WO2012155238A1

WO2012155238A1 - Method of fermenting a sugar stream to produce an alcohol stream

Info

Publication number: WO2012155238A1
Application number: PCT/CA2012/000434
Authority: WO
Inventors: Murray J. Burke; Bradley Saville; Daniel Jing LIAO
Original assignee: Mascoma Canada Inc.
Priority date: 2011-05-18
Filing date: 2012-05-08
Publication date: 2012-11-22

Abstract

The present disclosure includes a method of fermenting a sugar stream to produce an alcohol stream in the presence of a nitrogen compound and an alkaline agent.

Description

TITLE: METHOD OF FERMENTING A SUGAR STREAM TO PRODUCE AN

ALCOHOL STREAM

FIELD

[0001] This application relates to a method for fermenting a sugar stream to produce an alcohol stream, wherein the sugar stream comprises fermentable sugars and a weak acid. More specifically, this application relates to conducting the fermentation in the presence of a nitrogen source and an alkaline agent.

BACKGROUND

[0002] Although biomass has long shown promise as a renewable source of fuel energy, there remains a need for more efficient means of transforming biomass into suitable biofuels. Plant materials are a significant source of fermentable sugars, such as glucose that can be transformed into biofuels. However, the sugars in plant materials are contained in long polymeric chains of cellulose and hemicellulose. Utilizing current fermentation processes, it is necessary to break down these polymeric chains into monomeric sugars, prior to the fermenting step.

[0003] Methods of converting plant biomass into fermentable sugars are known in the art and in general, comprise two main steps: a pretreatment step to loosen the plant structure, and an enzymatic or chemical hydrolysis step to convert the polymeric chains of cellulose and hemicellulose into monomeric sugars. Several approaches have been used for the pretreatment step, e.g., autohydrolysis, acid hydrolysis, ammonia activation, kraft pulping, organic solvent pulping, hot water pretreatment, ammonia percolation, lime pretreatment, caustic solvent pulping, and alkali peroxide pretreatment. Each pretreatment technology has a different mechanism of action on the plant structure, inducing either physical and/or chemical modifications. However, the main objective of the pretreatment is to provide accessibility of the plant material to the enzymes. In the autohydrolysis process, the acetyl groups attached to hemicelluloses are broken down by steam and pressure releasing organic acids, e.g., acetic acid, giving the conditions for a mild acid hydrolysis process. Although a simple process, the yield of fermentable sugars is poor, in addition to the process requiring a significant amount of energy.

[0004] Enzymatic hydrolysis, using enzymes such as hemicellulases and cellulases, may be used to catalyze the hydrolysis of hemicellulose or cellulose to simple sugars, which can then be subjected to fermentation to produce ethanol. The production of inhibitors, such as acetic acid, during the autohydrolysis and enzymatic hydrolysis significantly decreases the efficiency of the subsequent fermentation.

[0005] Overall, the production process is complex and has low conversion rates. Cellulosic ethanol processes, namely processes that produce ethanol from sugars obtained by breaking down the cellulose and/or hemicellulose from non- corn plant fiber (i.e. plant fiber that excludes corn kernels), typically produce a raw alcohol stream having an ethanol content of about 2 - 6% v/v. Accordingly, ethanol from plant stalks and similar biomass may not be cost competitive with ethanol made from corn kernels.

SUMMARY

[0006] This application relates to a method of fermenting a sugar stream to produce an alcohol stream wherein the sugar stream comprises fermentable sugars and a weak acid. The fermentation is conducted the presence of a nitrogen compound and an alkaline agent wherein the amount and type of nitrogen compound and an alkaline agent are selected to maintain the pH of the sugar stream at a level at which the weak acid at least partially dissociates. The pH adjustment of the fermentation medium, which alters the ratio of the weak acid and its conjugate base, in combination with nitrogen containing compounds significantly enhances the fermentation rate of the sugar stream.

[0007] This application also relates to a two-stage enzymatic process to prepare the sugar stream from a feedstock derived from plant materials. The process and apparatus may result in the conversion of at least 60%, preferably more than 75% and more preferably over 90% of the cellulose and hemicelluloses to monomeric sugars, which can then be fermented to alcohol using the process of the application. The alcohol stream from the fermentation stage (i.e., the raw alcohol stream) may have an ethanol content of about 3 to about 22% v/v. Optional operating ranges include about 5 to about 15% and preferably about 5 to about 22% as well as about 8 to about 2%, preferably about 8 to about 15% and more preferably about 8 to about 22% (v/v). Such alcohol concentrations may be obtained without using com as a feedstock.

[0008] With the process and apparatus described in this application, cellulose ethanol plants may produce a raw alcohol stream having a comparable alcohol concentration to that obtained by corn based ethanol plants, namely plants that produce ethanol from sugars obtained from the starch in corn. Accordingly, one advantage of the process and apparatus of this invention is that the amount of water to be removed from the raw alcohol stream to produce a fuel ethanol stream having a comparable concentration to the concentration of a product stream from a corn based ethanol plant is substantially reduced compared to current cellulosic ethanol plant technology. As a fuel ethanol stream is typically produced by distillation, the process and apparatus described here therefore results in a substantial reduction in energy required for the distillation process and, optionally, a substantial reduction in the size (i.e., the diameter) of the distillation column compared to current cellulose ethanol plant technology. Furthermore, as the ethanol concentration increases in the raw ethanol stream, the fermentation volume decreases, representing a 2 to 3 times reduction when compared to current cellulosic ethanol plant technology. [0009] Another advantage of the process of the invention is the recovery and recycling of the enzymes used in each stage of the enzymatic process which permits enzymes that would otherwise be lost, being redeployed to an alternate stage where the enzymes may be efficaciously used. Accordingly, the amount of enzymatic hydrolysis occurring per unit of enzymes is increased. In one embodiment, at least some of the enzymes from each of the enzymatic stages and the fermentation process are recovered from product streams and recycled for further use in the enzymatic hydrolysis stages. In one embodiment, the product streams are subjected to ultrafiltration and/or diafiltration to recover the enzymes.

[0010] In one embodiment, the lignocellulosic feedstock is subjected to a first enzymatic hydrolysis process to preferentially solubilize xylose and obtain an effluent stream. During the first enzymatic hydrolysis process or stage, hemicellulose and cellulose are broken down, preferably to solubilize oligosaccharides of sugars. During this step, it is preferred to preferentially hydrolyze the hemicelluloses instead of the celluloses, and therefore solubilize xylose to obtain an effluent stream (e.g., preferentially acts on the hemicellulose relative to the cellobiose in the feedstock). For example, this process step may utilize an enzyme preparation comprising hemicellulase and cellulase activities. While it will be appreciated that a suitable enzyme preparation will typically contain enzymes that may act on the cellulose, it is preferred that only a portion of the celluloses will be converted.

[0011] Subsequently, the effluent stream from the first enzymatic hydrolysis process is subjected to a second enzymatic hydrolysis process or stage to preferentially solubilize cellulose and obtain a sugar rich process stream. The second enzymatic hydrolysis process preferably utilizes enzymes to hydrolyze cellulose as well as to convert the oligosaccharides to monomeric sugars suitable for fermentation. Preferably, this second enzyme preparation comprises beta- glucosidase activities. For example, the second enzyme preparation may have an activity to convert cellulose and cellobiose to monomers and cello- oligosaccharides. In this second enzymatic hydrolysis process, it is preferred that all, or essentially all, (e.g., preferably at least about 60, more preferably at least about 75 and most preferably at least about 90%) of the remaining cellulose and hemicelluloses, and their respective oligosaccharides, are converted, to the extent desired, but preferably to the extent commercially feasible, to monomeric sugars. In one embodiment, the second enzymatic hydrolysis process or stage also utilizes fermentation organisms to simultaneously ferment the sugar rich process stream to alcohol.

[0012] In one embodiment, the monomeric sugars produced as described above constitute the sugar stream that is used to conduct a fermentation to produce an alcohol stream. However, it will be understood that monomeric sugars or a sugar stream produced from any process can be utilized with the fermentation process of the present application.

[0013] Without being limited by theory, oligosaccharides, and in particular cellobiose, have an inhibitory effect on celiulase enzymes and, in particular, on endo-gluconases and cellobiohydrolases. Accordingly, in the first stage, the hemicelluloses, and optionally the cellulose, are treated with enzymes to produce soluble sugars, for example, xylose. However, the process is preferably conducted so as not to render a substantial portion of the cellulose into monomers or dimers, such as cellobiose. While it will be appreciated that enzymatic hydrolysis will result in the production of some monomers and cellobiose, the process is preferably conducted so as to prevent a substantial inhibition of the enzymes. Subsequently, in the second enzymatic process, the oligosaccharides are subjected to enzymatic hydrolysis to produce fermentable sugars (preferably monomers).

[0014] Preferably, the first enzyme preparation preferentially acts on the hemicellulose to solubilize the xylose. In accordance with this embodiment, without being limited by theory, it is believed that in such a first enzymatic process, the hemicellulose is broken down into oligomers and monomers that are removed from the fiber as soluble compounds in an aqueous medium (preferably water). This targeted enzymatic process opens up the fiber structure by the breakdown of the hemicellulose and the removal of the lower molecular weight compounds. The resultant more open fiber structure permits enzymes, such as cellulases, to more readily enter the fiber structure and hydrolyze the cellulose.

[0015] Accordingly, the second enzymatic hydrolysis step preferably uses enzymes that preferentially target cellulose relative to hemicellulose in the feedstock (e.g., the second enzyme preparation preferentially acts on the cellulose and cellobiose relative to xylans in the feedstock). It will be appreciated that the second enzymatic hydrolysis step may use an enzyme preparation that includes enzymes that target hemicelluloses. However, as most of the hemicelluloses may have already been treated in the first stage, a relatively large percentage of such enzymes may not be required in the second enzyme preparation.

[0016] In this application, the term preferentially hydrolyze means that a significant portion of the enzymes that are used target the hemicelluloses instead of the celluloses (or vise versa), even though some of the enzymes present may still target the celluloses. Preferred preferential hydrolysis in the first stage, include hydrolyzing about 60% or more, and preferably about 85% or more, of the hemicelluloses while, preferably, hydrolyzing less than about 25%, and more preferably less than about 15% of the celluloses.

[0017] The first enzyme preparation preferentially acts upon the β-1 ,4 linkage of the xylose residues of xylan and the p- ,4 linkage of the mannose residues of mannan. However, many commercial hemicellulase enzyme preparations also possess cellulase activity. As the hemicellulose is hydrolyzed, water is released from the fiber, in addition to the production of oligosaccharides and monomeric sugars. This hydrolysis results in the reduction in the length of hemicellulose and cellulose polymer chains.

