WO2016207147A1 - Procédé d'hydrolyse enzymatique d'une matière lignocellulosique et de fermentation de sucres - Google Patents

Procédé d'hydrolyse enzymatique d'une matière lignocellulosique et de fermentation de sucres Download PDF

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WO2016207147A1
WO2016207147A1 PCT/EP2016/064285 EP2016064285W WO2016207147A1 WO 2016207147 A1 WO2016207147 A1 WO 2016207147A1 EP 2016064285 W EP2016064285 W EP 2016064285W WO 2016207147 A1 WO2016207147 A1 WO 2016207147A1
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lignocellulosic material
enzyme
fermentation
enzymes
process according
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PCT/EP2016/064285
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English (en)
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Erik DE VOS BURCHART
Udo Karl Jozef LAMERS
Jeroen Leonardus DEN HOLLANDER
Rolf POLDERMANS
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Dsm Ip Assets B.V.
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Publication of WO2016207147A1 publication Critical patent/WO2016207147A1/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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the invention relates to a process for preparing succinic acid from lignocellulosic material by enzymatic hydrolysis and fermentation.
  • Lignocellulosic material is primarily composed of cellulose, hemicellulose and lignin and provides an attractive platform for generating alternative energy sources to fossil fuels.
  • the material is available in large amounts and can be converted into valuable products e.g. sugars or biofuel, such as bioethanol.
  • Producing fermentation products from lignocellulosic material is known in the art and generally includes the steps of pretreatment, hydrolysis, fermentation, and optionally recovery of the fermentation products.
  • cellulose present in the lignocellulosic material is partly (typically 30 to 95 %, dependable on enzyme activity and hydrolysis conditions) converted into sugars by cellulolytic enzymes.
  • the hydrolysis typically takes place during a process lasting 6 to 168 hours (see Kumar, S. , Chem. Eng. Technol. 32 (2009), 517-526) under elevated temperatures of 45 to 50°C and non-sterile conditions.
  • the sugars are then converted into valuable fermentation products such as ethanol by microorganisms like yeast.
  • the fermentation takes place in a separate, preferably anaerobic, process step, either in the same or in a different vessel.
  • the temperature during fermentation is adjusted to 30 to 33°C to accommodate growth and ethanol production by microorganisms, commonly yeasts.
  • the remaining cellulosic material is converted into sugars by the enzymes already present from the hydrolysis step, while microbial biomass and ethanol are produced.
  • the fermentation is finished once the cellulosic material is converted into fermentable sugars and all fermentable sugars are converted into ethanol, carbon dioxide and microbial biomass. This may take up to 6 days. In general, the overall process time of hydrolysis and fermentation may amount up to 13 days.
  • optimization of process design is a crucial tool to reduce overall costs of the production of sugar products and fermentation products.
  • An object of the invention is to provide an improved process for the preparation of succinic acid from lignocellulosic material.
  • the process is improved by detoxification of the lignocellulosic material after enzymatic hydrolysis.
  • Figure 1 sets out the plasmid map of pSUC223.
  • Figure 2 sets out a schematic depiction of the principle of PDC6 replacement by a FRD1 expression cassette by direct integration and in vivo recombination of the split Cre-recombinase construct. Expression of Cre-recombinase is regulated by the GAL1 promoter.
  • Figure 3 sets out a plasmid map of pSUC228.
  • the present invention relates to a process for the preparation of succinic acid from lignocellulosic material, comprising the steps of (a) enzymatic hydrolysis of the lignocellulosic material in one or more containers using an enzyme composition to obtain enzymatically hydrolysed lignocellulosic material, (b) subjecting the enzymatically hydrolysed lignocellulosic material to a detoxification step by contacting the enzymatically hydrolysed lignocellulosic material with activated carbon, (c) fermentation of the enzymatically hydrolysed lignocellulosic material to produce succinic acid, and (d) optionally, recovery of the succinic acid.
  • the ratio (based on dry matter weight) of the enzymatically hydrolysed lignocellulosic material:activated carbon is 1 :0.00001 to 1 : 1 , preferably 1 :0001 to 1 : 0.5, more preferably 1 :0.001 to 1 :0.2.
  • the enzymatically hydrolysed lignocellulosic material can be passed through a bed of activated carbon, such as a fixed bed, a moving bed, or a simulated moving bed.
  • the activated carbon can be added continuously or batch-wise to the enzymatically hydrolysed lignocellulosic material followed by removal of the activated carbon by techniques known to the skilled person, such as centrifugation, filtration, sedimentation, to name just a few. Contact times and temperatures can be adjusted to optimise the efficiency and/or the economics of the process.
  • the activated carbon is regenerated after the detoxification step.
  • Regeneration of activated carbon can be performed chemically or thermally. Chemical regeneration includes treatment with acids or bases.
  • the temperature of the detoxification step is between 15°C - 75°C, preferably between 20°C - 70°C, more preferably between 45°C - 65°C.
  • the enzymatically hydrolysed lignocellulosic material may be subjected to a solid/liquid separation before and/or after the detoxification step.
  • the process of the present invention further comprises the step of subjecting the enzymatically hydrolysed lignocellulosic material to a solid/liquid separation before the detoxification step to obtain a solid fraction and a liquid fraction and subjecting the liquid fraction to the detoxification step.
  • the detoxified material is subjected to a concentration step.
  • the detoxified enzymatically hydrolysed lignocellulosic material is subjected to a concentration step before fermentation.
  • the detoxified liquid fraction is subjected to a concentration step before fermentation.
  • the pH of the enzymatically hydrolysed lignocellulosic material is lowered.
  • the pH of the liquid fraction obtained after solid/liquid separation of the enzymatically hydrolysed lignocellulosic material is lowered.
  • the lowering of the pH may be done before and/or after the detoxification step.
  • the pH is lowered to 3.0 or lower.
  • the pH is lowered to 2.9 or lower, 2.8 or lower, 2.7 or lower, 2.6 or lower, 2.5 or lower, 2.4 or lower, 2.3 or lower, 2.2 or lower, 2.1 or lower, 2.0 or lower, 1 .9 or lower, 1 .8 or lower, 1 .7 or lower, 1 .6 or lower, 1 .5 or lower, 1.4 or lower, 1.3 or lower, 1.2 or lower, 1 .1 or lower, 1.0 or lower.
  • the pH is lowered to between 0.5 and 3.0, between 0.5 and 2.9, between 0.5 and 2.8, between 0.5 and 2.7, between 0.5 and 2.6, between 0.5 and 2.5, between 0.5 and 2.4, between 0.5 and 2.3, between 0.5 and 2.2, between 0.5 and 2.1 , between 0.5 and 2.0, between 0.5 and 1.9, between 0.5 and 1.8, between 0.5 and 1.7, between 0.5 and 1.6, between 0.5 and 1.5, between 0.5 and 1.4, between 0.5 and 1.3, between 0.5 and 1.2, between 0.5 and 1.1 , between 0.5 and 1.0.
  • acids can be used to lower the pH. Examples include, but are not limited to, hydrochloric acid and sulfuric acid. Sulfuric acid is normally preferred on an industrial scale to minimise corrosion of equipment and to minimise waste treatment costs.
  • the acid can be added directly to the hydrolysate or it can be added to a circulation loop.
  • the pH is typically measured inline in a container or in a withdrawal line. Alternatively, it can be measured offline in samples taken from the lignocellulosic material.
  • solid/liquid separation will depend on the type of lignocellulosic material used and are well within the scope of the skilled artisan. Examples include, but are not limited to, centrifugation, cyclonic separation, filtration, decantation, sieving and sedimentation. In a preferred embodiment the solid/liquid separation is performed by centrifugation or sedimentation. During solid/liquid separation, means and/or aids for improving the separation may be used.
  • the lignocellulosic material is subjected to a pretreatment step before the enzymatic hydrolysis. In an embodiment the lignocellulosic material is subjected to a washing step before the enzymatic hydrolysis. In an embodiment the lignocellulosic material is subjected to at least one solid/liquid separation before the enzymatic hydrolysis. So, before subjecting the lignocellulosic material to enzymatic hydrolysis, it can be subjected to at least one solid/liquid separation. The solid/liquid separation may be done before and/or after the pretreatment step. Suitable methods and conditions for a solid/liquid separation have been described above.
  • the solid fraction comprises between 1 and 80 wt% C6 sugars and/or the at least liquid fraction comprises between 1 and 80 wt% C6 sugars.
  • the solid fraction comprises between 5 and 75 wt% C6 sugars, between 10 and 70 wt% C6 sugars, between 15 and 65 wt% C6 sugars, between 20 and 60 wt% C6 sugars and/or the at least liquid fraction comprises between 2 and 70 wt% C6 sugars, between 3 and 65 wt% C6 sugars, between 4 and 60 wt% C6 sugars, between 5 and 55 wt% C6 sugars, between 6 and 50 wt% C6 sugars, between 7 and 45 wt% C6 sugars, between 8 and 40 wt% C6 sugars.
  • lignocellulosic material may be added to the one or more containers.
  • the enzyme composition is already present in the one or more containers before the lignocellulosic material is added.
  • the enzyme composition may be added to the one or more containers.
  • the lignocellulosic material is already present in the one or more containers before the enzyme composition is added.
  • both the lignocellulosic material and the enzyme composition are added simultaneously to the one or more containers.
  • the enzyme composition present in the one or more containers may be an aqueous composition.
  • the enzymatic hydrolysis comprises at least a liquefaction step wherein the lignocellulosic material is hydrolysed in at least a first container, and a saccharification step wherein the liquefied lignocellulosic material is hydrolysed in the at least first container and/or in at least a second container.
  • Saccharification can be done in the same container as the liquefaction (i.e. the at least first container), it can also be done in a separate container (i.e. the at least second container). So, in the enzymatic hydrolysis of the processes according to the present invention liquefaction and saccharification may be combined. Alternatively, the liquefaction and saccharification may be separate steps.
  • Liquefaction and saccharification may be performed at different temperatures, but may also be performed at a single temperature.
  • the temperature of the liquefaction is higher than the temperature of the saccharification.
  • Liquefaction is preferably carried out at a temperature of 60 - 75 °C and saccharification is preferably carried out at a temperature of 50 - 65 °C.
  • the enzymatic hydrolysis can be performed in one or more containers, but can also be performed in one or more tubes or any other continuous system. This also holds true when the enzymatic hydrolysis comprises a liquefaction step and a saccharification step.
  • the liquefaction step can be performed in one or more containers, but can also be performed in one or more tubes or any other continuous system and/or the saccharification step can be performed in one or more containers, but can also be performed in one or more tubes or any other continuous system.
