WO2014191267A1 - Procédé d'hydrolyse enzymatique d'un matériau lignocellulosique - Google Patents

Procédé d'hydrolyse enzymatique d'un matériau lignocellulosique Download PDF

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Publication number
WO2014191267A1
WO2014191267A1 PCT/EP2014/060392 EP2014060392W WO2014191267A1 WO 2014191267 A1 WO2014191267 A1 WO 2014191267A1 EP 2014060392 W EP2014060392 W EP 2014060392W WO 2014191267 A1 WO2014191267 A1 WO 2014191267A1
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enzyme
hydrolysis
protein
oxygen
cellulases
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PCT/EP2014/060392
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English (en)
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Bertus Noordam
Michael Petrus Jozef BERKHOUT
DE Johannes Hendrikus LOOFF
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Dsm Ip Assets B.V.
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Publication of WO2014191267A1 publication Critical patent/WO2014191267A1/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
    • 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
    • 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
    • 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)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • 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
    • 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
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • the invention relates to a process for the enzymatic hydrolysis of lignocellulosic material.
  • Ligno-cellulosic plant material herein also called feedstock or cellulose containing material
  • feedstock or cellulose containing material
  • cellulose containing material is a renewable source of energy in the form of sugars that can be converted into valuable products e.g. bio-fuel, such as bio-ethanol.
  • bio-fuel such as bio-ethanol.
  • (ligno or hemi)-cellulose present in the feedstock such as wheat straw, corn stover, rice hulls, etc.
  • (hemi)-cellulolytic enzymes which then are converted into valuable products such as ethanol by microorganisms like yeast, bacteria and fungi.
  • Glucan or cellulose is a polysaccharide of linked glucose units.
  • the conversion into reducing sugars is in general slow and incomplete.
  • enzymatic hydrolysis of untreated feedstock yields less than 20% sugars compared to the theoretical quantity.
  • the feedstock is hydrolysed to liberate sugars for further fermentation. Fermentation into ethanol is used herein to show the possibilities for the products of the hydrolysis process, but other fermentations or other uses of the hydrolysis products are equally known and applied such as biogas as will discussed hereinafter.
  • a typical ethanol yield from glucose, derived from pre-treated corn stover, is 40 gallons of ethanol per 1000 kg of dry corn stover (Badger, P, Ethanol from cellulose: a general review, Trends in new crops and new uses. 2002. J. Janick and A. Whipkey (eds.) ASHS Press, Alexandria, VA), or 0.3 g ethanol per gram feedstock.
  • Cellulolytic enzymes -most of them are produced by species like Trichoderma, Humicola and Aspergillus- are commercially used to convert pre-treated feedstock into a mash containing reducing sugars made from cellulose and hemicellulose, optionally containing lignin and/or other polymers. This mash is then used in a fermentation during which the reducing sugars are converted into for example yeast biomass (cells), carbon dioxide and ethanol. The ethanol produced in this way is called bio-ethanol.
  • the fermentation following the hydrolysis takes place in a separate anaerobic process step, either in the same or in a different vessel, in which temperature is adjusted to 30-33 °C (mesophilic process) to accommodate growth and ethanol production by microbial biomass, commonly yeasts.
  • microbial biomass commonly yeasts.
  • SSF Simultaneously Saccharification and Fermentation
  • the so obtained fermented mash consists of non-fermentable sugars, non- hydrolysable (hemi) cellulosic material, lignin, microbial cells (most common yeast cells), water, ethanol, dissolved carbon dioxide. During the successive steps, ethanol is distilled from the mash and further purified. The remaining solid suspension is dried and used as, for instance, burning fuel, fertilizer or cattle feed.
  • Thermostable cellulolytic enzymes derived from Talaromyces have been used for degrading ligno-cellulosic feedstock and these enzymes are known for their thermostability in WO2007091231 . However, no disclosure is given how to optimize the process of hydrolysis.
  • WO201 1/042437 an enzyme dosage of 6 mg of cellulase (per dram dry matter biomass) or less is disclosed which corresponds to an enzyme dosage of 17 mg of cellulase (per gram dry matter cellulose or glucan) or less.
  • An object of the invention is to provide a process for the hydrolysis of cellulose containing biomass which comprises
  • cellulases or the cellulase-containing composition comprises BG and another cellulase
  • the amount of BG is at least 4 wt% of the cellulases (on protein) or the amount of BG is at least 4 wt% of the protein present in the cellulases or a cellulase-containing composition added, preferably the amount of BG is at least 4 wt% of the cellulases (on protein).
  • the pretreated cellulose containing biomass is washed before the hydrolysis.
  • a sugar product is recovered from the saccharified or hydrolysed ligno-cellulosic material and/or the saccharified or hydrolysed ligno-cellulosic material is fermented to produce a fermentation product which step may be followed by the recovery of the fermentation product.
  • oxygen is added to the ligno-cellulosic material, more preferably during a part of the time of the enzymatic hydrolysis or saccharification, oxygen is added to the ligno-cellulosic material and during part of the time of the enzymatic hydrolysis less oxygen is added to the ligno-cellulosic material compared to the other part of the time of the enzymatic hydrolysis, preferably no oxygen is added to the ligno-cellulosic material.
  • the oxygen concentration in the liquid phase of the hydrolysis during the part of the time wherein oxygen is added is at least 2 times, preferably at least 4 times, more preferably at least 10 times the oxygen concentration in the liquid phase during the part of the time wherein less or no oxygen is added.
  • the oxygen concentration in the liquid phase, wherein the ligno-cellulosic material is present during the enzymatic hydrolysis is at least 0.001 mol/m 3 , preferably at least 0.002 mol/m 3 and most preferably at least 0.003 mol/m 3 and even more preferably more than 0.01 mol/m 3 , for example more than 0.02 mol/m 3 or 0.03 mol/m 3 .
  • DO values of below 0.01 mol/m 3 or 0.02 mol/m 3 will be obtained by slow stirring.
  • Vigorous mixing or stirring at such scale introduces part of the gas phase of the headspace into the reaction liquid.
  • the mixing or stirring may create a whirlpool that draws oxygen into the liquid.
  • flushing the headspace with oxygen (for example in the form of air) in combination with (vigorous) mixing or stirring will introduce sufficient oxygen into the cellulosic material in the hydrolysis reactor for reactors up to a size of 100 liter to 1 m 3 .
  • oxygen for example in the form of air
  • vigorous mixing or stirring will introduce sufficient oxygen into the cellulosic material in the hydrolysis reactor for reactors up to a size of 100 liter to 1 m 3 .
  • At larger scale for example in a reactor of 50 m 3 or more, for example 100 m 3 , so much energy is needed for vigorous stirring that from economic point of view this will not be applied in a commercially operating process.
  • stirring or mixing without introducing air or oxygen will result in DO values of less than 0.01 mol/m 3 .
  • the oxygen concentration in the liquid phase, wherein the ligno-cellulosic material is present during the enzymatic hydrolysis is preferably at most 80% of the saturation concentration of oxygen under the hydrolysis reaction conditions, more preferably at most 0.12 mol/m 3 , still more preferably at most 0.09 mol/m 3 , even more preferably at most 0.06 mol/m 3 , even still more preferably at most 0.045 mol/m 3 and most preferably at most 0.03 mol/m 3 .
  • Temperature and pressure will influence the DO.
  • the preferred and exemplary mol/m 3 values given above relate to normal atmospheric pressure and a temperature of about 62 °C. The skilled person in the art will appreciate favourable DO values on basis of the present teachings.
  • the reactor for the enzymatic hydrolysis has a volume of 1 m 3 or more.
  • the enzymatic hydrolysis time of the present process is preferably from 5 to 150 hours.
  • the enzyme composition is derived from a fungus, preferably a microorganism of the genus Rasamsonia or the enzyme composition comprises a fungal enzyme, preferably a Rasamsonia enzyme.
  • the dry matter content in the hydrolysis step c) is 10 wt% or more, preferably is 14 wt% or more and still more preferably is 14 to 33% wt%.
  • the enzymatic hydrolysis preferably takes place in a batch, fed batch and/or continuous culture reactor.
  • the oxygen that is introduced in the present process is an oxygen-containing gas such as air.
  • oxygen-containing gas such as air.
  • less oxygen is added to or is present in the ligno-cellulosic material during part of the time of the enzymatic hydrolysis is meant that at least 50% less, preferably at least 70% less, most preferably at least 90% less of oxygen (expressed in mol oxygen/m 3 ) is introduced, for example in bubble-form or is present than is added or is present during the other part of the time of the enzymatic hydrolysis wherein less oxygen is added.
  • oxygen is added in the form of (gaseous) bubbles.
  • the stable enzyme composition used retains activity for 30 hours or more.
  • the hydrolysis is preferably conducted at a temperature of 40°C or more, more preferably at a temperature of 50°C or more and most preferably at a temperature of 55°C or more. The process of the invention will be illustrated in more detail below.
  • the amount of enzyme used herein is the amount of protein determined by TCA Biuret (see Examples).
  • the amount of the individual proteins or enzymes is preferably determined using SDS page (see Examples).
