SIMULTANEOUS REPRODUCTION AND SACHARIFICATION OF YEAST

Description

[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.

[0002] Dwindling fossil fuel stocks have led the industry to seek alternative solutions, implementing renewable raw materials and less polluting processes whenever possible. These solutions include the production of bioethanol from plant biomass, biomass from plant waste, or even municipal waste. To be accepted, green versions of chemical compounds must be as efficient, or even more so, than existing versions, and their manufacturing processes must be economically competitive.

[0003] The applications are numerous: first-generation fuels, biodiesel or ethanol, derived from plant raw materials such as sugar cane, beet, wheat, corn or vegetable oil, second-generation fuels, biodiesel, bio-kerosene, cellulosic ethanol, derived from non-food plant biomass or crop residues, and other heavy or fine chemical products.

[0004] The raw material must be pretreated. Depending on its origin, the pretreatment is mechanical (grating, grinding, chopping, milling, pressing), thermal, or chemical. The raw pomace 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 using a solvent. Different stages of this process represent technological challenges and have been widely studied: pretreatment of the biomass to make it accessible to enzymes, definition of enzymatic mixtures for efficient hydrolysis of carbohydrate polymers or even alcoholic fermentation of the different sugars obtained (pentoses, hexoses).

[0005] The step of alcoholic propagation of the yeast, the subject of the present invention, is little described. As such, the present invention relates to a method of propagating yeasts, for use in the production of a fermentation product from lignocellulosic biomass, comprising the steps of: a) having a reactor b) bringing into contact in said reactor a population of yeasts capable of metabolizing pentoses and hexoses, at a rate of 0.2 to 2.0 g of dry yeast matter per kg of prepared complete medium, where the yeasts of the population are yeasts of a yeast strain chosen from the strains deposited at the CNCM under the following numbers: I-4624, I-4625, I-4626, I-4627 and I-4783, the raw marc resulting from the pretreatment of lignocellulosic biomass without hydrolysis or separation steps at a rate of dry matter (DM) content of between 8 and 15%, nutrients, cellulases, at a rate of 5 to 15 mg of protein per gram of DM, c) incubating the mixture at a temperature of between 25 and 38°C, preferably between 28 and 33°C, and in micro-aerobiosis, in which the saccharification of the raw marc and the growth of the yeasts are carried out simultaneously. Brief description of the figures:

[0006] ThereFigure 1 presents a process for the production of bioethanol comprising pretreatment, yeast propagation and alcoholic fermentation phases, detailing the different substrates that can be generated by the process, substrates that can be used for the yeast propagation and ethanol production stages. The sugar composition of the five substrates is as follows: (1) Cellulose (glucose polymer) and solubilized hemicellulose (monomers and oligomers of hexoses and pentoses). Solid substrate. (2) Hydrolyzed cellulose and hemicellulose (monomers and oligomers of hexoses and pentoses). Liquid substrate containing suspended solids. (3) Cellulose (glucose polymer). Solid substrate. (4) Solubilized hemicellulose (monomers and oligomers of hexoses and pentoses). Liquid substrate. (5) Hydrolyzed cellulose (glucose). Liquid substrate containing suspended solids. The Figure 2illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during propagation on lignocellulosic hydrolysate (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. Figure 4 illustrates the evolution of yeast concentrations (in cells / mL), substrates (sugars C5 and C6) and ethanol (in g / kg) during SSP propagation (simultaneous propagation and saccharification) according to the invention, at 10% DM and 10 mg enzymatic proteins / g DM. Figure 5 illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during SSP propagation according to the invention, at 12% DM and 10 mg enzymatic proteins / g DM. The Figure 6illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during SSP propagation according to the invention, at 10% DM and 7 mg enzymatic proteins / g DM. The Figure 7 illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during SSP propagation according to the invention, at 32°C. The Figure 8 compares the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during SSP propagation according to the invention, at 30 and 32°C. Figure 9 illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during SSP propagation according to the invention, at 10% DM and 7 mg enzymatic proteins / g DM, with and without the addition of acetate. Figure 10illustrates the evolution of yeast concentrations (in cells / mL), substrates (C5 and C6 sugars) and ethanol (in g / kg) during SSP propagation according to the invention, at 10% DM and 10 mg enzymatic proteins / g DM, with and without the addition of acetate. Figure 11 compares the evolution of yeast concentrations (in cells / mL) during propagation on lignocellulosic hydrolysate (SHF) and SSP propagation at 10% DM and 7 mg enzymatic proteins / g DM with and without the addition of acetate. Figure 12 compares the evolution of yeast concentrations (in cells / mL) during propagation on C5 juice and SSP propagation at 10% DM and 7 mg enzymatic proteins / g DM with and without the addition of acetate. Figure 13 illustrates the concentrations of sugars (C5 and C6) and ethanol (in g / kg) during SSCF fermentation on wheat straw with the addition of acetate (qsp 4 g / kg) inoculated with the yeast I-4783 propagated in SSP.

[0007] According to the Figure 1 , pretreated plant biomass can be used as a substrate in yeast propagation and / or alcoholic fermentation in five different forms: The most common solution consists of hydrolyzing the whole marc in the presence of cellulases, in order to obtain a liquid lignocellulosic hydrolysate (2) comprising a mixture of hexose (C6) and pentose (C5) monomers, as well as small quantities of oligomers. Another common substrate is the hemicellulosic hydrolysate also called "C5 juice" (4), which corresponds to the soluble fraction of the substrate at the end of the pretreatment. This extraction of the hemicellulosic hydrolysate (4) at the end of the pretreatment results in cellulose-rich cellulosic marc (3) which can be hydrolyzed using cellulases in the same way as the whole marc. In this case, the cellulosic hydrolysate obtained contains mainly glucose monomers (5). This cellulose-rich cellulose marc (3), which is in solid form, can also be used without prior hydrolysis.In this case, cellulases are added during fermentation and cellulose hydrolysis and glucose consumption are carried out simultaneously. Finally, the most advanced process integration is to use raw pretreated pomace (1) as a fermentation substrate without an intermediate hydrolysis or separation step. In this case, cellulases are added during fermentation and cellulose hydrolysis is carried out simultaneously with the consumption of xylose and hexoses. This last option for the alcoholic fermentation step is known as Simultaneous Saccharification and Co-Fermentation (SSCF).

