WO2013088003A1 - Method for producing alcohol from lignocellulosic biomass by complementation of the cellulolytic and hemicellulolytic enzymes of trichoderma reesei by the fungus podospora anserina - Google Patents

Method for producing alcohol from lignocellulosic biomass by complementation of the cellulolytic and hemicellulolytic enzymes of trichoderma reesei by the fungus podospora anserina Download PDF

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WO2013088003A1
WO2013088003A1 PCT/FR2012/000499 FR2012000499W WO2013088003A1 WO 2013088003 A1 WO2013088003 A1 WO 2013088003A1 FR 2012000499 W FR2012000499 W FR 2012000499W WO 2013088003 A1 WO2013088003 A1 WO 2013088003A1
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Prior art keywords
cellulolytic
residues
anserina
microorganism
enzymatic hydrolysis
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PCT/FR2012/000499
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French (fr)
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Laetitia POIDEVIN
Senta Blanquet
Eric Record
Jean-Guy BERRIN
Pedro M. Coutinho
Bernard Henrissat
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IFP Energies Nouvelles
C N R S
Universite Aix-Marseille
Institut National De La Recherche Agronomique
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Publication of WO2013088003A1 publication Critical patent/WO2013088003A1/en

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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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the simultaneous use of cellulolytic and hemicellulolytic enzymes from the microorganism Trichoderma reesei and proteins of the fungus Podospora anserina in the enzymatic hydrolysis step of a pretreated lignocellulosic material, particularly in the context of the production of ethanol from pretreated lignocellulosic materials.
  • Lignocellulosic biomass is one of the most abundant renewable resources on earth and constitutes an interesting alternative to fossil raw materials. Its use for energy purposes makes it possible to reduce greenhouse gases while minimizing energy dependence on oil-producing countries.
  • Lignocellulosic biomass is composed of three main polymers: cellulose at a content of between 35 and 50%, hemicelluloses which are polysaccharides essentially consisting of pentoses and hexoses at a content of between 20 and 30% and lignins at a concentration of content between 15 and 25% of the weight. Biomass degradation is difficult because plant wall polysaccharides (cellulose and hemicellulose) are intimately associated with lignin, which gives the walls their rigidity.
  • cellulose is the main source of fermentable sugars in ethanol because it consists of glucose, the latter being easily fermented in ethanol by the microorganism Saccharomyces cerevisiae in proven and efficient industrial processes. Pentoses contained in hemicelluloses are not efficiently converted to ethanol.
  • Other microorganisms of the genera Saccharomyces, Pichia, Candida, Pachysolen, Zymomonas, Klebsiella, Escherichia can be chosen to valorise the monomeric sugars derived from biomass into ethanol.
  • the process for converting lignocellulosic materials into ethanol comprises a physicochemical pretreatment step, followed by an enzymatic or chemical hydrolysis step, an ethanolic fermentation step of the released sugars and a recovery step of ethanol.
  • the purpose of the pretreatment step is to release the fermentable sugars contained in the hemicelluloses in the form of monomers, essentially pentoses, such as xylose and arabinose, and hexoses, such as galactose, mannose and glucose, and to improve the accessibility of cellulose, embedded in the matrix of lignin and hemicelluloses, to hydrolytic enzymes.
  • pentoses such as xylose and arabinose
  • hexoses such as galactose, mannose and glucose
  • the solid residue from the pretreatment stage consisting essentially of cellulose and lignin is hydrolysed by cellulolytic and hemicellulolytic enzymes produced by microorganisms.
  • Said microorganisms such as fungi belonging to the genera Trichoderma, Aspergillus, Penicillium or Schizophyllum, or anaerobic bacteria belonging for example to the genus Clostridium, produce these enzymes, said enzymes containing in particular cellulases and xylanases, suitable for the total hydrolysis of polysaccharides. contained in plants.
  • the enzymatic hydrolysis is conventionally carried out under mild conditions such as for example at a temperature of between 45 and 50 ° C. and at a pH of 4.8 and is effective.
  • the cost of the enzymes remains very high and a lot of works have been carried out to reduce this cost: in particular i) the increase of the production of enzymes, by selecting the hyperproducing strains and by improving the processes of fermentation, ii) reducing the amount of enzymes in hydrolysis, optimizing the composition of the enzyme cocktail or improving the specific activity of these enzymes.
  • the main work has focused on understanding the mechanisms of action of cellulases and enzyme expression in order to produce and secrete microorganisms the most appropriate enzyme complex for the hydrolysis of lignocellulosic substrates. modifying strains with molecular biology tools.
  • the most used microorganism for the industrial production of cellulases used for enzymatic hydrolysis is the T. reesei mushroom.
  • This filamentous fungus is capable of secreting, in the presence of an inducing substrate, cellulose, for example, significant concentrations of cellulases and hemicellulases.
  • the enzymatic cocktail enzymes contain three major types of activities: the endoglucanases that attack cellulose in the middle of the chains and preferably in the amorphous zones, the exoglucanases releasing glucose dimers, the cellobiose, from the ends of chains and the ⁇ -glucosidases which hydrolyze cellobiose to glucose.
  • xylanases proteins possessing properties indispensable for the hydrolysis of lignocellulosic materials are also secreted by the T. reesei microorganism, xylanases for example.
  • the presence of an inducing substrate is essential for the expression of said cellulolytic and / or hemicellulolytic enzymes and the nature of the carbon substrate has a strong influence on the composition of the secreted enzyme cocktail.
  • T. reesei strains such as the MCG77 strains described in Gallo-US Pat. No. 4,275,167, MCG 80 described in Allen, AL and Andreotti, RE, Biotechnology. Bioengi 1982, 12, 451-459 1982, RUT C30 described in Montenecourt, BS and Eveleigh, DE, Appl. About. Microbiol. 1977, 34, 777-782 and CL847 described in Durand et al, 1984, Proc. SFM Symposium "Genetics of industrial microorganisms". Paris. H. HESLOT Ed, pp 39-50.
  • the improvements have made it possible to obtain hyperproductive strains that are less sensitive to catabolic repression by monomeric sugars, in particular glucose, for example, compared with wild-type strains.
  • glycoside hydrolases which represents a number lower than the average of 211 GH in sequenced sordariomycetes, and only ten genes coding for endoglucanases and cellobiohydrolases.
  • This is the the smallest number of cellulases among the twelve fungi whose genome is currently available, capable of degrading the plant wall of the lignocellulosic material.
  • Said T. reesei microorganism also has the smallest number of genes coding for hemicellulases (16 genes), for pectin degradation enzymes and for proteins containing a CBM or "carbohydrate binding module" according to the English or French terminology.
  • the capacity of degradation of the plant walls of the T. reesei microorganism thus resides mainly in its ability to secrete large quantities of enzymes, up to one hundred grams of extracellular proteins per liter according to Cherry J. and Fidantsef A., Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol. 2003, 14 pp 438-43. But these enzymes are not very diversified and do not have a higher hydrolysis capacity than some other ascomycetes. Such an apparent imbalance may in fact reflect an optimization of the enzymatic hydrolysis process developed by the fungus in its natural habitat: the T. reesei microorganism produces essential enzymes in high proportion, and its proximity to other microorganisms allows it to to profit from the enzymatic activities secreted by them.
  • T. reesei secretome i.e., all of the proteins secreted by said microorganism, can be improved by addition of other enzymes for use in a method involving enzymatic hydrolysis.
  • Enzymes that can supplement the T. reesei secretome can come from any type of microorganism possessing said enzymes.
  • fungi are interesting organisms for obtaining proteins capable of improving the cellulolytic activity of T. reesei.
  • About 72,000 species of fungi have been described to date but Ainsworth et al. ("Dictionary of fungi", 1971) lists 200,000 and according to Hawksworth (The magnitude of fungal diversity: the 1.5 million species estimate revisited 2001, Mycological research, 105, 1422-1432), the number of species in the world would be potentially 1, 5 million.
  • This immense fungal diversity remains unexplored to this day, and gives hope for the discovery of new or better enzymatic activities for the hydrolysis of lignocellulosic biomass, according to the ecosystem where the fungal strains have evolved.
  • the patent application US2010 / 0159494A describes that the addition of individual enzymes can also improve the hydrolysis of lignocellulosic substrates: GH61 family enzymes from T. terrestris or Thermoascus aurianticus have a "boost" effect on cellulases of T Reesei for the hydrolysis of a lignocellulosic substrate, even if they alone have only weak hydrolytic activity (Quinlan et al., Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components, Proc Nat Sci USA 201 108 (37): 15079-84.
  • US2010 / 0124769A discloses that a 20% replacement of the cellulolytic cocktail with Aspergillus aculeatus xylanase and Myceliophthora thermophila cellobiohydrolase (Cel6) could improve the hydrolysis yield by 16%.
  • the applicant has discovered that the simultaneous use of cellulolytic and hemicellulolytic enzymes from the T. reesei microorganism and P. anserina mushroom proteins in the enzymatic hydrolysis step of a pretreated lignocellulosic material allowed the improving the hydrolysis capacity of said lignocellulosic material, particularly in the context of the production of alcohol and preferably of ethanol, from said pretreated lignocellulosic material.
  • An object of the present invention is therefore to provide a process for producing alcohol from pretreated lignocellulosic materials, comprising at least the following steps:
  • An advantage of the present invention is to improve the hydrolysis capacity of said lignocellulosic materials by the simultaneous use of cellulolytic and hemicellulolytic enzymes originating from the T. reesei microorganism and P. anserina mushroom proteins in the hydrolysis step. enzyme.
  • the present invention relates to a process for producing alcohol and preferably ethanol from pretreated lignocellulosic materials.
  • the lignocellulosic materials constituting the charge of the process according to the present invention are pretreated according to the methods known to those skilled in the art described above.
  • the lignocellulosic materials used in the process according to the present invention are chosen from agricultural residues such as cereal straws, sugarcane bagasse or any other type of wood residues, logging residues. such as forest thinning products, logging products such as wood chips, sawmill residues, dedicated crops such as miscanthus, short-rotation coppice, alcoholic, sugar and cereal plants, residues of the paper industry, vegetable wastes or transformation products of said lignocellulosic materials, alone or as a mixture.
