FR3015519A1 - PROCESS FOR PRODUCTION OF ETHANOL USING SUGARS OF 5 AND 6 CARBON ATOMS - Google Patents

Abstract

The present invention relates to a process for the production of ethanol using sugars with 5 and 6 carbon atoms.

Description

The present invention relates to a process for the production of ethanol using 5- and 6-carbon sugars. The use of fossil resources, especially oil since the advent of the industrial era in the early twentieth century, is accompanied by a continuous and increasing release of many elements in the atmosphere. The direct consequence of these discharges is in particular the increase of the carbon dioxide (CO2) content in the atmosphere. This rise in CO2 levels contributes to global warming, which has a clear and lasting impact on the environment. The share of oil used in the means of transport is in majority compared to the other uses, that is why many actors of the economic world sought to substitute a part, or even all, of the oil and its derivatives in the fuels intended for the vehicles. Ethanol produced by fermentation of sugars is already a molecule widely used as fuel in many countries and especially in Brazil. The first-generation fuel ethanol produced today comes almost exclusively from the fermentation of sugars, glucose and sucrose, derived from the agricultural exploitation of the soil. Succulent plants such as sugar beet or sugar cane and starchy plants such as wheat or maize, the main sources of sugars, are plants grown mainly to feed the populations. The main consequence of the growth of managed generation ethanol is the competition between the fuel and food sectors as far as the destiny of the produced sugars is concerned and indirectly, a competition as for the use of the grounds.

To overcome this duality, emerged the idea of the use of plants or agricultural co-products not intended for human consumption for the production of biofuels. While first-generation ethanol comes from the fermentation of sucrose and glucose from starch, these "new" raw materials are characterized by high productivity per hectare and a high total sugar content. These sugars consist mainly of glucose derived from cellulose and sugars from hemicelluloses. Cellulose and hemicelluloses are therefore sources of sugars alternatives to starch and sucrose. This is called second generation ethanol.

However, whereas the strains used for 1st generation alcoholic fermentation can use glucose from cellulose, they are unable to produce ethanol from hemicelluloses mainly composed of xylose and arabinose, five-atom sugars. carbon. The proportion of sugars from hemicelluloses is variable from one plant to another but can represent up to 40% of the sugars present in the plant. So these are so many sugars not used for the production of ethanol. At the industrial level, the processes of producing ethanol using glucose or sucrose as a carbon source almost exclusively use Saccharomyces cerevisiae, the latter being unable to use naturally occurring sugars with five carbon atoms. The search for strains capable of transforming pentoses into ethanol began in the 1970s. Scientists mainly focus on xylose because it constitutes between 10 and 40% of the sugars present in lignocellulosic biomass, while arabinose is at most 3% of the lignocellulosic biomass. A strain capable of fermenting xylose and glucose would increase the overall yield of alcoholic fermentation by about 25%. There are microorganisms that naturally ferment five-carbon sugars into ethanol. Among these microorganisms there are bacteria that ferment C5 and yeasts that ferment C5.

Several species of thermophilic bacteria such as Thermoanaerobacter ethanolicus, Clostridium thermohydrosulfuricum and Clostridium thermocellum are capable of carrying out naturally alcoholic fermentation on xylose. A yield of 0.42 ethanolylose and a productivity of 0.5 g.L-1.11-1 was reported for Thermoanaerobacter ethanolicus on a complex medium at 4 g.l -1 of xylose.

Their use has the advantage of reducing the risk of contamination due to the high temperature used, but the performance is too low for an industrial application. Other bacteria such as Escherichia coli, Klebsiella oxytoca, Erwinia chrysanthemi can be used for the production of ethanol, but a major disadvantage of bacteria is the high risk of contamination of the medium during fermentation. Bacterial contaminations are common and difficult to control in non-sterile conditions and the implementation of sterile conditions is not always applicable in industrial settings. By using yeasts for fermentation, exogenous bacterial contaminations are more easily controllable. In fact, the high alcoholic strengths are unfavorable to bacteria and it is easier, thanks to the fermentation conditions, in particular the pH, to prevent the appearance of a bacterial contamination. The alternative to the use of bacteria for the production of ethanol at the industrial stage is therefore the use of fungal organisms: yeasts.

Several yeast species, naturally fermenting xylose, have been identified as the most effective: Pichia stipitis, Candida shehatae, Candida tropicalis and Pachylosen tannophilus. Table 1: Fermentative performance of three yeasts on xylose Today many strains metabolizing pentoses exist, but these laboratory yeasts are not adapted to industrial conditions, because they do not tolerate the presence of inhibitors present in lignocellulose hydrolysates . In addition, the construction of genetically modified yeast strains capable of effectively converting xylose and arabinose into ethanol is a difficult goal, as it involves modifying yeast metabolic fluxes and energy equilibria. In addition, when the metabolizing strains, in addition to the hexoses, the pentoses, ferment said hexoses and pentoses to give ethanol, said strains, in the presence of both types of sugars, always consume the C6 sugars first and then only the C5 sugars, whether natural or genetically modified. This mechanism known as diauxie results in a C5 / C6 co-fermentation with the presence of C5 at the end of fermentation, which, because of the presence of alcohol and of conditions prevails. 1 50 90 20 6,2 26 56 21.5 5.0 150 20 50 cocup4.2ce 14. rather unfavorable for the strain (long residence time in the presence of alcohol, possibility of presence of organic acids produced during the fermentation by a contaminating flora), will not be consumed by strain. This results in a loss of sugar in the wine going to the ethanol isolation stage, especially by distillation.

Thus, an object of the present invention is to provide a method for optimal use of sugars from cellulose and hemicelluloses from lignocellulosic biomasses or agro-industrial co-products to produce ethanol. The subject of the invention is therefore the use of a propagation must comprising a microbial population for preparing ethanol from a raw material generating at least one pentose, in particular xylose and / or arabinose. , and at least one hexose, in particular glucose, by fermentation of said pentose and hexose by said microbial population, said raw material being extracted to give an extraction juice containing said pentose, in particular xylose and / or arabinose, and a marc, said marc giving, after having been liquefied, a liquéfiat, said liquéfiat being a source of the aforesaid hexose, in particular glucose, said propagation must being obtained by proliferation of an inoculum of said microbial population to of said extraction juice, in the presence of air, said propagation wort containing no ethanol, said microbial population being used both: - for the preparation of a first source of ethanol, by fermentation of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice by said microbial population and - for the preparation of a second source of ethanol, by fermenting the aforesaid hexose, in particular glucose, generated by said liquefied by said microbial population, said ethanol being isolated from the first and / or second sources of ethanol, in particular by distillation of said first and / or second sources of ethanol. Thus, as illustrated by FIG. 2 relating to a use according to the present invention, the raw material, in particular a lignocellulosic biomass, is extracted after having been in particular impregnated and pretreated. Said extraction makes it possible to obtain a liquid rich in pentoses (a juice rich in C5 sugars) and a solid rich in hexoses (a marc rich in C6 sugars).

Said liquid allows the propagation of microorganisms, in particular yeasts: it is a step of proliferation of said microorganisms, mainly on the C5 sugars contained in said liquid. After propagation, said microorganisms ferment the pentoses of said liquid into alcohol, obtained in the form of wine (derived from C5 sugars) corresponding to a first source of ethanol. Said solid is subjected to liquefaction (to obtain a liquifate) and to a fermentation using the microorganisms obtained after propagation and / or those isolated after the fermentation of the pentoses, to obtain the second source of ethanol (final wine) .

