WO2012019308A1

WO2012019308A1 - Process for co-fermenting glucose and xylose

Info

Publication number: WO2012019308A1
Application number: PCT/CA2011/050494
Authority: WO
Inventors: Jan-Maarten Geertman; Azher Razvi; Glenn Munkvold
Original assignee: Iogen Energy Corporation
Priority date: 2010-08-12
Filing date: 2011-08-12
Publication date: 2012-02-16

Abstract

The present invention provides a process for the fermentation of glucose and xylose to a fermentation product. The process comprises fermenting a primary feed stream comprising glucose and xylose, thereby producing a stream comprising the fermentation product. This stream is fed to a second-stage fermentation or a second fermentation vessel and the feed to this fermentation has a ratio of glucose to xylose fed in grams per hour of between about 0.1:1.0 and about 3.0:1.0. Further provided is a process that comprises continuously fermenting the glucose and xylose with a fermentation microorganism that converts both the sugars to the fermentation product and maintaining the q_G throughout this fermentation between values of between about 0.4 and about 2.8 g glucose-DCW^-1h^-1.

Description

PROCESS FOR CO-FERMENTING GLUCOSE AND XYLOSE

TECHNICAL FIELD

[0001] The present invention relates to a method for the production of a fermentation product. More specifically, the present invention relates to a method for the production of a fermentation product from a stream comprising glucose and xylose.

BACKGROUND

[0002] Lignocellulosic feedstock is a term commonly used to describe plant-derived biomass comprising cellulose, hemicellulose and lignin. Much attention and effort has been applied in recent years to the production of fuels and chemicals, primarily ethanol, from lignocellulosic feedstocks, such as agricultural wastes and forestry wastes, due to their low cost and wide availability. These agricultural and forestry wastes are typically burned or land-filled; thus using these lignocellulosic feedstocks for ethanol production offers an attractive alternative to disposal. Yet another advantage of these feedstocks is that the lignin byproduct, which remains after the cellulose conversion process, can be used as a fuel to power the process instead of fossil fuels. Several studies have concluded that, when the entire production and consumption cycle is taken into account, the production of ethanol from

lignocellulosic feedstocks generates close to zero greenhouse gases.

[0003] The first chemical processing step for converting lignocellulosic feedstock to ethanol, or other fermentation products, involves breaking down the fibrous lignocellulosic material to liberate sugar monomers from the feedstock for conversion to a fermentation product in a subsequent step of fermentation.

[0004] There are various known methods for producing fermentable sugars from lignocellulosic feedstocks, one of which involves an acid or alkali pretreatment followed by hydrolysis of cellulose with cellulase enzymes and β-glucosidase. The purpose of the pretreatment is to increase the cellulose surface area and convert the fibrous feedstock to a muddy texture, with limited conversion of the cellulose to glucose. Acid pretreatment typically hydrolyses the hemicellulose component of the feedstock to yield xylose, glucose, galactose, mannose and arabinose and this is thought to improve the accessibility of the cellulose to cellulase enzymes. The cellulase enzymes hydrolyse cellulose to cellobiose, which is then hydrolysed to glucose by β-glucosidase. Hydrolysis of the cellulose and hemicellulose can also be achieved with a single-step chemical treatment in which the lignocellulosic feedstock is contacted with a strong acid or alkali under conditions sufficient to hydrolyse both the cellulose and hemicellulose components of the feedstock to sugar monomers.

[0005] After production of a stream comprising fermentable sugar from the lignocellulosic feedstock, a solids separation may be conducted to remove lignin, followed by fermentation of the sugars to ethanol or other fermentation products. If glucose is the predominant substrate present, the fermentation is typically carried out with a Saccharomyces spp. yeast that converts this sugar and other hexose sugars present to ethanol.

[0006] However, if the hydrolysate contains significant proportions of pentose sugars, such as xylose and arabinose derived from hemicellulose, the fermentation is preferably carried out with a microbe that has the ability to ferment xylose and/or arabinose to ethanol or other product(s).

[0007] It is commonly accepted that wild-type Saccharomyces cerevisiae, a yeast that is typically used for glucose fermentation, cannot utilize xylose. However, many yeast, including Saccharomyces cerevisiae do utilize and ferment xylulose, which is an isomer of xylose. Thus, researchers have genetically modified this yeast by introducing genes encoding the enzymes that allow xylose to be converted to xylulose. In bacteria, and some anaerobic fungi, xylose is converted to xylulose in one step by xylose isomerase (XI). U.S. Patent Nos. 6,475,768 and 7,622,284 disclose Saccharomyces strains containing a heterologous fungal or bacterial xylose isomerase (XI) gene to convert xylose to xylulose.

[0008] By contrast, in most fungi, xylose is first converted to xylitol by xylose reductase (X ) and xylitol dehydrogenase (XD) then converts the xylitol to xylulose. Recombinant Saccharomyces strains containing xylose reductase (XR) and xylitol dehydrogenease (XDH) genes from Pichia stipitis are described in U.S. Patent Nos. 5,789,210, 5,866,382, 6,582,944 and 7,527,927 and European Patent No. 450 530.

[0009] Subsequently, xylulose is phosphorylated to xylulose-5-phosphate by xylulokinase (X ), which is an intermediate in the pentose phosphate pathway (PPP). The importance of the flux through the pentose phosphate pathway has been confirmed by the superior pentose utilization and ethanolic fermentation by strains in which the enzymes of the nonoxidative PPP have been overexpressed (Hahn-Hagerdal et al., 2007, Advances in Biochemical Engineering Biotechnology 108:147-177).

[0010] Further genetic modifications to yeast strains have been made, by genetic engineering and/or adaptive evolution techniques, to enhance the xylose conversion rate of ethanol yield from xylose. These modifications include overexpression of sugar transporters (US 2007/0082386), deletion of endogenous nonspecific aldose reductase GRE3 (U.S. Patent No. 6,410,302), enhancement in the pentose phosphate pathway (WO 2005/108552, US 2006/0216804 and US 2007/0082386),

overexpression of ZWF1 (Gorsich, 2006, Applied Microbiology and Biotechnology 71(3):339-349), altering co-factor affinity, manipulating intracellular concentrations and fluxes of cofactors to minimize xylitol formation and introducing

transhydrogenase function (Hahn-Hagerdal et al., supra).

[0011] One problem arising from the fermentation of xylose or other pentose sugars originating from lignocellulosic feedstocks is the presence of fermentation inhibitors in sugar-containing streams resulting from hydrolysis of the feedstock. For example, after pretreatment of a lignocellulosic feedstock with acid, the resulting aqueous hydrolysate stream will contain acetic acid, furfural and 5-hydroxymethylfurfural (HMF), which are highly inhibitory to the yeast. Other inhibitors that may be generated by pretreatment are described in Klinke et al., 2004, Applied Microbiology and Biotechnology 66(1): 10-26; Larsson et al., 2000, Applied Biochemistry and Biotechnology 84-86(l):617-632; Georgieva et al., 2008, Applied Biochemistry and Biotechnology 145(l-3):99-l 10; Sundstrom et al., 2009, Applied Biochemistry and Biotechnology 161(1-8): 106-115; and Helle et al., 2003, Enzyme and Microbial Technology 33(6):786-792. Various strategies have been proposed to remove inhibitors, such as overliming (U.S. Patent Nos. 2,203,360, 4,342,831, 6,737,258, 7,455,997 and Wooley et al., 1999, Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzyme Hydrolysis Current and Future Scenarios, Technical Report, National Renewable Energy Laboratory pp. 16-17) and ion exchange (Nilvebrant et al., 2001, Applied Biochemistry and Biotechnology 91-93:35-49 and Watson et al., 1984, Enzyme and Microbial Technology 6:451-456). However, these processes are often costly and are likely to result in an increase in overall costs for the production of ethanol or other desired fermentation product(s) from the lignocellulosic feedstock.

