LIGNOCELLULOSIC MATERIALS FIELD

[0001] This application relates to a method for treating plant materials to release fermentable sugars using enzymes, wherein the enzymes are subsequently recovered and recycled. More specifically, this application relates to a two-stage enzymatic hydrolysis process for treating lignocellulosic materials and producing a sugar rich process stream, wherein the enzymes are recovered and recycled, optionally from each stage of the enzymatic hydrolysis.

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.

[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 two-stage enzymatic process to prepare a sugar rich process stream from a feedstock derived from plant materials, and the recovery and recycling of the enzymes used in the enzymatic process. 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. The sugar rich process stream may subsequently be subjected to fermentation to produce an alcohol stream. 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 12%, preferably about 8 to about 15% and more preferably about 8 to about 22% (v/v). Such alcohol concentrations may be obtained without using corn as a feedstock.

[0007] 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.

[0008] Another advantage of the process of the invention is the recovery and recycling of the enzymes used in each stage of the enzymatic process 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 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. [0009] In one embodiment, the 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.

[0010] 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. [001 1] Without being limited by theory, oligosaccharides, and in particular cellobiose, have an inhibitory effect on cellulase 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).

[0012] 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.

[0013] 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.

[0014] 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. [0015] The first enzyme preparation preferentially acts upon the β-1 ,4 linkage of the xylose residues of xylan and the β-1 ,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.

[0016] 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.

[0017] 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

[0018] 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:

[0019] Figure 1 is a flow chart of the method according to embodiments that include sugar recovery and enzyme recovery subsequent to the first and second enzymatic hydrolysis stages;

[0020] Figure 2 is a flow chart of the method according to one embodiment that includes sugar recovery and enzyme recovery subsequent to the first enzymatic hydrolysis stage;

[0021] Figure 3 is a flow chart of the method according to one embodiment that includes sugar recovery and enzyme recovery subsequent to the second enzymatic hydrolysis stage;

[0022] Figure 4 is a flow chart of the method according to one embodiment that includes xylose recovery, enzyme recovery and removal of acetic acid subsequent to the first enzymatic hydrolysis stage;

[0023] Figure 5 is a flow chart of the method according to one embodiment that includes enzyme recovery subsequent to both the first and second enzymatic hydrolysis stages; and [0024] Figure 6 is a flow chart of the method according to one embodiment that includes simultaneous saccharification and fermentation and enzyme recovery during the second enzymatic hydrolysis stage.

DETAILED DESCRIPTION

[0025] This application relates generally to a method of treating a lignocellulosic feedstock to breakdown cellulose and hemicellulose in the feedstock into monomeric sugars such as glucose, which may be fermented 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 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.

[0026] 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.

[0027] 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 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 20 to produce an effluent stream 22. As described above, 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.

[0028] The effluent stream 22 may be subjected to a solid/liquid separation 24, for example by means of a filter press, to produce a solid stream 26 and a liquid/filtrate stream 38. 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 38, 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.

[0029] The solid stream 26, containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 28. As described above, 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 30, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose.

[0030] The sugar rich process stream is then subjected to a solid/liquid separation 32, such as by means of a filter press, to produce a second solid stream 34 and a second liquid/filtrate stream 40. Accordingly, the lignin may be removed from the sugar rich process stream 30 before the sugars (i.e., those in second liquid/filtrate stream 40) are subjected to fermentation. As lignin inhibits the yeast used in fermentation, the removal of lignin increases the yield of alcohol.

[0031] The second solid stream 34, containing lignin, ash and/or other insoluble components such as unhydrolyzed hemicellulose and/or cellulose, is optionally further processed 36 to obtain a purified lignin stream. This lignin stream may then be disposed of or used in a subsequent process.

[0032] One or both of the liquid/filtrate streams 38 and 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, micro filtration and/or ultrafiltration and/or diafiltration.

[0033] Referring to Figure , filtrate streams 38 and 40 are combined and then subjected to micro-filtration 44, which produces a residual solid stream 46 (retenate) and a particle reduced stream 48. Microfiltration removes fine particulate solids, which may be suspended in the liquid/filtrate streams 38 and 40. For example, residual hemicellulose and cellulose which has not been hydrolyzed may form part of the residual solid stream 46, and accordingly, the residual solid stream 46 is optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28 for further enzymatic hydrolysis. Particle reduced stream 48 comprises sugars (both monomeric and oligomeric) and enzymes.

[0034] The particle reduced stream 48 (raffinate) may then be subjected to another micro solid/liquid separation 50, such as ultrafiltration, to produce a recovered enzyme stream 52 and an enzyme reduced stream 54. When ultrafiltration is utilized, the ultrafiltration separation filters hemicellulose and/or cellulase enzymes from the particle reduced stream 48, which allows these enzymes to be recovered and/or recycled. Accordingly, the recovered enzyme stream 52 is then optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28.

[0035] The enzyme reduced stream 54 may then be subjected to a further micro solid/liquid separation 56, such as diafiltration, to produce a second recovered enzyme stream 58 and a sugar rich enzyme reduced stream 60. When diafiltration is utilized, the diafiltration process filters any remaining hemicellulase and/or cellulase enzymes. Accordingly, the second recovered enzyme stream 58 is then optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. Sugar rich enzyme reduced stream 60 is preferably essentially free of enzymes and lignin.