The second source of ethanol, and possibly the first source of ethanol, is (are) processed, in particular distilled to give ethanol. Indeed, said first source of ethanol can be distilled with the second source of ethanol and / or used during liquefaction. The liquid (clarified vinasse) isolated by clarification of the distillation residue (raw vinasse) is optionally joined to said raw material during the extraction. Interestingly, the sugars with five carbon atoms that are notably xylose and arabinose are used for the production of yeast cells in the presence of air, then for the production of ethanol, thus preserving the easily fermentable sugars that are sugars with six carbon atoms and in particular glucose for the production of ethanol.

The separation of the C5 and C6 streams and their use in two distinct stages make it possible to maximize the effectiveness of the sugars for the production of ethanol, in particular by limiting the diauxis phenomena. In addition, with the use of a single strain of microorganism capable of both growth on C5, ferment C5 to ethanol and then ferment C6 ethanol, it is no longer necessary to have two fermental infrastructures (one for the fermentation of C5 using a first strain, the other for the fermentation of C6, with the aid of a second strain) and as a result, the cross contamination of two C5 ethanol and C6 ethanol production lines are non-existent. By "propagation must" is meant a composition comprising a proliferated microbial population and a culture medium appropriate to said microbial population. "Appropriate culture medium" means an aqueous solution containing nutrients, in particular a source of carbon, a source of nitrogen, a source of trace elements and a source of growth factors necessary for metabolism and proliferation. of said microbial population.

Said culture medium comprises said extraction juice containing pentose, in particular xylose and / or arabinose, as well as a residual glucose content of less than 10 g / l. The propagation wort thus comprises the microbial population having proliferated and the culture medium at the end of the growth step of said microbial population, that is to say an aqueous solution in which the nutrients not consumed are present. by said microbial population and metabolites possibly excreted by the strain, said solution comprising said extraction juice. By "microbial population" is meant a set of microorganisms of the same strain, the mass of said set being likely to change during its use. By "raw material generating at least one pentose and at least one hexose" is meant a raw material capable of providing, after transformation (s), said pentose and said hexose in a form permitting their assimilation by said microbial population. By "extraction" is meant the action of mixing said raw material with an extraction solvent, in particular water, said mixture being followed by a liquid / solid separation: this is called liquid / solid extraction By "extraction juice" is meant the liquid obtained at the end of said liquid / solid separation relative to said extraction. From 50 to 98% of the sugars with five carbon atoms that can be generated by said raw material are in said extraction juice.

Indeed, the majority of pentoses, resulting from the destructuration of hemicellulose chains, migrate into the extraction solvent, in particular water. Said extraction juice contains from 1 to 50% by weight of C5. By "marc" is meant the solid obtained at the end of said liquid / solid separation relative to said extraction. From 80 to 99% of the six-carbon sugars likely to be generated by said raw material are in said pomace, mainly in the form of cellulose present in said pomace. In fact, cellulose remains largely insoluble and is concentrated in the solid fraction that is marc. Said liquid-solid separation is for example carried out on packet press or on a belt press.

By "liquefaction" is meant an operation of first hydrolysis of the marc making it possible to reduce the viscosity of said marc with a value of between 40,000 and 25,000 Pa.s at a value of less than 100 Pa.s. (AR Instrumentation Rheometer) , 33 ° C, speed 1s1, geometry: 40 mm plane, 1 mm gap), desirable for the subsequent fermentation of hexose, in particular glucose, generated by said liquéfiat.

A viscosity less than or equal to this value allows a better availability of water for the microbial population, to avoid the formation of foam, and to limit the need for agitation. Said liquefaction is in particular carried out in the presence of lignocellulolytic enzymes, said enzymes allowing a first hydrolysis of said marc, said enzymes being more particularly ligno-cellulolytic enzymes contained in commercial products such as Celic Cetec 2 (Genencor®) or Celluclast (Novozymes®). By "inoculum" is meant a preculture medium comprising a microbial population and a culture medium appropriate to said microbial population, said preculture medium for inoculating a culture medium as described above to obtain said fermentation must. By "the wort of propagation does not contain ethanol" is meant that said wort of propagation contains less than about 1 g.1-1 ethanol. is meant a liquid or a solid-liquid suspension By "ethanol source" containing ethanol. By "first source of ethanol" is meant a source of ethanol obtained by fermentation of pentoses, that is to say sugars with 5 carbon atoms. By "second source of ethanol" is meant a source of ethanol obtained by fermentation of hexoses, that is to say sugars with 6 carbon atoms.

In particular, said first and / or second sources of ethanol are substantially free of microorganisms from said microbial population. According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, wherein said raw material is a lignocellulosic biomass, in particular chosen from: - cereal straw, said cereals being in particular selected from wheat, barley, triticale, sorghum and maize, - cereal bran, said cereals being selected in particular from wheat, barley, triticale, sorghum and maize, - crops so-called energetics, chosen in particular from switchgrass, miscanthus and arundo donax, and - forest crops, chosen in particular from wood, short rotation coppices, very short rotation coppices, and forest chips.

According to an advantageous embodiment, said raw material is, prior to said extraction, pretreated. Advantageously it is impregnated and then pretreated. Any means of pretreatment, in particular impregnation and pretreatment, known to those skilled in the art may be suitable, insofar as it can provide a liquid and a separable solid, the liquid containing most of the C5 and the solid the most of the C6. The impregnation consists in preparing said raw material with a view to its pretreatment by, for example, penetrating into its matrix, in particular lignocellulosic, an acidic or basic chemical catalytic agent, in particular an acidic catalyst, in particular sulfuric acid.

The pretreatment of said raw material consists, for example, of steam cooking or explosion in the presence of said acidic or basic catalyst, in particular the acid catalyst, in particular sulfuric acid, having previously impregnated said raw material. Said pretreatment allows in particular the hydrolysis of the hemicelluloses contained in said raw material as well as the preparation for the hydrolysis of the cellulose also contained in said raw material. The pentoses, resulting in particular from the destructuration of the hemicellulose chains of said raw material, are rendered soluble by the pretreatment, and are thus capable of migrating into the extraction solvent, in particular water, during said extraction.

According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, wherein said ethanol is isolated either from the first source of ethanol, or from the second source of ethanol, or first and second sources of ethanol, by distillation of said first and / or second sources of ethanol.

According to an advantageous embodiment, the invention relates to the use of a propagation must as described above, wherein said liquefaction of said grounds is carried out in the presence of all or part of said first source of ethanol. According to a particularly advantageous embodiment, the invention relates to the use of a propagation wort as described above, in which said liquefaction of said marc is carried out in the presence of all or part of said first source of ethanol, said first ethanol source being substantially free of microorganisms from said microbial population. Indeed, the yeasts can be separated from said first source of ethanol before recycling, because of their intolerance to the temperatures used in liquefaction.

The fact of devoting part of said first source of ethanol to liquefaction makes it possible in particular to reduce the total volume of the vat room required for the second fermentation. According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, wherein said distillation, in addition to said ethanol, generates a residue, which contains substantially no ethanol, all or part of the soluble elements, water and insolubles is added to said raw material during said extraction of said raw material. The recycling of all or part of said soluble elements during said extraction makes it possible in particular to minimize the amount of extraction solvent, in particular water, used during said extraction. Said residue comprises in particular the first and / or second sources of ethanol, exhausted in alcohol, non-sugars and the microbial population at the end of the second fermentation.