[0012] Moreover, strategies that require a single microorganism to convert both glucose and xylose to a femientation product can suffer from low xylose consumption rates. Even when yeast cells are modified genetically to use xylose, they

preferentially ferment glucose before switching to the much slower xylose fermentation. This is because sugar transport specific to xylose does not naturally exist in yeast. Therefore, aspecific transport of glucose, xylose and other sugars into the cell occurs via transmembrane transporters like intermediate glucose transporters or galactose transporters. In most cases, these transporters have a higher affinity for hexose than for pentose sugars. Thus, when glucose is present at high concentrations in the medium, xylose has less competitive advantage with respect to transport into the cell and this invariably leads to low xylose uptake. This is especially problematic when the fermentation is operated in batch mode since the hexose transporter is saturated with both sugars at one time. Moreover, not all hexose transporters are able to translocate pentose sugars across the cellular membrane and expression of these transporters varies with the state of fermentation.

[0013] Energy metabolism plays an important role in the fermentation of sugars to ethanol. Free energy in the form of ATP is the energy available to microorganisms for metabolic processes. These processes can be precursors for synthesis for cell growth, synthesis of proteins that enable transport or cellular responses to the environment (such as stress conditions and inhibitory compounds). As transcription and especially translation are energetically expensive processes (Warner, 1999, Trends in Biochemical Sciences 24:437-440) the depletion of the ATP concentration may also hinder protein synthesis, which in turn can reduce the ability of the yeast to withstand inhibitory conditions and its ability to sustain pentose fermentation rates.

[0014] The regeneration rate of ATP during a specific metabolic reaction can have a significant effect on its overall rate. Hexose fermentation is characterized by high carbon and energy flux rates, and thus high regeneration rates of ATP. By contrast, pentose fermentation, such as on xylose, is characterized by much lower rates of ATP generation (Kresnowati et al., 2008, FEBS Journal 275(22):5527-41 and Abbott, 2009, FEMS Yeast Research 9:349-357). Consequently, the ATP availability during xylose only fermentations is low and results in an energy-limited state.

[001 ] Moreover, some inhibitors generated during the production of lignocellulosic hydrolysate result in the loss of ATP available for the above-mentioned metabolic processes. In particular weak organic acids, such as acetic acid, can diffuse across the yeast cell membrane in the undissociated (protonated) form. Once inside the cell, the acid dissociates because of the higher intracellular pH and this causes acidification of the cytoplasm. To maintain the intracellular pH, the cells must pump protons out of the cell, which requires ATP.

[0016] The presence of inhibitors, slow xylose transport into the yeast cell and the lack of ATP availability all contribute to low xylose conversion rates. Low xylose conversion rates, in turn, further reduce the ATP availability, which can lead to further decreases in the xylose conversion rates. Although low conversion rates can be offset by increasing the fermentation time, this is not always a preferred option. Moreover, prolonged fermentation can lead to poor yeast health since under such conditions the cells are not able to revive quickly due to the presence of inhibitors.

[0017] There is thus a need to develop methods whereby the conversion rates of xylose from solutions containing both xylose and glucose may be further increased. Previous fermentation approaches to convert both glucose and xylose to ethanol and other biochemicals may not be able to achieve conversion rates necessary to make the process economically viable.

SUMMARY OF THE INVENTION

[0018] The present invention provides a method for producing a fermentation product. More specifically, the present invention relates to a method for the production of a fermentation product from a stream comprising xylose and glucose.

[0019] It is an object of the invention to provide an improved method for the production of a fermentation product from xylose.

[0020] The process of the invention provides improvements in the conversion of xylose to a fermentation product from solutions containing both glucose and xylose. [0021] According to a first aspect of the invention, there is provided a process for the fermentation of glucose and xylose to a fermentation product, the process comprising:

(i) fermenting a primary feed stream comprising glucose and xylose in a first- stage fermentation vessel, thereby producing a first-stage stream comprising the fermentation product, which primary feed stream has a weight ratio of glucose to xylose of between about 0.1 :1.0 and about 3.0:1.0 and wherein fermentation of about 30 wt% to about 90 wt% of the xylose occurs in said first-stage fermentation vessel;

(ii) withdrawing the first-stage stream from the first-stage fermentation vessel and feeding same to a second-stage fermentation vessel, wherein the feed to the second- stage fermentation vessel has a ratio of glucose to xylose fed in grams per hour of between about 0.1 :1.0 and about 3.0:1.0;

(iii) fermenting the glucose and xylose in the second-stage fermentation vessel; and

(iv) withdrawing a second-stage stream therefrom comprising the fermentation product, wherein steps (i) to (iv) are conducted continuously and wherein steps (i) and (iii) are carried out with a fermentation microorganism that converts both glucose and xylose to the fermentation product.

[0022] According to embodiments of the invention, the feed to the second-stage fermentation vessel may comprise both the first-stage stream and a glucose-containing stream. Without being limiting, the glucose-containing stream may be a portion of the primary feed stream. The primary feed stream may be a hydrolysate from a lignocellulosic feedstock. The hydrolysate may be produced by pretreating the lignocellulosic feedstock to hydrolyse hemicellulose, followed by hydrolysis of the cellulose by cellulase enzymes.

[0023] According to further embodiments of the invention, the ratio of glucose to xylose in the primary feed stream is between 1.0:1.0 and 3.0:1.0 wt:wt. [0024] The first-stage and second-stage reaction vessels may optionally be connected in series. In further embodiments, the second-stage stream is sent to a third-stage fermentation vessel and any remaining xylose in said second-stage stream is converted to the fermentation product therein.

[0025] The fermentation product may be selected from the group consisting of ethanol, lactic acid, citric acid, succinic acid, butanol, pyruvic acid, malic acid, itaconic acid and glycerol.

[0026] According to one embodiment of the invention, the fermentation

microorganism is a genetically modified Saccharomyces cerevisiae strain. The Saccharomyces cerevisiae may comprise genes encoding for xylose reductase (XR) and xylitol dehydrogenease (XDH). According to further embodiments of the invention, the Saccharomyces cerevisiae comprises a gene encoding for xylitol isomerase. The fermenting microorganism may be separated and recycled during the process.

[0027] According to further embodiments of the invention, the q_G during steps (i) to (iii) is maintained between about 0.4 and about 2.8.

