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    • 专利摘要:
    A yeast propagation method for use in the production of a fermentation product from lignocellulosic biomass, comprising the steps of: a. have a reactor, b. contacting in said reactor: a population of yeasts capable of metabolizing pentoses and hexoses, at a rate of 0.2 to 2.0 g / kg, of the crude marc resulting from the pretreatment of the lignocellulosic biomass, at the rate of dry matter content (DM) of between 8 and 15%, nutrients, and cellulases, at 5 to 15 mg protein per gram of DM, c. incubate the mixture at a temperature between 25 and 38 ° C, preferably between 28 and 33 ° C, in microaerobiosis, in which the saccharification of the crude marc and the growth of the yeasts are carried out simultaneously.
    公开号:FR3036709A1
    申请号:FR1554902
    申请日:2015-05-29
    公开日:2016-12-02
    发明作者:Flora Ghisoni
    申请人:Lesaffre et Cie SA；Institut National de la Recherche Agronomique INRA；Agro Industrie Recherches et Developpements ARD；
    IPC主号:
    专利说明:

    [0001] The present invention relates to the field of yeasts used in the manufacture of biofuels and other green chemistry compounds, produced by a fermentation process. The decline in fossil fuel stocks has led the industry to look for alternative solutions, implementing as much as possible renewable raw materials and less polluting processes. These solutions include the production of bioethanol from plant biomass, biomass from vegetable waste or even municipal waste. In order to be accepted, green versions of chemical compounds must be as effective or even more efficient than existing versions, and their manufacturing processes must be economically competitive. The applications are numerous: first-generation fuels, biodiesel or ethanol, derived from vegetable raw materials such as sugar cane, beetroot, wheat, maize or vegetable oil, second-generation fuels, biodiesel, biofuel kerosene, cellulosic ethanol, derived from non-food plant biomass or crop residues, and other products of heavy chemistry or fine chemistry. The raw material must be pretreated. According to its origin, the pre-treatment is mechanical (grating, grinding, grinding, mill, pressure), thermal, or chemical. The crude marc obtained is subjected to extraction and / or enzymatic hydrolysis. This leads to a fermentable substrate to which the fermenting microorganism is added. Finally, the fermentation product can be used to extract products of interest, for example by distillation or extraction by means of a solvent. Different stages of this process are technological locks and have been widely studied: pretreatment of biomass to make it accessible to enzymes, definition of enzyme mixtures for efficient hydrolysis of carbohydrate polymers or alcoholic fermentation of the various sugars obtained (pentoses, hexoses ). The alcoholic propagation step of the yeast, object of the present invention, is poorly described. As such, the present invention relates to a yeast propagation process comprising the steps of: a) having a reactor b) contacting in said reactor a yeast population capable of metabolizing pentoses and / or hexoses , at a rate of 0.2 to 2.0 g of yeast solids per kg of complete medium prepared, 3036709 2 - raw pre-treated marc composed of organic fibers, preferably derived from plant biomass at a concentration of dry matter (DM) between 8 and 15%, preferably between 10 and 12%, even more preferably 10%, 5 - nutrients, - a source of nitrogen such as yeast extracts, urea, ammonia, cellulases, 5 to 15 mg of enzyme protein per gram of DM, c) incubate the mixture at a temperature between 25 and 38 ° C, preferably between 28 and 33 ° C, and micro-aerobiosis, in which the saccharification of Raw marc and yeast growth are carried out simultaneously.
    [0002] BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a process for the production of bioethanol comprising the pretreatment, the yeast propagation and the alcoholic fermentation phases, by detailing the different substrates that can be generated by the process, usable substrates for yeast propagation steps and ethanol production.
    [0003] The sugar composition of the five substrates is as follows: (1) Cellulose (glucose polymer) and solubilized hemicellulose (hexose and pentose monomers and oligomers). Solid substrate. (2) Hydrolyzed cellulose and hemicellulose (hexose and pentose monomers and oligomers). Liquid substrate containing suspended solids (3) Cellulose (glucose polymer). Solid substrate. (4) Solubilized hemicellulose (hexose and pentose monomers and oligomers). Liquid substrate (5) Hydrolyzed cellulose (glucose). Liquid substrate containing suspended solids
    [0004] Figure 2 illustrates the evolution of yeast (in cells / mL), substrate (C5 and C6 sugars) and ethanol (in g / kg) concentrations during propagation on lignocellulosic hydrolyzate (or SHF for Separated Hydrolysis). and Fermentation). Figure 3 illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during propagation on C5 juice.
    [0005] Figure 4 illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during SSP propagation (simultaneous propagation and saccharification). according to the invention, at 10% MS and 10 mg enzyme proteins / g MS. FIG. 5 illustrates the evolution of yeast concentrations (in cells / ml), in substrates (sugars 05 and 06) and in ethanol (in g / kg) during the SSP propagation according to the invention, at 12%. MS and 10 mg enzyme protein / g MS. FIG. 6 illustrates the evolution of yeast concentrations (in cells / ml), in substrates (sugars 05 and 06) and in ethanol (in g / kg) during the SSP propagation according to the invention, at 10% MS. and 7 mg enzymatic proteins / g MS. Figure 7 illustrates the evolution of yeast concentrations (in cells / ml), substrates 10 (sugars 05 and 06) and ethanol (in g / kg) during the SSP propagation according to the invention, at 32 ° C. . FIG. 8 compares the evolution of yeast concentrations (in cells / ml), in substrates (sugars 05 and 06) and in ethanol (in g / kg) during the SSP propagation according to the invention, at 30 and 32. ° C.
    [0006] FIG. 9 illustrates the evolution of yeast concentrations (in cells / ml), in substrates (sugars 05 and 06) and in ethanol (in g / kg) during the SSP propagation according to the invention, at 10% MS and 7 mg enzyme proteins / g MS, with and without acetate addition. FIG. 10 illustrates the evolution of yeast concentrations (in cells / ml), in substrates (sugars 05 and 06) and in ethanol (in g / kg) during the SSP propagation according to the invention, at 10% MS and 10 mg enzymatic proteins / g MS, with and without acetate addition. Figure 11 compares the evolution of yeast concentrations (in cells / mL) during propagation on lignocellulosic hydrolyzate (SHF) and SSP propagation at 10% MS and 7 mg enzyme proteins / g MS with and without addition of 'acetate. Figure 12 compares the evolution of yeast concentrations (in cells / mL) during 05 propagation and SSP propagation at 10% MS and 7 mg enzyme proteins / g MS with and without addition of acetate. Figure 13 illustrates the concentrations of sugars (05 and 06) and ethanol (in g / kg) during fermentation SSCF on wheat straw with addition of acetate (qs 4 g / kg) seeded with yeast 1- 4783 propagated in SSP.