  • agricultural residues such as cereal straws, sugarcane bagasse or any other type of wood residues
  • logging residues such as forest thinning products, logging products such as wood chips, sawmill residues, dedicated crops such as miscanthus, short-rotation coppice, alcoholic, sugar and cereal plants, residues of the paper industry, vegetable wastes or transformation products of said lignocellulosic materials, alone or as a mixture.
  • step a) of the process according to the present invention the step of enzymatic hydrolysis of said pretreated lignocellulosic materials uses a mixture of cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism with P. anserina mushroom proteins. .
  • Patent FR 2 555 603 describes for example a process for producing cellulolytic enzymes.
  • said cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism are obtained from T. reesei hyperproductnce strain of cellulases, such as Rut C30, described by ontenecourt BS and Eveleigh DE Appl. About. Microbiol. 1977, 34 (p777) or CL847, described by Warzywoda M. Biotechnol. Lett. 1983, 5 (p.243).
  • Said cellulolytic and / or hemicellulolytic enzymes produced by said fungus are advantageously recovered in their culture medium and optionally packaged before use in the enzymatic hydrolysis step a) of the process according to the invention.
  • the P. anserina fungus is an ascomycete fungus whose habitat is quite atypical since in nature it grows exclusively on herbivore droppings. Said fungus appears in the late stages of degradation, after the digestion of the plants in the digestive tract of the animal and after the successive passage of several other fungal species.
  • the P. anserina fungus is characterized by its ability to feed on very recalcitrant substrates, with easily accessible carbonaceous substances being consumed previously.
  • the extracellular proteins of the P. anserina fungus are advantageously produced over a wide range of carbon sources.
  • said P. anserina mushroom proteins are produced on monomeric sugars, dimers, polymers or plant residues as carbon sources.
  • said proteins are obtained after growth of the fungus on a carbon source selected from xylose, arabinose, sucrose, cellobiose, lactose, Avicel, Solka-Floc cellulose, corn bran, birch xylan, beet pulp and wheat straw.
  • a carbon source selected from xylose, arabinose, sucrose, cellobiose, lactose, Avicel, Solka-Floc cellulose, corn bran, birch xylan, beet pulp and wheat straw.
  • the amount and nature of the secreted proteins are not the same.
  • said carbon sources from which P. anserina mushroom proteins are obtained are selected from wheat straw, Solka-Floc cellulose, Avicel, sucrose and beet pulp.
  • Said P. anserina mushroom proteins are advantageously recovered in their culture medium and optionally packaged before use in the enzymatic hydrolysis step a) of the process according to the invention, in admixture with the cellulolytic and / or hemicellulolytic enzymes originating from the microorganism T. reesei.
  • the cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism and said P. anserina mushroom proteins are advantageously mixed in proportions ranging from 40:60 to 95: 5. Preferably, they are mixed in 50:50 proportions.
  • a ⁇ -glucosidase enzyme such as the enzyme SP188 (Novozymes) may advantageously be added to said mixture used in the enzymatic hydrolysis step a). Such an addition makes it possible to accelerate the initial speed of the hydrolysis and to obtain higher hydrolysis yields.
  • Step a) of enzymatic hydrolysis is advantageously carried out at a temperature of between 30 ° C. and 50 ° C., and preferably between 37 ° and 45 ° C. Said step is advantageously carried out at a pH of between 4.5 and 5.5.
  • the enzymatic hydrolysis reaction may advantageously contain between 1 and 20% of lignocellulosic substrate, that is to say of dry matter, and the total enzymatic charge content is advantageously between 5 and 30 mg per gram of substrate (material dried).
  • Step a) enzymatic hydrolysis is advantageously carried out for a period of between 24 and 120h, and preferably between 72h and 96h.
  • the enzymatic hydrolysis reaction is followed by assaying the sugars released, in particular glucose by the DNS method known to those skilled in the art.
  • step b) of the process according to the invention the hydrolyzate from step a) undergoes an alcoholic fermentation step with an alcoholic microorganism so as to obtain a fermentation must.
  • the alcoholic fermentation step b) of the process according to the present invention is advantageously carried out under standard conditions known to those skilled in the art.
  • the sugar solution or hydrolyzate obtained is fermented under conditions well known to those skilled in the art, in the presence of an alcoholic microorganism such as the yeast Saccharomyces cerevisiae or such as the bacterium Zymomonas mobilis, at a temperature advantageously between 30 and 35 ° C.
  • an alcoholic microorganism such as the yeast Saccharomyces cerevisiae or such as the bacterium Zymomonas mobilis
  • step c) of the process according to the invention the alcohol obtained at the end of step b) of fermentation is separated from the fermentation must.
  • the alcohol obtained is separated from the fermentation broth and non-soluble residues by distillation and the residue consists of distillation vinasses.
  • step a) of enzymatic hydrolysis of the process according to the present invention using in mixture, cellulolytic and / or hemicellulolytic enzymes originating from the microorganism T. reesei and at least P. anserina mushroom protein and alcoholic fermentation step b) are carried out in a single step.
  • the process according to the present invention is a simultaneous saccharification and fermentation process known as the SSF method.
  • the operating conditions used in the said embodiment are identical to those described above in the case where the enzymatic hydrolysis step a) and the alcoholic fermentation step b) are carried out separately, with the differences being different. that the temperature is advantageously between 28 and 40 ° C, and the reaction is advantageously carried out for a period of between 50h and 300h.
  • the cultures of P. anserina S mat + are carried out with an M2 medium (KH 2 PO 4 0.25 gl -1 , K 2 HPO 4 0.3 gl -1 , MgSO 4 7H 2 O 0.25 gl -1 , urea 0.5 gl "1, thiamine 0.05 mgl" 1, biotin 0.25 pg L " ⁇ citric acid 2.5 mgl” 1, ZnS0 4 2.5 mgl "1, 4 CuS0 0.5 mg.l -1 , MnSO 4 125 Mg.l -1 , boric acid 25 ⁇ g -1 , sodium molybdate 25 Mg -1 -1 , iron alum 25 ⁇ g -1 , dextrin 5 ⁇ l.
  • M2 medium KH 2 PO 4 0.25 gl -1 , K 2 HPO 4 0.3 gl -1 , MgSO 4 7H 2 O 0.25 gl -1 , urea 0.5 gl "1, thiamine 0.05 mgl
  • preculture from 0.5 mm diameter agar slices milled for 30 sec at a power of 5 at Fastprep (MP Biomedicals) is carried out so as to seed 200 ml of medium M2 in Roux vial.
  • the mycelia of the 7 vials of Roux are recovered after 5 days of culture and crushed with the mixer for 20 seconds with a medium power.
  • This ground material makes it possible to inoculate 2 I of culture in a homogeneous way. All the cultures are carried out at 27 ° C. and at pH 7, 100 rpm, in 500 ml baffled flasks containing 100 ml / flask of M2.
  • Example 2 aims to characterize the enzymatic activities present in the secreted proteins of P. anserina.
  • the hydrolysis tests of simple sugars are carried out with 4 ⁇ l of P. anserina proteins in a volume of 100 ⁇ l of 50 mM pH 5 acetate buffer and 1 mM of substrate for 20 minutes. min at 37 ° C with stirring.
  • the substrates tested are 4-nitrophenyl- ⁇ -D-glucopyranoside ( ⁇ - ⁇ -D-glucopyranoside), ⁇ - ⁇ -D-lactopyranoside, ⁇ - ⁇ -D-cellobioside, ⁇ - ⁇ -D-xylopyranoside, pNP-D -arabinofuranoside, ⁇ - ⁇ -D-galactopyranoside and ⁇ - ⁇ -D-mannopyranoside.
  • the reaction is stopped by the addition of 130 ⁇ l of 1 M Na 2 CO 3 , and the absorbance at 410 nm is measured. A white realized with 100 ⁇ ⁇ H 2 0 is subtracted from the measurements and a range of 0.02 to 0.2 mM of 4-nitrophenol is assayed in parallel with each series of tests.
  • the enzymatic activity is based on the colorimetric assay of the pNP released into the medium under the enzymatic action. It is expressed in ⁇ of paranitrophenol released per minute and per mg of enzyme.
  • the hydrolyzed CMC complex substrates Avicel ® pH 101, citrus pectin, and birch xylan, xylan wheat, 1, 4 ⁇ D-mannan, galactomannan, beet pulp the arabinan and arabinogalactan larch, are made in a volume of 100 ⁇ of 50 mM acetate buffer pH 5 and 1% (weight / volume) of substrates with 12 ⁇ g of protein, for 1 hour at 37 ° C, with stirring. Reducing sugars released during hydrolysis
  • pNP-Glc 4-nitrophenyl ⁇ -D-glucopyranoside
  • pNP-Lac 4-nitrophenyl ⁇ -D-lactopyranoside
  • pNPCel 4-nitrophenyl ⁇ -D-cellobioside
  • pNP-Xyl ⁇ -D-xylopyranoside
  • pNP-Gal 4-nitrophenyl ⁇ -D-galactopyranoside
  • pNP-Man 4-nitrophenyl ⁇ -D-mannopyranoside
  • nd activity not detected in the test conditions.
  • Table 2 Enzymatic activities of the different secretomas of P. anserina on polymers present in the plant wall (cellulose, hemicelluloses and pectin).
  • CMC Carboxymethyl cellulose
  • X Xylan
  • XI insoluble Xylan
  • Man mannan
  • GalMan Galactomannan
  • Ara Arabinan
  • AraGal ArabinoGalactan.
  • nd activity not detected in the test conditions.
  • the tests are carried out with 100 ⁇ l of a suspension of wheat straw pretreated with steam explosion at a rate of 1% (weight / volume) in 50 mM acetate buffer pH 5, supplemented with cycloheximide 30 mg. 1 and tetracycline 40 mg.l -1 .