  • the cellulases or the cellulase-containing composition further comprise GH61 , EG, CBH1 and/or CBH2, more preferably GH61 and/or EG, CBH1 and CBH2.
  • the cellulases can be added separately or combined as a cellulase-containing composition.
  • cellulase on protein
  • cellulose containing biomass on dry matter
  • cellulase on protein
  • about 0.4 g cellulose is present in 1 g cellulose containing biomass (on dry matter) such as corn stover.
  • the amount of BG is between 4 and 20 wt% of the cellulases (on protein) and more preferably between 4 and 10 wt% of the cellulases (on protein) added in the process of the invention. Even more preferably the amount of BG is between 5 and 10 wt% of the cellulases (on protein), between 6 and 10 wt% of the cellulases (on protein) or between 7 and 10 wt% of the cellulases (on protein) added in the process of the invention.
  • the amount of BG is between 4 and 8 wt% of the cellulases (on protein), 5 and 8 wt% of the cellulases (on protein), 6 and 8 wt% of the cellulases (on protein) or 7 and 8 wt% of the cellulases (on protein) added in the process of the invention.
  • Figure 1 the effect of enzyme dosage on cellobiose concentration after 140 hours of incubation.
  • the cellobiose concentration (g/l) in the hydrolysis process is given as function of the dosage of TEC-210 (mg enzyme / g feedstock on dry matter).
  • FIG. 2 the effect of extra BG on cellulose hydrolysis, glucose concentration (g/l) is shown as function of time (hours), - ⁇ - 1 .25 mg TEC-210; - A- 2.50 mg TEC- 210; - ⁇ - 1 .25 mg TEC-210 +1 .25 mg BG.
  • Figure 3 the effect of BG addition on glucose production.
  • the present invention is directed to reduce operational costs such as enzyme costs, reactor costs and energy costs, in a process for the hydrolysis of cellulose containing biomass.
  • Enzyme costs can be reduced by using less enzyme and at same time maintaining high production results such as high levels of sugars, ethanol, biogas or other desired products, all compared to known hydrolysis processes.
  • Reactor cost can be reduced by selecting process conditions which result in lower total reactor volume compared to known hydrolysis processes. Energy savings can be obtained according to the invention by a lower need of energy supply to the process of the invention.
  • high-amount enzyme dosage is meant the use of at least 10 or 12 mg cellulase (on protein) per gram cellulose present in the cellulose containing biomass (on dry matter).
  • a cellulase-containing composition used wherein BG (beta-glucosidase or ⁇ -glucosidase) is present in at least 4 wt% of the cellulases (on protein) or the amount of BG is at least 4 wt% of the protein present in the cellulases or a cellulase-containing composition added, preferably the amount of BG is at least 4 wt% of the cellulases (on protein) is favourable for the glucose production.
  • cellulases or a cellulase-containing composition whereby the amount of BG ( ⁇ -glucosidase) is at least 4 wt% of the cellulases (on protein) or the amount of BG is at least 4 wt% of the protein present in the cellulases or a cellulase-containing composition added, preferably the amount of BG is at least 4 wt% of the cellulases (on protein).
  • BG ⁇ -glucosidase
  • the amount of BG is between 4 and 20 wt%, preferably between 4 and 10 wt%, more preferably between 4 and 8 wt% of the cellulases (on protein) added or the amount of BG is between 4 and 20 wt%, preferably between 4 and 10 wt%, more preferably between 4 and 8 wt% of the protein present in the cellulases or a cellulase-containing composition added, preferably the amount of BG is between 4 and 20 wt%, preferably between 4 and 10 wt%, more preferably between 4 and 8 wt% of the cellulases (on protein) added in the process of the invention.
  • the cellulases or the cellulase-containing composition further comprise GH61 , EG (endo-glucanase), CBH1 and/or CBH2 (cellobiohydrolase), preferably GH61 and/or EG, CBH1 and CBH2.
  • An enzyme composition can be made suitable for the process of the invention by adding, if needed, extra BG to a cellulase composition.
  • BG can be produced by fermenting suitable host cells or microorganisms.
  • the suitable cells or microorganisms can be of any suitable source or can be a mutant or recombinant strain which is mutated, selected or designed for this purpose.
  • the enzyme composition is produced by one microorganism which is suitable to produce the (complete) enzyme composition for use in the process of the invention.
  • This microorganism can be of any suitable source or can be a mutant or recombinant strain which is mutated, selected or designed for this purpose.
  • the cellulases or the cellulase-containing composition comprise at least one cellulase which is thermostable, preferably at least a BG which is thermostabile and more preferably at least 80% of the cellulases (on protein) is thermostable.
  • the hydrolysis of the present invention shows results in an improved reaction product that gives higher amounts of (reduced) sugar products and/or desired fermentation products in the fermentation following the hydrolysis as compared to a process wherein no oxygen is added.
  • an increase of the glucose conversion is observed of 5 to 15 w/w%, or even up to 25 w/w%.
  • Oxygen can be added in several ways. For example oxygen can be added as oxygen gas, oxygen enriched gas such as oxygen enriched air or air (example of oxygen containing gas). Oxygen can be added continuously or dis-continuously.
  • oxygen “is added” is meant that oxygen is added to the liquid phase (comprising the ligno-cellulosic material) in the hydrolysis reactor and not that oxygen is present in the headspace in the reactor above the liquid phase (in combination with slow or no stirring) whereby the oxygen has to diffuse from the headspace to the liquid phase. So preferably the oxygen is added as bubbles, most preferably as small bubbles.
  • oxygen-containing gas can be introduced, for example blown, into the liquid hydrolysis reactor contents of cellulolytic material.
  • oxygen-containing gas is introduced into the liquid cellulolytic material stream that will enter the hydrolysis reactor.
  • oxygen containing gas is introduced together with the cellulolytic material that enters the hydrolysis reactor or with part of the liquid reactor contents that passes an external loop of the reactor.
  • the addition of oxygen before entering the hydrolysis reactor is not sufficient enough and oxygen addition may be done during the hydrolysis as well.
  • the gaseous phase present in the upper part of the reactor (head space) is continuously or dis-continuously refreshed with the oxygen-containing gas.
  • (vigorous) mixing or stirring is needed to get the oxygen as bubbles and/or by diffusion into the liquid reactor contents preferably in combination with over-pressure in the reactor.
  • flushing the headspace with air in combination with (vigorous) mixing or stirring may introduce sufficient oxygen into the cellulosic material in the hydrolysis reactor for reactors up to a size of 100 liter to 1 m 3 .
  • At larger scale for example in a reactor of 50 m 3 or more, for example 100 m 3 , so much energy is needed for vigorous stirring that from economic point of view this will not be applied in a commercially operating process.
  • the oxygen may be added during part of the hydrolysis step.
  • the addition of oxygen during only part of the hydrolysis may be done for example in case of oxidation damage of the enzyme(s) occurs.
  • oxygen addition may be done except for the last part of the hydrolysis and thus (most of) the oxygen is consumed before the hydrolysed biomass enters the fermentation reactor.
  • the oxygen preferably in the form of (gaseous) bubbles, is added in the last part of the hydrolysis step.
  • the inventors have also noticed that aeration during an enzymatic hydrolysis process in the beginning of the hydrolysis process results in an increased glucose production during the hydrolysis.
  • the part of the time wherein less or preferably no oxygen is added is 10 to 80 %, preferably 20 to 80%, more preferably 30 to 80% and most preferably 40 to 80% of the total enzymatic hydrolysis time.
  • the part of the time wherein more oxygen is added is 2 to 80 %, preferably 4 to 60%, more preferably 8 to 50% and most preferably 10 to 50% of the total enzymatic hydrolysis time.
  • the oxygen concentration in the liquid phase during the part of the time wherein oxygen is added is at least 2 times, preferably at least 4 times, more preferably at least 10 times the oxygen concentration in the liquid phase during the part of the time wherein less or no oxygen is added.
  • the oxygen concentration (DO) in the liquid phase wherein the ligno-cellulosic material is present during the enzymatic hydrolysis is at least 0.001 mol/m 3 , preferably at least 0.002 mol/m 3 , more preferably at least 0.003 mol/m 3 and even more preferably more than 0.01 mol/m 3 , for example more than 0.02 mol/m 3 or 0.03 mol/m 3 .
  • DO values of below 0.01 mol/m 3 or 0.02 mol/m 3 will be obtained by slow stirring.
  • Vigorous mixing or stirring at such scale introduces part of the gas phase of the headspace into the reaction liquid.
  • the mixing or stirring may create a whirlpool that draws oxygen into the liquid.
  • flushing the headspace with air in combination with (vigorous) mixing or stirring will introduce sufficient oxygen into the cellulosic material in the hydrolysis reactor for reactors up to a size of 100 liter to 1 m 3 .
  • At larger scale for example in a reactor of 50 m 3 or more, for example 100 m 3 , so much energy is needed for vigorous stirring that from economic point of view this will not be applied in a commercially operating process.
  • stirring or mixing without introducing air or oxygen will result in DO values of less than 0.01 mol/m 3 .