[0028] According to a second aspect of the invention, there is provided a process for the fermentation of glucose and xylose to ethanol, the process comprising:

(i) fermenting a portion of a primary feed stream comprising glucose and xylose in a first-stage fermentation vessel, thereby producing a first-stage stream comprising ethanol, which primary feed stream has a weight ratio of glucose to xylose of between 0.1:1.0 and 3.0:1.0 and wherein fermentation of 50 wt% to 90 wt% of the xylose occurs in said first-stage fermentation vessel;

(ii) withdrawing the first-stage stream from the first-stage fermentation vessel and feeding same to a second-stage fermentation vessel;

(iii) feeding a portion of the primary feed stream to the second-stage fermentation vessel, such that the combined feed from the first-stage stream and the portion of the primary feed stream fed to the second-stage fermentation has a ratio of glucose to xylose fed in grams per hour of between 0.1:1 and 3.0:1; (iv) fermenting the glucose and xylose in the second-stage fermentation vessel; and

(v) withdrawing a second-stage stream therefrom comprising the ethanol, wherein steps (i) to (v) are conducted continuously and wherein steps (i) and (iv) are carried out using a fermentation microorganism that is capable of converting both glucose and xylose to ethanol.

[0029] According to a third aspect of the invention, there is provided a process for the fermentation of glucose and xylose to a fermentation product, the process comprising:

(i) introducing a feed stream comprising glucose and xylose to a fermentation system comprising one or more stages, which feed stream has a weight ratio of glucose to xylose of between 0.1 :1.0 and 3.0:1.0;

(ii) continuously fermenting the glucose and xylose with a fermentation microorganism that converts both the glucose and xylose to the fermentation product;

(iii) maintaining the qo throughout said fermentation between about 0.4 and about 2.8; and

(iv) recovering the fermentation product thus produced, wherein the fermentation is performed under anaerobic conditions.

[0030] The "qo" is defined as the glucose fed to the fermentation minus the glucose out of the fermentation divided by the cell concentration (measured in dry cell weight per liter (DCW-L^"1). This quotient is multiplied by the dilution rate (L^"1). The units of this measurement are g glucose-g DCW^_I-h^_1.

[0031] According to the fourth aspect, there is provided a process for the fermentation of glucose and xylose to a fermentation product, the process comprising:

(i) fermenting a primary feed stream comprising glucose and xylose in a fermentation vessel, thereby producing a stream comprising the fermentation product, which primary feed stream is produced from a lignocellulosic feedstock; (ii) withdrawing the stream comprising the fermentation product from the fermentation vessel and feeding same to a second fermentation vessel, wherein the feed to the second fermentation vessel has a ratio of glucose to xylose fed in grams per hour of between about 0.1 :1.0 and about 3.0:1.0;

(iii) fermenting the glucose and xylose in the second fermentation vessel wherein steps (i) to (iii) are conducted continuously and wherein steps (i) and (iii) are carried out with a fermentation microorganism that converts both glucose and xylose to the fermentation product.

[0032] By operating the fermentation in accordance with the invention, xylose uptake rates can be substantially higher than in a corresponding, single-stage batch fermentation or a continuous fermentation without glucose present. Without being bound by any particular theory, a number of mechanisms could provide for the improvements in conversion rates observed. Firstly, the continuous fermentation of the invention could allow for the co-consumption of both glucose and xylose as the culture is not saturated with all available sugar at one time as is typical in batch culture. By operating the fermentation in this mode, xylose has more competitive advantage with respect to transport into the yeast cell by high affinity glucose transporters. Moreover, the presence of glucose in both stages of the fermentation may have a "priming effect" upon the oxidative stage of the pentose phosphate pathway, which is not present with xylose alone.

[0033] Without being limiting, another feature that is believed to contribute to the improvements in conversion rates is the higher ATP availability for xylose metabolism. That is, by increasing the carbon flux rates of xylose metabolism through co-fermentation of glucose, higher regeneration rates of ATP can be achieved to facilitate xylose fermentation. Additionally, by operating a continuous process, a reduction in the concentration of inhibitors can be achieved to facilitate xylose fermentation. This can further increase ATP availability since, as mentioned previously, inhibitors generated during the production of fermentable sugar, such as acetic acid can reduce ATP availability. [0034] Regardless of the proposed mechanism(s), the present invention provides a significant advance with respect to maximizing the utilization of all sugars present in lignocellulosic hydrolysates or other streams comprising both xylose and glucose.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGURE 1 is a schematic of a system for co-fermenting glucose and xylose according to an embodiment of the invention.

[0036] FIGURE 2 is a schematic of a lab-scale setup used to investigate the influence glucose consumption rate (qo) has on xylose consumption rate (qx) in a two-stage microaerobic continuous fermentation performed to co-ferment xylose and glucose to ethanol.

[0037] FIGURE 3 is a plot of qx versus qo for one and two-stage microaerobic continuous fermentation experiments performed to co-ferment xylose and glucose to ethanol.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect. The headings provided are not meant to be limiting of the various embodiments of the invention. Terms such as "comprises", "comprising", "comprise", "includes", "including" and "include" are not meant to be limiting. In addition, the use of the singular includes the plural, and "or" means "and/or" unless otherwise stated. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0039] The primary feed stream to the first-stage fermentation vessel may contain fermentable sugar derived from a lignocellulosic feedstock. Lignocellulosic feedstock includes any type of plant biomass such as, but not limited to, non- woody plant biomass, cultivated crops such as, but not limited to grasses, for example, but not limited to, C4 grasses, such as switch grass, cord grass, rye grass, miscanthus, reed canary grass, or a combination thereof, sugar processing residues, for example, but not limited to, baggase, such as sugar cane bagasse, beet pulp, or a combination thereof, agricultural residues, for example, but not limited to, soybean stover, corn stover, rice straw, rice hulls, barley straw, sugar cane straw, corn cobs, wheat straw, canola straw, oat straw, oat hulls, corn fiber, or a combination thereof, forestry biomass for example, but not limited to, recycled wood pulp fiber, sawdust, hardwood, for example aspen wood, softwood, or a combination thereof

Furthermore, the lignocellulosic feedstock may comprise cellulosic waste material or forestry waste materials such as, but not limited to, newsprint, cardboard and the like. Lignocellulosic feedstock may comprise one species of fiber or, alternatively, lignocellulosic feedstock may comprise a mixture of fibers that originate from different lignocellulosic feedstocks. Moreover, new lignocellulosic feedstock varieties may be produced from any of those species listed above by plant breeding or by genetic engineering.

[0040] Lignocellulosic feedstocks comprise cellulose in an amount greater than about 20%, more preferably greater than about 30%, more preferably greater than about 40%) (w/w). For example, the lignocellulosic material may comprise from about 20% to about 50% (w/w) cellulose, or any amount therebetween. The lignocellulosic feedstock also comprises lignin in an amount greater than about 10%, more typically in an amount greater than about 15% (w/w). The lignocellulosic feedstock may also comprise small amounts of sucrose, fructose and starch. Additionally, the feedstock may contain pectin.

[0041] The lignocellulosic feedstock may be pretreated. Pretreatment methods are intended to deliver a sufficient combination of mechanical and chemical action so as to disrupt the fiber structure and increase the surface area of feedstock to make it accessible to hydrolytic enzymes such as cellulases. Mechanical action typically includes the use of pressure, grinding, milling, agitation, shredding,

compression/expansion and chemical action includes the use of heat (often steam), acid or alkali, and solvents.