  • the amounts of protein are 10 or 20 ⁇ g of T. reesei protein (E508) alone or 10 ⁇ g of E508 supplemented with 10 ⁇ g of P. anserina protein.
  • the reaction mixture also contains 25 ⁇ of ⁇ -glucosidase per g of dry matter (SP188, Novozyme).
  • the hydrolysis is carried out at a temperature of 37 ° C., the enzymes are filtered and the reducing sugars are assayed at DNS at several times between 2 h and 96 h of hydrolysis. After 96 hours of hydrolysis, 24 mM of reducing sugars released with 10 g are obtained as with 20 ⁇ g of E508 enzymes per mg of substrate. The amount of total sugars hydrolysed after 24 hours and 96 h is increased with the addition of P. anserina enzymes induced on Avicel (17% additional reducing sugars) and on Solka-Floc, beet pulp and wheat straw (13%, 9% and 5% reducing sugars) released additional).
  • FIG. 1 also illustrates the results obtained by hydrolysis of wheat straw pretreated with T. reesei cocktail E508 supplemented with ⁇ -glucosidase and supplemented with P. anserina secretomas, produced on Solka-Floc cellulose, Avicel, pulp of beet and wheat straw.

Abstract

The invention describes a method for producing alcohol from pretreated lignocellulosic materials, comprising at least the following steps: a) a step of enzymatic hydrolysis of said pretreated lignocellulosic materials using, as a mixture, cellulolytic and/or hemicellulolytic enzymes originating from the microorganism Trichoderma reesei and proteins of the fungus Podospora anserina; b) a step of alcoholic fermentation, by an alcohol-generating microorganism, of the hydrolysate resulting from step a) and obtaining of a fermentation must, and c) a step of separation of the alcohol from the fermentation must.

Description

PROCÉDÉ DE PRODUCTION D'ALCOOL A PARTIR DE BIOMASSE LIGNOCELLULOSIQUE PAR COMPLEMENTATION DES ENZYMES CELLULOLYTIQUES ET HEMICELLULOLYTIQUES DE TRICHODERMA REESEl PAR LE CHAMPIGNON PODOSPORA ANSERINA  PROCESS FOR THE PRODUCTION OF ALCOHOL FROM LIGNOCELLULOSIC BIOMASS BY COMPLEMENTATION OF CELLULOLYTIC AND HEMICELLULOLYTIC ENZYMES OF TRICHODERMA REESEl BY FUNGUS PODOSPORA ANSERINA
Domaine technique Technical area
La présente invention concerne l'utilisation simultanée d'enzymes cellulolytiques et hémicellulolytiques provenant du microorganisme Trichoderma reesei et de protéines du champignon Podospora anserina dans l'étape d'hydrolyse enzymatique d'un matériel lignocellulosique prétraité, en particulier dans le cadre de la production d'éthanol à partir de matériaux ligno-cellulosiques prétraités.  The present invention relates to the simultaneous use of cellulolytic and hemicellulolytic enzymes from the microorganism Trichoderma reesei and proteins of the fungus Podospora anserina in the enzymatic hydrolysis step of a pretreated lignocellulosic material, particularly in the context of the production of ethanol from pretreated lignocellulosic materials.
État de la technique  State of the art
La biomasse lignocellulosique est une des ressources renouvelables les plus abondantes sur terre et constitue une alternative intéressante aux matières premières fossiles. Son utilisation à des fins énergétiques permet en effet de réduire les gaz à effet de serre, tout en minimisant la dépendance énergétique vis-à-vis des pays producteurs de pétrole.  Lignocellulosic biomass is one of the most abundant renewable resources on earth and constitutes an interesting alternative to fossil raw materials. Its use for energy purposes makes it possible to reduce greenhouse gases while minimizing energy dependence on oil-producing countries.
Depuis les années 1970, la transformation de la biomasse ligno-cellulosique en éthanol, après hydrolyse des polysaccharides constitutifs en sucres fermentescibles, a fait l'objet de nombreux travaux.  Since the 1970s, the transformation of lignocellulosic biomass into ethanol, after hydrolysis of the constituent polysaccharides into fermentable sugars, has been the subject of numerous studies.
La biomasse lignocellulosique est composée de trois principaux polymères : la cellulose à une teneur comprise entre 35 et 50%, les hémicelluloses qui sont des polysaccharides essentiellement constitués de pentoses et d'hexoses à une teneur comprise entre 20 et 30% et les lignines à une teneur comprise entre 15 et 25% du poids. La dégradation de la biomasse se révèle difficile car les polysaccharides de la paroi végétale (cellulose et hémicelluloses) sont intimement associés à de la lignine, qui confère aux parois leur rigidité.  Lignocellulosic biomass is composed of three main polymers: cellulose at a content of between 35 and 50%, hemicelluloses which are polysaccharides essentially consisting of pentoses and hexoses at a content of between 20 and 30% and lignins at a concentration of content between 15 and 25% of the weight. Biomass degradation is difficult because plant wall polysaccharides (cellulose and hemicellulose) are intimately associated with lignin, which gives the walls their rigidity.
De ces trois polymères, la cellulose est la principale source de sucres fermentescibles en éthanol car elle est constituée de glucose, ce dernier étant facilement fermenté en éthanol par le microorganisme Saccharomyces cerevisiae dans des procédés industriels éprouvés et performants. Les pentoses contenus dans les hémicelluloses ne sont pas efficacement convertis en éthanol. D'autres microorganismes parmi les genres Saccharomyces, Pichia, Candida, Pachysolen, Zymomonas, Klebsiella, Escherichia, peuvent être choisis pour valoriser les sucres monomères issus de la biomasse en éthanol. Le procédé de transformation des matériaux ligno-cellulosiques en éthanol comprend une étape de prétraitement physico-chimique, suivie d'une étape d'hydrolyse enzymatique ou chimique, d'une étape de fermentation éthanolique des sucres libérés et d'une étape de récupération de l'éthanol. Of these three polymers, cellulose is the main source of fermentable sugars in ethanol because it consists of glucose, the latter being easily fermented in ethanol by the microorganism Saccharomyces cerevisiae in proven and efficient industrial processes. Pentoses contained in hemicelluloses are not efficiently converted to ethanol. Other microorganisms of the genera Saccharomyces, Pichia, Candida, Pachysolen, Zymomonas, Klebsiella, Escherichia can be chosen to valorise the monomeric sugars derived from biomass into ethanol. The process for converting lignocellulosic materials into ethanol comprises a physicochemical pretreatment step, followed by an enzymatic or chemical hydrolysis step, an ethanolic fermentation step of the released sugars and a recovery step of ethanol.
L'étape de prétraitement a pour objet de libérer les sucres fermentescibles contenus dans les hémicelluloses sous forme de monomères, essentiellement des pentoses, comme le xylose et l'arabinose, et des hexoses, comme le galactose, le mannose et le glucose, et d'améliorer l'accessibilité de la cellulose, incrustée dans la matrice de lignine et hémicelluloses, aux enzymes hydrolytiques. De nombreuses technologies existent : cuissons acides, cuissons alcalines, explosion à la vapeur, procédés organosolv, etc. L'efficacité du prétraitement se mesure par le taux de récupération des hémicelluloses et par la susceptibilité à l'hydrolyse du résidu cellulosique. Les prétraitements de type acide en condition douce et par explosion à la vapeur sont les mieux adaptés. Ils permettent une récupération totale des pentoses et une bonne accessibilité de la cellulose à l'hydrolyse.  The purpose of the pretreatment step is to release the fermentable sugars contained in the hemicelluloses in the form of monomers, essentially pentoses, such as xylose and arabinose, and hexoses, such as galactose, mannose and glucose, and to improve the accessibility of cellulose, embedded in the matrix of lignin and hemicelluloses, to hydrolytic enzymes. Many technologies exist: acid cooking, alkaline cooking, steam explosion, organosolv processes, etc. The effectiveness of pretreatment is measured by the hemicellulose recovery rate and the susceptibility to hydrolysis of the cellulosic residue. Pretreatments of the acidic type under mild conditions and by steam explosion are the most suitable. They allow total recovery of pentoses and good accessibility of cellulose to hydrolysis.
Le résidu solide issu de l'étape de prétraitement, composé essentiellement de cellulose et de lignine est hydrolysé par des enzymes cellulolytiques et hémicellulolytiques produites par des microorganismes. Lesdits microorganismes, comme les champignons appartenant aux genres Trichoderma, Aspergillus, Pénicillium ou Schizophyllum, ou les bactéries anaérobies appartenant par exemple au genre Clostridium, produisent ces enzymes, lesdites enzymes contenant notamment les cellulases et les xylanases, adaptées à l'hydrolyse totale des polysaccharides contenus dans les végétaux.  The solid residue from the pretreatment stage, consisting essentially of cellulose and lignin is hydrolysed by cellulolytic and hemicellulolytic enzymes produced by microorganisms. Said microorganisms, such as fungi belonging to the genera Trichoderma, Aspergillus, Penicillium or Schizophyllum, or anaerobic bacteria belonging for example to the genus Clostridium, produce these enzymes, said enzymes containing in particular cellulases and xylanases, suitable for the total hydrolysis of polysaccharides. contained in plants.
L'hydrolyse enzymatique s'effectue classiquement dans des conditions douces telles que par exemple à une température comprise entre 45 et 50°C et à un pH de 4,8 et est efficace. En revanche, le coût des enzymes reste très élevé et beaucoup de travaux ont été conduits pour réduire ce coût : notamment i) l'augmentation de la production d'enzymes, en sélectionnant les souches hyperproductrices et en améliorant les procédés de fermentation, ii) la diminution de la quantité d'enzymes en hydrolyse, en optimisant la composition du cocktail enzymatique ou en améliorant l'activité spécifique de ces enzymes. Au cours de la dernière décennie, les principaux travaux se sont attachés à comprendre les mécanismes d'action des cellulases et d'expression des enzymes afin de faire produire et sécréter aux microorganismes le complexe enzymatique le plus approprié à l'hydrolyse des substrats lignocellulosiques en modifiant les souches avec les outils de biologie moléculaire.  The enzymatic hydrolysis is conventionally carried out under mild conditions such as for example at a temperature of between 45 and 50 ° C. and at a pH of 4.8 and is effective. On the other hand, the cost of the enzymes remains very high and a lot of works have been carried out to reduce this cost: in particular i) the increase of the production of enzymes, by selecting the hyperproducing strains and by improving the processes of fermentation, ii) reducing the amount of enzymes in hydrolysis, optimizing the composition of the enzyme cocktail or improving the specific activity of these enzymes. Over the last decade, the main work has focused on understanding the mechanisms of action of cellulases and enzyme expression in order to produce and secrete microorganisms the most appropriate enzyme complex for the hydrolysis of lignocellulosic substrates. modifying strains with molecular biology tools.