[0018] During the first stage enzymatic hydrolysis processes, acetyl groups are removed from the hemicellulose. In an aqueous medium, these form acetic acid. Acetic acid reduces the pH of the mixture in the reactor, e.g., from about 4.9 to about 4.4. This pH reduction has an inhibitory effect on the first stage enzyme preparation. Therefore, in accordance with a preferred embodiment, acetic acid is treated or removed from the process. For example, the acetic acid may be neutralized by the addition of a neutralizing agent (e.g., urea, anhydrous ammonia, aqueous ammonia, sodium hydroxide, potassium hydroxide) and/or acetic acid may be removed from the process, such as by operating under vacuum. As acetic acid is relatively volatile, it may be drawn off by vacuum as it is produced. Further, as the first stage enzymatic process reduces the viscosity of the mixture in the reactor, the mixture is more easily induced to flow, e.g., due to stirring, and the acetic acid has a greater chance to reach the surface of the mixture and volatilize.

[0019] Further aspects and advantages of the embodiments described herein will appear from the following description taken together with the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] For a better understanding of the embodiments described herein and to show more clearly how they may be carried into effect, reference will now be made, by way of example only, to the accompanying drawing which shows at least one exemplary embodiment, and in which:

[0021] Figure 1 is a flow chart of the method according to embodiments that include a two-stage enzymatic hydrolysis process followed by enzyme recovery and fermentation with the addition of a nitrogen source and an alkaline agent; [0022] Figure 2 is a flow chart of the method according to embodiments that include a two-stage enzymatic hydrolysis process followed by fermentation with the addition of a nitrogen source and an alkaline agent, and subsequent enzyme recovery;

[0023] Figure 3 is a flow chart of the method according to embodiments that include the addition of an alkaline agent during a two-stage enzymatic hydrolysis process followed by fermentation with the addition of a nitrogen source and an alkaline agent;

[0024] Figure 4 is a graph illustrating the ethanol concentration at different time points during fermentation with addition of different nitrogen sources and alkaline agents using the method according to embodiments of the disclosure;

[0025] Figure 5 is a graph illustrating the ethanol productivity at different time points during fermentation with addition of different nitrogen sources and alkaline agents using the method according to different embodiments of the disclosure;

[0026] Figure 6 is a graph illustrating the ethanol productivity at different time points during fermentation with addition of different nitrogen sources and alkaline agents using the method according to different embodiments of the disclosure;

[0027] Figure 7 is a graph illustrating the ethanol concentration at different time points during fermentation with addition of different nitrogen sources and alkaline agents using the method according to different embodiments of the disclosure;

[0028] Figure 8 is a graph illustrating the ethanol concentration at different time points during fermentation with addition of different nitrogen sources and alkaline agents using the method according to different embodiments of the disclosure;

[0029] Figure 9 is a graph illustrating the ethanol productivity at different time points during fermentation with addition of different nitrogen sources and alkaline agents using the method according to different embodiments of the disclosure; [0030] Figure 10 is a graph illustrating the concentration of various components during a fermentation;

[0031] Figure 1 1 is a graph illustrating the pH at different time points during fermentation with addition of a nitrogen source and different alkaline agents using the method according to different embodiments of the disclosure; and

[0032] Figure 12 is a graph illustrating the pH at different time points during fermentation with addition of a nitrogen source and different alkaline agents using the method according to different embodiments of the disclosure.

DETAILED DESCRIPTION

[0033] This application relates generally to a method of fermenting a sugar stream to produce an alcohol stream, in which the sugar stream comprises fermentable sugars and a weak acid. In particular, the fermentation is conducted in the presence of a nitrogen compound and an alkaline agent, wherein the amount and type of nitrogen compound and an alkaline agent are selected to maintain the pH of the sugar stream at a level at which the weak acid at least partially dissociates.

[0034] The application also includes a process for treating a lignocellulosic feedstock to breakdown cellulose and hemicellulose in the feedstock into monomeric sugars such as glucose, which may be fermented using the method of the present application to produce alcohol, and also to recover and recycle the enzymes used to hydrolyze the cellulose and hemicellulose. The product streams from one stage and, preferably, each stage of the enzymatic hydrolysis and the fermentation are treated such that at least some of the viable enzymes used in the enzymatic hydrolysis of hemicellulose and cellulose are recovered and optionally, can be recycled for use in further enzymatic hydrolysis reactions. [0035] In an optional embodiment, the applicants have found that activating and/or physically modifying the feedstock prior to the enzymatic hydrolysis process results in an increased yield of fermentable sugars in the process stream and/or a faster reaction rate.

[0036] Figure 1 exemplifies a schematic of different embodiments of the invention. The processes to be discussed may be used singularly or in any particular combination or sub-combination. The lignocellulosic feedstock 10 is optionally first subjected to a pretreatment and optional steam explosion 12 to produce an activated feedstock 14, and then subsequently an optional disc refining step 16 to produce a fine particulate stream 8. It will be appreciated that neither of these optional steps, or one or both of these optional steps, may be utilized. The fine particulate stream is then subjected to a first enzymatic hydrolysis stage 20 to produce an effluent stream 22. The first enzymatic hydrolysis stage 20 preferentially hydrolyzes the hemicelluloses in the feedstock to produce monomeric sugars of xylose and glucose. The effluent stream may also contain short oligosaccharides and other higher molecular weight oligosaccharides that have not been fully hydrolyzed in the first enzymatic hydrolysis stage. The effluent stream 22 also contains unhydrolyzed cellulose, which is preferentially hydrolyzed in the second enzymatic hydrolysis stage 28. The effluent stream further contains enzyme inhibitors, such as acetic acid and monomeric sugars (end product inhibition), which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and cellulases used in the first and/or second enzymatic hydrolysis stages 20 and 28.

[0037] The effluent stream 22 may be subjected to a solid/liquid separation 24, for example by means of a filter press, a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone to produce a solid stream 26 and a liquid/filtrate stream 30. The solid stream 26 contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage 20. The liquid/filtrate stream 30, which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage 20, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, enzyme inhibitors (inhibitors to the enzymes used in a second enzymatic hydrolysis stage 28) are removed from the solid stream 26 before being subjected to the second enzymatic hydrolysis stage 28.

[0038] The solid stream 26, containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 28. The second enzymatic hydrolysis stage 28 preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose. The second enzymatic hydrolysis stage 28 produces a sugar rich process stream 32, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose.

[0039] The sugar rich process stream 32 is then subjected to a solid/liquid separation 34, such as by means of a filter press, a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone to produce a second solid stream 38 and a second liquid/filtrate stream 36. Accordingly, the lignin may be removed from the sugar rich process stream 32 before the sugars (i.e., those in second liquid/filtrate stream 36) are subjected to fermentation. As lignin inhibits the yeast used in fermentation, the removal of lignin increases the yield of alcohol.

[0040] The second solid stream 38, containing lignin, ash and/or other insoluble components such as unhydrolyzed hemicellulose and/or cellulose, is optionally further processed to obtain a purified lignin stream. This lignin stream may then be disposed of or used in a subsequent process.

[0041] The liquid/filtrate streams 30 and 36 are optionally combined to form sugar stream 40, which contains fermentation inhibitors, such as weak acids, for example acetic acid or succinic acid, produced from the enzymatic hydrolysis processes 20 and 28. Sugar stream 40 may then be optionally subjected to micro solid/liquid separation. The streams may be treated separately or, optionally combined and treated concurrently. The streams may be subjected to one or more micro solid/liquid separation, for example, ultrafiltration and/or diafiltration. Referring to Figure 1 , sugar stream 40 is subjected to micro solid/liquid separation 42, such as ultrafiltration, to produce a recovered enzyme stream 44 and an enzyme reduced stream 46. When ultrafiltration is utilized, the ultrafiltration separation filters hemicellulose and/or cellulase enzymes from sugar stream 40 , which allows these enzymes to be recovered and/or recycled. Accordingly, the recovered enzyme stream 44 is then optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28.

[0042] The enzyme reduced stream 46 may then be subjected to a further micro solid/liquid separation 48, such as diafiltration, to produce a second recovered enzyme stream 50 and a sugar rich enzyme reduced stream 52. When diafiltration is utilized, the diafiltration process filters any remaining hemicellulase and/or cellulase enzymes. Accordingly, the second recovered enzyme stream 50 is then optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. Sugar rich enzyme reduced stream 52 is preferably essentially free of enzymes and lignin.

[0043] In one embodiment, the recovered enzyme stream 44 and the second recovered enzyme stream 50 are combined before being recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. It will be understood that one or both of the above micro solid/liquid separation processes (for example, ultrafiltration and diafiltration) may be used to recover the hemicellulase and/or cellulase enzymes.

[0044] The sugar rich enzyme reduced stream 52 may then be subjected to fermentation 54 to produce an alcohol stream 60, such as ethanol, from the sugars in the sugar rich enzyme reduced stream 52. The removal of lignin from the liquid/filtrate stream 32, which inhibits the organisms in the fermentation stage, also increases the amount of alcohol that is produced as compared to when the lignin is not removed, such as in a simultaneous saccharification and fermentation.

[0045] During fermentation 44, a nitrogen source stream 46 and an alkaline agent stream 48 are added to the fermentation broth to maintain the pH of the fermentation at a level at which weak acids present in the sugar rich enzyme reduced stream 52 (carried over from the enzymatic hydrolysis processes 20 and 28) at least partially dissociates, improving the yield of the alcohol produced in the fermentation 44.

[0046] Figure 2 exemplifies a schematic of different embodiments of the invention. The processes to be discussed may be used singularly or in any particular combination or sub-combination. The lignocellulosic feedstock 1 10 is optionally first subjected to a pretreatment and optional steam explosion 1 2 to produce an activated feedstock 1 14, and then subsequently an optional disc refining step 1 16 to produce a fine particulate stream 18. It will be appreciated that neither of these optional steps, or one or both of these optional steps, may be utilized. The fine particulate stream is then subjected to a first enzymatic hydrolysis stage 120 to produce an effluent stream 122. The first enzymatic hydrolysis stage 120 preferentially hydrolyzes the hemicelluloses in the feedstock to produce monomeric sugars of xylose and glucose. The effluent stream may also contain short oligosaccharides and other higher molecular weight oligosaccharides that have not been fully hydrolyzed in the first enzymatic hydrolysis stage. The effluent stream 122 also contains unhydrolyzed cellulose, which is preferentially hydrolyzed in the second enzymatic hydrolysis stage 128. The effluent stream further contains enzyme inhibitors, such as acetic acid and monomeric sugars (end product inhibition), which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and cellulases used in the first and/or second enzymatic hydrolysis stages 120 and 128. [0047] The effluent stream 122 may be subjected to a solid/liquid separation 24, for example by means of a filter press, a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone to produce a solid stream 126 and a liquid/filtrate stream 130. The solid stream 126 contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage 120. The liquid/filtrate stream 130, which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage 120, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, enzyme inhibitors (inhibitors to the enzymes used in a second enzymatic hydrolysis stage 128) are removed from the solid stream 126 before being subjected to the second enzymatic hydrolysis stage 128.