  • Examples of containers to be used in the present invention include, but are not limited to, fed-batch stirred containers, batch stirred containers, continuous flow stirred containers with ultrafiltration, and continuous plug-flow column reactors. Stirring can be done by one or more impellers, pumps and/or static mixers.
  • the enzymes used in the enzymatic hydrolysis may be added before and/or during the enzymatic hydrolysis. As indicated above, when the lignocellulosic material is subjected to a solid/liquid separation before enzymatic hydrolysis, the enzymes used in the enzymatic hydrolysis may be added before the solid/liquid separation. Alternatively, they may also be added after solid/liquid separation or before and after solid/liquid separation. The enzymes may also be added during the enzymatic hydrolysis. In case the enzymatic hydrolysis comprises a liquefaction step and saccharification step, additional enzymes may be added during and/or after the liquefaction step. The additional enzymes may be added before and/or during the saccharification step. Additional enzymes may also be added after the saccharification step.
  • the total enzymatic hydrolysis time is 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, 30 hours or more, 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, 1 10 hours or more, 120 hours or more, 130 hours or more, 140 hours or more, 150 hours or more, 160 hours or more, 170 hours or more, 180 hours or more, 190 hours or more, 200 hours or more.
  • the total enzymatic hydrolysis time is 10 to 300 hours, 16 to 275 hours, preferably 20 to 250 hours, more preferably 30 to 200 hours, most preferably 40 to 150 hours.
  • the viscosity of the lignocellulosic material in the one or more containers used for the enzymatic hydrolysis is kept between 10 and 4000 cP, between 10 and 2000 cP, preferably between 10 and 1000 cP.
  • the viscosity of the lignocellulosic material in the liquefaction step is kept between 10 and 4000 cP, between 10 and 2000 cP, preferably between 10 and 1000 cP and/or the viscosity of the lignocellulosic material in the saccharification step is kept between 10 and 1000 cP, between 10 and 900 cP, preferably between 10 and 800 cP.
  • the viscosity can be determined with a Brookfield DV III Rheometer at the temperature used for the hydrolysis.
  • oxygen is added during the enzymatic hydrolysis. In an embodiment oxygen is added during at least a part of the enzymatic hydrolysis. Oxygen can be added continuously or discontinuously during the enzymatic hydrolysis. In an embodiment oxygen is added one or more times during the enzymatic hydrolysis. In an embodiment oxygen may be added before the enzymatic hydrolysis, during the addition of lignocellulosic material to a container used of enzymatic hydrolysis, during the addition of enzyme to a container used of enzymatic hydrolysis, during a part of the enzymatic hydrolysis, during the whole enzymatic hydrolysis or any combination thereof. Oxygen is added to the one or more containers used in the enzymatic hydrolysis.
  • Oxygen can be added in several forms.
  • oxygen can be added as oxygen gas, oxygen-enriched gas, such as oxygen-enriched air, or air.
  • Oxygen may also be added by means of in situ oxygen generation.
  • oxygen may be generated by electrolysis, oxygen may be produced enzymatically, e.g. by the addition of peroxide, or oxygen may be produced chemically, e.g. by an oxygen generating system such as KHS0 5 .
  • oxygen is produced from peroxide by catalase.
  • the peroxide can be added in the form of dissolved peroxide or generated by an enzymatic or chemical reaction.
  • catalase is used as enzyme to produce oxygen
  • catalase present in the enzyme composition for the hydrolysis can be used or catalase can be added for this purpose.
  • Examples how to add oxygen include, but are not limited to, addition of oxygen by means of sparging, electrolysis, chemical addition of oxygen, filling the one or more containers used in the enzymatic hydrolysis from the top (plunging the hydrolysate into the tank and consequently introducing oxygen into the hydrolysate) and addition of oxygen to the headspace of said one or more containers.
  • oxygen is added to the headspace of the container(s)
  • sufficient oxygen necessary for the hydrolysis reaction may be supplied.
  • the amount of oxygen added to the container(s) can be controlled and/or varied. Restriction of the oxygen supplied is possible by adding only oxygen during part of the hydrolysis time in said container(s).
  • oxygen at a low concentration for example by using an mixture of air and recycled air (air leaving the container) or by "diluting" air with an inert gas.
  • Increasing the amount of oxygen added can be achieved by addition of oxygen during longer periods of the hydrolysis time, by adding the oxygen at a higher concentration or by adding more air.
  • Another way to control the oxygen concentration is to add an oxygen consumer and/or an oxygen generator. Oxygen can be introduced, for example blown, into the liquid hydrolysis container contents of lignocellulosic material. It can also be blown into the headspace of the container.
  • oxygen is added to the one or more containers used in the enzymatic hydrolysis before and/or during and/or after the addition of the lignocellulosic material to said one or more containers.
  • the oxygen may be introduced together with the lignocellulosic material that enters the hydrolysis container(s).
  • the oxygen may be introduced into the material stream that will enter the container(s) or with part of the container(s) contents that passes an external loop of the container(s).
  • the container(s) used in the enzymatic hydrolysis and/or the fermentation have a volume of at least 1 m 3 .
  • the containers have a volume of at least 1 m 3 , at least 2 m 3 , at least 3 m 3 , at least 4 m 3 , at least 5 m 3 , at least 6 m 3 , at least 7 m 3 , at least 8 m 3 , at least 9 m 3 , at least 10 m 3 , at least 15 m 3 , at least 20 m 3 , at least 25 m 3 , at least 30 m 3 , at least 35 m 3 , at least 40 m 3 , at least 45 m 3 , at least 50 m 3 , at least 60 m 3 , at least 70 m 3 , at least 75 m 3 , at least 80 m 3 , at least 90 m 3 , at least 100 m 3 , at least 200 m 3 , at least 300 m 3 , at
  • the container(s) will be smaller than 3000 m 3 or 5000 m 3 .
  • the container(s) used for the liquefaction step and the container(s) used for the saccharification step may have the same volume, but also may have a different volume.
  • the enzyme composition is from a fungus, preferably a filamentous fungus.
  • the enzymes in the enzyme composition are derived from a fungus, preferably a filamentous fungus or the enzymes comprise a fungal enzyme, preferably a filamentous fungal enzyme.
  • the fungus is Rasamsonia, with Rasamsonia emersonii being most preferred.
  • the enzymes used in the enzymatic hydrolysis of the processes of the present invention are derived from a fungus or the enzymes used in the enzymatic hydrolysis of the processes of the present invention comprise a fungal enzyme.
  • Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth ef a/. , In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • the filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligatory aerobic.
  • Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Beauvaria, Cephalosporium, Ceriporiopsis, Chaetomium paecilomyces, Chrysosporium, Claviceps, Cochiobolus, Coprinus, Cryptococcus, Cyathus, Emericella, Endothia, Endothia mucor, Filibasidium, Fusarium, Geosmithia, Gilocladium, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Podospora, Pyricularia, Rasamsonia, Rhizomucor, Rhizopus, Scylatidium, Schizophyllum, Stagonospora, Talarom
  • strains of filamentous fungi are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • ATCC American Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the enzymatic hydrolysis of the processes of the present invention are advantageously applied in combination with enzymes derived from a microorganism of the genus Rasamsonia or the enzymes used in the enzymatic hydrolysis of the processes of the present invention comprise a Rasamsonia enzyme.
  • thermostable cellulolytic enzymes are preferred.
  • a "thermostable” enzyme as used herein means that the enzyme has a temperature optimum of 50°C or higher, 60°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher. They may for example be isolated from thermophilic microorganisms or may be designed by the skilled person and artificially synthesized. In one embodiment the polynucleotides may be isolated or obtained from thermophilic or thermotolerant filamentous fungi or isolated from non-thermophilic or non-thermotolerant fungi, but are found to be thermostable.
  • thermophilic fungus is meant a fungus that grows at a temperature of 50°C or higher.
  • theotolerant fungus is meant a fungus that grows at a temperature of 45°C or higher, having a maximum near 50°C.
  • thermophilic or thermotolerant fungal cells may be a Humicola, Rhizomucor,
  • thermophilic or thermotolerant fungi are Humicola grisea var.
  • thermoidea Humicola lanuginosa, Myceliophthora thermophila, Papulaspora thermophilia, Rasamsonia byssochlamydoides, Rasamsonia emersonii, Rasamsonia argillacea, Rasamsonia eburnean, Rasamsonia brevistipitata, Rasamsonia cylindrospora, Rhizomucor pusillus, Rhizomucor miehei, Talaromyces bacillisporus, Talaromyces leycettanus, Talaromyces thermophilus, Thermomyces lenuginosus, Thermoascus crustaceus, Thermoascus thermophilus Thermoascus aurantiacus and Thielavia terrestris.
  • Thermophilic fungi are not restricted to a specific taxonomic order and occur all over the fungal tree of life. Examples are Rhizomucor in the Mucorales, Myceliophthora in Sordariales and Talaromyces, Thermomyces and Thermoascus in the Eurotiales (see Mouchacca, 1997). The majority of Talaromyces species are mesophiles, but exceptions are species within sections Emersonii and Thermophila. Section Emersonii includes Talaromyces emersonii, Talaromyces byssochlamydoides, Talaromyces bacillisporus and Talaromyces leycettanus, all of which grow well at 40°C.
  • Talaromyces bacillisporus is thermotolerant
  • Talaromyces leycettanus is thermotolerant to thermophilic
  • Talaromyces emersonii and Talaromyces byssochlamydoides are truly thermophilic (see Stolk and Samson, 1972).
  • the sole member of Talaromyces section Thermophila, Talaromyces thermophilus grows rapidly at 50°C (see Stolk and Samson, 1972).
  • the current classification of these thermophilic Talaromyces species is mainly based on phenotypic and physiological characters, such as their ability to grow above 40°C, ascospore color, the structure of ascornatal covering and the formation of a certain type of anamorph.
  • 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 Talaromyces emersonii, Talaromyces byssochlamydoides, Talaromyces eburneus, Geosmithia argillacea and Geosmithia cylindrospora to Rasamsonia gen. nov.
  • thermophilic fungi are Rasamsonia byssochlamydoides, Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus, Thermoascus crustaceus, Thermoascus thermophilus and Thermoascus aurantiacus, with Rasamsonia emersonii being most preferred.
  • Talaromyces emersonii, Penicillium geosmithia emersonii and Rasamsonia emersonii are used interchangeably herein.
  • process steps are preferably done under septic conditions to lower the operational costs.
  • Contamination and growth of contaminating microorganisms can therefore occur and result in undesirable side effects, such as lactic acid, formic acid and acetic acid production, yield losses of fermentation product on substrate, production of toxins and extracellular polysaccharides. These effects may affect production costs significantly.
  • a high process temperature and/or a short process time limits the risk on contamination during hydrolysis and fermentation.
