Methods and compositions for treatment of cellulosic biomass, and products produced thereby

Abstract

To provide methods for treating cellulosic biomass to produce cellulosic sugars, and products thereof.SOLUTION: A two-step method for activating cellulosic feedstock is described. The feedstock is subjected to a first high-temperature activation step at a temperature above 190°C. and a second activation step at a lower temperature under alkaline conditions. Also described are methods and compositions for the enzymatic hydrolysis of activated cellulose using one or more cellulase enzymes, a surfactant and polyaspartic acid. Also described are products of the methods.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO PRIOR APPLICATIONS This application claims priority under the Paris Convention to U.S. Patent Application No. 62 / 145,785, filed April 10, 2015, and U.S. Patent Application No. 62 / 246,271, filed October 26, 2015, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present application relates to methods for treating cellulosic biomass to produce cellulosic sugars. In one aspect, methods for activating cellulosic feedstock and / or enzymatic hydrolysis to produce glucose are provided. Also provided are activated cellulosic feedstocks and products produced from the enzymatic hydrolysis of the activated cellulosic feedstock. [Background technology]

[0003] The production of sugars such as glucose from cellulosic biomass has been the focus of much research and development, however the high cost and low conversion rates of many processes limit the widespread adoption of cellulosic sugar technology.

[0004] A number of different methods for converting cellulosic biomass into sugars are known in the art. These typically involve a pretreatment step in which the cellulosic biomass is physically and / or chemically altered to open up the structure of the polysaccharides contained in the cellulosic biomass, as well as an enzymatic or chemical hydrolysis step in which the polysaccharides are broken down into monosaccharides.

[0005] Although high yields of glucose based on cellulose (>90%) have been reported, these yields are usually obtained at low concentrations of glucose, typically 2-5%. Even with high enzyme inputs, methods that result in both high yields and high concentrations of glucose are difficult to achieve because the presence of glucose usually reduces the activity of cellulase enzymes. Cellulase enzyme activity decreases over time, necessitating the addition of new enzyme to the enzymatic hydrolysis reaction to maintain yield. However, the high cost of cellulase enzymes can be prohibitive. Cellulase enzymes can also bind to recalcitrant cellulose and / or lignin, making them unavailable for further hydrolysis of cellulose to glucose. This unproductive binding also prevents enzyme reuse, which is desirable to reduce enzyme usage and lower costs.

[0006] A variety of different processes have been developed for the pretreatment of cellulosic feedstocks, and various enzymatic hydrolysis processes have been developed to convert the treated cellulosic feedstocks into sugars. For example, Parekh (PCT Publication No. WO2014 / 026154) describes a two-stage pretreatment process for lignocellulosic biomass under primarily acidic conditions. Schiffino et al. (US Patent Publication No. 2011 / 0250645) describe a method to improve the release of monosaccharides from alkali-treated biomass. Liu et al. (US Patent Publication No. 2011 / 0300586) describe a two-stage pretreatment process for lignocellulosic biomass with the goal of reducing cellulose crystallinity and to dissociate hemicellulose-cellulose complexes. An embodiment includes mild steam treatment or autohydrolysis followed by hydrolysis with dilute acid or hot water. Summary of the Invention

[0007] summary This summary is intended to guide the reader to the more detailed description that follows, and is not intended to limit or define any claimed or yet unclaimed inventions. One or more inventions may exist in any combination or sub-combination of the elements or process steps disclosed in any part of this document, including the claims and drawings.

[0008] In one broad aspect, a method is provided for activating a cellulosic feedstock to increase the chemical and / or enzymatic reactivity of cellulose in the feedstock. The activated cellulose can then be converted to cellulosic sugars, such as by subjecting the activated cellulose to enzymatic hydrolysis.

[0009] In this aspect, the cellulosic feedstock may be subjected to a first high temperature activation step followed by a second activation step under alkaline conditions at a lower temperature. In this embodiment, the method may include subjecting the feedstock to a first activation step in which the feedstock is treated at a temperature above 190° C. and a pressure above 200 psig to produce a first activated cellulose stream comprising cellulose II and insoluble solids. The insoluble solids may comprise components of the feedstock other than cellulose, such as lignin. The first activated cellulose stream may then be subjected to a second activation step in which the first activated cellulose stream is treated with alkali at a lower temperature than the first activation step to produce a second activated cellulose stream comprising cellulose IV. Preferably, the first activation step is carried out in the presence of water.

[0010] Without being limited by theory, it is believed that the first activation step changes the crystalline state of the cellulose in the cellulosic feedstock, producing a first activated cellulose stream containing a higher proportion of cellulose II compared to the amount of cellulose II in the cellulosic feedstock. It is believed that the second activation step further changes the crystalline state of the cellulose in the first activated cellulose stream, producing a second activated cellulose stream containing a higher proportion of cellulose IV compared to the amount of cellulose IV in the first activated cellulose stream. In one embodiment, the two-step activation method described herein produces a mixture of cellulose II, hydrated cellulose II and alkali cellulose IV. Optionally, the cellulosic material can be treated, e.g., washed and / or filtered, after one or each activation step to remove soluble non-cellulosic components.

[0011] It has also been determined that the cellulose activation methods described herein result in activated cellulose with increased levels of glucan and / or decreased levels of non-cellulosic components of the cellulosic feedstock, such as lignin. For example, in one embodiment, the methods described herein produce activated cellulose that contains at least 60%, at least 70%, or at least 75% glucan. In one embodiment, the methods described herein produce activated cellulose that contains less than 25%, less than 20%, or less than 15% lignin.

[0012] In another broad aspect, methods and compositions are provided for stabilizing enzymes, maintaining enzyme activity during enzymatic hydrolysis, and / or obtaining an enzyme recycle stream.

[0013] In another broad aspect, there is provided an enzymatic hydrolysis mixture suitable for use in the enzymatic hydrolysis of cellulose. In a preferred embodiment, the enzymatic hydrolysis mixture is contacted with activated cellulose produced according to the methods described herein.

[0014] In these aspects, one or more cellulase enzymes are provided in combination with a surfactant and / or dispersing agent for the enzymatic hydrolysis of cellulose. Without being limited by theory, it is believed that the cellulase enzymes form complexes with the surfactant and / or dispersing agent, which may stabilize the enzyme, aid in maintaining enzymatic activity, prevent enzymatic degradation, and / or facilitate recovery of the enzyme after enzymatic hydrolysis. It is also believed that the presence of a dispersing agent, such as an oligopeptide, helps prevent non-productive binding of the cellulase enzymes due to interactions with lignin and / or other non-cellulosic components. In a preferred embodiment, the surfactant is a non-ionic surfactant, such as a polysorbate surfactant. In another preferred embodiment, the surfactant is a mixture of surfactants, such as Tween™, alkoxylated glycerides, and nonylphenol. In one embodiment, the dispersing agent is a non-enzymatic oligopeptide, optionally a polyamino acid, optionally having a molecular weight of 500-10,000, 1000-5000, or 3500-4500. In a preferred embodiment, the polyamino acid is polyaspartic acid.

[0015] The methods and compositions described herein exhibit many advantages with respect to cellulose activation and / or cellulosic sugar production, which may be obtained from some embodiments. For example, in some embodiments, the use of the methods and compositions described herein may produce a glucose-rich sugar stream that contains more than about 12% glucose, more than about 14% glucose, more than about 16% glucose, or more than about 18% glucose. Furthermore, in these or other embodiments, the methods and compositions described herein may produce a high yield of monosaccharides. For example, in some embodiments, the methods and compositions described herein may produce a glucose yield of more than about 70%, more than about 80%, more than about 85%, more than about 90%, or more than about 95% of the theoretical yield of glucose. The theoretical yield of glucose in an enzymatic hydrolysis reaction may be determined based on the amount of glucan in the activated cellulosic material subjected to enzymatic hydrolysis. In some preferred embodiments, the methods and compositions described herein may produce a glucose-rich sugar stream that has both a high yield and a high concentration of glucose. For example, in one embodiment, the glucose-rich sugar stream has greater than about 12% glucose and a yield of greater than 70%, or greater than 14% glucose and a yield of greater than 80%, or greater than 16% glucose and a yield of greater than 90%.