Le microorganisme le plus utilisé pour la production industrielle de cellulases utilisées pour l'hydrolyse enzymatique est le champignon T. reesei. Ce champignon filamenteux est capable de sécréter, en présence d'un substrat inducteur, la cellulose par exemple, des concentrations importantes de cellulases et hémicellulases. Les enzymes du cocktail enzymatique contiennent trois grands types d'activités : les endoglucanases qui attaquent la cellulose en milieu des chaînes et de préférence dans les zones amorphes, les exoglucanases libérant des dimères de glucose, le cellobiose, à partir des extrémités de chaînes et les β-glucosidases qui hydrolysent le cellobiose en glucose. D'autres protéines possédant des propriétés indispensables à l'hydrolyse des matériaux ligno-cellulosiques sont également sécrétées par le microorganisme T. reesei, les xylanases par exemple. La présence d'un substrat inducteur est indispensable à l'expression desdites enzymes cellulolytiques et/ou hémicellulolytiques et la nature du substrat carboné a une forte influence sur la composition du cocktail enzymatique sécrété. C'est le cas du xylose, qui, associé à un substrat carboné inducteur comme la cellulose ou le lactose, permet d'augmenter significativement l'activité dite xylanase dans le mélange. Du point de vue du procédé, il est nécessaire dans un premier temps de récupérer les enzymes produites par ledit champignon dans le milieu de culture et éventuellement de les conditionner avant leur utilisation dans l'hydrolyse enzymatique. The most used microorganism for the industrial production of cellulases used for enzymatic hydrolysis is the T. reesei mushroom. This filamentous fungus is capable of secreting, in the presence of an inducing substrate, cellulose, for example, significant concentrations of cellulases and hemicellulases. The enzymatic cocktail enzymes contain three major types of activities: the endoglucanases that attack cellulose in the middle of the chains and preferably in the amorphous zones, the exoglucanases releasing glucose dimers, the cellobiose, from the ends of chains and the β-glucosidases which hydrolyze cellobiose to glucose. Other proteins possessing properties indispensable for the hydrolysis of lignocellulosic materials are also secreted by the T. reesei microorganism, xylanases for example. The presence of an inducing substrate is essential for the expression of said cellulolytic and / or hemicellulolytic enzymes and the nature of the carbon substrate has a strong influence on the composition of the secreted enzyme cocktail. This is the case of xylose, which, combined with an inducing carbon substrate such as cellulose or lactose, makes it possible to significantly increase the so-called xylanase activity in the mixture. From the point of view of the process, it is necessary firstly to recover the enzymes produced by said fungus in the culture medium and possibly to condition them before their use in enzymatic hydrolysis.
Les techniques de génétique classique par mutation ont permis la sélection de souches de T. reesei hyperproductrices de cellulases telles que les souches MCG77, décrites dans le brevet Gallo - US 4275 167, MCG 80 décrites dans Allen, A.L. et Andreotti, R.E., Biotechnol-Bioengi 1982, 12, 451-459 1982, RUT C30 décrites dans Montenecourt, B.S. et Eveleigh, D.E., Appl. Environ. Microbiol. 1977, 34, 777-782 et CL847 décrites dans Durand et al, 1984, Proc. Colloque SFM "Génétique des microorganismes industriels". Paris. H. HESLOT Ed, pp 39-50. Les améliorations ont permis d'obtenir des souches hyperproductrices, moins sensibles à la répression catabolique par les sucres monomères notamment, glucose par exemple, par rapport aux souches sauvages.  Conventional genetic mutation techniques have allowed the selection of cellulosic hyperproducing T. reesei strains such as the MCG77 strains described in Gallo-US Pat. No. 4,275,167, MCG 80 described in Allen, AL and Andreotti, RE, Biotechnology. Bioengi 1982, 12, 451-459 1982, RUT C30 described in Montenecourt, BS and Eveleigh, DE, Appl. About. Microbiol. 1977, 34, 777-782 and CL847 described in Durand et al, 1984, Proc. SFM Symposium "Genetics of industrial microorganisms". Paris. H. HESLOT Ed, pp 39-50. The improvements have made it possible to obtain hyperproductive strains that are less sensitive to catabolic repression by monomeric sugars, in particular glucose, for example, compared with wild-type strains.
Cependant, la dégradation de substrats inducteurs complexes, comme la biomasse végétale, nécessite un grand nombre d'activités enzymatiques différentes. Or, le séquençage récent du génome du microorganisme T. reesei (34 méga paires de bases, environ 9100 gènes) a révélé des caractéristiques inattendues décrites dans Martinez et al., Génome sequencing and analysis of the biomass-degrading fungus T. reesei (syn. Hypocrea jecorina). Nat Biotechnol 2008, 26 pp 553-560. Environ 2,8% des régions codantes de son génome coderaient pour des enzymes de dégradation des sucres, loin derrière les 4 % trouvés chez d'autres champignons filamenteux. En effet, une recherche portée sur son génome a permis de dénombrer 200 glycoside hydrolases (GH), ce qui représente un nombre plus faible que la moyenne de 211 GH chez les sordariomycètes séquencées, et seulement dix gènes codant pour des endoglucanases et des cellobiohydrolases. Ceci est le plus petit nombre de cellulases parmi les douze champignons dont le génome est actuellement disponible, capables de dégrader la paroi végétale de la matière lignocellulosique. Ledit microorganisme T. reesei possède également le plus petit nombre de gènes codant pour des hémicellulases (16 gènes), pour des enzymes de dégradation de la pectine et pour des protéines contenant un CBM ou "carbohydrate binding module" selon la terminologie anglo-saxonne ou module de fixation aux polysaccharides ( artinez et al., Génome sequencing and analysis of the biomass-degrading fungus T. reesei (syn. Hypocrea jecorina). Nat Biotechnol 2008, 26 pp 553-560). However, the degradation of complex inducing substrates, such as plant biomass, requires a large number of different enzymatic activities. However, the recent genome sequencing of the T. reesei microorganism (34 mega base pairs, approximately 9100 genes) has revealed unexpected features described in Martinez et al., Genome sequencing and analysis of the biomass-degrading fungus T. reesei (syn Hypocrea jecorina). Nat Biotechnol 2008, 26 pp 553-560. About 2.8% of the coding regions of his genome would encode sugar-degrading enzymes, far behind the 4% found in other filamentous fungi. Indeed, a search carried out on its genome made it possible to count 200 glycoside hydrolases (GH), which represents a number lower than the average of 211 GH in sequenced sordariomycetes, and only ten genes coding for endoglucanases and cellobiohydrolases. This is the the smallest number of cellulases among the twelve fungi whose genome is currently available, capable of degrading the plant wall of the lignocellulosic material. Said T. reesei microorganism also has the smallest number of genes coding for hemicellulases (16 genes), for pectin degradation enzymes and for proteins containing a CBM or "carbohydrate binding module" according to the English or French terminology. polysaccharide binding module (artinez et al., Genome sequencing and analysis of the biomass-degrading fungus T. reesei (Syn. Hypocrea jecorina) Nat Biotechnol 2008, 26 pp. 553-560).
On trouve de plus une diversité de glycosides hydrolases réduite, certaines familles étant totalement absentes chez le microorganisme T. reesei. En outre, il ne possède pas non plus de gènes codant pour des tannases ou des féruloyl estérases, faits qui soulignent l'handicap probable de T. reesei face à la dégradation des hémicelluloses.  In addition, there is a reduced diversity of glycoside hydrolases, some families being totally absent in the T. reesei microorganism. In addition, it does not have either genes encoding tannases or feruloyl esterases, facts that highlight the likely handicap of T. reesei against the degradation of hemicelluloses.
La capacité de dégradation des parois végétales du microorganisme T. reesei réside donc principalement dans son aptitude à sécréter de grandes quantités d'enzymes, jusqu'à cent grammes de protéines extracellulaires par litre d'après Cherry J. et Fidantsef A., Directed évolution of industrial enzymes: an update. Curr Opin Biotechnol. 2003, 14 pp 438- 43. Mais ces enzymes sont peu diversifiées et ne présentent pas de capacité d'hydrolyse supérieure à certains autres ascomycètes. Un tel déséquilibre apparent peut en fait refléter une optimisation du processus d'hydrolyse enzymatique développé par le champignon dans son habitat naturel : le microorganisme T. reesei produit les enzymes essentielles en forte proportion, et sa proximité à d'autres micro-organismes lui permet de tirer profit des activités enzymatiques sécrétées par ceux-ci.  The capacity of degradation of the plant walls of the T. reesei microorganism thus resides mainly in its ability to secrete large quantities of enzymes, up to one hundred grams of extracellular proteins per liter according to Cherry J. and Fidantsef A., Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol. 2003, 14 pp 438-43. But these enzymes are not very diversified and do not have a higher hydrolysis capacity than some other ascomycetes. Such an apparent imbalance may in fact reflect an optimization of the enzymatic hydrolysis process developed by the fungus in its natural habitat: the T. reesei microorganism produces essential enzymes in high proportion, and its proximity to other microorganisms allows it to to profit from the enzymatic activities secreted by them.
