WO2012155240A1 - Élimination d'inhibiteurs d'enzymes au cours de l'hydrolyse enzymatique d'une matière première lignocellulosique - Google Patents

Élimination d'inhibiteurs d'enzymes au cours de l'hydrolyse enzymatique d'une matière première lignocellulosique Download PDF

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WO2012155240A1
WO2012155240A1 PCT/CA2012/000436 CA2012000436W WO2012155240A1 WO 2012155240 A1 WO2012155240 A1 WO 2012155240A1 CA 2012000436 W CA2012000436 W CA 2012000436W WO 2012155240 A1 WO2012155240 A1 WO 2012155240A1
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stream
effluent stream
enzyme
enzymatic hydrolysis
reduced
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PCT/CA2012/000436
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English (en)
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Murray J. Burke
Bradley Saville
Daniel Jing LIAO
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Mascoma Canada Inc.
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Publication of WO2012155240A1 publication Critical patent/WO2012155240A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C11/00—Regeneration of pulp liquors or effluent waste waters
    • D21C11/0007—Recovery of by-products, i.e. compounds other than those necessary for pulping, for multiple uses or not otherwise provided for
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This application relates to a method for the removal of enzyme inhibitors during the enzymatic hydrolysis of lignocellulosic materials.
  • the application relates to a two-stage enzymatic hydrolysis process for treating lignocellulosic materials and producing a sugar rich process stream, wherein enzyme inhibiting compounds are removed after the first stage of the enzymatic hydrolysis.
  • 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.
  • 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.
  • Methods of converting plant biomass into fermentable sugars 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.
  • a pretreatment step to loosen the plant structure
  • 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.
  • the main objective of the pretreatment is to provide accessibility of the plant material to the enzymes.
  • 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.
  • organic acids e.g., acetic acid
  • 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.
  • This application relates to a two-stage enzymatic process to prepare a sugar rich process stream from a feedstock derived from plant materials, in which the effluent stream from the first hydrolysis is treated to reduce the level of at least one hydrolysis inhibiting compound optionally with 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
  • 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.
  • 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.
  • 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.
  • Another advantage of the process of the invention is the removal of at least one hydrolysis inhibiting compound after the first enzymatic hydrolysis process, which increases the efficiency of the second enzymatic hydrolysis process as a result of there being a reduced concentration of compounds which inhibit the enzymes in the second enzymatic hydrolysis process.
  • a method for treating a lignocellulosic feedstock comprising cellulose, hemicellulose, and lignin to produce a sugar rich process stream wherein the method comprises:
  • Another advantage of the process of the invention is the recovery and recycling of the enzymes used in each stage of the enzymatic process which permits enzymes that would otherwise be lost, being redeployed to an alternate stage where the enzymes may be efficaciously used. Accordingly, the amount of enzymatic hydrolysis occurring per unit of enzymes is increased.
  • 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.
  • the product streams are subjected to ultrafiltration and/or diafiltration to recover the enzymes.
  • the feedstock is subjected to a first enzymatic hydrolysis process to preferentially solubilize xylose and obtain an effluent stream.
  • hemicellulose and cellulose are broken down, preferably to solubilize oligosaccharides of sugars.
  • 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.
  • 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.
  • this second enzyme preparation comprises beta- glucosidase activities.
  • the second enzyme preparation may have an activity to convert cellulose and cellobiose to monomers and cello- oligosaccharides.
  • the 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.
  • the second enzymatic hydrolysis process or stage also utilizes fermentation organisms to simultaneously ferment the sugar rich process stream to alcohol.
  • oligosaccharides and in particular cellobiose, have an inhibitory effect on cellulase enzymes and, in particular, on endo-gluconases and cellobiohydrolases.
  • the hemicelluloses, and optionally the cellulose are treated with enzymes to produce soluble sugars, for example, xylose.
  • the process is preferably conducted so as not to render a substantial portion of the cellulose into monomers or dimers, such as cellobiose.
  • the process is preferably conducted so as to prevent a substantial inhibition of the enzymes.
  • the oligosaccharides are subjected to enzymatic hydrolysis to produce fermentable sugars (preferably monomers).
  • the first enzyme preparation preferentially acts on the hemicellulose to solubilize the xylose.