[0008] The propagation of yeasts that will be used for the fermentation stage involves different issues, particularly in terms of oxygen transfer.

[0009] It is classically carried out on C5 juice (4). Thus, patent application US2014 / 0065700 mentions propagation carried out on C5 juice and application WO2009 / 155633 indicates the prior step of hydrolysis of cellulose as essential.

[0010] Propagation is little discussed in the scientific literature, and 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 hydrolysate obtained by prior hydrolysis of the entire pretreated plant. After propagation, 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., 1992; Biotechnol Bioeng, 40 (3): 353-8; Holder et al., 1989; Biological Wastes, 28 (4): 239-46; Gonzalez-Valdes & Moo-Young, 1981; Biotechnology Letters, 3 (3): 143-8). Bellissimi & Richards (Bellissimi E, Richards C: Yeast propagation. In The alcohol textbook, a reference for the beverage, fuel and industrial alcohol industries, 5th edition, Edited by Ingledew WM, Kelsall DR, Austin GD, Kluhspies C.Nottingham: University Press; 2009:145-159) indicate that the method of production of industrial yeasts is aerobic propagation, in which there is no production of alcohol and a maximum rate of . cells is reached. Indeed, for yeast, the capacity to grow for a long period and in strictly anaerobic conditions is limited.

[0011] One of the disadvantages of raw pre-treated pomace before liquefaction or hydrolysis is its viscosity. For this reason, the liquefaction / solubilization or hydrolysis step is assumed to be essential. No document describes or suggests a process for simultaneous saccharification and propagation.

[0012] Patent application WO2011 / 56991 A1 describes a process for simultaneous saccharification and fermentation, with optionally aerated propagation, in parallel, in the liquefied medium rich in hexoses which will be used for fermentation. Patent application WO 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 process for aerobic propagation in a lignocellulosic hydrolysate (used as a carbon source), in which the hydrolysate is added in "fed-batch" mode so as to obtain and maintain a given pH in the reactor. Patent application US2014 / 0273167 A1 describes an aerobic process for propagating yeasts, with stirring and aeration, on a substrate rich in hexoses resulting from hydrolysis.Patent application US2014 / 0273166 A1 describes a method for propagating yeasts on a substrate derived from the transformation of plant biomass, a substrate preferably rich in pentoses. The yeasts subjected to propagation, in this case, are transformed yeasts, capable of metabolizing pentoses.

[0013] The C5 juice propagation process requires a complex step of extracting the liquid fraction from the pretreated pomace. The C6-rich hydrolyzate propagation process (or one comprising a C5-C6 mixture) also requires a specific hydrolysis step. Such a step is costly and time-consuming. Thus, there remains a desirable improvement of the process towards a more integrated version, maintaining satisfactory performance in terms of yield, productivity, and multiplication rate.

[0014] The present invention proposes a process for simultaneous saccharification and propagation. Contrary to the prior art on the essential nature of a hydrolysis or extraction step prior to obtaining a substrate suitable for yeast propagation, the Applicant proposes a process using raw pretreated marc as a yeast propagation substrate, without prior hydrolysis or separation steps. Said process combines the saccharification of the raw marc by cellulases and the growth of yeasts from the available C5 sugar(s) and the C6 sugar(s) released by enzymatic hydrolysis.

[0015] The simultaneous saccharification and propagation process according to the invention will subsequently be abbreviated to SSP (according to its Anglo-Saxon name Simultaneous Saccharification and Propagation).

[0016] An advantage of the propagation according to the invention is the reduction of time and cost thanks to the integration of the process by removing a step.

[0017] Another advantage of the invention is the limitation in fermentable sugars due to the enzymatic hydrolysis carried out simultaneously which makes it possible to achieve a high biomass production yield without imposing a fed-batch protocol (also called fed discontinuous fermentation).

[0018] Another advantage of the invention is that the continuously low glucose level promotes the consumption of xylose, usually inhibited in the presence of glucose.

[0019] Another advantage of the invention is that the high final biomass content (around 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 the inoculation which only represents 3% of the SSCF fermentation volume.

[0020] Another advantage of the invention is that the impact of inhibitors present in the pretreated marc on growth performance is significantly reduced.

[0021] Finally, another advantage of the invention is that the consumption of glucose as it is released by enzymatic hydrolysis limits the risks of contamination. Detailed description of the invention

[0022] The simultaneous saccharification and propagation process according to the invention is applied to a pretreated biomass. Said biomass is a lignocellulosic material, in other words a material that contains lignocellulose. The lignocellulosic material may contain other constituents such as cellulosic material (cellulose, hemicellulose), as well as sugars, fermentable or not, and pectins. Generally speaking, the lignocellulosic material is derived from plant material: stems, leaves, hulls, plant envelopes, leaves, branches or tree wood. The lignocellulosic material may also be derived from herbaceous material, agricultural residues, forest residues, municipal solid waste or paper mill effluents.

[0023] The biomass used in the process can come from miscanthus, poplar, or wheat straw.

[0024] Lignocellulosic material must be pretreated to break down the lignin and cellulose crystalline structure. This facilitates the solubilization of hemicellulose and cellulose and their accessibility for enzymes likely to be used in biomass processing. Any means of pretreatment, in particular impregnation followed by pretreatment, known to those skilled in the art, may be suitable. Schematically, pretreatment may be chemical, mechanical or biological. Chemical pretreatment includes treatment with an acid-base catalytic agent, in particular sulfuric acid, or with organic solvents, sulfur dioxide or carbon dioxide. Oxidation in a liquid medium and hydrothermolysis at controlled pH are also considered chemical treatments. Mechanical pretreatment corresponds to any mechanical or physical treatment such as grinding, irradiation, high-pressure or high-temperature explosion (steam explosion).According to some embodiments, the chemical and mechanical treatments may be combined, sequentially or simultaneously.