    [0007] According to FIG. 1, the pretreated plant biomass can be used as substrate for yeast propagation and / or alcoholic fermentation in five different forms: The most widespread solution consists in carrying out a hydrolysis of the whole marc in the presence of cellulases, in order to obtaining a liquid lignocellulosic hydrolyzate (2) comprising a mixture of hexose (06) monomers and pentoses (05), as well as oligomers in a small amount. Another common substrate is the hemicellulose hydrolyzate also called "juice of 05" (4), which corresponds to the soluble fraction of the substrate at the pre-treatment outlet. This extraction of the hemicellulosic hydrolyzate (4) at the pre-treatment outlet results in cellulosic cellulose-rich marc (3) which can be hydrolysed by means of cellulases in the same way as full-fat marc. In this case, the cellulose hydrolyzate obtained contains predominantly glucose monomers (5). This cellulosic cellulose-rich marc (3), which is in solid form, can also be used without prior hydrolysis. In this case, the cellulases are added in fermentation and the hydrolysis of the cellulose and the glucose consumption are carried out simultaneously. Finally, the integration of the most advanced process consists of using the crude pretreated marc (1) as a fermentation substrate without an intermediate step of hydrolysis or separation. In this case, the cellulases are added in fermentation and the hydrolysis of the cellulose is carried out simultaneously with the consumption of xylose and hexoses. This latter option for the alcoholic fermentation step is known as simultaneous saccharification and co-fermentation of xylose and hexoses (Simultaneous Saccharification and Co-Fermentation or SSCF). The propagation of the yeasts that will be used for the fermentation stage is 20 different problems, especially in terms of oxygen transfer. It is classically performed on C5 (4) juice. Thus, patent application U52014 / 0065700 mentions a propagation carried out on C5 juice and the application WO2009 / 155633 indicates as essential the preliminary step of hydrolysis of the cellulose. Propagation is poorly treated in the scientific literature, and the propagation carried out simultaneously with hydrolysis has never been described. The substrate used is either the soluble fraction at the end of pretreatment (or C5 juice), or a lignocellulosic hydrolyzate obtained by prior hydrolysis of the totality of the pretreated vegetable. After propagation, the yeasts are used either for fermentation (with (SSF) or without saccharification), or to produce proteins of interest (Duarte et al., 2008; Applied Biochem Biotechnol, 148: 119-29; Meyer et al. Biotechnol Bioeng, 40 (3): 353-8, Holder et al., 1989, Biological VVastes, 28 (4): 239-46, Gonzalez-Valdes & Moo-Young, 1981, Biotechnology Letters, 3 (1992). 3): 143-8). Bellissimi & Richards (Bellissimi E, Richards C: Yeast Propagation in The alcohol textbook, 5th edition, Edited by Ingledew VVM, Kelsall DR, Austin GD, Kluhspies C. Nottingham: University Press; 35 2009: 145-159) indicate that the method of production of industrial yeasts is aerobic propagation, in which there is no alcohol production and a maximum rate of 3036709 cells is reached. Indeed, for yeast, the growth capacity for a long time and under strictly anaerobic conditions is limited. One of the disadvantages of raw pretreated marc before liquefaction or hydrolysis is its viscosity. For this reason, the liquefaction / solubilization or hydrolysis step is presumed essential. No document describes or suggests a simultaneous saccharification and propagation process. The patent application WO2011 / 56991 A1 describes a simultaneous saccharification and fermentation process, with possibly an aerated propagation, in parallel, in the liquefied medium rich in hexoses which will be used for fermentation. Patent application VVO 2010/014817 A2 describes a process for improving the quality and / or quantity of the fermenting organism (yeast) during the fermentation phase. Patent Application WO2014 / 72232 A1 describes a method of aerobic propagation in a lignocellulosic hydrolyzate (used as a carbon source), in which the hydrolyzate is added in "fed-batch" mode so as to obtain and maintain a given pH in the reactor. US2014 / 0273167 A1 discloses an aerobic yeast propagation method, with agitation and aeration, on a hexose-rich substrate resulting from hydrolysis. The patent application US2014 / 0273166 A1 describes a method for propagating yeasts on a substrate resulting from the transformation of a plant biomass, which substrate is preferably rich in pentoses. Yeasts subject to propagation, in this case, are transformed yeasts capable of metabolizing pentoses. The juice propagation method of 05 requires a complex step of extracting the liquid fraction of the pretreated marc. The 06-rich hydrolysate propagation method (or comprising a 05-06 mixture) also requires a specific hydrolysis step. Such a step is expensive and time consuming. Thus, there remains a desirable improvement of the process to a more integrated version, retaining satisfactory performance in terms of yield, productivity, and multiplication rate. The present invention provides a method of simultaneous saccharification and propagation. In contrast to the prior art on the essential character of a hydrolysis or extraction step prior to obtaining a substrate suitable for the propagation of yeasts, the Applicant proposes a process using the pretreated marc. crude as yeast propagation substrate, without prior steps of hydrolysis or separation. Said method combines the saccharification of crude marc with cellulases and the growth of yeasts from the available sugar (s) and the sugar (s) liberated by enzymatic hydrolysis.
    [0008] The simultaneous saccharification and propagation process according to the invention will thereafter be abbreviated SSP (according to its Anglo-Saxon name Simultaneous Saccharification and Propagation). One advantage of propagation according to the invention is the reduction of time and cost by the integration of the process by eliminating a step. Another advantage of the invention is the limitation in fermentable sugars due to the simultaneous enzymatic hydrolysis which makes it possible to achieve a high biomass production yield without imposing a fed-batch protocol (also called fed batch fermentation).
    [0009] Another advantage of the invention is that the continuously low glucose level promotes the consumption of xylose, usually inhibited in the presence of glucose. Another advantage of the invention is that the high final biomass content (of the order of 17.4 g / kg) makes it possible to limit the size of the yeast propagation unit, as well as the dilution of the fermentation must. due to seeding which accounts for only 3% of SSCF fermentation volume. Another advantage of the invention is that the impact of the inhibitors present in the pretreated marc on the growth performance is significantly reduced. Finally, another advantage of the invention is that glucose consumption as it is released by enzymatic hydrolysis limits the risk of contamination.
    [0010] DETAILED DESCRIPTION OF THE INVENTION The simultaneous saccharification and propagation method according to the invention is applied to a pretreated biomass. Said biomass is a lignocellulosic material, that is, a material which contains lignocellulose. The lignocellulosic material may contain other constituents such as cellulosic material (cellulose, hemicellulose), as well as sugars, fermentable or not, and pectins. In general, the lignocellulosic material is derived from plant material: stems, leaves, shells, plant envelopes, leaves, antlers or wood of trees. The lignocellulosic material may also be derived from herbaceous material, agricultural residues, forest residues, municipal solid waste or paper mill effluents. According to one embodiment of the invention, the biomass used in the process is derived from miscanthus, poplar, or wheat straw. The lignocellulosic material must be pretreated to break the lignin and crystalline structure of the cellulose. This facilitates the solubilization of hemicellulose and cellulose and their accessibility for enzymes that may be used in the treatment of biomass. Any pretreatment means, in particular impregnation and pretreatment, known to those skilled in the art, may be suitable. Schematically, the pretreatment can be chemical, mechanical or biological. The chemical pretreatment comprises treatment with a basic acidic catalytic agent, especially sulfuric acid, or with organic solvents, sulfur dioxide or carbon dioxide. Liquid oxidation and pH controlled hydrothermolysis are also considered chemical treatments. Mechanical pretreatment is any mechanical or physical treatment such as grinding, irradiation, explosion at high pressure or at high temperature (steam explosion). In some embodiments, chemical and mechanical treatments may be combined, sequentially or simultaneously. According to an advantageous embodiment, the pretreatment of the raw material comprises the following steps: impregnation in the presence of an acidic or basic chemical catalytic agent, in particular an acid catalyst, preferentially sulfuric acid, in proportions comprised between 0.1 and 2.0% by weight, preferably 0.5%. Advantageously, said impregnation is carried out at a temperature of between about 50 ° C. and about 80 ° C., in particular between about 60 ° C. and 70 ° C., preferentially at about 65 ° C. injecting water vapor at a temperature of between about 120 ° C. and about 250 ° C., in particular between about 170 ° C. and about 190 ° C., preferably at about 180 ° C., at a pressure of between about And about 15 bar, in particular between about 8 and about 10 bar, preferably at about 9 bar, and for a time between 1 and 10 min, preferably 5 min.