  • the oxygen concentration in the liquid phase wherein the ligno- cellulosic material is present during the enzymatic hydrolysis is during the part of the time wherein oxygen is added preferably at most 80% of the saturation concentration of oxygen under the hydrolysis reaction conditions, more preferably at most 0.12 mol/m 3 , still more preferably at most 0.09 mol/m 3 , even more preferably at most 0.06 mol/m 3 , even still more preferably at most 0.045 mol/m 3 and most preferably at most 0.03 mol/m 3 .
  • Temperature and pressure will influence the DO.
  • the preferred and exemplary mol/m 3 values given above relate to normal atmospheric pressure and a temperature of about 62 °C. The skilled person in the art will appreciate favourable DO values on basis of the present teachings.
  • the oxygen concentration in the liquid phase, wherein the ligno-cellulosic material is present during the enzymatic hydrolysis is during the part of the time wherein less or no oxygen is added less than 0.02 mol/m 3 , preferably less than 0.01 mol/m 3 , more preferably less than 0.005 mol/m 3 , and most preferably less than 0.001 mol/m 3 .
  • the oxygen addition in the form of air or other oxygen-containing gas according to the invention may also be used to at least partially stir or mix the hydrolysis reactor contents.
  • the present process of the invention shows especially on pilot plant and industrial scale advantages.
  • the hydrolysis reactor has a volume of 1 m 3 or more, preferably of more than 10 m 3 and most preferably of 50 m 3 or more.
  • the hydrolysis reactor will be smaller than 3000 m 3 or 5000 m 3
  • the inventors pose the theory that especially at large scale insufficient oxygen is available for the hydrolysis which might be due to oxygen transfer limitations in the reactor for example in the cellulolytic biomass. On lab-scale experiments this oxygen-insufficiency may play a less important role.
  • the surface area (or oxygen contact area of the reactor content) to reactor volume ratio is more favourable for small scale experiments than in large scale experiments. Moreover mixing in small scale experiments is relatively easier than at large scale.
  • the transport of oxygen from the headspace of the hydrolysis reactor is faster than compared to the situation in large scale experiments.
  • This theory is only given as possible explanation of the effect noticed by the inventors, and the present invention does not fall or stands with the correctness of this theory.
  • the addition of oxygen may be used to control at least partially the hydrolysis process.
  • Lignocellulosic material herein includes any lignocellulosic and/or hemicellulosic material and is composed mainly of cellulose, hemicellulose and lignin.
  • Lignocellulosic material suitable for use as feedstock or cellulose containing material in the invention includes biomass, e.g. virgin biomass and/or non-virgin biomass such as agricultural biomass, commercial organics, construction and demolition debris, municipal solid waste, waste paper and yard waste.
  • biomass include trees, shrubs and grasses, wheat, wheat straw, sugar cane bagasse, switch grass, miscanthus, corn, corn stover, corn husks, corn cobs, canola stems, soybean stems, sweet sorghum, corn kernel including fiber from kernels, products and by-products from milling of grains such as corn, wheat and barley (including wet milling and dry milling) often called "bran or fibre” as well as municipal solid waste, waste paper and yard waste.
  • the biomass can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues.
  • Agricultural biomass includes branches, bushes, canes, corn and corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short rotation woody crops, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, and hard and soft woods (not including woods with deleterious materials).
  • agricultural biomass includes organic waste materials generated from agricultural processes including farming and forestry activities, specifically including forestry wood waste. Agricultural biomass may be any of the aforementioned singularly or in any combination or mixture thereof.
  • the feedstock may optionally be pre-treated with heat, mechanical, microbial, enzymatic and/or chemical modification or any combination of such methods in order to to enhance the accessibility of the substrate to enzymatic hydrolysis and/or hydrolyse the hemicellulose and/or solubilize the hemicellulose and/or cellulose and/or lignin, in any way known in the art (see for example P. Kumar, Ind. Eng. Chem. Res., 2009, 48 (8), pp 3713-3729).
  • the pre-treatment is conducted treating the lignocellulose with steam explosion, hot water treatment or treatment with dilute acid or dilute base.
  • the process according to the invention comprises a washing step.
  • the optional washing step may be used to remove water soluble compounds that may act as inhibitors for the fermentation step.
  • the washing step may be conducted in known manner.
  • the washing step may be done for example before an optional pretreatment, between an optional pretreatment and a hydrolysis, between an optional liquefaction and saccharification, between a hydrolysis and a fermentation or during the hydrolysis.
  • Carbohydrate degradation can be performed in a separate liquefaction step followed by a sugar production step, or both steps can be performed simultaneously for example in one reactor.
  • the liquefaction step is at least partly done before the sugar producing step, different enzymes or enzyme compositions can be used for the liquefaction and the sugar producing step or saccharification.
  • the liquefaction step is combined with the sugar production step in general one enzyme composition will be used.
  • sugars including mono sugars
  • saccharification is made.
  • the liquefaction is directed to make the (pretreated) feedstock more liquid and the saccharification or hydrolysis step is directed to produce mono sugars.
  • the process according to the invention comprises the liquefaction of the feedstock.
  • optionally pre-treated and optionally washed ligno-cellulosic material is brought into contact with a first enzyme or (first) enzyme composition.
  • the liquefaction can be done separate from the saccharification or done as part of the saccharification.
  • the different reaction conditions e.g. temperature, enzyme dosage, hydrolysis reaction time and dry matter concentration, may be adapted by the skilled person in order to achieve a desired conversion of lignocellulose.
  • a first enzyme or (first) enzyme composition is added to liquefy at least part of the solids present in the biomass and to keep the viscosity of the cellulose containing biomass in this step below 1000 cP, preferably below 800 cP, more preferably below 600 cP.
  • the cellulose containing biomass in this step has preferably a dry matter content of 5 wt% or higher, 8 wt% or higher, 10 wt% or higher, 1 1 wt% or higher, 12 wt% or higher, 13 wt% or higher, 14 wt% or higher, 15 wt% or higher, 20 wt% or higher, 25 wt% or higher, 30 wt% or higher, 35 wt% or higher or 40 wt% or higher and preferably less than 42 wt%.
  • the dry matter content in the liquefaction step is 14 wt% or more, 15 wt% or more, 16 wt% or more, 17 wt% or more, 18 wt% or more, 19 wt% or more, 20 wt% or more, 21 wt% or more, 22 wt% or more, 23 wt% or more, 24 wt% or more, 25 wt% or more, 26 wt% or more, 27 wt% or more, 28 wt %, 29 wt% or more, 30 wt% or more, 31 wt% or more, 32 wt% or more, or 33 wt% or more. More preferably the dry matter content in the liquefaction step is between 12 and 35 wt%, more preferably between 14 and 33 wt%, and most preferably between 15 and 30 wt%.
  • the cellulose containing biomass will have a viscosity of higher than 1000 cP, for example 2000 cP before liquefaction and/or saccharification.
  • a first enzyme or (first) enzyme composition is added to liquefy at least part of the solids present in the biomass to obtain a viscosity reduction factor of at least 2, at least 4, at least 6, at least 10, at least 15 or at least 20 in the liquefaction step.
  • the viscosity reduction factor will be less than 50.
  • viscosity reduction factor is meant the viscosity of the biomass which enters the liquefaction step divided by the viscosity of the biomass that leaves the liquefaction step (cP / cP).
  • the liquefaction reactor may contain water and cellulose containing biomass (and the first enzyme or (first) enzyme composition) in an amount that the viscosity is lower than 1000 cP, preferably below 800 cP, more preferably below 600 cP.
  • the viscosity is decreased due to enzyme added, more cellulose containing biomass can be added and thus the dry matter content in the liquefaction step will increase.
  • This start-up procedure can be done until the desired dry matter content in the liquefication reactor is reached. Subsequently the liquefaction step can be continued in fed-batch, semi-continuous or continuous mode.
  • Another way of starting up is a procedure wherein the cellulose containing biomass in the liquefaction step has initially a viscosity of higher than 500 cP.
  • the first enzyme or (first) enzyme composition has decreased the viscosity below 1000 cP, preferably below 800 cP, more preferably below 600 cP
  • the liquefaction step is continued in fed-batch, semi-continuous or continuous mode.
  • more first enzyme or (first) enzyme composition is dosed (per dry matter amount of cellulose containing biomass) in the start-up phase than in the subsequent fed-batch, semi-continuous or continuous mode.
  • the first enzyme or (first) enzyme composition which is added in the liquefaction step may be different from or the same as the (second) enzyme composition used in the saccharification step.
  • the first enzyme or (first) enzyme composition may comprise an endoglucanase (EG) or GH61 , preferably a thermostabile EG or GH61 .
  • the first enzyme or (first) enzyme composition may comprise more endogluconase than the (second) enzyme composition (expressed in cellulase protein).
  • the liquefaction step is conducted at a temperature of 50°C or more, 55°C or more, 60°C or more, 65°C or more, or 70°C or more.
  • the high temperature during liquefaction has many advantages, which include working at the optimum temperature of the enzyme composition, the reduction of risk of (bacterial) contamination, higher enzyme activity, smaller amount of cooling water required, use of cooling water with a higher temperature, re-use of the enzymes and more.