[0036] In one embodiment, the recovered enzyme stream 52 and the second recovered enzyme stream 58 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.

[0037] The sugar rich enzyme reduced stream 60 may then be subjected to fermentation 62 to produce an alcohol stream 64, such as ethanol, from the sugars in the sugar rich enzyme reduced stream 60. As a result of the removal of enzymatic inhibitors before the solid stream 26 is subjected to the second enzymatic hydrolysis stage 28, the amount of monomeric sugars is increased as compared to when the inhibitors are not removed. In addition, the removal of lignin from the liquid/filtrate stream 40, 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. [0038] As shown in Figure 1 using dashed arrows, there are other optional processes that may be utilized in combination with any of the preceding processes. In one embodiment, rather than combining the liquid/filtrate streams 38 and 40, each liquid/filtrate stream is subjected to micro liquid/solid separation individually. Accordingly, in one embodiment, each liquid/filtrate stream 66 and 86 is subjected to micro solid/liquid separation 68 and 88, such as micro-filtration, which produces a residual solid stream (70 and 90) and a particle reduced stream 72 and 92. Microfiltration removes any fine particulate solids, which may be suspended in the liquid/filtrate streams 66 and 86. For example, residual hemicellulose and cellulose, which has not been hydrolyzed, may form part of the residual solid streams 70 and 90, and accordingly, the residual solid streams 70 and 90 are optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28 for further enzymatic hydrolysis. Alternatively, if both processes are simultaneously conducted, residual solid streams 70 and 90 may be combined before being recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. The particle reduced streams 72 and 92 may then be subjected to another micro solid/liquid separation 74 and 94, such as ultrafiltration, to produce recovered enzyme streams 76 and 96 and enzyme reduced streams 78 and 98. When ultrafiltration is utilized, the ultrafiltration separation filters hemicellulose and/or cellulase enzymes from the particle reduced streams 72 and 92, which allows these enzymes to be recovered and/or recycled. Accordingly, the recovered enzyme streams 76 and 96 may then be optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. Alternatively, if both processes are simultaneously conducted, recovered enzyme streams 76 and 96 may then be combined before being recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. The enzyme reduced streams 78 and 98 may then be subjected to a further micro solid/liquid separation 80 and 100, such as diafiltration, to produce second recovered enzyme streams 82 and 102 and sugar rich enzyme reduced streams 84 and 104. When diafiltration is utilized, the diafiltration process filters any remaining hemicellulase and/or cellulase enzymes. Accordingly, the second recovered enzyme streams 82 and 102 may then be optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. The sugar rich enzyme reduced streams 84 and 104 may then be subjected to fermentation individually, or alternatively, or are combined before being subjected to fermentation.

[0039] In an embodiment, the recovered enzyme streams 76 and 96 and the second recovered enzyme streams 82 and 102 may be combined before being recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. Alternatively, any of the streams 76, 82, 96 and 102 may be combined or they may be individually recycled to the first and/or second enzymatic hydrolysis stages. In an embodiment, at least some, optionally all, of recovered enzyme streams 76 and 82 (separately or combined) are recycled to the second enzymatic hydrolysis process 28. In an embodiment, at least some, or optionally all, of recovered enzyme streams 96 and 102 (separately or combined) are recycled to the first enzymatic hydrolysis process 20.

[0040] In yet another embodiment shown in Figure 1 , which may be used with any of the preceding process options, one or more micro solid/liquid separations may be conducted after fermentation 62. In this embodiment, if any enzymes are present in stream 60, such as if not all of the enzymes are removed as shown in Figure 1 , 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 62, as the hemicellulase and cellulase enzymes are still active. In this embodiment, the sugar rich enzyme reduced stream 60 is subjected to the fermentation stage 62 resulting in an alcohol stream 64 and a residual fermentation stream 106. The residual fermentation stream 106 may be subjected to one or more micro solid/liquid separations steps, such as micro- filtration 108, which produces a residual solid stream 1 10 (retenate) and a particle reduced stream 1 12. Microfiltration removes any fine particulate solids, such as unhydrolyzed hemicellulose and/or cellulose, in addition to microorganisms such as yeast used in the fermentation stage 62. The residual solid stream 1 10 may then be optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28 for further enzymatic hydrolysis. The particle reduced stream 1 12 (raff ^' mate) may then be subjected to another micro solid/liquid separation 1 14, such as ultrafiltration, to produce a recovered enzyme stream 1 16 and an enzyme reduced stream 1 18. When ultrafiltration is utilized, the ultrafiltration separation filters hemicellulose and/or cellulase enzymes from the particle reduced stream 1 12, which allows these enzymes to be recovered and/or recycled. Accordingly, the recovered enzyme stream 16 may then be optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. The enzyme reduced stream 8 may then be subjected to a further micro solid/liquid separation 120, such as diafiltration, to produce a second recovered enzyme stream 122. When diafiltration is utilized, the diafiltration process filters any remaining hemicellulase and/or cellulase enzymes. Accordingly, the second recovered enzyme stream 122 may then be optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28. In one embodiment, the recovered enzyme stream 1 16 and the second recovered enzyme stream 122 are combined before being recycled to the first and/or second enzymatic hydrolysis stages 20 and 28.