According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, in which: said liquefaction of said marc is carried out in the presence of all or part of said first source of ethanol; said distillation, in addition to said ethanol, generates a residue, which contains substantially no ethanol, all or part of the soluble elements, water and insolubles is added to said raw material during said extraction of said raw material. The flows described hereinafter have been numbered according to Scheme 3, having use according to the present invention. According to an advantageous embodiment, the invention relates to the use of a propagation wort (13) as described above, comprising a microbial population for preparing ethanol from a raw material generating at least one pentose , in particular xylose and / or arabinose, and at least one hexose, in particular glucose, by fermentation of said pentose and hexose by said microbial population, said raw material being extracted to give an extraction juice (9) containing of the aforesaid pentose, in particular xylose and / or arabinose, and a marc (10), said marc giving, after having been liquefied, a liquéfiat (23), said liquéfiat being a source of the aforesaid hexose, in particular glucose, said propagation must (13) being obtained by proliferation of an inoculum of said microbial population using said extraction juice (11), in the presence of air, said propagation wort containing no ethanol , said propagation must (13) is subjected to a solid / liquid separation to give a propagation juice (16) and a fresh microbial population (17), said fresh microbial population (17) is separated into two parts, to obtain a first part (17a) and a second portion (17b) of said fresh microbial population (17), said first portion of said fresh microbial population (17a) is used for the preparation of a fermentation broth (18) by fermentation of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12), by said first part of the fresh microbial population (17a), said fermentation wort (18) giving after liquid / solid separation said first source of ethanol (19) and all or part of the recyclable microbial population (20), said second source of ethanol (24) is obtained by fermentation of the aforesaid hexose, in particular glucose, generated by said liquéfiat (23), all or part of said recyclable microbial population (20) and said second portion of the fresh microbial population (17b). Said liquid / solid separation making it possible to obtain the first source of ethanol and a recyclable microbial population is such that said first source of ethanol is substantially free of microorganisms originating from said microbial population.

By "substantially free of microorganisms" is meant that the microorganism concentration of said first source of ethanol (19) is less than 10% of that of said fermentation must (18). According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, said wort of propagation containing no ethanol, wherein said microbial population comprises yeasts of Candida shehatae strain, Pichia stipitis, Pichia guilliermondii, Candia tropicalis or Pachysolen tannophilus, said microbial population including in particular yeasts of Candida shehatae strain. According to an advantageous embodiment, the invention relates to the use of a propagation wort (13) as described above, comprising a microbial population comprising yeasts of Candida shehatae, Pichia stipitis, Pichia guilliermondii, Candia tropicalis or Pachysolen strain. tannophilus, in particular Candida shehatae, for preparing ethanol from a lignocellulosic biomass generating at least one pentose, in particular xylose and / or arabinose, and at least one hexose, in particular glucose, by fermentation said pentose and hexose by said microbial population, said lignocellulosic biomass being extracted to give an extraction juice (9) containing the aforesaid pentose, in particular xylose and / or arabinose, and a pomace (10), said pomace. giving, after having been liquefied, a liquéfiat (23), said liquéfiat being a source of the aforesaid hexose, in particular glucose, said propagation must (13) being obtained p proliferation of an inoculum of said microbial population using said extraction juice (11), in the presence of air, said propagation wort containing no ethanol, said microbial population being used at the same time: for the preparation of a first source of ethanol (19), by fermentation of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12) by said microbial population and - for the preparation of a second source of ethanol (24), by fermentation of the aforesaid hexose, in particular glucose, generated by said liquéfiat by said microbial population, said ethanol being isolated from the first (19) and / or second (23) ) sources of ethanol by distillation of said first and / or second ethanol sources, said liquefaction of said pomace (10) being carried out in the presence of all or part of said first source of ethanol (19), said propagation mash (13). ) being subject to a solid / liquid paration to provide a propagation juice (16) and a fresh microbial population (17), said fresh microbial population (17) being split into two parts to obtain a first portion (17a) and a second portion (17b) of said fresh microbial population, said first part of said fresh microbial population (17a) being used for the preparation of a fermentation broth (18) by fermentation of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12), by said first portion of the fresh microbial population (17a), said fermentation wort (18) giving after said liquid / solid separation said first source of ethanol (19) and all or part of the recyclable microbial population (20), said second source of ethanol (24) is obtained by fermentation of the aforesaid hexose, in particular glucose, generated by said liquéfiat, by all or part of said recyclable microbial population (20) and said second portion of the fresh microbial population (17b). According to an advantageous embodiment, the invention relates to the use of a propagation wort (13) as described above, comprising a microbial population comprising yeasts of Candida shehatae, Pichia stipitis, Pichia guilliermondii, Candia tropicalis or Pachysolen strain. tannophilus, in particular Candida shehatae, for preparing ethanol from a lignocellulosic biomass generating at least one pentose, in particular xylose and / or arabinose, and at least one hexose, in particular glucose, by fermentation said pentose and hexose by said microbial population, said lignocellulosic biomass being extracted to give an extraction juice (9) containing the aforesaid pentose, in particular xylose and / or arabinose, and a pomace (10), said pomace. giving, after having been liquefied, a liquéfiat (23), said liquéfiat being a source of the aforesaid hexose, in particular glucose, said propagation must (13) being obtained p proliferation of an inoculum of said microbial population using said extraction juice (11), in the presence of air, said propagation wort containing no ethanol, said microbial population being used at the same time: for the preparation of a first source of ethanol (19), by fermentation of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12) by said microbial population and - for the preparation of a second source of ethanol (24), by fermentation of the aforesaid hexose, in particular glucose, generated by said liquéfiat by said microbial population, said ethanol being isolated from the first (19) and / or second (23) ) sources of ethanol by distillation of said first and / or second sources of ethanol, said distillation, in addition to said ethanol, generating a residue (26), which contains substantially no ethanol, all or part of the soluble elements, water and i nsolubles is added to said lignocellulosic biomass during said extraction of said lignocellulosic biomass, said liquefaction of said pomace (10) being carried out in the presence of all or part of said first source of ethanol (19), said propagation mash (13) being subjected to solid / liquid separation to provide a propagation juice (16) and a fresh microbial population (17), said fresh microbial population (17) being separated into two parts, to obtain a first portion (17a) and a second portion (17b) of said fresh microbial population, said first portion of said fresh microbial population (17a) being used for the preparation of a fermenting must (18) by fermentation of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12), by said first portion of the fresh microbial population (17a), said fermentation wort (18) giving after sep liquid / solid ation said first source of ethanol (19) and all or part of the recyclable microbial population (20), said second source of ethanol (24) is obtained by fermentation of the aforesaid hexose, in particular glucose, generated by said liquefied, all or part of said recyclable microbial population (20) and said second portion of the fresh microbial population (17b). Since said first source of ethanol is substantially free of microorganisms from said microbial population, said liquefaction of said pomace is carried out in the absence of microorganisms from said microbial population. According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, wherein said extraction juice containing said pentose, in particular xylose and / or arabinose, is detoxified before said proliferation and the preparation of said first source of ethanol. The detoxification treatment allows a reduction of the fermentation inhibitors that are in particular 5-HMF, furfural and especially the products of degradation of lignin. Said detoxification is in particular: - an alkaline treatment, more particularly with calcium hydroxide or with ammonia, - the addition of acetaldehyde, making it possible to reduce the lag phase and the yeast mortality rate, - a extraction of inhibitors on ion exchange resin or activated carbon, - biological and enzymatic treatments, - extractions with ether or ether acetate, or - a treatment by adding gas or steam in the product ("stripping").

According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, in which said raw material is subjected, after said impregnation and said pretreatment, to an additional hydrolysis treatment. . Said additional hydrolysis treatment follows the pretreatment allowing in particular the hydrolysis of the hemicelluloses contained in said raw material. Said additional hydrolysis treatment is in particular thermal or enzymatic, and allows the hydrolysis of C5 oligomers to monomeric sugars. According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, in which said liquefier is hydrolysed before said fermentation of the aforesaid hexose, in particular glucose, generated by the hydrolysis of said liquefier . Said hydrolysis of said liquéfiat thus constitutes a second hydrolysis, the first being the liquefaction, that is to say a first hydrolysis, said marc, to give said liquéfiat.