  • the total or sum of the cellulase(s) added is less than 10 mg cellulase (on protein) per gram cellulose present in the cellulose containing biomass (on dry matter) and whereby the amount of BG is added at least 4 wt% of the cellulases (on protein) or the amount of BG is at least 4 wt% of the protein present in the cellulases or a cellulase-containing composition added, preferably the amount of BG is at least 4 wt% of the cellulases (on protein).
  • the total or sum of the cellulase(s) added is less than 7 mg, preferably less than 5 mg, more preferably less than 4 mg, even more preferably less than 3 mg, most preferably between 3 and 0.2 mg per gram cellulose present in the cellulose containing biomass (on dry matter) in the process of the invention.
  • the liquefaction time is 10 hours or less, 5 hours or less, 3 hours or less, or 2 hours or less.
  • the hydrolysis reaction time is 10 hours to 1 minute, 5 hours to 3 minutes, or 3 hours to 5 minutes. Due to the stability of the enzyme or enzyme composition, the enzyme or enzyme composition may remain active in the next (saccharification) step.
  • the pH during liquefaction may be chosen by the skilled person.
  • the pH during the liquefaction may be 3.0 - 6.4.
  • the stable enzymes of the invention may be chosen to have a broad pH range of up to 2 pH units, up to 3 pH units, up to 5 pH units. The optimum pH may lay within the limits of pH 2.0 to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to 6.0 3.5 to 5.0, 3.5 to 4.5, 4.0 to 4.5 or is about 4.2.
  • a process of the invention may be carried out using high levels of dry matter (of the lignocellulosic material) in the liquefaction step.
  • the invention may be carried out with a dry matter content of 5 wt% or higher, 8 wt% or higher, 10 wt% or higher, 1 1 wt% or higher, 12 wt% or higher, 13 wt% or higher, 14 wt% or higher, 15 wt% or higher, 20 wt% or higher, 25 wt% or higher, 30 wt% or higher, 35 wt% or higher or 40 wt% or higher and preferably less than 42 wt%.
  • the dry matter content in the liquefaction step is 14 wt% or more, 15 wt% or more, 16 wt% or more, 17 wt% or more, 18 wt% or more, 19 wt% or more, 20 wt% or more, 21 wt% or more, 22 wt% or more, 23 wt% or more, 24 wt% or more, 25 wt% or more, 26 wt% or more, 27 wt% or more, 28 wt %, 29 wt% or more, 30 wt% or more, 31 wt% or more, 32 wt% or more, or 33 wt% or more. More preferably the dry matter content in the liquefaction step is between 12 and 35 wt%, more preferably between 14 and 33 wt%, and most preferably between 15 and 30 wt%.
  • the liquefaction reactor has a volume of 1 m 3 or more, preferably of more than 10 m 3 and most preferably of 50 m 3 or more. In general the liquefaction reactor will be smaller than 3000 m 3 or 5000 m 3 .
  • the process according to the invention comprises an enzymatic saccharification step.
  • the enzymatic hydrolysis includes, but is not limited to hydrolysis for the purpose of releasing sugar from the liquefied feedstock.
  • this step optionally pre-treated and optionally washed ligno-cellulosic material is maintained, after liquefaction, into contact with the (second) enzyme composition.
  • the different reaction conditions e.g. temperature, enzyme present or optionally dosed, hydrolysis reaction time and dry matter concentration, may be adapted by the skilled person in order to achieve a desired conversion of lignocellulose to sugar. Some indications are given hereafter.
  • a (second) enzyme composition is added to form oligomeric and/or monomeric sugars; and the (second) enzyme composition comprises a cellulase.
  • the (second) composition preferably comprises at least two different cellobiohydrolases and a beta-glucosidase and/or GH61.
  • the saccharification is conducted at a temperature of 50°C or more, 55°C or more, 60°C or more, 65°C or more, or 70°C or more.
  • the high temperature during hydrolysis has many advantages, which include working at the optimum temperature of the enzyme composition, the reduction of risk of (bacterial) contamination, smaller amount of cooling water required, use of cooling water with a higher temperature, re-use of the enzymes and more.
  • the amount of (second) enzyme composition optionally added (herein also called enzyme dosage or enzyme load) is low. Low enzyme dosage is possible, since because of the activity and stability of the enzymes, it is possible to increase the saccharification reaction time.
  • the saccharification reaction time in the saccharification step is 40 hours or more, 50 hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90 hours or more, 100 hours or more, 120 hours or more, 130 h or more.
  • the hydrolysis reaction time is 40-130 hours, 50-120 hours, 60-120 hours, 60-1 10 hours, 60-100 hours, 70-100 hours, 70-90 hours or 70-80 hours. Due to the stability of the enzyme composition longer hydrolysis reaction times are possible with corresponding higher sugar yields.
  • the pH during hydrolysis in the saccharification step may be chosen by the skilled person.
  • the pH during the hydrolysis may be 3.0 - 6.4.
  • the stable enzymes of the invention may have a broad pH range of up to 2 pH units, up to 3 pH units, up to 5 pH units.
  • the optimum pH may lie within the limits of pH 2.0 to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to 6.0 3.5 to 5.0, 3.5 to 4.5, 4.0 to 4.5 or is about 4.2.
  • the saccharification step is conducted until 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more of available sugar in lignocellulosic material is released.
  • a process of the invention may be carried out using high levels of dry matter (of the lignocellulosic material) in the saccharification step.
  • the invention may be carried out with a dry matter content of 5 wt% or higher, 8 wt% or higher, 10 wt% or higher, 1 1 wt% or higher, 12 wt% or higher, 13 wt% or higher, 14 wt% or higher, 15 wt% or higher, 20 wt% or higher, 25 wt% or higher, 30 wt% or higher, 35 wt% or higher or 40 wt% or higher and preferably less than 42 wt%.
  • the dry matter content in the saccharification step is 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt% or more or 14-33 wt%.
  • the saccharification reactor has a volume of 1 m 3 or more, preferably of more than 10 m 3 and most preferably of 50 m 3 or more.
  • the saccharification reactor will be smaller than 3000 m 3 or 5000 m 3
  • thermostable enzymes under optimal temperature conditions
  • the invention relates to the use of a thermostable enzyme such as a cellulolytic enzyme of Talaromyces in a saccharification process, optionally a separate liquefaction and saccharification (SLS) process for the production of reducing sugars from pre-treated ligno-cellulosic feedstock in, but not limiting to, ethanol production.
  • a thermostable enzyme such as a cellulolytic enzyme of Talaromyces in a saccharification process, optionally a separate liquefaction and saccharification (SLS) process for the production of reducing sugars from pre-treated ligno-cellulosic feedstock in, but not limiting to, ethanol production.
  • SLS separate liquefaction and saccharification
  • thermostable cellulolytic enzymes such as from Talaromyces
  • thermophilic ethanol-producing microorganisms thermophilic ethanol-producing microorganisms
  • fermentation times will be shorter as cellulolytic enzymes of Talaromyces release the reducing sugars faster at higher temperatures than at mesophilic temperatures.
  • the product concentration (g/L) is dependent on the amount of glucose produced, but this is not visible since sugars are converted to product in the SSF, and product concentrations can be related to underlying glucose concentration by multiplication with the theoretical maximum yield.
  • Rasamsonia is a new genus comprising thermotolerant and thermophilic Talaromyces and Geosmithia species (J.Houbraken et al vida supra). Based on phenotypic, physiological and molecular data, Houbraken et al proposed to transfer the species T. emersonii, T. byssochlamydoides, T. eburneus, G. argillacea and G. cylindrospora to Rasamsonia gen. nov. Talaromyces emersonii, Penicillium geosmithia emersonii and Rasamsonia emersonii are used interchangeably herein.
  • the (second) enzyme composition comprises a cellulase, preferably at least 2 cellulases, more preferably two different cellobiohydrolases, BG and optionally GH61 .
  • a cellulase preferably at least 2 cellulases, more preferably two different cellobiohydrolases, BG and optionally GH61 .
  • at least one, more preferably at least two, even more preferably at least three of the at least three different enzymes are thermostable.
  • thermostable enzyme means that the enzyme has a temperature optimum 60°C or higher, for example 70°C or higher, such as 75°C or higher, for example 80°C or higher such as 85°C or higher.
  • suitable polynucleotides may for example be isolated from thermophilic microorganisms, or may be designed by the skilled person and artificially synthesized.
  • the polynucleotides may be isolated from thermophilic or thermotolerant filamentous fungi or isolated from non-thermophilic or non-thermotolerant fungi but are found to be thermostable.
  • carbohydrate degrading enzyme and/or carbohydrate hydrolysing enzyme is any polypeptide which is capable of degrading and/or hydrolysing of carbohydrate or enhancing the degrading and/or hydrolysing of carbohydrate.
  • carbohydrate degrading and/or carbohydrate hydrolysing enzymes are cellulase and hemicellulase and which include enzymes having cellulase enhancing activity (such as GH61 ) or hemicellulase enhancing activity.
  • a cellulase is any polypeptide which is capable of degrading or modifying cellulose.
  • a polypeptide which is capable of degrading cellulose is one which is capable of catalysing the process of breaking down cellulose into smaller units, either partially, for example into cellodextrins, or completely into glucose monomers.