[0041] Figure 2 exemplifies a schematic of one embodiment of the invention wherein the product stream from the first enzymatic hydrolysis process is treated before being subjected to the second enzymatic hydrolysis process. In this embodiment, the recovered enzyme stream is preferably recycled to the second enzymatic hydrolysis process. 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 an effluent stream 222. The effluent stream is then subjected to solid/liquid separation 224 to produce a solid stream 226 and a liquid/filtrate stream 230, wherein the solid stream 226 is subjected to the second enzymatic hydrolysis stage 228. The liquid/filtrate stream 230 is then subjected to micro solid/liquid separation 232 to produce a recovered enzyme stream 234 (retentate) and an enzyme reduced sugar rich stream 236 (raffinate). The recovered enzyme stream 234 is recycled to the second enzymatic hydrolysis stage 228, while the enzyme reduced sugar rich stream 236 is subjected to fermentation 238 to produce ethanol. The recovered enzyme stream 234 is preferably recycled to the second enzymatic hydrolysis stage 228 since the stream may predominantly contain unused enzymes that will preferentially hydrolyze cellulose.

[0042] Figure 3 exemplifies a schematic of another embodiment of the invention wherein the product stream from the second enzymatic hydrolysis process is treated before being subjected a fermentation process. In this embodiment, the recovered enzyme stream is preferably recycled to the first enzymatic hydrolysis process. The lignocellulosic feedstock 310 is again optionally first subjected to a pretreatment and optional steam explosion 312 to produce an activated feedstock 314, and then subsequently an optional disc refining step 316 to produce a fine particulate stream 318. 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 318 is then subjected to a first enzymatic hydrolysis stage 320 to produce an effluent stream 322. The effluent stream 322 is then subjected to a second enzymatic hydrolysis stage 324, resulting in a sugar rich process stream 326. The sugar rich process stream 326 is then subjected to solid/liquid separation 328, resulting in a solid stream 330 and a liquid/filtrate stream 332. The liquid/filtrate stream 332 is then subjected to micro solid/liquid separation 334 to produce a recovered enzyme stream 336 (retentate) and an enzyme reduced sugar rich stream 338 (raffinate). The recovered enzyme stream 336 is recycled to the first enzymatic hydrolysis stage 320, while the enzyme reduced sugar rich stream 338 is subjected to fermentation 340 to produce ethanol. The recovered enzyme stream 336 is preferably recycled to the first enzymatic hydrolysis stage 320 since the stream may predominantly contain unused enzymes that will preferentially hydrolyze hemicellulose.

[0043] Figure 4 exemplifies a schematic of one embodiment of the invention wherein inhibitory compounds are removed prior to fermentation. The lignocellulosic feedstock 410 is optionally first subjected to a pretreatment and optional steam explosion 4 2 to produce an activated feedstock 414, and then subsequently an optional disc refining step 416 to produce a fine particulate stream 418. 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 418 is then subjected to a first enzymatic hydrolysis stage 420 to produce an effluent stream 422. The effluent stream 422 is then subjected to solid/liquid separation 424 to produce a solid stream 426 and a liquid/filtrate stream 430, wherein the solid stream 426 is subjected to the second enzymatic hydrolysis stage 428. The liquid/filtrate stream 430 is then subjected to micro solid/liquid separation 432 to produce a recovered enzyme stream 434 (retentate) and an enzyme reduced sugar rich stream 436 (raffinate). The recovered enzyme stream 434 is recycled to the second enzymatic hydrolysis stage 428, while the enzyme reduced sugar rich stream 436 is subjected to a chromatographic separation 438 to remove acetic acid and/or other inhibitor compounds 440, and produce a liquid sugar stream 442. The liquid sugar stream 442 is then subjected to fermentation 444 to produce ethanol. Chromatographic separation 438 may be used in the embodiments of Figures 1 , 2 or 3. [0044] Figure 5 exemplifies a schematic of one embodiment of the invention wherein the recovered enzymes from each enzymatic hydrolysis stage is recycled to the other enzymatic hydrolysis stage. The lignocellulosic feedstock 510 is optionally first subjected to a pretreatment and optional steam explosion 512 to produce an activated feedstock 514, and then subsequently an optional disc refining step 516 to produce a fine particulate stream 518. 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 518 is then subjected to a first enzymatic hydrolysis stage 520 to produce an effluent stream 522. The effluent stream 522 is then subjected to solid/liquid separation 524 to produce a liquid/filtrate stream 526 and a solid stream 528. The solid stream 528 is subjected to the second enzymatic hydrolysis stage 530 to produce a sugar rich process stream 532, which is subsequently subjected to solid/liquid separation 534 to produce a second solid stream 536 and a second liquid/filtrate stream 538. The liquid/filtrate streams 526 and 538 are then subjected to micro solid/liquid separation 540, 542 to produce a recovered enzyme stream 544, 546 (retentate) and an enzyme reduced sugar rich stream 548, 550 (raffinate). The recovered enzyme stream 544, 546 is then recycled to either the first enzymatic hydrolysis stage 520, the second enzymatic hydrolysis stage 530, or both enzymatic stages 520 and 530, while the enzyme reduced sugar rich stream 548, 550 is subjected to fermentation 552 to produce ethanol.