[0042] The pretreatment is preferably a chemical treatment involving the addition of acid or alkali. This includes any acid or alkali that is suitable for disrupting fiber structure of the lignocellulosic feedstock and increasing accessibility of the lignocellulosic feedstock to being hydrolysed in a subsequent enzymatic hydrolysis. Non-limiting examples of suitable acid and alkali for such purpose include sulfuric acid, nitric acid, hydrochloric acid, sulfurous acid, phosphoric acid, ammonia, ammonium hydroxide, sodium hydroxide, potassium hydroxide, lime and magnesium hydroxide.

[0043] Pretreatment with acid hydrolyses the hemicellulose, or a portion thereof, that is present in the lignocellulosic feedstock to the monomeric sugars including, but not limited to, xylose, arabinose, mannose, and/or galactose, and organic acids, such as acetic acid, galacturonic acid and glucuronic acid. Sucrose, fructose and starch may also be present in the sugar hydrolysate. Preferably, the acid pretreatment is performed so that nearly complete hydrolysis of the hemicellulose and a small amount of conversion of cellulose to glucose occurs. The cellulose is hydrolysed to glucose in a subsequent step that uses cellulase enzymes. Typically a dilute acid, at a concentration from about 0.02% (w/v) to about 2% (w/v), or any amount

therebetween, (measured as the percentage weight of pure acid in the total weight of dry feedstock plus aqueous solution) is used for the pretreatment. Preferably, the acid pretreatment is carried out at a temperature of about 180°C to about 250°C, or any temperature therebetween, for a time of about 60 seconds to about 600 seconds, or any time therebetween, at a pH of about 0.8 to about 2.0, or any pH therebetween.

[0044] After pretreatment, the lignocellulosic feedstock may be separated to obtain a solids stream comprising the pretreated feedstock and an aqueous stream comprising soluble components. This may be carried out by washing the pretreated feedstock with an aqueous solution to produce a wash stream, and a solids stream comprising the pretreated feedstock. Alternatively, the pretreated feedstock is subjected to a solids-liquid separation, using known methods such as centrifugation, microfiltration, plate and frame filtration, crossflow filtration, pressure filtration, vacuum filtration and the like. When an acidic pretreatment is employed, the aqueous phase comprises sugars produced by the hydrolysis of hemicellulose, as well as the acid added during the pretreatment and any organic acids liberated during the pretreatment.

[0045] The pH of the pretreated feedstock is typically adjusted so that it is within a range that is optimal for the cellulase enzymes used. Generally, the pH of the pretreated feedstock is adjusted to within a range of about 3.0 to about 7.0, or any pH therebetween. For example, the pH may be within a range of about 4.0 to about 6.0, or any pH therebetween, between about 4.5 and about 5.5, or any pH therebetween.

[0046] The temperature of the pretreated feedstock is adjusted so that it is within the optimum range for the activity of the cellulase enzymes. Generally, a temperature of about 45 °C to about 55°C, or any temperature therebetween, is suitable for most cellulase enzymes, or any temperature therebetween.

[0047] The cellulase enzymes and the β-glucosidase enzyme are added to the pretreated feedstock, prior to, during, or after the adjustment of the temperature and pH of the aqueous slurry after pretreatment. Preferably the cellulase enzymes and the β-glucosidase enzyme are added to the pretreated lignocellulosic feedstock after the adjustment of the temperature and pH of the slurry.

[0048] By the term "cellulase enzymes" or "cellulases," it is meant a mixture of enzymes that hydrolyse cellulose. The mixture may include cellobiohydrolases (CBH), endoglucanases (EG) and beta-glucosidase. In a non-limiting example, a cellulase mixture may include CBH, EG and beta-glucosidase enzymes. By the term "beta-glucosidase", it is meant any enzyme that hydrolyses the glucose dimer, cellobiose, to glucose. The activity of the beta-glucosidase enzyme is defined by its activity by the Enzyme Commission as EC 3.2.1.21.

[0049] Any type of cellulase enzymes suitable for hydrolysing cellulose to glucose can be utilized, regardless of their source. Non-limiting examples of cellulases which may be used in the practice of the invention include those obtained from fungi of the genera Aspergillus, Humicola, Chrysosporium, Myceliopthora, Penicillium, Neurospora, Thielavia, Sporotrichum and Trichoderma, and from bacteria of the genera Bacillus and Thermobifida.

[0050] The cellulase enzyme dosage is chosen to convert the cellulose of the pretreated feedstock to glucose. For example, an appropriate cellulase dosage can be about 0.1 to about 100.0 Filter Paper Units (FPU or IU) per gram of cellulose, or any amount therebetween.

[0051] The enzymatic hydrolysis with cellulase enzymes produces a solution comprising glucose, unconverted cellulose and lignin. Other components that may be present in the hydrolysate slurry include xylose, arabinose, mannose and galactose, acetic acid, glucuronic acid and galacturonic acid, as well as silica, insoluble salts and other compounds.

[0052] The primary feed stream comprising glucose and xylose fed to the first-stage fermentation vessel may arise from one or more of the foregoing stages in the processing of the lignocellulosic feedstock that yield streams containing fermentable sugar. For example, a hemicellulose hydrolysate separated from a solids stream comprising the pretreated feedstock may be the primary feed stream submitted to the first-stage fermentation vessel. This sugar hydrolysate will typically comprise xylose, glucose, arabinose, mannose and galactose. Alternatively, a stream of pretreated feedstock comprising cellulose as well as monomeric sugars resulting from hemicellulose hydrolysis is subjected to enzymatic hydrolysis with cellulase enzymes. The resultant stream is then sent to the first-stage fermentation vessel. Treating the feedstock in this manner yields a stream comprising sugars liberated from

hemicellulose during pretreatment, as well as glucose resulting from the enzymatic hydrolysis of cellulose. In a further embodiment, a hemicellulose hydrolysate is separated from the pretreated feedstock and then is added to the stream comprising glucose obtained from the enzymatic hydrolysis of cellulose, thereby producing a stream comprising both pentose sugars derived from hemicellulose and glucose, which in turn is sent to the first-stage fermentation vessel.

[0053] In yet a further embodiment of the invention, the primary feed stream fed to the first-stage fermentation vessel is obtained by a complete acid or alkali hydrolysis of a lignocellulosic feedstock in which both the cellulose and hemicellulose components are hydrolysed to their monomeric constituents in a single step.

[0054] Sugar streams derived from lignocellulosic feedstocks contain a number of compounds that may or may not be inhibitory to the microorganism in the

fermentation. Furan derivatives such as 2-furaldehyde (furfural) and 5- hydroxymethyl-2-furaldehyde (HMF) are inhibitory compounds that originate from the breakdown of the carbohydrate fraction, namely xylose and glucose, respectively, although other inhibitory compounds can be present in sugar streams as set forth below. These compounds can be degraded further by pretreatment or hydrolysis into organic acids including acetic acid, as well as formic, and levulinic acids that are also inhibitory. Additional organic acids found in the sugar stream that may be inhibitory to yeast or other microorganisms include galacturonic acid, lactic acid, glucuronic acid, 4-O-methyl-D-glucuronic acid or a combination thereof. Inhibiting phenolic compounds are also produced by the degradation of lignin, which include vanillin, syringaldeyhde, and hydroxybenzylaldehyde. In particular, vanillin and

syringaldehyde are produced via the degradation syringyl propane units and guaiacylpropane units of lignin (Jonsson et al., 1998, Applied Microbiology and Biotechnology 49:691).