  • the hemicellulose is broken down into oligomers and monomers that are removed from the fiber as soluble compounds in an aqueous medium (preferably water).
  • 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.
  • 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.
  • the term preferentially hydrolyze or solubilize 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.
  • the first enzyme preparation preferentially acts upon the ⁇ -1 ,4 linkage of the xylose residues of xylan and the ⁇ -1 ,4 linkage of the mannose residues of mannan.
  • 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.
  • 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.
  • a neutralizing agent e.g., urea, anhydrous ammonia, aqueous ammonia, sodium hydroxide, potassium hydroxide
  • acetic acid may be removed from the process, such as by operating under vacuum.
  • acetic acid 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.
  • Figure 1 is a flow chart of the method according to embodiments that includes the removal of at least one hydrolysis inhibiting compound and removal of acetic acid;
  • Figure 2 is a flow chart of the method according to embodiments that includes the removal of at least one hydrolysis inhibiting compound and enzyme recovery;
  • Figure 3 is a flow chart of the method according to embodiments that includes the removal of at least one hydrolysis inhibiting compound and enzyme recovery, enzyme recovery and removal of acetic acid;
  • Figure 4 is a flow chart of the method according to embodiments that includes the removal of at least one hydrolysis inhibiting compound, and enzyme recovery after the first and second enzymatic hydrolysis stages.
  • 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 reduce the level of at least one hydrolysis inhibiting compound after the first enzymatic hydrolysis stage.
  • the product streams from one stage and, preferably, each stage of the enzymatic hydrolysis are preferably 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.
  • 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.
  • FIG. 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.
  • 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 22 further contains enzyme inhibitors, such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, 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.
  • enzyme inhibitors such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and cellula
  • the effluent stream 22 may be subjected to a solid/liquid separation 24, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, to produce an inhibitor reduced stream (a solid stream) 26 and a treated effluent stream (a liquid stream) 30.
  • the inhibitor reduced stream 26 contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage 20.
  • the treated effluent stream 30, which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage 20, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, enzyme inhibitors (inhibitors to the enzymes used in a second enzymatic hydrolysis stage 28) are removed from the inhibitor reduced stream 26 before being subjected to the second enzymatic hydrolysis stage 28.
  • the inhibitor reduced stream 26, containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 28.
  • the second enzymatic hydrolysis stage 28 preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose.
  • the second enzymatic hydrolysis stage 28 produces a sugar rich process stream 32, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose.
  • the second sugar rich stream 38 is then subjected to fermentation 40.
  • FIG. 2 exemplifies a schematic of different embodiments of the invention.
  • the lignocellulosic feedstock 10 is optionally first subjected to a pretreatment and optional steam explosion 1 12 to produce an activated feedstock 1 14, and then subsequently an optional disc refining step 1 16 to produce a fine particulate stream 118. It will be appreciated that neither of these optional steps, or one or both of these optional steps, may be utilized.
  • the fine particulate stream is then subjected to a first enzymatic hydrolysis stage 120 to produce an effluent stream 122.
  • the first enzymatic hydrolysis stage 120 preferentially hydrolyzes the hemicelluloses in the feedstock to produce monomeric sugars of xylose and glucose.
  • the effluent stream may also contain short oligosaccharides and other higher molecular weight oligosaccharides that have not been fully hydrolyzed in the first enzymatic hydrolysis stage.
  • the effluent stream 122 also contains unhydrolyzed cellulose, which is preferentially hydrolyzed in the second enzymatic hydrolysis stage 128.
  • the effluent stream 122 further contains enzyme inhibitors, such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and cellulases used in the first and/or second enzymatic hydrolysis stages 120 and 128.
  • enzyme inhibitors such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and cell
  • the effluent stream 122 may be subjected to a solid/liquid separation 124, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, to produce an inhibitor reduced stream (a solid stream) 126 and a treated effluent stream (a liquid stream) 130.
  • the inhibitor reduced stream 126 contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage 120.
  • the treated effluent stream 130 which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage 120, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, enzyme inhibitors (inhibitors to the enzymes used in a second enzymatic hydrolysis stage 128) are removed from the inhibitor reduced stream 126 before being subjected to the second enzymatic hydrolysis stage 128.
  • the inhibitor reduced stream 126 containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 128.