[0025] 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, preferably sulfuric acid, in proportions of between 0.1 and 2.0% by weight, preferably 0.5%. Advantageously, said impregnation is carried out at a temperature of between approximately 50°C and approximately 80°C, in particular of between approximately 60°C and 70°C, preferably at approximately 65°C. injection of water vapor at a temperature of between approximately 120°C and approximately 250°C, in particular of between approximately 170°C and approximately 190°C, preferably at approximately 180°C, at a pressure of between approximately 5 and approximately 15 bars, in particular of between approximately 8 and approximately 10 bars, preferably at approximately 9 bars, and for a time of between 1 and 10 min, preferably 5 min.

[0026] The pretreated plant biomass can then be used as a substrate for yeast propagation and / or in alcoholic fermentation, as indicated in Figure 1 described above.

[0027] 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 pentose-rich, hexose-rich, or a mixture of pentoses and hexoses. Pentoses are sugars with 5 carbon atoms, also called C5 sugars or simply C5. The main natural monomeric representatives of pentoses are D-xylose and L-arabinose. By analogy, hexoses are sugars with 6 carbon atoms, also called C6 sugars or simply C6. The main representatives of hexoses in monomeric form are glucose, fructose, mannose, and galactose.

[0028] The SSP propagation (simultaneous saccharification and propagation) according to the present invention is aimed at yeasts, capable of transforming both one or more pentoses and one or more hexoses.

[0029] The term "yeast strain" refers to a homogeneous population of yeast cells. 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.

[0030] The term "derived yeast strain" means a yeast strain derived by one or more crosses and / or by mutation and / or by genetic transformation.

[0031] A yeast strain derived by crossbreeding can be obtained by interspecific crossbreeding or not. A yeast strain derived by mutation can 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 can affect the phenotype or not. The expression "mutagenesis" refers to the process of occurrence of a mutation. Classically, two methods are possible, random mutagenesis and insertional or site-directed mutagenesis. The first consists of the application of a physical treatment (for example UV radiation) or treatment with mutagenic chemical agents, 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 ( iepromoter, gene, terminator, etc.), either in any region of the genome or at a specific locus. A locus is 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 a DNA sequence has been introduced, preferably delivered by a plasmid or integrated directly into the genome.

[0032] 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 likened to a period of adaptation; it is followed by the "growth phase" during which the cells multiply according to the maximum growth rate 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 added to the medium, 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 industrial settings due to their cost. The faster and shorter the growth phase, the more microbial contamination can be avoided. Excessive contamination during propagation will lead to yield losses during the subsequent fermentation stage. To limit contamination, antimicrobials and antibiotics such as penicillin or virginiamycin, or acidic hop extracts, can be used.

[0033] The propagation by SSP according to the invention must be carried out in microaerobiosis. This means that the medium is aerated but the quantity of oxygen supplied is limiting. The dissolved oxygen pressure is zero, unlike aerobiosis. The dissolved oxygen in the fermentation medium is measured using an oxygen probe according to a method known to those skilled in the art. Microaerobiosis in the process according to the invention is obtained by moderate aeration and agitation. Preferably, the aeration is 0.1 VVM (volume of air / volume of medium / minute, or 60 mL for a reactor containing 600 mL of medium per minute) and the agitation 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 equipment, the volume of said equipment, and the acceptable energy expenditure. The higher the work volume, the lower the agitation.

[0034] The biomass obtained can then be used in a fermentation process. The fermentation is preferably carried out at 32°C, with moderate stirring, for example 90 rpm. The stirring 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. This may be a direct measurement of the ethanol produced or an indirect measurement. viaa parameter correlated with ethanol production, such as mass loss. For example, ethanol production can be measured by chromatography, in particular by HPLC (High Performance Liquid Chromatography), by an enzymatic kit, or by a potassium dichromate assay. The quantity of xylose and / or glucose present in the medium is measured by any suitable means known to those skilled in the art, preferably by chromatography, in particular by HPLC.

[0035] The person skilled in the art knows how to determine the appropriate conditions for alcoholic fermentation.

[0036] As an example, one can refer to the alcoholic fermentation conditions described in the reference book "Yeast Technology", 2nd edition, 1991, G. Reed and TW Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8.

[0037] The fermentation medium comprises the following elements: at least one source of fermentable carbon, at least one source of nitrogen, at least one source of sulfur, at least one source of phosphorus, at least one source of vitamins and / or at least one source of minerals.

[0038] The carbon source is, for example, provided in the form of a sugar immediately assimilated by the yeast, such as xylose, arabinose, glucose, fructose or galactose, a sucrose-type disaccharide and / or a mixture of these sugars.

[0039] These sugars can be provided in the form of syrup, molasses, EP2 (Poor Sewage from the 2nd crystallization of sugar), hydrolysates of all or part of a plant material and / or a mixture of these.

[0040] The nitrogen source is provided, for example, in the form of yeast extracts, ammonium sulfate, ammonium hydroxide, diammonium phosphate, ammonia, urea and / or a combination of these.

[0041] The sulfur source is provided, for example, in the form of ammonium sulfate, magnesium sulfate, sulfuric acid and / or a combination of these.

[0042] The phosphorus source is provided, for example, in the form of phosphoric acid, potassium phosphate, diammonium phosphate, monoammonium phosphate, and / or a combination of these.

[0043] The vitamin source is provided, for example, in the form of corn steep liquor, molasses, yeast hydrolysate, pure vitamin solution or a mixture of pure vitamins and / or a combination of these. The vitamin source provides the yeast with all the vitamins in quantities at least equivalent to those recommended in reference works. Several vitamin sources can be combined.