[0045] Figure 6 exemplifies a schematic of one embodiment of the invention wherein the second enzymatic hydrolysis process or stage also utilizes fermentation organisms to simultaneously ferment the sugar rich process stream to alcohol. In this embodiment, the enzymes from both enzymatic hydrolysis stages are preferably recovered and preferably recycled to the other enzymatic hydrolysis stage (the first enzymatic hydrolysis stage or the SSF). However, any of the processes described herein can be utilized with a simultaneous saccharification and fermentation process. [0046] As exemplified, the lignocellulosic feedstock 610 is optionally first subjected to a pretreatment and optional steam explosion 612 to produce an activated feedstock 614, and then subsequently an optional disc refining step 616 to produce a fine particulate stream 618. 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 618 is then subjected to a first enzymatic hydrolysis stage 620 to produce an effluent stream 622. The effluent stream 622 is then subjected to solid/liquid separation 624 to produce a liquid/filtrate stream 626 and a solid stream 628. The liquid/filtrate stream 626 is then subjected to micro solid/liquid separation 640 to produce a recovered enzyme stream 644 (retentate) and an enzyme reduced sugar rich stream 648 (raffinate). The recovered enzyme stream 644 is then recycled to either the first enzymatic hydrolysis stage 620, the second enzymatic hydrolysis stage 630, or both enzymatic stages 620 and 630, while the enzyme reduced sugar rich stream 648 is subjected to fermentation 652 to produce ethanol. The solid stream 628 is subjected to the second enzymatic hydrolysis stage 630, which also contains fermentation organisms to ferment the sugars produced from the hydrolysis, to produce an alcohol stream 632. Alcohol stream 632 is then subject to solid/liquid separation 634 to produce a second solid stream 636, and a liquid/filtrate alcohol stream 638. The liquid/filtrate alcohol stream 638 is then subjected to micro solid/liquid separation 642 to produce a recovered enzyme stream 646 (retentate) and an enzyme reduced alcohol stream 650 (raffinate).

INPUT FEEDSTOCK

[0047] The lignocellulosic feedstock 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.

[0048] 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. 25 to about 0.5 inches in length.

[0049] 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

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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., 150 - 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%.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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 [0062] The applicants herein describe a method for efficiently breaking down a lignocellulosic feedstock into fermentable sugars using enzymatic hydrolysis, and then recovering and recycling the enzymes for further use. 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.

[0063] 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.

[0064] 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. [0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 rich process 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 rich process stream described in the present application has a total solids content of greater than about 15%. In a further embodiment, the sugar rich process stream has a total solids content from about 15 to about 30%. In a further embodiment, the sugar rich process stream may have a total solids content up to about 50% (e.g., about 15 to about 50%, preferably about 30 to about 50%).

[0070] 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.

[0071 ] 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 rich process stream. In one embodiment, at least one of the effluent stream and the sugar rich process 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.

[0072] 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%.

[0073] 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.

[0074] 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.

[0075] 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. [0076] 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

[0077] In the second enzymatic hydrolysis process, the effluent stream is treated with a second enzyme preparation to produce a sugar rich process 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] The resulting sugar rich process 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 10 to about 45%, preferably about 15 to about 45% and more preferably about 25 to about 45%. The sugar rich process stream optionally also contains a total solids content of between about 0% to about 60%.

[0082] 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 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. As such, the cellulose present in the 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.

[0083] 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

[0084] The applicants have found that the recovery and recycling of the hemicellulase and cellulase enzymes from the first and/or second enzymatic hydrolysis stages is advantageous as the recycling of the enzymes used in one enzymatic hydrolysis stage to the enzymatic hydrolysis other 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, and subsequently recycling the enzymes into either the first and/or second enzymatic hydrolysis stages, significant cost savings are obtained.

[0085] 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 rich process stream. In one embodiment, at least one of the effluent stream and the sugar rich process 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 to preferentially solubilize cellulose to obtain a sugar rich process 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.

[0086] 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.

[0087] In one embodiment, the sugar rich process 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. 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.

[0088] 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) 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. [0089] The effluent stream, the sugar rich process stream 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 effluent stream, the sugar rich process stream and the alcohol stream is filtered to obtain the recovered enzyme stream. In one embodiment, at least one of the effluent stream, the sugar rich process stream and the alcohol stream is subjected to at least one membrane filtration process. In one embodiment, at least one of the effluent stream, the sugar rich process and the alcohol stream is subjected to at least one of ultrafiltration and diafiltration. In one embodiment, at least one of the effluent stream, the sugar rich process stream 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.

[0090] 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 monomeric sugar rich stream and a monomeric sugar reduced stream and wherein the monomeric sugar reduced stream (preferably a solid stream - e.g., a filtrate) is subjected to the second enzymatic hydrolysis process. In one embodiment, the monomeric sugar rich 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 monomeric rich 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.

[0091] 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 of acetic 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, formic acid, glycerol, furfural and hydroxymethylfurfural.