  • the second enzymatic hydrolysis stage 128 preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose.
  • Treated effluent stream 30 is subjected to a separation step 132, for example a filtration step, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis, to obtain a recovered first stage enzyme stream 134 and a first enzyme reduced effluent stream 136.
  • the recovered first stage enzyme stream 34 containing enzymes from the first enzymatic hydrolysis stage 120 is recycled to the first and/or second enzymatic hydrolysis stages 120 and 128.
  • the first enzyme reduced effluent stream 136 comprises sugars which is then subjected to fermentation 138.
  • the second enzymatic hydrolysis stage 128 produces a sugar rich process stream 140, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose and may be subjected to a solid/liquid separation 142, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, to produce a solid lignin stream 144 and a sugar rich liquid/filtrate effluent stream 146.
  • the sugar rich liquid/filtrate stream 146 may then be treated to recover enzymes utilized in the second enzymatic hydrolysis process.
  • the sugar rich liquid/filtrate stream 146 is subjected to a separation step 148, for example a filtration step, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis, to obtain a recovered second stage enzyme stream 152 and a second enzyme reduced effluent stream 136.
  • the recovered second stage enzyme stream 152 containing enzymes from the second enzymatic hydrolysis stage 128 is recycled to the first and/or second enzymatic hydrolysis stages 120 and 128.
  • the second enzyme reduced effluent stream 150 comprises monomeric sugars which is then subjected to fermentation 138.
  • FIG. 3 exemplifies a schematic of different embodiments of the invention.
  • 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 first enzymatic hydrolysis stage 220 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 222 also contains unhydrolyzed cellulose, which is preferentially hydrolyzed in the second enzymatic hydrolysis stage 228.
  • the effluent stream 222 further contains enzyme inhibitors, such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and cellulases used in the first and/or second enzymatic hydrolysis stages 220 and 228.
  • enzyme inhibitors such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and
  • the effluent stream 222 may be subjected to a solid/liquid separation 224, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, to produce an inhibitor reduced stream (a solid stream) 226 and a treated effluent stream (a liquid stream) 230.
  • the inhibitor reduced stream 226 contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage 220.
  • the treated effluent stream 230 which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage 220, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, enzyme inhibitors (inhibitors to the enzymes used in a second enzymatic hydrolysis stage 228) are removed from the inhibitor reduced stream 226 before being subjected to the second enzymatic hydrolysis stage 228.
  • the inhibitor reduced stream 226, containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 228.
  • the second enzymatic hydrolysis stage 228 preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose.
  • Treated effluent stream 230 is subjected to a separation step 232, for example a filtration step, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis, to obtain a recovered first stage enzyme stream 234 and a first enzyme reduced effluent stream 236.
  • the recovered first stage enzyme stream 234 containing enzymes from the first enzymatic hydrolysis stage 220 is recycled to the first and/or second enzymatic hydrolysis stages 220 and 228.
  • the first enzyme reduced effluent stream 236 is then subjected to a separation step 246, for example by means of a chromatographic separation to remove enzyme inhibitors, such as acetic acid, and obtain an inhibitor rich stream 248 and a second sugar rich stream 250.
  • the second sugar rich stream 250 is then subjected to fermentation 252.
  • the second enzymatic hydrolysis stage 228 produces a sugar rich process stream 238, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose and may be subjected to a solid/liquid separation and membrane separation 240, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, followed by a membrane separation, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis, to obtain a recovered second stage enzyme stream 242 and a second enzyme reduced effluent stream 244.
  • a solid/liquid separation and membrane separation 240 for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, followed by a membrane separation, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis
  • the recovered second stage enzyme stream 242 containing enzymes from the second enzymatic hydrolysis stage 228 is recycled to the first and/or second enzymatic hydrolysis stages 220 and 228.
  • the second enzyme reduced effluent stream 244 is then subjected to a separation step 246, for example by means of a chromatographic separation to remove enzyme inhibitors, such as acetic acid, and obtain an inhibitor rich stream 248 and the sugar rich stream 250.
  • FIG. 4 exemplifies a schematic of different embodiments of the invention.
  • the lignocellulosic feedstock 310 is 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 witl 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 320 to produce an effluent stream 322.