Ainsi, il paraît probable que le sécrétome de T. reesei, c'est-à-dire de l'ensemble des protéines sécrétées par ledit microorganisme, peut être amélioré par ajout d'autres enzymes pour une utilisation dans un procédé impliquant une hydrolyse enzymatique de la lignocellulose  Thus, it seems likely that T. reesei secretome, i.e., all of the proteins secreted by said microorganism, can be improved by addition of other enzymes for use in a method involving enzymatic hydrolysis. lignocellulose
Des enzymes pouvant compléter le sécrétome de T. reesei peuvent venir de tout type de microorganismes possédant lesdites enzymes. Notamment, les champignons sont des organismes intéressants pour obtenir des protéines susceptibles d'améliorer l'activité cellulolytique de T. reesei. Environ 72 000 espèces de champignons ont été décrites à ce jour mais Ainsworth et al. ( « Dictionary of fungi », 1971 ) en répertorient 200 000 et selon Hawksworth (The magnitude of fungal diversity: the 1.5 million species estimate revisited 2001 , Mycological research, 105, 1422-1432), le nombre d'espèces dans le monde serait potentiellement de 1 ,5 millions. Cette immense diversité fongique reste à ce jour inexplorée, et laisse espérer la découverte d'activités enzymatiques nouvelles ou plus performantes pour l'hydrolyse de la biomasse lignocellulosique, selon l'écosystème où ont évolué les souches fongiques. Enzymes that can supplement the T. reesei secretome can come from any type of microorganism possessing said enzymes. In particular, fungi are interesting organisms for obtaining proteins capable of improving the cellulolytic activity of T. reesei. About 72,000 species of fungi have been described to date but Ainsworth et al. ("Dictionary of fungi", 1971) lists 200,000 and according to Hawksworth (The magnitude of fungal diversity: the 1.5 million species estimate revisited 2001, Mycological research, 105, 1422-1432), the number of species in the world would be potentially 1, 5 million. This immense fungal diversity remains unexplored to this day, and gives hope for the discovery of new or better enzymatic activities for the hydrolysis of lignocellulosic biomass, according to the ecosystem where the fungal strains have evolved.
Des exemples récents illustrent en effet la faisabilité d'une supplémentation des cellulases de T. reesei par des enzymes venant d'autres champignons :  Recent examples illustrate the feasibility of supplementing T. reesei cellulases with enzymes from other fungi:
L'étude de Rosgaard et al. (Efficiency of new fungal cellulase Systems in boosting enzymatic dégradation of barley straw lignocellulose. Biotechnol Prog 22, 493-498) montre que la quantité de sucres réducteurs libérés est plus importante si des protéines produites par différentes espèces, telles que Chaetomium globosum, Thielavia terrestris ou Penicillum funiculosum, sont ajoutées aux enzymes produites par T. reesei. The study by Rosgaard et al. (Efficiency of new fungal cellulase Systems in boosting enzymatic degradation of barley straw lignocellulose, Biotechnol Prog 22, 493-498) shows that the amount of reducing sugars released is greater if proteins produced by different species, such as Chaetomium globosum, Thielavia terrestris or Penicillum funiculosum, are added to the enzymes produced by T. reesei.
La demande de brevet US2010/0159494A décrit que l'ajout d'enzymes individuelles peut également améliorer l'hydrolyse de substrats lignocellulosiques : des enzymes de famille GH61 provenant de T. terrestris ou Thermoascus aurianticus ont un effet "boost" sur les cellulases de T. reesei pour l'hydrolyse d'un substrat lignocellulosique, même si elles seules ne possèdent qu'une faible activité hydrolytique (Quinlan et al., Insights into the oxidative dégradation of cellulose by a copper metalloenzyme that exploits biomass components, Proc. Nat Sci USA 201 1 108 (37): 15079-84. The patent application US2010 / 0159494A describes that the addition of individual enzymes can also improve the hydrolysis of lignocellulosic substrates: GH61 family enzymes from T. terrestris or Thermoascus aurianticus have a "boost" effect on cellulases of T Reesei for the hydrolysis of a lignocellulosic substrate, even if they alone have only weak hydrolytic activity (Quinlan et al., Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components, Proc Nat Sci USA 201 108 (37): 15079-84.
La demande de brevet US2010/0124769A décrit qu'un remplacement de 20% du cocktail cellulolytique par une xylanase d'Aspergillus aculeatus et une cellobiohydrolase (Cel6) de Myceliophthora thermophila pouvait améliorer le rendement d'hydrolyse de 16%.  US2010 / 0124769A discloses that a 20% replacement of the cellulolytic cocktail with Aspergillus aculeatus xylanase and Myceliophthora thermophila cellobiohydrolase (Cel6) could improve the hydrolysis yield by 16%.
II existe donc un potentiel de renforcer l'efficacité des cellulases sécrétées par T. reesei dans le but d'améliorer la capacité d'hydrolyse d'un substrat lignocellulosique. There is therefore a potential to enhance the efficiency of cellulases secreted by T. reesei in order to improve the hydrolysis capacity of a lignocellulosic substrate.
Dans ce contexte, la demanderesse a découvert que l'utilisation simultanée d'enzymes cellulolytiques et hémicellulolytiques provenant du microorganisme T. reesei et de protéines du champignon P. anserina dans l'étape d'hydrolyse enzymatique d'un matériel lignocellulosique prétraité permettait l'amélioration de la capacité d'hydrolyse dudit matériel lignocellulosique, en particulier dans le cadre de la production d'alcool et de préférence d'éthanol, à partir dudit matériel lignocellulosique prétraité.  In this context, the applicant has discovered that the simultaneous use of cellulolytic and hemicellulolytic enzymes from the T. reesei microorganism and P. anserina mushroom proteins in the enzymatic hydrolysis step of a pretreated lignocellulosic material allowed the improving the hydrolysis capacity of said lignocellulosic material, particularly in the context of the production of alcohol and preferably of ethanol, from said pretreated lignocellulosic material.
Un objet de la présente invention est donc de fournir un procédé de production d'alcool à partir de matériaux ligno-cellulosiques prétraités, comprenant au moins les étapes suivantes : An object of the present invention is therefore to provide a process for producing alcohol from pretreated lignocellulosic materials, comprising at least the following steps:
a) une étape d'hydrolyse enzymatique desdits matériaux lignocellulosiques prétraités utilisant, en mélange, des enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei et de protéines du champignon P. anserina ; b) une étape de fermentation alcoolique par un microorganisme alcooligène de l'hydrolysat issu de l'étape a) et obtention d'un moût de fermentation et a) a step of enzymatic hydrolysis of said pretreated lignocellulosic materials using, in a mixture, cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism and P. anserina mushroom proteins; b) a step of alcoholic fermentation with an alcoholic microorganism of the hydrolyzate from step a) and obtaining a fermentation must and
c) une étape de séparation de l'alcool du moût de fermentation. Un avantage de la présente invention est d'améliorer la capacité d'hydrolyse desdits matériaux lignocellulosique par l'utilisation simultanée d'enzymes cellulolytiques et hémicellulolytiques provenant du microorganisme T. reesei et de protéines du champignon P. anserina dans l'étape d'hydrolyse enzymatique. Description détaillée de l'invention  c) a step of separating the alcohol from the fermentation must. An advantage of the present invention is to improve the hydrolysis capacity of said lignocellulosic materials by the simultaneous use of cellulolytic and hemicellulolytic enzymes originating from the T. reesei microorganism and P. anserina mushroom proteins in the hydrolysis step. enzyme. Detailed description of the invention
La présente invention concerne un procédé de production d'alcool et de préférence d'éthanol, à partir de matériaux lignocellulosiques prétraités.  The present invention relates to a process for producing alcohol and preferably ethanol from pretreated lignocellulosic materials.
Conformément à l'invention, les matériaux lignocellulosiques constituants la charge du procédé selon la présente invention sont prétraités selon les méthodes connues de l'homme du métier décrites plus haut.  According to the invention, the lignocellulosic materials constituting the charge of the process according to the present invention are pretreated according to the methods known to those skilled in the art described above.
De préférence, les matériaux lignocellulosiques utilisés dans le procédé selon la présente invention sont choisis parmi les résidus d'exploitation agricole tels que les pailles de céréales, la bagasse de canne à sucre ou tout autre type de résidus ligneux, les résidus d'exploitation forestière tels que les produits résultant de la première éclaircie des forêts, les produits de l'exploitation forestière tels que les copeaux de bois, les résidus de scieries, les cultures dédiées telles que par exemple le miscanthus, les taillis à courte rotation, les résidus de plantes alcooligènes, sucrières et céréalières, les résidus de l'industrie papetière, les déchets végétaux ou les produits de transformation desdits matériaux lignocellulosiques, pris seuls ou en mélange.  Preferably, the lignocellulosic materials used in the process according to the present invention are chosen from agricultural residues such as cereal straws, sugarcane bagasse or any other type of wood residues, logging residues. such as forest thinning products, logging products such as wood chips, sawmill residues, dedicated crops such as miscanthus, short-rotation coppice, alcoholic, sugar and cereal plants, residues of the paper industry, vegetable wastes or transformation products of said lignocellulosic materials, alone or as a mixture.
Conformément à l'étape a) du procédé selon la présente invention, l'étape d'hydrolyse enzymatique desdits matériaux lignocellulosiques prétraités utilise un mélange d'enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei avec des protéines du champignon P. anserina. According to step a) of the process according to the present invention, the step of enzymatic hydrolysis of said pretreated lignocellulosic materials uses a mixture of cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism with P. anserina mushroom proteins. .
Lesdites enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei sont avantageusement produites de manière connues de l'homme du métier. Le brevet FR 2 555 603 décrit par exemple un procédé de production d'enzymes cellulolytiques.  Said cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism are advantageously produced in a manner known to those skilled in the art. Patent FR 2 555 603 describes for example a process for producing cellulolytic enzymes.