  • Thermostable enzymes like those of Rasamsonia, are capable of hydrolysing lignocellulosic material at temperatures of higher than 60°C. At these temperatures, the risk that a contaminating microorganism will cause undesired side effects is little to almost zero.
  • temperatures are typically between 30 to 38°C and are preferably not raised because of production losses.
  • the risks and effects of contamination and/or growth of contaminants are reduced as much as possible.
  • stable enzymes like those of Rasamsonia, a short fermentation time can be applied and thus risks of contamination and/or growth of contaminants are reduced as much as possible.
  • the first step after thermal pretreatment is to cool the pretreated material to temperatures wherein the enzymes have an optimal activity. On large scale, this is typically done by adding (cooled) water, which, besides decreasing the temperature, reduces the dry matter content.
  • thermostable enzymes like those of Rasamsonia, cost reduction can be achieved, because (i) less cooling of the pretreated material is required since higher temperatures are allowed during hydrolysis, and (ii) less water is added, which increases the dry matter content during hydrolysis and fermentation and thus increase the ethanol production capacity (amount produced per time unit per volume) of an ethanol plant.
  • cost reduction may also be achieved by using cooling water having a higher temperature than the water that is used in a process with non-thermostable enzyme.
  • 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 techniques including, but not limited to, ultra- and microfiltration, centrifugation, cantation, sedimentation, with or without first adsorption of the enzyme to a carrier of any kind. For example, after hydrolysis of pretreated material with 0.175 ml/g material dry matter enzyme load for 20h, 50% of the theoretical maximum amount of reducing sugars is liberated and after the same hydrolysis for 72h, 90% of the theoretical maximum amount of reducing sugars is liberated.
  • the process including enzyme recycling after hydrolysis can be combined with recycling of the microorganism producing the fermentation product after fermentation and with the use of the reducing sugars containing filtrate as a substrate (purified and/or concentrated or diluted) in the propagation and cultivation of the microorganism producing the fermentation product and the microorganism producing the enzymes for hydrolysis.
  • the reducing sugars containing filtrate can also be used as a substrate (purified and/or concentrated or diluted) in the production of the fermentation product by the fermentation product producing microorganism and the production of the enzymes for hydrolysis by the enzyme producing microorganism.
  • thermostability 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 pretreated material conversion into for example ethanol.
  • the thin stillage can be used either in concentrated or (un)diluted form and/or purified and with or without additional enzyme supplementation.
  • an amount of enzyme is supplemented into the thin stillage, before its re-use in a new process cycle, equal to the amount of activity lost during the three successive process steps of the previous process cycle. In this way over dosage of enzyme is avoided and thus most efficient use of enzyme is obtained. Moreover, by providing high enzyme dosage in the first process cycle, and supplementing enzyme equal to the amount of activity lost during the three successive process steps in the following process cycles, highest possible hydrolysis rates can be obtained in each process cycle resulting in short hydrolysis times of less than 48h in combination with most efficient use of enzymes.
  • An advantage of expression and production of the enzymes for example at least two, three or four different cellulases
  • a suitable microorganism may be a high enzyme composition yield which can be used in the processes of the present invention.
  • enzyme compositions are used.
  • the compositions are stable.
  • “Stable enzyme compositions” as used herein means that the enzyme compositions retain activity after 30 hours of hydrolysis reaction time, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of its initial activity after 30 hours of hydrolysis reaction time.
  • the enzyme composition retains activity after 40, 50, 60, 70, 80, 90 100, 150, 200, 250, 300, 350, 400, 450, 500 hours of hydrolysis reaction time.
  • the enzymes may be prepared by fermentation of a suitable substrate with a suitable microorganism, e.g. Rasamsonia emersonii or Aspergillus niger, wherein the enzymes are produced by the microorganism.
  • the microorganism may be altered to improve or to make the enzymes.
  • the microorganism may be mutated by classical strain improvement procedures or by recombinant DNA techniques. Therefore, the microorganisms mentioned herein can be used as such to produce the enzymes or may be altered to increase the production or to produce altered enzymes which might include heterologous enzymes, e.g. cellulases, thus enzymes that are not originally produced by that microorganism.
  • a fungus more preferably a filamentous fungus is used to produce the enzymes.
  • a thermophilic or thermotolerant microorganism is used.
  • a substrate is used that induces the expression of the enzymes by the enzyme producing microorganism.
  • the enzymes are used to release sugars from lignocellulosic material, that comprises polysaccharides.
  • the major polysaccharides are cellulose (glucans), hemicelluloses (xylans, heteroxylans and xyloglucans).
  • hemicellulose may be present as giucomannans, for example in wood-derived lignocellulosic material.
  • sugar product is meant the enzymatic hydrolysis product of the lignocellulosic material.
  • the sugar product comprises soluble sugars, including both monomers and multimers. Preferably, it comprises glucose.
  • sugars examples include cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and other hexoses and pentoses.
  • the sugar product may be used as such or may be further processed for example recovered and/or purified.
  • pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins).
  • Lytic polysaccharide monooxygenases, endoglucanases (EG) and exo- cellobiohydrolases (CBH) catalyze the hydrolysis of insoluble cellulose to products such as cellooligosaccharides (cellobiose as a main product), while ⁇ -glucosidases (BG) convert the oligosaccharides, mainly cellobiose and cellotriose, to glucose.
  • Xylanases together with other accessory enzymes, for example oL- arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases, and ⁇ - xylosidases catalyze the hydrolysis of hemicellulose.
  • An enzyme composition for use in the process of the current invention comprises preferably at least two activities, although typically a composition will comprise more than two activities, for example, three, four, five, six, seven, eight, nine or even more activities.
  • an enzyme composition for use in the processes of the current invention comprises at least two cellulases.
  • the at least two cellulases may contain the same or different activities.
  • the enzyme composition for use in the processes of the current invention may also comprises at least one enzyme other than a cellulase.
  • the at least one other enzyme has an auxiliary enzyme activity, i.e. an additional activity which, either directly or indirectly leads to lignocellulose degradation. Examples of such auxiliary activities are mentioned herein and include, but are not limited, to hemicellulases.
  • composition for use in the processes of the current invention may comprise lytic polysaccharide monooxygenase activity, endoglucanase activity and/or cellobiohydrolase activity and/or beta-glucosidase activity.
  • a composition for use in the invention may comprise more than one enzyme activity per activity class.
  • a composition for use in the invention may comprise two endoglucanase activities, for example, endo-1 ,3(1 ,4)- glucanase activity and endo- ⁇ -1 ,4-glucanase activity.
  • a composition for use in the processes of the current invention may be derived from a fungus, such as a filamentous fungus, such as Rasamsonia, such as Rasamsonia emersonii.
  • a core set of (lignocellulose degrading) enzyme activities may be derived from Rasamsonia emersonii. Rasamsonia emersonii can provide a highly effective set of activities as demonstrated herein for the hydrolysis of lignocellulosic material. If needed, the set of activities can be supplemented with additional enzyme activities from other sources. Such additional activities may be derived from classical sources and/or produced by genetically modified organisms.
  • the activities in a composition for use in the processes of the current invention may be thermostable.
  • this means that the activity has a temperature optimum of 60°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher.
  • Activities in a composition for use in the processes of the current invention will typically not have the same temperature optima, but preferably will, nevertheless, be thermostable.
  • low pH indicates a pH of 5.5 or lower, 5 or lower, 4.9 or lower, 4.8 or lower, 4.7 or lower, 4.6 or lower, 4.5 or lower, 4.4 or lower, 4.3 or lower, 4.2 or lower, 4.1 or lower, 4.0 or lower 3.9 or lower, 3.8 or lower, 3.7 or lower, 3.6 or lower, 3.5 or lower.
  • Activities in a composition for use in the processes of the current invention may be defined by a combination of any of the above temperature optima and pH values.
  • the enzyme composition for use in the processes of the current invention may comprise a cellulase and/or a hemicellulase and/or a pectinase from a source other than Rasamsonia. They may be used together with one or more Rasamsonia enzymes or they may be used without additional Rasamsonia enzymes being present.
  • enzymes for use in the processes of the current invention may comprise a beta-glucosidase (BG) from Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 02/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigatus, such as the one disclosed as SEQ ID NO:2 in WO 2005/047499 or SEQ ID NO:5 in WO 2014/130812 or an Aspergillus fumigatus beta-glucosidase variant, such as one disclosed in WO 2012/044915, such as one with the following substitutions: F100D, S283G, N456E, F512Y (using SEQ ID NO: 5 in WO 2014/130812 for numbering), or Aspergillus aculeatus, Aspergillus niger or Aspergillus kawachi.
  • BG beta-glucosidase
  • beta- glucosidase is derived from Penicillium, such as Penicillium brasilianum disclosed as SEQ ID NO:2 in WO 2007/019442, or from Trichoderma, such as Trichoderma reesei, such as ones described in US 6,022,725, US 6,982,159, US 7,045,332, US 7,005,289, US 2006/0258554 US 2004/0102619. In an embodiment even a bacterial beta-glucosidase can be used.
  • the beta-glucosidase is derived from Thielavia terrestris (WO 201 1/035029) or Trichophaea saccata (WO 2007/019442).
  • enzymes for use in the processes of the current invention may comprise an endoglucanase (EG) from Trichoderma, such as Trichoderma reesei; from Humicola, such as a strain of Humicola insolens; from Aspergillus, such as Aspergillus aculeatus or Aspergillus kawachii; from Erwinia, such as Erwinia carotovara; from Fusarium, such as Fusarium oxysporum; from Thielavia, such as Thielavia terrestris; from Humicola, such as Humicola grisea var.
  • EG endoglucanase
  • thermoidea or Humicola insolens from Melanocarpus, such as Melanocarpus albomyces; from Neurospora, such as Neurospora crassa; from Myceliophthora, such as Myceliophthora thermophila; from Cladorrhinum, such as Cladorrhinum foecundissimum and/or from Chrysosporium, such as a strain of Chrysosporium lucknowense.
  • a bacterial endoglucanase can be used including, but are not limited to, Acidothermus cellulolyticus endoglucanase (see WO 91/05039; WO 93/15186; US 5,275,944; WO 96/02551 ; US 5,536,655, WO 00/70031 , WO 05/093050); Thermobifida fusca endoglucanase III (see WO 05/093050); and Thermobifida fusca endoglucanase V (see WO 05/093050).
  • enzymes for use in the processes of the current invention may comprise a cellobiohydrolase I from Aspergillus, such as Aspergillus fumigatus, such as the Cel7A CBH I disclosed in SEQ ID NO:6 in WO 201 1/057140 or SEQ ID NO:6 in WO 2014/130812, or from Trichoderma, such as Trichoderma reesei.