    [0011] The pretreated plant biomass can then be used as a substrate for the propagation of yeasts and / or in alcoholic fermentation, as indicated in Figure 1 described above. Propagation is also called multiplication, proliferation or biomass production. The goal is to obtain an optimal amount of biomass for fermentation. The propagation medium from the pretreated biomass can be rich in pentoses, rich in hexoses or a mixture of pentoses and hexoses. By pentoses is meant sugars having 5 carbon atoms, also called C5 or more simply C5 sugars. The main natural monomeric representatives of pentoses are D-xylose and Larabinose. By analogy, hexoses are sugars with 6 carbon atoms, also called C6 or more simply C6 sugars. The main representatives of the 35 hexoses in monomeric form are glucose, fructose, mannose and galactose.
    [0012] The SSP propagation (simultaneous saccharification and propagation) according to the present invention is directed to yeasts, able to transform both one or more pentoses and one or more hexoses. The term "yeast strain" refers to a homogeneous population of yeast cells.
    [0013] A yeast strain is obtained from the isolation of a clone. A clone gives rise to a population of cells obtained from a single yeast cell. By the term "derived yeast strain" is meant a yeast strain derived by one or more crosses and / or mutation and / or genetic transformation. A yeast strain derived by crossing can be obtained by interspecific crossing or not. A yeast strain derived by mutation may be a yeast strain that has undergone at least one spontaneous mutation in its genome or at least one mutation induced by mutagenesis. The mutation (s) of a derived strain may or may not affect the phenotype. By the term "mutagenesis" is meant the process of appearance of a mutation. Classically, two methods are possible, random mutagenesis and insertional or directed mutagenesis. The first is the application of physical treatment (eg UV radiation) or treatment with mutagenic chemicals, which will randomly induce mutations in the genome of the organism studied. The second will use molecular biology methods to bring about a precise modification (i.e., promoter, gene, terminator, etc.) either in any region of the genome or at a specific locus. Locus refers to the precise and invariable physical location of a gene on a chromosome. A yeast strain derived by genetic transformation is a yeast strain into which has been introduced a DNA sequence which is preferably provided by a plasmid or integrated directly into the genome. Schematically, it is possible to distinguish four phases during the propagation of a yeast strain: the so-called "latency" phase during which no growth is detectable and which can be assimilated to an adaptation period; it is followed by the "growth phase" during which the cells multiply according to the maximum rate of growth then the "stationary phase" in which the fermenting organism enters when the period of maximum growth decreases and then ceases. And finally, the decline phase during which the number of viable cells will decrease. Propagation is generally an aerated process. Aerobiosis, or aeration of the propagation medium, guarantees a much better biomass production yield than anaerobiosis. Similarly, nutrients can be brought into the environment, such as a source of nitrogen, a source of phosphorus, minerals. Vitamins and organic compounds such as amino acids or nucleic acids are rarely added in an industrial setting because of their cost. The faster and shorter the growth phase, the more microbial contaminations will be avoided. Too much contamination of the propagation will lead to losses of production yield during the subsequent fermentation stage. To limit contaminations, antimicrobials and penicillin or virginiamycin antibiotics, or acid extracts of hops, may be used.
    [0014] The SSP propagation according to the invention must be carried out in microaerobiosis. This means that the medium is aerated but the amount of oxygen supplied is limiting. The dissolved oxygen pressure is zero, unlike the aerobiosis. The oxygen dissolved in the fermentation medium is measured using an oxygen probe according to a method known to those skilled in the art. The microaerobiosis in the process according to the invention is obtained by moderate aeration and stirring. Preferably, the aeration is 0.1 VVM (volume of air / volume of medium / minute, ie 60 mL for a reactor containing 600 mL of medium per minute) and the stirring is set around 500 rpm . Concretely, the agitation depends on the scale at which the process is implemented; in other words, the person skilled in the art adapts according to the material, the volume of said equipment, and the acceptable energy expenditure. The higher the volume of work, the lower the agitation. The biomass obtained can then be invested in a fermentation process. The fermentation is preferably carried out at 32 ° C., with moderate stirring, for example 90 rpm. The agitation is moderate so as not to be oxygenating. The pH of the fermentation medium is preferably controlled, for example by the buffering capacity of an acid / base pair. The preferred target pH in the process according to the invention is 5.0. When the fermentation is intended to produce ethanol, the amount of ethanol present in the fermentation medium is measured by any suitable means known to those skilled in the art. It can be a direct measurement of the ethanol produced or an indirect measurement via a parameter correlated to the production of ethanol, such as mass loss. For example, ethanol production can be measured by chromatography, such as HPLC (High Performance Liquid Chromatography), an enzyme kit, or potassium dichromate assay. The amount of xylose and / or glucose present in the medium is measured by any appropriate means known to those skilled in the art, preferably by chromatography, in particular by HPLC.
    [0015] The person skilled in the art knows how to determine the appropriate conditions for an alcoholic fermentation. By way of example, reference may be made to the alcoholic fermentation conditions described in the reference book "Yeast Technology", 2nd edition, 1991, G. Reed and T.W. Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8.
    [0016] The fermentation medium comprises the following elements: at least one fermentable carbon source, at least one nitrogen source, at least one source of sulfur, at least one source of phosphorus, at least one source of vitamins and at least one source of or at least one source of minerals. The carbon source is, for example, provided in the form of a sugar that can be immediately assimilated by the yeast, such as xylose, arabinose, glucose, fructose or galactose, a saccharose disaccharide and / or a mixture. of these sugars. These sugars may be provided in the form of syrup, molasses, EP2 (Poor Sewer resulting from the 2nd crystallisation of sugar), hydrolysates of all or part of a plant material and / or a mixture of these. this. The nitrogen source is, for example, provided in the form of yeast extract, ammonium sulfate, ammonium hydroxide, di-ammonium phosphate, ammonia, urea and / or a combination thereof. The source of sulfur is for example provided in the form of ammonium sulfate, magnesium sulfate, sulfuric acid and / or a combination thereof. The phosphorus source is, for example, provided in the form of phosphoric acid, potassium phosphate, di-ammonium phosphate, mono-ammonium phosphate, and / or a combination thereof. The source of vitamins is, for example, provided in the form of corn steep liquor, molasses, yeast hydrolyzate, pure vitamin solution or a mixture of pure vitamins and / or a combination thereof. The source of vitamins provides the yeast 20 with all the vitamins in amounts at least equivalent to those recommended in the reference works. Several sources of vitamins can be associated. The source of minerals is, for example, provided in the form of molasses, a mixture of mineral salts and / or their combination. The source of minerals provides the yeast with all the macronutrients and trace elements in amounts at least equivalent to those recommended in the reference works. Several sources of minerals can be associated. The same substance can bring several different elements. The propagation according to the invention is characterized in that saccharification and propagation are carried out simultaneously. The crude pretreated marc is used at a dry matter content of between 8 and 15%, preferably between 10 and 12%, advantageously at 10%. According to one embodiment of the invention, the pomace comprises about 1/3 soluble solids (type "juice of 05) and 2/3 dry matter insoluble (type lignocellulosic fibers).
