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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
  • C12P19/02—Monosaccharides
• • 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
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • C—CHEMISTRY; METALLURGY
  • C13—SUGAR INDUSTRY
  • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
  • C13K1/00—Glucose; Glucose-containing syrups
  • C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
• • 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

• the present invention relates to a method of producing glucose from a cellulosic biomass using enzymatic hydrolysis.
• the present invention also relates to a device for performing such method.
• the conventional batch approach can operate at high solids content in enzymatic hydrolysis; the resulting mixture is difficult to mix and the enzyme loading cannot be lowered without impacting overall sugar yields.
• the solids can be elevated with less agitation impact in the enzymatic hydrolysis process, but lowering the enzyme loading without impacting the sugars concentrations and yields is not possible.
• a method of producing glucose from a cellulosic biomass using enzymatic hydrolysis wherein said enzymatic hydrolysis is performed in a multistage hydrolysis process in a plurality of hydrolysis vessels, said plurality of hydrolysis vessels being serially arranged such that there is a first hydrolysis vessel and, optionally, one or several hydrolysis vessels in between, wherein during said multistage process, each hydrolysis vessel contains a solid fraction SF which is biomass and a liquid fraction LF which is an aqueous solution, said multistage process having a front-end at said first hydrolysis vessel and a back-end at said last hydrolysis vessel, said multistage process involving an initial addition of enzyme to each hydrolysis vessel, said multistage hydrolysis process further involving in each hydrolysis stage the feeding of cellulosic biomass and enzyme at the front-end, the removal of en
• the inventions relates to a method of producing glucose from a cellulosic biomass using enzymatic hydrolysis, said method comprising the steps: a) providing n hydrolysis vessels HV l5 HV 2 , HV 3 , ..., HV n-1 , HV n , n being an integer from 2 to 100, preferably 2-50, more preferably 2-20, even more preferably 2-10, b) adding to each hydrolysis vessel, in any order, a defined amount of cellulosic biomass, a defined volume of water and a defined amount of enzyme, c) performing an enzymatic hydrolysis stage in all n hydrolysis vessels HYi, HV 2 , HV 3 , ..., HV flavour, d) separating, for each hydrolysis vessel, a solid fraction from a liquid fraction, such separation resulting in solid fraction SFi, SF 2 , SF 3 , SF n-1 , and SF n and in liquid fractions LF], LF 2 ,
• step f) for all remaining solid fractions SF n-2 - SF ls if any, which are preferably located in hydrolysis vessel HV n-2 - HVn, respectively, by transferring said remaining solid fractions into hydrolysis vessels HV n-1 - HV 2 , respectively h) adding a specified amount of cellulosic biomass to hydrolysis vessel HVi i) taking all or a specified partial volume of liquid fraction LF ls preferably from hydrolysis vessel HV l5 if not already separate therefrom, and storing it as final product, said final product containing sugar(s) dissolved in said liquid fraction LFi j) adding all or a specified partial volume of liquid fraction LF 2 to hydrolysis vessel HVi k) performing step j) for all remaining liquid fractions LF 3 - LF n , if any, by adding them to hydrolysis vessels HV
• step e) adding a specified volume of water to hydrolysis vessel HV n m) repeating steps c)-l) n-1 times wherein, for each repetition, additional enzyme is added to hydrolysis vessel HV l5 wherein, preferably, for each repetition, additional enzyme is added to hydrolysis vessel ⁇ only.
• said specified partial amount discarded in step e) and said specified partial amounts transferred in steps f)-g) and said specified amount added in step h) are the same.
• said specified partial volumes of steps i)-k) and said specified volume of step 1) are the same.
• said specified partial amounts of steps e)-g) and said specified amount of step h), respectively is 0.5-100% of the defined amount of cellulosic biomass added to each hydrolysis vessel during step b).
• said specified partial volumes of steps i)-k) and said specified volume of step 1), respectively, is 0.5%-100% of the defined volume added to each hydrolysis vessel during step b).
• each hydrolysis stage is performed for a time period of 0.5-36 hours and/or at a temperature of 40°C - 60°C, preferably 45°C - 57°C, more preferably 48°C - 55°C.