[0055] Acetic acid is a component of sugar streams produced from lignocellulosic material that is highly inhibitory to yeast. The acetate arises from acetyl groups attached to xylan and lignin that are liberated as acetic acid and/or acetate by exposure to acid or other chemicals that hydrolyse the feedstock. (Abbott et al., 2007, FEMS Yeast Research 7:819-833; Hu et al., 2009, Bioresource Technology 100:4843-4847; Taherzadeh et al, 1997, Chemical Engineering Science, 52:2653-5659.). Acetic acid has a pKa of about 4.75 (Ka of 1.78 x 10^"5) so that at pH 4.0, about 14.8 mole% of the acid is present as acetate. Thus, the species present in the sugar stream will depend on the pH of the solution. Although it should be appreciated that the practice of the invention is not limited by the pH of the sugar stream, the fermentation is typically conducted at a pH at which acetate is the dominant species in solution. However, the term "acetate" as used herein encompasses acetic acid species. Acetate may be present in the sugar stream at a concentration of between about 0.1 and about 50 g/L, about 0.1 and about 20 g/L, about 0.5 and about 20 g/L or about 1.0 and about 15 g/L.

[0056] The inhibitory compounds set forth above are representative of the compounds present in a sugar stream produced from a lignocellulosic feedstock. It will be appreciated that the inhibitory compounds present depend on both the raw material and the pretreatment that is employed.

[0057] In a preferred embodiment, the primary feed stream to the first-stage fermentation is substantially free of undissolved solids, such as lignin and other unhydrolysed components. This is particularly advantageous in embodiments of the invention employing a subsequent step of separating and recycling the yeast from the fermentation broth since it is desirable to avoid any significant recycle of undissolved solids along with the yeast. The separation may be carried out by known techniques, including centrifugation, microfiltration, plate and frame filtration, crossflow filtration, pressure filtration, vacuum filtration and the like.

[0058] The primary feed stream has a weight ratio of glucose to xylose of between about 0.1 :1.0 and about 3.0:1.0. Streams having such a range of glucose to xylose weight ratios are typical of that produced by pretreatment and/or enzymatic hydrolysis. In a further embodiment of the invention, the primary feed stream has a ratio of glucose to xylose of between 1.5:1.0 and 2.25:1.0.

[0059] During the first-stage fermentation, between about 30% and up to about 90% (w/w) of the xylose is consumed relative to that in the primary feed stream. In one embodiment of the invention, between about 30% and about 90% or between about 50% and 90% or between about 60% and about 90% of the xylose is consumed during the first-stage fermentation relative to that present in the primary feed stream. This includes all ranges therebetween, including sub-ranges having numerical limits of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and 90%.

Higher levels of xylose consumption, i.e., 95% and up to 100% could be attained as well. In one embodiment of the invention, the glucose is "substantially consumed" during the first-stage fermentation. By "substantially consumed", it is meant that less than 5 wt% of the glucose in the primary feed stream remains in the first-stage stream withdrawn from the first-stage fermentation.

[0060] After the first-stage fermentation, a first-stage stream is withdrawn from the first-stage fermentation vessel. This stream is then fed to the second-stage fermentation vessel where the second-stage fermentation is conducted. The feed to the second-stage fermentation vessel has a ratio of glucose to xylose fed in grams per hour of between about 0.1 : 1 and about 3.0:1. In one example of the invention, the feed to the second-stage fermentation vessel has a ratio of glucose to xylose fed in grams per hour of between about 0.1 :1 and about 2.0:1 or between about 0.1:1 and about 1.5 : 1 or between about 0.1:1 and about 1.0:1 or between about 0.1:1 and about 0.5:1, or any range therebetween. The combined glucose to xylose weight ratio entering the second-stage fermentor can be measured by known techniques readily available to those of ordinary skill in the art. [0061] In those embodiments where most or all of the glucose is consumed in the first-stage fermentation, the feed to the second-stage fermentation vessel may be supplemented with a glucose-containing stream in order to attain a combined feed having a ratio of glucose fed (g-h^"1) to xylose fed (g-bf¹) of between about 0.1:1 and about 3.0: 1. That is, the stream withdrawn from the first-stage fermentation vessel and fed to the second-stage fermentation vessel and the glucose-containing stream fed to the second-stage fermentation vessel should together supply a combined feed ratio (g-h ¹) in this range. The amount of feed supplemented from the glucose-containing stream may be varied as required to achieve a desired ratio of glucose to xylose (wt:wt) during the second-stage fermentation. In a particularly advantageous embodiment of the invention, the glucose-containing stream fed to the second-stage vessel is the primary feed stream.

[0062] In. those embodiments where a significant amount of glucose and xylose remains after the first-stage fermentation, the feed to the second-stage fermentor need not be supplemented with the glucose-containing stream. That is, the desired the feed ratio (g-h^-1) of glucose and xylose may be attained by feeding only the first stage stream containing both xylose and glucose to the second-stage fermentation vessel.

[0063] The second-stage stream withdrawn from the second-stage fermentation vessel may be sent to one or more additional fermentation vessels. Schemes similar to those employed for the second-stage fermentation may be utilized for subsequent fermentation stages. Alternatively, any sugar remaining after the fermentation may be converted to yeast cell mass in subsequent vessels.

[0064] It should be appreciated that an additional fermentation stage(s) can be interposed between the first and second stages or an additional stage(s) can be carried out before the first stage and/or after the second stage.

[0065] Although a two-stage, continuous fermentation has been described, the fermentation may alternatively be conducted in a single fermentation reactor.

[0066] The specific glucose consumption during fermentation referred to herein as "qG" may range between about 0.4 and about 2.8 g glucose-g DCW^-h^"1 or between about 0.6 and about 2.0 g glucose-g DCW^-h^"1. [0067] The fermentation may be performed at or near the temperature and pH optima of the fermentation microorganism. The temperature range for the fermentation may be between about 10°C to about 70°C, although the temperature may be higher if the yeast is naturally or genetically modified to be thermostable. For example, the temperature may be from about 10°C to about 55°C, or any temperature

therebetween, or about 15°C to about 45°C, or any temperature therebetween. The pH of the fermentation may be between about 3 and about 6, or any pH therebetween, for example, a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or any pH therebetween. The dose of the fermentation microorganism will depend on other factors, such as the activity of the fermentation microorganism, the desired fermentation time, the volume of the reactor and other parameters. It will be appreciated that these parameters may be adjusted as desired by one of skill in the art to achieve optimal fermentation conditions.

[0068] The fermentation may also be supplemented with additional nutrients required for growth and fermentation performance of the fermentation microorganism. For example, yeast extract, specific amino acids, phosphate, nitrogen sources, salts, trace elements and vitamins may be added to the sugar hydrolysate to support growth and optimize productivity of the microorganism. (See also Verduyn et al., 1992, Yeast 8(7):501-170, fergensen, 2009, Applied Biochemistry and Biotechnology, 153:44-57 and Zhao et al., 2009, Journal of Biotechnology, 139:55-60, which are each incorporated herein by reference).

[0069] The fermentation is conducted under anaerobic conditions, which includes microaerobic fermentations.

[0070] A suitable method for ensuring that anaerobic or microaerobic conditions are maintained during the fermentation (i.e., that a culture is not respiring) is to measure the amount of oxygen consumption and the carbon dioxide production and then calculate a respiratory quotient (RQ), which is the ratio of C0₂ produced to 0₂ consumed. A suitable RQ for the fermentation can be determined with ease by those of ordinary skill in the art. [0071] As used herein, the term anaerobic means that the Q is maintained in a range between about 2 and about 50. Preferably, the RQ is maintained in a range between about 5 and about 30.