    [0017] The crude pretreated marc is contacted with a population of yeasts capable of metabolizing pentoses and hexoses. The yeasts are added, preferably in dry form, at a rate of 0.2 to 2 g / kg, in other words at a rate of 0.2 to 2 g of yeast solids per kilogram of complete prepared medium. The raw pretreated marc and yeast mixture is supplemented with a combination of cellulases and hemicellulases which allow saccharification. Saccharification is the hydrolysis of polysaccharides to soluble monomeric sugars. This means that the concentration of simple sugars would increase in the medium if they were not consumed by the yeasts for propagation, in parallel with their release by the enzymes. Cellulases thus allow the hydrolysis of cellulose to obtain glucose. Exocellulases or cellobiohydrolases act at the ends of the cellulose to form the cellobiose disaccharide. Endoglucanases act by cleaving the internal bonds of the cellulose to form cellulosic oligosaccharides. Cellobiases or betaglucosidases hydrolyze cellulosic oligopolymers and cellobiose by their reducing end by releasing glucose. Concretely, we group under the term cellulases a mixture of enzymatic proteins.
    [0018] Preferably, the enzymes are used in a proportion of 5 to 15 mg of proteins (enzymes) per gram of dry matter. Advantageously, they are used in a proportion of 7 to 10 mg of proteins (enzymes) per gram of dry matter, preferably 7 mg of enzyme proteins per gram of dry matter. To allow for a proper understanding and comparison between the activities of different compositions exhibiting cellulase-type activity, the Filter Paper Unit (FPU) activity can be used as a reference. The biotechnology commission of the International Union of Pure and Applied Chemistry (IUPAC) recommends the following procedure: FPU activity is measured on VVhatman # 1 paper at the initial concentration of 50 g.L-1. The aim is to determine by colorimetric assay (with dinitrosalicylic acid, DNS) the amount of reduced sugars from VVhatman # 1 paper. By way of example, the test portion of the enzymatic solution to be analyzed which releases the equivalent of 2 g of glucose in 60 minutes is determined. Specific activities are obtained by dividing the activities expressed in IU.mL-1 by protein concentration; they are expressed in IU.mg-1. Advantageously, the combination of cellulases and hemicellulases used in a process according to the invention correspond to an enzymatic composition having one or more activity (s) improved (s) relative to a composition containing proteins produced by the native mushroom. Such cellulases are known to those skilled in the art, for example described by Durand et al., 1988 (Enzyme Microb Technol., 10: 341346). According to a preferred embodiment of the invention, the cellulases correspond to an enzymatic composition as described in application WO2010029259 A1, in particular an enzymatic composition produced by filamentous fungi, preferentially Trichoderma reesei. The mixture is then incubated, in micro-aerobiosis, at a temperature between 25 and 38 ° C, preferably between 28 and 33 ° C, preferably between 30 and 32 ° C.
    [0019] Advantageously, the pH of the solution is around 5.0. The incubation is maintained between 24 and 50 hours, particularly between 28 and 50 hours, more preferably between 30 and 42 hours. Advantageously, the target cell concentration (at the end of propagation) is between 5.0 × 10 8 and 1.0 × 10 9 cells per milliliter.
    [0020] According to a particular embodiment of the invention, the transformed yeasts capable of metabolizing both pentoses and hexoses are obtained according to processes described in patent applications VV02010000464A1, VV02011128552A1 and VV02012072793A1. Advantageously, said strains are also resistant to acetic acid, obtained by a process as described in application VV02013178915A1. According to one embodiment of the invention, the yeast strain used metabolizes preferentially xylose and glucose. In other words, according to a particular embodiment, the invention relates to a process for fermenting sugars derived from lignocellulosic biomass, preferentially pentoses and / or hexoses, using a fermenting microorganism, characterized in that said microorganism was produced directly on raw pretreated marc, according to a simultaneous saccharification and propagation method. According to a preferred embodiment of the invention, the yeast strain subjected to simultaneous propagation and saccharification according to the invention corresponds to one of the strains deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on May 24, 2012 under the numbers 1-4624, 1-4625, 1-4626, 1-4627 or the strain filed on June 26, 2013, under number I4783.
    [0021] According to a preferred embodiment of the invention, the propagation by SSP precedes a fermentation step. The present invention also relates to a process for producing at least one fermentation product comprising a fermentation step, under anaerobic or semi-aerobic conditions, by a yeast propagated on crude pretreated marc according to a method in which saccharification and propagation are carried out simultaneously.
    [0022] The fermentation product is in particular chosen from ethanol, a metabolite obtained from ethanol or a secondary metabolite. A preferred fermentation product according to the invention is ethanol.
    [0023] The invention can be better understood in the light of the following examples which are in no way limiting. EXAMPLE 1 Pretreatment Conditions and Analysis of the Composition of the Substrate The substrate used for these tests is crude marc of wheat straw resulting from a pretreatment according to the following method: the ground straw is impregnated in acidic water between 0.1 and 2.0% by weight of H 2 SO 4, then pretreated by steam explosion continuously at approximately 50% dry matter for 1 to 10 min at 170 to 190 ° C, preferably at 180 ° C. This crude straw marc was analyzed by high performance liquid chromatography (HPLC or HPLC) and the contents of sugars and inhibitors are shown in Table 1. Table 1: Composition of straw marc used for the present study. Concentrations in g / kg. Raw Marc concentration in g / kg Content in MS (%) 46.02% Sugars Cellulose 170 Cellobiose 4 Glucose 9.7 Xylose 93.8 Galactose NQ Arabi nose 10.9 Mannose 2.1 Inhibitory compounds Lactic acid 0.7 Acetic acid 3.6 Formic acid 0.6 5-HMF 0.4 Furfural 0.2 3036709 14 NQ means unquantified ie not measured. Substrate analysis showed conventional acetic and furfural levels. The dry matter content of raw straw marc is 46.0%. In most subsequent propagation tests, it was used at 10% dry matter, which corresponds to a dilution of a factor of 4.6 of the concentrations given above at the time of setting-up. in the reactor, while the other two substrates shown in the table, namely the hydrolyzate and the 05 juice, were used without further dilution. The following experiments were carried out with the yeast Saccharomyces cerevisiae 10 deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) June 26, 2013, under the number 1-4783. The methods used are described below. For comparison, another yeast strain capable of metabolizing pentoses and hexoses would give similar results. The reference methods use liquid substrates, namely lignocellulosic hydrolyzate, which requires a step of hydrolysis of the pre-treated marc prior to propagation, or C5 juice (also called hemicellulosic hydrolyzate), which requires a step of separation of the sugars. soluble in the marc at the end of pre-treatment. In contrast, the Concurrent Saccharification and Propagation (SSP) protocol according to the invention uses the crude pretreated marc solid substrate.