[0044] The source of minerals is provided, for example, in the form of molasses, a mixture of mineral salts and / or their combination.

[0045] The mineral source provides the yeast with all the macroelements and trace elements in quantities at least equivalent to those recommended in reference works. Several mineral sources can be combined.

[0046] The same substance can provide several different elements.

[0047] The propagation according to the invention is characterized in that saccharification and propagation are carried out simultaneously.

[0048] The raw pre-treated marc is used at a dry matter content of between 8 and 15%, preferably between 10 and 12%, advantageously 10%. According to one embodiment of the invention, the marc comprises approximately 1 / 3 soluble dry matter (type "C5 juice") and 2 / 3 insoluble dry matter (type lignocellulosic fibers).

[0049] The raw pre-treated marc is brought into contact 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 dry yeast matter per kilogram of prepared complete medium.

[0050] The mixture of raw pretreated pomace and yeast is supplemented with a combination of cellulases and hemicellulases that allow saccharification. Saccharification corresponds to the hydrolysis of polysaccharides into 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. Exo-cellulases or cellobiohydrolases act at the ends of cellulose to form the disaccharide cellobiose. Endoglucanases act by cleaving the internal bonds of cellulose to form cellulose oligosaccharides. Cellobiases or beta-glucosidases hydrolyze cellulose oligopolymers and cellobiose at their reducing end, releasing glucose.

[0051] Concretely, the term cellulases refers to a mixture of enzymatic proteins. Preferably, the enzymes are used at a rate of 5 to 15 mg of proteins (enzymes) per gram of dry matter. Advantageously, they are used at a rate of 7 to 10 mg of proteins (enzymes) per gram of dry matter, preferably 7 mg of enzymatic proteins per gram of dry matter. To allow a correct understanding and comparison between the activities of different compositions exhibiting cellulase-type activity, the FPU (Filter Paper Unit) activity can be used as a reference. The biotechnology commission of the international organization IUPAC (International Union of Pure and Applied Chemistry) recommends the following procedure: the FPU activity is measured on Whatman No. 1 paper at an initial concentration of 50 gL -1< .The aim is to determine by colorimetric assay (with dinitrosalicylic acid, DNS) the quantity of reduced sugars from Whatman paper No. 1. As an example, the test portion of the enzymatic solution to be analyzed is determined, which releases the equivalent of 2 gL -1< of glucose in 60 minutes. The specific activities are obtained by dividing the activities expressed in IU.mL -1< by the protein concentration; they are expressed in IU.mg -1< .

[0052] Advantageously, the combination of cellulases and hemicellulases used in a method according to the invention corresponds to an enzymatic composition having one or more improved activity(ies) compared to a composition containing proteins produced by the native fungus. Such cellulases are known to those skilled in the art, for example described by Durand et al., 1988 (Enzyme Microb. Technol., 10: 341-346). 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.

[0053] 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.

[0054] Advantageously, the pH of the solution is around 5.0.

[0055] Incubation is maintained between 24 and 50 hours, particularly between 28 and 50 hours, more preferably between 30 and 42 hours.

[0056] Advantageously, the target cell concentration (at the end of propagation) is between 5.0 x 10 8< and 1.0 x 10 9< cells per milliliter.

[0057] Transformed yeasts capable of metabolizing both pentoses and hexoses can be obtained according to methods described in patent applications WO2010000464A1, WO2011128552A1 and WO2012072793A1. Advantageously, said strains are also resistant to acetic acid, obtained according to a method as described in application WO2013178915A1.

[0058] According to one embodiment of the invention, the yeast strain used preferentially metabolizes xylose and glucose. In other words, according to a particular embodiment, the invention relates to a method for fermenting sugars derived from lignocellulosic biomass, preferentially pentoses and hexoses, using a fermenting microorganism, characterized in that said microorganism was produced directly on raw pretreated marc, according to a method of simultaneous saccharification and propagation.

[0059] In the context of the invention, the yeast strain subjected to simultaneous propagation and saccharification according to the invention is 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 numbers I-4624, I-4625, I-4626, I-4627 or the strain deposited on June 26, 2013, under number I-4783.

[0060] According to a preferred embodiment of the invention, propagation by SSP precedes a fermentation step.

[0061] The present invention also relates to a method for producing at least one fermentation product comprising a fermentation step, under anaerobic or semi-aerobic conditions, by a yeast propagated on raw pretreated marc according to a method in which the saccharification and the propagation are carried out simultaneously.

[0062] The fermentation product is in particular chosen from ethanol, a metabolite obtained from ethanol or a secondary metabolite.

[0063] A preferred fermentation product according to the invention is ethanol.

[0064] The invention can be better understood in light of the following examples which are in no way limiting. Example 1: Pretreatment conditions and analysis of substrate composition

[0065] The substrate used for these tests is raw wheat straw marc resulting from a pretreatment according to the following method: the crushed straw is impregnated in acidic water between 0.1 and 2.0% by weight of H 2 SO 4 , then pretreated by continuous steam explosion at approximately 50% dry matter for 1 to 10 min between 170 and 190°C, preferably at 180°C.

[0066] This raw straw marc was analyzed by high performance liquid chromatography (HPLC) and the sugar and inhibitor contents are indicated in Table 1. Table 1: Composition of the straw marc used for this study. Concentrations in g / kg. Raw marc concentrations in g / kg MS content (%) 46,02% Sugars Cellulose 170 Cellobiose 4 Glucose 9,7 Xylose 93,8 Galactose NQ Arabinose 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

[0067] NQ means unquantified, in other words it has not been measured.

[0068] Analysis of the substrates showed typical acetic acid and furfural contents. The dry matter content of the raw straw pomace was 46.0%. In most subsequent propagation tests, this was used at 10% dry matter, which corresponds to a dilution of a factor of 4.6 of the concentrations given above when implemented in the reactor, while the other two substrates shown in the table, namely the hydrolysate and the C5 juice, were used without further dilution.