  • the first enzymatic hydrolysis stage 320 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 322 also contains unhydrolyzed cellulose, which is preferentially hydrolyzed in the second enzymatic hydrolysis stage 328.
  • the effluent stream 322 further contains enzyme inhibitors, such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, 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 320 and 328.
  • enzyme inhibitors such as acetic acid, furfural, hydroxymethylfurfural, phenolic compounds and monomeric sugars, such as glucose and xylose (end product inhibition), as well as gluco- oligosaccharides, xylo-oligosachharides, soluble lignin and organic acids, which are products of the enzymatic hydrolysis and which inhibit the activity of the hemicellulases and
  • the effluent stream 322 may be subjected to a solid/liquid separation 324, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, to produce an inhibitor reduced stream (a solid stream) 326 and a treated effluent stream (a liquid stream) 330.
  • the inhibitor reduced stream 326 contains insoluble solids, such as cellulose, which were not solubilized in the first enzymatic hydrolysis stage 320.
  • the treated effluent stream 330 which in an embodiment is a clear filtrate stream, contains any of the soluble compounds that were produced during the first enzymatic hydrolysis stage 320, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors. Accordingly, enzyme inhibitors (inhibitors to the enzymes used in a second enzymatic hydrolysis stage 328) are removed from the inhibitor reduced stream 326 before being subjected to the second enzymatic hydrolysis stage 328.
  • the inhibitor reduced stream 326 containing insoluble compounds such as cellulose and lignin, may then be subjected to the second enzymatic hydrolysis stage 328.
  • the second enzymatic hydrolysis stage 328 preferentially hydrolyzes the celluloses in the feedstock to produce monomeric sugars, for example, glucose.
  • the treated effluent stream 330 containing soluble compounds that were produced during the first enzymatic hydrolysis stage 320, such as soluble monomeric sugars, short oligosaccharides and enzyme inhibitors, is then treated to recover enzymes utilized in the first enzymatic hydrolysis process to obtain a recovered first stage enzyme stream 334 and a first enzyme reduced effluent stream 338.
  • the recovered first stage enzyme stream 334 may then be recycled to the first and/or second enzymatic hydrolysis stages 320 and/or 328 (as shown with the dashed arrow in Figure 4).
  • Treated effluent stream 330 is subjected to a separation step 332, for example a filtration step, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis, to obtain a recovered first stage enzyme stream 334 and a first enzyme reduced effluent stream 338.
  • the separation step 332 comprises at least one membrane separation step wherein the step uses a membrane having a molecular weight cut-off of less than 10 kDa, or less than 5 kDa (the membrane will not allow compounds having a molecular weight of higher than 10 kDa, or 5 kDa, to pass through the membrane).
  • the separation step comprises ultrafiltration followed by diafiltration, or ultrafiltration.
  • the recovered first stage enzyme stream 334 may then be subjected to at least one membrane separation step 336, for example diafiltration, to obtain an inhibitor reduced recovered enzyme stream 340 and a second enzyme reduced effluent stream 342.
  • the inhibitor reduced recovered enzyme stream 340 may then be recycled to the first and/or second enzymatic hydrolysis stages 320 and/or 328.
  • the first enzyme reduced effluent stream 338 and second enzyme reduced effluent stream 342 are then combined to form a combined enzyme reduced effluent stream 344, which may be directly subjected to fermentation 350.
  • the combined enzyme reduced effluent stream 344 is treated to reduce fermentation inhibitory compounds and obtain an inhibitor reduced effluent stream 348 which is subjected to fermentation.
  • the combined enzyme reduced effluent stream 344 may be subjected to a at least one separation step 346, such as for example a chromatographic separation or at least one membrane separation step, such as nanofiltration, ultrafiltration, diafiltration and/or reverse osmosis.
  • a separation step 346 such as for example a chromatographic separation or at least one membrane separation step, such as nanofiltration, ultrafiltration, diafiltration and/or reverse osmosis.
  • the second enzymatic hydrolysis stage 328 produces a sugar rich process stream 352, which contains soluble monomeric sugars, and other insoluble components such as lignin, ash and unhydrolyzed hemicellulose and cellulose and may be subjected to a solid/liquid separation 354, for example by means of a filter press, or a decanting centrifuge, a belt filter, a vibratory screen and/or a hydrocyclone, to produce a solid lignin stream 358 and a sugar rich liquid/filtrate effluent stream 356.