  • a cellulase according to the invention may give rise to a mixed population of cellodextrins and glucose monomers when contacted with the cellulase. Such degradation will typically take place by way of a hydrolysis reaction.
  • GH61 glycoside hydrolase family 61 or sometimes referred to EGIV
  • PMO oxygen-dependent polysaccharide monooxygenase
  • GH61 may (also) have a function in the liquefaction of cellulosic materials or liberation of cellulosic materials and/or saccharification of cellulosic materials.
  • GH61 was originally classified as endogluconase based on measurement of very weak endo-1 ,4-3-d-glucanase activity in one family member.
  • the term "GH61 " as used herein, is to be understood as a family of enzymes, which share common conserved sequence portions and foldings to be classified in family 61 of the well-established CAZY GH classification system (http://www.cazy.org/GH61 .html).
  • the glycoside hydrolase family 61 is a member of the family of glycoside hydrolases EC 3.2.1 . GH61 is used herein as being part of the cellulases.
  • a hemicellulase is any polypeptide which is capable of degrading or modifying hemicellulose. That is to say, a hemicellulase may be capable of degrading or modifying one or more of xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan.
  • a polypeptide which is capable of degrading a hemicellulose is one which is capable of catalysing the process of breaking down the hemicellulose into smaller polysaccharides, either partially, for example into oligosaccharides, or completely into sugar monomers, for example hexose or pentose sugar monomers.
  • a hemicellulase according to the invention may give rise to a mixed population of oligosaccharides and sugar monomers when contacted with the hemicellulase. Such degradation will typically take place by way of a hydrolysis reaction.
  • a pectinase is any polypeptide which is capable of degrading or modifying pectin.
  • a polypeptide which is capable of degrading pectin is one which is capable of catalysing the process of breaking down pectin into smaller units, either partially, for example into oligosaccharides, or completely into sugar monomers.
  • a pectinase according to the invention may give rise to a mixed population of oligosacchardies and sugar monomers when contacted with the pectinase. Such degradation will typically take place by way of a hydrolysis reaction.
  • a cellobiohydrolase (EC 3.2.1 .91 ) is any polypeptide which is capable of catalysing the hydrolysis of 1 ,4-3-D-glucosidic linkages in cellulose or cellotetraose, releasing cellobiose from the ends of the chains.
  • This enzyme may also be referred to as cellulase 1 ,4-3-cellobiosidase, 1 ,4-3-cellobiohydrolase, 1 ,4-3-D-glucan cellobiohydrolase, avicelase, exo-1 ,4-3-D-glucanase, exocellobiohydrolase or exoglucanase.
  • an endo-3-1 ,4-glucanase (EC 3.2.1 .4) is any polypeptide which is capable of catalysing the endohydrolysis of 1 ,4-3-D-glucosidic linkages in cellulose, lichenin or cereal ⁇ -D-glucans. Such a polypeptide may also be capable of hydrolyzing 1 ,4-linkages in ⁇ -D-glucans also containing 1 ,3-linkages.
  • This enzyme may also be referred to as cellulase, avicelase, ⁇ -1 ,4-endoglucan hydrolase, ⁇ -1 ,4-glucanase, carboxymethyl cellulase, celludextrinase, endo-1 ,4 ⁇ -D-glucanase, endo-1 ,4-3-D- glucanohydrolase, endo-1 ,4 ⁇ -glucanase or endoglucanase.
  • a ⁇ -glucosidase (EC 3.2.1 .21 ) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing ⁇ -D-glucose residues with release of ⁇ -D-glucose.
  • Such a polypeptide may have a wide specificity for ⁇ -D-glucosides and may also hydrolyze one or more of the following: a ⁇ -D-galactoside, an oL-arabinoside, a ⁇ -D-xyloside or a ⁇ -D-fucoside.
  • This enzyme may also be referred to as amygdalase, ⁇ -D-glucoside glucohydrolase, cellobiase or gentobiase.
  • a ⁇ -(1 ,3)(1 ,4)-glucanase (EC 3.2.1 .73) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4 ⁇ -D-glucosidic linkages in ⁇ -D-glucans containing 1 ,3- and 1 ,4-bonds.
  • Such a polypeptide may act on lichenin and cereal ⁇ -D- glucans, but not on ⁇ -D-glucans containing only 1 ,3- or 1 ,4-bonds.
  • This enzyme may also be referred to as licheninase, 1 ,3-1 ,4-3-D-glucan 4-glucanohydrolase, ⁇ -glucanase, endo- ⁇ -1 ,3-1 ,4 glucanase, lichenase or mixed linkage ⁇ -glucanase.
  • An alternative for this type of enzyme is EC 3.2.1 .6, which is described as endo-1 ,3(4)-beta-glucanase.
  • This type of enzyme hydrolyses 1 ,3- or 1 ,4-linkages in beta-D-glucanse when the glucose residue whose reducing group is involved in the linkage to be hydrolysed is itself substituted at C-3.
  • Alternative names include endo-1 ,3-beta-glucanase, laminarinase, 1 ,3-(1 ,3;1 ,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates include laminarin, lichenin and cereal beta-D-glucans.
  • a hemicellulase may be used in the process of the invention , for example, an endoxylanase, a ⁇ -xylosidase, a a-L-arabionofuranosidase, an a-D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase, an a-galactosidase, a ⁇ -galactosidase, a ⁇ -mannanase or a ⁇ -mannosidase.
  • an endoxylanase (EC 3.2.1.8) is any polypeptide which is capable of catalyzing the endohydrolysis of 1 ,4 ⁇ -D-xylosidic linkages in xylans.
  • This enzyme may also be referred to as endo-1 ,4 ⁇ -xylanase or 1 ,4 ⁇ -D-xylan xylanohydrolase.
  • An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyse 1 ,4 xylosidic linkages in glucuronoarabinoxylans.
  • a ⁇ -xylosidase (EC 3.2.1 .37) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4 ⁇ -D-xylans, to remove successive D-xylose residues from the non-reducing termini. Such enzymes may also hydrolyze xylobiose. This enzyme may also be referred to as xylan 1 ,4 ⁇ -xylosidase, 1 ,4 ⁇ -D-xylan xylohydrolase, exo-1 ,4 ⁇ -xylosidase or xylobiase.
  • an oL-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on oL-arabinofuranosides, oL-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans.
  • This enzyme may also be referred to as oN-arabinofuranosidase, arabinofuranosidase or arabinosidase.
  • This enzyme may also be referred to as alpha- glucuronidase or alpha-glucosiduronase.
  • These enzymes may also hydrolyse 4-0- methylated glucoronic acid, which can also be present as a substituent in xylans.
  • xylan alpha-1 ,2-glucuronosidase which catalyses the hydrolysis of alpha-1 ,2-(4-0-methyl)glucuronosyl links.
  • an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide which is capable of catalyzing the deacetylation of xylans and xylo-oligosaccharides.
  • Such a polypeptide may catalyze the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically, not from triacetylglycerol.
  • Such a polypeptide typically does not act on acetylated mannan or pectin.
  • the saccharide may be, for example, an oligosaccharide or a polysaccharide. It may typically catalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyi) group from an esterified sugar, which is usually arabinose in 'natural' substrates, p-nitrophenol acetate and methyl ferulate are typically poorer substrates.
  • This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It may also be referred to as a hemicellulase accessory enzyme, since it may help xylanases and pectinases to break down plant cell wall hemicellulose and pectin.
  • the saccharide may be, for example, an oligosaccharide or a polysaccharide.
  • This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzyme also falls within EC 3.1.1 .73 so may also be referred to as a feruloyi esterase.
  • an a-galactosidase (EC 3.2.1 .22) is any polypeptide which is capable of catalyzing the hydrolysis of of terminal, non-reducing oD-galactose residues in a-D- galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans.
  • Such a polypeptide may also be capable of hydrolyzing a-D- fucosides.
  • This enzyme may also be referred to as melibiase.
  • a ⁇ -galactosidase (EC 3.2.1.23) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing ⁇ -D-galactose residues in ⁇ -D- galactosides. Such a polypeptide may also be capable of hydrolyzing a-L-arabinosides. This enzyme may also be referred to as exo-(1 ->4)-3-D-galactanase or lactase.
  • a ⁇ -mannanase (EC 3.2.1 .78) is any polypeptide which is capable of catalyzing the random hydrolysis of 1 ,4-3-D-mannosidic linkages in mannans, galactomannans and glucomannans. This enzyme may also be referred to as mannan endo-1 ,4-3-mannosidase or endo-1 ,4-mannanase.
  • a ⁇ -mannosidase (EC 3.2.1.25) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing ⁇ -D-mannose residues in ⁇ -D- mannosides. This enzyme may also be referred to as mannanase or mannase.
  • An enzyme composition may comprise any pectinase, for example an endo polygalacturonase, a pectin methyl esterase, an endo-galactanase, a beta galactosidase, a pectin acetyl esterase, an endo-pectin lyase, pectate lyase, alpha rhamnosidase, an exo-galacturonase, an expolygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, a rhamnogalacturonan galacturonohydrolase, a xylogalacturonase.