[0069] The following experiments were performed with yeast Saccharomyces cerevisiaedeposited at the CNCM (National Collection of Microorganism Cultures, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on June 26, 2013, under number I-4783. The processes implemented are described below. For comparison, another yeast strain capable of metabolizing pentoses and hexoses would give similar results.

[0070] The reference processes use liquid substrates, namely lignocellulosic hydrolysate, which requires a hydrolysis step of the pretreated marc prior to propagation, or C5 juice (also called hemicellulosic hydrolysate), which requires a step of separation of the soluble sugars from the marc at the end of pretreatment.

[0071] On the contrary, The Simultaneous Saccharification and Propagation (SSP) protocol according to the invention uses the solid substrate of raw pretreated marc. Example 2: Reference processes 2.1. Protocols

[0072] Antibacterials and nutrients were added respectively to the C5 juice and to the lignocellulosic hydrolysate in quantities adapted to the yeast deposited at the CNCM under number I-4783 used in these tests, namely: NH 4 OH 25% Urea H 3 PO 4 85% Mineral Mixture Antibacterial

[0073] The composition of the mineral mixture is presented in Table 2. Those skilled in the art will know how to adapt the proportions for optimal effectiveness. Table 2: Composition of the mineral mixture Compound MgSO 4 .7H 2 O CuSO 4 .2H 2 O MnCl 2 .4H 2 O ZnSO 4 .7H 2 O

[0074] The reactors were inoculated with 0.4 g / kg of dry yeast and maintained at a temperature of 30°C.

[0075] The pH was maintained at 5.0 by the addition of KOH and H 2 SO 4 .

[0076] For microaerobic conditions, the air flow rate was set at 0.1 VVM (air volume / medium volume / minute) and the agitation was set at 500 rpm.

[0077] As a guide, an air flow rate at 0.1 VVM is 60 mL / min for a reactor containing 600 mL of medium. 2.2. Results 2.2.1. Propagation on lignocellulosic hydrolysate (SHF)

[0078] The kinetics of yeast growth, substrate consumption and ethanol production during yeast propagation under microaerobic conditions (0.1 VVM) on lignocellulosic hydrolysate are presented in Figure 2 .

[0079] This propagation test lasted 46.1 h, but the Figure 2shows that yeast growth was complete after approximately 27 h of culture. The final biomass content produced is estimated at 3.9.10 8< cells / mL. Glucose was consumed preferentially over xylose, as is commonly the case in excess glucose. 18 g / kg of ethanol was produced. Note: Yeast viability during propagation is not shown in the figure for clarity. After the first few hours of culture, it is greater than 95% for all the tests presented in these examples. 2.2.2. Propagation on C5 juice

[0080] The kinetics of yeast growth, substrate consumption and ethanol production during yeast propagation under micro-aerobic conditions (0.1 VVM) on C5 juice are presented on the Figure 3 .

[0081] This propagation test on C5 juice shows a longer lag phase (than that of the test on lignocellulosic hydrolysate), 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.

[0082] The difference in final biomass content has already been observed. Generally speaking, yeast production yields are higher when propagated on C5 juice than when propagated on lignocellulosic hydrolysate, which is mainly composed of C6 sugar (glucose). Example 3: Simultaneous saccharification and propagation (SSP) 3.1. SSP method according to the invention

[0083] The Simultaneous Saccharification and Propagation process is also carried out in a reactor comprising: Water, pretreated raw cellulosic marc (solid substrate) used at a rate of 10% or 12% dry matter (DM) respectively in most tests, and nutrients as indicated abovefor reference processes.

[0084] Propagation was initiated by the simultaneous addition of cellulases (at 7 and 10 mg of protein per gram of dry matter, respectively) and dry yeast at 0.4 g / kg. The enzymes used in the present examples can be replaced by commercial enzymes in equivalent quantities. For comparison, the FPase activity (see above the mention of the units (Filter Paper Unit) specific to 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 IU.mg -1< ).

[0085] The temperature was maintained at 30°C or 32°C respectively.

[0086] The pH was maintained at 5.0 by the addition of KOH and H 2 SO 4 .

[0087] For microaerobic conditions, the air flow rate was set at 0.1 VVM (air volume / medium volume / minute) and the agitation was set at 500 rpm.

[0088] Samples were taken during the various propagation trials to count yeasts and quantify sugars and fermentation products by High Performance Liquid Chromatography (HPLC).

[0089] Total enzymatic hydrolyses were performed on the final samples to determine the unhydrolyzed cellulose content at the end of propagation. 3.2. Dry matter (DM) content-enzyme dose pairs

[0090] Different MS-enzyme dose pairs were tested in SSP. The prerequisites were: (i) a concentration of fermentable sugars that must allow reaching a biomass content of around 15 g / kg at the end of propagation; (ii) cellulose hydrolysis kinetics that must limit the amount of glucose, so that (1) the carbon flow is directed towards biomass production rather than ethanol production, which could take place, even in the presence of oxygen, if the sugar concentration is too high, and (2) the use of xylose is favored without penalizing productivity; and (3) the viscosity of the mixture that must allow moderate agitation and micro-aeration of the medium.

[0091] The MS contents and enzyme doses tested are shown in Table 3. Table 3: MS contents and doses of enzymes tested in SSP Essay MS content % Enzyme dose mg prot. / g DM 1 10 10 2 12 10 3 10 7

[0092] Reminder: as for the reference processes (supra), These propagation tests were inoculated with 0.4 g / kg of dry yeast, then conducted at pH 5.0, at 30°C, with moderate agitation at 500 rpm and micro-aeration of 0.1 VVM. 3.2.1. Propagation in SSP at 10% DM and 10 mg of enzymatic proteins / g DM

[0093] The yeast I-4783 was propagated under micro-aerobic conditions (0.1 VVM) on raw straw marc (at 10% DM), in the presence of cellulases (10 mg protein / g DM) which allow simultaneous hydrolysis of 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 the Figure 4 .