  • the sugar rich liquid/filtrate stream 356 may then be treated to recover enzymes utilized in the second enzymatic hydrolysis process.
  • the sugar rich liquid/filtrate stream 356 is subjected to a separation step 360, for example a filtration step, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis, to obtain a recovered second stage enzyme stream 364 and a second enzyme reduced effluent stream 362.
  • the recovered second stage enzyme stream 364 containing enzymes from the second enzymatic hydrolysis stage 328 is recycled to the first and/or second enzymatic hydrolysis stages 320 and 328.
  • the second enzyme reduced effluent stream 362 comprises monomeric sugars which is then subjected to fermentation 350.
  • the lignocellulosic feedstock is derived from plant materials.
  • 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.
  • the feedstock may be derived from trees, preferably deciduous trees such as poplar (e.g., wood chips).
  • 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.
  • the lignocellulosic feedstock comprises agricultural residues and wood biomass, more preferably wood biomass and most preferably deciduous.
  • the feedstock may be any feedstock that does not contain edible agricultural produce, however such material may be used.
  • 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.
  • 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.
  • 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.
  • the lignocellulosic feedstock is optionally subjected to one or more activation steps prior to the feedstock being subject to enzymatic hydrolysis.
  • 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.
  • the lignocellulosic feedstock may also be subjected to chemical or physical modification pretreatment, extraction or hydrolysis.
  • 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.
  • 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.
  • a chemical agent or catalyst such as sulphuric 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.
  • the autohydrolysis is conducted in a steam explosion digester, which is known in the art.
  • 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.
  • a catalyst such as sulphuric acid.
  • 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.
  • acidic e.g. 3.0 - 4.0
  • hemicellulose is partially hydrolyzed to xylose, soluble xylo-oligosaccharides and other pentosans.
  • the yield may be up to about 75%.
  • 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.
  • 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.
  • 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.
  • 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.
  • the water and heat impregnation step can be performed in the same vessel as the hydrolyser.
  • 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.
  • 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.
  • 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.
  • the moisture content of the fiber passing through the disc refiner is about 50 to about 99% by weight.
  • a disc refiner can be used with a lignocellulosic feedstock at a range of different particle sizes.
  • 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.
  • 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.
  • 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.
  • 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.
  • the first enzyme preparation i.e., a hemicellulase enzyme preparation
  • the first enzyme preparation 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.
  • the hemicellulase preferentially acts upon the ⁇ -1 ,4 linkage of the xylose residues of xylan to solubilize the xylans and the ⁇ -1 ,4 linkage of the mannose residues of mannan.
  • Cellulose is a linear polymer of glucose, wherein the glucose residues are held together by beta (1 ⁇ 4) 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.
  • Beta-glucosidase 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.
  • enzymes may exhibit a range of different activities on different substrates.
  • 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.
  • a hemicellulase would preferentially act on hemicellulose to produce pentosans relative to its activity for cellulose to produce glucose.
  • 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.
  • 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.
  • total solids content refers to the total amount of soluble and insoluble material in the 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.
  • 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%).
  • the removal of at least one hydrolysis inhibiting compound such as glucose, gluco- oligosaccharides, xylose, xylooligosaccharides, furfural, hydroxymethylfurfural, soluble lignin, organic acids, and/or phenolic compounds, increases the efficiency of the hydrolytic enzymes.
  • at least one hydrolysis inhibiting compound such as glucose, gluco- oligosaccharides, xylose, xylooligosaccharides, furfural, hydroxymethylfurfural, soluble lignin, organic acids, and/or phenolic compounds
  • 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 treated to reduce the level of at least one hydrolysis inhibiting compound to obtain an inhibitor reduced stream and a treated effluent stream.
  • the treated effluent stream is then subjected to a second enzymatic hydrolysis process to preferentially solubilize cellulose and to obtain a sugar rich process stream.
  • 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.
  • the first enzymatic hydrolysis stage uses a first enzyme preparation that preferably comprises hemicellulase.
  • the hemicellulase preparation will also possess cellulase activity.
  • the first enzyme preparation is a xylanase enzyme cocktail such as Dyadic XBPTM.
  • the first enzyme preparation is AlternaFuel 00LTM. It will be understood by a person skilled in the art that combinations of the enzyme preparations may be used.