De préférence, lesdites enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei, appelées cocktail d'enzymes de T. reesei, sont obtenues à partir d'une souche T. reesei hyperproductnce de cellulases, telles que Rut C30, décrite par ontenecourt B.S. et Eveleigh D.E. Appl. Environ. Microbiol. 1977, 34 (p. 777) ou CL847, décrite par Warzywoda M. Biotechnol. Lett. 1983, 5 (p.243). Preferably, said cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism, called T. reesei enzyme cocktail, are obtained from T. reesei hyperproductnce strain of cellulases, such as Rut C30, described by ontenecourt BS and Eveleigh DE Appl. About. Microbiol. 1977, 34 (p777) or CL847, described by Warzywoda M. Biotechnol. Lett. 1983, 5 (p.243).
Lesdites enzymes cellulolytiques et/ou hémicellulolytiques produites par ledit champignon sont avantageusement récupérées dans leur milieu de culture et éventuellement conditionnées avant leur utilisation dans l'étape a) d'hydrolyse enzymatique du procédé selon l'invention.  Said cellulolytic and / or hemicellulolytic enzymes produced by said fungus are advantageously recovered in their culture medium and optionally packaged before use in the enzymatic hydrolysis step a) of the process according to the invention.
Le champignon P. anserina est un champignon ascomycète dont l'habitat est tout à fait atypique puisque dans la nature, il se développe exclusivement sur des excréments d'herbivore. Ledit champignon apparaît dans les étapes tardives de dégradation, après la digestion des végétaux dans le tractus digestif de l'animal et après le passage successif de plusieurs autres espèces fongiques. Le champignon P. anserina se caractérise par sa capacité à se nourrir de substrats très récalcitrants, les substances carbonées facilement accessibles étant consommées auparavant.  The P. anserina fungus is an ascomycete fungus whose habitat is quite atypical since in nature it grows exclusively on herbivore droppings. Said fungus appears in the late stages of degradation, after the digestion of the plants in the digestive tract of the animal and after the successive passage of several other fungal species. The P. anserina fungus is characterized by its ability to feed on very recalcitrant substrates, with easily accessible carbonaceous substances being consumed previously.
Les protéines extracellulaires du champignon P. anserina sont avantageusement produites sur une large gamme de sources de carbone. De préférence, lesdites protéines du champignon P. anserina sont produites sur des sucres monomères, dimères, polymères ou résidus de végétaux comme sources de carbone. De préférence, lesdites protéines sont obtenues après croissance du champignon sur une source de carbone choisie parmi le xylose, l'arabinose, le saccharose, le cellobiose, le lactose, l'Avicel, la cellulose Solka-Floc, le son de maïs, le xylane de bouleau, la pulpe de betterave et la paille de blé. Selon le substrat utilisé, la quantité et la nature des protéines sécrétées ne sont pas les mêmes. The extracellular proteins of the P. anserina fungus are advantageously produced over a wide range of carbon sources. Preferably, said P. anserina mushroom proteins are produced on monomeric sugars, dimers, polymers or plant residues as carbon sources. Preferably, said proteins are obtained after growth of the fungus on a carbon source selected from xylose, arabinose, sucrose, cellobiose, lactose, Avicel, Solka-Floc cellulose, corn bran, birch xylan, beet pulp and wheat straw. Depending on the substrate used, the amount and nature of the secreted proteins are not the same.
De manière très préférée, lesdites sources de carbone à partir desquelles les protéines du champignon P. anserina sont obtenues sont choisies parmi la paille de blé, la cellulose Solka-Floc, Avicel, le saccharose et la pulpe de betterave.  Very preferably, said carbon sources from which P. anserina mushroom proteins are obtained are selected from wheat straw, Solka-Floc cellulose, Avicel, sucrose and beet pulp.
Lesdites protéines du champignon P. anserina sont avantageusement récupérées dans leur milieu de culture et éventuellement conditionnées avant leur utilisation dans l'étape a) d'hydrolyse enzymatique du procédé selon l'invention, en mélange avec les enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei.  Said P. anserina mushroom proteins are advantageously recovered in their culture medium and optionally packaged before use in the enzymatic hydrolysis step a) of the process according to the invention, in admixture with the cellulolytic and / or hemicellulolytic enzymes originating from the microorganism T. reesei.
Les enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei et de lesdites protéines du champignon P. anserina sont avantageusement mélangées dans des proportions allant de 40:60 jusqu'à 95:5. De préférence, elles sont mélangées dans des proportions 50:50. Une enzyme β-glucosidase, tel que l'enzyme SP188 (Novozymes) peut avantageusement être ajoutée au dit mélange utilisé dans l'étape a) d'hydrolyse enzymatique. Un tel ajout permet d'accélérer la vitesse initiale de l'hydrolyse et d'obtenir des rendements d'hydrolyse plus importants. The cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism and said P. anserina mushroom proteins are advantageously mixed in proportions ranging from 40:60 to 95: 5. Preferably, they are mixed in 50:50 proportions. A β-glucosidase enzyme, such as the enzyme SP188 (Novozymes) may advantageously be added to said mixture used in the enzymatic hydrolysis step a). Such an addition makes it possible to accelerate the initial speed of the hydrolysis and to obtain higher hydrolysis yields.
L'étape a) d'hydrolyse enzymatique est avantageusement effectuée à une température comprise entre 30°C et 50°C, et de préférence entre 37 et 45°C. Ladite étape est avantageusement effectuée à un pH compris entre 4,5 et 5,5. La réaction d'hydrolyse enzymatique peut avantageusement contenir entre 1 et 20% de substrat lignocellulosique, c'est-à-dire de matière sèche, et la teneur en charge enzymatique totale est avantageusement comprise entre 5 et 30 mg par gramme de substrat (matière sèche). Step a) of enzymatic hydrolysis is advantageously carried out at a temperature of between 30 ° C. and 50 ° C., and preferably between 37 ° and 45 ° C. Said step is advantageously carried out at a pH of between 4.5 and 5.5. The enzymatic hydrolysis reaction may advantageously contain between 1 and 20% of lignocellulosic substrate, that is to say of dry matter, and the total enzymatic charge content is advantageously between 5 and 30 mg per gram of substrate (material dried).
L'étape a) d'hydrolyse enzymatique est avantageusement effectuée pendant une durée comprise entre 24 et 120h, et de préférence entre 72h et 96h. La réaction d'hydrolyse enzymatique est suivie par dosage des sucres libérés, notamment le glucose par la méthode DNS connue de l'homme du métier.  Step a) enzymatic hydrolysis is advantageously carried out for a period of between 24 and 120h, and preferably between 72h and 96h. The enzymatic hydrolysis reaction is followed by assaying the sugars released, in particular glucose by the DNS method known to those skilled in the art.
Conformément à l'étape b) du procédé selon l'invention, l'hydrolysat issu de l'étape a) subit une étape de fermentation alcoolique par un microorganisme alcooligène de manière à obtenir un moût de fermentation. According to step b) of the process according to the invention, the hydrolyzate from step a) undergoes an alcoholic fermentation step with an alcoholic microorganism so as to obtain a fermentation must.
L'étape b) de fermentation alcoolique du procédé selon la présente invention est avantageusement mise en œuvre dans des conditions classiques connues de l'homme du métier.  The alcoholic fermentation step b) of the process according to the present invention is advantageously carried out under standard conditions known to those skilled in the art.
De préférence, la solution sucrée ou hydrolysat obtenue est fermentée dans des conditions bien connues de l'homme de l'art, en présence d'un microorganisme alcooligène tel que la levure Saccharomyces cerevisiae ou tel que la bactérie Zymomonas mobilis, à une température avantageusement comprise entre 30 et 35°C.  Preferably, the sugar solution or hydrolyzate obtained is fermented under conditions well known to those skilled in the art, in the presence of an alcoholic microorganism such as the yeast Saccharomyces cerevisiae or such as the bacterium Zymomonas mobilis, at a temperature advantageously between 30 and 35 ° C.
Conformément à l'étape c) du procédé selon l'invention, l'alcool obtenu à l'issue de l'étape b) de fermentation est séparé du moût de fermentation. L'alcool obtenu est séparé du moût de fermentation et des résidus non solubles par distillation et le résidu est constitué des vinasses de distillation. According to step c) of the process according to the invention, the alcohol obtained at the end of step b) of fermentation is separated from the fermentation must. The alcohol obtained is separated from the fermentation broth and non-soluble residues by distillation and the residue consists of distillation vinasses.
Selon un mode de réalisation préférée du procédé selon l'invention, l'étape a) d'hydrolyse enzymatique du procédé selon la présente invention, utilisant en mélange, des enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei et au moins une protéine du champignon P. anserina et l'étape b) de fermentation alcoolique sont réalisées en une seule étape. Selon ce mode de réalisation préféré, le procédé selon la présente invention est un procédé de saccharification et fermentation simultanées dit procédé SSF. Les conditions opératoires mises en œuvre dans ledit mode de réalisation sont identiques à celle décrites ci-dessus dans le cas ou l'étape a) d'hydrolyse enzymatique et l'étape b) de fermentation alcoolique sont mises en oeuvre séparément, aux différences prés que la température est avantageusement comprise entre 28 et 40°C, et la réaction est avantageusement réalisée pendant une durée comprise entre 50h et 300h. According to a preferred embodiment of the process according to the invention, step a) of enzymatic hydrolysis of the process according to the present invention, using in mixture, cellulolytic and / or hemicellulolytic enzymes originating from the microorganism T. reesei and at least P. anserina mushroom protein and alcoholic fermentation step b) are carried out in a single step. According to this preferred embodiment, the process according to the present invention is a simultaneous saccharification and fermentation process known as the SSF method. The operating conditions used in the said embodiment are identical to those described above in the case where the enzymatic hydrolysis step a) and the alcoholic fermentation step b) are carried out separately, with the differences being different. that the temperature is advantageously between 28 and 40 ° C, and the reaction is advantageously carried out for a period of between 50h and 300h.
Les exemples ci-dessous illustrent l'invention sans en limiter la portée. The examples below illustrate the invention without limiting its scope.