The microbial population is thus brought into contact with the aforesaid hexose, generated by the hydrolysis of said liquéfiat: this type of fermentation decoupled from hydrolysis is called: hydrolysis and separate fermentation (SHF: separate saccharification and fermentation). Said hydrolysis is in particular carried out in the presence of lignocellulolytic enzymes, more particularly ligno-cellulolytic enzymes contained in commercial products such as Celic Cetec 2 (Genencor®) or Celluclast (Novozymes®). In particular, said hydrolysis makes it possible at the same time to transform the cellulose of said liquéfiat into cellobiose, then into hexose, more particularly into glucose. According to an advantageous embodiment, the invention relates to the use of a propagation wort as described above, in which said liquefier is hydrolysed, said hydrolysis and said fermentation of the aforesaid hexose, in particular glucose, generated by the hydrolysis of said liquéfiat being concomitant. This type of fermentation coupled with hydrolysis is called Simultaneous Saccharification and Fermentation (SSF). Thus, said microbial population is brought into contact with the liquefier, during the hydrolysis of said liquefied liquor. According to an advantageous embodiment, liquefaction of said grounds (that is to say a first hydrolysis of said grounds) to obtain said liquéfiat and hydrolysis of said liquéfiat (that is to say a second hydrolysis) are carried out by the action of the same enzymes, in particular with cellulolytic action.

According to another advantageous embodiment, the liquefaction of said grounds (that is to say a first hydrolysis of said grounds) to obtain said liquéfiat and the hydrolysis of said liquéfiat (that is to say a second hydrolysis) are performed by the action of the same enzymes, in particular cellulolytic action, said microbial population being brought into contact with the liquefied, during the hydrolysis of said liquefied. The invention also relates to a process for preparing ethanol from a raw material generating at least one pentose, in particular xylose and / or arabinose, and at least one hexose, in particular glucose, comprising: ) a step of extracting said material, to obtain an extraction juice containing the aforesaid pentose, in particular xylose and / or arabinose, and a marc, b) a liquefaction step of said marc obtained at preceding step, to obtain a liquéfiat, said liquéfiat being a source of the aforesaid hexose, in particular glucose, c) a step of proliferation of an inoculum of a microbial population with the aid of said extraction juice containing the aforesaid pentose , in particular xylose and / or arabinose, in the presence of air, in order to obtain a propagation must comprising said microbial population, said propagation wort containing no ethanol, d) a first fermentation step of the aforesaid pentose, in particular of xylose and / or arabinose, contained in said extraction juice, by said microbial population, to obtain a first source of ethanol, e) a second fermentation step of the aforesaid hexose, in particular glucose, generated by said liquéfiat, by said microbial population, to obtain a second source of ethanol, f) a step of isolating said ethanol from the first and / or second sources of ethanol, in particular by distillation of said first and / or second sources of ethanol. ethanol.

During said extraction, the liquid-solid separation is for example carried out on a package press, in particular when the mass of raw material to be treated is of the order of about ten kg, or on a belt press, in particular when the mass of raw material to be treated is of the order of a hundred kg. The extraction provides an extraction juice whose composition varies according to the raw material treated and pretreatment. An example of composition is given in the example below. These modes of preparation and the juices obtained are widely described in the literature. The liquid-solid separation is preferably carried out on a belt press.

According to an advantageous embodiment, the temperature of the wort varies from about 20 ° C to about 40 ° C, in particular from about 28 to about 32 ° C. In another advantageous embodiment, the pH of the wort is controlled to vary from about 4 to about 7, in particular from about 5 to about 6.

According to another advantageous embodiment, the wort of propagation is such that the oxygen partial pressure in said wort is greater than or equal to about 20%, in particular greater than or equal to about 30%. By oxygen partial pressure is meant the oxygen concentration (O 2) dissolved in the liquid phase. This partial pressure is expressed in% of the saturating oxygen concentration in the working conditions under consideration, such as, without limitation, temperature, pressure, agitation. According to an advantageous embodiment, the temperature during said first fermentation varies from about 20 ° C to about 40 ° C, in particular from about 28 to about 32 ° C.

According to another advantageous embodiment, the pH during said first fermentation is regulated so as to vary from about 4 to about 7, in particular from about 5 to about 6. According to another advantageous embodiment, the partial pressure in oxygen at said first fermentation is about 0%.

This oxygen partial pressure of about 0% can in particular be obtained despite an aeration flow of about 0.2 VVM. According to an advantageous embodiment, the temperature during said liquefaction ranges from about 25 to about 75 ° C., in particular from about 40 to about 60 ° C., the temperature being more particularly about 50 ° C.

According to an advantageous embodiment, the temperature during said second fermentation ranges from about 20 ° C to about 40 ° C, in particular from about 28 to about 35 ° C. According to another advantageous embodiment, the pH during said second fermentation is regulated so as to vary from about 4 to about 7, in particular from about 5 to about 6. According to another advantageous embodiment, the partial pressure in oxygen during said second fermentation is about 0%. This oxygen partial pressure of about 0% can in particular be obtained despite a ventilation rate of about 0.4 VVM.

According to an advantageous embodiment, said liquefaction is carried out in the presence of lignocellulolytic enzymes, at a concentration varying from approximately 5 to approximately 500 mg / g of cellulose, in particular from approximately 10 to approximately 80 mg / g of cellulose, more particularly at about 40 mg / g of cellulose.

According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, wherein said raw material is, prior to said extraction, impregnated and pretreated. According to a particularly advantageous embodiment, said impregnation is carried out in the presence of an acidic or basic chemical catalytic agent, in particular an acidic catalyst, especially sulfuric acid, said catalytic agent impregnating said raw material. According to another particularly advantageous embodiment, said impregnation is carried out at a temperature of approximately 50 to approximately 80 ° C, in particular of approximately 60 to approximately 70 ° C, more particularly to approximately 65 ° C.

According to an advantageous embodiment, said pretreatment is carried out by injecting steam at a temperature of about 120 to about 250 ° C., in particular about 170 to about 190 ° C., more particularly to about 180 ° C. and at a pressure of about 5 to about 15 bar, particularly about 8 to about 10 bar, more preferably about 9 bar.

At the end of this pretreatment step, the lignocellulosic matrix impacted by said pretreatment is then reactive to the liquefaction enzymes. The combination of thermal, chemical and possibly mechanical treatment implies a strong increase in the digestibility of the cellulose. According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, in which the ethanol is isolated either from the first source of ethanol, or from the second source of ethanol, or from first and second sources of ethanol, by distillation of said first and / or second sources of ethanol. According to an advantageous embodiment, the invention relates to a process for the preparation of ethanol as described above, in which said step of liquefying said grounds is carried out in the presence of all or part of said first source of ethanol obtained from the from the first fermentation step of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice.

According to an advantageous embodiment, the invention relates to a process for the preparation of ethanol as described above, in which said step of liquefying said grounds is carried out in the presence of all or part of said first source of ethanol obtained from the from the first fermentation step of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice, said first source of ethanol being substantially free of microorganisms from said microbial population. Indeed, the yeasts can be separated from said first source of ethanol before recycling, because of their intolerance to the temperatures used in liquefaction.

According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, wherein said distillation generates, in addition to said ethanol, a residue, which contains substantially no ethanol, all or part of which soluble elements, water and insoluble elements, is added to said raw material during said extraction of said raw material.