[0072] The first and second stages of the fermentation are continuous. By the term "continuously" it is meant that the streams fed to the fermentation vessels and exiting the fermentation vessels are continuously fed and withdrawn from the vessels.

Typically, the streams are fed and withdrawn at equal volumetric rates from the fermentation vessels. Stages subsequent to the second-stage fermentation may be operated in either continuous, batch or fed-batch mode.

[0073] The dilution rate employed during the fermentation will depend on the growth rate of the yeast and the concentration of yeast cells in the fermentation, which in turn depends on whether or not yeast recycle is employed. A suitable dilution rate may be selected by those of ordinary skill in the art taking into account such variables.

Without being limiting, the dilution rate may be selected so that it is greater than the growth rate of the fastest growing contaminant in the fermentation. In such embodiments, the dilution rate may be relatively low since with no recycle populations of contaminating microbes may not be allowed to establish. With recycle, higher dilution rates may be employed such as between 0.5 and 2.5 h^~l.

[0074] The fermentation reactors may be arranged in series, parallel or a combination of these two arrangements. In one embodiment of the invention, the fermentation reactors are agitated lightly with mixing. The fermentation configuration may also include tanks for conditioning the yeast. Any one of a number of known yeasts may be used to convert the xylose and glucose to ethanol or other fermentation products. This includes, but is not limited to yeast from the genera Saccharomyces, Hansenula, Pichia, Kluyveromyces and Candida. The yeast may be genetically engineered to ferment both hexose and pentose sugars to ethanol. Alternatively, the yeast may be a strain that has been made capable of xylose fermentation by one or more non- recombinant methods, such as adaptive evolution or random mutagenesis and selection.

[0075] Non-limiting examples of fermentation products include an alcohol, such as ethanol or butanol, organic acids, such as lactic acid, citric acid, itaconic acid, pyruvic acid, malic acid or succinic acid and glycerol. For example, see Geertman et al., 2006, Metabolic Engineering, 8(6):532-542, which describes high yields of glycerol from glucose by Saccharomyces cerevisiae; and Van Maris et al., 2004, Applied and Environmental Microbiology, 70(1):159-166, which describes the production of pyruvic acid from glucose using Saccharomyces cerevisiae. The production of succinic acid from fumaric acid by recombinant yeast or filamentous fungi that contain NAD(H) dependent fumarate reductase has been described (WO

2009/065778). Moreover, recombinant microbes have been produced that comprise a nucleotide sequence encoding malate synthase which catalyzes the conversion of glyoxylic acid to malic acid (WO 2009/101180).

[0076] Byproduct formation is typically avoided. For example, xylitol production should be avoided as it reduces the yield of ethanol from xylose. According to one embodiment of the invention, less than 0.20 g xylitol is produced per gram of xylose converted. In embodiments of the invention, less than 0.20, 0.15, 0.10, 0.05 or 0.01 g xylitol is produced per gram of xylose converted.

[0077] The recombinant Saccharomyces yeast strain may be a strain that has been made capable of xylose fermentation by recombinant incorporation of (a) genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) (see for example, U.S. Patent Nos. 5,789,210, 5,866,382, 6,582,944 and 7,527,927 and EP 450 530) and/or (b) gene(s) encoding one or more xylose isomerase (XI) (see for example, U.S. Patent Nos. 6,475,768 and 7,622,284). In addition, the modified yeast strain may also overexpress an endogenous or heterologous gene encoding xylulokinase.

[0078] Other yeast besides Saccharomyces cerevisiae can ferment hexose and pentose sugars to ethanol. This includes, but is not limited to other yeast of the genera Saccharomyces, and those of Hansenula, Pichia, Kluyveromyces and Candida. WO 2008/130603 discloses Hansenula polymorpha strains with increased production of ethanol from xylose. Moreover, Pichia stipitis and Candida shehatae mutants have been isolated by the method disclosed in U.S. Patent No. 5,126,266.

[0079] In embodiments of invention, yeast recycle is employed. This is achieved by separating the yeast from the fermentation broth, such as by centrifugation, and then re-circulating the yeast back to the same or other fermentation vessels in the configuration. By re-introducing yeast back into the fermentation vessel(s) in this manner, the concentration of yeast in the fermentor(s) is continuously maintained at a high level, without significant diversion of sugars to cell growth and away from the desired fermentation product. Yeast recycle may be employed at any stage of the fermentation process.

[0080] The yeast are separated from the broth by known separation techniques to produce a yeast slurry. Examples of suitable separation techniques include, but are not limited to, centrifugation, microfiltration, plate and frame filtration, crossflow filtration, pressure filtration, settling, vacuum filtration and the like.

[0081] Bacterial contamination can reduce productivity during yeast fermentation. To reduce or eliminate bacterial contamination during the fermentation, an oxidizing agent can be introduced to the fermentation that selectively kills microbial contaminants. Preferably, the oxidant is added to the yeast slurry separated from the fermentation broth prior to its recycle to a fermentation vessel. (See co-pending and co-owned WO 2009/026706, which is incorporated herein by reference). Suitable antimicrobial agents that reduce the concentration of unwanted microbes include oxidants selected from the group consisting of ozone, chlorine, chlorine dioxide, hydrogen peroxide and potassium permanganate. Preferably, the oxidant is chlorine dioxide. This oxidant destroys microbial cells via the oxidation of aromatic and sulfur-containing amino acids of the intracellular enzymes. Chlorine dioxide is particularly effective since bacteria are more susceptible to its effects than yeast. This is because most bacterial enzymes are located just inside the cell membrane while most yeast enzymes reside deeper inside the cell structure. However, the invention is not limited by the particular antimicrobial agent that is selected for decontamination.

[0082] In one exemplary embodiment of the invention, the oxidant possesses maximum effectiveness at reducing bacterial contaminants at a pH of less than 4.0, or less than 2.5. (See co-pending and co-owned U.S. Serial No. 61/308,028, which is incorporated herein by reference). A suitable oxidant can be selected by those of ordinary skill in the art by routine experimentation. Without being limiting, the oxidant may be chlorine dioxide or ozone. [0083] If ethanol is the product of the fermentation, it may be recovered from the fermentation broth by distillation. After distillation, further removal of water may be carried out using molecular sieves or other expedients.

[0084] Referring now to Figure I, there is shown a non-limiting example of the

fermentation conducted in accordance with embodiments of the invention. Figure 1 is for illustrative purposes only and should not be construed to limit the current

invention in any manner.

[0085] According to this embodiment of the invention, a primary feed stream 10 comprising at least xylose and glucose is fed to a first-stage fermentation vessel 20.

Optionally, the primary feed stream 10 also feeds into downstream fermentation vessels, as discussed below. This stream 10 will additionally comprise other sugars derived from the lignocellulosic feedstock, such as galactose, mannose and arabinose. Nutrients 30 can be added to the primary feed stream prior to its introduction to the first-stage fermentation vessel 20.