  • Aspergillus such as Aspergillus fumigatus
  • Cel7A CBH I disclosed in SEQ ID NO:6 in WO 201 1/057140 or SEQ ID NO:6 in WO 2014/130812
  • Trichoderma such as Trichoderma reesei.
  • enzymes for use in the processes of the current invention may comprise a cellobiohydrolase II from Aspergillus, such as Aspergillus fumigatus, such as the one in SEQ ID NO:7 in WO 2014/130812 or from Trichoderma, such as Trichoderma reesei, or from Thielavia, such as Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.
  • Aspergillus such as Aspergillus fumigatus
  • Trichoderma such as Trichoderma reesei
  • Thielavia such as Thielavia terrestris
  • cellobiohydrolase II CEL6A from Thielavia terrestris.
  • enzymes for use in the processes of the current invention may comprise a
  • GH61 polypeptide (a lytic polysaccharide monooxygenase) from Thermoascus, such as Thermoascus aurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO:2 and SEQ ID NO: 1 in WO2014/130812 and in WO 2010/065830; or from Thielavia, such as Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 8 or SEQ ID NO:4 in WO2014/130812 and in WO 2008/148131 , and WO 201 1/035027; or from Aspergillus, such as Aspergillus fumigatus, such as the one described in WO 2010/138754 as SEQ ID NO:2 or SEQ ID NO: 3 in WO2014/130812; or from Penicillium, such as Penicillium emersonii, such as the one disclosed as SEQ ID NO
  • GH61 polypeptides include, but are not limited to, Trichoderma reesei (see WO 2007/089290), Myceliophthora thermophila (see WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868), Penicillium pinophilum (see WO 201 1/005867), Thermoascus sp. (see WO 201 1/039319), and Thermoascus crustaceous (see WO 201 1/041504).
  • the GH61 polypeptide is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043, e.g.
  • the GH61 polypeptide is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated cellulosic material such as pretreated corn stover.
  • cellulolytic enzymes that may be used in the processes of the present invention are described in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481 , WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/0521 18, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/1 17432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, US 5,457,046, US 5,648,263, and US 5,686,593, to name just a few.
  • examples of xylanases useful in the processes of the present invention include, but are not limited to, xylanases from Aspergillus aculeatus (see WO 94/21785), Aspergillus fumigatus (see WO 2006/078256), Penicillium pinophilum (see WO 201 1/041405), Penicillium sp. (see WO 2010/126772), Thielavia terrestris NRRL 8126 (see WO 2009/079210), and Trichophaea saccata GH10 (see WO 201 1/057083).
  • beta-xylosidases useful in the processes of the present invention include, but are not limited to, beta-xylosidases from Neurospora crassa and Trichoderma reesei.
  • acetylxylan esterases useful in the processes of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (see WO 2010/108918), Chaetomium globosum, Chaetomium gracile, Humicola insolens DSM 1800 (see WO 2009/073709), Hypocrea jecorina (see WO 2005/001036), Myceliophtera thermophila (see WO 2010/014880), Neurospora crassa, Phaeosphaeria nodorum and Thielavia terrestris NRRL 8126 (see WO 2009/042846).
  • arabinofuranosidases useful in the processes of the present invention include, but are not limited to, arabinofuranosidases from Aspergillus niger, Humicola insolens DSM 1800 (see WO 2006/1 14094 and WO 2009/073383) and M. giganteus (see WO 2006/1 14094).
  • alpha-glucuronidases useful in the processes of the present invention include, but are not limited to, alpha-glucuronidases from Aspergillus clavatus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Humicola insolens (see WO 2010/014706), Penicillium aurantiogriseum (see WO 2009/068565) and Trichoderma reesei.
  • An enzyme composition for use in the processes of the current invention may comprise one, two, three, four classes or more of cellulase, for example one, two, three or four or all of a lytic polysaccharide monooxygenas (LPMO) , an endoglucanase (EG), one or two exo- cellobiohydrolase (CBH) and a beta-glucosidase(BG).
  • LPMO lytic polysaccharide monooxygenas
  • EG endoglucanase
  • CBH exo- cellobiohydrolase
  • BG beta-glucosidase
  • An enzyme composition for use in the processes of the current invention may comprise one type of cellulase activity and/or hemicellulase activity and/or pectinase activity provided by a composition as described herein and a second type of cellulase activity and/or hemicellulase activity and/or pectinase activity provided by an additional cellulase/hemicellulase/pectinase.
  • 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 catalyzing 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. Such degradation will typically take place by way of a hydrolysis reaction.
  • Lytic polysaccharide monooxygenases are recently classified by CAZy in family AA9 (Auxiliary Activity Family 9) or family AA10 (Auxiliary Activity Family 10). As mentioned above, lytic polysaccharide monooxygenases are able to open a crystalline glucan structure. Lytic polysaccharide monooxygenases may also affect cello-oligosaccharides. GH61 (glycoside hydrolase family 61 or sometimes referred to EGIV) proteins are (lytic) oxygen-dependent polysaccharide monooxygenases (PMO's/LPMO's) according to the latest literature (see Isaksen et al., Journal of Biological Chemistry, vol. 289, no. 5, pp.
  • GH61 was originally classified as endoglucanase based on measurement of very weak endo-1 ,4- -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 folding 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 are recently now reclassified by CAZy in family AA9 (Auxiliary Activity Family 9).
  • GH61 is used herein as being part of the cellulases.
  • CBM33 family 33 carbohydrate-binding module
  • CAZy has recently reclassified CBM33 in AA10 (Auxiliary Activity Family 10).
  • 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 catalyzing 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. 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 catalyzing 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. Such degradation will typically take place by way of a hydrolysis reaction.
  • an enzyme composition for use in the processes of the current invention may comprise any cellulase, for example, a lytic polysaccharide monooxygenase (e.g. GH61 ), a cellobiohydrolase, an endo- -1 ,4-glucanase, a beta-glucosidaseor a -(1 ,3)(1 ,4)-glucanase.
  • a lytic polysaccharide monooxygenase e.g. GH61
  • a cellobiohydrolase e.g. a cellobiohydrolase
  • an endo- -1 ,4-glucanase e.g. a cellobiohydrolase
  • an endo- -1 ,4-glucanase e.g. a cellobiohydrolase
  • an endo- -1 ,4-glucanase e.g. a cellobiohydrolase
  • a cellobiohydrolase (EC 3.2.1.91 ) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4- -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- ⁇ - cellobiosidase, 1 ,4- -cellobiohydrolase, 1 ,4- -D-glucan cellobiohydrolase, avicelase, ⁇ -1 ,4- ⁇ - D-glucanase, exocellobiohydrolase or exoglucanase.
  • an endo ⁇ -1 ,4-glucanase (EC 3.2.1.4) is any polypeptide which is capable of catalyzing the endohydrolysis of 1 ,4 ⁇ -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 ⁇ -D-glucanohydrolase, endo-1 , 4 ⁇ -glucanase or endoglucanase.
  • a beta-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) ) ⁇ 3 ⁇ 35 ⁇ is any polypeptide which is capable of catalysing 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- -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 composition for use in the processes of the current invention may comprise any hemicellulase, for example, an endoxylanase, a ⁇ -xylosidase, a oL-arabionofuranosidase, an o D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase, an o galactosidase, a ⁇ -galactosidase, a ⁇ -mannanase or a ⁇ -mannosidase.
  • hemicellulase for example, an endoxylanase, a ⁇ -xylosidase, a oL-arabionofuranosidase, an o D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase, an o galactos
  • an endoxylanase (EC 3.2.1.8) is any polypeptide which is capable of catalysing 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 hydrolyze 1 ,4 xylosidic linkages in glucuronoarabinoxylans.
  • a ⁇ -xylosidase (EC 3.2.1.37) is any polypeptide which is capable of catalysing 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-O-methylated glucoronic acid, which can also be present as a substituent in xylans.
  • An alternative is EC 3.2.1.131 : 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 catalysing the deacetylation of xylans and xylo-oligosaccharides.
  • 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 catalyse the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) 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 hydroxycinnamoyi 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 feruloyl esterase.
  • an ogalactosidase (EC 3.2.1.22) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing oD-galactose residues in oD-galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing oD-fucosides. This enzyme may also be referred to as melibiase.
  • a ⁇ -galactosidase (EC 3.2.1.23) is any polypeptide which is capable of catalysing the hydrolysis of terminal non-reducing ⁇ -D-galactose residues in ⁇ -D-galactosides. Such a polypeptide may also be capable of hydrolyzing oL-arabinosides. This enzyme may also be referred to as exo-(1->4)- -D-galactanase or lactase.
  • a ⁇ -mannanase (EC 3.2.1.78) is any polypeptide which is capable of catalysing the random hydrolysis of 1 ,4- -D-mannosidic linkages in mannans, galactomannans and glucomannans.
  • This enzyme may also be referred to as mannan endo-1 ,4- -mannosidase or endo-1 ,4-mannanase.
  • a ⁇ -mannosidase (EC 3.2.1.25) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing ⁇ -D-mannose residues in ⁇ -D-mannosides.
  • This enzyme may also be referred to as mannanase or mannase.
  • a composition for use in the processes of the current invention 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 pec
  • an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide which is capable of catalysing 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 catalysing the endohydrolysis of 1 ,4- -D-galactosidic linkages in arabinogalactans.
  • the enzyme may also be known as arabinogalactan endo-1 , 4- -galactosidase, endo-1 , 4- -galactanase, galactanase, arabinogalactanase or arabinogalactan 4- -D-galactanohydrolase.
  • a pectin acetyl esterase is defined herein as any enzyme which has an acetyl esterase activity which catalyses 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 catalysing the eliminative cleavage of (1 ⁇ 4)-oD-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 irans-eliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1 -*4)-6-0- methyl-oD-galacturonan lyase.
  • a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalysing the eliminative cleavage of (1 ⁇ 4)-oD-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 irans-eliminase, polygalacturonic acid irans-eliminase or (1 ⁇ 4)-oD-galacturonan lyase.
  • an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which is capable of catalysing the hydrolysis of terminal non-reducing a-L-rhamnose residues in oL- rhamnosides or alternatively in rhamnogalacturonan.
  • This enzyme may also be known as oL- rhamnosidase T, oL-rhamnosidase N or oL-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-ogalacturonosidase, 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-oD-galacturonide) galacturonohydrolase.
  • exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptide capable of catalysing 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-irans-eliminase, PATE, exo-PATE, exo-PGL or (1 ⁇ 4)-oD- galacturonan reducing-end-disaccharide-lyase.
  • rhamnogalacturonan hydrolase is any polypeptide which is capable of hydrolyzing the linkage between galactosyluronic 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-Rhap-(1 ⁇ 4)-oD-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 encfo-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 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.
  • endo-arabinanase (EC 3.2.1.99) is any polypeptide which is capable of catalysing endohydrolysis of 1 ,5-oarabinofuranosidic linkages in 1 ,5-arabinans.