[0092] 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.

[0093] 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.

[0094] In one embodiment, the cellulose, hemicellulose and lignin containing material is subjected to hydrolysis to obtain the feedstock.

[0095] 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. Likewise, in one embodiment, the first and/or second recovered enzyme streams are recycled to the second and/or first enzymatic hydrolysis processes, respectively. For example,

(i) in one embodiment, the effluent stream from the first enzymatic hydrolysis stage is subjected to solid/liquid separation to produce a solid stream and a liquid/filtrate stream. Subsequently, the liquid/filtrate stream is subjected to micro solid/liquid separation to produce a recovered enzyme stream (retentate) and an enzyme reduced effluent stream (raffinate). At least some of the first recovered enzyme stream is then recycled to the second enzymatic hydrolysis stage, or at least some of the first recovered enzyme stream is then recycled to the first and second enzymatic stages;

(ii) in another embodiment, the sugar rich process stream produced from the second enzymatic hydrolysis stage is subjected to solid/liquid separation to produce a solid stream and a liquid/filtrate stream. Subsequently, the

liquid/filtrate stream is subjected to micro solid/liquid separation to produce a second recovered enzyme stream (retentate) and an enzyme reduced sugar rich process stream (raffinate). At least some of the second recovered enzyme stream is then recycled to the first enzymatic stage, or at least some of the second recovered enzyme stream is then recycled to the first and second enzymatic stages;

(iii) in another embodiment, the liquid/filtrate streams from both the first and second enzymatic hydrolysis stages are combined (after solid/liquid separation of the effluent stream and the sugar rich process stream) and subsequently subjected to micro solid//liquid separation to produce a recovered enzyme stream (retentate) and an enzyme reduced sugar rich process stream (raffinate). The recovered enzyme stream can then either be recycled to the first and/or second enzymatic stage;

(iv) in another embodiment, the recovery of the enzymes is conducted after both the first and second enzymatic hydrolysis stages. Accordingly, the effluent stream from the first enzymatic hydrolysis stage is subjected to solid/liquid separation to produce a solid stream and a liquid/filtrate stream. Subsequently, the liquid/filtrate stream is subjected to micro solid//liquid separation to produce a recovered enzyme stream (retentate) and an enzyme reduced effluent stream (raffinate). The recovered enzyme stream is then recycled to the second enzymatic stage, or the first and second enzymatic hydrolysis stages. Further, the sugar rich process stream produced from the second enzymatic hydrolysis stage is subjected to solid/liquid separation to produce a solid stream and a liquid/filtrate stream. Subsequently, the liquid/filtrate stream is subjected to micro solid//liquid separation to produce a second recovered enzyme stream (retentate) and an enzyme reduced sugar rich process stream (raffinate). The second recovered enzyme stream is then recycled to the first enzymatic stage, or the first and second enzymatic hydrolysis stages; and

(v) in one embodiment, the effluent stream from the first enzymatic hydrolysis stage is subjected to solid/liquid separation to produce a solid stream and a liquid/filtrate stream. Subsequently, the solid stream is subjected to a simultaneous saccharification and fermentation process to produce an alcohol stream, which is subjected to solid/liquid separation to obtain a solid stream and a liquid/filtrate alcohol stream. Subsequently, the liquid/filtrate alcohol stream is subjected to micro solid/liquid separation to produce a recovered enzyme stream (retentate) and an enzyme reduced alcohol stream, (raffinate). The recovered enzyme stream is then recycled to the first enzymatic stage, or the first enzymatic hydrolysis and the SSF process.

INHIBITORY COMPOUNDS

[0096] 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.

[0097] 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 hydrolize and solubilize cellulose to obtain a sugar rich process 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.

[0098] In one embodiment, the effluent stream is subjected to solid/liquid separation to obtain a liquid stream comprising the inhibitor reduced stream and a solid stream comprising the treated effluent 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.

[0099] In one embodiment, the effluent stream from the first hydrolysis stage is subjected to solid/liquid separation to obtain a liquid/filtrate stream and a 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 solid stream and a liquid/filtrate stream. The solid stream contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage. The liquid/filtrate 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 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 solid stream is subjected to the second enzymatic hydrolysis stage increases the sugar output as compared to when the inhibitors are not removed.

LIGNIN [00100] 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 product stream before the stream is fermented for ethanol production.

[00101 ] Accordingly, in one embodiment, there is included an optional process to treat the sugar rich process 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.

[00102] In one embodiment, the sugar rich process 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.

[00103] 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.

[00104] In one embodiment, the 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 sugar rich process stream, which contains the soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose. The sugar rich process 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/filtrate 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 sugar rich process 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.

NITROGEN SOURCE AND ALKALINE AGENT FOR FERMENTATION

[00105] The applicants have found that when the sugar rich process stream 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. Accordingly, preferably, the pH is adjusted to a level at which acetic acid, or another weak acid that may be present, dissociates, or partially dissociates.

[00106] 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. In one embodiment, the fermentation of the sugar rich process stream is conducted at an elevated pH of between about 5.0 and 6.0, or optionally 5.0 to 5.5, or 5.5 to 6.0, in which the pH of the fermentation process in raised using an alkaline agent, such as calcium hydroxide. At such a level, acetic acid will dissosiate. 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 acetic acid to form calcium acetate, which is not very soluble in the fermentation broth. As a result, the calcium acetate precipitates from the broth. Accordingly 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.