[0094] Result: This propagation test lasted 41 h at the end of which 5.6.10 8< cells / mL were obtained. 11 g / kg of ethanol were produced, then partially consumed during this propagation in SSP on raw straw marc. Furthermore, the DM content used allowed the addition of the entire substrate to the initial starter while maintaining a low viscosity allowing moderate agitation and micro-aeration of the culture medium. The experiment was repeated by extending the growth beyond 41 h. 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 those obtained previously under the same operating conditions. 3.2.2. Propagation in SSP at 12% DM and 10 mg of enzyme proteins / g DM

[0095] The yeast I-4783 was propagated under micro-aerobic conditions (0.1 VVM) on raw straw marc (at 12% DM), in the presence of cellulases (10 mg protein / g DM) which allow simultaneous hydrolysis of 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 the Figure 5 .

[0096] Result: This propagation test lasted 46.3 h at the end of which 5.8.10 8< cells / mL were obtained. 15 g / kg of ethanol were produced, then partially consumed during this propagation in SSP on raw straw marc.

[0097] Increasing the MS content from 10% to 12% did not cause a significant increase in viscosity that would disrupt moderate agitation and micro-aeration of the culture medium.

[0098] 3.2.3. Propagation in SSP at 10% DM and 7 mg of enzymatic proteins / g of DM The yeast I-4783 was subjected to propagation in micro-aerobic conditions (0.1 VVM) on raw straw marc (at 10% DM), in the presence of cellulases (7 mg enzymatic proteins / g of DM) which allow simultaneous hydrolysis of cellulose. The kinetics of yeast growth and ethanol production, as well as the evolution of glucose and xylose concentrations during yeast propagation are represented on the Figure 6 .

[0099] Result: This propagation test lasted 43.9 h at the end of which 8.3.10 8< cells / mL were obtained. 9.3 g / kg of ethanol were produced, then completely consumed during this propagation in SSP on raw straw marc. Discussion

[0100] Comparison of the performances obtained for the SSP tests carried out at different MS contents and enzyme doses shows that: Increasing the DM content from 10 to 12% for trials conducted with 10 mg protein / kg DM leads to an increase in ethanol production but has no positive impact on yeast growth. Decreasing the enzyme dose from 10 to 7 mg enzyme protein / g DM for trials conducted at 10% DM leads to a stronger glucose limitation which results in a faster consumption of xylose and a carbon flow oriented more towards biomass production. The difference in hydrolysis yield due to the decrease in the enzyme dose is less than 2% at the end of propagation. 3.3. Effect of temperature

[0101] In order to observe the effect of temperature on the propagation efficiency in SSP, the yeast I-4783 was propagated at 32°C under micro-aerobic conditions (0.1 VVM) on raw straw marc (at 10% DM) in the presence of cellulases (at 10 mg protein / g DM). The kinetics of yeast growth and ethanol production, as well as the evolution of glucose and xylose concentrations during this propagation test are represented on the Figure 7 .

[0102] Result: This propagation trial lasted 42.6 hours. A slowdown in growth was observed at the end of the culture. 12 g / kg of ethanol was produced, then partially consumed. The final biomass was estimated at 5.7.10 8< cells / mL.

[0103] There Figure 8compares 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 protein / g DM.

[0104] No positive effect is observed on yeast growth.

[0105] It appears that the increase in temperature can promote the uptake of xylose, which results in faster ethanol production kinetics.

[0106] Increasing the temperature improves enzymatic hydrolysis and increases the amount of fermentable sugars (by 12% in this case). If the propagation must is transferred in its entirety to inoculate 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 high acetate concentration

[0107] To evaluate the robustness of the SSP process, tests were performed with the addition of acetate to the medium (QSP (quantity sufficient for) 3 g / kg) to simulate higher toxicity of the pretreated pomace. 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 in 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 pomace from 3.6 g / kg to 13.8 g / kg.

[0108] This latter concentration leaves significant room for increasing the volatile compound content of pretreated substrates when moving to industrial scale.

[0109] 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 acetate at 10% DM and 7 mg protein / g DM are illustrated in the Figure 9 .

[0110] Comparison of SSP kinetics performed respectively in the presence of 0.7 g / kg or 3.0 g / kg of acetic acid shows that increasing acetate concentration slows down xylose utilization and causes a visible growth delay 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.

[0111] The yeast growth kinetics, as well as the evolution of xylose and ethanol concentrations during propagation tests carried out with and without the addition of acetate at 10% DM and 10 mg protein / g DM are illustrated in the Figure 10 .

[0112] The comparison of the SSP kinetics 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 acetate concentration causes a visible growth delay up to 30 h of culture and that lecarbon flux is slightly diverted towards ethanol production. At the end of propagation, the biomass content produced in the presence of 3 g / kg of acetate exceeds the reference: 6.9.10 8< cells / mL were obtained in 46.7 h compared to 6.5.10 8< cells / mL in 48.1 h for the test without the addition of acetate. These results show that the significant increase in the amount of acetic acid in the pretreated straw marc does not significantly degrade the performance of the SSP propagation process. Such robustness cannot be expected from the yeast propagation process on C5 juice, as xylose fermentation is affected much more strongly than glucose fermentation by the toxicity of the culture medium.