  • pectinase for example an endo polygalacturonase, a pectin methyl esterase, an
  • an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide which is capable of catalyzing the random hydrolysis of 1 ,4-oD-galactosiduronic linkages in pectate and other galacturonans.
  • This enzyme may also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly-a-1 ,4-galacturonide glycanohydrolase, endogalacturonase; endo-D-galacturonase or poly(1 ,4-a-D-galacturonide) glycanohydrolase.
  • the enzyme may also been known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinoesterase or pectin pectylhydrolase.
  • an endo-galactanase (EC 3.2.1 .89) is any enzyme capable of catalyzing the endohydrolysis of 1 ,4-3-D-galactosidic linkages in arabinogalactans.
  • the enzyme may also be known as arabinogalactan endo-1 ,4-3-galactosidase, endo-1 ,4-3- galactanase, galactanase, arabinogalactanase or arabinogalactan 4- ⁇ - ⁇ - galactanohydrolase.
  • a pectin acetyl esterase is defined herein as any enzyme which has an acetyl esterase activity which catalyzes the deacetylation of the acetyl groups at the hydroxyl groups of GalUA residues of pectin
  • an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable of catalyzing the eliminative cleavage of (1 ⁇ *4)-a-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-0-methyl-a-D-galact-4-enuronosyl groups at their non- reducing ends.
  • the enzyme may also be known as pectin lyase, pectin frans-eliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1 ⁇ 4)-6-0-methyl-a-D-galacturonan lyase.
  • a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzing the eliminative cleavage of (1 ⁇ 4)-a-D-galacturonan to give oligosaccharides with 4-deoxy- oD-galact-4-enuronosyl groups at their non-reducing ends.
  • the enzyme may also be known polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectic lyase, a-1 ,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N, endo-a-1 ,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin frans-eliminase, polygalacturonic acid frans-eliminase or (1 ⁇ 4)-a-D- galacturonan lyase.
  • an alpha rhamnosidase (EC 3.2.1 .40) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing a-L-rhamnose residues in a-L- rhamnosides or alternatively in rhamnogalacturonan.
  • This enzyme may also be known as a-L-rhamnosidase T, a-L-rhamnosidase N or a-L-rhamnoside rhamnohydrolase.
  • exo-galacturonase (EC 3.2.1.82) is any polypeptide capable of hydrolysis of pectic acid from the non-reducing end, releasing digalacturonate.
  • the enzyme may also be known as exo-poly-a-galacturonosidase, exopolygalacturonosidase or exopolygalacturanosidase.
  • the enzyme may also be known as galacturan 1 ,4-ogalacturonidase, exopolygalacturonase, poly(galacturonate) hydrolase, exo-D-galacturonase, exo-D- galacturonanase, exopoly-D-galacturonase or poly(1 ,4-a-D-galacturonide) galacturonohydrolase.
  • exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptide capable of catalyzing eliminative cleavage of 4-(4-deoxy-a-D-galact-4-enuronosyl)-D-galacturonate from the reducing end of pectate, i.e. de-esterified pectin.
  • This enzyme may be known as pectate disaccharide-lyase, pectate exo-lyase, exopectic acid transeliminase, exopectate lyase, exopolygalacturonic acid-frans-eliminase, PATE, exo-PATE, exo-PGL or (1 ⁇ »4)-a-D-galacturonan reducing-end-disaccharide-lyase.
  • rhamnogalacturonan hydrolase is any polypeptide which is capable of hydrolyzing the linkage between galactosyluronic acid acid and rhamnopyranosyl in an endo-fashion in strictly alternating rhamnogalacturonan structures, consisting of the disaccharide [(1 ,2-alpha-L-rhamnoyl-(1 ,4)-alpha-galactosyluronic acid].
  • rhamnogalacturonan lyase is any polypeptide which is any polypeptide which is capable of cleaving a-L- hap-(1 ⁇ 4)-a-D-GalpA linkages in an endo-fashion in rhamnogalacturonan by beta-elimination.
  • rhamnogalacturonan acetyl esterase is any polypeptide which catalyzes the deacetylation of the backbone of alternating rhamnose and galacturonic acid residues in rhamnogalacturonan.
  • rhamnogalacturonan galacturonohydrolase is any polypeptide which is capable of hydrolyzing galacturonic acid from the non-reducing end of strictly alternating rhamnogalacturonan structures in an exo-fashion.
  • xylogalacturonase is any polypeptide which acts on xylogalacturonan by cleaving the ⁇ -xylose substituted galacturonic acid backbone in an enc/o-manner.
  • This enzyme may also be known as xylogalacturonan hydrolase.
  • an oL-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on a-L-arabinofuranosides, oL-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans.
  • This enzyme may also be referred to as a-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.
  • endo-arabinanase (EC 3.2.1 .99) is any polypeptide which is capable of catalyzing endohydrolysis of 1 ,5-oarabinofuranosidic linkages in 1 ,5-arabinans.
  • the enzyme may also be know as endo-arabinase, arabinan endo-1 ,5-oL-arabinosidase, endo-1 ,5-ol_-arabinanase, endo-a-1 ,5-arabanase; endo-arabanase or 1 ,5-oL-arabinan 1 ,5-oL-arabinanohydrolase.
  • An enzyme composition may comprise at least one cellulase and optionally at least one hemicellulase and optionally at least one pectinase (one of which is a polypeptide according to the invention).
  • An (second) enzyme composition may comprise a GH61 , a cellobiohydrolase, an endoglucanase and/or a ⁇ -glucosidase.
  • Such a composition may also comprise one or more hemicellulases and/or one or more pectinases.
  • one or more (for example two, three, four or all) of an amylase, a protease, a lipase, a ligninase, a hexosyltransferase, a glucuronidase or an expansin or a cellulose induced protein or a cellulose integrating protein or like protein may be used in the process of the invention (these are referred to as auxiliary activities above).
  • proteases includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other moieties, such as sugars (glycopeptidases). Many proteases are characterized under EC 3.4, and are suitable for use in the invention incorporated herein by reference. Some specific types of proteases include, cysteine proteases including pepsin, papain and serine proteases including chymotrypsins, carboxypeptidases and metalloendopeptidases.
  • Lipase includes enzymes that hydrolyze lipids, fatty acids, and acylglycerides, including phospoglycerides, lipoproteins, diacylglycerols, and the like. In plants, lipids are used as structural components to limit water loss and pathogen infection. These lipids include waxes derived from fatty acids, as well as cutin and suberin.
  • Liganase includes enzymes that can hydrolyze or break down the structure of lignin polymers. Enzymes that can break down lignin include lignin peroxidases, manganese peroxidases, laccases and feruloyl esterases, and other enzymes described in the art known to depolymerize or otherwise break lignin polymers. Also included are enzymes capable of hydrolyzing bonds formed between hemicellulosic sugars (notably arabinose) and lignin.
  • Ligninases include but are not limited to the following group of enzymes: lignin peroxidases (EC 1.1 1.1.14), manganese peroxidases (EC 1 .1 1 .1 .13), laccases (EC 1.10.3.2) and feruloyl esterases (EC 3.1 .1 .73).
  • “Hexosyltransferase” (2.4.1 -) includes enzymes which are capable of catalyzing a transferase reaction, but which can also catalyze a hydrolysis reaction, for example of cellulose and/or cellulose degradation products.
  • An example of a hexosyltransferase which may be used in the invention is a ⁇ -glucanosyltransferase.
  • Such an enzyme may be able to catalyze degradation of (1 ,3)(1 ,4)glucan and/or cellulose and/or a cellulose degradation product.
  • Glucuronidase includes enzymes that catalyze the hydrolysis of a glucoronoside, for example ⁇ -glucuronoside to yield an alcohol.
  • Many glucuronidases have been characterized and may be suitable for use in the invention, for example ⁇ - glucuronidase (EC 3.2.1.31 ), hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl- disulfoglucosamine glucuronidase (3.2.1.56), glycyrrhizinate ⁇ -glucuronidase (3.2.1 .128) or a-D-glucuronidase (EC 3.2.1 .139).
  • a composition for use in the invention may comprise an expansin or expansin- like protein, such as a swollenin (see Salheimo et al., Eur. J. Biohem. 269, 4202-421 1 , 2002) or a swollenin-like protein.
  • an expansin or expansin- like protein such as a swollenin (see Salheimo et al., Eur. J. Biohem. 269, 4202-421 1 , 2002) or a swollenin-like protein.
  • Expansins are implicated in loosening of the cell wall structure during plant cell growth. Expansins have been proposed to disrupt hydrogen bonding between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, they are thought to allow the sliding of cellulose fibers and enlargement of the cell wall. Swollenin, an expansin-like protein contains an N-terminal Carbohydrate Binding Module Family 1 domain (CBD) and a C-terminal expansin-like domain.
  • CBD Carbohydrate Binding Module Family 1 domain
  • an expansin-like protein or swollenin-like protein may comprise one or both of such domains and/or may disrupt the structure of cell walls (such as disrupting cellulose structure), optionally without producing detectable amounts of reducing sugars.