  • the enzyme may also be known 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 for use in the processes of the current invention will typically comprise at least two cellulases and optionally at least one hemicellulase and optionally at least one pectinase.
  • a composition for use in the processes of the current invention may comprise a lytic polysaccharide monooxygenases (such as GH61 ), a cellobiohydrolase, an endoglucanase and/or a beta-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, an expansin, a cellulose induced protein or a cellulose integrating protein or like protein may be present in a composition for use in the processes of the current 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 processes of the current invention. 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 catalysing 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 oD-glucuronidase (EC 3.2.1.139).
  • a composition for use in the processes of the current invention may comprise an expansin or expansin-like protein, such as a swollenin (see Salheimo ef a/., Eur. J. Biochem. 269, 4202-421 1 , 2002) or a swollenin-like protein.
  • an expansin or expansin-like protein such as a swollenin (see Salheimo ef a/., Eur. J. Biochem. 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 composition for use in the processes of the current invention may comprise a cellulose induced protein, for example the polypeptide product of the cipl or c; 2 gene or similar genes (see Foreman ef a/., J. Biol. Chem. 278(34), 31988-31997, 2003), a cellulose/cellulosome integrating protein, for example the polypeptide product of the cipA or cipC gene, or a scaffoldin or a scaffoldin-like protein.
  • Scaffoldins and cellulose integrating proteins are multi-functional integrating subunits which may organize cellulolytic subunits into a multi-enzyme complex. This is accomplished by the interaction of two complementary classes of domain, i.e.
  • scaffoldin subunit also bears a cellulose-binding module (CBM) that mediates attachment of the cellulosome to its substrate.
  • CBM cellulose-binding module
  • a scaffoldin or cellulose integrating protein for the purposes of this invention may comprise one or both of such domains.
  • a composition for use in the processes of the current invention may also comprise a catalase.
  • catalase means a hydrogen-peroxide: hydrogen-peroxide oxidoreductase (EC 1.1 1.1.6 or EC 1.1 1.1.21 ) that catalyzes the conversion of two hydrogen peroxides to oxygen and two waters.
  • Catalase activity can be determined by monitoring the degradation of hydrogen peroxide at 240 nm based on the following reaction: 2H 2 0 2 ⁇ 2H 2 0 + 0 2 . The reaction is conducted in 50 mM phosphate pH 7.0 at 25°C with 10.3 mM substrate (H 2 0 2 ) and approximately 100 units of enzyme per ml.
  • One catalase activity unit can be expressed as one micromole of H 2 0 2 degraded per minute at pH 7.0 and 25°C.
  • a composition for use in the processes of the current invention may be composed of 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).
  • a composition for use in the processes of the current invention may be composed of enzymes from (1 ) commercial suppliers; (2) cloned genes expressing enzymes; (3) 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 material.
  • the enzyme may be produced in a fermentation that uses (pretreated) lignocellulosic material (such as corn stover or wheat straw) to provide nutrition to an organism that produces an enzyme(s).
  • plants that produce the enzymes may themselves serve as a lignocellulosic material and be added into lignocellulosic material.
  • compositions described above may be provided concomitantly (i.e. as a single composition per se) or separately or sequentially.
  • the enzyme composition is in the form of a whole fermentation broth of a fungus, preferably Rasamsonia.
  • the whole fermentation broth can be prepared from fermentation of non-recombinant and/or recombinant filamentous fungi.
  • the filamentous fungus is a recombinant filamentous fungus comprising one or more genes which can be homologous or heterologous to the filamentous fungus.
  • the filamentous fungus is a recombinant filamentous fungus comprising one or more genes which can be homologous or heterologous to the filamentous fungus wherein the one or more genes encode enzymes that can degrade a cellulosic substrate.
  • the whole fermentation broth may comprise any of the polypeptides described above or any combination thereof.
  • the enzyme composition is a whole fermentation broth wherein the cells are killed.
  • the whole fermentation broth may contain organic acid(s) (used for killing the cells), killed cells and/or cell debris, and culture medium.
  • the filamentous fungi is cultivated in a cell culture medium suitable for production of enzymes capable of hydrolyzing a cellulosic substrate.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art.
  • suitable culture media, temperature ranges and other conditions suitable for growth and cellulase and/or hemicellulase and/or pectinase production are known in the art.
  • the whole fermentation broth can be prepared by growing the filamentous fungi to stationary phase and maintaining the filamentous fungi under limiting carbon conditions for a period of time sufficient to express the one or more cellulases and/or hemicellulases and/or pectinases.
  • the whole fermentation broth can be used.
  • the whole fermentation broth of the present invention may comprise filamentous fungi.
  • the whole fermentation broth comprises the unfractionated contents of the fermentation materials derived at the end of the fermentation.
  • the whole fermentation broth comprises the spent culture medium and cell debris present after the filamentous fungi is grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (particularly, expression of cellulases and/or hemicellulases and/or pectinases).
  • the whole fermentation broth comprises the spent cell culture medium, extracellular enzymes and filamentous fungi.
  • the filamentous fungi present in whole fermentation broth can be lysed, permeabilized, or killed using methods known in the art to produce a cell-killed whole fermentation broth.
  • the whole fermentation broth is a cell-killed whole fermentation broth, wherein the whole fermentation broth containing the filamentous fungi cells are lysed or killed.
  • the cells are killed by lysing the filamentous fungi by chemical and/or pH treatment to generate the cell-killed whole broth of a fermentation of the filamentous fungi.
  • the cells are killed by lysing the filamentous fungi by chemical and/or pH treatment and adjusting the pH of the cell- killed fermentation mix to a suitable pH.
  • the whole fermentation broth comprises a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least 6 or more carbon organic acid and/or a salt thereof.
  • the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or any combination thereof and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or any combination thereof.
  • whole fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
  • whole fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g. , expression of enzymes by host cells) and secretion into cell culture medium.
  • the whole fermentation broth is unfractionated and comprises spent cell culture medium, extracellular enzymes, and microbial, preferably non-viable, cells.
  • the whole fermentation broth can be fractionated and the one or more of the fractionated contents can be used.
  • the killed cells and/or cell debris can be removed from a whole fermentation broth to provide a composition that is free of these components.
  • the whole fermentation broth may further comprise a preservative and/or anti-microbial agent.
  • a preservative and/or anti-microbial agent are known in the art.
  • the whole fermentation broth as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified whole fermentation broth.
  • the whole fermentation broth may be supplemented with one or more enzyme activities that are not expressed endogenously, or expressed at relatively low level by the filamentous fungi, to improve the degradation of the cellulosic substrate, for example, to fermentable sugars such as glucose or xylose.
  • the supplemental enzyme(s) can be added as a supplement to the whole fermentation broth and the enzymes may be a component of a separate whole fermentation broth, or may be purified, or minimally recovered and/or purified.
  • the whole fermentation broth comprises a whole fermentation broth of a fermentation of a recombinant filamentous fungi overexpressing one or more enzymes to improve the degradation of the cellulosic substrate.
  • the whole fermentation broth can comprise a mixture of a whole fermentation broth of a fermentation of a non-recombinant filamentous fungus and a recombinant filamentous fungus overexpressing one or more enzymes to improve the degradation of the cellulosic substrate.
  • the whole fermentation broth comprises a whole fermentation broth of a fermentation of a filamentous fungi overexpressing beta-glucosidase.
  • the whole fermentation broth for use in the present methods and reactive compositions can comprise a mixture of a whole fermentation broth of a fermentation of a non-recombinant filamentous fungus and a whole fermentation broth of a fermentation of a recombinant filamentous fungi overexpressing a beta-glucosidase.
  • Enzymes are present in the liquefaction step and in the saccharification step of the enzymatic hydrolysis. These enzymes may be the same or may be different. Furthermore, as described above, additional enzymes are added during the liquefaction step and the saccharification step of the integrated processes according to the present invention. The enzymes added may be enzymes that are already present in the liquefaction step and in the saccharification step. Alternatively, they may be different enzymes. Moreover, the additional enzymes added during the liquefaction step may differ or may be the same as the additional enzymes added during the saccharification step of the integrated processes according to the present invention.
  • Lignocellulosic material as used herein includes any lignocellulosic and/or hemicellulosic material.
  • Lignocellulosic material suitable for use in the processes of the current 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 e.g. virgin biomass and/or non-virgin biomass
  • Common forms of biomass include trees, shrubs and grasses, wheat, wheat straw, sugar cane, cane straw, sugar cane bagasse, sugar cane trash, switch grass, miscanthus, energy cane, 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 lignocellulosic material is selected from the group consisting of sugar cane, bagasse, energy cane, cane straw and sugar cane trash.
  • Cellulose is an organic compound with the formula (C 6 H 10 O5)n, a polysaccharide consisting of a linear chain of several hundred to over ten thousand ⁇ (1 ⁇ 4) linked D-glucose units.
  • a glucan molecule is a polysaccharide of D-glucose monomers linked by glycosidic bonds.
  • glucan and cellulose are used interchangeably for a polysaccharide of D-glucose monomers linked by glycosidic bonds.
  • Methods for the quantitative analysis of glucan or polysaccharide compositions are well-known and described in the art and are for example summarized in Carvalho de Souza et al., Carbohydrate Polymers 95 (2013) 657-663. In general, 50 to 70% of the glucan is crystalline cellulose, the remainder is amorphous cellulose.
  • the lignocellulosic material is pretreated before and/or during the enzymatic hydrolysis.
  • Pretreatment methods are known in the art and include, but are not limited to, heat, mechanical, chemical modification, biological modification and any combination thereof.
  • Pretreatment is typically performed in order to enhance the accessibility of the lignocellulosic material to enzymatic hydrolysis and/or hydrolyse the hemicellulose and/or solubilize the hemicellulose and/or cellulose and/or lignin, in the lignocellulosic material.
  • the pretreatment comprises treating the lignocellulosic material with steam explosion, hot water treatment or treatment with dilute acid or dilute base.
  • pretreatment methods include, but are not limited to, steam treatment (e.g. treatment at 100-260°C, at a pressure of 7-45 bar, at neutral pH, for 1-10 minutes), dilute acid treatment (e.g. treatment with 0.1 - 5% H 2 S0 4 and/or S0 2 and/or HN0 3 and/or HCI, in presence or absence of steam, at 120-200°C, at a pressure of 2- 15 bar, at acidic pH, for 2-30 minutes), organosolv treatment (e.g. treatment with 1 - 1.5% H 2 S0 4 in presence of organic solvent and steam, at 160-200°C, at a pressure of 7-30 bar, at acidic pH, for 30-60 minutes), lime treatment (e.g.