Exemple 1 : Culture du champignon P. anserina Example 1: Culture of the P. anserina mushroom
Les cultures de P. anserina S mat+ sont réalisées avec un milieu M2 (KH2P04 0,25 g.l"1, K2HP04 0,3 g.l'1, MgS04 7H20 0,25 g.l"1, urée 0,5 g.l"1, thiamine 0,05 mg.l"1, biotine 0,25 pg.l" \ acide citrique 2,5 mg.l"1, ZnS04 2,5 mg.l"1, CuS04 0,5 mg.l"1, MnS04 125 Mg.l"1, acide borique 25 pg.l"1, molybdate de sodium 25 Mg.l"1, alun de fer 25 pg.l"1, dextrine 5 g.l'1, extrait de levure 10 g.l"1. Ajuster à pH7 avec une solution de KH2P04). En phase de croissance, le mycélium pousse de 7 mm par jour à 27°C sur gélose de M2 (M2 + agar 12,5 g.l'1). The cultures of P. anserina S mat + are carried out with an M2 medium (KH 2 PO 4 0.25 gl -1 , K 2 HPO 4 0.3 gl -1 , MgSO 4 7H 2 O 0.25 gl -1 , urea 0.5 gl "1, thiamine 0.05 mgl" 1, biotin 0.25 pg L "\ citric acid 2.5 mgl" 1, ZnS0 4 2.5 mgl "1, 4 CuS0 0.5 mg.l -1 , MnSO 4 125 Mg.l -1 , boric acid 25 μg -1 , sodium molybdate 25 Mg -1 -1 , iron alum 25 μg -1 , dextrin 5 μl. 1 , yeast extract 10 μl "1. Adjust to pH7 with a solution of KH 2 PO 4 ) In the growth phase, the mycelium grows 7 mm per day at 27 ° C. on M2 agar (M2 + agar 12). , 5 gl '1 ).
Pour les cultures liquides, une préculture à partir de 5 rondelles de gélose de 0,5 mm de diamètre broyées pendant 30 sec à puissance 5 au Fastprep (MP Biomedicals) est réalisée permettant d'ensemencer 200 ml de milieu M2 en fiole de Roux. Les mycelia des 7 fioles de Roux sont récupérés après 5 jours de culture et broyés au mixeur pendant 20 secondes à une puissance moyenne. Ce broyât permet d'ensemencer 2 I de culture de façon homogène. Toutes les cultures sont réalisées à 27°C et à pH 7, 100 rpm, en fioles bafflées de 500 ml contenant 100 ml/fiole de M2. Une culture de quatre jours sur dextrine comme substrat de carbone permet d'obtenir 70 mg I"1 de protéines extracellulaires. A partir des substrats complexes, la concentration en protéine produite est plus forte. La paille de blé micronisée permet d'obtenir 306 mg.l"1 et la pulpe de betterave 220 mg I"1 de protéines. Exemple 2 : Activités enzymatiques présentes dans les sécrétomes de P. anserina For liquid cultures, preculture from 0.5 mm diameter agar slices milled for 30 sec at a power of 5 at Fastprep (MP Biomedicals) is carried out so as to seed 200 ml of medium M2 in Roux vial. The mycelia of the 7 vials of Roux are recovered after 5 days of culture and crushed with the mixer for 20 seconds with a medium power. This ground material makes it possible to inoculate 2 I of culture in a homogeneous way. All the cultures are carried out at 27 ° C. and at pH 7, 100 rpm, in 500 ml baffled flasks containing 100 ml / flask of M2. A four-day dextrin culture as a carbon substrate yields 70 mg I- 1 of extracellular proteins, and the concentration of protein produced is higher from the complex substrates Micronized wheat straw gives 306 mg 1 and the beet pulp 220 mg I- 1 of protein Example 2: Enzymatic activities present in the secretomas of P. anserina
L'exemple 2 vise à caractériser les activités enzymatiques présentes dans les protéines sécrétées de P. anserina.  Example 2 aims to characterize the enzymatic activities present in the secreted proteins of P. anserina.
Les tests d'hydrolyse de sucres simples sont réalisés avec 4 ig de protéines de P. anserina dans un volume de 100 μΙ de tampon acétate 50 mM pH 5 et 1 mM de substrat, pendant 20 min à 37°C sous agitation. Les substrats testés sont les 4-nitrophenyl-p-D-glucopyranoside (ρΝΡ-β-D-glucopyranoside), ρΝΡ-β-D-lactopyranoside, ρΝΡ-β-D-cellobioside, ρΝΡ-β-D- xylopyranoside, pNP- -D-arabinofuranoside, ρΝΡ-α-D-galactopyranoside et ρΝΡ-β-D- mannopyranoside. La réaction est stoppée par l'ajout de 130 μΙ de Na2C03 1 M, et 5 l'absorbance à 410 nm est mesurée. Un blanc réalisé avec 100μ Ι d'H20 est soustrait aux mesures et une gamme de 0,02 à 0,2 mM de 4-nitrophenol est dosée en parallèle de chaque série de tests. L'activité enzymatique est basée sur le dosage colorimétrique du pNP libéré dans le milieu sous l'action enzymatique. Elle est exprimée en μηιοΙ de paranitrophénol libéré par minute et par mg d'enzyme. The hydrolysis tests of simple sugars are carried out with 4 μl of P. anserina proteins in a volume of 100 μl of 50 mM pH 5 acetate buffer and 1 mM of substrate for 20 minutes. min at 37 ° C with stirring. The substrates tested are 4-nitrophenyl-β-D-glucopyranoside (ρΝΡ-β-D-glucopyranoside), ρΝΡ-β-D-lactopyranoside, ρΝΡ-β-D-cellobioside, ρΝΡ-β-D-xylopyranoside, pNP-D -arabinofuranoside, ρΝΡ-α-D-galactopyranoside and ρΝΡ-β-D-mannopyranoside. The reaction is stopped by the addition of 130 μl of 1 M Na 2 CO 3 , and the absorbance at 410 nm is measured. A white realized with 100μ Ι H 2 0 is subtracted from the measurements and a range of 0.02 to 0.2 mM of 4-nitrophenol is assayed in parallel with each series of tests. The enzymatic activity is based on the colorimetric assay of the pNP released into the medium under the enzymatic action. It is expressed in μηιοΙ of paranitrophenol released per minute and per mg of enzyme.
10 Les hydrolyses des substrats complexes CMC, Avicel® pH-101 , pectine d'agrume, et xylane de bouleau, xylane de blé, 1 ,4 β-D mannane, le galactomannane, l'arabinane de pulpe de betterave et l'arabinogalactane de mélèze, sont réalisées dans un volume de 100 μΙ de tampon acétate 50 mM pH 5 et 1 % (poids/volume) de substrats avec 12 μg de protéines, pendant 1 h à 37°C, sous agitation. Les sucres réducteurs libérés au cours de l'hydrolyse10 The hydrolyzed CMC complex substrates, Avicel ® pH 101, citrus pectin, and birch xylan, xylan wheat, 1, 4 β D-mannan, galactomannan, beet pulp the arabinan and arabinogalactan larch, are made in a volume of 100 μΙ of 50 mM acetate buffer pH 5 and 1% (weight / volume) of substrates with 12 μg of protein, for 1 hour at 37 ° C, with stirring. Reducing sugars released during hydrolysis
15 sont quantifiés par le dosage DNS. 15 are quantified by the DNS assay.
Activités sur substrats liés au pNP (en Ul.mg'1) Activities on substrates related to pNP (in Ul.mg '1 )
Inductions pNP-Glc /?NP-Lac NP-Cell pNP-Xyl pNP-Ara pNP-Gal >NP-Man  Inductions pNP-Glc / NP-Lac NP-Cell pNP-Xyl pNP-Ara pNP-Gal NP-Man
Avicel 395,6 169, 1 128,7 nd nd 155,4 nd Avicel 395.6 169, 1 128.7 n / a N / A 155.4 N / A
Solka-Floc 242,4 79,2 59,8 nd nd 86,5 nd Solka-Floc 242.4 79.2 59.8 n / a N / A 86.5 N / A
Betterave 0,7 nd nd nd nd 24,8 nd paille de blé native 0,05 nd nd nd nd 8,5 nd Beetroot 0.7 n / a N / A N / A N / A 24.8 N / A Wheat straw Native 0.05 N / A N / A N / A N / A 8.5 N / A
Saccharose 24,2 nd nd nd nd nd ndSucrose 24.2 na na na na na na
Tableau 1 : Activités des productions enzymatiques de P. anserina sur les substrats Table 1: Activities of enzymatic productions of P. anserina on substrates
synthétiques marqués au pNP.  synthetic materials marked with pNP.
Abréviations : pNP-GIc = 4-nitrophenyl β-D-glucopyranoside, pNP-Lac = 4-nitrophenyl β-D- lactopyranoside, pNPCel = 4-nitrophenyl β-D-cellobioside, pNP-Xyl = β-D-xylopyranoside, Abbreviations: pNP-Glc = 4-nitrophenyl β-D-glucopyranoside, pNP-Lac = 4-nitrophenyl β-D-lactopyranoside, pNPCel = 4-nitrophenyl β-D-cellobioside, pNP-Xyl = β-D-xylopyranoside,
■>0 pNP-Gal = 4-nitrophenyl α-D-galactopyranoside, pNP-Man = 4-nitrophenyl β-D- mannopyranoside. nd : activité non détectée dans les conditions du test. ■> 0 pNP-Gal = 4-nitrophenyl α-D-galactopyranoside, pNP-Man = 4-nitrophenyl β-D-mannopyranoside. nd: activity not detected in the test conditions.