[0016] In another broad aspect, a method is provided for the enzymatic hydrolysis of activated cellulose to produce cellulosic sugars, such as glucose. In this embodiment, the enzymatic hydrolysis can be carried out as a batch or continuous process. The enzymatic hydrolysis can be carried out using an enzymatic hydrolysis mixture and / or activated cellulose as disclosed herein.

[0017] In another aspect, a method is provided for treating a glucose-rich sugar stream to remove enzymes used in the enzymatic hydrolysis. Removal and / or reuse of enzymes used in the enzymatic hydrolysis can reduce the amount of enzymes required for enzymatic hydrolysis, thus reducing the costs associated with the production of cellulosic sugars. For example, the methods and compositions described herein can be used to recover at least 60%, at least 70%, at least 80%, or at least 85% of the cellulase enzyme activity in the enzyme recycle stream after enzymatic hydrolysis. The enzyme recycle stream can be reused to continue to process activated cellulose and / or can be used to process new activated cellulose, such as a second activated cellulose stream as described herein. In some embodiments, the glucose-rich sugar stream is subjected to multiple enzyme removal treatments, either the same enzyme removal treatment repeated two or more times, or different enzyme removal treatments.

[0018] In another aspect, a glucose-rich sugar stream produced by a method as described herein is provided. In one embodiment, the sugar stream contains more than 12%, more than 14%, more than 16%, or more than 18% glucose. In one embodiment, the sugar stream contains polyaspartic acid. In some embodiments, the polyaspartic acid is present at a concentration of 1 ppb to 10000 ppm.

[0019] In another aspect, a method for producing a glucose-rich sugar stream is provided, comprising: (a) providing an activated cellulose containing a mixture of cellulose II, hydrated cellulose II, and alkali cellulose IV; and subjecting the activated cellulose to enzymatic hydrolysis with one or more cellulase enzymes, surfactants, and dispersants to produce a glucose-rich sugar stream. Optionally, the activated cellulose is produced using a method described herein.

[0020] In another aspect, a glucose-rich sugar stream is provided further comprising non-glucose sugars, wherein the non-glucose sugars are one or more of xylose, xylooligosaccharides, and xylan. In one embodiment, the non-glucose sugars comprise about 3-8%, about 4-7%, or about 5-6% of the dry matter of the composition. In a particular embodiment, the glucose-rich sugar stream contains about 5% non-glucose sugars.

[0021] In a further aspect of the invention, there is provided a fructose-rich sugar stream prepared by conversion of glucose in the glucose-rich sugar stream of the invention to fructose. The fructose-rich sugar stream further contains non-fructose sugars, where the non-fructose sugars are one or more of xylose, xylooligosaccharides and xylans. In one embodiment, the non-fructose sugars comprise about 1-8%, about 2-7% or about 3-6% of the dry matter of the composition. In a particular embodiment, the fructose-rich sugar stream contains about 5% non-glucose sugars.

[0022] In another aspect of the invention, there is provided a lower glycemic index glucose syrup or a lower glycemic index fructose syrup, which contains about 1-8%, about 2-7% or about 3-6% of one or more of xylose, xylooligosaccharides and xylan, and which has a glycemic index lower than the glycemic index of conventional glucose syrup or fructose syrup produced by conventional methods.

[0023] Other features and advantages of the present disclosure will become apparent from the detailed description below. However, it should be understood that the detailed description and specific examples, while illustrating embodiments of the disclosure, are given for illustrative purposes only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. In particular, it will be recognized that any method may employ all aspects disclosed herein, or any specific combination or sub-combination of aspects. [Brief description of the drawings]

[0024] The drawings included in this application are for the purpose of illustrating various embodiments of the methods and compositions of the teachings herein and are not intended to limit the scope of the teachings in any way. The present disclosure will hereinafter be described in connection with the following drawings: [Figure 1] FIG. 1 is a flow diagram of a process according to a preferred embodiment, including two-stage activation of a cellulosic feedstock, enzymatic hydrolysis of the activated feedstock, and an enzyme removal process to obtain an enzyme recycle stream and a glucose-rich, enzyme-poor sugars stream. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Hereinafter, various methods and compositions are described, providing examples of embodiments of each claimed invention. The embodiments described hereafter do not limit any claimed invention, and any claimed invention may include methods and compositions different from those described hereafter. The claimed invention is not limited to methods and compositions having all the features of any one method and composition described hereafter, or to features common to more than one or all of the methods and compositions described below. A method or composition described hereafter may not be an embodiment of any claimed invention. Any inventions disclosed in the methods or compositions described hereafter that are not claimed herein may be the subject of a separate protective document, e.g., a continuing patent application, and it is not the intention of the applicant, inventor, or owner to relinquish, relinquish, or dedicate to the public any such invention by its disclosure herein.

[0026] Various methods and compositions useful for processing cellulosic biomass to produce cellulosic sugars are described herein. In one embodiment, a method is provided for activating cellulosic feedstock to produce activated cellulose. It has been determined that subjecting cellulosic feedstock to a first activation step at high temperature and pressure, followed by a second activation step at a lower temperature than the first activation step using alkali, produces activated cellulose with chemical and / or physical properties that are favorable for hydrolysis of cellulose into monosaccharides.

[0027] The methods disclosed herein use a cellulosic feedstock 10. The cellulosic feedstock 10 can be any feedstock known in the cellulosic carbohydrate art. For example, the cellulosic feedstock can include one or more of straw, corn stover, bagasse, hardwood, softwood, energy crops, and the like.

[0028] Raw agricultural produce supplied to the plant may be processed by crushing, grinding, milling, etc., or otherwise treated to remove rocks, soil and other material present in the raw agricultural produce and to reduce the size of the raw agricultural produce or forest-derived material supplied to the process.

[0029] 1, cellulosic feedstock 10 can be fed to reactor 14 where cellulosic feedstock 10 is subjected to a first activation step to produce a first activated cellulose stream 16. In the first activation step, cellulosic feedstock 10 can be treated at high temperature and pressure to produce a first activated cellulose stream 16 containing cellulose II and insoluble solids.

[0030] Reactor 14 can be a batch reactor or a continuous process reactor. If it is a batch reactor, cellulosic feedstock 10 is fed into reactor 14, which can be a stirred tank reactor, and the reactor can be brought up to operating conditions for a desired period of time. If reactor 14 is a continuous flow reactor, it can be a steam-exposed reactor as known in the art, and can be maintained at the desired operating conditions.

[0031] The first activation step may be carried out under conditions that increase the amount of cellulose II in the first activated cellulose stream compared to the amount of cellulose II in the feedstock.

[0032] The temperature may be greater than 190° C., optionally greater than 210° C., preferably greater than 220° C., and less than about 250° C. Thus, the process may be carried out at a temperature in the range of 190° C. to 250° C., 210° C. to 250° C., 220° C. to 240° C., or 222° C. to 230° C.

[0033] The pressure can be greater than 200 psig, and optionally less than 500 psig. The pressure in the reactor scales with the temperature, at a minimum, via saturated steam thermodynamics. In one embodiment, the pressure can be increased above that value by adding pressurized gas or by adding superheat.

[0034] The cellulosic feedstock 10 may be subjected to the first activation step for less than 30 minutes, less than 20 minutes, less than 10 minutes, or less than 5 minutes. The length of treatment time will vary depending on many factors, including the extent of the activation step, e.g., the temperature and pressure of the reactor 14.

[0035] It will be understood that temperature, pressure and treatment time may be combined in any desired combination. Thus, for example, the first activation step may involve exposing the feedstock to a pressure of 200-500 psig and a temperature of 200-250° C. for 1-30 minutes, or to a pressure of 200-500 psig and a temperature of 190-215° C. for less than 4 minutes.