  • steam treatment e.g. treatment at 100-260°C, at a pressure of 7-45 bar, at neutral pH, for 1-10 minutes
  • dilute acid treatment e.g. treatment with 0.1 - 5% H 2 S0 4 and/or S0 2 and/or HN0 3 and
  • ARP treatment e.g. treatment with 5 - 15% NH 3 , at 150-180°C, at a pressure of 9-17 bar, at alkaline pH, for 10-90 minutes
  • AFEX treatment e.g. treatment with > 15% NH 3 , at 60-140°C, at a pressure of 8-20 bar, at alkaline pH, for 5-30 minutes).
  • the lignocellulosic material may be washed.
  • the lignocellulosic material may be washed after the pretreatment.
  • the washing step may be used to remove water soluble compounds that may act as inhibitors for the fermentation and/or hydrolysis step.
  • the washing step may be conducted in manner known to the skilled person.
  • the enzymatically hydrolysed lignocellulosic material is washed and/or detoxified.
  • the solid fraction and/or the liquid fraction obtained after solid/liquid separation of the enzymatically hydrolysed lignocellulosic material is washed and/or detoxified.
  • the liquid fraction obtained after solid/liquid separation of the enzymatically hydrolysed lignocellulosic material is subjected to a detoxification step and/or a concentration step.
  • the detoxification step has been described in detail above.
  • the concentration step can be done by methods well known to a person skilled in the art including, but not limited to, centrifugation.
  • the enzyme composition used in the process of the invention can extremely effectively hydrolyze lignocellulosic material, for example corn stover, wheat straw, cane straw, sugar cane, sugar cane trash and/or bagasse, which can then be further converted into a product, such as ethanol, biogas, butanol, a plastic, an organic acid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical feedstock.
  • a product such as ethanol, biogas, butanol, a plastic, an organic acid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical feedstock.
  • intermediate products from a process following the hydrolysis for example lactic acid as intermediate in biogas production, can be used as building block for other materials.
  • the amount of enzyme added (herein also called enzyme dosage or enzyme load) is low.
  • the amount of enzyme is 10 mg protein / g dry matter weight or lower, 9 mg protein / g dry matter weight or lower, 8 mg protein / g dry matter weight or lower, 7 mg protein / g dry matter weight or lower, 6 mg protein / g dry matter weight or lower, 5 mg protein / g dry matter or lower, 4 mg protein / g dry matter or lower, 3 mg protein / g dry matter or lower, 2 mg protein / g dry matter or lower, or 1 mg protein / g dry matter or lower (expressed as protein in mg protein / g dry matter).
  • the amount of enzyme is 5 mg enzyme / g dry matter weight or lower, 4 mg enzyme / g dry matter weight or lower, 3 mg enzyme / g dry matter weight or lower, 2 mg enzyme / g dry matter weight or lower, 1 mg enzyme / g dry matter weight or lower, 0.5 mg enzyme / g dry matter weight or lower, 0.4 mg enzyme composition / g dry matter weight or lower, 0.3 mg enzyme / g dry matter weight or lower, 0.25 mg enzyme / g dry matter weight or lower, 0.20 mg enzyme / g dry matter weight or lower, 0.18 mg enzyme / g dry matter weight or lower, 0.15 mg enzyme / g dry matter weight or lower or 0.10 mg enzyme / g dry matter weight or lower (expressed as total of cellulase enzymes in mg enzyme / g dry matter).
  • enzyme may be added before and/or during only one of the steps or before and/or during both steps.
  • the pH during the enzymatic hydrolysis may be chosen by the skilled person.
  • the pH during the hydrolysis may be 3.0 to 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, 2.5 to 7.5, 3.0 to 7.0, 3.5 to 6.5, 4.0 to 5.0, 4.0 to 4.5 or is about 4.2.
  • the pH used in the liquefaction step of the enzymatic hydrolysis and the saccharification step of the enzymatic hydrolysis may differ or may be the same. In case different enzymes are used during the liquefaction step and the saccharification step, the optimum pH of said enzymes may differ or may be the same.
  • the hydrolysis 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 the lignocellulosic material is released.
  • a process of the invention may be carried out using high levels of dry matter
  • the dry matter content at the end of the enzymatic hydrolysis is 5 wt% or higher, 6 wt% or higher, 7 wt% or higher, 8 wt% or higher, 9 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, 16 wt% or higher, 17 wt% or higher, 18 wt% or higher, 19 wt% or higher, 20 wt% or higher, 21 wt% or higher, 22 wt% or higher, 23 wt% or higher, 24 wt% or higher, 25 wt% or higher, 26 wt% or higher, 27 wt% or higher, 28 wt% or higher, 29 wt% or higher, 30 wt% or higher, 31 wt% or higher,
  • the dry matter content at the end of the enzymatic hydrolysis is between 5 wt% - 40 wt%, 6 wt% - 40 wt%, 7 wt% - 40 wt%, 8 wt% - 40 wt%, 9 wt% - 40 wt%, 10 wt% - 40 wt%, 1 1 wt% - 40 wt%, 12 wt% - 40 wt%, 13 wt% - 40 wt%, 14 wt% - 40 wt%, 15 wt% - 40 wt%, 16 wt% - 40 wt%, 17 wt% - 40 wt%, 18 wt% - 40 wt%, 19 wt% - 40 wt%, 20 wt% - 40 wt%, 21 wt% - 40 wt%, 22 wt% - 40 wt%,
  • the dry matter content at the end of the liquefaction step of the enzymatic hydrolysis is 5 wt% or higher, 6 wt% or higher, 7 wt% or higher, 8 wt% or higher, 9 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, 16 wt% or higher, 17 wt% or higher, 18 wt% or higher, 19 wt% or higher, 20 wt% or higher, 21 wt% or higher, 22 wt% or higher, 23 wt% or higher, 24 wt% or higher, 25 wt% or higher, 26 wt% or higher, 27 wt% or higher, 28 wt% or higher, 29 wt% or higher, 30 wt% or higher, 31 wt% or higher, 32 wt% or higher, 33 wt% or
  • the dry matter content at the end of the liquefaction step of the enzymatic hydrolysis is between 5 wt% - 40 wt%, 6 wt% - 40 wt%, 7 wt% - 40 wt%, 8 wt% - 40 wt%, 9 wt% - 40 wt%, 10 wt% - 40 wt%, 1 1 wt% - 40 wt%, 12 wt% - 40 wt%, 13 wt% - 40 wt%, 14 wt% - 40 wt%, 15 wt% - 40 wt%, 16 wt% - 40 wt%, 17 wt% - 40 wt%, 18 wt% - 40 wt%, 19 wt% - 40 wt%, 20 wt% - 40 wt%, 21 wt% - 40 wt%, 22 wt%
  • the dry matter content at the end of the saccharification step of the enzymatic hydrolysis is 5 wt% or higher, 6 wt% or higher, 7 wt% or higher, 8 wt% or higher, 9 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, 16 wt% or higher, 17 wt% or higher, 18 wt% or higher, 19 wt% or higher, 20 wt% or higher, 21 wt% or higher, 22 wt% or higher, 23 wt% or higher, 24 wt% or higher, 25 wt% or higher, 26 wt% or higher, 27 wt% or higher, 28 wt% or higher, 29 wt% or higher, 30 wt% or higher, 31 wt% or higher, 32 wt% or higher, 33 wt% or higher
  • the dry matter content at the end of the saccharification step of the enzymatic hydrolysis is between 5 wt% - 40 wt%, 6 wt% - 40 wt%, 7 wt% - 40 wt%, 8 wt% - 40 wt%, 9 wt% - 40 wt%, 10 wt% - 40 wt%, 1 1 wt% - 40 wt%, 12 wt% - 40 wt%, 13 wt% - 40 wt%, 14 wt% - 40 wt%, 15 wt% - 40 wt%, 16 wt% - 40 wt%, 17 wt% - 40 wt%, 18 wt% - 40 wt%, 19 wt% - 40 wt%, 20 wt% - 40 wt%, 21 wt% - 40 wt%, 22 wt% -
  • the present invention also relates to a process for the preparation of succinic acid from lignocellulosic material.
  • the fermentation is performed in one or more containers.
  • the fermentation can be done in the same container(s) wherein the enzymatic hydrolysis is performed.
  • Fermentation by a succinic acid producing microorganism to produce succinic acid can be combined with fermentation by an alcohol producing microorganism to produce alcohol.
  • the fermentations can be performed in one or more separate containers, but may also be done in one or more of the same containers.
  • the fermentation is done by a yeast.
  • the succinic acid producing microorganism is a yeast.
  • Compositions of fermentation media for growth of microorganisms such as yeasts are well known in the art.
  • the succinic acid producing microorganism is able to ferment at least a C6 sugar.
  • the alcohol producing microorganism and the organic acid producing microorganism are different microorganisms.
  • the alcohol producing microorganism and the organic acid producing microorganism are the same microorganism, i.e. the succinic acid producing microorganism is also able to produce alcohol.
  • the invention thus includes fermentation processes in which a microorganism is used for the fermentation of a carbon source comprising sugar(s).
  • 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.
  • the fermentation time is 100 hours or less, 90 hours or less, 80 hours or less, 70 hours or less. In an embodiment the fermentation time of the succinic acid production is between 20 and 70 hours.
  • 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 + .
  • anaerobic fermentation process pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as succinic acid.
  • 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. Under aerobic conditions, usually the biomass yield is higher than under anaerobic conditions. As a consequence, usually under aerobic conditions, the expected product yield is lower than under anaerobic conditions.
  • the fermentation process is under oxygen-limited conditions.
  • the fermentation process may be 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.
  • an alcohol fermentation process is anaerobic, while a succinic acid fermentation process is aerobic, but done under oxygen-limited conditions.
  • the fermentation process is preferably run at a temperature that is optimal for the microorganism used.
  • the fermentation process is performed at a temperature which is less than 42°C, preferably 38°C or lower.
  • 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.
  • an alcohol fermentation step and a succinic acid fermentation step are performed between 25°C and 35°C.
  • the fermentations are conducted with a fermenting microorganism.
  • the alcohol (e.g. ethanol) fermentations are conducted with a C5 fermenting microorganism.
  • the alcohol (e.g. ethanol) fermentations are conducted with a C5 fermenting microorganism and/or a commercial C6 fermenting microorganism.
  • yeast suitable for ethanol production include, but are not limited to, BIOFERMTM AFT and XR (NABC— North American Bioproducts Corporation, GA, USA), ETHANOL REDTM yeast (Fermentis/Lesaffre, USA), FALITM (Fleischmann's Yeast, USA), FERMIOLTM (DSM Specialties), GERT STRANDTM (Gert Strand AB, Sweden), and SUPERSTARTTM and THERMOSACCTM fresh yeast (Ethanol Technology, Wl, USA).