According to an advantageous embodiment, the invention relates to a process for the preparation of ethanol as described above, in which: said liquefaction of said grounds is carried out in the presence of all or part of said first source of ethanol, said distillation , in addition to said ethanol, generates a residue, which contains substantially no ethanol, all or part of the soluble elements, water and insoluble elements, is added to said raw material during said extraction of said raw material. According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, wherein said raw material is a lignocellulosic biomass, in particular chosen from: - cereal straw, said cereals being in particular chosen from wheat, barley, triticale, sorghum and maize, - cereal bran, said cereals being chosen in particular from wheat, barley, triticale, sorghum and maize, - so-called energetic crops , selected in particular from switchgrass, miscanthus and arundo donax, and - forest crops, chosen in particular from wood, short rotation coppices, very short rotation coppices, and forest chips.

The flows described hereinafter were numbered according to Scheme 3, showing a method according to the present invention. According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, comprising a microbial population for preparing ethanol from a raw material generating at least one pentose, in particular xylose and / or arabinose, and at least one hexose, in particular glucose, by fermentation of said pentose and hexose by said microbial population, said raw material being extracted to give an extraction juice (9) containing the aforesaid pentose, in particular xylose and / or arabinose, and a marc (10), said marc giving, after being liquefied, a liquefied (23), said liquéfiat being a source of the aforesaid hexose, in particular glucose, said wort of propagation (13) being obtained by proliferation of an inoculum of said microbial population with the aid of said extraction juice (11), in the presence of air, said propagation wort containing no ethanol, said moû t of propagation (13) being subjected to a solid / liquid separation to give a propagation juice (16) and a fresh microbial population (17), said propagation wort containing no ethanol, said fresh microbial population (17) being separated into two parts, to obtain a first portion (17a) and a second portion (17b) of said fresh microbial population (17), said first portion of said fresh microbial population (17a) being used for the preparation of a must fermenting (18) by fermentation of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12), by said fresh microbial population, said fermentation wort (18) giving after separation liquid / solid said first source of ethanol (19) and all or part of the recyclable microbial population (20), said second source of ethanol (24) being obtained by fermentation of the aforesaid hexose, in particular the glucose, generated by said liquéfiat (23), all or part of said recyclable microbial population (20) and said second portion of the fresh microbial population (17b). Said liquid / solid separation making it possible to obtain the first source of ethanol and a recyclable microbial population is such that said first source of ethanol is substantially free of microorganisms originating from said microbial population.

According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, in which said microbial population comprises yeasts of the Candida shehatae, Pichia stipitis, Pichia guilliermondii, Candia tropicalis or Pachysolen tannophilus strain, said population microbial including in particular yeasts of Candida shehatae strain. According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, comprising: a) a step of extracting a lignocellulosic biomass, to obtain an extraction juice (9) containing said pentose, in particular xylose and / or arabinose, and a marc (10), b) a step of liquefying said marc (10) obtained in the preceding step in the presence of all or part of said first source of ethanol (19) obtained at the end of the first fermentation step f), to obtain a liquéfiat (23), said liquéfiat being a source of the aforesaid hexose, in particular glucose, c) a step of proliferation of a inoculum comprising yeasts of Candida shehatae, Pichia stipitis, Pichia guilliermondii, Candia tropicalis or Pachysolen tannophilus strain, in particular Candida shehatae, with the aid of said extraction juice (11) containing the aforesaid pentose, in particular xylose and / or arabinose, in pr lack of air, to obtain a propagation must (13) comprising a population of said yeasts, said propagation wort containing no ethanol, d) a solid / liquid separation step relative to said propagation wort (13), to give a propagation juice (16) and a population of said fresh yeasts (17), e) a step of separating said fresh yeast population into two parts, to obtain a first portion (17a) and a second portion (17b) ) of said population of fresh yeasts, f) a first fermentation step of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12), by said first part of the population of said fresh yeasts (17a), to obtain after liquid / solid separation a first source of ethanol (19) and a population of said yeasts, recyclable (20), g) a second fermentation step of the aforesaid hexose, in particular glucose, generated by said liquefier (23), said population of said recyclable yeasts (20) and said second portion of said population of said fresh yeasts (17b), to obtain a second source of ethanol (24), h) a step of isolating said ethanol first (19) and / or second sources (24) of ethanol by distillation of said first and / or second sources of ethanol.

According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, comprising: a) a step of extracting a lignocellulosic biomass and all or part of the soluble elements, water and insoluble elements, the residue (26) obtained at the end of the distillation step h), to obtain an extraction juice (9) containing the above pentose, in particular xylose and / or arabinose , and a marc (10), b) a step of liquefying said marc (10) obtained in the preceding step in the presence of all or part of said first source of ethanol (19) obtained at the end of the first step fermentation f), to obtain a liquéfiat (23), said liquéfiat being a source of the aforesaid hexose, in particular glucose, c) a step of proliferation of an inoculat comprising yeasts of the strain Candida shehatae, Pichia stipitis, Pichia guilliermondii , Candia tropicalis or Pachysolen tannophilus, in particular Candida shehatae, using said extraction juice (11) containing said pentose, in particular xylose and / or arabinose, in the presence of air, to obtain a propagation wort (13) comprising a population of said yeasts, said propagation wort containing no ethanol, d) a solid / liquid separation step relative to said propagation wort (13), to give a propagation juice (16) and a population of said yeasts, fresh (17), e) a step of separating said population of fresh yeasts into two parts, to obtain a first portion (17a) and a second portion (17b) of said population of fresh yeasts, f) a first fermentation step of said pentose, in particular xylose and / or arabinose, contained in said extraction juice (12), by said first portion of the population of said fresh yeasts (17a), to obtain after liquid / solid separation a first source ethanol (19) and a population of said yeasts, recyclable (20), g) a second fermentation step of the aforesaid hexose, in particular glucose, generated by said liquéfiat (23), said population of said recyclable yeasts (20) and said second part of said population said fresh yeasts (17b), to obtain a second source of ethanol (24), h) a step of isolating said ethanol from the first (19) and / or second sources (24) of ethanol by distillation of said first and / or or second sources of ethanol, said distillation generating, in addition to said ethanol, a residue (26), which contains substantially no ethanol.

Since said first source of ethanol is substantially free of microorganisms from said microbial population, said liquefaction of said pomace is carried out in the absence of microorganisms from said microbial population. According to an advantageous embodiment, the invention relates to a process for the preparation of ethanol as described above, in which said extraction juice containing said pentose, in particular xylose and / or arabinose, is detoxified before said proliferation and the preparation of said first source of ethanol. According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, wherein said raw material is subjected, after said impregnation and said pretreatment, to an additional hydrolysis treatment. According to an advantageous embodiment, the invention relates to a process for preparing ethanol as described above, in which said liquefier is hydrolysed before said fermentation of the aforesaid hexose, in particular glucose, generated by the hydrolysis of said liquefier. According to an advantageous embodiment, the invention relates to a process for the preparation of ethanol as described above, in which said liquefier is hydrolysed, said hydrolysis and said fermentation of the aforesaid hexose, in particular glucose, generated by the hydrolysis of said liquéfiat being concomitant.

According to an advantageous embodiment, liquefaction of said grounds (that is to say a first hydrolysis of said grounds) to obtain said liquéfiat and hydrolysis of said liquéfiat (that is to say a second hydrolysis) are carried out by the action of the same enzymes, in particular with cellulolytic action. According to another advantageous embodiment, the liquefaction of said grounds (that is to say a first hydrolysis of said grounds) to obtain said liquéfiat and the hydrolysis of said liquéfiat (that is to say a second hydrolysis) are performed by the action of the same enzymes, in particular cellulolytic action, said microbial population being brought into contact with the liquefied, during the hydrolysis of said liquefied.

DESCRIPTION OF THE FIGURES Figure 1 shows the metabolic pathways involved in the use of glucose and xylose by microorganisms. Figure 2 shows the general diagram of a use or a method according to the invention.