[00107] In one embodiment, the fermentation of the sugar rich process is conducted at a pH of at least 5.0 in the presence of sodium hydroxide. In another embodiment, the fermentation of the sugar rich process is conducted at a pH of at least 5.5 in the presence of calcium hydroxide. In another embodiment, the fermentation of the sugar rich process is conducted at a pH of at least 5.5 in the presence of a calcium hydroxide and sodium hydroxide.

[00108] In one embodiment, the fermentation of the sugar rich process stream is also conducted in the presence of a nitrogen source. In one embodiment, the nitrogen source is urea, a urea derivative, nitrates, or ammonia derivatives, such ammonium hydroxide. 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. 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 an alkaline or pH adjustment agent.

[00109] 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 of between about 5.0 and 6.0, or optionally 5.0 to 5.5, or 5.5 to 6.0, in which the pH of the fermentation process in raised using calcium hydroxide and also in the presence of urea.

RECOVERY OF ENZYMES FROM FERMENTATION PROCESS

[001 10] 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 rich process stream. Accordingly, the recovery and recycling of hemice!lulase and cellulase enzymes may be used by itself or in combination with any other process or processes disclosed herein.

[001 1 1 ] 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.

[001 12] As exemplified in Figure 1 , the sugar rich enzyme reduced stream may be subjected to the fermentation stage resulting in an alcohol stream and a residual fermentation stream. The residual fermentation stream may be subjected to a micro solid/liquid separation, such as micro-filtration, which produces a residual solid stream (retenate) and a particle reduced stream. Microfiltration may be used to remove fine particulate solids, such as unhydrolyzed hemicellulose and/or cellulose, in addition to microorganisms such as yeast used in the fermentation stage. The residual solid stream is optionally recycled to the first and/or second enzymatic hydrolysis stages 20 and 28 for further enzymatic hydrolysis. The particle reduced stream (raffinate) may then be subjected to another micro solid/liquid separation, such as ultrafiltration and/or diafiltration, to produce a recovered enzyme stream and an enzyme reduced stream. When ultrafiltration is utilized, the ultrafiltration separation filters hemicellulose and/or cellulase enzymes from the particle reduced stream, which allows these enzymes to be recovered and/or recycled. Accordingly, the recovered enzyme stream is then optionally recycled to the first and/or second enzymatic hydrolysis stages. The enzyme reduced stream may then be subjected to a further micro solid/liquid separation, such as diafiltration, to produce a second recovered enzyme stream. When diafiltration is utilized, the diafiltration process filters remaining hemicellulase and/or cellulase enzymes. Accordingly, the second recovered enzyme stream is then optionally recycled to the first and/or second enzymatic hydrolysis stages. In one embodiment, the recovered enzyme stream and the second recovered enzyme stream are combined before being recycled to the first and/or second enzymatic hydrolysis stages.

[001 13] 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. 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 are optionally recovered from the SSF process.

[001 14] 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.

[001 15] In another embodiment, as exemplified in Figure 6, the product stream 632 containing alcohol from the simultaneous saccharification and fermentation of the second enzymatic hydrolysis process is treated to recover enzymes from this SSF process. In one embodiment, at least some of the recovered enzymes 646 are recycled to the first enzymatic hydrolysis process. The product stream 632 from the SSF {second enzymatic hydrolysis process and fermentation) is treated by any process which is able to separate the enzymes contained in the product stream. In one embodiment, the product stream 632 is subjected to solid/liquid separation 634 to obtain a solid stream 636 and a liquid/filtrate alcohol stream 638. In one embodiment, the liquid/filtrate alcohol stream is filtered to obtain the recovered enzyme stream 646. In one embodiment, the liquid/filtrate alcohol stream is subjected to at least one membrane filtration process. In one embodiment, the liquid/filtrate alcohol 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 stream, the enzymes are retained by the filter membrane, while the alcohol product, for example ethanol, passes through the filter membrane, allowing for recovery and recycling of the enzymes.

OTHER EMBODIMENTS

[001 16] 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.

[001 17] 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.

[001 18] 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

[001 19] 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: Recovery and Assay of Enzymes from Poplar Enzymatic Hydrolysis 1.1 Material

[00120] Two pails of hydrolyzate centrate (from solid-liquid separation by centrifugation) were collected from Tank A (middle-tank) and Tank B respectively, during an enzymatic hydrolysis semi-continuous trial.

1.2 Material initial condition

[00121] Precipitate was observed in both Tank A and B centrate, especially in the former one, despite the fact that they both went through a solid removal process. The precipitate did not dissolve after heating. Small bubbles appeared constantly in the surface of the hydrolyzate from both sources. It appeared that fermentation was proceeding in the pails.

1.3 Pre-treatment

[00122] The hydrolyzate was centrifuged at 6000-7000rpm, and then filtered through a Whatman No.1 filter paper.