• said defined amount of enzyme added initially in step b) to each hydrolysis vessel is in the range of from 0.01 wt.% - 5 wt.%, preferably 0.1 wt.% - 3 wt.%, more preferably 0.5 - 1.5% with reference to the weight of dry biomass in said vessel.
• said additional enzyme added to hydrolysis vessel HV ! in step m) for each repetition is in the range of from 0.5wt.% to 1.5 wt.%, with reference to the weight of dry biomass in said hydrolysis vessel HVj .
• the solid contents during the hydrolysis in each hydrolysis vessel is in the range of from 12% to 33%, preferably 15% - 25%, more preferably, 18% to 23%.
• the specified partial amount transferred or discarded is in the range of from 5% to 100% of said respective solid fraction, e. g. 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% and 100% and any value in between of said respective solid fraction.
• the pH is adjusted in the respective hydrolysis vessel to the working range of the respective enzyme employed, preferably to a pH in the range of from 4-7.5, preferably 4.5-7, more preferably 4.8-5.5.
• the enzyme used for hydrolysis is selected from cellulase, hemicellulase, cellulase mixtures, hemicellulase mixtures and combinations of any of the foregoing.
• step d) said separating is performed by centrifugation, filtration, sieving, decantation, pressing, plate and frame filtration, vacuum filter belt, basket centrifugation, disk stack centrifugation, and sedimentation.
• centrifugation or plate and frame filtration are the preferred liquid solid separation technologies to be used.
• the invention relates to a device for performing the method according to the present invention, said device comprising a plurality of hydrolysis vessels, preferably serially arranged, means to add cellulosic biomass, water and enzyme to each hydrolysis vessel, means to perform an enzymatic hydrolysis stage in each hydrolysis vessel, means to add and remove solid fractions and liquid fractions from each hydrolysis vessel, means to separate a solid fraction from a liquid fraction for each hydrolysis vessel, means to transfer solid fractions or parts thereof from one hydrolysis vessel to another, means to transfer liquid fractions or parts thereof from one hydrolysis vessel to another, means to add enzyme to one or several hydrolysis vessels, and means to store liquid fractions or parts thereof as final product, hi one embodiment, this device also comprises a computer allowing a user to perform the method according to the present invention, which computer controls the performance of the steps of the method according to the present invention.
• said computer allows a user to define parameters of the method according to the present invention, such as, but not limited to number and duration of hydrolysis steps, temperature of the hydrolysis step(s), amount of cellulosic biomass, water, enzyme, solid fraction(s), liquid fraction(s) that is added or removed or transferred, respectively, during each step. .
• the inventors have surprisingly found that by performing a multistage counter current enzymatic hydrolysis process, a substantial increase in sugar concentration can be achieved in the final product whilst at the same time, the enzyme dose used for such hydrolysis can be reduced. Also, the biomass hydrolysis time can be reduced. Overall, the process is more efficient, consumes less enzyme and results in higher product concentrations.
• a multistage hydrolysis process which is performed in a plurality of hydrolysis vessels.
• a plurality of hydrolysis reactions are performed in parallel, and after conclusion of such parallel performed hydrolysis reactions, a transfer of components takes place in such a manner that solid fractions and liquid fractions are moved from one hydrolysis vessel to another in opposite directions.
• the plurality of hydrolysis vessels as constituting a multistage process with a front-end and a back-end, such front-end being represented by the first hydrolysis vessel and the back-end being represented by the last hydrolysis vessel, in such multistage hydrolysis process, there is a repeated feeding of cellulosic biomass and enzyme at the front end, a repeated removal of enzymatically treated biomass at the back-end, a repeated addition of water at the back-end, a repeated removal of aqueous liquid at the front-end, and a repeated transfer of solid fractions towards the back- end and a repeated transfer of liquid fractions towards the front-end.
• Such a multi stage hydrolysis process in a plurality of hydrolysis vessels, involving the repeated transfer of solid fractions and liquid fractions in opposite directions is sometimes herein also referred to as "continuous counter current enzymatic hydrolysis".
• What is removed at the back-end are the remains of the solid fractions which have been digested by the enzyme(s).
• What is removed at the front-end is an aqueous solution containing dissolved sugar(s), mainly in the form of glucose and xylose, resulting from the digestion of the cellulosic biomass by the appropriate enzymes.