Figure 3 shows the detailed diagram of a use or a method of the invention. In Figure 3, the flows and operations are as follows: REF. DESIGNATIONS DESCRIPTIONS PT PRETREATMENT operation \ saying to make cellulose available to the action of cellulolytic enzymes EXTC5 EXTRACTION OF C5 operation Nsant to separate the soluble fraction of the insoluble fraction of the product output of the STEAM operation (separate operation in 2 phases : 1st: dilution, 2nd separation) IF LIQUID / SOLID SEPARATION operation to separate the soluble fraction from the insoluble fraction of the product from the STEAM operation (separate operation in 2 phases: 1st: dilution, 2nd separation) N ° 1 PROPA PROPAGATION OF YEAST operation allowing the growth (yeast multiplication) sufficiently, in order to produce the yeast for the fermentation of sugars in C6 (glucose) on one side and the fermentation of C5 (xylose) on the other side . Ethanol is produced jointly during this propagation step. S2 SEPARATION LIQUID / SOLID concentration operation of fresh yeasts for use in SSF No. 2 FE C5 ALCOHOLIC FERMENTATION OF C5 fermentation operation of C5 (xylose) in ethanol. The presence of C6 (glucose) is not negligible but they are in the minority presence. These are also fermented in ethanol. S3 LIQUID / SOLID SEPARATION operation of concentration of recycled yeasts for use in alcoholic fermentation of C5 (xylose) and SSF (glucose) N ° 3 ENZ PRODUCTIN ENZYMES operation transforming C5 juice into suitable enzymes for the hydrolysis of cellulose and hemicellulose LQ LIQUEFACTION operation to reduce the viscosity of the C6 marc and to bring about a state of flow sufficient for the fermentation (availability of water for the yeast, to avoid the formation of foam, to limit the needs stirring ...) SSF SACCHARIFICATION AND FERMENTATION OF SIMULTANEOUS C6 operation during which the cellulose is hydrolysed to glucose and the glucose is mainly converted into ethanol and CO2 DIST DISTILLATION separation operation by distillation of ethanol and aqueous phase S4 SEPARATION LIQUID / SOLID Clarification operation of raw vinasse to extract ligno-cellulosic residues and valorise the vinasse cl arified in dilution water, extraction ... N ° 4 REF. DESCRIPTIONS DESCRIPTIONS 1 straw or lignocellulosic substrate (mainly comprising cellulose, hemicellulose and lignin) and little concentrated in water 7 unwashed marc the unwashed marc corresponds to the lignocellulosic biomass which has undergone the acidic and acid pretreatment. It contains solubilized hemicelluloses, cellulose more available for the action of enzymes and lignins (as well as various degradation products). 8 diluted marc The extraction of solubilized hemicelluloses (C5) is carried out by a diluent supply counter-current to the advance of the marc. To simulate this step from a mass balance point of view, it is necessary to separate it into two phases (phase 1: dilution, phase 2: separation). In the real process, these two phases are carried out simultaneously. The diluted marc has the same distribution C5 C6 only its water content is higher than the unwashed marc (possible difference related to the recycling of vinasse by diluting the extraction of C5) 9 jus C5 the juice of C5 corresponds to the soluble fraction extracted from the extraction step of C5 and S1. This fraction is rich in C5 and poor in C6, but traces of C6 are still present. 10 washed marc washed marc corresponds to the insolubles which remained in the C5 extraction system. The washed marc is mainly composed of Cellulose and lignin 11 juice C5 propa the composition of the juice C5 propa is the same as the juice C5; this flow corresponds only to a volume fraction of the flow of juice C5 which is sent specifically towards the propagation of the yeasts 12 juice C5 FE the composition of the juice C5 FE is the same as the juice C5; this flow corresponds only to a volume fraction of the flow of C5 juice which is sent specifically to the fermentation of the C5 in ethanol. 13 ml propa the wort propa corresponds to the suspension of yeasts after growth from juice C5 FE 14 mout propa A the Propa wort A corresponds to the volume fraction directly fed to C5 fermentation for inoculating fermentors for the production of ethanol from C5 juice. 15 propa propa B propa B is the volume fraction sent to a solid liquid separation plant (disinfector, centrifuge) to obtain a yeast cream (17) for inoculation of the flow of C6 16 propa juice propa juice corresponds to the supernatant of the separation step S2 (centrifugation); this stream contains ethanol and a residual concentration of C5; It is sent in a fermentation tank of C5 17 fresh yeasts the le.urea f! aiches are creams of yeasts obtained from the workshop and crocagation of yeasts from a juice of sugars in 05 and growth - '- They are emo, in alcoholic fermentation of fiu ..! of 05 18 must FEC5 The FEC5 must corresponds to wine ex 05. this stream contains yeasts, ethanol and water. The C5s were normally consumed and the residual C5 concentration should be very low. FEC5 juice The C5 FE juice corresponds to the F05. e. separation W r,: in .. galleon: this is all or part of dilution water of the ll: - tiefacticr; The e nnual d ity rjrcl.ise enz-- ,. -the cellulose, 20 recyclable yeasts recyclable yeasts are derived from the must FEC5 and the separating apparatus S3. They are reintroduced for a first part (21) in fermentation of C5 and for a second part (22) in fermentation of the C6 (SSF) 21 yeasts recycled to FEC5 these yeasts - ::: c: enent wort FEC5 and have ù! .E. Concentrated by S3 (they were in contact with a C5 juice), and then they were returned with inoculation of the C6 FE juice. 1) to take in cc: -. Inc purge] 22 yeasts recycled in SSF these yeasts come from the must FEC5 and were concentrated by 53 (they have already been in contact with a juice of C5 and have already produced ethanol) They are sent inoculation of the fermentation of the CG (SSF) [it will also be necessary to take into account a purge] 23 liquefied! L liciteflat corres, Doncl to the marc cellulosioue 4 lignine on leel it is made to act a cocktail enzymatioue sc.écifioue oem The purpose of this experiment was to reduce the spe- cies and to remove the cellulose in the form of the FEC5 juice. :: CLW lalic: uefaction:.-_, FLi- .: ie lio.,: Efle: t also contains ethanol while the leeks have acted as 24 fermentation must SSF SSF fermentation corresponds to the wine C6, it contains ethanol ex C6, but also ethanol ex C5, lignins and yeasts. Ethanol = this edible ethanol flow at the outlet of the distillate mill 26 raw vinasse the raw vinasse corresponds to the SSF fermentation broth (24) from which the ethanol was distilled off. The vinasses contain water, residual sugars. of cellulose residues that have not been hydrolysed. lignin residues ... The high rate of suspended matter limits its recycling as it is. It will be necessary to post-treat it to separate the insolubles from the solubles. The lignocellulosulphide residue corresponds to the lignocellulosic residuum of the crude fraction (the inset part of which is massaged). e.,: ed *: The i: 15-: ole this residue will be increased by oressage so da ii iiier his pop.cir calo.:- 7 ': 7: years e out of the valo: iser in boiler o.co/ generation oe, fear and u electricity 28 clear vinasses the clear vinasses mainly correspond to the soluble part of the raw vinasses, They come from the separation 4 and can be used in recylcage water in extraction of the C5 for example , 29, The flow 29 cc-esoonP to iapocint totie by diluting to carry out the extraction of the 05 Its value is function c ta C5 total C5 extraction for extraction Cs in the juices: -: -: -: . :: ue I on snl-laite o: hold 30 EFI This flow corresponds to a part of the clear vinasse that could not be valorized (if excess of vinasse production compared to u rate of incorporation into the flow of diluent for the extraction of C5) 31 net soopin for excration 05 ce. flis,.: corresponds to a net entry of water. To enter the composition of the flow 32 32 C6 juice for the production of enzymes The composition of the EC juice for the enzyme de-activation is the same as the C5 juice: this flow corresponds only to a volume fraction of the flow. C5 juice which is sent specifically to the laboratory of production of enzymes 33 'us PE C6 to Pistler this fiu .: a -ee the composition that the flu, 19 I Corresocnc the P-ie Pe -5 Peie, that Do not return to water this way of liquorice. IIE.i .--- ante: s .-. i, it ti - -. In this case, the flux corresponds to the fraction of the light spirits sent as the extrusion mud of the volume fraction of the stream. FIG. 4 shows the kinetics of hydrolysis of the cellulose of the straw impregnated and pretreated versus straw impregnated without acid and pretreated. EXAMPLE Unless otherwise indicated, the flows described below were numbered according to Scheme 3, showing a method according to the present invention. Example 1: Implementation of the process with a strain not producing ethanol in the propagation phase: Candida shehatae: The substrate used is a wheat straw whose composition is as follows: Plant straws TMS / ha 3.0 Minerals 7 , 0% Protein 3.2% Cellulose 39.1% Lignin 17.9% Hemicellulose 25.5% Arabinane 2.9% Galactan 0.5% Mannane 0.3% Xylane 21.8% Uronates 1.9% Starch Others 5.3% MS 88.0% Table 2: composition of a straw Experte 2011 Impregnation in dilute phase / batch process: An expert straw of composition as described in Table 2 is impregnated by soaking in a reactor of 10001. The soaking is carried out by completely immersing the straw for 12 hours, after contacting the sulfuric acid solution previously heated to 65 ° C. The formula of the straw mixture and sulfuric acid solution is such that the final dry matter reaches 5%. For the preparation of the sulfuric acid solution, the ratio sulfuric acid (dry weight) per 100 g of final solution is 0.51%. After one night of tempering, the temperature that has not been regulated is only 35 ° C. The reactor is then emptied of its liquid content, this draining is achieved by the opening of the bottom valve, the output is filled with a selection gate. At the end of this dripping, the impregnated straw is introduced into the pre-treatment reactor.