  • a member of each of the classes of enzymes mentioned above, several members of one enzyme class, or any combination of these enzymes classes or helper proteins i.e. those proteins mentioned herein which do not have enzymatic activity per se, but do nevertheless assist in lignocellulosic degradation.
  • enzymes may be used from (1 ) commercial suppliers; (2) cloned genes expressing enzymes; (3) complex broth (such as that resulting from growth of a microbial strain in media, wherein the strains secrete proteins and enzymes into the media; (4) cell lysates of strains grown as in (3); and/or (5) plant material expressing enzymes.
  • Different enzymes in a composition of the invention may be obtained from different sources.
  • the enzymes can be produced either exogenously in microorganisms, yeasts, fungi, bacteria or plants, then isolated and added, for example, to lignocellulosic feedstock.
  • the enzymes are produced, but not isolated, and crude cell mass fermentation broth, or plant material (such as corn stover or wheat straw), and the like may be added to, for example, the feedstock.
  • the crude cell mass or enzyme production medium or plant material may be treated to prevent further microbial growth (for example, by heating or addition of antimicrobial agents), then added to, for example, a feedstock.
  • These crude enzyme mixtures may include the organism producing the enzyme.
  • the enzyme may be produced in a fermentation that uses (pre-treated) feedstock (such as corn stover or wheat straw) to provide nutrition to an organism that produces an enzyme(s).
  • feedstock such as corn stover or wheat straw
  • plants that produce the enzymes may themselves serve as a lignocellulosic feedstock and be added into lignocellulosic feedstock.
  • the process according to the invention may further comprise a fermentation step.
  • the invention optionally includes a fermentation process in which a microorganism is used for the fermentation of a carbon source comprising sugar(s), e.g. glucose, L-arabinose and/or xylose.
  • the carbon source may include any carbohydrate oligo- or polymer comprising L-arabinose, xylose or glucose units, such as e.g. lignocellulose, xylans, cellulose, starch, arabinan and the like.
  • carbohydrases for release of xylose or glucose units from such carbohydrates, appropriate carbohydrases (such as xylanases, glucanases, amylases and the like) may be added to the fermentation medium or may be produced by the modified host cell. In the latter case the modified host cell may be genetically engineered to produce and excrete such carbohydrases.
  • An additional advantage of using oligo- or polymeric sources of glucose is that it enables to maintain a low(er) concentration of free glucose during the fermentation, e.g. by using rate-limiting amounts of the carbohydrases. This, in turn, will prevent repression of systems required for metabolism and transport of non-glucose sugars such as xylose.
  • the modified host cell ferments both the L-arabinose (optionally xylose) and glucose, preferably simultaneously in which case preferably a modified host cell is used which is insensitive to glucose repression to prevent diauxic growth.
  • the fermentation medium will further comprise the appropriate ingredient required for growth of the modified host cell.
  • Compositions of fermentation media for growth of microorganisms such as yeasts or filamentous fungi are well known in the art.
  • the fermentation time may be shorter than in conventional fermentation at the same conditions, wherein part of the enzymatic hydrolysis still has to take part during fermentation.
  • the fermentation time is 100 hours or less, 90 hours or less, 80 hours or less, 70 hours or less, or 60 hours or less, for a sugar composition of 50g/l glucose and corresponding other sugars from the lignocellulosic feedstock (e.g. 50 g/l xylose, 35 g/l L-arabinose and 10 g/l galactose.
  • a sugar composition of 50g/l glucose and corresponding other sugars from the lignocellulosic feedstock (e.g. 50 g/l xylose, 35 g/l L-arabinose and 10 g/l galactose.
  • the fermentation time may correspondingly be reduced.
  • the fermentation process may be an aerobic or an anaerobic fermentation process.
  • An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors.
  • NADH produced in glycolysis and biomass formation cannot be oxidised by oxidative phosphorylation.
  • many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD + .
  • pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, lactic acid, 3- hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1 ,3-propane-diol, ethylene, glycerol, butanol, methane or biogas, a ⁇ -lactam antibiotics and a cephalosporin.
  • the fermentation process is anaerobic.
  • An anaerobic process is advantageous since it is cheaper than aerobic processes: less special equipment is needed.
  • anaerobic processes are expected to give a higher product yield than aerobic processes.
  • aerobic conditions usually the biomass yield is higher than under anaerobic conditions.
  • the expected product yield is lower than under anaerobic conditions.
  • the fermentation process is under oxygen-limited conditions. More preferably, the fermentation process is aerobic and under oxygen-limited conditions.
  • An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing/mass transfer properties of the fermentation equipment used.
  • the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.
  • the fermentation process is preferably run at a temperature that is optimal for the modified cell.
  • the fermentation process is performed at a temperature which is less than 42°C, preferably less than 38°C.
  • the fermentation process is preferably performed at a temperature which is lower than 35, 33, 30 or 28°C and at a temperature which is higher than 20, 22, or 25°C.
  • the fermentation is conducted with a microorganism that is able to ferment at least one C5 sugar.
  • the process is a process for the production of ethanol whereby the process comprises the step d) comprises fermenting a medium containing sugar(s) with a microorganism that is able to ferment at least one C5 sugar, whereby the host cell is able to ferment glucose, L-arabinose and xylose to ethanol.
  • the microorganism that is able to ferment at least one C5 sugar is a yeast.
  • the yeast is belongs to the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae .
  • the fermentation process for the production of ethanol is anaerobic. Anaerobic has already been defined earlier herein. In another preferred embodiment, the fermentation process for the production of ethanol is aerobic. In another preferred embodiment, the fermentation process for the production of ethanol is under oxygen-limited conditions, more preferably aerobic and under oxygen-limited conditions. Oxygen-limited conditions have already been defined earlier herein.
  • the volumetric ethanol productivity is preferably at least 0.5, 1.0, 1 .5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre per hour.
  • the ethanol yield on L- arabinose and optionally xylose and/or glucose in the process preferably is at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 98%.
  • the ethanol yield is herein defined as a percentage of the theoretical maximum yield, which, for glucose and L-arabinose and optionally xylose is 0.51 g. ethanol per gram glucose or xylose.
  • the optional fermentation process leading to the production of ethanol has several advantages by comparison to known ethanol fermentations processes:
  • the strain used may be able to use L-arabinose and optionally xylose.
  • this process is a co-fermentation process.
  • All preferred embodiments of the fermentation processes as described above are also preferred embodiments of this co-fermentation processe: identity of the fermentation product, identity of source of L-arabinose and source of xylose, conditions of fermentation (aerobical or anaerobical conditions, oxygen-limited conditions, temperature at which the process is being carried out, productivity of ethanol, yield of ethanol).
  • the fermentation process may be carried out without any requirement to adjust the pH during the process. That is to say, the process is one which may be carried out without the addition of any acid(s) or base(s). However, this excludes a pretreatment step, where acid may be added.
  • the composition of the invention is capable of acting at low pH and, therefore, there is no need to adjust the pH of acid of an acid pretreated feedstock in order that saccharification may take place. Accordingly, a method of the invention may be a zero waste method using only organic products with no requirement for inorganic chemical input. Overall reaction time
  • the overall reaction time (i.e the reaction time of the liquefaction and hydrolysis step, and optionally the fermentation step together may be reduced.
  • the overall reaction time is 150 hours or less, 140 hours or less, 130 or less, 120 hours or less, 1 10 hours or less, 100 hours of less, 90 hours or less, 80 hours or less, 75 hours or less, or about 72 hours at 90% glucose yield.
  • lower overall times may be reached at lower glucose yield. This is independent on the mode in which the processes are conducted in SLH mode.
  • Fermentation products which may be produced according to the invention include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels such as methane or biogas and ethanol, or other organic polymers, lactic acid, including fuel ethanol (the term "ethanol” being understood to include ethyl alcohol or mixtures of ethyl alcohol and water).
  • Specific value-added products that may be produced by the methods of the invention include, but not limited to, biofuels (including ethanol and butanol); lactic acid; 3-hydroxy-propionic acid; acrylic acid; acetic acid; 1 ,3-propane-diol; ethylene; glycerol; a plastic; a specialty chemical; an organic acid, including citric acid, succinic acid and maleic acid; a solvent; an animal feed supplement; a pharmaceutical such as a ⁇ -lactam antibiotic or a cephalosporin; a vitamin; an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid; an enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductase, a transferase or a xy
  • the process according to the invention optionally comprises recovery of fermentation product.
  • a fermentation product may be separated from the fermentation broth in any known manner.
  • the skilled person will thus be able to select a proper separation technique.
  • ethanol may be separated from a yeast fermentation broth by distillation, for instance steam distillation/vacuum distillation in conventional way.
  • the enzyme in solution can be separated from the solution containing reducing sugars and other hydrolysis products from the enzymatic actions.
  • This separation can be done by, but not limiting to, (ultra and micro)filtration, centrifugation, sedicantation, sedimentation, with or without first adsorption of the enzyme to a carrier of any kind.
  • the enzymes used in the liquefaction can be recycled in a similar way as described hereinabove for the recycling after hydrolysis (liquefaction and saccharification).