• This aqueous sugar solution is then subsequently used for further downstream processing purposes.
• fresh i. e.
• enzymatically non- digested cellulosic biomass is fed into the process in the first hydrolysis vessel, i. e. a the front-end of the process, and digested cellulosic biomass, i. e. cellulosic biomass which has been exposed to enzymatic hydrolysis is removed at the back-end, i. e. typically from the last hydrolysis vessel.
• the number of hydrolysis vessels may vary and typically is in the range of from 2 - 100, preferably 2 - 50, more preferably 2 - 20, even more preferably 2 - 10, even more preferably 3 - 6, and most preferably 4.
• the cellulosic biomass which is undergoing enzymatic hydrolysis in the method according to the present invention is a cellulosic biomass that has previously under- gone a treatment by phosphoric acid or other mineral acid so as to separate the lignin- components from the cellulosic and hemicellulosic components.
• Processes for such pretreat- ment of cellulosic biomass are disclosed e.g. in WO 2007/111605 and WO 2009/114843.
• the lignocellulosic biomass is exposed to an acidic solvent to perform a solvation and/or dissolution process subsequent to which the resultant product is transferred into a separate reactor or several reactors to be further processed.
• the process results in a pretreated cellulosic biomass which can then be subjected to the method according to the present invention.
• Such procedure for pretreatment is sometimes also referred to as cellulose solvent- and solvent-based ligno cellulose fractionation (COSLIF).
• an enzyme for enzymatic hydrolysis, is used selected from cellulase, hemicellulase, mixtures of different cellulases and/or hemicellu- lases and combinations thereof.
• the cellulases and other suitable enzymes may also be recombinant enzymes which have been optimized for their function.
• the pH is adjusted to the preferred working range of the enzyme(s) used. Typically, such working range is in the range of from 4 - 7.5, preferably 4.5-7, more preferably 4.8 - 5.5.
• pH adjustment can be achieved by any suitable means, such as addition of a base or acid, as necessary, but also through the use of an appropriate buffer system.
• Suitable bases for adjusting the pH are selected from ammonium hydroxide, sodium hydroxide, potassium hydroxide, urea, lime, calcium hydroxide, sodium carbonate, potassium carbonate and others.
• Suitable buffer systems are known to persons skilled in the art, and useful examples are ammonium hydroxide, potassium hydroxide, sodium hydroxide, citrate buffer, and phosphate buffers.
• the transfer of components is initiated by separating, for each hydrolysis vessel, a solid fraction from a liquid fraction.
• Such separation can be achieved by various techniques, including, but not limited to centrifugation, filtration, decantation, pressing, sieving, plate and frame filtration, vacuum filter belt, basket centrifugation, disk stack centrifugation, and sedimentation.
• centrifugation or plate and frame filtration are the most likely liquid solid separation technologies to be used.
• Such separation may be performed in such a manner that the solid fraction remains in the respective hydrolysis vessel whereas the liquid fraction is retained separately from each respective hydrolysis vessel.
• the separation may, however, also be performed such that the liquid fraction remains in the hydrolysis vessel, whereas the solid fraction is obtained separate therefrom.
• the whole hydrolysis reaction after completion at the end of the hydrolysis stage, is transferred into a separate vessel, and the two fractions, i. e. the solid fraction and the liquid fraction are obtained separate from and outside of the respective hydrolysis vessel.
• the entire solid fraction or a part thereof is removed therefrom and discarded, and the other solid fractions resulting from hydrolysis in the other hydrolysis vessels are used to serially replenish the back-end of the hydrolysis vessel system.
• all solid fractions move up one position by one hydrolysis vessel, and the first hydrolysis vessel is filled/replenished with fresh, i. e. enzymatically undigested cellulosic biomass. It should be noted, however, that during the transfer just described, there may also occur only a partial transfer, in that only a part of each solid fraction is transferred to the next hydrolysis vessel.
• Such part may be a suitable percentage of the entire respective solid fraction, e. g. 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and any intervening percentage.
• the solid fractions or defined parts thereof from each hydrolysis vessel are transferred upstream by one hydrolysis vessel at a time, and the respective liquid fractions or defined parts thereof are transferred downstream one hydrolysis vessel at a time.