Pretreatment: The impregnated straw is put in a reactor heated by steam injection at 180 ° C and maintained under pressure at 9 bar for 5 minutes.

The internal temperature measured at the beginning of the reaction is 167 ° C., and 174 ° C. at the end of the reaction. At the end of this pretreatment step, the reactor is brought back to atmospheric pressure. The lignocellulosic matrix impacted by this pretreatment, is then reactive to liquefaction enzymes. The combination of thermal, chemical and mechanical treatments implies a strong increase in the digestibility of cellulose. Thus, at the end of the liquefaction, 90% of the constituent glucose of the cellulose is available for conversion to ethanol. FIG. 4 shows the kinetics of hydrolysis of the cellulose of the impregnated and pretreated straw versus straw treated without acid and pretreated.

Extraction of C5: At the end of pretreatment, the product is mixed with water (extraction solvent in this example) to allow the diffusion and then the extraction of C5. Once hydrated, the pretreated straw (stream 8) can undergo a liquid-solid extraction. It aims to separate sugars into 5 carbons (mostly solubilized) sugars into 6 carbons (mostly insoluble). The pentoses, resulting from the destructuration of the hemicellulose chains, are rendered soluble by the pretreatment, and migrate into the extraction solvent. As for cellulose, although hydrolysable, it remains mostly insoluble, and is concentrated in the solid fraction. For a large quantity of straw to be treated (250kg / h), the liquid-solid separation is carried out on a belt press. The pretreated straw (stream 7, FIG. 3) is distributed uniformly over the lower fabric and progressively advances from a drainage zone to a pressing zone. In this area, pretreated straw is under increasing pressure and shear forces. Under the action of these two factors, the liquid fraction is extracted from the solid residue which sees its dry matter increased.

Example of a balance sheet resulting from the pretreatment: 1000 T of straw at 88% DM (stream 1) makes it possible to obtain 1 754 T of crude marc at 50% of MS (stream 7) containing 372 T of potential glucose and 177 T of xylose.

C5 extraction is maximized to recover 95% of the C5 in the C5 juice stream (stream 9). The remaining 5% are found in the washed C6 marc (stream 10). In the example, the 1,754 T of crude marc (stream 7) are washed with 2,335 T of diluent (stream 29). This stream 29 can be composed of make-up water (stream 31) and / or clarified clarified stream 34. The separation leads to the production of 2643 T of C5 juice (stream 9) containing 95% of the C5 ( xylose) and having glucose and xylose concentrations of 1.2% and 6.4% (w / w), respectively. The other flow corresponds to 1 446 T of marc washed at 44% MS (stream 10) and contains 5% of the C5 as well as the majority of potential C6 (355 T versus 33 T in the C5 juice). Preculture stage The use of yeasts in fermenters requires the establishment of a chain of precultures. The first preculture consists of inoculating with the desired strain (here Candida shehatae) a tube containing the following medium: - Yeast extract 10 g / kg - Bacto peptone: 10 g / kg - Xylose: 30 g / kg - NaCl: 9 g / kg The incubation conditions of this first preculture are the following: - Temperature: 30 ° C - pH: 5 - agitation: 130 rpm After 12 hours of incubation under the previous conditions, the first preculture serves as inoculum for the second preculture performed in Erlenmeyer flask. The medium of the second preculture is shown in Table 3 below: Pantathern acid Pr a, The medium also contains 30 g / kg of xylose. The conditions and the duration of incubation are identical to those followed during the first preculture (30 ° C, pH 5, 130 RPM for 12 hours).

Stage of propagation of yeasts on C5 juice The precultures carried out previously make it possible to inoculate the yeast propagation fermenter. The propagation medium corresponds to stream 11. The latter is complemented in various elements listed in Table 3 above. The respective glucose and xylose concentrations of stream 11 are 7 to 12 g / kg and 45 to 64 g / kg. The mass fraction of the flow 11 dedicated to propagation is here 5%. The culture conditions of the propagation are the following: - Temperature 30 ° C - pH 5.5 regulated with potash (KOH) and ortho-phoshoric acid - agitation and aeration sufficient to maintain P02 at more than 30 % and this without dome pressure After 48 hours of culture under the above conditions, the culture makes it possible to obtain 22 g / kg of yeasts from 48.2 g / kg of xylose. The recovery yield of the substrate (xylose) in yeast (Yx / s) reached 46% / g). Co-products such as ethanol, glycerol, xylitol are also present at the following concentrations: Ethanol: 0.38 g / kg Glycerol: 0.4 g / kg Xylitol: 0.15 g / kg The conversion efficiency of the substrate in ethanol is therefore 1.8%.

Step of fermentation of juice C5 The juice of C5 (stream 9) is sent to about 80% to the alcoholic fermentation of juice C5 (stream 12). The medium is supplemented according to the following formula: - Yeast extract at 5 g / kg, - Urea at 0.4 g / kg The culture conditions for the fermentation of C5 juice are the following: - Temperature: 30 ° C - pH: 5.5 - Aeration: oxygen limitation.