  • propagation of the alcohol producing microorganism and/or the succinic acid producing microorganism is performed in one or more propagation containers.
  • the propagation can be done by fermentation of the lignocellulosic material, the enzymatically hydrolysed lignocellulosic material, the liquid fraction and/or the solid fraction.
  • the alcohol producing microorganism and/or the succinic acid producing microorganism may be added to one or more fermentation containers.
  • the propagation of the alcohol producing microorganism and/or the succinic acid producing microorganism is combined with the fermentation of the lignocellulosic material, the enzymatically hydrolysed lignocellulosic material, the liquid fraction and/or the solid fraction by the alcohol producing microorganism and/or the succinic acid producing microorganism to produce alcohol and/or succinic acid, respectively.
  • the alcohol producing microorganism is a microorganism that is able to ferment at least one C5 sugar. Preferably, it also is able to ferment at least one C6 sugar.
  • the succinic acid producing microorganism is a microorganism that is able to ferment at least one C6 sugar.
  • the fermentation can be done with a microorganism that is able to ferment at least one C6 sugar.
  • the alcohol producing microorganisms may be a prokaryotic or eukaryotic organism.
  • the microorganism used in the process may be a genetically engineered microorganism.
  • suitable alcohol producing organisms are yeasts, for instance Saccharomyces, e.g. Saccharomyces cerevisiae, Saccharomyces pastorianus or Saccharomyces uvarum, Hansenula, Issatchenkia, e.g. Issatchenkia orientalis, Pichia, e.g. Pichia stipites or Pichia pastoris, Kluyveromyces, e.g. Kluyveromyces fagilis, Candida, e.g.
  • Candida pseudotropicalis or Candida acidothermophilum Pachysolen, e.g. Pachysolen tannophilus or bacteria, for instance Lactobacillus, e.g. Lactobacillus lactis, Geobacillus, Zymomonas, e.g. Zymomonas mobilis, Clostridium, e.g. Clostridium phytofermentans, Escherichia, e.g. E. coli, Klebsiella, e.g. Klebsiella oxytoca.
  • 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 yeast e.g. Saccharomyces cerevisiae, used in the processes according to the present invention is capable of converting hexose (C6) sugars and pentose (C5) sugars.
  • the yeast e.g. Saccharomyces cerevisiae, used in the processes according to the present invention can anaerobically ferment at least one C6 sugar and at least one C5 sugar.
  • the yeast is capable of using L-arabinose and xylose in addition to glucose anaerobically.
  • the yeast is capable of converting L-arabinose into L- ribulose and/or xylulose 5-phosphate and/or into a desired fermentation product, for example into ethanol.
  • Organisms for example Saccharomyces cerevisiae strains, able to produce ethanol from L-arabinose may be produced by modifying a host yeast introducing the araA (L-arabinose isomerase), araB (L-ribuloglyoxalate) and araD (L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes may be introduced into a host cell in order that it is capable of using arabinose. Such an approach is given is described in WO2003/095627.
  • araA, araB and araD genes from Lactobacillus plantarum may be used and are disclosed in WO2008/041840.
  • the araA gene from Bacillus subtilis and the araB and araD genes from Escherichia coli may be used and are disclosed in EP1499708.
  • araA, araB and araD genes may derived from of at least one of the genus Clavibacter, Arthrobacter and/or Gramella, in particular one of Clavibacter michiganensis, Arthrobacter aurescens, and/or Gramella forsetii, as disclosed in WO 200901 1591.
  • the yeast may also comprise one or more copies of xylose isomerase gene and/or one or more copies of xylose reductase and/or xylitol dehydrogenase.
  • the yeast may comprise one or more genetic modifications to allow the yeast to ferment xylose.
  • genetic modifications are introduction of one or more xy/A-gene, XYL1 gene and XYL2 gene and/or XKS7-gene; deletion of the aldose reductase (GRE3) gene; overexpression of PPP-genes TALI, TKL1, RPE1 and RKI1 to allow the increase of the flux through the pentose phosphate pathway in the cell.
  • GRE3 aldose reductase
  • PPP-genes TALI, TKL1, RPE1 and RKI1 examples of genetically engineered yeast are described in EP1468093 and/or WO2006/009434.
  • RN1016 is a xylose and glucose fermenting Saccharomyces cerevisiae strain from DSM, the Netherlands.
  • the fermentation process for the production of ethanol is anaerobic.
  • Anaerobic has already been defined earlier herein.
  • the fermentation process for the production of ethanol is aerobic.
  • 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 fermentation processes described above are also preferred embodiments of this co- fermentation process: identity of the fermentation product, identity of source of L-arabinose and source of xylose, conditions of fermentation (aerobic or anaerobic conditions, oxygen-limited conditions, temperature at which the process is being carried out, productivity of ethanol, yield of ethanol).
  • the succinic acid producing microorganisms may be a prokaryotic or eukaryotic organism.
  • the microorganism used in the process may be a genetically engineered microorganism.
  • suitable succinic acid producing organisms are yeasts, for instance Saccharomyces, e.g.
  • Saccharomyces cerevisiae fungi for instance Aspergillus strains, such as Aspergillus niger and Aspergillus fumigatus, Byssochlamys nivea, Lentinus degener, Paecilomyces varioti and Penicillium viniferum; and bacteria, for instance Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannhei succiniciproducers MBEL 55E, Escherichia coli, Propionibacterium species, Pectinatus sp., Bacteroides sp., such as Bacteroides amylophilus, Ruminococcus flavefaciens, Prevotella ruminicola, Succcinimonas amylolytica, Succinivibrio dextrinisolvens, Wolinella succinogenes, and Cytophaga succinicans.
  • the succinic acid producing microorganism that is able to ferment at least one C6 sugar is a yeast.
  • the yeast is belongs to the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae.
  • the yeast, e.g. Saccharomyces cerevisiae, used in the production processes of succinic acid according to the present invention is capable of converting hexose (C6) sugars.
  • the yeast, e.g. Saccharomyces cerevisiae, used in the processes according to the present invention can anaerobically ferment at least one C6 sugar.
  • the overall reaction time (or the reaction time of hydrolysis step and fermentation step together) may be reduced.
  • the overall reaction time is 300 hours or less, 200 hours or less, 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 reaction times may be reached at lower glucose yield.
  • Fermentation products can be any substance derived from fermentation. They include, but are not limited to, alcohol (such as arabinitol, butanol, ethanol, glycerol, methanol, 1 ,3- propanediol, sorbitol, and xylitol); organic acid (such as acetic acid, acetonic acid, adipic acid, ascorbic acid, acrylic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3- hydroxypropionic acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); ketones (such as acetone); amino acids (such as aspartic acid, glutamic acid, glycine, lysine, serine
  • the fermentation product can also be a protein, a vitamin, a pharmaceutical, an animal feed supplement, a specialty chemical, a chemical feedstock, a plastic, a solvent, ethylene, an enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductase, a transferase or a xylanase.
  • succinic acid and/or an alcohol is prepared in the fermentation processes of the present invention.
  • the alcohol, the succinic acid, the enzymes, the enzyme producing microorganism, the alcohol producing microorganism and/or the succinic acid producing microorganism are recovered.
  • the processes according to the invention may comprise recovery of all kinds of products made during the processes including fermentation products such as ethanol and succinic acid.
  • a fermentation product may be separated from the fermentation broth in manner know to the skilled person. Examples of techniques for recovery include, but are not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For each fermentation product the skilled person will thus be able to select a proper separation technique. For instance, ethanol may be separated from a yeast fermentation broth by distillation, for instance steam distillation/vacuum distillation in conventional way.
  • the processes of the invention also produce energy, heat, electricity and/or steam.
  • the solid fraction obtained after solid/liquid separation of the enzymatically hydrolysed lignocellulosic material and the waste obtained after purification/recovery of the fermentation product can be used in the production of electricity.
  • Electricity can be made by incineration of any one of the above-mentioned materials.
  • the electricicty can be used in any one of the steps of the processes according to the present invention.
  • the beneficial effects of the present invention are found for several lignocellulosic materials and therefore believed to be present for the hydrolysis of all kind of lignocellulosic materials. This beneficial effects of the present invention are found for several enzymes and therefore believed to be present for all kind of hydrolysing enzyme compositions.
  • a part of the enzymatically hydrolysed lignocellulosic material is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism.
  • the part of the enzymatically hydrolysed lignocellulosic material that is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism is the liquid fraction obtained after solid/liquid separation of the enzymatically hydrolysed lignocellulosic material.
  • a part of the enzymatically hydrolysed lignocellulosic material and a part of the lignocellulosic material is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism. This means that a part of the enzymatically hydrolysed lignocellulosic material and/or a part of the lignocellulosic material is added to the enzyme producing microorganism before and/or during propagation and/or before and/or during production of enzymes by the enzyme producing microorganism.
  • the enzyme producing microorganism can also be added to the part of the enzymatically hydrolysed lignocellulosic material and/or the part of the lignocellulosic material and/or the pretreated lignocellulosic material.
  • the lignocellulosic material and/or the pretreated lignocellulosic material used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism has not undergone enzymatic hydrolysis.
  • part of the lignocellulosic material and/or the pretreated lignocellulosic material that is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism has not been subjected to a solid/liquid separation.
  • part of the lignocellulosic material and/or the pretreated lignocellulosic material that is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism has been subjected to a solid/liquid separation.
  • the solid fraction obtained after solid/liquid separation of the lignocellulosic material and/or the pretreated lignocellulosic material is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism.
  • the enzymes produced by the enzyme producing microorganism are used in the enzymatic hydrolysis of the lignocellulosic material and/or the pretreated lignocellulosic material to obtain enzymatically hydrolysed lignocellulosic material.
  • the propagation of the enzyme producing microorganism and the production of enzymes by the enzyme producing microorganism are a single step, meaning that during propagation of the enzyme producing microorganism enzymes are already produced by the microorganism.
  • the enzymatically hydrolysed lignocellulosic material that is added to the enzyme producing microorganism before and/or during propagation of the enzyme producing microorganism and/or before and/or during production of enzymes by the enzyme producing microorganism can be concentrated before addition.
  • the part of the enzymatically hydrolysed lignocellulosic material that is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism has been subjected to a solid/liquid separation.
  • the liquid fraction obtained after solid/liquid separation of the enzymatically hydrolysed lignocellulosic material may be used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism.
  • liquid fraction may be subjected to a concentration step before it is used in the propagation of the enzyme producing microorganism and/or the production of enzymes by the enzyme producing microorganism.
  • the lignocellulosic material and/or the pretreated lignocellulosic material that is added to the enzyme producing microorganism before and/or during propagation of the enzyme producing microorganism and/or before and/or during production of enzymes by the enzyme producing microorganism can be washed before addition.