[0036] Optionally, the first activation step is carried out in the presence of water. Water may be introduced into reactor 14 by one or more of what is present in cellulosic feedstock 10, what is present in reactor 14 as the cellulosic feedstock is introduced into reactor 14, and by being introduced by feed stream 12. The total amount of water introduced into the reactor may be at least 30% and can reach 90%. In certain embodiments, 50% water is in the reactor.

[0037] The water present in reactor 14 may be in the form of steam or liquid water, and is preferably in the form of liquid water. It will be appreciated that the temperature and pressure of the first activation step may be selected such that liquid water is present in reactor 14.

[0038] The first activated cellulose stream 16 may have a solids content of about 30% to 50% by weight. The solids contain primarily cellulose, which may then be subjected to a second activation step. The solids may further contain lignin, hemicellulose, and minor components such as ash, protein, or extractives.

[0039] Optionally, the cellulosic material may be subjected to one or more washing steps, either under the same or different conditions, after the first and / or second activation steps. To this end, the first activated cellulose stream 16 may be subjected to one or more washing steps prior to the second activation step to remove hemicellulose and some soluble non-cellulosic components such as ash, extractives and lignin. The first wash removes these solubles, and because the solubles are at an acidic pH, the washing steps also reduce the need for alkali in the second alkaline activation stage.

[0040] As illustrated in FIG. 1, the first activated cellulose stream 16 and wash water 20 can be introduced into a wash reactor 18 to produce a wastewater 22 and a washed first activated cellulose stream 24 .

[0041] The wash water 20 can be hot water, such as water at a temperature of about 40° C. to 100° C., or about 50° C. to 95° C. Wastewater stream 22 can be treated and reused within the process or elsewhere, or can be discarded.

[0042] The wash reactor 18 can be of any design known in the art. Optionally, the wash reactor 18 can be operated countercurrently and can be a countercurrent belt filter. Other filtration or separation methods can also be used, such as a filter press, twin wire press, twin roll press, rotary vacuum filter, or centrifugation.

[0043] As illustrated in Figure 1, the washed first activated cellulose stream 24 is fed to a reactor 26 where the washed first activated cellulose stream 24 is subjected to a second activation step to produce a second activated cellulose stream 30. In another embodiment, some or all of the first activated cellulose stream may be introduced into the reactor 26. The following description is based on Figure 1, which illustrates the use of a first wash step. In the second activation step, the washed first activated cellulose stream 24 may be treated with alkali at a lower temperature than the first activation step to produce a second activated cellulose stream containing cellulose IV.

[0044] Reactor 26 can be a batch reactor or a continuous process reactor. If a batch reactor, the washed first activated cellulose stream 24 is fed to reactor 26, which can be a stirred tank reactor, and the reactor can be brought up to operating conditions for a desired period of time. If reactor 26 is a continuous flow reactor, it can be a steam-exposed reactor as known in the art, and can be maintained at the desired operating conditions.

[0045] The second activation step may be carried out under conditions that increase the amount of cellulose IV in the second activated cellulose stream compared to the amount of cellulose IV in the washed first activated cellulose stream 24.

[0046] The temperature can optionally be greater than 60° C. and less than about 180° C., less than about 160° C., less than about 140° C., less than about 120° C., less than about 100° C., or less than about 80° C. Thus, the process can be carried out at a temperature in the range of 60° C. to 180° C., 60° C. to 160° C., 60° C. to 140° C., 60° C. to 120° C., 60° C. to 100° C., or 60° C. to 80° C.

[0047] Optionally, the second activation step is carried out at superatmospheric pressure, which can be, for example, a pressure of about 0.1 to 400 psig.

[0048] The washed first activated cellulose stream 24 may be subjected to a second activation step for less than 180 minutes, less than 120 minutes, less than 90 minutes, or less than 60 minutes, optionally more than 15 minutes, more than 30 minutes, or more than 45 minutes. The length of treatment time will vary depending on many factors, including the extent of the activation step, e.g., the temperature and pressure of the reactor 26.

[0049] It will be understood that temperature, pressure and treatment time can be combined in any desired combination. Thus, for example, the second activation step can include exposing the first activated cellulose stream to a temperature of 60-240° C. at a pressure of 0-500 psig for 15-120 minutes, or to a temperature of 80-150° C. at a pressure of 0-300 psig for at least 60 minutes.

[0050] 1, the second activation step preferably involves treating the first activated cellulose stream in the presence of an alkali. The alkali may be introduced into reactor 26 in any manner. For example, as illustrated, alkali stream 28 is introduced separately into reactor 26. It will be understood that alkali stream 28 may be introduced into reactor 26 prior to, simultaneously with, or after the introduction of washed first activated cellulose stream 24 into reactor 26. Alternatively, alkali stream 28 may be introduced into washed first activated cellulose stream 24 and then the combined streams may be introduced into reactor 26.

[0051] The alkali may include one or more of sodium hydroxide, potassium hydroxide, magnesium hydroxide, and ammonia. In one embodiment, the alkali is sodium hydroxide. In one embodiment, the alkali is dosed at about 10% to 1%, about 7% to 2%, or preferably less than 6% of the total insoluble solids in the first activated cellulose stream 24. The alkali swells the cellulose and also breaks the inter- and intra-molecular hydrogen bonds of the cellulose, thereby further altering the crystal structure. Optionally, a second activation step can be carried out in the presence of an oxidizing agent and / or an enzyme, such as, for example, a laccase and / or a lignin modifying enzyme.

[0052] Examples of oxidizing agents suitable for use in the second activation step include, but are not limited to, hydrogen peroxide (H2O2). In one embodiment, the oxidizing agent is dosed at a concentration of less than about 2% and / or greater than 0.0001% of the total insoluble solids in the first activated cellulose stream 16 / 24. In one embodiment, the oxidizing agent is dosed at less than about 1%, less than about 0.1%, or less than about 0.001%, optionally between about 1% and 0.0001%, of the total insoluble solids in the first activated cellulose stream 16 / 24. Examples of enzymes suitable for use in the second activation step include, but are not limited to, lignin modifying enzymes, such as laccase oxidase.

[0053] The second activated cellulose stream 30 may have a solids content of about 5% to 50% solids by weight, preferably about 20% to 35% solids. The solids comprise primarily cellulose, which may optionally be recovered and recycled. Other components are hemicellulose and lignin, both less than 20%.

[0054] Optionally, the second activated cellulose stream 30 may be subjected to one or more washing steps after activation to remove the alkali and solubilized lignin.

[0055] The second activated cellulose stream 30 and wash water 34 may be introduced into a wash reactor 32 to produce wastewater 36 and a washed second activated cellulose stream 38. The wash reactor 32 may be operated in a similar manner to the wash reactor 18 or in a different manner.

[0056] The wash water 34 can be hot water, such as water at a temperature of about 50° C. to 95° C. or about 60° C. to 95° C. Wastewater stream 36 may be treated and reused within the process or elsewhere, or it may be discarded.

[0057] The wash reactor 32 can be of any design known in the art. Optionally, the wash reactor 32 can be operated countercurrently and can be a countercurrent belt filter. Other filtration or separation methods can also be used, such as a filter press, twin wire press, twin roll press, rotary vacuum filter, or centrifugation.

[0058] The benefit of subjecting a cellulosic feedstock to a first activation step at high temperature followed by a second activation step under alkaline conditions at a lower temperature has been shown to increase the level of glycans and decrease the level of lignin in the second activated cellulose stream 30 compared to a feedstock that has only undergone a first high temperature activation step. Without being limited by theory, it is believed that the two-step activation process described herein alters the crystallinity of the cellulose in the feedstock and improves the physical and / or chemical properties of the cellulose for enzymatic hydrolysis. In one embodiment, the two-step activation process produces activated cellulose, including cellulose II and alkaline cellulose IV.