1.4 Membrane Separation Protocol

[00123] The hydrolyzate was subjected to microfiltration, which resulted in a permeate and a retentate. The permeate was then further subjected to ultrafiltration with a molecular weight cut-off of 5 kDa, resulting in an enzyme rich retentate and a permeate.

1.5 Material balance [00124] Shown in Table 1 is the material balance after both microfiltration and ultrafiltration of the hydrolyzate for Tank A and Tank B.

1.6 Protein content

[00125] Protein content in each portion of the hydrolyzates, retentates and permeates was measured using a Bradford protein assay and is shown in Table 2. Data in the table show that the microfiltration permeate and retentate had very similar protein concentration, which means the protein distribution was not impacted by the microfiltration process. Higher protein concentration was observed in the ultrafiltration retentate than in the ultrafiltration permeate. However, when taking the volume of each portion into account, there was still more total protein in the ultrafiltration permeate than the retentate. The enzyme cocktail used consisted of a mixture of Novozymes NS22074, Novozymes Novo188, and Genencor's Accellerase 1500.

1.7 Sugar content

[00126] Glucose and xylose concentration in the Tank A and Tank B hydrolyzate, as well as the ultrafiltration permeate and retentate were analyzed and shown in Table 3. Microfiltration and ultrafiltration did not appear to change the sugar distribution profile.

1.8 Enzyme activity assay

[00127] The hydrolyzate filtrate from both Tank A and Tank B were tested for enzyme activity. Sigmacell and xylan were used as substrates against the original hydrolyzate filtrate, ultrafiltration permeate and retentate. Samples were taken at certain time intervals to measure the glucose and xylose concentration using HPLC. Data were recorded as the percentage increase in glucose or xylose concentration during the trial, relative to the original glucose or xylose concentration in the sample. The test results are listed in Table 4.

[00 28] The assays were conducted with 1 part of test solution mixed with 4 parts substrate solution that contained either 1 % or 2% substrate. Buffer (pH 5.0) without substrate was also used as a control to confirm that the glucose/xylose being generated were hydrolysis products from the fresh substrate.

[00129] From the control tests, it was found that the digestible substrate was exhausted. This is confirmed by the fact that there was no measurable change in xylose or glucose concentration during the control studies. But glucose and xylose could be generated when the hydrolyzate was provided with new substrates, as a result of the cellulases and xylanases that remained active.

Example 2: Recovery and Assay of Enzymes from Poplar Enzymatic Hydrolysis 2.1 Material

[00130] Activity tests and protein assays were conducted with some Tank B filtrate (by filter press), the same as used in Example 1 . The filtrate was further processed such that it was passed through a sterilization cartridge with pore size of 0.2 μιη to completely remove the solid and bacteria, followed by ultrafiltration and diafiltration.

2.2 Material initial condition

[00 31 ] The hydrolyzate filtrate was clear and residue free.

2.3 Pre-treatment

[00132] No pre-treatment was performed this time.

2.4 Membrane Separation Protocol

[00133] The hydrolyzate was subjected to ultrafiltration with a molecular weight cut-off of 5kDa, which resulted in a permeate and a retentate. The retentate was then further subjected to diafiltration with a molecular weight cut-off of 5 kDa, resulting in an enzyme rich retentate and a permeate.

2.5 Material balance

[00134] Shown in Table 5 is the material balance after both ultrafiltration and diafiltration of the hydrolyzate.

2.6 Protein content

[00135] Protein content was measured using the Bradford protein assay. About half of the total proteins were recovered in the diafiltration retentate. This time, the ultrafiltration achieved much higher protein concentration in its retentate than in Example 1 . Almost half of the proteins in the original hydrolyzate filtrate were recovered in the diafiltration retentate. The sterilization step and near immediate use of the hydrolyzate prevented fermentation from occurring. Preventing growth of these fermentation organisms limited the generation of low molecular weight proteins and amino acids (i.e., "junk proteins") that would otherwise tend to appear in the ultrafiltration filtrate. The protein content data is shown in Table 6.

2.7 Sugar content

[00136] Sugar content of each portion was measured using HPLC. The results are listed in Table 7. After these filtrations, over 80% of the sugar remained in the ultrafiltration permeate and the amount of sugar found in the diafiltration retentate (the protein portion), was minimal. In this regard, the sugar- protein separation was reasonably achieved, which meant a relatively successful removal of sugar inhibitors in the protein recovery process.

2.8 Enzyme activity assay

[00137] Enzyme activity tests were conducted on four hydrolyzate filtrate samples collected from Tank B at different times over the period of one week. The average protein content of the hydrolyzate filtrates was around 0.1 16mg/ml. The results after 24 hour of each hydrolysis test and its replicate are listed in the Table 8. 12 ml of 1 % or 2% sigmacell and 12ml of 1 % or 2% xylan were mixed with 3 ml of testing material in all the tests.