[0048] The solid stream 126, containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 128. The second enzymatic hydrolysis stage 128 preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose. The second enzymatic hydrolysis stage 128 produces a sugar rich process stream 132, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose.

[0049] The sugar rich process stream 132 is then subjected to a solid/liquid separation 134, such as by means of a filter press, a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone to produce a second solid stream 138 and a second liquid/filtrate stream 136. Accordingly, the lignin may be removed from the sugar rich process stream 132 before the sugars (i.e., those in second liquid/filtrate stream 136) are subjected to fermentation, or further processing. As lignin inhibits the yeast used in fermentation, the removal of lignin increases the yield of alcohol. [0050] The second solid stream 138, containing lignin, ash and/or other insoluble components such as unhydrolyzed hemicellulose and/or cellulose, is optionally further processed to obtain a purified lignin stream. This lignin stream may then be disposed of or used in a subsequent process.

[0051] The liquid/filtrate streams 130 and 136 are optionally combined to form sugar stream 140, which contains fermentation inhibitors, such as weak acids, for example acetic acid or succinic acid, produced from the enzymatic hydrolysis processes 120 and 128. Sugar stream 140 is then subjected to fermentation 142, in which a nitrogen source stream 144 and an alkaline agent stream 146 are added to the fermentation broth to maintain the pH of the fermentation at a level at which weak acids present in the sugar stream 140 (carried over from the enzymatic hydrolysis processes 120 and 128) at least partially dissociate, improving the yield of the alcohol stream 148 produced in the fermentation 142.

[0052] Alcohol stream 148 may then be optionally subjected to a solid/liquid separation 1 50, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, to produce a solid stream 152 and a treated alcohol stream (a liquid stream) 154. Solid stream 152 contains recovered fermentation organisms such as yeast, and also the alkaline agent, which may be recycled to fermentation 142. Treated alcohol stream 1 54 may then be subjected to one or more micro solid/liquid separation, for example, ultrafiltration and/or diafiltration. Referring to Figure 2, treated alcohol stream 1 54 is subjected to micro solid/liquid separation 156, such as ultrafiltration, to produce a recovered enzyme stream 160 and an enzyme reduced stream 158. When ultrafiltration is utilized, the ultrafiltration separation filters hemicellulose and/or cellulase enzymes from treated alcohol stream 154, which allows these enzymes to be recovered and/or recycled . Accordingly, the recovered enzyme stream 160 is then optionally recycled to the first and/or second enzymatic hydrolysis stages 120 and 128. [0053] The enzyme reduced stream 158 may then be subjected to a further micro solid/liquid separation 162, such as diafiltration, to produce a second recovered enzyme stream 64 and an enzyme reduced alcohol stream 166. When diafiltration is utilized, the diafiltration process filters any remaining hemicellulase and/or cellulase enzymes. Accordingly, the second recovered enzyme stream 164 is then optionally recycled to the first and/or second enzymatic hydrolysis stages 120 and 128. Enzyme reduced alcohol stream 166 is preferably essentially free of enzymes and lignin and may be further processed, such as in a distillation to purify the alcohol.

[0054] Figure 3 exemplifies a schematic of different embodiments of the invention. The processes to be discussed may be used singularly or in any particular combination or sub-combination. The lignocellulosic feedstock 210 is optionally first subjected to a pretreatment and optional steam explosion 212 to produce an activated feedstock 214, and then subsequently an optional disc refining step 216 to produce a fine particulate stream 218. It will be appreciated that neither of these optional steps, or one or both of these optional steps, may be utilized. The fine particulate stream is then subjected to a first enzymatic hydrolysis stage 220 to produce a first effluent stream 222. The first enzymatic hydrolysis stage 220 preferentially hydrolyzes the hemicelluloses in the feedstock to produce monomeric sugars of xylose and glucose. The first effluent stream may also contain short oligosaccharides and other higher molecular weight oligosaccharides that have not been fully hydrolyzed in the first enzymatic hydrolysis stage. The first effluent stream 222 also contains unhydrolyzed cellulose, which is preferentially hydrolyzed in the second enzymatic hydrolysis stage 228. The effluent stream further contains enzyme inhibitors, such as acetic acid and monomeric sugars (end product inhibition), which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and cellulases used in the first and/or second enzymatic hydrolysis stages 220 and 228. [0055] The first effluent stream 222 may be subjected to a solid/liquid separation 224, for example by means of a filter press, a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone to produce a first solid stream 226 and a first liquid sugar stream 230. The first solid stream 226 contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage 220. The first liquid sugar stream 230, which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage 220, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, enzyme inhibitors (inhibitors to the enzymes used in a second enzymatic hydrolysis stage 228) are removed from the first solid stream 226 before being subjected to the second enzymatic hydrolysis stage 228.

[0056] The first solid stream 226, containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 228. The second enzymatic hydrolysis stage 228 preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose. The second enzymatic hydrolysis stage 228 produces a second effluent stream 232, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose.

[0057] The second effluent stream 232 is then subjected to a solid/liquid separation 234, such as by means of a filter press, a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone to produce a second solid stream 236 and a second liquid sugar stream 238. Accordingly, the lignin may be removed from the second effluent stream 232 before the sugars (i.e., those in second liquid sugar stream 238) are subjected to fermentation, or further processing. As lignin inhibits the yeast used in fermentation, the removal of lignin increases the yield of alcohol.

[0058] The second solid stream 236, containing lignin, ash and/or other insoluble components such as unhydrolyzed hemicellulose and/or cellulose, is optionally further processed to obtain a purified lignin stream. This lignin stream may then be disposed of or used in a subsequent process.

[0059] The first and second liquid sugar streams 230 and 238 are optionally combined to form sugar stream 240, which contains fermentation inhibitors, such as weak acids, for example acetic acid, lactic acid or succinic acid, produced from the enzymatic hydrolysis processes 220 and 228. Sugar stream 240 is then subjected to fermentation 242, in which a nitrogen source stream 244 and an alkaline agent stream 246 are added to the fermentation broth to maintain the pH of the fermentation at a level at which weak acids present in the sugar stream 240 (carried over from the enzymatic hydrolysis processes 220 and 228) at least partially dissociate, improving the yield of the alcohol stream 248 produced in the fermentation 242. Alcohol stream 248 may then be optionally further processed, such as in a distillation to purify the alcohol.

NITROGEN SOURCE AND ALKALINE AGENT FOR FERMENTATION

[0060] The applicants have found that when a sugar stream comprising fermentable sugars and a weak acid is fermented using yeast in the presence of a nitrogen source and an alkaline agent, the process unexpectedly provides a synergistic benefit, which reduces the duration of the fermentation process. As described above, one of the inhibitors from a pre-hydrolysis step, such as autohydrolysis, and/or the first enzymatic hydrolysis stage, is acetic acid, which is produced as a result of the breakdown of acetyl groups attached to the hemicellulose and cellulose. As a result of the acetic acid, which is carried through the solid/liquid separations, the pH of the sugar rich process stream is between about 4.0 and 5.0, optionally 4.0 to 4.6 or optionally 4.5 to 4.9. Without being bound by theory, the applicants believe that acetic acid is easily absorbed by fatty acids present in the cell walls of the yeast during fermentation, which poisons the yeast and causes a significant decrease and/or slow-down in ethanol production. However, upon addition of an alkaline agent, such as an alkali hydroxide or alkaline earth hydroxide, the acetic acid is converted to its corresponding acetate ion, which as a result of its non-lipophilic property is not absorbed by the fatty acids of the cell wall, and therefore does not poison the yeast cells. Other weak acids, which are a poison to yeast, may also be present, such as lactic acid and/or succinic acid. Accordingly, preferably, the pH is adjusted to a level, using an alkaline agent, at which acetic acid, or another weak acid that may be present, dissociates, or partially dissociates.

[0061] Accordingly, in one embodiment, the sugar rich process stream obtained from the processes as described above is fermented using yeast in the presence of a nitrogen source and an alkaline agent wherein the amount and type of nitrogen compound and the alkaline agent are selected to maintain a level of nutrients in the sugar stream below a level at which the weak acid at least partially dissociates. In one embodiment, the nitrogen compound and the alkaline agent include chemicals that are nutrients for a fermentation organism and the amount and type of the nitrogen compound and the alkaline agent are selected to maintain a level of nutrients in the sugar stream below a level at which the nutrients are toxic to the fermentation organism. In another embodiment, the use of an alkaline agent is also advantageous to inhibit or reduce scaling of process equipment downstream from the fermentation vessel.

[0062] In another embodiment, the weak acid produced during the first and/or second enzymatic hydrolysis processes has a single dissociation product and a pKa, and the method comprises maintaining the pH of the sugar stream at a level above the pKa of the weak acid. For example, acetic acid has a single dissociation product, acetate ion, with a pKa of 4.75. Lactic acid, another acid produced during the enzymatic hydrolysis processes has a single dissociation product (lactate ion), and a pKa of 3.86.

[0063] In another embodiment, the weak acid has at least two dissociation products, such as succinic acid, and a pKal and the method comprises maintaining the pH of the sugar stream at a level above the first pKa (pKal ) of the weak acid. Succinic acid has a pKal of 4.21 . In one embodiment, the method further comprises maintaining the pH of the sugar stream at a level at which at least 75% of the weak acid dissociates.

[0064] In another embodiment, the concentration of the weak acid prior to fermentation is > 0.5 wt %, based on the total weight of the sugar stream.

[0065] In another embodiment, the nitrogen compound and the alkaline agent are used to buffer acid that is produced during the fermentation.

[0066] In another embodiment, the nitrogen compound and the alkaline agent are used to increase the pH (making the pH alkaline) of the sugar stream such that the weak acid dissociates prior to the acid contacting a fermentation organism, and thereby preventing the weak acid from rendering its toxic effect. Accordingly, the method comprises adding an amount of the nitrogen compound and the alkaline agent to increase the pH of the sugar stream to a level at which the weak acid dissociates, optionally to produce a first dissociation product, prior to the acid contacting a fermentation organism. Without being bound by theory, it is believed that the nitrogen source acts as a nutrient for the yeast and improves the rate of sugar conversion to alcohol, increasing the rate of the fermentation. In one embodiment, an advantage of using urea is that the dissociation of urea in the broth will increase the pH. Accordingly urea may act as a nutrient source to increase the rate of the fermentation and as an alkaline or pH adjustment agent.