[0059] Those skilled in the art will appreciate that cellulose exists in a number of different crystal structures, corresponding to the location of inter- and intra-chain hydrogen bonds. For example, naturally occurring cellulose found in cellulosic biomass has Structure I α and I βThe cellulose in regenerated cellulose fibers is usually cellulose II. Regenerated cellulose fibers refer to fibers produced by the viscose process for the production of viscose from cellophane or rayon. The conversion of cellulose I to cellulose II is irreversible. Cellulose III and cellulose IV structures can be produced by various chemical treatments. Different crystalline forms of cellulose can be identified by their unique X-ray diffraction patterns. Cellulose and different crystalline structures of cellulose are further described in Perez and Samain, “Structure and Engineering of Cellulose” Advances in Carbohydrate Chemistry and Biochemistry, Vol. 64, Elsevier (2010), the entire contents of which are incorporated herein by reference.

[0060] The washed second activated cellulose stream 38 may be subjected to enzymatic hydrolysis with one or more cellulase enzymes 42 in an enzymatic hydrolysis reactor 40 to produce a glucose-rich sugars stream 44. It will be understood that some or all of the second activated cellulose stream 30 may be subjected to enzymatic hydrolysis and thus only some or none of the second activated cellulose stream 30 may be subjected to washing. The following description may apply to the second activated cellulose stream 30 whether or not it has been subjected to a washing step.

[0061] Surprisingly, it has been determined that activated cellulose containing cellulose II (which may be a combination of cellulose II and hydrated cellulose II) and cellulose IV (which may be alkali cellulose IV), which may be produced by the two-step activation process disclosed herein, is particularly susceptible to enzymatic hydrolysis. In particular, the activated cellulose has demonstrated a surprising ability to adsorb cellulase enzymes. Contacting the activated cellulose with one or more cellulase enzymes in a glucose-rich sugar stream may initially result in the enzymes being adsorbed to the activated cellulose. The cellulose may then be removed from the glucose-rich sugar stream and optionally introduced into the enzymatic hydrolysis reactor 40.

[0062] Thus, the activated cellulose produced by any of the methods disclosed herein can be subjected to enzymatic hydrolysis to break down the cellulose into cellulosic sugars such as glucose. Alternatively, the enzymatic hydrolysis processes disclosed herein can be used with any conventional enzymatically hydrolyzed cellulosic feedstock.

[0063] Thus, the activated cellulose, optionally a second activated cellulose stream as described herein, may be contacted with one or more cellulase enzymes to produce a glucose-rich sugar stream. As illustrated in FIG. 1, the washed second activated cellulose stream 30 and an enzyme stream 42 are introduced into an enzymatic hydrolysis reactor 40 to produce a glucose-rich sugar stream 44. The washed second activated cellulose stream 30 may be introduced into the enzymatic hydrolysis reactor 40 prior to, simultaneously with, or after the introduction of the enzyme stream 42 into the reactor 40. Alternatively, or in addition, the enzyme stream 42 may be introduced into the washed second activated cellulose stream 30 and the combined streams introduced into the enzymatic hydrolysis reactor 40.

[0064] The enzymatic hydrolysis reactor 40 may be any enzymatic hydrolysis reactor known in the art and may be operated batchwise or continuously. The enzymatic hydrolysis reactor 40 may be operated at any conventional temperature and pressure, cellulose dosage, enzyme dosage, etc. For example, the enzymatic hydrolysis reactor 40 may be operated at a temperature range of 40° C. to 55° C.

[0065] The cellulase enzyme may be selected to degrade cellulose into monosaccharides. For example, the cellulase enzyme may be selected to hydrolyze 1,4-β-D-glycosidic bonds into monosaccharides. The one or more cellulase enzymes may include enzymes having at least one of cellobiohydrolase, endoglucanase and β-glucosidase activity. Cellulase enzyme preparations may be isolated from a number of sources, such as natural cultures of bacteria, yeast or fungi, although those skilled in the art will recognize the use of enzymes produced using recombinant technology. Examples of commercially available enzymes suitable for use in the methods described herein include, but are not limited to, Ctec 2 or 3 from Novozymes, Rohament from AB Enzymes.

[0066] The one or more cellulase enzymes may be added at a dosage of 0.1-120 mg, 0.2-60 mg, or 1-30 mg enzyme protein per gram of glucan. In one embodiment, the cellulase enzyme is added at a dosage of 0.1-5 mg enzyme protein per gram of glucan in the activated cellulose. In one embodiment, the one or more cellulase enzymes are added at a dosage of about 2 to about 60 Filter Paper Units (FPU) / g glucan, or optionally about 2-30 or 1-15 FPU / g glucan to the activated cellulose. The one or more cellulase enzymes may be added independently directly to the activated cellulose or may be first combined with a surfactant and / or dispersant as described below.

[0067] To convert the cellulose to monosaccharides by enzymatic hydrolysis, one or more cellulase enzymes can be contacted with the activated cellulose for a suitable length of time (e.g., 24-144 hours, 48-144 hours, 48-60 hours, or 24-72 hours).

[0068] In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% of the theoretical yield of glucose based on the glycan content of the activated cellulose is converted to glucose during the enzymatic hydrolysis to produce a glucose-rich sugar stream. In some embodiments, the enzymatic hydrolysis is carried out for a predetermined time or until a predetermined glucose yield is obtained. After a certain time, the rate of glucose production from the enzymatic hydrolysis of cellulose may decrease because the cellulose substrate is depleted or because the presence of glucose inhibits the activity of cellulase enzymes.

[0069] Optionally, the activated cellulose can be contacted with one or more cellulase enzymes in the presence of a surfactant and / or a dispersing agent. In a preferred embodiment, the dispersing agent is polyaspartic acid.

[0070] Surprisingly, it has been determined that subjecting activated cellulose, particularly the activated cellulose disclosed herein, to enzymatic hydrolysis in the presence of a surfactant and / or dispersing agent such as polyaspartic acid exhibits numerous advantages in the production of monosaccharides. For example, the presence of a surfactant and / or dispersing agent may improve the stability of the cellulase enzyme, help protect the cellulase enzyme from degradation, prevent irreversible binding, and / or improve the activity of the cellulase enzyme. The presence of a surfactant and / or dispersing agent is also believed to improve the recovery of cellulase enzyme after enzymatic hydrolysis to the enzyme recycle stream. For example, in some embodiments, the cellulase enzyme may be used in the enzymatic hydrolysis of the activated cellulose and removed from the resulting glucose-rich sugar stream and recycled to the enzymatic hydrolysis reactor 40 or contacted with fresh activated cellulose for further enzymatic hydrolysis. In some embodiments, the cellulase enzyme may be used and reused for at least 3-4 enzymatic hydrolysis cycles, with each cycle lasting 48-72 hours.

[0071] The surfactant may be a non-ionic surfactant, optionally a polysorbate surfactant such as Tween. The surfactant may also be a mixture of surfactants. In a preferred embodiment, the surfactant is a mixture of Tween 80, alkoxylated glycerides and nonylphenol. In one embodiment, the surfactant is present at a dosage of less than about 2% and / or greater than about 0.01%. In one embodiment, the surfactant is present at a dosage of 1% to 0.01%, 0.5% to 0.05%, or about 0.1% to 0.2% of the weight of the cellulose content in the activated cellulose.

[0072] The dispersing agent can be an oligopeptide, optionally a non-enzymatic polypeptide, having a molecular weight of 500-10,000 or 1000-5000. The oligopeptide can be polyaspartic acid. The polyaspartic acid can have a molecular weight of 500-10,000, 1000-5000, or 3500-4500. The polyaspartic acid can be present at a dosage of less than about 2% and / or greater than about 0.001% by weight of the cellulose content in the activated cellulose. In some embodiments, the polyaspartic acid can be present at a dosage of 1%-0.001%, 0.25%-0.025%, or about 0.1% by weight of the cellulose content in the activated cellulose.

[0073] Optionally, the ratio of surfactant to dispersing agent (eg, polyaspartic acid) in the enzymatic hydrolysis mixture is from 0.1:1 to 10:1, optionally from 0.5:1 to 2:1. Optionally, the molar ratio of dispersing agent (eg, polyaspartic acid) to the one or more cellulase enzymes is 0.01-10:1.