    [0024] Example 2: Reference Methods 2.1. Protocols Antibacterials and nutrients were respectively added to the C5 juice and the lignocellulosic hydrolyzate in amounts adapted to the yeast deposited at the CNCM under the number 1-4783 used in these tests, namely: NH 4 OH % Urea H3PO4 85% Antibacterial mineral mixture The composition of the mineral mixture is shown in Table 2. Those skilled in the art will be able to adapt the proportions for optimum efficiency.
    [0025] Table 2: Composition of the mineral mixture Compound MgSO 4 · 7H 2 O CuSO 4 · 2H 2 O MnCl 2 · 4H 2 0 ZnSO 4 · 7H 2 O The reactors were inoculated at 0.4 g / kg dry yeast and maintained at a temperature of 30 ° C. vs.
    [0026] The pH was maintained at 5.0 by addition of KOH and H 2 SO 4. For the micro-aerobic conditions, the air flow was set at 0.1 VVM (air volume / volume of medium / minute) and the agitation was set at 500 rpm. As an indication, an air flow rate of 0.1 VVM is 60 mL / min. for a reactor containing 600 ml of medium. 2.2 2.2. Results 2.2.1. Propagation on lignocellulosic hydrolyzate (SHF) The yeast growth, substrate consumption and ethanol production kinetics during the propagation of yeasts under micro-aerobic conditions (0.1 VVM) on the lignocellulosic hydrolyzate are presented Figure 2. This propagation test lasted 46.1 h, but Figure 2 shows that yeast growth was complete after about 27 h of culture. The final biomass content produced is estimated at 3.9 × 10 8 cells / ml. Glucose has been consumed preferentially with xylose, as is commonly the case in excess of glucose. 18 g / kg of ethanol were produced.
    [0027] Note: Yeast viability during propagation is not shown in the figure for readability. After the first hours of culture, it is greater than 95% for all the tests presented in these examples. 2.2.2. Propagation on C5 juice The kinetics of yeast growth, substrate consumption and ethanol production during the propagation of yeasts under micro-aerobic conditions (0.1 VVM) on C5 juice are presented in FIG. 3. This C5 juice propagation assay shows a longer lag phase (than that of the lignocellulosic hydrolyzate assay), then the biomass grows rapidly to reach 7.5 × 10 8 cells / ml after 41 h of culture. 10 g / kg of ethanol were produced.
    [0028] The difference in final biomass content has already been observed. In general, the yeast production yields are higher during propagation on 05 juice than during propagation on lignocellulosic hydrolyzate, mainly composed of sugar in 06 (glucose).
    [0029] EXAMPLE 3 Simultaneous Saccharification and Propagation (SSP) 3.1. SSP Process According to the Invention The Simultaneous Saccharification and Propagation Process is also carried out in a reactor comprising: water, pre-treated crude cellulosic marc (solid substrate) used respectively at 10% or 12% dry matter (DM) in most tests, and nutrients as indicated supra for the reference methods. Propagation was initiated by simultaneous addition of cellulases (at 7 and 10 mg protein per gram DM, respectively) and dry yeast at 0.4 g / kg. The enzymes used in the present examples can be replaced by commercial enzymes in an equivalent amount. By way of comparison, the FPase activity (see above the mention of the Filter Paper Unit units) specific for the cellulases used in the examples is between 0.8 and 1.5 IU.mg-1. They can be replaced by commercial enzymes in the same quantities (in I U.mg-1). The temperature was maintained at 30 ° C or 32 ° C, respectively. The pH was maintained at 5.0 by addition of KOH and H2504. For the micro-aerobic conditions, the air flow was set at 0.1 VVM (air volume / volume of medium / minute) and the agitation was set at 500 rpm.
    [0030] Samples were taken during the various propagation tests in order to enumerate yeasts and to quantify sugars and fermentation products by high performance liquid chromatography (HPLC). Total enzyme hydrolyses were performed on the final samples to determine the non-hydrolysed cellulose content at the end of propagation. 3.2. Couples dry matter content (DM) -dose of enzymes Different MS-dose couples of enzymes were tested in SSP. The prerequisites were: (i) a concentration of fermentable sugars which should make it possible to reach a biomass content of the order of 15 g / kg at the end of propagation; (ii) cellulose hydrolysis kinetics which must limit the amount of glucose, so that (1) the carbon flux is directed towards biomass production rather than ethanol production which could have even in the presence of oxygen, if the concentration of sugars is too high, and (2) the use of xylose is favored without productivity being penalized; and (3) the viscosity of the mixture which should allow moderate agitation and micro-aeration of the medium.
    [0031] The contents of MS and the enzyme doses tested are shown in Table 3. Table 3: DM contents and enzyme doses tested in SSP Test MS content Dose of enzymes mg protig MS 1 10 10 2 12 10 Recall: As for the reference methods (supra), these propagation tests were inoculated with 0.4 g / kg of dry yeast and then conducted at pH 5.0 at 30 ° C. with stirring. moderate at 500 rpm and a micro-aeration of 0.1 VVM. 3.2.1. SSP propagation at 10% DM and 10 mg enzyme protein / g MS Yeast 1-4783 was propagated in a microaerobic condition (0.1 VVM) on raw straw marc (at 10 % of MS), in the presence of cellulases (10 mg proteins / g of MS) which make it possible simultaneously to hydrolyze the cellulose. The kinetics of yeast growth and ethanol production, as well as the evolution of glucose and xylose concentrations during the propagation of yeasts are shown in Figure 4. Result: this propagation test lasted 41 hours at after which 5.6.108 cells / ml were obtained. 11 g / kg of ethanol were produced, then partially consumed during this propagation in SSP on raw straw marc. Moreover, the content of MS employed made it possible to add the entire substrate in the initial stock while maintaining a low viscosity allowing moderate agitation and micro-aeration of the culture medium. The experiment was repeated by extending the growth beyond 41h. The results (not shown) were as follows: 6.5 × 10 8 cells / ml were obtained after 48 h of culture and the growth kinetics were superimposed on that obtained previously under the same operating conditions. 3036709 18 3.2.2. SSP spread at 12% DM and 10 mg enzyme protein / g MS Yeast 1-4783 was propagated under micro-aerobic conditions (0.1 VVM) on raw straw marc (at 12% of MS), in the presence of cellulases (10 mg proteins / g of MS) which make it possible simultaneously to carry out hydrolysis of the cellulose. The kinetics of yeast growth and ethanol production, as well as the evolution of glucose and xylose concentrations during yeast propagation are shown in Figure 5. Result: this propagation test lasted 46.3 h after which 5.8 × 10 8 cells / ml were obtained. 15 g / kg of ethanol were produced and then partially consumed during this propagation in SSP on raw straw marc. Increasing the DM content from 10% to 12% did not cause a significant increase in viscosity that could interfere with moderate agitation and microaeration of the culture medium. 3.2.3. SSP spread at 10% MS and 7 mg enzyme protein / g DM Yeast 1-4783 was propagated under micro-aerobic conditions (0.1 VVM) on raw straw marc (at 10% of MS), in the presence of cellulases (7 mg enzymatic proteins / g of MS) which make it possible simultaneously to perform hydrolysis of the cellulose.