[0067] In another embodiment, the nitrogen compound is selected from at least one of urea, a urea derivative, a nitrate, ammonia or an ammonia derivative (such as ammonium hydroxide), suitably urea or a derivative, or urea. In another embodiment, the nitrogen compound comprises a compound that dissociates to produce at least two cation radicals in solution, each of which has a different pKa. [0068] In another embodiment, the alkaline agent comprises a compound that dissociates to produce at least two cation radicals in solution, each of which has a different pKa. In one embodiment, the alkaline agent comprises an alkaline earth metal alkaline hydroxide. In one embodiment, the alkaline agent comprises an alkaline metal hydroxide or an alkaline earth metal hydroxide. In one embodiment, the alkaline metal or the alkaline earth metal is a nutrient for a fermentation organism.

[0069] In another embodiment, the alkaline agent comprises an alkaline metal hydroxide and the alkaline metal hydroxide is sodium hydroxide. In one embodiment, the alkaline agent comprises an alkaline earth metal hydroxide and the alkaline earth metal hydroxide is calcium hydroxide.

[0070] In one embodiment, the method of the present application comprises subjecting a lignocellulosic feedstock to hydrolysis and obtaining a hydrolyzed feedstock stream wherein the sugar stream comprises the hydrolyzed feedstock stream. In one embodiment, the method comprises subjecting a lignocellulosic feedstock to hydrolysis and obtaining a hydrolyzed feedstock stream and subjecting the hydrolyzed feedstock stream to solid liquid separation and obtaining the sugar stream.

[0071] In one embodiment, the sugar stream is maintained at an elevated pH above 3.5, or optionally above 5.5. In one embodiment, the sugar stream is maintained at a pH between 5.5 to 7.0, in which the pH of the fermentation process in maintained using an alkaline agent, such as calcium hydroxide. At such a level, the weak acid present in the sugar stream, such as acetic acid, will dissociate. In one embodiment, the alkaline agent is a combination of calcium hydroxide and/or sodium hydroxide. In another embodiment, the alkaline agent is ammonium hydroxide. Without being bound by theory, it is thought that the alkaline agent, such as calcium hydroxide, reacts with the weak acid, such as acetic acid, to form the salt of the conjugate base of the weak acid (in this case calcium acetate), which is not very soluble in the fermentation broth. As a result, the salt of the conjugate base precipitates from the broth. Accordingly, the weak acid, such as acetic acid, is not present to poison the yeast. In addition, and without being bound by theory, an alkaline agent such as calcium hydroxide, may act as a nutrient source for the yeast, thereby increasing the speed of the fermentation reaction.

[0072] In one embodiment, the fermentation of the sugar rich process is conducted at a pH of above 3.5, or optionally above 5.5, in the presence of sodium hydroxide. In another embodiment, the fermentation of the sugar rich process is conducted at a pH of above 3.5, or optionally above 5.5, in the presence of calcium hydroxide. In another embodiment, the fermentation of the sugar rich process is conducted at a pH of above 3.5, or optionally above 5.5, in the presence of a calcium hydroxide and sodium hydroxide.

[0073] .In one embodiment, calcium hydroxide is used as the alkaline agent and urea is used as the nitrogen source. Accordingly, in one embodiment, the fermentation of the sugar rich process stream is conducted at an elevated pH above 3.5, or optionally above 5.5, or optionally between 5.5 and 7.0, in which the pH of the fermentation process in raised using calcium hydroxide and also in the presence of urea.

INPUT FEEDSTOCK

[0074] The lignocellulosic feedstock used to produce the sugar stream sugars through enzymatic hydrolysis is derived from plant materials. As used herein, a "lignocellulosic feedstock" refers to plant fiber containing cellulose, hemicellulose and lignin. The applicants contemplate other sources of plant materials comprising cellulose, hemicellulose and lignin for use in deriving lignocellulosic feedstocks and any of those may be used. In some embodiments, the feedstock may be derived from trees, preferably deciduous trees such as poplar (e.g., wood chips). Alternately or in addition, the feedstock may also be derived from agricultural residues such as corn stover, wheat straw, barley straw, rice straw, switchgrass, sorghum, bagasse, rice hulls and/or corn cobs. Preferably, the lignocellulosic feedstock comprises agricultural residues and wood biomass, more preferably wood biomass and most preferably deciduous. Accordingly, the feedstock may be any feedstock that does not contain edible agricultural produce, however such material may be used.

[0075] The lignocellulosic feedstock is preferably cleaned, e.g., to remove ash, silica, metal strapping (e.g., from agricultural products), stones and dirt. The size of the components of the lignocellulosic feedstock may also be reduced. The size of the components of the feedstock may be from about 0.05 to about 2 inches, preferably from about 0.1 to about 1 inch, and more preferably from about 0.125 to about 0.5 inches in length.

[0076] It will be appreciated that if the optional activation, extraction, hydrolysis or physical modification is not utilized, the feedstock may be further crushed, ground or otherwise modified so as to decrease the average particle size of the components and increase the surface area of the material in the feedstock. Accordingly, the size of the feedstock may be from about 0.0625 to about 2 inches, preferably from about 0.125 to about 1 inch and more preferably from about 0.125 to about 0.5 inches. Any process machinery that is able to crush, grind or otherwise decrease the particle size may be utilized. The feedstock that is fed to the optional disc refiner that is immediately upstream of the first enzymatic hydrolysis stage is preferably comprises from 1 % to 60% wt total solids.

ACTIVATION

[0077] The lignocellulosic feedstock is optionally subjected to one or more activation steps prior to the feedstock being subject to enzymatic hydrolysis. As used herein an "activated" feedstock refers to a feedstock that has been treated so as to increase the susceptibility of cellulose and hemicellulose in the feedstock to subsequent enzymatic hydrolysis. In addition, the lignocellulosic feedstock may also be subjected to chemical or physical modification pretreatment, extraction or hydrolysis.

[0078] The applicants have found that certain processes for treating lignocellulosic feedstocks are surprisingly beneficial for preparing the feedstocks for enzymatic hydrolysis. Without being limited by theory, the applicant's believe that activation involves the chemical activation of hydrogen bond sites in the hemicellulose and cellulose polymer chains.

[0079] Methods of activation, extraction, hydrolysis, and chemical or physical modification include, but are not limited to, autohydrolysis, acid- hydrolysis, ammonia activation, disc refining, kraft pulping, organic solvent pulping, hot water pretreatment, ammonia percolation, lime pretreatment, caustic solvent pulping and alkali peroxide pretreatment, one or more of which may be used. Any process equipment known in the art may be used. Preferably, at least one of disc refining and autohydrolysis is utilized and more preferably, both are utilized.

[0080] In some embodiments, the feedstock is subjected to autohydrolysis. Autohydrolysis is a process of breaking down hemicellulose and cellulose by exposure to high temperatures, steam and pressure, preferably in the presence of a chemical agent or catalyst, such as sulphuric acid. When performed in the presence of an acid, an autohydrolysis process is known as an acid hydrolysis. Autohydrolysis often results in the release of acetic acid from the breakdown of acetylated hemicellulose, which further helps the hydrolysis process.

[0081] Preferably, the autohydrolysis is conducted in a steam explosion digester, which is known in the art. For example, feedstock having a moisture content of about 45% to about 55% by weight may be fed to an autohydrolysis digester wherein the biomass is hydrolyzed under steam at high pressure (e.g. 100-400 psig) and temperature (e.g., 50 - 250°C), optionally in the presence of a catalyst, such as sulphuric acid. In autohydrolysis, the acetyl groups are hydrolyzed from the plant structure producing acetic acid. The release of acetic acid decreases the pH of the reaction mixture in the digester from, e.g., neutral, to acidic (e.g., 3.0 - 4.0) supplying acid conditions for a mild acid hydrolysis reaction. During the autohydrolysis step, hemicellulose is partially hydrolyzed to xylose, soluble xylo-oligosaccharides and other pentosans. The yield may be up to about 75%.

[0082] During autohydrolysis, the degree of polymerization of cellulose and hemicellulose may be reduced from about 10,000 to about 1 ,500-1 ,000. This process is preferably carried out above the glass transition temperature of lignin (120 - 160°C). Depending upon the severity of the reaction, degradation products may still be produced, such as furfural, hydroxyl-methylfurfural, formic acid, levulinic acid and other organic compounds.

[0083] At the instant of release from the digester (steam explosion), the biomass exits the high temperature, high pressure hydrolyzer into a reduced pressure, preferably atmospheric pressure and, more preferably into a vacuum. The pressure in the digester is suddenly released, e.g., in less than 1 second and preferably instantaneously. The rapid decrease in pressure results in the biomass separating into individual fibres or bundles of fibres. This step opens the fibre structure and increases the surface area. The lignin remains in the fibre along with cellulose and residual hemicellulose, which are then subjected to enzymatic hydrolysis for recovery of fermentable sugars from this residual cellulose and hemicellulose.

[0084] In one embodiment, a lignocellulosic feedstock is fed into a water and heat impregnator, where water and/or catalyst may be added to the feedstock. The heating is preferably carried out without steam addition to avoid the random and uncontrollable addition of moisture. The feedstock may be assayed for moisture content in order to carefully control the amount of amount water added to the feedstock. In a preferred embodiment, the moisture content of the feedstock is from about 45% to about 55% by weight before the start of autohydrolysis. The moist feedstock is then subject to autohydrolysis in a hydrolyser. In some embodiments, the water and heat impregnation step can be performed in the same vessel as the hydrolyser.

[0085] The resulting autohydrolysed feedstock may enter a solid/vapor separation unit to produce a vapor stream and a solid stream. The separation unit may be operated at vacuum to remove acetic acid, furfural and other volatile compounds. The vapor stream may be passed to a scrubber to remove volatile products, including water, some of which may be recycled.

[0086] The resulting autohydrolyzed solid stream is then preferably subjected to disc refining prior to enzymatic hydrolysis and fermentation. Any disc refiner known in the art may be used. Passing the chemically hydrolyzed lignocellulosic feedstock through a disc refiner further activates the feedstock and increases the susceptibility of the feedstock to enzymatic hydrolysis. The use of a disc refiner also reduces the size of the particles in the feedstock as well as increasing the total available surface area of the particles in the feedstock.

[0087] The temperature in the disc refiner is preferably maintained at less than 65°C. Above this temperature, sugar degradation may occur decreasing the sugar content in the material. Preferably, the moisture content of the fiber passing through the disc refiner is about 50 to about 99% by weight.