I Activité sur substrats complexes (en Ul.mg'1) I Activity on complex substrates (in Ul.mg '1 )
X de X of
Inductions CMC Avicel X de blé [Vian GalMan Pectine Ara AraGal bouleau  Inductions CMC Avicel X Wheat [Vian GalMan Pectin Ara AraGal Birch
Avicel 0,55 nd 0,84 2,63 1,98 4,03 nd nd nd Avicel 0.55 n / a 0.84 2.63 1.98 4.03 na na na
Solka-Floc 0,47 nd 3,72 5,10 1,12 2,40 nd nd nd Solka-Floc 0.47 n / a 3.72 5.10 1.12 2.40 N / A N / A N / A
Betterave nd 0,18 nd 0,38 0,29 0,79 0,36 2,82 1,00 Beetroot n / a 0,18 n / a 0,38 0,29 0,79 0,36 2,82 1,00
Son de maïs nd nd nd 0,17 0,07 0,35 nd 1,60 0,32 Corn bran n / a n / a 0,17 0,07 0,35 n / a 1,60 0,32
X de bouleau nd nd 3,69 4,12 nd nd nd nd nd Birch X N / A N / A 3,69 4,12 N / A N / A N / A N / A N / A
Paille de blé Wheat straw
nd 0,13 nd 0,22 0,25 2,48 0,95 4,55 1,19 native  n / a 0,13 n / a 0,22 0,25 2,48 0,95 4,55 1,19 native
Glucose nd 0,01 0,13 0,01 0,06 nd 0,10 nd 0,21 Glucose n0 0.01 0.13 0.01 0.06 nd 0.10 nd 0.21
Dextrine nd nd 0,14 0,12 0,02 0,04 0,06 nd 0,18 Dextrin na na 0.14 0.12 0.02 0.04 0.06 na 0.18
Tableau 2 : Activités enzymatiques des différents sécrétomes de P. anserina sur des polymères présents dans la paroi végétale (cellulose, hémicelluloses et pectine). Table 2: Enzymatic activities of the different secretomas of P. anserina on polymers present in the plant wall (cellulose, hemicelluloses and pectin).
Abréviations : CMC = Carboxymethyl cellulose, X = Xylane, XI = Xylane Insoluble, Man = mannane, GalMan = GalactoMannane, Ara = Arabinane, AraGal = ArabinoGalactane. nd = activité non détectée dans les conditions du test. Abbreviations: CMC = Carboxymethyl cellulose, X = Xylan, XI = insoluble Xylan, Man = mannan, GalMan = Galactomannan, Ara = Arabinan, AraGal = ArabinoGalactan. nd = activity not detected in the test conditions.
Exemple 3 : Hydrolyse de la paille prétraitée avec ajout de β-glucosidase Example 3 Hydrolysis of Pretreated Straw with Addition of β-Glucosidase
Les tests sont effectués avec 100 μ\ d'une suspension de paille de blé prétraitée à l'explosion à la vapeur à raison de 1 % (poids/volume) dans du tampon acétate 50 mM pH 5, supplémentés de cycloheximide 30 mg.l"1 et tetracycline 40 mg.l"1. Les quantités de protéines sont de 10 ou 20 μg de protéines de T. reesei (E508) seules ou de 10 pg de E508 supplémentées de 10 pg de protéines de P. anserina. Le mélange réactionnel contient également 25U de β-glucosidase par g de matière sèche (SP188, Novozyme). L'hydrolyse est réalisée à une température de 37°C, les enzymes sont filtrées et les sucres réducteurs sont dosés au DNS à plusieurs temps entre 2h et 96h d'hydrolyse. Après 96 heures d'hydrolyse, on obtient 24 mM de sucres réducteurs libérés avec 10 g comme avec 20 μg d'enzymes E508 par mg de substrat. La quantité de sucres totaux hydrolysés après 24 h et 96 h est augmentée avec l'ajout des enzymes de P. anserina induites sur Avicel (17 % de sucres réducteurs supplémentaires) et sur Solka-Floc, pulpe de betterave et paille de blé (13 %, 9 % et 5 % de sucres réducteurs libérés supplémentaires). The tests are carried out with 100 μl of a suspension of wheat straw pretreated with steam explosion at a rate of 1% (weight / volume) in 50 mM acetate buffer pH 5, supplemented with cycloheximide 30 mg. 1 and tetracycline 40 mg.l -1 . The amounts of protein are 10 or 20 μg of T. reesei protein (E508) alone or 10 μg of E508 supplemented with 10 μg of P. anserina protein. The reaction mixture also contains 25 μ of β-glucosidase per g of dry matter (SP188, Novozyme). The hydrolysis is carried out at a temperature of 37 ° C., the enzymes are filtered and the reducing sugars are assayed at DNS at several times between 2 h and 96 h of hydrolysis. After 96 hours of hydrolysis, 24 mM of reducing sugars released with 10 g are obtained as with 20 μg of E508 enzymes per mg of substrate. The amount of total sugars hydrolysed after 24 hours and 96 h is increased with the addition of P. anserina enzymes induced on Avicel (17% additional reducing sugars) and on Solka-Floc, beet pulp and wheat straw (13%, 9% and 5% reducing sugars) released additional).
La Figure 1 illustre également les résultats obtenus par hydrolyse de la paille de blé prétraitée par le cocktail E508 de T. reesei additionné de β-glucosidase et complémenté par les sécrétomes de P. anserina, produits sur cellulose Solka-Floc, Avicel, pulpe de betterave et paille de blé.  FIG. 1 also illustrates the results obtained by hydrolysis of wheat straw pretreated with T. reesei cocktail E508 supplemented with β-glucosidase and supplemented with P. anserina secretomas, produced on Solka-Floc cellulose, Avicel, pulp of beet and wheat straw.

Claims

REVENDICATIONS
1. Procédé de production d'alcool à partir de matériaux ligno-cellulosiques prétraités, comprenant au moins les étapes suivantes : A process for producing alcohol from pretreated lignocellulosic materials, comprising at least the following steps:
a) une étape d'hydrolyse enzymatique desdits matériaux lignocellulosiques prétraités utilisant, en mélange, des enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei et de protéines du champignon P. anserina ;  a) a step of enzymatic hydrolysis of said pretreated lignocellulosic materials using, in a mixture, cellulolytic and / or hemicellulolytic enzymes from the T. reesei microorganism and P. anserina mushroom proteins;
b) une étape de fermentation alcoolique par un microorganisme alcooligène de l'hydrolysat issu de l'étape a) et obtention d'un moût de fermentation ; et  b) a step of alcoholic fermentation with an alcoholic microorganism of the hydrolyzate from step a) and obtaining a fermentation must; and
c) une étape de séparation de l'alcool du moût de fermentation,  c) a step of separating the alcohol from the fermentation must,
caractérisé en ce que les protéines du champignon P. anserina sont produites sur des sucres monomères, dimères ou polymères ou résidus de végétaux choisis parmi la paille de blé, la pulpe de betterave, Avicel, Solka-Floc cellulose ou le saccharose.  characterized in that the P. anserina mushroom proteins are produced on monomeric, dimeric or polymeric sugars or vegetable residues selected from wheat straw, beet pulp, Avicel, Solka-Floc cellulose or sucrose.
2. Procédé selon la revendication 1 dans lequel ledit procédé est un procédé de production d'éthanol. The process of claim 1 wherein said process is a process for producing ethanol.
3. Procédé selon la revendication 1 ou 2 dans lequel lesdits matériaux lignocellulosiques sont choisis parmi les résidus d'exploitation agricole, la bagasse de canne à sucre ou tout autre type de résidus ligneux, les résidus d'exploitation forestière, les produits de l'exploitation forestière, les résidus de scieries, les cultures dédiées, les résidus de plantes alcooligènes, sucrières et céréalières, les résidus de l'industrie papetière, les déchets végétaux et les produits de transformation desdits matériaux lignocellulosiques, pris seuls ou en mélange. 3. Method according to claim 1 or 2 wherein said lignocellulosic materials are selected from agricultural residues, sugarcane bagasse or any other type of wood residues, logging residues, products of the logging, sawmill residues, dedicated crops, residues of alcoholic, sugar and cereal plants, residues of the paper industry, plant wastes and transformation products of such lignocellulosic materials, alone or as a mixture.
4. Procédé selon la revendication 1 à 3 dans lequel lesdites enzymes cellulolytiques et/ou hémicellulolytiques sont récupérées dans leur milieu de culture avant leur utilisation dans ladite étape a) d'hydrolyse enzymatique. 4. Method according to claim 1 to 3 wherein said cellulolytic and / or hemicellulolytic enzymes are recovered in their culture medium prior to their use in said enzymatic hydrolysis step a).
5. Procédé selon l'une des revendications 1 à 4 dans lequel les enzymes cellulolytiques et/ou hémicellulolytiques provenant du microorganisme T. reesei et lesdites protéines du champignon P. anserina sont mélangées dans des proportions allant de 40:60 jusqu'à 95:5. 5. Method according to one of claims 1 to 4 wherein the cellulolytic and / or hemicellulolytic enzymes from the microorganism T. reesei and said P. anserina mushroom proteins are mixed in proportions ranging from 40:60 to 95: 5.
6. Procédé selon l'une des revendications 1 à 5 dans lequel une enzyme β-glucosidase est ajoutée audit mélange utilisé dans l'étape a) d'hydrolyse enzymatique. 6. Method according to one of claims 1 to 5 wherein an β-glucosidase enzyme is added to said mixture used in step a) enzymatic hydrolysis.
7. Procédé selon l'une des revendications 1 à 6 dans lequel l'étape a) d'hydrolyse enzymatique est effectuée à une température comprise entre 30°C et 50°C, à un pH compris entre 4,5 et 5,5 et pendant une durée comprise entre 24 et 120h. 7. Method according to one of claims 1 to 6 wherein the step a) enzymatic hydrolysis is carried out at a temperature between 30 ° C and 50 ° C, at a pH between 4.5 and 5.5 and for a period of between 24 and 120h.
8. Procédé selon l'une des revendications 1 à 7 dans lequel l'étape a) d'hydrolyse enzymatique et l'étape b) de fermentation alcoolique sont réalisées en une seule étape à une température entre 28 et 40°C pendant 50 à 300h. 8. Method according to one of claims 1 to 7 wherein the step a) of enzymatic hydrolysis and the alcoholic fermentation step b) are carried out in a single step at a temperature between 28 and 40 ° C for 50 to 50 minutes. 300h.
PCT/FR2012/000499 2011-12-13 2012-11-30 Method for producing alcohol from lignocellulosic biomass by complementation of the cellulolytic and hemicellulolytic enzymes of trichoderma reesei by the fungus podospora anserina WO2013088003A1 (en)

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