    [0032] The kinetics of yeast growth and ethanol production, as well as the evolution of glucose and xylose concentrations during the propagation of yeasts are shown in FIG. 6. Result: this propagation test lasted 43.9. h after which 8.3.108 cells / ml were obtained. 9.3 g / kg of ethanol were produced and then totally consumed during this SSP propagation on raw straw marc. Discussion The comparison of the performances obtained for the SSP tests carried out at different levels of DM and enzyme doses shows that: The increase in the DM content of 10 to 12% for the tests carried out with 10 mg proteins / kg DM leads to an increase in ethanol production but does not have a positive impact on yeast growth. Decreasing the enzyme dose of 10 to 7 mg enzyme proteins / g MS for the 10% MS tests results in a higher glucose limitation which results in faster xylose consumption and directed carbon flow. 3036709 19 more towards the production of biomass. The difference in hydrolysis yield due to the drop in the enzyme dose is less than 2% at the end of propagation. 3.3. Effect of Temperature In order to observe the effect of temperature on the efficiency of SSP propagation, yeast 1-4783 was propagated at 32 ° C in a microaerobic condition (0.1 VVM) on the crude marc straw (at the rate of 10% of MS) in the presence of cellulases (at the rate of 10 mg proteins / g of MS). The kinetics of yeast growth and ethanol production, as well as the evolution of glucose and xylose concentrations during this propagation assay are shown in Figure 7. Result: This propagation test lasted 42.6 hours. A slowdown in growth was observed at the end of the crop. 12 g / kg of ethanol were produced and then partially consumed. The final biomass was estimated at 5.7 × 10 8 cells / m L.
    [0033] FIG. 8 compares the evolution of the yeast population, as well as that of the xylose and ethanol concentrations, for the SSP tests carried out respectively at 30 ° C. and 32 ° C., at 10% DM and 10 mg proteins. MS. No positive effect is observed on yeast growth. It appears that the increase in temperature may promote the uptake of xylose, which results in faster ethanol production kinetics. The increase in temperature improves the enzymatic hydrolysis and increases the amount of fermentable sugars (12% in this case). If the propagation must is transferred integrally to seed the alcoholic fermentations, the residual sugars, whatever their form, represent a small proportion and will be used during the alcoholic fermentation. 3.4. Process robustness test: SSP tests in the presence of acetate in high concentration In order to evaluate the robustness of the SSP process, tests were carried out with the addition of acetate in the medium (QSP (sufficient quantity for 3 g / kg) to simulate higher toxicity of pretreated marc. These tests were performed at pH 5.0, 30 ° C, with 10% DM and enzyme / substrate ratios equal to 7 mg protein / g DM and 10 mg protein / g DM. The increase of the acetate content in the culture medium from 0.7 g / kg to 3 g / kg corresponds to an increase in the acetic acid content of the pretreated marc from 3.6 g / kg 35 to 13.8 g / kg.
    [0034] This last concentration leaves an important margin for the increase in the content of volatile compounds of the pretreated substrates during the industrial scale transition. The growth kinetics of yeasts, as well as the evolution of xylose and ethanol concentrations during propagation tests carried out with and without the addition of 10% 5 MS and 7 mg protein / g DM, are illustrated in FIG. Figure 9. The comparison of the kinetics of SSP performed respectively in the presence of 0.7 g / kg or 3.0 g / kg of acetic acid shows that the increase in the concentration of acetate slows the use of xylose and generates a growth retardation visible up to 25 h of culture. The carbon flux is slightly diverted towards ethanol production. However, the difference in biomass concentration disappears at the end of culture: 8.0 × 10 8 cells / ml were obtained in 42.5 h for the test at 3.0 g / kg of acetate while 8.3 × 10 8 cells / mL were obtained in 43.9 h for the test at 0.7 g / kg of acetate. Yeast growth kinetics, as well as the evolution of xylose and ethanol concentrations during propagation assays performed with and without the addition of acetate to 10% DM and 10 mg protein / g DM are illustrated in FIG. FIG. 10. The comparison of the kinetics of SSP carried out respectively in the presence of 0.7 g / kg or 3.0 g / kg of acetic acid shows that the increase in the concentration of acetate causes a visible growth retardation up to 30 h of culture and that carbon flux is slightly deviated to ethanol production. At the end of propagation, the biomass content produced in the presence of 3 g / kg of acetate exceeds the reference: 6.9.108 cells / mL were obtained in 46.7 h against 6.5.108 cells / mL in 48.1 h for the test without adding acetate. These results show that the significant increase in the amount of acetic acid in pretreated straw marc does not significantly degrade the performance of the SSP propagation method. Such robustness can not be expected from the process of yeast propagation on C5 juice, the fermentation of xylose being affected much more strongly than the fermentation of glucose by the toxicity of the culture medium. Again, two enzyme / substrate ratios were tested. The results are consistent with those obtained previously (supra), namely that the decrease in the enzyme dose improves the performance of the SSP process. EXAMPLE 4 Comparison of the Growth Performance of the SSP According to the Invention with those Obtained with the Reference Methods The evolution of the yeast population is compared with that obtained for the reference processes under temperature conditions, pH, micro-aeration, moderate shaking and identical seeding rates. The quantities of fermentable sugars are of the same order of magnitude. 4.1. Comparison with Propagation on the Lignocellulosic Hydrolyzate (SHF) 5 Evolution of the yeast population during propagation on lignocellulosic hydrolyzate and SSP tests, with and without added acetate, at 10% DM and 7 mg protein / g MS is shown in Figure 11. Result and Discussion The respective substrates of the lignocellulosic hydrolyzate propagation assay and the SSP propagation assay without acetate addition are identical, with one difference: one has been hydrolysed beforehand. The growth is slower on the hydrolyzate, and this from the beginning of the propagation, which can be explained by a slightly higher acetate content due to the further hydrolysis of the substrate at the beginning of culture (1, 0 g / kg vs. 0.7 g / kg SSP); the osmotic pressure due to sugars is also greater. In addition to its better kinetics, the SSP propagation makes it possible to obtain a much greater amount of biomass (8.3 × 10 8 cells / ml vs. 3.9 × 10 8 cells / ml). In addition, it is surprising to note that the SSP propagation test performed in the presence of 3 g / kg of acetate is also better than the reference test. 4.2. Comparison with the spread on C5 juice The evolution of the yeast population during propagation on 05 juice and SSP tests, with and without added acetate, at 10% DM and 7 mg enzyme proteins / MS is shown in Figure 12.