[0088] The applicants have found that a disc refiner can be used with a lignocellulosic feedstock at a range of different particle sizes. Preferably, the size of the particles fed to the disc refiner is from 0.0625 to 2 inches, more preferably 0.125 to 1 inch and most preferably 0.125 to 0.5 inches.

FIRST ENZYMATIC HYDROLYSIS STEP [0089] Also included in the application is a method for efficiently breaking down a lignocellulosic feedstock into a sugar stream comprising fermentable sugars using enzymatic hydrolysis. In addition, the application also includes recovering and recycling the enzymes for further use from the enzymatic hydrolysis stages and the fermentation. Lignocellulosic feedstocks generally comprise cellulose, hemicellulose and lignin and have a high degree of polymerization. Hemicellulose is covalently linked to lignin, which in turn may be cross-linked to other polysaccharides such as cellulose resulting in a matrix of lignocellulosic material. Lignin is a hydrophobic cross-linked aromatic polymer and one of the major constituents of the cell walls of plants representing about one-quarter to one-third of the dry mass of wood.

[0090] Hemicellulose is a branched heteropolymer with a random, amorphous structure that includes a number of different sugar molecules such as xylose, glucose, mannose, galactose, rhamnose, and arabinose. Xylose is the most common sugar molecule present in hemicellulose. Xylose and arabinose are both pentosans, which are polymeric 5-carbon sugars present in plant material.

[0091] Hemicellulase enzymes break down the hemicellulose structure and solubilize the xylose. The use of hemicellulase enzymes results in the breakdown of the xylan backbone and side chains into pentosans such as xylose, mannose, galactose and arabinose as well as other sugars and polysaccharides. It will be apparent to those skilled in the art that most commercial preparations of hemicellulase enzyme also possess cellulase activity. Therefore, the first enzyme preparation (i.e., a hemicellulase enzyme preparation) used in the present disclosure, may possess about 10% to about 90% hemicellulase activity, preferably about 30% to about 90% hemicellulase activity and, more preferably about 50% or more (e.g., to about 90%) hemicellulase activity. In an embodiment, the hemicellulase preferentially acts upon the β-1 ,4 linkage of the xylose residues of xylan to solubilize the xylans and the β-1 ,4 linkage of the mannose residues of mannan.

[0092] Cellulose is a linear polymer of glucose, wherein the glucose residues are held together by beta (1 Π4) glycosidic bonds. Cellulase enzymes catalyze the hydrolysis of cellulose into smaller polymeric units by breaking beta- glycosidic bonds. Endo-cellulase enzymes generally cleave internal glycosidic bonds in cellulose to create smaller polysaccharide chains, while exo-cellulase enzymes are able to cleave off 2-4 units of glucose from the ends of cellulose chains. Cellulase enzymes are not generally capable of cleaving cellulose into individual glucose molecules.

[0093] In contrast, cellobiase or beta-glucosidase enzymes catalyze the hydrolysis of a beta-glycosidic linkages resulting in the release of at least one glucose molecule. Beta-glucosidase is therefore able to cleave cellobiose, which consists of two molecules of glucose joined together by a beta-glycosidic bond.

[0094] A person skilled in the art will appreciate that enzymes may exhibit a range of different activities on different substrates. As used herein, an enzyme preparation "preferentially acts" on a substrate when the relative activity of the enzyme for that substrate is greater than for other possible substrates. For example, a hemicellulase would preferentially act on hemicellulose to produce pentosans relative to its activity for cellulose to produce glucose.

[0095] An enzyme preparation may be a single enzyme or a combination of multiple enzymes. While enzyme preparations may be isolated from a number of sources such as natural cultures of bacteria, yeast or fungi a person skilled in the art will appreciate using enzymes produced using recombinant techniques.

[0096] In some embodiments, the two-stage enzymatic hydrolysis process described in the present application is able to increase the total solids content of the resulting sugar stream. As used herein, "total solids content" refers to the total amount of soluble and insoluble material in the feedstock. For example, in a lignocellulosic feedstock, soluble material would include monomeric sugars, some oligosaccharides, organic acids, extractives and low molecular weight compounds resulting from the autohydrolysis. Insoluble materials would include cellulose, lignin and hemicellulose. Suspensions with a high content of insoluble materials are generally difficult to process due to their high viscosity. Further, high-viscosity mixtures are difficult, if not impossible, to mix or handle through conventional pumping processes. In some embodiments, the sugar stream described in the present application has a total solids content of greater than about 15%. In a further embodiment, the sugar stream has a total solids content from about 15 to about 30%. In a further embodiment, the sugar stream may have a total solids content up to about 50% (e.g., about 1 5 to about 50%, preferably about 30 to about 50%).

[0097] While not limited by a particular theory, the applicants note that by performing the enzymatic hydrolysis in two stages, the hemicellulase enzymes and in particular xylanase are not exposed to inhibitory concentrations of sugar monomers and dimers, and in particular glucose and cellobiose, that are produced during the second enzymatic hydrolysis stage.

[0098] Accordingly, in one embodiment, the lignocellulosic feedstock is subjected to a first enzymatic hydrolysis process to preferentially solubilize xylose to obtain an effluent stream. The effluent stream is then subjected to a second enzymatic hydrolysis process to preferentially solubilize cellulose and to obtain a sugar stream. In one embodiment, at least one of the effluent stream and the sugar stream is treated to recover enzymes utilized in at least one of the first enzymatic hydrolysis process and the second enzymatic hydrolysis process to obtain a recovered enzyme stream.

[0099] The first enzymatic hydrolysis stage uses a first enzyme preparation that preferably comprises hemicellulase. As will be known by those skilled in the art, the hemicellulase preparation will also possess cellulase activity. In one embodiment, the first enzyme preparation is a xylanase enzyme cocktail such as Dyadic XBP™. In a further embodiment, the first enzyme preparation is AlternaFuel 100L™. It will be understood by a person skilled in the art that combinations of the enzyme preparations may be used. In an embodiment, the first enzyme preparation will possess hemicellulase activity from about 10% to about 90% and cellulase activity from about 90% to about 10%. In an embodiment, the hemicellulase activity will be from about 30% to about 90% and the cellulase activity will be from about 70% to about 10%. In a further embodiment, the hemicellulase activity will be from about 50% to about 90% and the cellulase activity will be from about 50 to about 10%.

[00100] In one embodiment, the pH of the process is adjusted using an acid stream or a base stream such that the pH of the feedstock is in a range suitable for enzymatic activity. In a preferred embodiment, the pH is adjusted to be between about 4.5 to about 6.0.

[00101] The temperature of the first enzymatic process may also be controlled. In one embodiment the temperature of the process is adjusted to be between about 20°C to about 70 °C. In a further embodiment, the first enzymatic process is conducted between about 30°C to about 70°C. The process may be cooled using indirect cooling water, or warmed using indirect steam heating or by other methods known in the art.

[00102] The result of the first enzymatic process on the feedstock is an effluent stream that may comprise xylans, cellobiose, glucose, xylose, lignin, ash, and organic acids, in addition to the enzymes used for the enzymatic process. Generally, the action of the first enzyme preparation results in the production of short-chain polysaccharides (oligosaccharides) such as cellobiose but not large quantities of individual glucose molecules. Without being bound by theory, this is thought to prevent the hemicellulase enzymes in the first enzyme preparation from being inhibited by glucose molecules. [00103] In one optional embodiment, the first enzymatic process is performed under vacuum and results in a volatile components stream, which can be removed from the low viscosity effluent stream. In one embodiment, the volatile component stream includes at least one yeast, fungi, bacteria or one or more enzyme inhibiting compounds present during the first enzymatic hydrolysis process and the volatile component stream that is drawn off includes at least one inhibiting compound. In another embodiment, the inhibiting compound in the volatile component stream may be one or more of acetic acid, furfural, formic acid, and any other volatile organic compounds.

SECOND ENZYMATIC HYDROLYSIS STEP

[00104] In the second enzymatic hydrolysis process, the effluent stream is treated with a second enzyme preparation to produce a sugar stream high in fermentable sugars such as glucose. In one embodiment, the second enzymatic hydrolysis process alternately, or in addition, contains fermentation organisms to simultaneously ferment the fermentable sugars and obtain an alcohol stream, such as ethanol, in which the process contains a nitrogen compound and an alkaline agent as described above.

[00105] The second enzyme preparation preferably primarily includes cellulase activity. In another embodiment, the second enzyme preparation comprises beta-glucosidase activity to convert disaccharides and other small polymers of glucose into monomeric glucose. In one embodiment, the second enzyme preparation is Novozym 188™, available from Novozymes™. In another embodiment, the second enzyme preparation is NS50073™. It will be understood by those in the art that combinations of the enzyme preparations may be used.

[00106] In one embodiment, the pH of the second hydrolysis process is adjusted using an acid stream or a base stream such that the pH of the feedstock slurry is in a range suitable for enzymatic activity. In a preferred embodiment, the pH is adjusted to be between about 4.5 to about 5.4. In an embodiment, the acid stream comprises any mineral acid. In another embodiment, the acid stream comprises nitric acid, sulphuric acid, phosphoric acid, acetic acid and/or hydrochloric acid. In an embodiment, the base stream comprises potassium hydroxide, sodium hydroxide, ammonium hydroxide, urea and/or ammonia.

[00107] The temperature of the second enzymatic process may also be controlled. In one embodiment the temperature of the process adjusted to be between about 20 to about 70 °C. In a further embodiment, the second enzymatic process is conducted between about 30 to about 70°C. The process may be cooled using indirect cooling water, or warmed using indirect steam heating or by other methods known in the art.

[00108] The resulting sugar stream contains between about 5 to about 45% w/w fermentable sugars. Optional ranges include about 5 to about 30%, preferably about 10 to about 30% and more preferably about 15 to about 25%, as well as about 1 0 to about 45%, preferably about 1 5 to about 45% and more preferably about 25 to about 45%. The sugar stream optionally also contains a total solids content of between about 10% to about 60%.

[00109] In another embodiment, which may be used by itself or in combination with any other process or processes disclosed herein, the recovery and recycling of hemicellulase and cellulase enzymes is also used in a simultaneous saccharification and fermentation (SSF) process, in which the first solid stream from the first enzymatic hydrolysis stage (after solid/liquid separation) is subjected to the second hydrolysis process and fermented in the same reaction vessel, in the presence of a nitrogen compound and an alkaline agent as described above. As such, the cellulose present in the first solid stream from the first enzymatic hydrolysis process is hydrolyzed in the reaction vessel using cellulases, and the monomeric sugars produced from the hydrolysis are directly fermented by yeast that are also present in the vessel. When such an SSF process is used, the cellulase enzymes from the reaction vessel can therefore be recovered and recycled to the first and/or second enzymatic hydrolysis processes. In addition, the yeast present in the vessel is optionally recovered from the SSF process.