Table 1

Tank A

Tank A j Microfiltration Microfiltration Ultrafiltration filtrate (kg) Permeate (kg)

(kg) (starting material for

j ultrafiltration)

(kg) Permeate Retentate Permeate] Retentate

21.56 20.39 1.17 20.00 16.54 3.46 ^{* *} Determined by difference

Tank B

^* Determined by difference

Table 2

Sample Name Protein Content (rr ig/ml) Total protein (mg)

Tank A hydrolyzate filtrate 0.122 2630

Tank A microfiltration permeate 0.106 ^{7 "} 2161

Tank A microfiltration retentate 0.131 153

Tank A ultrafiltration permeate 0.099 ; 1638

Tank A ultrafiltration retentate 0.175 606

Tank B hydrolyzate filtrate 0.128 2483

Tank B microfiltration permeate 0.1 17 21 19

Tank B microfiltration retentate 0.1 19 154

Tank B ultrafiltration permeate 0.087 ^: 1275

Tank B ultrafiltration retentate 0.163 448

Table 3

Original Hydrolyzate

Glucose Xylose Glucose Xylose (g/100ml) (g/100ml) (g/100ml) (g/100ml)

4.349 1 .360 3.148 1.733

Tank A 4.169 1 .305 Tank B 3.035 1.701

2.178 1 .130 3.160 1.801

2.202 1 .281 2.959 1.718

Average 4.259 1.269 3.075 1.738

Ultrafiltration Permeate

Glucose Xylose Glucose Xylose (g/100ml) (g/100ml) (g/100ml) (g/100ml)

4.014 1 .235 3.328 1.686

4.237 1 .265 3.453 1.673

3.994 1.991

Tank A Tank B 3.950 1.988

3.937 1.962

4.187 1.989

3.830 1.996

3.660 1.899

Average 4.126 1.250 3.792 1.898

Ultrafiltration Retentate

Glucose Xylose Glucose Xylose (g/100ml) (g/100ml) (g/100ml) (g/100ml)

3.929 1.203

3.832 1.428

2.949 1.554

2.962 1.672

3.037 1.642

Tank A Tank B

3.604 2.003

3.599 2.039

3.636 2.073

3.740 2.109

3.618 2.001

3.479 1 .951

Average 3.881 1.316 3.403 1.894 Table 4

Cellulase Xylanase

Incubation Glucose Xylose

Enzyme Activity (g Activity (g

Substrate Duration Increase Increase

Source glucose/hr/mg xylose/hr/mg

(hr) (%) (%) protein) protein)

Tank A 18 6.2 3.2 0.00122 0.00020 hydrolyzate 18 10.7 9.2 0.00106 0.00047

Tank B 18 9.5 5.2 0.00130 0.00039 hydrolyzate 18 1 1.2 4.6 0.00153 0.00036

Tank A

1 wt% ultrafiltration

sigma permeate 18 10.6 9.6 0.00238 0.00066 cell

Tank A

ultrafiltration

retentate 18 7.3 1 1 .0 0.00091 0.00042

Tank B 18 -6.7 -2.4

ultrafiltration 24 -0.6 -0.3

permeate 24 2.1 2.5 0.00043 0.00024

1 wt%

sigma

cell 24 6.2 4.0 0.00047 0.00016

2 wt% Tank B 24 14.1 5.0 0.00129 0.00025 sigma ultrafiltration

cell retentate 24 15.8 6.9 0.00145 0.00036

1 wt% 24 8.2 2.5 0.00076 0.00013 sigma

cell 24 10.8 6.4 0.00096 0.00032

Tank A 18 -2.7 12.1 0.00072 hydrolyzate 18 -6.5 -3.7

Tank B 18 2.5 10.1 0.00033 0.00075 hydrolyzate 18 -0.5 -1.9

Tank A

ultrafiltration

permeate 18 1.7 4.6 0.00040 0.00033

1 wt% Tank A

xylan ultrafiltration

retentate 18 1.5 -4.3 0.00018

Tank B 18 -7.3 -1 .3

ultrafiltration 24 0.03 -0.02 0.00051

permeate 24 4.5 2.3 0.00079 0.00021

Tank B

ultrafiltration

retentate 24 2.2 4.0 0.00002 0.00017 Table 5

Tank B Ultrafiltration Ultrafiltration Water Diafiltration filtrate (kg) Retentate Addition (kg)

(kg) (starting material (kg)

for diafiltration)

(kg)

Permeate Retentate Permeate Retentate

29.3 25.51 3.79 3.6 32.50 32.53 ^" 3.57 ^*

^* Determined by difference

Table 6

Sample Name Protein Content (mg/ml) Total protein (mg)

Tank B hydroiyzate filtrate 0.154 4510

ultrafiltration permeate 0.051 1300

ultrafiltration retentate 0.772 2930

diafiltration permeate 0.020 650

diafiltration retentate 0.575 2050

Table 7

Sugar Concentration Weight of Total Sugar

Cellobiose Glucose Xylose

Sample Name (g/100ml) (g/100m l) (g/100m l) Cellobiose (g) Glucose (g) Xylose (g)

Hydroiyzate

Filtrate 0.57 1 1 .60 3.12 168 3400 914

Ultrafiltration

Permeate 0.55 1 1 .09 2.94 139 2829 749

Ultrafiltration

Retentate 0.52 1 1 .51 3.08 20 436 1 1 7

Diafiltration

Permeate 0.05 1 .60 0.52 16 521 170

Diafiltration

Retentate 0.00 0.06 0.02 0 2 1 Table 8

^* % conversion from sigma cell/xylan to glucose/xylose