    [0035] Result and discussion The propagation carried out on juice of 05 has a growth kinetics significantly slower than the propagation tests in SSP on crude marc, however the increase of the speed of growth at the end of propagation enables it to reach a level of final biomass equivalent to SSP propagations. However, if the propagation time was reduced compared to the presented test, the advantage of the SSP propagation over the C5 juice propagation would increase. The C5 juice used for this propagation test was derived from the same cellulosic wheat straw that was used for the SSP propagation tests. It was obtained by suspending the cellulosic marc in water, followed by solid / liquid separation. This process for obtaining C5 juices actually extracts all the soluble elements of the pretreated marc; Therefore, it can be considered and it has been verified that the inhibitor content is proportional to the xylose concentration, which makes the juice the most concentrated inhibitor substrate. On the other hand, the fact that xylose uptake is more affected by the toxicity of the medium than glucose consumption and that the increased toxicity of the pre-treated marc leads to a more rapid increase in inhibitor content in the juice. (because proportional to the xylose content) make the advantage of the SSP process will increase with the toxicity of the pretreated marc. Thus, increasing the acetate content from 0.7 g / kg to 3.0 g / kg in the SSP stock, which slightly degrades growth kinetics in SSP, corresponds to an increase in the content of SSP. in acetate from 1.6 g / kg to about 7 g / kg in the juice of 05, which strongly penalizes the growth of the yeast. 4.3. Synthesis and Comparison of Propagation Performance Table 4 provides for each propagation test carried out: The concentration of sugars used in the culture medium (total amount and fermentable amount for the tests carried out in SSP), the final biomass concentration obtained (in cells / ml), The yield of biomass production (referred to the fermentable sugar content and relative to the DM content), An estimate of the yield of yeast production in g yeast / g of fermentable sugars. Table 4: amounts of sugars used, biomass concentrations obtained and biomass production yields for the various propagation tests (cell means cells, mass and thin mass, respectively mean initial mass and final mass).
    [0036] Juice C5, 30T SHF, 10% MS, 30T 1.41E + 5.88E + 09 0.29 0.12 607.4 593.7 53.8 65.6 41 46.1 7.50E + 08600 3.90E + 08 600 64.5 64.5 64.5 64.5 77.3 64.5 64.5 41 48.1 42.6 43.9 46.3 42.5 46.7 Potential sugar g / kg Fermented sugar. g / kg Mas Biomass is final mi. Cell / mL Mas Yield ends. Cell / g fermented sugars. Y Yield / g sugars Conditions Duration h 10% DM, 10 mg prot / g DM, 30T 10% DM, 10 mg prot / g DM, 30T 10% DM, 10 mg prot / g DM, 32T 10% DM, 7 mg prot / g MS, 30T 12% MS, 10 mg prot / g MS, 30T 10% MS, 7 mg prot / g MS, 30 ° C, 3g / kg acetate 10% MS, 10 mg prot / g MS , 30T, 3g / kg of acetate 53.3 5,60E + 08 600 53,3 6,50E + 08,600 59,9 5,70E + 08,600 8,00E + 08,600 6,90E + 08,600 9, 16E + 09 1.13E + 10 9.78E + 09 1.60E + 10 8.37E + 09 1.53E + 10 1.20E + 10 523.0 557.0 616.3 604.9 571.5 599, 3 610.4 52.25 58.7 0.19 0.24 0.20 0.32 0.25 3036709 23 Remarks: Sugar potential is calculated assuming equal hydrolysis yield of cellulose 100%. The amount of fermentable sugars is calculated by estimating the amount of residual cellulose by total enzymatic hydrolysis on a sample taken at the end of culture. For the estimation of the production yield of biomass in g of yeast / g of fermentable sugars, the conversion is made considering that 1 g of yeast contains 4.8.1019 cells (measured at the end of propagation on juice of 05).
    [0037] The final mass measured is abnormally low for propagation tests carried out at 10% DM with 10 mg of protein at 30 ° C, which penalizes them when calculating the yields. Results and Discussion Table 4 shows that the highest final concentration of biomass was obtained with the SSP method carried out at 10% DM and 7 mg protein / g DM. The final biomass content obtained on the juice of 05 is close to that obtained with the SSP method under the best conditions, whereas the propagation on lignocellulosic hydrolyzate results in a final biomass content that is significantly lower than all the other tests. Yield yield calculation equals 1.6.1019 cells / g of fermentable sugars for the best performing test performed on SSP with 10% DM and 7 mg protein / g MS at 30 ° C (and 1, 5.1019 cells / g of fermentable sugars for the test carried out with an increased concentration of acetic acid). The propagation yield on 25 05 juice is slightly lower: 1.4.1019 cells / g of fermentable sugars were obtained. The other SSP conditions tested have yeast production yields close to 1.0 × 10 19 cells / g of fermentable sugars, whereas the reference test carried out on lignocellulosic hydrolyzate has a yield of 5.9 × 10 9 cells / g of fermentable sugars. about 3 times less than the best performing SSP test.
    [0038] In order to compare with known references, the biomass yield is estimated in g of yeast / g of fermentable sugar by considering a conversion number of cells / g of MS obtained at the end of the alcoholic propagation on a juice of 05. According to known data. those skilled in the art, the reference propagation on lignocellulosic hydrolyzate has a yield of the order of 0.12 g / g.
    [0039] The best SSP condition yielded 0.33 g yeast / g fermentable sugars. The multiplication rate of the yeast in propagation is then greater than 40 (final concentration estimated 17.4 g / kg of yeast). In terms of productivity, the propagation process in SSP is also the most efficient, in fact: the average volume productivity is estimated at 0.38 g of yeast / kg of must / h for the SSP process, against 0.37 respectively. g / kg / h and 0.17 g / kg / h for propagations on 05 juice and on lignocellulosic hydrolyzate. The average volume productivity over the first 30 hours of culture is estimated at 0.33 g of yeast / kg of must / h for the SSP process, against 0.26 g / kg / h for propagation on juice of 05 and on lignocellulosic hydrolyzate. EXAMPLE 5 Validation of the SSP propagation method: performance of the yeast propagated in SSCF fermentation The present invention is an essential intermediate link in a global industrial process of alcoholic fermentation. The present example is intended to validate that the yeast obtained at the end of a propagation according to the invention is effective in alcoholic fermentation on lignocellulosic substrate.
    [0040] Yeast 1-4783 propagated in SSP was used to seed SSCF (Simultaneous Saccharification and CoFermentation) fermentation; both crops were grown on raw straw marc. SSCF was run at 24% DM with 10 mg protein / g DM, with acetate added to the medium (QSP 4 g / kg). The reactor was inoculated with 2.4 x 10 7 cells / ml (ie approximately 0.5 g / kg of yeast) and the medium was maintained at pH 5.5 and 33 ° C. for 142.5 h. The evolution of the concentrations of glucose, xylose and ethanol during this fermentation is illustrated in FIG. 13. This SSCF fermentation exhibits a coherent kinetics with what is usually obtained. The yeast consumes very quickly the glucose released by the enzymes so that the glucose concentration is zero in the first hours of fermentation. The xylose released is mainly consumed in 72 hours; the kinetics of ethanol production is then limited by enzymatic hydrolysis. The final content of ethanol is equal to 67.4 g / kg, which corresponds to a difference of less than 5% with the concentration obtained at the end of the SSCFs carried out without addition of acetate with yeast 1-4783 propagated on juice This result makes it possible to conclude that the propagation method according to the invention does not degrade the performance of the yeast produced with respect to a C5 juice propagation method conventionally used.
    权利要求:
    Claims (11)
    [0001]
    REVENDICATIONS1. A yeast propagation method for use in the production of a fermentation product from lignocellulosic biomass comprising the steps of: a. have a reactor, b. contacting in said reactor: a population of yeasts capable of metabolizing pentoses and hexoses, at a rate of 0.2 to 2.0 g of yeast dry matter per kg of prepared complete medium, crude marc resulting from the pretreatment of lignocellulosic biomass, with a solids content (DM) of between 8 and 15%, nutrients and cellulases, at a rate of 5 to 15 mg of protein per gram of DM, c. incubate the mixture at a temperature between 25 and 38 ° C, preferably between 28 and 33 ° C, in microaerobiosis, in which the saccharification of the crude marc and the growth of the yeasts are carried out simultaneously.