[0086] Fermentation of the xylose and glucose in the primary feed stream 10 to

ethanol is then conducted in the first-stage fermentation vessel 20. In this non- limiting embodiment of the invention, the glucose in the primary feed stream 10 is almost or completely converted to ethanol so that a first-stage product stream 40 withdrawn from the first-stage fermentation vessel 20 is substantially devoid of

glucose. Xylose conversion to ethanol may be less complete, i.e., between 50 and 90 wt% may remain. The first-stage stream 40 is then fed via pump 50 to a second-stage fermentation vessel 60. Because glucose is now depleted in the first-stage stream 40, the ratio of glucose to xylose fed into the second-stage fermentation vessel 60 is controlled by the rate of fresh feed from the primary feed stream 10 containing both glucose and xylose.

[0087] After fermentation of xylose and glucose to ethanol in the second-stage

fermentation vessel 60, a second-stage stream 70 is withdrawn comprising ethanol, xylose, but that is mostly depleted of glucose. The second-stage stream 70 is pumped via pump 80 to a third stage fermentation vessel 90. This fermentation vessel 90 is also fed with the primary feed stream 10 to control the ratio of glucose to xylose that is fed. The stream 100 withdrawn from the third-stage fermentation vessel 90 is then pumped via pump 105 to a centrifuge 110 or other solid-liquid separation device. The aqueous stream 120 containing ethanol is sent to a beer tank 140 and then to distillation to concentrate the ethanol. After distillation, the ethanol may be further concentration by molecular sieves, membrane extraction or other techniques for removing the small amounts of water remaining. A yeast slurry 130 obtained from the solids-liquid separation step is then treated with an oxidant, such as chlorine dioxide, to reduce the concentration of microbial contaminants. Prior to chlorine dioxide treatment, the yeast slurry may be treated with sulfuric acid to reduce the pH of the slurry to below about 3.0. As described previously in co-pending and commonly owned PCT/CA2011/000220, which is incorporated herein by reference, this serves to increase the efficacy of the chlorine dioxide. The oxidant-treated yeast slurry is then recycled back to fermentation vessels 20, 60 and 90.

[0088] It should be appreciated that yeast slurry from the solid-liquid separator need not be sent back to each fermentation reactor in the system, as shown in Figure 1. If desired, the yeast slurry need only be re-circulated to one or two fermentation reactors in the system.

EXAMPLES

Example 1: Single pass, single-stage anaerobic continuous fermentation

Feed Preparation

[0089] Lignocellulosic hydrolysate was prepared by pretreating wheat straw with sulfuric acid as set forth in U.S. Patent No. 4,461,648 (Foody). The pretreated feedstock was adjusted to pH 5.0 with alkali and subsequently hydrolysed with cellulase enzymes secreted by Trichoderma reesei to produce a lignocellulosic hydrolysate comprising glucose, xylose and other sugars derived from hemicellulose.

[0090] For each continuous stirred tank reactor (CSTR), three flasks were prepared in an identical manner. One glycerol stock stored at -80°C was used to inoculate a 2 L baffled flask containing 1000 mL of media prepared as set forth in Verduyn et a!., 1992, Yeast 8(7):501-570, which is incorporated herein by reference, plus 60 g/L glucose. Cells were cultivated for 48 h in a shaker incubator at 30°C and 160 rpm. Cells were then spun down and transferred to a flask containing 500 mL of lignocellulosic hydrolysate described previously adjusted to pH 6. The cells were incubated for 72 h in a shaker incubator at 30°C and 160 rpm with an initial pH of 6. The conditioned cells were again spun down and re-suspended in the vitamins and traces required for the batch start-up phase of the CSTR experiment (see Verduyn et al., supra). The cells were aseptically transferred to a 60 mL sterile syringe and needle assembly. The syringe and its contents were then used to inoculate 14 L fermentors where the CSTR experiments took place.

CSTR Experiments

[0091] A series of continuous culture experiments were performed under microaerated conditions at varying dilution rates. The microaerated conditions were maintained with no air flow to the fermentor, as only the oxygen available in the headspace of the fermentor would be transferred. Feed for all experiments was lignocellulosic hydrolysate obtained as described previously. The CSTR experiments were performed at a pH set point of 5 maintained via addition of 10% (w/v) ammonium hydroxide, a temperature of 30°C, and an agitation rate of 250 rpm. A working volume of 7.5 L was targeted and the level was maintained with a draft tube set at a specific height in the fermentor.

Sample Analysis

[0092] Once at steady-state, which was attained after 5 turnovers of the vessel volume, 3 to 4 samples were taken over the period of one additional turnover. The analyses of these samples were averaged. From this sample, a 2 mL aliquot was centrifuged at 14,000 x g for 3 minutes and the supernatant filtered through a 0.2 μιη syringe and filter assembly. Dilutions of the supernatant were prepared in a 5 mM sulfuric acid solution and filtered again. Sample dilutions were analyzed via HPLC using an Agilent 1100 Series HPLC system. Glucose, ethanol, xylose and xylitol were quantified using an Agilent 1100 Series Refractive Index Detector while acetic acid was quantified using an Agilent 1200 Series Variable Wavelength Detector. The HPLC system was also equipped with an 1100 Series Autosampler and Pumping System. The controlling software for the HPLC was Chemstation. A Varian® column maintained at 50°C was used for separation. The eluent was a 5 mM aqueous sulfuric acid solution with a flow rate of 0.600 mL-min^"1. [0093] Biomass determination was performed via dry cell weight (DCW) measurement. According to this method, a 10 niL aliquot was filtered through a pre- weighed FisherBrand® Glass fiber filter circle (G6). The filter paper and its contents were dried using a Procter Silex® Model #35038 microwave set at 450 Watts for 15 minutes at 40% power. Samples were checked daily for contamination via microscope. As previously mentioned, three dilution rates were studied.

[0094] Table 2 summarizes the results of the experiments performed.

Table 2: Concentration and yield results from the three anaerobic steady-states

Xylose Ethanol Cell Y_E Y_c q_x

0.0746 15.8 29.3 3.05 0.44 0.046 0.347

0.110 11.9 32.2 4.32 0.46 0.062 0.461

Batch - 39.1 12.0 0.46 0.041 0.083

[0095] The results presented in Table 2 show enhanced xylose consumption anaerobically with co-consumption of glucose and xylose as compared to typical diauxic consumption kinetics observed in batch culture or when compared to a xylose only feed for continuous fermentation. For example, compared to batch fermentation consumption rates of xylose in the co-fermentation, the physiological specific consumption of xylose is usually in the range of 0.05-0.08 g xylose-g DCW^'^h^"1. This is an order of magnitude less than what was observed in the CSTR experiments performed herein, which have q_x = 0.25-0.45 g-xylose-g DCW^-h^"1 (based on 30 g-L^"1 xylose in the feed).

Example 2: Single pass, two-stage microaerobic continuous fermentation

[0096] The experiments described herein illustrate how an increase in the specific glucose consumption rate increases the specific xylose consumption rate.

Experiments were performed in two-stage, microaerobic, continuous culture to illustrate the relationship that exists between qo and qx. A schematic of the experimental setup is shown in Figure 2.

[0097] Two fermentors were set up in series with all of the effluent from the first stage being pumped into the second stage. The fermentor volumes were maintained in the same manner as described in Example 1. Conditions for the experiments were the same as Example 1 with the exception of pH which was 5.5 for these experiments. Lignocellulosic hydrolysate prepared in the same manner as that in Example 1 was simultaneously fed to both the first stage and second stages as shown. The stage 1 glucose and xylose feed concentrations were 61 and 32 g-L^"1, respectively. For stage 2, the feed composition varied.