[0074] Thus, the enzymatic hydrolysis mixture containing one or more cellulase enzymes, one or more surfactants and one or more dispersing agents may be used in any enzymatic hydrolysis step or in conjunction with any activation and enzymatic hydrolysis step disclosed herein. The enzymatic hydrolysis mixture is particularly suitable for the enzymatic hydrolysis of activated cellulose, including cellulose II and cellulose IV, as described herein.

[0075] One or more cellulase enzymes, surfactants, and dispersants may be introduced into the enzymatic hydrolysis reactor 40 alone or in combination or subcombinations. For example, they may each be combined individually with the activated cellulose prior to introducing stream 38 into reactor 40 (e.g., each may be added sequentially to stream 38, or stream 38 may be split into three streams and the cellulase enzymes, surfactants, and dispersants may each be added to one of the split streams). Alternatively, one or more cellulase enzymes, surfactants, and dispersants may be mixed together to form stream 42 prior to combining the mixture with the activated cellulose (e.g., stream 42 may be introduced into reactor 40, or stream 42 may be introduced into stream 38 prior to introducing stream 38 into reactor 40). It is believed that mixing the enzymes, surfactants, and dispersants together prior to contacting the activated cellulose promotes the formation of a ternary complex that helps stabilize the enzymes and prevents enzyme degradation. Thus, one or more cellulase enzymes may be mixed with the surfactants and dispersants prior to subjecting the activated cellulose to enzymatic hydrolysis. For example, the one or more cellulase enzymes can be mixed with the surfactants and dispersants for at least 5 seconds, at least 10 seconds, at least 30 seconds, or at least 1 minute before contacting them with the activated cellulose (e.g., stream 38) or before subjecting the activated cellulose to enzymatic hydrolysis.

[0076] 1, the glucose-rich sugar stream 44 can be subjected to an enzyme removal step to obtain a glucose-rich, enzyme-poor sugar stream 48 and an enzyme recycle stream 50. The enzyme removal step can be any enzyme removal step known in the art and can be carried out in any equipment known in the art. Optionally, the enzyme removal step includes contacting the glucose-rich sugar stream 44, for example for a limited time, with cellulose, which can be activated cellulose produced by any of the methods disclosed herein.

[0077] For example, the enzyme removal step may include the following steps: (a) contacting a glucose-rich sugar stream containing an enzyme with cellulose to obtain cellulose having the enzyme adsorbed thereon; and (b) subjecting the glucose-rich sugar stream to a cellulose removal step to obtain a glucose-rich, enzyme-poor sugar stream having reduced cellulose levels and an enzyme recycle stream.

[0078] Optionally, step (a) includes contacting the glucose-rich sugar stream with activated cellulose, and optionally a second activated cellulose stream produced according to the methods described herein.

[0079] Without being limited by theory, it is believed that the enzymes in the glucose-rich sugar stream adsorb onto the cellulose and that removing the cellulose from the glucose-rich sugar stream removes the enzymes from the stream, resulting in a glucose-rich, enzyme-poor sugar stream 48 and an enzyme recycle stream 50. In a particularly preferred embodiment, the cellulase enzymes in the glucose-rich sugar stream are in the presence of surfactants and dispersants and the enzymes in the glucose-rich sugar stream 44 are removed by contacting the glucose-rich sugar stream 44 with activated cellulose 16, 24, 30, 38 produced using the methods described herein.

[0080] Thus, the cellulose stream 52 may be introduced into the reactor 46. The reactor 46 may include any reactor that allows the glucose-rich sugar stream 44 and the cellulose to contact one another so that the enzymes can be recovered from the solution and the cellulose along with the enzymes adsorbed thereon can be separated. Thus, for example, the reactor 46 may include a stirred tank reactor or a plug flow reactor for mixing the glucose-rich sugar stream and the cellulose to produce the mixed stream 54.

[0081] The glucose-rich sugar stream and the cellulose can be contacted together for less than about 2 hours, less than about 90 minutes, or less than about 60 minutes, and can be contacted together for about 10 minutes to 60 minutes, or about 30 minutes to 90 minutes.

[0082] The mixed stream 54 is then subjected to a solid-liquid separation step in separator 56. Separator 56 can be any separator known in the art. Separator 56 can use any separation technique known in the art, such as filtration, decantation, gravity separation, centrifugation, or the use of an expeller. For example, separator 56 can include a filter, an expeller, and optionally a twin screw press, twin wire press, or twin roll press.

[0083] The enzyme recycle stream 50 can be a high solids stream. For example, the enzyme recycle stream can contain greater than about 30%, greater than about 40%, or greater than about 50% cellulosic solids.

[0084] The enzyme recycle stream 50 may be used to perform enzymatic hydrolysis on the freshly activated cellulose. Alternatively, the enzyme recycle stream 50 may be recycled to the reactor 40. Thus, when the reactor 40 is operated in batch mode, a purge stream of the glucose-rich sugar stream may be recovered and treated to obtain the recycle stream 50. Surprisingly, it has been determined that the enzymes recycled by this process typically maintain their activity even after being recycled one, two, three or four times. As a result, the glucose-rich sugar stream 48 may contain more than about 12% glucose, more than about 14% glucose, more than about 16% glucose or more than about 18% glucose. Furthermore, a glucose yield of more than about 70%, more than about 80%, more than about 85%, more than about 90% or more than about 95% of the theoretical yield of glucose may be obtained.

[0085] Optionally, the glucose-rich sugar stream 48 may contain detectable levels of polyaspartic acid, hi one embodiment, the sugar stream contains between about 1 ppb and 10,000 ppm of polyaspartic acid.

[0086] Surprisingly, it has been found that the glucose-rich sugar stream resulting from the enzymatic hydrolysis of activated cellulose disclosed herein contains about 5% non-glucose sugars that are one or more of xylose, oligomers of xylose (xylooligosaccharides) and xylan. As described herein, xylooligosaccharides refer to polymers of xylose having a degree of polymerization (dp) of about 2 to about 10. As described herein, xylan refers to polymers of xylose having a degree of polymerization (dp) of >10.

[0087] In certain embodiments, the enzymatic hydrolysis of activated cellulose can be carried out using a hydrolysis mixture as disclosed herein. In further embodiments, a glucose-rich sugar stream is prepared using the cellulose activation and / or enzymatic hydrolysis methods described herein.

[0088] Glucose obtained using standard methods from sources such as corn is also known to have about 5% non-glucose sugars, however the non-glucose sugars found in corn glucose are sugars with higher glycemic indexes such as maltose, maltotriose and the higher molecular weight dextrose sugars.

[0089] In one embodiment, the non-glucose sugars found in the glucose-rich sugar streams disclosed herein comprise about 1-8%, about 2-7%, or about 3-6% of the dry matter of the composition and are one or more of xylose, xylooligosaccharides, and xylan. In a particular embodiment, the dry matter of the glucose-rich sugar stream was found to contain 95% glucose, 4% xylose, and 1% xylooligosaccharides.

[0090] The glucose obtained from the methods disclosed herein can be converted to fructose using known methods, such as glucose isomerization to fructose as described, for example, by SZ Dziedzic et al. in "Handbook of starch hydrolysis products and their derivatives" December 31, 1995, pages 55-58, which is incorporated herein by reference. The fructose obtained by conversion of the glucose obtained from the methods disclosed herein also contains about 3-5% non-glucose sugars, which were found to be xylose and / or oligomers of xylose.

[0091] In a further aspect of the invention, there is provided a fructose-rich sugar stream prepared by conversion of glucose in the glucose-rich sugar stream disclosed herein to fructose. The fructose-rich sugar stream also contains non-fructose sugars, which are one or more of xylose, xylooligosaccharides, and xylan. In one embodiment, the non-fructose sugars comprise about 1-8%, about 2-7%, or about 3-6% of the dry matter of the composition.