  • the first enzyme preparation will possess hemicellulase activity from about 10% to about 90% and cellulase activity from about 90% to about 10%.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • the treated effluent stream containing soluble compounds that were produced during the first enzymatic hydrolysis stage is then treated to recover enzymes utilized in the first enzymatic hydrolysis process to obtain a recovered first stage enzyme stream and a first enzyme reduced effluent stream.
  • the recovered first stage enzyme stream may then be recycled to the first and/or second enzymatic hydrolysis stages.
  • the treated effluent stream is subjected to a separation step, for example a filtration step, for example a membrane separation such as nanofiltration, ultrafiltration, diafiltration or reverse osmosis, to obtain a recovered first stage enzyme stream and a first enzyme reduced effluent stream.
  • the separation step comprises at least one membrane separation step wherein the step uses a membrane having an average pore size less than 0,000MW, or less than 1 ,00MW.
  • the separation step comprises ultrafiltration followed by diafiltration, or ultrafiltration.
  • the recovered first stage enzyme stream may then be subjected to at least one membrane separation step, for example diafiltration, to obtain an inhibitor reduced recovered enzyme stream and a second enzyme reduced effluent stream.
  • the inhibitor reduced recovered enzyme stream may then be recycled to the first and/or second enzymatic hydrolysis stages and/or.
  • the first enzyme reduced effluent stream and second enzyme reduced effluent stream are then combined to form a combined enzyme reduced effluent stream, which may be directly subjected to fermentation.
  • the combined enzyme reduced effluent stream is treated to reduce fermentation inhibitory compounds and obtain an inhibitor reduced effluent stream which is subjected to fermentation.
  • the combined enzyme reduced effluent stream may be subjected to a at least one separation step, such as for example a chromatographic separation or at least one membrane separation step, such as nanofiltration, ultrafiltration, diafiltration and/or reverse osmosis.
  • 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.
  • 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.
  • 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.
  • the inhibitor reduced stream is treated with a second enzyme preparation to produce a sugar rich process stream high in fermentable sugars such as glucose.
  • 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.
  • the second enzyme preparation preferably primarily includes cellulase activity.
  • the second enzyme preparation comprises beta-glucosidase activity to convert disaccharides and other small polymers of glucose into monomeric glucose.
  • the second enzyme preparation is Novozym 188TM, available from NovozymesTM.
  • the second enzyme preparation is NS50073TM. It will be understood by those in the art that combinations of the enzyme preparations may be used.
  • 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.
  • the acid stream comprises any mineral acid.
  • the acid stream comprises nitric acid, sulphuric acid, phosphoric acid, acetic acid and/or hydrochloric acid.
  • the base stream comprises potassium hydroxide, sodium hydroxide, ammonium hydroxide, urea and/or ammonia.
  • 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.
  • 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 10% to about 60%.
  • the process is used in a simultaneous saccharification and fermentation (SSF) process, in which the inhibitor reduced stream (a 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.
  • SSF simultaneous saccharification and fermentation
  • 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.
  • the cellulase enzymes from the reaction vessel can therefore be recovered and recycled to the first and/or second enzymatic hydrolysis processes.
  • the yeast present in the vessel is optionally recovered from the SSF process.
  • 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.
  • 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.
  • the product stream is subjected to solid/liquid separation to obtain a solid stream and a liquid/filtrate alcohol stream.
  • the liquid/filtrate alcohol stream is filtered to obtain the recovered enzyme stream.
  • 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.
  • 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.
  • 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.
  • inhibitory compounds which can inhibit both the enzymatic hydrolysis enzymes, as well as the yeast during the fermentation of the monomeric sugars.
  • 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.
  • products from the first enzymatic hydrolysis stage have a negative feedback (end product inhibition) on enzymes used in the first enzymatic hydrolysis stage.
  • inhibitory compounds include, but are not limited to, glucose, gluco- oligosaccharides, xylose, xylo-oligosaccharides, formic acid, glycerol furfural, hydroxymethylfurfural, organic acids, and phenolic compounds. Accordingly, 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.
  • 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.
  • 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 (inhibitor reduced stream) and a liquid/filtrate stream (treated effluent 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.