• the amounts transferred upstream are the same with respect to each other.
• the volumes transferred downstream are, in one embodiment, the same with respect to each other.
• the liquid fraction is used as the final product and represents an aqueous solution of sugar(s) typically glucose, but also, possibly, pentose(s).
• the liquid fraction from the second hydrolysis vessel is used to replenish the first hydrolysis vessel
• the liquid fraction or parts thereof form the third hydrolysis vessel is used to replenish the second hydrolysis vessel
• the liquid fraction of the nth hydrolysis vessel is used to replenish the n-lth hydrolysis vessel.
• water is added, preferably, at the same volume that was used to replenish the n-lth hydrolysis vessel.
• the system is ready for the next hydrolysis stage which is, again, performed for a defined period of time, typically in the range of from 0.5 h - 36 h and/or at a temperature of 40°C - 60°C, preferably 45°C - 57°C, more preferably 48°C - 55°C, even more preferably at the optimum temperature of the respective enzyme that is used for the enzymatic hydrolysis.
• the amount of enzyme that is initially used for the first enzymatic hydrolysis is in the range of from 0.01 wt.% - 5 wt.%, preferably 0.1 wt.% - 3 wt.%, more preferably 0.5 wt.% - 1.5 wt.% , with reference to the weight of dry biomass in said vessel.
• the solids content in each hydrolysis vessel is in the range of from 12%> to 33%.
• a high solids content can be used which thereby contributes to the good yield in final product(s).
• the number of hydrolysis stages and vessels n is 3 or 4
• the solids content in each hydrolysis vessel is 18% to 21%
• the amount of enzyme added initially and in subsequent hydrolysis stages is in the range of from 1.0 wt.% to 1.5 wt.%>, with respect to the weight of dry biomass in the first hydrolysis vessel.
• dry solids transfer also abbreviated as “DST” refers to the percentage of solids transferred from one hydrolysis vessel to the next between two hydrolysis stages
• biomass residence time also abbreviated as “BRT” refers to the amount of time the biomass spent in each reactor from its input at the front-end to its exit at the back-end.
• FIG. 1 shows a flow diagram for a multi-stage continuous counter current enzymatic hydrolysis (herein also sometimes abbreviated as CCCEH) in accordance with an embodiment of the present invention, showing few hydrolysis vessels.
• CCCEH continuous counter current enzymatic hydrolysis
• Figure 2 shows a batch hydrolysis versus a 2-stage counter current enzymatic hydrolysis with a biomass residence time of 2 days (48h) at 18% solids content and 1.5% wt enzyme dosage
• Figure 3 shows a batch hydrolysis versus a 3 -stage counter current enzymatic hydrolysis with a biomass residence time of 3 days (72 h) at 18% solids content and 1.5% wt enzyme dosage
• Figure 4 shows a batch hydrolysis versus a 4-stage counter current enzymatic hydrolysis with a biomass residence time of 4 days (96 h) at 18% solids content and 1.5% wt enzyme dosage
• Figure 5 shows a summary of batch hydrolysis versus 2,3, 4-stage counter current enzymatic hydrolysis at 18% solids content and 1.5% wt enzyme dosage
• Figure 6 shows a summary of batch hydrolysis versus 2,3, 4-stage counter current enzymatic hydrolysis at 18% solids content and 1.0% wt enzyme dosage
• Figure 7 shows a summary of 2,3, 4-stage counter current enzymatic hydrolysis at 18% solids content with 1.0% wt and 1.5% wt enzyme dosage
• Figure 8 shows a summary of a batch hydrolysis versus a 4-stage counter current enzymatic hydrolysis at 18% solids content at 1.0 % wt enzyme dosage
• Figure 9 shows a summary of a batch hydrolysis versus a 4-stage counter current enzymatic hydrolysis at 21% solids content at 1.0% wt enzyme dosage.
• Figure 10 shows a summary of batch hydrolysis versus 4-stage counter current enzymatic hydrolysis at 18% solids content, 21% solids content, 1.0% wt enzyme dosage and 3.0% wt enzyme dosage.