[00110] Accordingly, in one embodiment, the product stream containing ethanol from the SSF process (second enzymatic hydrolysis process and fermentation) is treated to recover enzymes utilized in the enzymatic hydrolysis stage of the SSF and to obtain a recovered enzyme stream. In one embodiment, at least some of the recovered enzyme stream is recycled to the first enzymatic hydrolysis process, or alternatively, the first and/or second enzymatic hydrolysis processes. The product stream from the SSF is treated by any process, which is able to separate the enzymes contained in the product stream. In one embodiment, the product stream is subjected to solid/liquid separation to obtain a solid stream and a liquid/filtrate alcohol stream. In one embodiment, the liquid/filtrate alcohol stream is filtered to obtain the recovered enzyme stream. In one embodiment, the liquid/filtrate alcohol stream is subjected to at least one membrane filtration process. In one embodiment, the liquid/filtrate stream is subjected to at least one of ultrafiltration and diafiltration. In one embodiment, the liquid/filtrate alcohol stream is sequentially subjected to ultrafiltration and diafiltration. When filtration is utilized to separate the enzymes from the liquid/filtrate alcohol stream, the enzymes are retained by the filter membrane, while the ethanol product, for example, passes through the filter membrane, allowing for recovery and recycling of the enzymes.

RECOVERY AND RECYCLING OF ENZYMES

[00111] The applicants have found that the recovery and recycling of the hemicellulase and cellulase enzymes from the first and/or second enzymatic hydrolysis stages, as well as the fermentation process, is advantageous as the recycling of the enzymes used in one enzymatic hydrolysis stage to the other enzymatic hydrolysis stage enables more of the enzymes to be utilized thereby increasing the amount of fermentable sugars that may be produced using a given amount of enzymes. As a significant portion of the expense of industrial scale ethanol processes is due to the high cost of the enzymes. Accordingly, the applicants have found that by recovering the enzymes from the first and/or second enzymatic hydrolysis stages, or the fermentation, and subsequently recycling the enzymes into either the first and/or second enzymatic hydrolysis stages, significant cost savings are obtained.

[001 12] Accordingly, in one embodiment, the lignocellulosic feedstock is subjected to a first enzymatic hydrolysis stage to preferentially solubilize xylose and obtain an effluent stream. The effluent stream is then subjected to a second enzymatic stage process to preferentially solubilize cellulose and obtaining a sugar stream, which is then subjected to fermentation. In one embodiment, at least one of the effluent stream and the sugar stream is treated to recover enzymes utilized in at least one of: (i) the first enzymatic hydrolysis process to obtain a first recovered enzyme stream; and (ii) the second enzymatic hydrolysis process to obtain a second recovered enzyme stream, wherein at least some of the first recovered enzyme stream is recycled to the second enzymatic hydrolysis and/or at least some of the second recovered enzyme stream is recycled to the first enzymatic hydrolysis. In one embodiment, the effluent stream is subjected to a simultaneous saccharification and fermentation process (in the presence of a nitrogen compound and an alkaline agent as described above) to preferentially solubilize cellulose to obtain a sugar stream and simultaneously ferment the sugar stream to produce an alcohol stream, with the alcohol stream treated to recover enzymes, which are recycled to the first enzymatic hydrolysis process.

[00113] In one embodiment, the effluent stream is treated to recover enzymes utilized in the first enzymatic hydrolysis stage and obtain a first recovered enzyme stream and an enzyme reduced effluent stream, and wherein the enzyme reduced effluent stream is subjected to the second enzymatic hydrolysis process. In one embodiment, at least some of the first recovered enzyme stream is recycled to the second enzymatic hydrolysis process. In another embodiment, at least some of the first recovered enzyme stream is recycled to the first enzymatic hydrolysis process. In another embodiment, at least some of the first recovered enzyme stream from the first enzymatic hydrolysis is recycled to the first and second enzymatic hydrolysis processes.

[00114] In one embodiment, the sugar stream is treated to recover enzymes utilized in the second enzymatic hydrolysis process whereby a second recovered enzyme stream and an enzyme reduced sugar rich process stream are obtained and wherein the enzyme reduced sugar rich process stream is subsequently fermented using the method of the present application. In one embodiment, at least some of the second recovered enzyme stream from the second enzymatic hydrolysis is recycled to the first enzymatic hydrolysis processes. In another embodiment, at least some of the second recovered enzyme stream from the second enzymatic hydrolysis is recycled to the second enzymatic hydrolysis process. In another embodiment, at least some of the second recovered enzyme stream from the second enzymatic hydrolysis is recycled to the first and second enzymatic hydrolysis processes.

[00115] In one embodiment, the first and second liquid sugar streams from the first and second enzymatic hydrolysis stages respectively, are combined and treated to recover enzymes used in the hydrolysis processes, whereby the recovered enzyme stream is subsequently recycled to the first and/or second enzymatic hydrolysis stages. In another embodiment, the first and second liquid sugar streams are fermented using the method of the present application to produce an alcohol, and wherein the alcohol stream is treated to recover enzymes used in the hydrolysis processes, whereby the recovered enzyme stream is subsequently recycled to the first and/or second enzymatic hydrolysis stages. [001 16] In one embodiment, when the effluent stream is subjected to simultaneous saccharification and fermentation, the alcohol stream from the SSF (second enzymatic hydrolysis process, and fermentation using the method of the present application) is treated to recover enzymes utilized in the SSF process whereby a second recovered enzyme stream and an enzyme reduced alcohol process stream are obtained. In one embodiment, at least some of the second recovered enzyme stream from the SSF is recycled to the first enzymatic hydrolysis processes. In another embodiment, at least some of the second recovered enzyme stream from the SSF is recycled to the SSF. In another embodiment, at least some of the second recovered enzyme stream from the SSF is recycled to the first and SSF processes.

[00117] The first and second liquid sugar streams and the alcohol stream may be treated using any processes, which are able to separate the hemicellulase and/or cellulase enzymes contained in the streams. In one embodiment, at least one of the first and second liquid sugar streams and the alcohol stream is filtered to obtain the recovered enzyme stream. In one embodiment, at least one of the first and second liquid sugar streams and the alcohol stream is subjected to at least one membrane filtration process. In one embodiment, at least one of the first and second liquid sugar streams and the alcohol stream is subjected to at least one of ultrafiltration and diafiltration. In one embodiment, at least one of the first and second liquid sugar streams and the alcohol stream is sequentially subjected to ultrafiltration and diafiltration. When filtration is utilized to separate the hemicellulase and/or cellulase enzymes from any of the streams, for example ultrafiltration or diafiltration, the enzymes are retained by the filter membrane, while the solution, for example containing monomeric sugars, passes through the filter membrane, allowing for recovery and recycling of the enzymes.

[001 18] After the first enzymatic hydrolysis process, the product mixture (effluent stream) will contain soluble monomeric sugars, such as xylan, as well as other insoluble lignocellulosic material that has not been hydrolyzed by the enzymes. In addition, the mixture will also contain the hemicellulase enzymes. Accordingly, in one embodiment, the effluent stream from the first enzymatic hydrolysis process is treated to obtain a first liquid sugar stream and a first solid stream and wherein the first solid stream is subjected to the second enzymatic hydrolysis process. In one embodiment, the first liquid sugar stream is obtained by subjecting the effluent stream to at least one of a decanting centrifuge, a filter press, a belt filter, a hydrocyclone and a vibratory screen. In one embodiment, the first liquid sugar stream is treated to recover enzymes utilized in the first enzymatic hydrolysis process to obtain a first recovered enzyme stream and an enzyme reduced effluent stream and subsequently recycling at least some of the first recovered enzyme stream to the second enzymatic hydrolysis process. In one embodiment, the enzyme reduced effluent stream is subjected to fermentation using the method of the present application.

[00119] As a result of the optional pre-treatment step, such as autohydrolysis, and the first enzymatic hydrolysis process, compounds which inhibit the yeast, which ferment the monomeric sugars, are produced. Such compounds include acetic acid, lactic acid, succinic acid, formic acid, glycerol, furfural and hydroxymethylfurfural, in addition to the product monomeric sugars themselves (end product inhibition). Accordingly, in one embodiment, the enzyme reduced effluent stream is treated to remove at least one of acetic acid, lactic acid, succinic acid, formic acid, glycerol, furfural and hydroxymethylfurfural.

[00120] In one embodiment, the cellulose, hemicellulose and lignin containing material is subjected to autohydrolysis to obtain the feedstock. In one embodiment, the autohydrolysis has a severity of from 3.6 to 4.5.

[00121] In one embodiment, the cellulose, hemicellulose and lignin containing material is subjected to hydrolysis followed by disc refining to obtain the feedstock for the enzymatic hydrolysis process. [00122] In one embodiment, the cellulose, hemicellulose and lignin containing material is subjected to hydrolysis to obtain the feedstock.

[00123] It will be understood that the recovery of enzymes, for example, hemicellulase and/or cellulase, can be performed after either, or both, of the first or second enzymatic hydrolysis processes to produce recovered enzyme streams. In addition, the recovery of enzymes can also be performed after fermentation. In one embodiment, the first and/or second recovered enzyme streams are recycled to the second and/or first enzymatic hydrolysis processes, respectively.

INHIBITORY COMPOUNDS

[00124] As described above, the optional pre-treatment steps, such as autohydrolysis optionally in the presence of sulphuric acid, results in the release of inhibitory compounds, which can inhibit both the enzymatic hydrolysis enzymes, as well as the yeast during the fermentation of the monomeric sugars. In addition, during the first enzymatic hydrolysis stage, acetyl groups are removed from the hemicellulose, which in an aqueous medium form acetic acid. The corresponding drop in pH as a result of the production of acetic acid has an inhibitory effect on the enzymes in the second enzymatic hydrolysis stage, and therefore reduces the monomeric sugar output from this stage. Moreover, products from the first enzymatic hydrolysis stage have a negative feedback (end product inhibition) on enzymes used in the first enzymatic hydrolysis stage. Such inhibitory compounds include, but are not limited to, glucose, gluco- oligosaccharides, xylose, xylo-oligosaccharides, formic acid, glycerol furfural, hydroxymethylfurfural, organic acids, and phenolic compounds. Preferably, at least some of these compounds are removed prior to the second enzymatic hydrolysis stage. Alternately, or in addition, at least some of these compounds are removed subsequent to the second enzymatic hydrolysis stage and prior to fermentation.