  • the solid fraction obtained after solid/liquid separation of the enzymatically hydrolysed lignocellulosic material is recycled back to the enzymatic hydrolysis container and subjected to another round of enzymatic hydrolysis.
  • Enzymatic hydrolysis was started by adding an enzyme composition (100 mg eWB-CE enriched in BG/g DM) to the hydrolysis container.
  • TEC-210 cellulase-containing composition was produced as described in WO 201 1/000949.
  • the whole broth of the TEC-210 cellulase-containing composition (eWB-CE) comprised 44 mg protein/g whole broth.
  • the hydrolysis was performed with pretreated sugar cane straw (pSCS) at a final concentration of 20% w/w total solids.
  • the hydrolysis was performed at 62°C at pH 4.5 for 120 hours.
  • the hydrolysed lignocellulosic material (8316 gram) was subjected to a solid/liquid separation by centrifugation for 20 minutes at a relative centrifugal force (RCF) of 10722 at 5°C to obtain a first liquid fraction (5191 gram) and a first solid fraction.
  • the pH of the first liquid fraction was 4.5.
  • the first solid fraction was washed with an equal volume of water, resuspended and again centrifuged for 20 minutes at a relative centrifugal force (RCF) of 10722 at 5°C to obtain a second liquid fraction (2350 gram) and a second solid fraction.
  • RCF relative centrifugal force
  • the first and second liquid fraction were combined to give a liquid fraction of 7541 gram.
  • This fraction comprises 8.5% (w/w) glucose (as measured by HPLC analysis).
  • the detoxified liquid after centrifugation (containing 8.5% (w/w) glucose) was concentrated in a rotavapor (pressure: 200-240 mbar; temperature: 75°C) to a detoxified concentrated liquid (1 151 gram) comprising 50% (w/w) glucose.
  • the detoxified and not detoxified concentrated liquid was used in further experimentation as described below.
  • yeast strain SUC-947 was contructed as follows.
  • the Saccharomyces cerevisiae strain SUC-632 was constructed as described in WO2013/004670. Strain SUC-632 was used as a starting point to construct strain SUC-947.
  • Strain SUC-708 is a mutant of strain SUC-632 obtained by classical strain improvement.
  • a fumarase gene of E. coli was transformed to strain SUC-708 as described below.
  • SEQ ID NO: 1 describes the fumarase (fumB) protein sequence from Escherichia coli (E.C. 4.2.1.2, UniProt accession number P14407).
  • the gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO 2008/000632.
  • the stop codon TAA was modified to TAAG.
  • Expression of the FUM_01 gene is controlled by the TDH1 promoter (600 bp directly before the start codon of the TDH1 gene) and the TDH1 terminator (300 bp directly after the stop codon of the TDH1 gene).
  • TDH1 promoter and TDH1 terminator sequences controlling expression of FUM_01 are native sequences derived from Saccharomyces cerevisiae S288C.
  • the synthetic promoter-gene sequence including appropriate restriction sites was synthesized by GenArt (Regensburg, Germany). This synthetic fragment is part of plasmid pSUC223 ( Figure 1 ), whose sequence is described in SEQ ID NO: 7. Plasmid pSUC223 contains a KanMX marker with allows for selection for growth in the presence of G418.
  • the KanMX marker flanked by lox66 and lox71 sites (Albert ef a/., Plant Journal, 7(4), 649-659), can be removed by the action of Cre-recombinase, as described by Gueldender ef al, (Nucleic Acids Res. 1996 Jul 1 ;24(13):2519-24).
  • the TD H 1 p-f u m B-TD H 11 and the lox66-KanMX-lox71 sequences were flanked by sequences that allow integration by double cross-over at the YPRCtau3 locus, which is located on chromosome XVI.
  • Plasmid pSUC223 was restricted using restriction enzymes ApaU and Xho ⁇ .
  • a 5.408 bp fragment containing the 5' YPRCtau3 flank, the synthetic fumB construct, the KanMX selection marker flanked by lox66 and lox71 sites and the 3' YPRCtau3 flank was excised from an agarose gel and purified using the ZymocleanTM Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions.
  • the fragment was transformed to strain SUC-708.
  • Transformants were selected on Yeast Extract BactoPeptone (YEP) 2% galactose plates supplemented with 200 ⁇ g G418/milliliter for selection of transformants containing the KanMX marker, yielding multiple transformants. Presence of the introduced fumB gene was confirmed by PCR using primer sequences that can anneal to the coding sequences of the ORF encoded by SEQ ID NO: 2.
  • SUC-708 FUM_01 #3 was named SUC-813.
  • the S. cerevisiae Frd1 protein has multiple possible localizations.
  • the variant omitting the 19 aa signal peptide is described in SEQ ID NO: 3, which was subjected to the codon-pair method as disclosed in WO 2008/000632 for expression in S. cerevisiae.
  • the stop codon TAA was modified to TAAG.
  • Expression of the FRD1 gene is controlled by the TDH3 promoter (600 bp directly before the start codon of the TDH3 gene) and the TDH3 terminator (300 bp directly after the stop codon of the TDH3 gene).
  • TDH3 promoter and TDH3 terminator sequences controlling expression of FRD1 are native sequences derived from Saccharomyces cerevisiae S288C.
  • the synthetic promoter-gene sequence including appropriate restriction sites was synthesized by DNA2.0 (Menlo Park, CA, USA) and is described in SEQ ID NO: 4.
  • the modified FRD1 gene was transformed into strain SUC-813 as depicted in Figure 2.
  • the modified FRD1 gene replaced the PDC6 gene in strain SUC-813 using the "split Cre recombinase integration” or "direct Cre recombinase integration” (DCI) approach as described in PCT/EP2013/055047.
  • PCR fragments were generated using Phusion DNA polymerase (New England Biolabs, USA) according to manufacturer's instructions.
  • PCR fragment 1 was generated by using the primer sequences described in SEQ ID NO: 5 and SEQ ID NO: 6, using a cloning plasmid of DNA 2.0 containing SEQ ID NO: 4 as template.
  • SEQ ID NO: 5 contains a 66 bp overlap with the 5' region of the PDC6 gene, directly located before the start codon of the PDC6 gene.
  • Plasmid pSUC228 is a modified version of pSUC227, which is described in PCT/EP2013/055047. Plasmid pSUC227 contains a KanMX marker, which is replaced by a nourseothricin (natMX4) marker (Goldstein and McCusker, Yeast. 1999 Oct; 15(14): 1541-53) resulting in pSUC228.
  • KanMX4 nourseothricin
  • PCR fragment 3 was generated by using the primer sequences described in SEQ ID NO: 10 and SEQ ID NO: 1 1 , using pSUC225 (described in PCT/EP2013/055047) as template.
  • SEQ ID NO: 1 1 contains a 64 bp overlap with the 3' region of the PDC6 gene, directly located after the stop codon of the PDC6 gene.
  • the size of the PCR fragments was checked with standard agarose electrophoresis techniques. PCR amplified DNA fragments were purified using the ZymocleanTM Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions.
  • Yeast transformation was done by a method known by persons skilled in the art.
  • S. cerevisiae strain SUC-813 was transformed with purified PCR fragments 1 , 2 and 3.
  • PCR fragment 1 contained an overlap with PCR fragment 2 at its 3' end.
  • PCR fragment 3 contained an overlap with PCR fragment 2 at its 5' end.
  • PCR fragment 2 contained an overlap at its 5' end with PCR fragment 1 and at its 3' end with PCR fragment 3, such that this allowed homologous recombination of all three PCR fragments ( Figure 2).
  • the 5' end of PCR fragment 1 and the 3' end of PCR fragment 3 were homologous to the PDC6 locus and enabled integration of all three PCR fragments in the PDC6 locus. This resulted in one linear fragment consisting of PCR fragments 1 to 3 integrated in the PDC6 locus ( Figure 2). This method of integration is described in patent application WO 2013/076280.
  • Transformation mixtures were plated on YPD-agar (per liter: 10 grams of yeast extract, 20 grams per liter peptone, 20 grams per liter dextrose, 20 grams of agar) containing 100 ⁇ g nourseothricin (Jena Bioscience, Germany) per ml. After three to five days of growth at 30 ° C, individual transformants were re-streaked on fresh YPD-agar plates containing 100 ⁇ g nourseothricin per ml.
  • the marker cassette and Cre-recombinase are effectively out- recombination by the method described in PCT/EP2013/055047, resulting in replacement of the PDC6 gene by SEQ ID NO: 4 and leaving a lox72 site as a result of recombination between the lox66 and lox71 sites. Due to the activity of Cre-recombinase, the KanMX marker flanked by lox66 and lox71 sites, which was introduced into genomic DNA to create strain SUC-813 was also efficiently out-recombined. The resulting markerfree strain was named SUC-947.
  • Strain SUC-947 was able to grow on YPD-agar plates, but unable to grow on YPD-agar plates supplemented with either 200 ⁇ g G418/ml or 100 ⁇ g/ml nourseothricin or both, confirming out- recombination of both the KanMX and the natMX4 marker.
  • the medium was based on Verduyn ef al. (Verduyn C, Postma E, Scheffers WA, Van Dijken JP. Yeast, 1992 Jul;8(7):501-517), with modifications in the carbon and nitrogen sources, as described herein below.
  • Table 1 Preculture medium composition
  • the pH was controlled at 5.0 by addition of ammonia (10 wt%). Temperature was controlled at 30°C. p0 2 was controlled at 25% (relative to air saturation) by adjusting the stirrer speed. Total airflow applied was 18 NL/h. Glucose concentration was kept limited by controlled feed to the fermenter (exponent of 0.15 was applied).

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Abstract

L'invention concerne un procédé de préparation d'acide succinique à partir de matière lignocellulosique, par hydrolyse enzymatique et fermentation. Ledit procédé comprend une étape consistant à soumettre la matière lignocellulosique hydrolysée par voie enzymatique à une étape de détoxification consistant à mettre en contact ladite matière avec du charbon actif.
PCT/EP2016/064285 2015-06-22 2016-06-21 Procédé d'hydrolyse enzymatique d'une matière lignocellulosique et de fermentation de sucres WO2016207147A1 (fr)

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WO2010011675A1 (fr) * 2008-07-23 2010-01-28 Novozymes A/S Procédés de production de charbon de bois et leurs utilisations
WO2011130725A2 (fr) * 2010-04-16 2011-10-20 Myriant Technologies Inc Production d'acides organiques à partir d'hydrolysat riche en xylose par fermentation bactérienne
WO2013004670A1 (fr) * 2011-07-01 2013-01-10 Dsm Ip Assets B.V. Procédé de préparation d'acides dicarboxyliques à l'aide de cellules fongiques
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