[0092] In another aspect of the invention, there is provided a lower glycemic index glucose product or a lower glycemic index fructose product, the glucose product or fructose product containing about 1-8%, about 2-7% or about 3-6%, preferably 5%, non-glucose or non-fructose sugars, the non-glucose or non-fructose sugars being one or more of xylose, xylooligosaccharides and xylan.

[0093] In a further embodiment, the glycemic index of the glucose or fructose product is lower than the glycemic index of a conventional glucose or fructose syrup produced by a conventional method. Glycemic index (GI) can be measured using methods known in the art, for example, as described in "In vitro method for predicting glycemic index of foods using simulated digestion and an artificial neural network" RL Magaletta et al., Cereal Chemistry vol. 87, no. 4, 2010.

[0094] The glucose or fructose products obtained by the method described herein can be used in the manufacture of various foods and beverages to replace glucose or fructose with higher glycemic index to provide products with lower glycemic index.Foods and beverages with lower glycemic index can provide health benefits in the management of blood sugar and insulin levels, thus reducing the risk of heart disease and / or diabetes.Foods with lower glycemic index can also be useful in controlling appetite and weight loss.

[0095] It will be understood that one or more of the embodiments described herein for the activation of cellulosic feedstock may be used in conjunction with one or more of the embodiments described herein for the enzymatic hydrolysis of cellulose to produce cellulosic sugars from the cellulosic feedstock.

[0096] What has been described above is intended to be illustrative and non-limiting of the present invention, and those skilled in the art will appreciate that other changes and modifications may be made without departing from the scope of the present invention as defined in the claims appended hereto. The claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

[0097] Although the above disclosure generally describes the present application, a more complete understanding can be obtained by referring to the following specific examples. These examples are set forth merely for illustrative purposes and are not intended to limit the scope of the disclosure. Modifications in form and substitution of equivalents are contemplated where the circumstances suggest or are appropriate. Although specific terms are used herein, such terms are intended to be descriptive and not limiting. The following non-limiting examples are illustrative of the present disclosure. EXAMPLES

[0098] [Example 1] Treatment of sugarcane bagasse to activate cellulose. Various methods for activating cellulose were investigated using sugarcane bagasse, which was subjected to either a first steaming step at 220 °C for 5 min residence time followed by a hot water wash with water at 80 °C, or a first steaming step followed by treatment with alkaline hydrogen peroxide at 90 °C for 60 or 120 min and an alkaline wash with 1% peroxide dosed as solids.

[0099] As shown in Table 1, the use of a two-step treatment with a steam treatment step followed by alkaline hydrogen peroxide significantly increased the level of glucan and decreased the amount of lignin compared to treatment with steam and hot water washing.

[0100] [Table 1]

[0101] [Example 2] Enzymatic hydrolysis of activated sugarcane bagasse. The water-insoluble cellulose component prepared in Example 1 was then subjected to enzymatic hydrolysis for 72 hours. Comet additive S-001, containing a 1:1 ratio mixture of surfactant Tween 80 and dispersant polyaspartic acid having a molecular weight of 3500-4500, was also added to the enzymatic hydrolysis mixture for the alkali-treated sugarcane bagasse.

[0102] As shown in Table 2, enzymatic hydrolysis of alkali-treated sugarcane bagasse in the presence of Comet additive S-001 resulted in a glucose yield of 105.1 grams of glucose per gram of glucan, close to the theoretical yield of ~110 grams of glucose per gram of glucan.

[0103] [Table 2]

[0104] Furthermore, as shown in Table 3, enzymatic hydrolysis in the presence of Comet additive S-001 did not alter the yield of monomeric xylose compared to alkali-treated sugarcane bagasse subjected to enzymatic hydrolysis without the additive. Thus, the additive did not adversely affect the yield.

[0105] [Table 3]

[0106] Analysis of the percentage of total protein content present in the supernatant after 72 hours of enzymatic hydrolysis is shown in Table 4. The use of Comet additive S-001 resulted in a higher percentage of total protein, indicating higher levels of enzyme in the supernatant and improved enzyme stability.

[0107] [Table 4]

[0108] Alkali-treated sugarcane bagasse was subjected to multiple recycle hydrolysis in the presence of Comet additive S-001. As shown in Table 5, recycle hydrolysis was able to produce high yields and high concentrations of glucose over repeated periods of enzymatic hydrolysis with minimal enzyme loss.

[0109] [Table 5]

[0110] [Example 3] Treatment of wheat straw to activate cellulose. The method for activating cellulosic raw materials was investigated using wheat straw, which was subjected to a first steam treatment step at 220°C with a residence time of 5 minutes, followed by a hot water wash at 80°C, followed by an alkaline wash at 90°C for 60 minutes and 1% peroxide solid dosage. As shown in Table 6, the use of a two-step alkaline hydrogen peroxide treatment resulted in water-insoluble components, including high levels of glucan (75.1%).

[0111] [Table 6]

[0112] [Example 4] Enzymatic hydrolysis of activated wheat straw. The water-insoluble cellulose components of steam-treated wheat straw or steam-treated followed by alkali-treatment of wheat straw were subjected to enzymatic hydrolysis as shown in Tables 7 to 9. The surfactant Tween 80 was also added as indicated in the tables.

[0113] [Table 7]

[0114] [Table 8]

[0115] [Table 9]

[0116] Further studies of the enzymatic hydrolysis of steam-treated followed by alkaline peroxide-treated wheat straw with the addition of a surfactant (Tween 80) were carried out as shown in Tables 10-12.

[0117] [Table 10]

[0118] [Table 11]

[0119] [Table 12]

[0120] [Example 5] The cellulosic glucose products produced by the activation and enzymatic conversion methods described herein were prepared and measured to have the following specifications:

[0121] Chemical and Physical Data Total solids 50-70% Moisture content: 30-50%

[0122] Composition (based on dry matter): Glucose 95% Xylose 4% Xylooligosaccharides 1% Ash content <0.01%

[0123] pH: 3~5 Conductivity: (30% DS)50 μs / cm Specific gravity: 1.2 Appearance: Clear solution Smell: Sweet

[0124] Mineral Ash Content (PPM) Chloride 16 Sulfate <1 Calcium 5 Potassium <1 Magnesium <1 Sodium 2 Rin 2

[0125] Glycemic Index Data A cellulosic glucose product having the above composition was found to have a glycemic index (GI) of 72. In comparison, glucose alone is known to have a glycemic index of 100. Dextrose is also known to have a glycemic index of 100, while maltose and maltodextrin are known to have glycemic indexes of 105 and 110, respectively.

[0126] While the present disclosure has been described with respect to what are presently considered to be embodiments, it is to be understood that the present disclosure is not limited to the disclosed embodiments. On the contrary, the present disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0127] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. 1. A two-step method for activating a cellulosic feedstock, comprising: (a) subjecting the feedstock to a first activation step at a temperature greater than 190° C. and a pressure greater than 200 psig to produce a first activated cellulose stream containing cellulose II and insoluble solids; (b) subjecting the first activated cellulose stream to a second activation step of treatment with alkali at a lower temperature than the first activation step to produce a second activated cellulose stream containing cellulose IV. The above method.

2. 2. The method of claim 1, wherein the first activation step is carried out in the presence of water.

3. 3. The process of claim 1 or 2, wherein the first activation step comprises exposing the feedstock to a pressure of 200 to 500 psig and a temperature of 200 to 250° C. for 1 to 30 minutes.

4. 3. The process of claim 1 or 2, wherein the first activation step comprises exposing the feedstock to a pressure of 200-500 psig and a temperature of 190-215°C for less than 4 minutes.

5. 5. The method of any one of claims 1 to 4, further comprising washing the first activated cellulose stream to remove soluble non-cellulosic components prior to the second activation step.

6. 6. The method of claim 5, wherein the step of washing the first activated cellulose stream comprises using water at about 50-95°C, optionally using a countercurrent wash.