[00125] In one embodiment therefore, there is an optional process for treating the effluent stream to reduce the level of at least one of the hydrolysis inhibiting compounds and obtaining an inhibitor reduced stream and a treated effluent stream. Subsequently, the treated effluent stream is then subjected to a second enzymatic hydrolysis stage to preferentially hydrolyze and solubilize cellulose to obtain a sugar stream. It will be understood that the steps recited herein to reduce the level of at least one of the hydrolysis inhibiting compounds are used in addition to the processes recited above to recover and recycle the enzymes.

[00126] In one embodiment, the effluent stream is subjected to solid/liquid separation to obtain a first liquid sugar stream and a first solid stream comprising the inhibitor reduced stream. The solid/liquid separation comprises at least one of a decanting centrifuge, a filter press, a belt filter, a vibratory screen and a hydrocyclone.

[00127] In one embodiment, the effluent stream from the first hydrolysis stage is subjected to solid/liquid separation to obtain a first liquid sugar stream and a first solid stream. The solid/liquid separation comprises at least one of a decanting centrifuge, a filter press, a belt filter, a vibratory screen and a hydrocyclone. The first enzymatic hydrolysis stage preferentially hydrolyzes the hemicelluloses in the feedstock to produce monomeric sugars of xylose and glucose. The effluent stream may also contain short oligosaccharides and other higher molecular weight oligosaccharides that have not been fully hydrolyzed in the first enzymatic hydrolysis stage. The effluent stream also contains unhydrolyzed cellulose which is preferentially hydrolyzed in the second enzymatic hydrolysis stage. The effluent stream further contains enzyme inhibitors (as described above), such as acetic acid and monomeric sugars (end product inhibition), which are products of the enzymatic hydrolysis (or pre- hydrolysis) and which inhibit the activity of the hemicellulases and cellulases used in the first and/or second enzymatic hydrolysis stages. The solid/liquid separation produces a first solid stream and a first liquid sugar stream. The first solid stream contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage. The first liquid sugar stream, which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, the enzyme inhibitors are removed from the first solid stream before the solid stream is subjected to the second enzymatic hydrolysis stage, or the SSF stage (second enzymatic hydrolysis and fermentation). As such, the removal of inhibitors and monomeric sugars before the first solid stream is subjected to the second enzymatic hydrolysis stage increases the sugar output as compared to when the inhibitors are not removed.

LIGNIN

[00128] As described above, lignin is a hydrophobic cross-linked aromatic polymer and one of the major constituents of the cell walls of plants representing about one-quarter to one-third of the dry mass of wood. Hemicellulases and cellulases do not hydrolyze the lignin present in the lignocellulosic material and therefore, the lignin carries through the solid streams throughout the first and second enzymatic hydrolysis processes. However, the presence of lignin during the fermentation of the monomeric sugars reduces the amount of ethanol produced because lignin inhibits the fermentation yeast. Accordingly, in one embodiment, it is advantageous to remove the lignin from the sugar stream before the stream is fermented for ethanol production.

[00129] Accordingly, in one embodiment, there is included an optional process to treat the second liquid sugar stream from the second enzymatic hydrolysis process to reduce the level of lignin and obtain a lignin stream and a lignin reduced sugar rich process stream. The lignin reduced sugar rich process stream is subsequently fermented to produce ethanol. It will be understood that the steps recited herein to reduce the level lignin may be used in addition to the processes recited above to recover and recycle the enzymes.

[00130] In one embodiment, the second liquid sugar stream is treated by solid/liquid separation to obtain the lignin stream and the lignin reduced sugar rich process stream. In one embodiment, the solid/liquid separation to remove the lignin comprises at least one of a decanting centrifuge, a filter press, a vibratory screen, a hydrocyclone, and a belt filter.

[00131] In addition, the lignin removed from the second enzymatic hydrolysis process can be purified and is useful for several products, for example as a fuel source or other polymeric materials.

[00132] In one embodiment, the first solid stream that is subjected to the second enzymatic hydrolysis stage contains insoluble compounds such as cellulose and lignin. As described above, the second enzymatic hydrolysis stage preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose. The second enzymatic hydrolysis stage produces a second liquid/filtrate stream, which contains the soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose. The second liquid/filtrate stream is then is then subjected to a solid/liquid separation, such as a filter press, to produce a second solid stream and a second liquid sugar stream. The second solid stream, containing lignin, ash and/or other insoluble components such as unhydrolyzed hemicellulose and/or cellulose, is optionally further processed to separate and purify lignin. Accordingly, the lignin is removed from the second liquid/filtrate stream before the sugars are subjected to fermentation. As lignin has been found by the Applicants to inhibit yeast during fermentation, the removal of lignin increases the yield of alcohol as compared to when lignin is present in the fermentation stage. RECOVERY OF ENZYMES FROM FERMENTATION PROCESS

[00133] The Applicants have also found that hemicellulases and cellulases are recoverable after the fermentation stage (including a simultaneous saccharfication process), and therefore, the micro solid/liquid separations as described above may be conducted after fermentation of the sugar stream.

[00134] In addition, any hemicellulose and/or cellulose which has not been hydrolyzed by the enzymes, such as oligosaccharides or other unhydrolyzed hemicellulose and/or cellulose, may be further hydrolyzed in the fermentation stage, if some enzymes remain as the hemicellulase and cellulase enzymes are still active.

[00135] In another embodiment, which may be used by itself or in combination with any other process or processes disclosed herein, the recovery and recycling of hemicellulase and cellulase enzymes is also used in simultaneous saccharification and fermentation (SSF) processes, in which the lignocellulosic material is both hydrolyzed and fermented in the same reaction vessel. As such, the lignocellulosic material is hydrolyzed in the reaction vessel using hemicellulases and cellulases, and the monomeric sugars produced from the hydrolysis are directly fermented by yeast that are also present in the vessel (also containing a nitrogen compounds and an alkaline agent). Accordingly, when an SSF process is utilized , it is also beneficial to recover the hemicellulase and cellulase enzymes from the fermentation vessel, which can therefore be recycled and used in further SSF processes. In addition, the yeast present in the vessel is optionally recovered from the SSF process.

[00136] Accordingly, in one embodiment, the product stream containing ethanol from the fermentation process is treated to recover enzymes utilized in at least one of the first enzymatic hydrolysis stage and the second enzymatic hydrolysis stage and to obtain a recovered enzyme stream. In one embodiment, at least some of the recovered enzyme stream is recycled to the SSF process, or alternatively, the first and/or second enzymatic hydrolysis processes. The product stream from the SSF is treated by any process, which is able to separate the enzymes contained in the product stream. In one embodiment, the product stream is subjected to solid/liquid separation to obtain a solid stream and a liquid/filtrate stream. In one embodiment, the liquid/filtrate stream is filtered to obtain the recovered enzyme stream. In one embodiment, the liquid/filtrate stream is subjected to at least one membrane filtration process. In one embodiment, the liquid/filtrate stream is subjected to at least one of ultrafiltration and diafiltration. In one embodiment, the liquid/filtrate stream is sequentially subjected to ultrafiltration and diafiltration. When filtration is utilized to separate the enzymes from the liquid/filtrate stream, the enzymes are retained by the filter membrane, while the ethanol product, for example, passes through the filter membrane, allowing for recovery and recycling of the enzymes.

OTHER EMBODIMENTS

[00137] In some embodiments, the sugar rich process stream is used to produce sugar derived products. In one embodiment of the invention, the sugar rich process stream is used to produce alcohol through fermentation. The fermentable sugars such as glucose and xylose may be fermented to alcohol after yeast addition. In an embodiment, the alcohol produced is methanol, ethanol and/or butanol.

[00138] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments or separate aspects, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment or aspect, may also be provided separately or in any suitable sub-combination. [00139] Although the invention has been described in conjunction with specific embodiments thereof, if is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

EXAMPLES

[00140] The operation of the invention is illustrated by the following representative examples. As is apparent to those skilled in the art, many of the details of the examples may be changed while still practicing the disclosure described herein.

Example 1 : Addition of Nitrogen Source and Alkaline Agent to Fermentation Hydrolysate

[00141] The hydrolysate used in this example was produced as follows: Poplar wood feedstock was subjected to steam explosion autohydrolysis pretreat ent followed by enzymatic hydrolysis. The hydrolyzed slurry was then filtered until a clear filtrate was obtained. This clear filtrate was stored at room temperature for 10 days. When analyzed by HPLC, the hydrolysate contained 101 .7 g/L of glucose, 25.9 g/L of xylose, 3.1 g/L of mannose, 4.74 g/L of cellobiose, and 1 1 .6 g/L of acetic acid, plus xylo- and gluco-oligosaccharides that brought the aggregate dry matter content to nearly 210 g/L. The measured pH of the hydrolysate ranged from 4.55 to 4.71 . From mass-balance calculations, it was determined that the hydrolysate contained approximately 4.6 g/L of NaOH. The dry content of the hydrolysate was measured at 21 %. Yeast strains

[00142] Superstart™ dry active Saccharomyces cerevisiae was supplied by Lallemand Inc. (Rexdale, ON, Canada). All the yeast used in this study was from a single carton.

Antibiotics

[00143] 1 mg/L of antibiotics was added to each fermentation batch prior to the addition of yeast. Antibiotics used were Lactoside 247™ supplied by Lallemand Inc dissolved in deionized water at a concentration of 1 mg/mL before addition to the hydrolysate.

Nutrients

[00144] Lallemand AYF 1000™ nutrient blend was used in a number of batches in the first set of trials at a level of 0.33 g/L

Reagents

[00145] Ca(OH)₂ in dry powder form (95% purity), NaOH in dry ground pellet form (97% purity), urea in dry pellet form (98% purity), NH₄OH at 14.8M concentration, and phosphoric acid at 85% wt/wt concentration were all used as is without dissolution or further dilution.

Preparation and Fermentation

[00146] 150 g of defrosted hydrolysate (γ = 1 .09) was weighed into each Erienmeyer flask and 0. 5 mL of antibiotic solution was added promptly to each flask. Reagents that were added to each flask are shown in Table 1 . Overliming was performed on flasks 4 and 5, where the pH was raised to 10, held for 2 hours, and readjusted back to 5.5 with phosphoric acid. In all other trials, the pH was increased to 5.5 by addition of the specified reagent(s), except for trial #1 , which served as a control, and trial #2, which replicated trial #1 except that nutrients were added.

[00147] In the next set of trials (Table 2), certain conditions identified in Table 1 were replicated. Several combinations of urea and Ca(OH)₂ were tested, along with trials involving supplementation with NaOH and urea, and with urea alone.

[00148] In trial number 3, different methods for pH adjustment were explored, including Ca(OH)₂, NH₄OH, and excess NH₄OH followed by readjustment with phosphoric acid, as shown in the reagent flasks of Table 3.