• the pretreatment was quenched by adding 300 kg of 95% ethanol in the reactor, the resulting slurry was pumped into a plate and frame filtration system where the pretreated biomass was washed via counter current washing with 524 kg of 95% ethanol followed by 866 kg of water. After the washing process the washed pretreated biomass was pressed in a hydraulic press to elevate the solids to approximately 38%. The washed pretreated biomass (with a moisture content of approximately 58%) was then conveyed into hydrolysis vessels where various counter current enzymatic hydrolysis experiments were performed. Hydrolysis procedure
• Figure 2 shows that the glucose concentration achieved with the batch enzymatic hydrolysis after 2 days was 73 g/L whereas steady state was achieved for the counter current enzymatic hydrolysis with 2 days biomass residence time (BRT) after approximately 5 days with an average glucose concentration of 99 g/L.2-stage continuous counter current enzymatic hydrolysis shows a 36% increase in glucose concentration compared to batch hydrolysis.
• Figure 3 shows that the glucose concentration achieved with the batch enzymatic hydrolysis after 3 days was 84 g/L whereas steady state was achieved for the counter current enzymatic hydrolysis with 3 days biomass residence after approximately 4 days with an average glucose concentration of 113 g/L.
• 3 -stage continuous counter current enzymatic hydrolysis show a 35% increase in glucose concentration compare to batch hydrolysis
• the hydrolysis vessels HV1, HV2 and HV3 were centrifuged, the liquid fractions labeled LFl (200 g), LF2 (257 g) LF3 (239 g) and LF4 (237 g) were separated from the solids fractions SF1 (72 g dry), SF2 (72 g dry), SF3 (72 g dry) and SF4 (72 g dry) that remained in their respective hydrolysis vessels.
• the liquid transfer was performed as follows:
• Figure 4 shows that the glucose concentration achieved with the batch enzymatic hydrolysis after 4 days was 86 g/L whereas steady state was achieved for the counter current enzymatic hydrolysis with 4 days biomass residence after approximately 5 days with an average glucose concentration of 127 g/L.
• 4-stage continuous counter current enzymatic hydrolysis show a 47% increase in glucose concentration compare to batch hydrolysis.
• the initial enzyme added in all hydrolysis vessel at the beginning of enzymatic hydrolysis was 0.72 g.
• the make-up enzyme added in HV1 after each transfer was 0.72 g.
• Figure 6 shows that the glucose concentration achieved with the batch enzymatic hydrolysis after 2, 3 and 4 days was respectively 79, 80, and 88 g/L whereas steady state was achieved for the counter current enzymatic hydrolysis with respectively 2, 3 and 4 days biomass residence after 7, 6 and 6 days with an average glucose concentration of respectively 100, 109 and 116 g/L.
• the liquid transfer was performed as follows:
• the hydrolysis vessels HV1, HV2 and HV3 were centrifuged, the liquid fractions labeled LF1 (186 g), LF2 (185 g) LF3 (189 g) and LF4 (192 g) were separated from the solids fractions SF1 (84 g dry), SF2 (84 g dry), SF3 (84 g dry) and SF4 (84 g dry) that remained in their respective hydrolysis vessels.
• the liquid transfer was performed as follows:
• Figure 8 shows that the glucose concentration achieved with the batch enzymatic hydrolysis at 18% solids content after 4 days was 88 g/L whereas steady state was achieved for the counter current enzymatic hydrolysis at 18% solids content with 4 days biomass residence after approximately 7 days with an average glucose concentration of 124 g/L.
• 4-stage continuous counter current enzymatic hydrolysis shows a 40% increase in glucose concentration compared to batch hydrolysis.
• Figure 9 shows that the glucose concentration achieved with the batch enzymatic hydrolysis at 2P/o solids content after 4 days was 112 g/L whereas steady state was achieved for the counter current enzymatic hydrolysis at 21% solids content with 4 days biomass residence after approximately 7 days with an average glucose concentration of 136 g/L.
• 4-stage continuous counter current enzymatic hydrolysis shows a 22% increase in glucose concentration compared to batch hydrolysis.
• % MC refers to moisture content.
• % TS refers to total solids.
• Total solids content in HYD refers to the amount of dry solids that will be present in the hydrolysis vessel(s) during enzymatic hydrolysis.

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