[0113] Again, two enzyme / substrate ratios were tested. The results are consistent with those obtained previously ( above ), namely that reducing the enzyme dose improves the performance of the SSP process. Example 4: Comparison of the growth performances of the SSP according to the invention with those obtained with the reference methods

[0114] The evolution of the yeast population is compared to that obtained for the reference processes, under identical conditions of temperature, pH, micro-aeration, moderate agitation and seeding rates. The quantities of fermentable sugars are of the same order of magnitude. 4.1. Comparison with propagation on lignocellulosic hydrolysate (SHF)

[0115] The evolution of the yeast population during propagation on lignocellulosic hydrolysate and SSP tests, with and without the addition of acetate, at 10% DM and 7 mg protein / g DM is shown in Figure 11 . Result and discussion

[0116] The respective substrates of the propagation test on lignocellulosic hydrolysate and the SSP propagation test without acetate addition are identical, with one difference: one has been previously hydrolyzed. Growth is slower on the hydrolysate, and this from the beginning of propagation, which can be explained by a slightly higher acetate content due to the more extensive hydrolysis of the substrate at the beginning of culture (1.0 g / kg vs. 0.7 g / kg in SSP); the osmotic pressure due to sugars is also higher. In addition to its better kinetics, propagation in SSP allows to obtain a much higher quantity of biomass (8.3.10 8< cells / mL vs. 3.9.10 8< cells / mL).

[0117] Furthermore, it is surprising to note that the SSP propagation test carried out in the presence of 3 g / kg of acetate is also better than the reference test. 4.2. Comparison with propagation on C5 juice

[0118] The evolution of the yeast population during propagation on C5 juice and SSP tests, with and without the addition of acetate, at 10% DM and 7 mg enzymatic proteins / g DM is shown in Figure 12 . Result and discussion

[0119] Propagation carried out on C5 juice has significantly slower growth kinetics than SSP propagation trials on raw pomace, however the increase in growth rate at the end of propagation allows it to reach a final biomass content equivalent to SSP propagations. However, if the propagation duration were reduced compared to the presented trial, the advantage of SSP propagation over propagation on C5 juice would increase.

[0120] The C5 juice used for this propagation test was derived from the same wheat straw cellulose pomace as that used for the SSP propagation tests. It was obtained by suspending the cellulose pomace in water, followed by solid / liquid separation. This process for obtaining C5 juice actually extracts all the soluble elements from the pretreated pomace; it can therefore be considered and it has been verified that the inhibitor content is proportional to the xylose concentration, which makes C5 juice the substrate with the highest concentration of inhibitors. Furthermore, the fact that xylose uptake is more affected by the toxicity of the environment than glucose consumption and that the increase in the toxicity of the pretreated pomace leads to a more rapid increase in the inhibitor content in the C5 juice (because it is proportional to the xylose content) means that the advantage of the SSP process will increase with the toxicity of the pretreated pomace.Thus, the increase in acetate content from 0.7 g / kg to 3.0 g / kg in the SSP starter culture, which slightly degrades the growth kinetics in SSP, corresponds to an increase in acetate content from 1.6 g / kg to approximately 7 g / kg in the C5 juice, which strongly penalizes yeast growth. 4.3. Synthesis and comparison of propagation performances

[0121] Table 4 provides for each propagation test carried out: The sugar concentration used in the culture medium (total quantity and fermentable quantity for tests carried out in SSP), The final biomass concentration obtained (in cells / mL), The biomass production yield (in relation to the fermentable sugar content and in relation to the DM content), An estimate of the yeast production yield in g of yeast / g of fermentable sugars. Table 4: quantities of sugars used, biomass concentrations obtained and biomass production yields for the different propagation tests (cell means cells, mass ini. and mass fin. mean initial mass and final mass respectively). Terms Duration h Sugar potential g / kg Fermented sugars. g / kg Final biomass Cell / mL But se ini. 9 But it ends. 9 Yield Cell / g fermented sugars. Yield g yeast / g sugars Juice C5, 30°C 41 53,8 7,50E+08 600 607,4 1,41E+10 0,29 SHF, 10% MS, 30°C 46,1 65,6 3,90E+08 600 593,7 5,88E+09 0,12 10% MS, 10 mg prot / g MS, 30°C 41 64,5 53,3 5,60E+08 600 523,0 9,16E+09 0,19 10% MS, 10 mg prot / g MS, 30°C 48,1 64,5 53,3 6,50E+08 600 557,0 1,13E+10 0,24 10% MS, 10 mg prot / g MS, 32°C 42,6 64,5 59,9 5,70E+08 600 616,3 9,78E+09 0,20 10% DM, 7 mg protein / g DM, 30°C 43,9 64,5 52,25 8,30E+08 600 604,9 1,60E+10 0,33 12% MS, 10 mg prot / g MS, 30°C 46,3 77,3 66,0 5,80E+08 600 571,5 8,37E+09 0,17 10% MS, 7 mg prot / g MS, 30°C, 3g / kg acetate 42,5 64,5 52,25 8,00E+08 600 599,3 1,53E+10 0,32 10% MS, 10 mg prot / g MS, 30°C, 3g / kg acetate 46,7 64,5 58,7 6,90E+08 600 610,4 1,20E+10 0,25 Remarks :

[0122] The sugar potential is calculated by assuming a cellulose hydrolysis yield of 100%. The quantity of fermentable sugars is calculated by estimating the quantity of residual cellulose by total enzymatic hydrolysis on a sample taken at the end of the culture. To estimate the biomass production yield in g of yeast / g of fermentable sugars, the conversion is made by considering that 1 g of yeast contains 4.8.10 10< cells (measured at the end of propagation on C5 juice). The final mass measured is abnormally low for propagation tests carried out at 10% DM with 10 mg of proteins at 30°C, which penalizes them when calculating yields. Results and Discussion

[0123] Table 4 shows that the highest final biomass concentration was obtained with the SSP process implemented at 10% DM and 7 mg protein / g DM. The final biomass content obtained on the C5 juice is close to that obtained with the SSP process under the best conditions, while propagation on lignocellulosic hydrolysate results in a final biomass content significantly lower than all other tests.