  • Enzyme recycling after hydrolysis in combination with enzyme production and yeast-cell recycling with stable enzymes can be combined with recycling of the ethanol producing microorganism after fermentation and with the use of the reducing sugars containing filtrate as a substrate (purified and/or concentrated or diluted) in enzyme-production fermentation and as substrate for the cultivation of the ethanol-producing microorganism.
  • thermo stability of enzymes causes remaining cellulolytic activity after hydrolysis, fermentation and vacuum distillation in the thin stillage.
  • the total activity of the enzyme is reduced during the three successive process steps.
  • the thin stillage obtained after vacuum distillation can thus be re-used as a source of enzyme for a newly started hydrolysis-fermentation-distillation process cycle of pre-treated wheat straw conversion into ethanol.
  • the thin stillage can be used either in concentrated or (un)diluted form and/or purified and with or without additional enzyme supplementation.
  • Thin stillage, optionally purified can be re-used for liquefaction or hydrolysis of cellulosic material.
  • Suitable strains can be fermented and equally used in the present examples to show the effect and advantages of the invention.
  • TEC-101 , TEC-142, TEC-192, TEC- 201 or TEC-210 are suitable Rasamsonia strains wich are described in WO201 1/000949.
  • TEC-210 cellulase-containing composition was produced according to the procedures such as inoculation and fermentation as described in WO201 1/000949.
  • Beta-glucosidase is produced by overexpression of EBA4 in Aspergillus niger as described in WO201 1/098577 followed by fermentation of the Aspergillus niger transformant.
  • EBA4 is a Rasamsonia emersonii (Talaromyces emersonii) BG and is identified in WO201 1/098577 as T. emersonii beta-glucosidase (BG) and represented by SEQ ID NO: 5 in WO201 1/098577.
  • Dilute-acid pre-treated corn stover (aCS) was obtained as described in Schell, D.J., Applied Biochemistry and Biotechnology (2003), vol. 105-108, pp 69-85.
  • a pilot scale pretreatment reactor was used operating at steady state conditions of 190°C, 1 min residence time and an effective H2S04 acid concentration of 1.45% (w/w) in the liquid phase.
  • the method was a combination of precipitation of protein using trichloro acetic acid (TCA) to remove disturbing substances and allow determination of the protein concentration with the colorimetric Biuret reaction.
  • TCA trichloro acetic acid
  • a copper (II) ion is reduced to copper (I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution.
  • a violet color indicates the presence of proteins.
  • the intensity of the color, and hence the absorption at 546 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.
  • the standardisation was performed using BSA (Bovine Serum Albumine) and the protein content was expressed in g protein as BSA equivalent/L or mg protein as BSA equivalent /ml.
  • the protein content was calculated using standard calculation protocols known in the art, by plotting the OD 546 versus the concentration of samples with known concentration, followed by the calculation of the concentration of the unknown samples using the equation generated from the calibration line.
  • the following samples preparation was performed. To 10 ⁇ sample 40 ⁇ MilliQ water and 50 ⁇ TCA (20%) was added to dilute the sample five times ( ⁇ 1 mg/ml) and precipitate the proteins. After 1 hour on ice the sample was centrifuged (10 minutes, 14000 rpm). The pellet was washed with 500 ⁇ Aceton and centrifuged (10 minutes, 14000 rpm). The pellet was treated as described below.
  • the pellet was dissolved in 65 ⁇ of the MilliQ water, 25 ⁇ NuPAGETM LDS sample buffer (4x) Invitrogen and 10 ⁇ NuPAGETM Sample Reducing agent (10x) Invitrogen. Prior to the the deanuarion step the sample was diluted 5 timnes using a mix of MilliQ; NuPAGETM LDS sample buffer and 10 ⁇ NuPAGETM Sample Reducing in the ratio of 65:25:10. After mixing, the samples were incubated in a thermo mixer for 10 minutes at 70°C. The sample solutions were applied on a 4-12% Bis-Tris gel (NuPAGETM BisTris, Invitrogen). A sample (1 ⁇ ) of marker M12 (Invitrogen) was also applied on the gel.
  • the gel was run at 200 V for 50 minutes, using the XCELL Surelock, with 600 ml 20 x diluted SDS buffer in the outer buffer chamber and 200 ml 20 x diluted SDS buffer, containing 0.5 ml of antioxidant (NuPAGETM Invitrogen) in the inner buffer chamber. After running, the gel was rinsed twice with demineralised water the gels were fixed with 50% methanol/7% acetic acid solution for one hour and stained with Sypro Ruby (50 ml per gel) overnight. An image was made using the Typhoon 9200 (610 BP 30, Green (532 nm), PMT 600V, 100 micron) after washing the gel with MilliQ water.
  • the ratio between protein bands within a lane was determined using standard methods known in the art. The sample was applied in triplicate and the gray values were determined using the program Image quant. Values are expressed as relative % protein to the total protein, calculated using the gray value of the selected protein band relative to the total gray value all the protein bands.
  • a solution of 20 w/w% feedstock was prepared by diluting acid pretreated corn stover (NREL) with water, NH40H titrant and enzyme. Incubations were carried out using 500 g solution in 2000 ml Schott bottles. Six different enzyme dosages were tested (0.5-1.25-2.50- 3.75-5.0-7.5 mg protein/g feedstock dry matter). The used enzyme for the experiment was TEC-210 cellulase-containing composition.
  • the Schott bottles were placed in the a shaking Incubator and the incubations were conducted at a temperature of 62°C ⁇ 1 °C, initial pH of 4.5 and shaking speed of 120 rpm. During the enzymatic hydrolysis, samples were taken daily for carbohydrate analysis (glucose, cellobiose) and pH measurement.
  • a solution of 20 w/w% feedstock was prepared by diluting acid pretreated corn stover (NREL) with water, NH40H titrant and enzyme. Incubations were carried out using 500 g solution in 2000 ml Schott bottles. The used enzyme for the experiment was TEC-210 cellulase-containing composition.
  • the BG used had a protein purity of 93 %.
  • the Schott bottles were placed in the a shaking Incubator and the incubations were conducted at a temperature of 62°C ⁇ 1 °C, initial pH of 4.5 and shaking speed of 120 rpm. During the enzymatic hydrolysis, samples were taken daily for carbohydrate analysis (glucose, cellobiose) and pH measurement
  • Figure 2 clearly shows a significant rate of hydrolysis improvement due to the addition of extra BG. This improvement is visible by higher glucose levels which were formed during the early phase of the process. Addition of extra BG (an overdose) resulted in a large increase in reaction speed (about 5 times faster) during the initial phase of the process. The effect of additional BG was even larger than a double dose of the complete cellulase-containing composition TEC-210.
  • the cellobiose concentrations were about 4 to 6 g/l after 100 hours of incubation for 1 .25 mg TEC-210 and 2.50 mg TEC-210, whereas for the 1 .25 mg TEC-210 + 1.25 mg BG the cellobiose concentration was 0.2 g/l.
  • a solution of 20 w/w% feedstock was prepared by diluting the acid pretreated corn stover (NREL) with water, NH3 titrant and enzyme. Incubations were done using 1000 g of prepared feedstcok in a 1500 ml reactor.
  • the BG enrichment amounted to 2%, 4%, 6%, 8% and 10% beta-glucosidase (93 % pure) on top of enzyme compositionTEC 210.
  • Table 1 glucose production in g/l from acid pretreated corn stover enzymatic hydrolysis
  • a solution of 20 w/w% feedstock was prepared by diluting the acid pretreated corn stover with water, NH3 titrant and enzyme. Incubations were done using 1000 g of prepared feedstock in a 1500 ml reactor.

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Abstract

Cette invention concerne un procédé d'hydrolyse d'une biomasse cellulosique comprenant • - une étape de prétraitement de ladite biomasse cellulosique : et • - une étape de saccharification consistant à convertir la biomasse cellulosique prétraitée en glucose. Dans l'étape de saccharification, les cellulases GH61 et/ou EG, CBH1, CBH2 et bêta- glucosidase (BG) sont ajoutées à la biomasse cellulosique prétraitée, de façon que moins de 10 mg de cellulase (sur une base de protéine) soient ajoutés par gramme de cellulose présente dans la biomasse cellulosique (sur une base de matières sèches) et que la quantité de BG soit d'au moins 4 % en poids des cellulases (sur une base de protéine) ou que la quantité de BG soit d'au moins 4 % en poids de la protéine présente dans les cellulases ou une composition contenant des cellulases ajoutée, la quantité de BG étant de préférence d'au moins 4 % en poids des cellulases (sur une base de protéine).
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WO2019006448A1 (fr) * 2017-06-30 2019-01-03 Danisco Us Inc Composition et procédé pour déterminer la quantité de glucose pouvant être dérivé de constituants cellulosiques d'une charge d'alimentation
WO2019229108A1 (fr) * 2018-05-30 2019-12-05 Dsm Ip Assets B.V. Procédé de production de sucres à partir de matières glucidiques
CN112204151A (zh) * 2018-05-30 2021-01-08 帝斯曼知识产权资产管理有限公司 从碳水化合物材料产生糖的方法
CN112204151B (zh) * 2018-05-30 2024-06-11 维尔萨利斯股份公司 从碳水化合物材料产生糖的方法

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