7. The method of any one of claims 1 to 6, wherein the first activated cellulose stream has a solids content of about 30 to 50% (wt / wt).

8. The method of any one of claims 1 to 7, wherein the second activation step comprises exposing the first activated cellulose stream to a temperature of from 60 to 240°C for a period of from 15 to 120 minutes.

9. 9. The method of claim 8, wherein the second activation step comprises exposing the first activated cellulose stream to a temperature of from 80 to 150° C. for at least 60 minutes.

10. 10. The method of any one of claims 1 to 9, wherein the second activation step is carried out at superatmospheric pressure, optionally from about 0.1 to 400 psig.

11. 11. The method of any one of claims 1 to 10, wherein the alkali in the second activation step comprises one or more of sodium hydroxide, potassium hydroxide, magnesium hydroxide and ammonia.

12. 12. The method of any one of claims 1 to 11, wherein the alkali in the second activation step comprises sodium hydroxide, and the sodium hydroxide is dosed at about 10% to 1%, or preferably less than about 6%, of the insoluble solids in the first activated cellulose stream.

13. The method according to any one of claims 1 to 12, wherein the second activation step is carried out in the presence of an oxidizing agent and / or an enzyme, optionally a laccase or a lignin modifying enzyme.

14. 14. The method of claim 13, wherein the oxidizing agent is hydrogen peroxide, optionally dosed at less than 1% of the insoluble solids in the first activated cellulose stream.

15. The method of any one of claims 1 to 14, wherein the second activated cellulose stream contains a mixture of cellulose II, hydrated cellulose II and alkali cellulose IV.

16. The method of any one of claims 1 to 15, further comprising washing the second activated cellulose stream to remove soluble non-cellulosic components.

17. 17. The method of claim 16, wherein the step of washing the second activated cellulose stream comprises using water at about 60-95°C, optionally using a countercurrent wash.

18. 18. A method for producing a glucose-rich sugar stream, comprising contacting the second activated cellulose stream obtained from the method of any one of claims 1 to 17 with one or more cellulase enzymes to produce a glucose-rich sugar stream.

19. 20. The method of claim 18, wherein the one or more cellulase enzymes are selected to hydrolyze 1,4-β-D-glycosidic linkages into monosaccharides.

20. The method of claim 18 or 19, wherein the one or more cellulase enzymes include enzymes having at least one of cellobiohydrolase, endoglucanase and β-glucosidase activity, optionally commercially available as Ctec 2 or 3 from Novozymes, Rohament from AB Enzymes.

21. The method of any one of claims 18 to 20, further comprising contacting the second activated cellulose stream with a surfactant and / or dispersant.

22. 22. The method of claim 21, wherein the dispersing agent is an oligopeptide, optionally a non-enzymatic polypeptide having a molecular weight of 500 to 10,000 or 1000 to 5000.

23. 23. The method of claim 22, wherein the polypeptide is polyaspartic acid.

24. 24. The method of claim 22 or 23, wherein the polypeptide is added to the second activated cellulose stream at a dosage of less than 2%, between 1% and 0.001%, between 0.25 and 0.025%, or about 0.1% by weight of the cellulose content of the second activated stream.

25. A method according to any one of claims 21 to 24, wherein the surfactant is a non-ionic surfactant, optionally a polysorbate surfactant such as Tween™.

26. The method of any one of claims 21 to 25, wherein the surfactant is a mixture of surfactants comprising Tween, alkoxylated glyceride and nonylphenol.

27. 27. The method of claim 25 or 26, wherein the surfactant is added to the second activated cellulose stream at a dosage of less than 2%, between 1% and 0.01%, between 0.5% and 0.05%, or between about 0.1% and 0.2% by weight of the cellulose content of the second activated stream.

28. The method of any one of claims 21 to 27, wherein the one or more cellulase enzymes, surfactants and dispersants are added separately to the second activated cellulose stream.

29. 28. The method of any one of claims 21 to 27, wherein the one or more cellulase enzymes are mixed with the surfactant and dispersant, optionally for at least 10 seconds, prior to contacting the second activated cellulose stream.

30. 30. The method of claim 29, wherein the one or more cellulase enzymes form a ternary complex with the surfactant and the dispersing agent.

31. 31. The method of any one of claims 18 to 30, wherein one or more cellulase enzymes are added to the second activated cellulose stream at a dosage of 0.1 to 120, optionally 0.1 to 5 mg of enzyme protein per g of glucan.

32. 32. The method of claim 31 , wherein the one or more cellulase enzymes are added to the second activated cellulose stream at a dosage of about 2 to 60 FPU / g glucan.

33. 33. The method of any one of claims 18 to 32, wherein the second activated cellulose stream is contacted with one or more cellulase enzymes for a period of from 24 to 144 hours, from 48 to 144 hours, from 48 to 60 hours, from 24 to 96 hours, less than 60 hours, or less than 48 hours to produce a glucose-rich sugar stream.

34. 34. The method of any one of claims 18 to 33, further comprising subjecting the glucose-rich sugars stream to an enzyme removal treatment to obtain a glucose-rich, enzyme-poor sugars stream and an enzyme recycle stream.

35. The enzyme removal process (a) contacting a glucose-rich sugar stream containing an enzyme with cellulose to obtain cellulose having the enzyme adsorbed thereon; and (b) subjecting the glucose-rich sugar stream to a cellulose removal step to obtain a glucose-rich, enzyme-poor sugar stream having reduced cellulose levels and an enzyme recycle stream.

35. The method of claim 34, comprising:

36. 36. The method of claim 35, wherein the glucose-rich sugar stream is contacted with the activated cellulose, and optionally a second activated cellulose stream produced according to the method of any one of claims 1 to 17, optionally for less than about 60 minutes.

37. 37. The method of claim 35 or 36, wherein the cellulose removal step comprises filtration, and optionally the enzyme recycle stream contains greater than about 40 wt % cellulose solids.

38. 38. The method of any one of claims 34 to 37, further comprising combining the enzyme recycle stream with fresh activated cellulose.

39. 39. The method of any one of claims 35 to 38, further comprising repeating the cellulose removal step of claim 35.

40. 40. The method of any one of claims 18-39, wherein the glucose-rich sugar stream contains greater than about 12% glucose, greater than about 14% glucose, greater than about 16% glucose, or greater than about 18% glucose.

41. 41. The method of any one of claims 18-40, wherein the method results in a yield of glucose that is greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% of the theoretical yield of glucose.

42. A method for producing a glucose-rich sugar stream from activated cellulose, the method comprising the step of subjecting the activated cellulose to enzymatic hydrolysis using one or more cellulase enzymes, a surfactant and polyaspartic acid to produce a glucose-rich sugar stream.

43. 43. The method of claim 42, wherein the one or more cellulase enzymes are selected to hydrolyze 1,4-β-D-glycosidic linkages into monosaccharides.

44. 44. The method of claim 42 or 43, wherein the one or more cellulase enzymes include enzymes having at least one of cellobiohydrolase, endoglucanase and β-glucosidase activity.

45. The method of any one of claims 42 to 44, wherein the polyaspartic acid has a molecular weight of 500 to 10,000.

46. The method according to any one of claims 42 to 44, wherein the polyaspartic acid has a molecular weight of 1000 to 5000.

47. 45. The method of any one of claims 42 to 44, wherein the polyaspartic acid has a molecular weight of 3500 to 4500.

48. 48. The method of any one of claims 42-47, wherein the polyaspartic acid is present at a dosage of less than 2%, between 1% and 0.001%, between 0.25 and 0.025%, or about 0.1% by weight of the cellulose content of the second activated stream.

49. 49. The method of any one of claims 44 to 48, wherein the surfactant is a non-ionic surfactant, optionally a polysorbate surfactant such as Tween™.

50. 50. The method of any one of claims 44 to 49, wherein the surfactant is a mixture of surfactants comprising Tween, alkoxylated glyceride and nonylphenol.