  • the hemicellulase and cellulase enzymes from the first and/or second enzymatic hydrolysis stages are recovered and recycled to the first and/or second enzymatic hydrolysis stages.
  • the recovery and recycling of enzymes is advantageous as the recycling of the enzymes 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, 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.
  • 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 treated to reduce the level of at least one hydrolysis inhibiting compound and obtaining an inhibitor reduced stream and a treated effluent stream.
  • the inhibitor reduced stream is then subjected to a second enzymatic hydrolysis process to preferentially solubilize cellulose and obtaining a sugar rich process stream.
  • At least one of the treated 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.
  • the inhibitor reduced 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.
  • the treated effluent stream is treated to recover enzymes utilized in the first enzymatic hydrolysis stage and obtain a recovered first stage enzyme stream and a first enzyme reduced effluent stream.
  • at least some of the first recovered enzyme stream is recycled to the second enzymatic hydrolysis process.
  • at least some of the first recovered enzyme stream is recycled to the first enzymatic hydrolysis process.
  • at least some of the first recovered enzyme stream from the first enzymatic hydrolysis is recycled to the first and second enzymatic hydrolysis processes.
  • 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.
  • at least some of the second recovered enzyme stream from the second enzymatic hydrolysis is recycled to the first enzymatic hydrolysis processes.
  • at least some of the second recovered enzyme stream from the second enzymatic hydrolysis is recycled to the second enzymatic hydrolysis process.
  • at least some of the second recovered enzyme stream from the second enzymatic hydrolysis is recycled to the first and second enzymatic hydrolysis processes.
  • 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.
  • at least some of the second recovered enzyme stream from the SSF is recycled to the first enzymatic hydrolysis processes.
  • at least some of the second recovered enzyme stream from the SSF is recycled to the SSF.
  • at least some of the second recovered enzyme stream from the SSF is recycled to the first and SSF processes.
  • the treated 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.
  • at least one of the treated effluent stream, the sugar rich process stream and the alcohol stream is filtered to obtain the recovered enzyme stream.
  • 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.
  • at least one of the treated effluent stream, the sugar rich process stream and the alcohol stream is subjected to at least one of ultrafiltration and diafiltration.
  • At least one of the treated effluent stream, the sugar rich process stream and the alcohol stream is sequentially subjected to ultrafiltration and diafiltration.
  • 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.
  • 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).
  • the enzyme reduced effluent stream is treated to remove at least one of acetic acid, formic acid, glycerol, furfural and hydroxymethylfurfural.
  • the cellulose, hemicellulose and lignin containing material is subjected to autohydrolysis to obtain the feedstock.
  • the autohydrolysis has a severity of from 3.6 to 4.5.
  • the cellulose, hemicellulose and lignin containing material is subjected to hydrolysis followed by disc refining to obtain the feedstock for the enzymatic hydrolysis process.
  • the cellulose, hemicellulose and lignin containing material is subjected to hydrolysis to obtain the feedstock.
  • the recovery of enzymes can be performed after either, or both, of the first or second enzymatic hydrolysis processes to produce recovered enzyme streams.
  • the first and/or second recovered enzyme streams are recycled to the second and/or first enzymatic hydrolysis processes, respectively.
  • 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.
  • 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 reduce the level of at least one hydrolysis inhibiting compound after the first enzymatic hydrolysis stage.
  • the sugar rich process stream is treated by solid/liquid separation to obtain the lignin stream and the lignin reduced sugar rich process stream.
  • 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.
  • 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.
  • the solid stream (inhibitor reduced stream) that is subjected to the second enzymatic hydrolysis stage contains insoluble compounds such as cellulose and lignin.
  • 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.
  • a solid/liquid separation such as a filter press
  • 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.
  • the lignin is removed from the sugar rich process stream before the sugars are subjected to fermentation.
  • 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.
  • the process unexpectedly provides a synergistic benefit, which reduces the duration of the fermentation process.
  • 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.
  • 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.
  • 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.
  • an alkaline agent such as an alkali hydroxide or alkaline earth hydroxide
  • the acetic acid is converted to its corresponding acetate ion, which as a result of its non-lipophilic property is not absorbed by the fatty acids of the cell wall, and therefore does not poison the yeast cells.
  • Other weak acids which are a poison to yeast, may also be present such as lactic acid or succinic acid. 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.
