperature after approximately 130 hours the glucose concentration increased again, which showed that the enzymes were still active and the hydrolysis rate benefited from the low level of glucose inhibition obtained by the conversion of the glucose to ethanol by an intermediate fermentation step.

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 several 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 control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

The invention is further defined by the following paragraphs:

Paragraph 1. A process of producing a fermentation product from lignocellulose-containing material, comprising the steps of pretreating lignocellulose-containing material (step (a)); hydrolyzing pretreated lignocellulose-containing material (step (b)), and fermenting pretreated, hydrolyzed lignocellulose-containing material (step (c)), wherein the initially pretreated, fermented lignocellulose-containing material is subjected to further hydrolysis (step (d)) and further fermentation (step (e)), and optionally recovery of the fermentation product (step (f)).

Paragraph 2. A process of paragraph 1 , comprising the steps of:

(a) pre-treating lignocellulose-containing material;

(c) fermenting using a fermenting organism;

(d) hydrolyzing at a temperature in the range from 25-70°C;

(e) fermenting using a fermenting organism;

(f) optionally recovering the fermentation product.

Paragraph 3. A process of paragraph 1 , comprising the steps of:

(a) pre-treating lignocellulose-containing material;

(c) fermenting using a fermenting organism;

(i) recovering the fermentation product;

(e) fermenting the using a fermenting organism;

(f) optionally recovering the fermentation product.

Paragraph 4. The process of paragraph 3, further comprising a step (step (g)) wherein the recovered fermentation product in step (f) is transferred to the recovering step (c)(i).

Paragraph 5. The process of paragraph 3 or 4, further comprising a step (step (h)) wherein the liquid fraction from the step (c)(ii) is recycled to step (b).

Paragraph 6. The process of any of paragraphs 1 -5, wherein initial hydrolysis (step (b)) runs until the degree of hydrolysis is in the range from 25-70%. Paragraph 7. The process of any of paragraphs 1 -6, wherein the dry matter content during initial hydrolysis (step (b)) is from 10-30% (w/w).

Paragraph 8. The process of any of paragraphs 1-7, wherein the temperature during initial hydrolysis (step (b)) is from 45-60°C.

Paragraph 9. The process of any of paragraphs 1-8, wherein the dry matter content during further hydrolysis (step (d)) is from 10-30% (w/w).

Paragraph 10. The process of any of paragraphs 1-9, wherein the temperature during further hydrolysis (step (d)) is from 45-60°C.

Paragraph 1 1. The process of any of paragraphs 1 -10, wherein the initially pretreated, hydrolyzed lignocellulose-containing material (step (b)) is cooled to a suitable temperature for the fermenting organism (step (c)), in particular a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C.

Paragraph 12. The process of any of paragraphs 1-1 1 , wherein the further pretreated, hydrolyzed lignocellulose-containing material (step (d)) is cooled to a suitable temperature for the fermenting organism (e.g., step (e)), in particular a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C.

Paragraph 13. The process of any of paragraphs 1 -12, wherein the initial hydrolysis (step (b)) and the initial fermentation (step (c)) are carried out either separately or simultaneously.

Paragraph 14. The process of paragraph 13, wherein the initial simultaneous hydrolysis and fermentation (steps (b) and (c)) are carried out at a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C.

Paragraph 15. The process of any of paragraphs 1 -14, wherein the initial hydrolysis (step (b)) is carried out at a temperature from 45-70°C when it is done separately from the initial fermentation step (step (c)). Paragraph 16. The process of any of paragraphs 1 -15, wherein the further hydrolysis (step (d)) and the further fermentation (step (e)) are carried out either separately or simultaneously.

Paragraph 17. The process of paragraph 16, wherein the further hydrolysis and fermentation (steps (d) and (e)) are carried out at a temperature in the range from 20-40°C, preferably 25- 35°C, especially around 32°C.

Paragraph 18. The process of any of paragraphs 1 -17, wherein the further hydrolysis (step (d)) is carried out at a temperature from 45-70°C when it is done separately from the further fermentation step (step (e)).