[00149] Each set of nine flasks were capped under non-sterile conditions and placed in an incubator with the temperature set to 34.7°C with stirring at 200 RPM. 1 .2 mL samples were withdrawn with a micropipette at various time points for analysis.

Discussion

[00150] In Trial #1 , fermentations that employed calcium hydroxide and urea attained the highest ethanol productivity, and were faster in reaching the maximum ethanol concentration. Flasks 3, 4, 5, 8, and 9 all reached maximum ethanol concentrations of between 76 g/L and 82 g/L, as can be seen in Figure 4. However, flasks 8 and 9, which contained 2.5 g of urea and pH-adjusted to 5.5 and 6.2 with Ca(OH)₂, achieved maximum ethanol productivities of 2.44 g(L^*hr)^~1 and 2.39 g(L^*hr)^"1 within the first 28 hours, respectively. Trial productivities are shown in Figure 5. Twenty-eight hours after addition of yeast to flasks 8 and 9, average glucose levels were 4.37 g/L and 5.27 g/L, respectively. The 28-hour fractional conversion with respect to glucose is 95.7% in flask 8 and 94.8% in flask 9. Fermentation of overlimed samples led to lower ethanol productivities and lower glucose conversions at 28 hours. At the 120-hour time point, flasks 3, 4, 5, 8, and 9 all attained complete conversion of glucose. Flask 2, which had only nutrients and yeast added to it, did not ferment at all. No fermentation was observed in flasks 6 and 7, which contained 2.5 g of urea or 2.5 g of urea and nutrients, respectively.

[00151] During Trial #2, more data points were collected. Fermentations that employed calcium hydroxide and urea attained higher ethanol productivity versus those flasks that had Ca(OH)2 as the sole reagent, as can be seen in Figure 6. Flasks 2 to 7 all reached maximum ethanol concentrations of between 74 g/L and 79 g/L, as can be seen in Figure 7. However, flask 5, which contained 1 g of urea and pH-adjusted to 5.5 with Ca(OH)₂, achieved a maximum ethanol productivity of 3.23 g(L^*hr)^"1 over the first 15.5 hours of fermentation. The ethanol productivity for flask 3 between 15.5 and 21 hours was 2.40 g(L^*hr)^"1 , which is consistent with results seen from Trial #1 . The 41 -hour fractional conversion with respect to glucose was 98.3% in flask 3, 98.0% in flask 4, and 100% in flask 5. Maximum ethanol productivities for flasks 6 and 7, containing Ca(OH)₂ but no urea were 1 .43 g(L^*hr)^"1 and 1 .47 g(L^*hr)^"1 , respectively. No fermentation was observed in flasks 8 and 9, which used large amounts of urea to adjust the pH to 5.5.

[00152] For Trial #3, hydrolysate was fermented with ammonium hydroxide in combination with either phosphoric acid or calcium hydroxide at pH = 5.5. Flasks 4 to 9 all reached maximum ethanol concentrations of between 73 g/L and 76 g/L, as can be seen in Figure 8. Ethanol productivities were also very consistent with one another, as can be seen in Figure 9. The 41 .5-hour fractional conversion with respect to glucose is 86.9% in flask 4, 94.1 % in flask 5, and 94.7% in flask 6, 94.5% in flask 7, 91.3% in flask 8, and 92.2% in flask 9. Slow fermentation was observed in flasks 2 and 3, in which 57% of the acetic acid was removed through boiling at reduced pressure and diluted back to the original sugar concentration two consecutive times. Collectively, these results indicate that the acetic acid concentration, although important, is not the sole determinant of fermentation rate, nor is it sufficient to simply adjust the pH to alter the acetate:acid ratio. [00153] A large scale experiment with the same hydrolysate, pH-unadjusted (Trial #4), led to fermentation only after a long lag-phase, and incomplete conversion of glucose, as shown in Figure 10. After 264 hours of fermentation, the residual glucose concentration was 7.7 g/L.

[00154] These fermentation trials confirm acetic acid as an inhibitor of fermentation. Hydrolysate that was twice-evaporated contained 6.83 g/L of acetic acid, but without additional pH adjustment, realized average ethanol productivities of 1 .14 g(L*hr)^"1 over the first 18 hours of fermentation. By comparison, untreated hydrolysate with 1 5.9 g/L of acetic acid was unfermentable. While improvements in ethanol productivity following pH adjustment, it is also apparent that the pH change does not account for all of the benefits observed in these trials. pH adjustment with different compounds (or combinations of compounds) led to different ethanol productivities, even though the resulting pH of the hydrolyzate was essentially the same at the start of fermentation.

[00155] It was observed during Trial #2 and Trial #3 that fermentations containing urea had a stable pH throughout the entire fermentation, whereas in the latter set of trials, when reagent combinations containing ammonium hydroxide were used, the pH decreased as fermentation proceeded. This observation is illustrated in Figures 1 and 12. While the final time points between the two trials are more than 40 hours apart, there is a definite upward trend in the urea-enriched fermentations while fermentations conducted with ammonium hydroxide resulted in an overall pH drop. This indicates that certain reagents may be able to provide natural pH buffering beyond that observed during the initial pH adjustment.

[00156] A small addition of urea resulted in improvements in fermentability, as can be seen from Trial #2. Flask 5 was supplemented with 1 g of urea, while employing similar amounts of Ca(OH)₂ as flasks 6 and 7. The presence of urea in Flask #5 led to a productivity that was more than double that of flasks 6 and 7, which contained only Ca(OH)₂.

Table 1 : Reagent flasks of Trial #1

Table 2: Reagent flasks of Trial #2

Table 3: Reagent flasks of Trial #3

Claims

A method of fermenting a sugar stream to produce an alcohol stream, comprising:

(a) providing a sugar stream comprising fermentable sugars and a weak acid;

(b) conducting the fermentation in the presence of a nitrogen compound and an alkaline agent wherein the amount and type of nitrogen compound and an alkaline agent are selected to maintain the pH of the sugar stream at a level at which the weak acid at least partially dissociates.

The method of claim 1 , wherein at least one of the nitrogen compound and the alkaline agent include chemicals that are nutrients for a fermentation organism and the amount and type of the nitrogen compound and of the alkaline agent are selected to maintain a level of nutrients in the sugar stream below a level at which the nutrients are toxic to the fermentation organism.

The method of claim 1 or 2, wherein the amount and type of the alkaline agent is selected to inhibit scaling of process equipment downstream from the fermentation.

The method of any one of claims 1 - 3, wherein the weak acid has a single dissociation product and a pKa and the method further comprises maintaining the pH of the sugar stream at a level above the pKa of the weak acid.

The method of any of claims 1 - 4 wherein the weak acid comprises acetic acid and/or lactic acid

The method of any one of claims 1 - 3, wherein the weak acid has at least two dissociation products and a pKal and the method further comprises maintaining the pH of the sugar stream at a level above the pKal of the weak acid.

7. The method of any of claims 1 - 3 and 6 wherein the weak acid comprises succinic acid.

8. The method of any one of claims 1 - 7, wherein the concentration of the weak acid prior to fermentation is > 0.5 wt %, based on the total weight of the sugar stream.

9. The method of any one of claims 1 - 8, further comprising maintaining the pH of the sugar stream at a level at which at least 75% of the weak acid dissociates.

10. The method of any one of claims 1 - 9 further comprising selecting at least one of the nitrogen compound and the alkaline agent to buffer acid produced during the fermentation.

1 1 . The method of any one of claims 1 - 5 and 8-10 further comprising adding an amount of the nitrogen compound and the alkaline agent to increase the pH of the sugar stream to a level at which the weak acid dissociates prior to the acid contacting a fermentation organism.

12. The method of any one of claims 1 - 3 and 6- 0 further comprising adding an amount of the nitrogen compound and the alkaline agent to increase the pH of the sugar stream to a level at which the weak acid dissociates to produce a first dissociation product prior to the acetic acid contacting a fermentation organism.

13. The method of any one of claims 10 - 12, further comprising selecting the nitrogen compound from at least one of urea, a urea derivative, a nitrate, ammonia or an ammonia derivative.

14. The method of any one of claims 10 - 13, wherein the nitrogen compound comprises urea or a urea derivative.

15. The method of any one of claims 10 - 14, wherein the nitrogen compound comprises urea.

16. The method of claim 13, further comprising selecting as the nitrogen compound a compound that dissociates to produce at least two cation radicals in solution, each of which has a different pKa.

17. The method of claim 13, wherein the nitrogen source comprises urea.

18. The method of claim 13, wherein the ammonia derivative comprises ammonium hydroxide.

19. The method of any one of claims 1 - 18, further comprising selecting as the alkaline agent a compound that dissociates to produce at least two cation radicals in solution, each of which has a different pKa.

20. The method of claim 19, wherein the alkaline agent comprises an alkaline earth metal alkaline hydroxide.

21 . The method of any one of claims 1 - 18, wherein the alkaline agent comprises an alkaline metal hydroxide or an alkaline earth metal hydroxide.

22. The method of claim 21 , wherein the alkaline metal or the alkaline earth metal is a nutrient for a fermentation organism.

23. The method of claim 21 , wherein the alkaline agent comprises an alkaline metal hydroxide and the alkaline metal hydroxide is sodium hydroxide.

24. The method of claim 21 , wherein the alkaline agent comprises an alkaline earth metal hydroxide and the alkaline earth metal hydroxide is calcium hydroxide.

25. The method of any one of claims 1 - 24, further comprising maintaining the pH of the sugar stream above 3.5.

26. The method of any one of claims 1 - 24, further comprising maintaining the pH of the sugar stream above 5.5.

27. The method of any one of claims 1 - 24, further comprising maintaining the pH of the sugar stream between 5.5 and 7.

28. The method of any one of claims 1 - 27 further comprising subjecting a lignocellulosic feedstock to hydrolysis and obtaining a hydrolyzed feedstock stream wherein the sugar stream comprises the hydrolyzed feedstock stream.

29. The method of claim 28, further comprising:

(a) subjecting the feedstock to a first enzymatic hydrolysis process to preferentially solubilize xylose and obtaining a first effluent stream;

(b) treating the first effluent stream to obtain a first liquid sugar stream and a first solid stream;

(c) subjecting the solid stream to a second enzymatic hydrolysis process to preferentially solubilize cellulose and obtaining a second effluent stream;

(d) treating the second effluent stream to obtain a second liquid sugar stream and a second solid stream; and,

(e) combining the first and second liquid sugar streams to obtain the sugar stream.

30. The method of any one of claims 1 - 27 further comprising subjecting a lignocellulosic feedstock to hydrolysis and obtaining a hydrolyzed feedstock stream and subjecting the hydrolyzed feedstock stream to solid liquid separation and obtaining the sugar stream.