  • 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.
  • the sugar rich process stream is maintained at an elevated pH of above 3.5, or optionally above 5.5, or optionally 5.5 to 7.0, in which the pH of the fermentation process in maintained using an alkaline agent, such as calcium hydroxide.
  • an alkaline agent such as calcium hydroxide.
  • acetic acid will dissociate.
  • the alkaline agent is a combination of calcium hydroxide and/or sodium hydroxide.
  • the alkaline agent is ammonium hydroxide.
  • the alkaline agent such as calcium hydroxide
  • the alkaline agent reacts with the acetic acid, or other weak acid, to form the salt of the conjugate base (in this case, calcium acetate), which is not very soluble in the fermentation broth.
  • the salt of the conjugate base calcium acetate
  • the weak acid acetic acid
  • an alkaline agent such as calcium hydroxide, may act as a nutrient source for the yeast, thereby increasing the speed of the fermentation reaction.
  • the fermentation of the sugar rich process stream is conducted at a pH above 3.5, or optionally above 5.5, or between 5.5 and 7.0, in the presence of sodium hydroxide.
  • the fermentation of the sugar rich process stream is conducted at a pH above 3.5, or optionally above 5.5, or between 5.5 and 7.0, in the presence of calcium hydroxide.
  • the fermentation of the sugar rich process stream is conducted at a pH above 3.5, or optionally above 5.5, or between 5.5 and 7.0, in the presence of a calcium hydroxide and sodium hydroxide.
  • the fermentation of the sugar rich process stream is also conducted in the presence of a nitrogen source.
  • the nitrogen source is urea, a urea derivative, nitrates, or ammonia derivatives, such ammonium hydroxide.
  • 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.
  • calcium hydroxide is used as the alkaline agent and urea is used as the nitrogen source. Accordingly, in one embodiment, the fermentation of the sugar rich process stream is conducted at an elevated pH above 3.5, or optionally above 5.5, or optionally between 5.5 and 7.0, in which the pH of the fermentation process in raised using calcium hydroxide and also in the presence of urea.
  • 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 hemicellulase and cellulase enzymes may be used by itself or in combination with any other process or processes disclosed herein.
  • 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.
  • 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.
  • 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.
  • the product stream is subjected to solid/liquid separation to obtain a solid stream and a liquid/filtrate stream.
  • the liquid/filtrate stream is filtered to obtain the recovered enzyme stream.
  • the liquid/filtrate stream is subjected to at least one membrane filtration process.
  • the liquid/filtrate stream is subjected to at least one of ultrafiltration and diafiltration.
  • the liquid/filtrate stream is sequentially subjected to ultrafiltration and diafiltration.
  • 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.
  • the sugar rich process stream is used to produce sugar derived products.
  • 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.
  • the alcohol produced is methanol, ethanol and/or butanol.
  • 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.
  • Table 1 Shown in Table 1 is the material balance after both ultrafiltration and diafiltration of the hydrolyzate.
  • Protein content was measured using the Bradford protein assay. About half of the total proteins were recovered in the diafiltration retentate. 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 2.
  • 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 4. 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.

Abstract

L'invention concerne un processus d'hydrolyse enzymatique en deux étapes pour traiter des matières lignocellulosiques et produire un flux de processus riche en sucres, des composés inhibant des enzymes étant éliminés après la première étape de l'hydrolyse enzymatique.
PCT/CA2012/000436 2011-05-18 2012-05-08 Élimination d'inhibiteurs d'enzymes au cours de l'hydrolyse enzymatique d'une matière première lignocellulosique WO2012155240A1 (fr)

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US61/487,585 2011-05-18

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CARTER B: "``Detoxification of a Lignocellulosic Biomass Slurry by Soluble Polyelectrolyte Adsorption for Improved Fermentation Efficiency", BIOTECHNOLOGY AND BIOENGINEERING, vol. 108, no. 9, September 2011 (2011-09-01), pages 2053 - 2060 *
REZAEI F ET AL.: "Selection of Conditions for Cellulase and Xylanase Extraction from Switchgrass Colonized by Acidothermus cellulolyticus", APPL BIOCHEM BIOTECHNOL, vol. 164, 12 February 2011 (2011-02-12), pages 793 - 803 *

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