Paragraph 19. The process of any of paragraphs 1 -18, wherein the further hydrolysis (step (d)) runs until the degree of hydrolysis is in the range from 70-100%.

Paragraph 20. The process of any of paragraphs 1-19, wherein the fermentation product is recovered by distillation (step (f)), in particular vacuum distillation.

Paragraph 21. The process of any of paragraphs 1-20, wherein the lignocellulose-containing material is chemically, mechanically and/or biologically pre-treated (step (a)).

Paragraph 22. The process of any of paragraphs 1-21 , wherein the lignocellulose-containing material is washed before being pre-treated.

Paragraph 23. The process of any of paragraphs 1-22, wherein the pretreated lignocelluloses- containing material is washed prior to hydrolysis, preferably after pretreatment and before hydrolysis.

Paragraph 24. The process of any of paragraphs 1 -23, wherein hemicellulose from the pretreated lignocellulose-containing material is removed before initial hydrolysis (step (b)).

Paragraph 25. The process of paragraph 24, wherein the cellulose content after hemicellulose removal is at least 40% (w/w), preferably at least 50% (w/w), more preferably at least 60% (w/w), more preferably at least 70% (w/w), more preferably at least 80% (w/w), more preferably at least 90% (w/w) of insoluble solids (IS). Paragraph 26. The process of any of paragraphs 1 -25, wherein the initial hydrolysis (step (b)) is carried out at a pH in the range from 4-6, preferably around 5, and preferably for 12 to 120 hours.

Paragraph 27. The process of any of paragraphs 1 -26, wherein the further hydrolysis (step (d)) is carried out at a pH in the range from 4-6, preferably around 5, and preferably for 12 to 120 hours.

Paragraph 28. The process of any of paragraphs 1 -27, wherein the initial fermentation (step (c)) is carried out for 24-120 hours.

Paragraph 29. The process of any of paragraphs 1 -28, wherein the further fermentation (step (e)) is carried out for 24-120 hours.

Paragraph 30. The process of any of paragraphs 1-29, wherein the pretreated lignocellulose- containing material is initially hydrolyzed (step (b)) by subjecting the material to a cellulolytic preparation.

Paragraph 31. The process of paragraph 30, wherein the cellulolytic preparation is derived from the genus Trichoderma, preferably Trichoderma reesei.

Paragraph 32. The process of paragraph 31 , wherein the cellulolytic preparation comprises a beta-glucosidase.

Paragraph 33. The process of any of paragraphs 29-32, wherein the cellulolytic preparation comprises a cellulolytic enzyme enhancing activity, in particular a GH61 molecule.

Paragraph 34. The process of any of paragraphs 1-33, wherein hydrolysis is carried out in the presence of a hemicellulase, in particular a xylanase.

Paragraph 35. The process of any of paragraphs 1-34, wherein the fermenting organism is a yeast, preferably a strain of the genus Saccharomyces, Pichia, or Kluyveromyces. Paragraph 36. The process of any of paragraphs 1 -35, wherein the fermenting organism is Saccharomyces cerevisae.

Paragraph 37. The process of any of paragraphs 1 -36, wherein the fermenting organism is a C6 and/or C5 fermenting organism.

Paragraph 38. The process of any of paragraphs 1 -37, wherein the lignocellulose-containing material is selected from the group consisting of herbaceous material, agricultural residues, forestry residues, municipal solid wastes, energy crops, waste paper, pulp and paper mill residues.

Paragraph 39. The process of any of paragraphs 1 -38, wherein the lignocellulose-containing material is selected from the group of corn fiber, corn cobs, corn stover, rice straw, pine wood, wood chips, poplar, bagasse, paper and pulp processing waste, hardwood, such as poplar and birch, softwood, cereal straw, such as wheat straw, switch grass, municipal solid waste (MSW), industrial organic waste, office paper, or mixtures thereof.

Paragraph 40. The process of any of paragraphs 1 -39, wherein the fermentation product is an alcohol, preferably ethanol.