[0098] A dilution rate of 0.075 h^"1 was targeted for all glucose to xylose ratios investigated. In order to vary the glucose to xylose ratio entering stage 2, the effluent rate and xylose concentration from stage 1 were used in combination with the concentration of glucose and xylose in the hydrolysate feed to calculate the feed rate and volume required in the second stage to achieve the desired ratio as well as sustain a constant dilution rate of 0.075 h^"1 over the range of ratios studied.

[0099] The relevant variables and equations used to determine the flow and volume are as follows:

D_x = D₂ = 0.075 h^"1

G_F = 60 g-L^"1

X_F = 30 g-L^"1

Vi = 8 L

Fi_{j In} = 0.6 L-h^"1

V₂ = unknown

F_2; i_n = unknown Equation 1 :

G_ Total glucose into stage 2

X Total xylose into stage 2

F ¹ l,Out G^J l,Out + ^τ F¹2,Ιη G^ Ρ

2

Equation 2:

[00100] There are two unknowns F_{2; ¾} and V₂ with two equations that can be solved for varying glucose to xylose concentrations. Table 3 below shows the volumes and flows required for achieving the desired ratios.

Table 3: Volumes and flows to achieve the desired glucose-to-xylose ratio (G/X)

(G X)₂ v₂ F₂

(g-g ¹) (L) (L-h-¹)

0.000 8.00 0.00

0.200 8.50 0.0373

0.400 9.12 0.0842

0.600 9.93 0.145

0.800 11.02 0.227

[00101] The glucose and xylose concentrations and DCW were determined as set out in the Sample Analysis section of Example 1.

[00102] Figure 3 illustrates the effect of glucose uptake rate on xylose uptake rate for the single pass, single stage microaerobic continuous fermentation (Example 1) and the single pass, two stage microaerobic fermentation (Example 2). Data from both stage 1 and stage 2 of the two stage microaerobic fermentation is presented. Open triangles represent data from the single stage microaerobic fermentation. Open squares and open diamonds represent data from stage 1 and stage 2 of the two-stage microaerobic fermentation, respectively.

Claims

1. A process for the fermentation of glucose and xylose to a fermentation product, the process comprising:

(i) fermenting a primary feed stream comprising glucose and xylose in a first- stage fermentation vessel, thereby producing a first-stage stream comprising the fermentation product, which primary feed stream has a weight ratio of glucose to xylose of between about 0.1:1.0 and about 3.0:1.0 and wherein fermentation of about 30 wt% to about 90 wt% of the xylose occurs in said first-stage fermentation vessel;

(ii) withdrawing the first-stage stream from the first-stage fermentation vessel and feeding same to a second-stage fermentation vessel, wherein the feed to the second- stage fermentation vessel has a ratio of glucose to xylose fed in grams per hour of between about 0.1:1.0 and about 3.0:1.0;

(iv) withdrawing a second-stage stream therefrom comprising the fermentation product,

wherein steps (i) to (iv) are conducted continuously and

wherein steps (i) and (iii) are carried out with a fermentation microorganism that converts both glucose and xylose to the fermentation product.

2. The process according to claim 1 , wherein the feed to the second-stage fermentation vessel comprises both the first-stage stream and a glucose-containing stream.

3. The process of claim 2, wherein the glucose-containing stream is a portion of the primary feed stream.

4. The process of claim 1, 2 or 3, wherein the ratio of glucose to xylose in the primary feed stream is between 1.0:1.0 and 3.0:1.0 w wt.

5. The process of any one of claims 1-4, wherein the second-stage stream is sent to a third-stage fermentation vessel and wherein any remaining xylose in said second- stage stream is converted to the fermentation product therein.

6. The process of any one of claims 1 -5, wherein the fermentation product is selected from the group consisting of ethanol, lactic acid, citric acid, succinic acid, butanol, pyruvic acid, malic acid, itaconic acid and glycerol.

7. The process of claim 6, wherein the fermentation product is ethanol.

8. The process of claim 7, wherein the fermentation microorganism is a genetically modified Saccharomyces cerevisiae strain.

9. The process of claim 8, wherein the Saccharomyces cerevisiae comprises genes encoding for xylose reductase (XR) and xylitol dehydrogenease (XDH).

10. The process of claim 8, wherein the Saccharomyces cerevisiae comprises a gene encoding for xylitol isomerase.

11. The process of any one of claims 1-10, wherein the first-stage and second- stage reaction vessels are connected in series.

12. The process of any one of claims 1-11, wherein primary feed stream is a hydrolysate from a lignocellulosic feedstock.

13. The process of claim 12, wherein the hydrolysate is produced by pretreating the lignocellulosic feedstock to hydrolyse hemicellulose, followed by hydrolysis of the cellulose by cellulase enzymes.

14. The process of any one of claims 1-13, wherein the fermenting microorganism is separated and recycled during the process.

15. The process of any one of claims 1-14, wherein the qo during steps (i) to (iii) is maintained between about 0.4 and about 2.8.

16. A process for the fermentation of glucose and xylose to a fermentation product, the process comprising:

(i) fermenting a portion of a primary feed stream comprising glucose and xylose in a first-stage fermentation vessel, thereby producing a first-stage stream comprising the fermentation product, which primary feed stream is produced from a

lignocellulosic feedstock and wherein fermentation of 50 wt% to 90 wt% of the xylose occurs in said first-stage fermentation vessel;

(iii) feeding a portion of the primary feed stream to the second-stage fermentation vessel, such that the combined feed from the first-stage stream and the portion of the primary feed stream fed to the second-stage fermentation has a ratio of glucose to xylose fed in grams per hour of between 0.1 :1 and 3.0:1 ;

(iv) fermenting the glucose and xylose in the second-stage fermentation vessel; and

(v) withdrawing a second-stage stream therefrom comprising the fermentation product,

wherein steps (i) to (v) are conducted continuously and wherein steps (i) and (iv) are carried out using a fermenting microorganism that is capable of converting both glucose and xylose to the fermentation product.

17. The process of claim 16, wherein the fermentation product is ethanol.

18. A process for the fermentation of glucose and xylose to a fermentation product, the process comprising:

(iii) maintaining the qo throughout said fermentation between about 0.4 and about 2.8; and (iv) recovering the fermentation product thus produced, wherein the fermentation is performed under anaerobic conditions.

19. A process for the fermentation of glucose and xylose to a fermentation product, the process comprising:

(i) fermenting a primary feed stream comprising glucose and xylose in a fermentation vessel, thereby producing a stream comprising the fermentation product, which primary feed stream is produced from a lignocellulosic feedstock;

(ii) withdrawing the stream comprising the fermentation product from the fermentation vessel and feeding same to a second fermentation vessel, wherein the feed to the second fermentation vessel has a ratio of glucose to xylose fed in grams per hour of between about 0.1:1.0 and about 3.0:1.0;

(iii) fermenting the glucose and xylose in the second fermentation vessel wherein steps (i) to (iii) are conducted continuously and

20. The process of any one of claims 1-7, 11, 12, 13 and 15-19, wherein the fermentation microorganism is a yeast.