    [0002]
    2. Propagation method according to claim 1 wherein the pentoses are xylose and / or arabinose.
    [0003]
    3. Propagation method according to claim 1 or 2 wherein the hexose is glucose.
    [0004]
    4. Propagation method according to one of claims 1 to 3, wherein the yeast strain is a transformed or unmodified strain, capable of consuming pentoses and resistant to fermentation inhibitors, particularly acetic acid.
    [0005]
    5. Propagation method according to one of claims 1 to 4, wherein the incubation in step c. is maintained until a cell concentration of between 5.0 x 108 and 1.0 x 109 cells per milliliter is achieved.
    [0006]
    6. Propagation method according to one of claims 1 to 5 wherein the yeast strains are selected from the strains deposited at the CNCM under the following numbers: 1-4624, 1-4625, 1-4626, 1-4627 and 1-4783.
    [0007]
    7. Propagation method according to claim 1, wherein the yeasts are seeded at a rate of 0.3 to 0.6 g / kg in the form of dry yeasts, the crude marc is used at a rate of 10% of DM and cellulases at a rate of 7 mg protein per gram of DM, the aeration rate is set at 0.1 VVM, the temperature is set at 30 ° C and the pH of the medium is set at pH 5.0. 3036709 26
    [0008]
    A process for producing a fermented product from a lignocellulosic biomass, comprising sequentially the steps of: a. pretreatment of lignocellulosic biomass to obtain a raw marc, b. contacting a fraction of the raw pretreated marc with 10 to 12% dry matter (DM), with (i) a population of yeasts capable of metabolizing pentoses and hexoses, (ii) cellulases with 5 to 15 mg of protein per gram of DM, (iii) optionally, nutrients, c. incubation of the mixture at a temperature between 25 and 38 ° C, preferably between 28 and 33 ° C, in a microaerobiosis, so as to obtain simultaneous saccharification and propagation, until a cell concentration of between , 0 x 108 and 1.0 x 109 cells per milliliter, d. transferring all or part of the propagated yeasts for contact with the fermentation must comprising a source of pentoses and a source of hexoses, e. carrying out the fermentation, under anaerobic or semi-aerobic conditions, f. obtaining the fermented product.
    [0009]
    9. The method of claim 8 wherein the lignocellulosic biomass is a biomass of plant origin. 20
    [0010]
    10. The method of claim 8 or 9 wherein the fermented product obtained is ethanol.
    [0011]
    11. Method according to one of claims 8 to 10, wherein the yeast population used is derived from the strain deposited at the CNCM under the number 1-4783.
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    同族专利:
    公开号 | 公开日
    WO2016193576A1|2016-12-08|
    EP3303600A1|2018-04-11|
    FR3036709B1|2019-06-07|
    US10837030B2|2020-11-17|
    CA2986784A1|2016-12-08|
    BR112017025727A2|2018-08-07|
    CN107771219A|2018-03-06|
    US20180142268A1|2018-05-24|
    AU2016272326B2|2020-06-04|
    AU2016272326A1|2017-12-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2009155633A1|2008-06-27|2009-12-30|Microbiogen Pty Ltd|Method of producing yeast biomass|
    WO2014072232A1|2012-11-07|2014-05-15|Dsm Ip Assets B.V.|pH CONTROLLED YEAST PROPAGATION|
    DE102008031350B4|2008-07-02|2011-02-10|Johann Wolfgang Goethe-Universität Frankfurt am Main|Prokaryotic xylose isomerase for the construction of xylose-fermenting yeasts|
    WO2010014817A2|2008-07-30|2010-02-04|Novozymes A/S|Producing fermentation products|
    FR2935986B1|2008-09-12|2012-11-23|Inst Francais Du Petrole|BETA-GLUCOSIDASE VARIANTS WITH IMPROVED ACTIVITY AND USES THEREOF|
    US8450094B1|2009-03-03|2013-05-28|Poet Research, Inc.|System for management of yeast to facilitate the production of ethanol|
    US9476068B2|2009-11-04|2016-10-25|Abengoa Bioenergy New Technologies, Llc|High efficiency process and high protein feed co-product|
    ES2665521T3|2010-04-14|2018-04-26|Lesaffre Et Compagnie|Industrial yeast, capable of producing ethanol from at least one pentose|
    FR2968313B1|2010-12-03|2014-10-10|Lesaffre & Cie|PROCESS FOR PREPARING INDUSTRIAL YEAST, INDUSTRIAL YEAST AND APPLICATION TO THE PRODUCTION OF ETHANOL FROM AT LEAST ONE PENTOSE|
    FR2991339B1|2012-06-01|2016-02-19|Lesaffre & Cie|YEAST STRAINS FOR METABOLIZING XYLOSE AND RESISTANT TO AT LEAST ONE FERMENTATION INHIBITOR, METHOD FOR OBTAINING THE SAME AND USE THEREOF|
    US9340767B2|2013-03-13|2016-05-17|Poet Research, Inc.|Propagating an organism and related methods and compositions|
    US9034631B2|2013-03-14|2015-05-19|Poet Research, Inc.|Systems and methods for yeast propagation|ES2713749T3|2016-02-22|2019-05-23|Versalis Spa|Process to propagate a yeast capable of fermenting glucose and xylose|
    PL3208340T3|2016-02-22|2020-07-13|Versalis S.P.A.|Process for propagating a yeast capable to ferment glucose and xylose|
    US20190264246A1|2018-02-23|2019-08-29|Domtar Paper Company, Llc|Propagation of yeast using cellulose as a carbon source|
    FR3083126B1|2018-06-27|2020-06-26|IFP Energies Nouvelles|LIGNO-CELLULOSIC BIOMASS TREATMENT PROCESS|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1554902|2015-05-29|
    FR1554902A|FR3036709B1|2015-05-29|2015-05-29|PROPAGATION OF YEAS SIMULTANEOUS TO SACCHARIFICATION|FR1554902A| FR3036709B1|2015-05-29|2015-05-29|PROPAGATION OF YEAS SIMULTANEOUS TO SACCHARIFICATION|
    CA2986784A| CA2986784A1|2015-05-29|2016-05-26|Yeast propagation simultaneous with saccharification|
    BR112017025727-0A| BR112017025727A2|2015-05-29|2016-05-26|yeast spread at the same time as saccharification|
    EP16733149.5A| EP3303600A1|2015-05-29|2016-05-26|Yeast propagation simultaneous with saccharification|
    US15/577,880| US10837030B2|2015-05-29|2016-05-26|Yeast propagation simultaneous with saccharification|
    PCT/FR2016/051237| WO2016193576A1|2015-05-29|2016-05-26|Yeast propagation simultaneous with saccharification|
    AU2016272326A| AU2016272326B2|2015-05-29|2016-05-26|Yeast propagation simultaneous with saccharification|
    CN201680036255.0A| CN107771219B|2015-05-29|2016-05-26|Yeast propagation with simultaneous saccharification|
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