Paragraph 41. The process of any of paragraphs 1-40, wherein the initial hydrolysis (step (b)) is carried out applying free fall mixing as described in WO 2006/056838.

Paragraph 42. The process of any of paragraphs 1-41 , wherein further hydrolysis (step (d)) is carried out applying free fall mixing as described in WO 2006/056838.

Claims

1. A process of producing a fermentation product from lignocellulose-containing material, comprising the steps of pretreating lignocellulose-containing material (step (a)); hydrolyzing pretreated lignocellulose-containing material (step (b)), and fermenting pretreated, hydrolyzed lignocellulose-containing material (step (c)), wherein the initially pretreated, fermented lignocellulose-containing material is subjected to further hydrolysis (step (d)) and further fermentation (step (e)), and optionally recovery of the fermentation product (step (f)).

2. A process of claim 1 , comprising the steps of:

(a) pre-treating lignocellulose-containing material;

(c) fermenting using a fermenting organism;

(d) hydrolyzing at a temperature in the range from 25-70°C;

(e) fermenting using a fermenting organism;

(f) optionally recovering the fermentation product.

3. A process of claim 1 , comprising the steps of:

(a) pre-treating lignocellulose-containing material;

(c) fermenting using a fermenting organism;

(i) recovering the fermentation product;

(e) fermenting the using a fermenting organism;

(f) optionally recovering the fermentation product.

4. The process of claim 3, further comprising a step (step (g)) wherein the recovered fermentation product in step (f) is transferred to the recovering step (c)(i).

5. The process of claim 3 or 4, further comprising a step (step (h)) wherein the liquid fraction from the step (c)(ii) is recycled to step (b).

6. The process of any of claims 1-5, wherein initial hydrolysis (step (b)) runs until the degree of hydrolysis is in the range from 25-70%.

7. The process of any of claims 1 -6, wherein the dry matter content during initial hydrolysis (step (b)) is from 10-30% (w/w).

8. The process of any of claims 1-7, wherein the temperature during initial hydrolysis (step (b)) is from 45-60°C.

9. The process of any of claims 1 -8, wherein the dry matter content during further hydrolysis (step (d)) is from 10-30% (w/w).

10. The process of any of claims 1-9, wherein the temperature during further hydrolysis (step (d)) is from 45-60°C.

1 1 . The process of any of claims 1-10, wherein the initially pretreated, hydrolyzed lignocellulose-containing material (step (b)) is cooled to a suitable temperature for the fermenting organism (step (c)), in particular a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C.

12. The process of any of claims 1-1 1 , wherein the further pretreated, hydrolyzed lignocellulose-containing material (step (d)) is cooled to a suitable temperature for the fermenting organism (e.g., step (e)), in particular a temperature in the range from 20-40°C, preferably 25-35°C, especially around 32°C.

13. The process of any of claims 1-12, wherein the initial hydrolysis (step (b)) and the initial fermentation (step (c)) are carried out either separately or simultaneously.

14. The process of claim 13, wherein the initial simultaneous hydrolysis and fermentation (steps (b) and (c)) are carried out at a temperature in the range from 20-40°C, preferably 25- 35°C, especially around 32°C.

15. The process of any of claims 1-14, wherein the initial hydrolysis (step (b)) is carried out at a temperature from 45-70°C when it is done separately from the initial fermentation step (step (c)).

16. The process of any of claims 1 -15, wherein the further hydrolysis (step (d)) and the further fermentation (step (e)) are carried out either separately or simultaneously.

17. The process of any of claims 1 -16, wherein the fermentation product is recovered by distillation (step (f)), in particular vacuum distillation.

18. The process of any of claims 1-17, wherein the pretreated lignocellulose-containing material is initially hydrolyzed (step (b)) by subjecting the material to a cellulolytic preparation.

19. The process of any of claims 1-18, wherein the lignocellulose-containing material is selected from the group consisting of herbaceous material, agricultural residues, forestry residues, municipal solid wastes, energy crops, waste paper, pulp and paper mill residues.

20. The process of claims 1-19, wherein the fermentation product is an alcohol, preferably ethanol.