51. 51. The method of claim 49 or 50, wherein the surfactant is present at a dosage of less than 2%, between 1% and 0.01%, between 0.5% and 0.05%, or between about 0.1% and 0.2% by weight of the cellulose content of the second activated stream.

52. The method of any one of claims 42 to 51, wherein the one or more cellulase enzymes, surfactants and polypeptides are mixed separately with the activated cellulose.

53. 52. The method of any one of claims 42 to 51, wherein the one or more cellulase enzymes are mixed with the surfactant and the polypeptide, optionally for at least 10 seconds, prior to subjecting the activated cellulose to enzymatic hydrolysis.

54. 54. The method of claim 53, wherein the one or more cellulase enzymes form a ternary complex with the surfactant and the polyaspartic acid.

55. 55. The method of any one of claims 42 to 54, wherein the one or more cellulase enzymes are present at a loading of 0.1 to 5 mg of enzyme protein per g of glucan.

56. 56. The method of claim 55, wherein the one or more cellulase enzymes are present at 2 to 60 FPU / g glucan.

57. 57. The method of any one of claims 42 to 56, further comprising subjecting the activated cellulose to enzymatic hydrolysis for a period of from 24 to 144 hours, from 48 to 144 hours, from 48 to 60 hours, from 24 to 96 hours, less than 60 hours or less than 48 hours.

58. 58. The method of any one of claims 41 to 57, further comprising subjecting the glucose-rich sugars stream to an enzyme removal treatment to obtain a glucose-rich, enzyme-poor sugars stream and an enzyme recycle stream, and recycling the enzyme recycle stream for enzymatic hydrolysis.

59. 59. The method of claim 58, wherein after introducing an enzyme recycle stream for use in enzymatic hydrolysis, the method results in a glucose yield of greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% of the theoretical yield.

60. (a) contacting a glucose-rich sugar stream with cellulose to obtain cellulose having an enzyme adsorbed thereon; and (b) subjecting the glucose-rich sugar stream to a cellulose removal step to obtain a glucose-rich, enzyme-poor sugar stream having reduced cellulose levels and an enzyme recycle stream.

60. The method of claim 58 or 59, comprising:

61. 61. The method of claim 60, wherein the glucose-rich sugar stream is contacted with activated cellulose, and optionally a second activated cellulose stream produced according to the method of any one of claims 1 to 17.

62. 62. The method of claim 60 or 61, wherein the cellulose removal step comprises filtration.

63. 63. The method of any one of claims 58 to 62, further comprising the step of combining the enzyme recycle stream with fresh activated cellulose.

64. 64. The method of any one of claims 58 to 63, further comprising repeating the cellulose removal step of claim 58.

65. 65. The method of any one of claims 42-64, wherein the glucose-rich sugar stream contains greater than about 12% glucose, greater than about 14% glucose, greater than about 16% glucose, or greater than about 18% glucose.

66. 66. The method of any one of claims 42-65, wherein the yield of glucose is greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90% or greater than about 95%.

67. 1. A method for producing a glucose rich sugar stream, comprising: (a) providing an activated cellulose containing a mixture of cellulose II, hydrated cellulose II and alkali cellulose IV; (b) subjecting the activated cellulose to enzymatic hydrolysis using one or more cellulase enzymes, surfactants and dispersing agents to produce a glucose-rich sugar stream. The above method.

68. 68. The method of claim 67, wherein the activated cellulose is produced by the method of any one of claims 1 to 17.

69. 69. The method of claim 67 or 68, wherein the dispersing agent is a non-enzymatic polypeptide having a molecular weight of less than 10,000.

70. 70. The method of any one of claims 67 to 69, wherein the dispersing agent is present at a loading of less than 2%, between 1% and 0.001%, between 0.25% and 0.025%, or about 0.1% by weight of the cellulose content of the activated cellulose.

71. 71. The method of claim 70, wherein the dispersing agent is polyaspartic acid.

72. 72. The method of claim 71, wherein the polyaspartic acid has a molecular weight of 1000 to 5000, optionally 3500 to 4500.

73. 73. The method of any one of claims 67 to 72, wherein the surfactant is present at a loading of less than 2%, between 1% and 0.01%, between 0.5% and 0.05%, or between about 0.1% and 0.2% by weight of the cellulose content of the activated cellulose.

74. 74. The method of claim 73, wherein the surfactant is a non-ionic surfactant, optionally a polysorbate surfactant such as Tween™.

75. 75. The method of claim 74, wherein the surfactant is a mixture of surfactants including Tween, alkoxylated glycerides and nonylphenol.

76. The method of any one of claims 67 to 75, wherein the one or more cellulase enzymes, surfactants and dispersing agents are added separately to the activated cellulose.

77. 76. The method of any one of claims 67 to 75, wherein the one or more cellulase enzymes are mixed with the surfactants and dispersants, optionally for at least 10 seconds, prior to subjecting the activated cellulose to enzymatic hydrolysis.

78. 78. The method of any one of claims 67-77, further comprising subjecting the glucose-rich sugars stream to an enzyme removal treatment to obtain a glucose-rich, enzyme-poor sugars stream and an enzyme recycle stream.

79. 79. The method of any one of claims 67-78, wherein the glucose-rich sugar stream contains greater than about 12% glucose, greater than about 14% glucose, greater than about 16% glucose, or greater than about 18% glucose.

80. 76. The method of any one of claims 67 to 75, wherein the yield of glucose is greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90% or greater than about 95%.

81. (a) one or more cellulase enzymes; (b) a surfactant or mixture of surfactants; and (c) Polyaspartic acid having a molecular weight of 500 to 10,000 1. An enzymatic hydrolysis mixture comprising:

82. 82. The enzymatic hydrolysis mixture of claim 81, wherein the one or more cellulase enzymes hydrolyze 1,4-β-D-glycosidic bonds into monosaccharides.

83. 83. The enzymatic hydrolysis mixture of claims 81 or 82, wherein the one or more cellulase enzymes comprise enzymes having one or more of cellobiohydrolase, endoglucanase and β-glucosidase activity.

84. 84. The enzymatic hydrolysis mixture according to any one of claims 81 to 83, wherein the polyaspartic acid has a molecular weight of from 1000 to 5000.

85. 85. The method of any one of claims 81 to 84, wherein the surfactant is a non-ionic surfactant, optionally a polysorbate surfactant such as Tween™.

86. 86. The enzymatic hydrolysis mixture according to any one of claims 81 to 85, wherein the ratio of surfactant to polyaspartic acid is from 0.1:1 to 10:1, optionally from 0.5:1 to 2:

1.

87. 81. A glucose-rich sugar stream produced by the method of any one of claims 18 to 80.

88. 88. The glucose-rich sugar stream of claim 87, wherein the sugar stream contains greater than 12% glucose, greater than 14% glucose, greater than 16% glucose, or greater than 18% glucose.

89. 89. The glucose-rich sugar stream of claim 87 or 88, wherein the sugar stream contains polyaspartic acid.

90. 90. The glucose-rich sugar stream of any one of claims 87-89, wherein the polyaspartic acid is present in a concentration from 1 ppb to 10000 ppm.

91. 90. The glucose-rich sugar stream of any one of claims 87 to 89, wherein the glucose-rich sugar stream contains non-glucose sugars.

92. 92. The glucose-rich sugar stream of claim 91, wherein the non-glucose sugars are one or more of xylose, xylooligosaccharides, or xylan.

93. 93. The glucose-rich sugar stream of claim 91 or 92, wherein the dry matter composition comprises about 95% glucose and about 5% non-glucose sugars.

94. 1. A glucose syrup containing about 95% glucose and about 5% non-glucose sugars, the non-glucose sugars being one or more of xylose, xylooligosaccharides and xylan.

95. 1. A fructose syrup containing about 95% fructose and about 5% non-fructose sugars, the non-fructose sugars being one or more of xylose, xylooligosaccharides and xylans.

96. 96. The fructose syrup of claim 95 prepared by glucose isomerization of the glucose-rich sugar stream of claim 93.

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