[0124] The calculation of the yeast production yield equals 1.6.10 10< cells / g of fermentable sugars for the best performing test carried out in SSP with 10% DM and 7 mg of protein / g of DM at 30°C (and 1.5.10 10< cells / g of fermentable sugars for the test carried out with an increased concentration of acetic acid). The yield of propagation on C5 juice is slightly lower: 1.4.10 10< cells / g of fermentable sugars were obtained. The other SSP conditions tested show yeast production yields close to 1.0.10 10< cells / g of fermentable sugars, while the reference test carried out on lignocellulosic hydrolysate shows a yield of 5.9.10 9< cells / g of fermentable sugars, i.e. approximately 3 times less than the best performing SSP test.

[0125] 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 DM obtained at the end of alcoholic propagation on C5 juice. According to data known to those skilled in the art, the reference propagation on lignocellulosic hydrolyzate has a yield of the order of 0.12 g / g.

[0126] The best SSP condition allowed to obtain 0.33 g of yeast / g of fermentable sugars. The multiplication rate of the yeast in propagation is then greater than 40 (estimated final concentration 17.4 g / kg of yeast).

[0127] In terms of productivity, the SSP propagation process is also the most efficient, in fact: The average volumetric productivity is estimated at 0.38 g of yeast / kg of must / h for the SSP process, compared to 0.37 g / kg / h and 0.17 g / kg / h for propagations on C5 juice and lignocellulosic hydrolysate respectively. The average volumetric productivity over the first 30 hours of culture is estimated at 0.33 g of yeast / kg of must / h for the SSP process, compared to 0.26 g / kg / h for propagations on C5 juice and lignocellulosic hydrolysate. Example 5: Validation of the SSP propagation process: performance of propagated yeast in SSCF fermentation

[0128] The present invention is an essential intermediate link in a global industrial alcoholic fermentation process. The purpose of this example is to validate that the yeast obtained following propagation according to the invention is effective in alcoholic fermentation on lignocellulosic substrate.

[0129] SSP-propagated yeast I-4783 was used to inoculate a SSCF (Simultaneous Saccharification and CoFermentation) fermentation; both cultures were grown on raw straw pomace. SSCF was conducted at 24% DM with 10 mg protein / g DM, with acetate added to the medium (QSP 4 g / kg). The reactor was inoculated with 2.4.10 7< cells / mL (i.e. approximately 0.5 g / kg yeast) and the medium was maintained at pH 5.5 and 33°C for 142.5 h. The evolution of glucose, xylose and ethanol concentrations during this fermentation is illustrated in Figure 13 .

[0130] This SSCF fermentation exhibits kinetics consistent with what is usually obtained. The yeast consumes the glucose released by the enzymes very quickly so that the glucose concentration is zero from the first hours of fermentation. The released xylose is mostly consumed in 72 h; the ethanol production kinetics is then limited by enzymatic hydrolysis. The final ethanol content 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 SSCF carried out without the addition of acetate with the I-4783 yeast propagated on C5 juice. This result allows us to conclude that the propagation process according to the invention does not degrade the performance of the yeast produced compared to a propagation process on C5 juice used usually.

Claims

1. A method for propagating yeast, for use in the production of a fermentation product from lignocellulosic biomass, comprising the steps of: a. providing a reactor, b. placing in contact in said reactor: - a population of yeasts capable of metabolising pentoses and hexoses, with 0.2 to 2.0 g of yeast dry matter per kg of prepared complete medium, where the yeasts of the population are yeasts of a yeast strain selected from among the strains deposited with the CNCM under the following numbers: I-4624, I-4625, 1-4626, I-4627 and 1-4783, - raw marc from the pre-treatment of the lignocellulosic biomass without any hydrolysis or separation steps, with a dry matter (DM) content between 8% and 15%, - nutrients, and - cellulases, with 5 to 15 mg protein per gram of DM, c. incubating the mixture at a temperature between 25°C and 38°C, preferably between 28°C and 33°C, in microaerobiosis, wherein the saccharification of the raw marc and the growth of the yeasts are carried out simultaneously.

2. The propagation method according to claim 1, wherein the pentoses are xylose and / or arabinose.

3. The propagation method according to claim 1 or claim 2, wherein the hexose is glucose.

4. The propagation method according to any one of claims 1 to 3, wherein the incubation in step c. is maintained until obtaining a cell concentration of between 5.0 x 108 and 1.0 x 109 cells per millilitre.

5. The 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 raw marc is used at a rate of 10% DM and the cellulases at a rate of 7 mg proteins per gramme of DM, the aeration rate is set at 0.1 air volume / medium volume / minute (VVM), the temperature is set at 30°C and the pH of the medium is set at pH 5.0.

6. A method for producing a fermentation product, from a lignocellulosic biomass, sequentially comprising the steps of: a. pre-treating the lignocellulosic biomass without any hydrolysis or separation steps to obtain a raw marc, b. placing in contact a fraction of the raw pre-treated marc, with 10% to 12% dry matter (DM), with (i) a population of yeasts capable of metabolising pentoses and hexoses, where the yeasts of the population are yeasts of a yeast strain selected from among the strains deposited with the CNCM under the following numbers: I-4624, 1-4625, 1-4626, 1-4627 and 1-4783, (ii) cellulases with 5 to 15 mg of protein per gram of DM, and (iii) optionally, nutrients, c. incubating the mixture at a temperature between 25°C and 38°C, preferably between 28°C and 33°C, in microaerobiosis, so as to obtain simultaneous saccharification and propagation, until obtaining a cell concentration between 5.0 x 108 and 1.0 x 109 cells per millilitre, d. transferring all or part of the propagated yeasts for contact with the fermentation wort comprising a source of pentoses and a source of hexoses, e. carrying out the fermentation, under anaerobic or semi-aerobic conditions, and f. obtaining the fermentation product.

7. The method according to claim 6, wherein the lignocellulosic biomass is a biomass of plant origin.

8. The method according to claim 6 or claim 7, wherein the obtained fermentation product is ethanol.

9. The method according to any one of claims 6 to 8, wherein the yeasts of the implemented population are yeasts of the strain deposited with the CNCM under the number 1-4783.