The culture conditions described above (oxygen limitation), allow the yeast population to produce ethanol with a yield of 28% to 33%. The ethanol concentration reaches 18.3 g / l (glucose: 7.2 g / l, xylose: 52.3 g / l) after 46 to 63 hours of culture. At the same time, there was yeast production with a yield of 6-8%. Finally, by-products were generated with the following yields: Yp (glycerol) / s) = 0.021 g / g-Yp (xylitol) / s) = 0.11 g / g-Yp (ribitol) / s) = 0.039 g / g Monitoring of the yeast population: The ethanolic fermentation workshop of C5 juice is inoculated with 1 g of yeast per kg of medium. In addition to this population added during inoculation, yeast yield is between 6 and 8% and allows yeasts to be produced in parallel with ethanol production. At the outlet of the ethanol fermentation workshop of C5 juice, the yeast concentration thus reaches 0.6%. The wort corresponds to the stream 18 can then be sent directly in dilution water of the liquefaction stage (hydrolysis 1) or undergo a separation step S3 to remove yeasts from the wine (stream 18). This separation operation S3 makes it possible to obtain a wine free from the majority of yeasts (stream 19) and yeast creams (stream 20). In the example, half of these yeast creams are recycled in the ethanolic fermentation step of the C5 juice (stream 21). The other half can be sent in fermentation of the C6 marc after the liquefaction stage (hydrolysis 1). This recycling of the yeasts makes it possible to substantially increase the yeast concentration in the stream 18. In fact, when the steady state is established, the yeast concentration at the outlet of the C5 fermentation plant is 1.2% against 0%. 6% without this recycling. Monitoring of ethanol production: Stream 12 (2075 T of C5 juice) contains 26 T of glucose and 132 T of xylose. The Yp (EtOH) / s yield of 28% to 33% thus makes it possible to produce 53.9 T of ethanol. In the example, the wine is rid of these yeasts and sent directly to the liquefaction workshop. This separation operation S3 is carried out without washing the creams, which generates a stream 19 containing 49.6 T of ethanol (the difference in the amount of ethanol corresponds to the ethanol contained in the recycled creams: stream 21 and stream 22 ). The mass concentration of ethanol flux 33 (C5 wine cleared yeasts) is 2.5%, which corresponds to a TAV of 3.2 °.

C6 fermentation step by hydrolysis and simultaneous or non-simultaneous fermentation The washing of the marc made it possible to obtain a first stream rich in sugars with 5 carbon atoms (stream 9) and a second stream rich in sugars with 6 atoms. of carbon (stream 10). This latter stream contains an equivalent of 25% potential glucose. However, this glucose equivalent is in the vast majority in the form of cellulose. It is not usable as such by yeasts. It must be subjected to hydrolysis steps by action of enzymatic cocktails with cellulolytic actions, which allows both to reduce the viscosity of the product (liquefaction or hydrolysis step 1) and both to transform cellulose into cellobiose then glucose (hydrolysis 2). These hydrolysis steps can be carried out either totally separate from the fermentation (we speak of SHF), or simultaneously with the action of yeasts (we speak of SSF). In the latter case, the yeasts can be introduced either directly during hydrolysis 1 (liquefaction) or during hydrolysis 2. In the example, it has been chosen to carry out a hydrolysis step 1 (liquefaction). in the absence of yeasts and a hydrolysis step 2 in the presence of yeasts (SSF step). Liquefaction stage The liquefaction stage consists in bringing into contact the washed marc containing the cellulose and the cellulolytic enzyme enzymatic cocktail. Dilution of the C6 marc flux is allowed by the integration of the entire flow 19 (wine ex-05 free of yeasts). This implementation makes it possible to obtain a cellulose concentration of 9.4%. The conditions of hydrolysis 1 (liquefaction) are as follows: - Temperature: 50 ° C - pH: 4.8 - Enzyme concentration: 40 mg / g cellulose - Duration from 2 to 12 h From a mass balance point of view therefore, 1 446 T of washed marc (stream 10) diluted with 1 956 T of ex-05 wine freed from yeasts (stream 19). The enzymatic cocktail flow is 14.2 T protein for 355 T of potential glucose.

Step fermentation of marc C6 To facilitate fermentation, the stream 23 is enriched in nutrients (yeast extract 5g / kg and urea 0.4g / kg). The potential glucose and xylose concentrations are 10-12% and 0.4-0.7%, respectively.

After the liquefaction stage (hydrolysis 1), the liquefied stream (stream 23) is inoculated by the yeast cream corresponding to stream 17b (fresh yeasts from the propagation workshop) and by the yeast cream corresponding to the stream 22 (yeast cream obtained at the end of the C5 juice fermentation workshop: recycled yeasts). The culture conditions used are the following: - Temperature: 30 ° C - pH 5.5 - aeration: oxygen limitation The culture conditions described above, allow the yeast population to produce ethanol with a yield of 28% -33%. At the same time, there was yeast production with Yx / s yield of 8.2%. Monitoring of the yeast population: According to the mass balance, the ethanol workshop of the C6 marc corresponds to a flow of 3 402 T liquefied with 10.4% potential glucose. These 3 402 T of juice are inoculated with 2.3 T MS of fresh yeasts obtained from the propagation of yeasts on C5 juice, but also from the yeasts resulting from the ethanol fermentation of C5 juice (stream 22) to height of 12.8 T MS. This amounts to inoculating the fermentation of the marc C6 with 4.3 g of yeast per kg of medium (including cream mass).

Monitoring of ethanol production: Stream 23 (3,402 T) contains 355 T of potential glucose and 9 T of xylose. The yield of hydrolysis of cellulose to glucose is 87% and the conversion efficiency of glucose to ethanol is 33.4%. The residual xylose (9 T) is also fermented in ethanol with the same yield. The ethanol production during the SSF stage (or during the SHF) is therefore 106 T. Moreover, the stream 24 also contains the ethanol that was produced during the fermentation of the C5 juice (49.6 T d). ethanol). All this makes it possible to reach a mass concentration of ethanol in wine (stream 24) of 4.66%, a TAV of 5.9 °.

Distillation step In the example, the distillation only recovers a single stream which corresponds to stream 24. The title of this wine entering the distillation column is 5.9 °. The vapor consumption associated with a wine having a TAV of 5.9 ° is about 2.67 kg of steam per kg of ethanol produced.

Vinasse treatment stage The vinasses at the distillation column outlet are clarified (stream 26) by an S4 separation plant. The lignocellulosic residues (stream 27) are treated so as to recover energy by carrying out their combustion (steam generation). The fraction of clarified vinasse (stream 28) is 100% recycled to the C5 extraction plant (stream 34). This recycling rate makes it possible to limit as much as possible the volumes of water introduced into the system (stream 31 = 239T).

Overall performance step of the example The overall performance of this configuration is as follows: For 1000 tons of straw at 87.7% of MS (or 877 kg of MS), the system produces 158.4 tons of ethanol, of which two third come from the fermentation of C6 and the remaining third from the fermentation of C5. The overall yield of the recovery of dry matter ethanol is 18.06% (m / m).

This configuration also allows to produce 4.7 tons of fresh yeasts divided into two equal streams towards the fermentation of C5 or fermentation of C6. The average TAV of the wines supplying the distillation workshop is 5.9 °.

Claims (10)

REVENDICATIONS1. Use of a propagation mash comprising a microbial population for preparing ethanol from a raw material generating at least one pentose, in particular xylose and / or arabinose, and at least one hexose, in particular the glucose, by fermentation of said pentose and hexose by said microbial population, said raw material being extracted to give an extraction juice containing said pentose, in particular xylose and / or arabinose, and a pomace, said pomace giving, after having been liquefied, a liquefier, said liquefier being a source of the aforesaid hexose, in particular glucose, said propagation must being obtained by proliferation of an inoculum of said microbial population with the aid of said extraction juice, in the presence of air, said propagation wort containing no ethanol, said microbial population being used both: - for the preparation of a first source of ethanol, by fermentati said pentose, in particular xylose and / or arabinose, contained in said extraction juice by said microbial population and - for the preparation of a second source of ethanol, by fermentation of the aforesaid hexose, in particular glucose, generated by said liquéfiat, by said microbial population, said ethanol being isolated from the first and / or second sources of ethanol, in particular by distillation of said first and / or second sources of ethanol, said liquefaction of said grounds being carried out in presence of all or part of said first source of ethanol, said first source of ethanol being, in particular, substantially free of microorganisms from said microbial population. A process for preparing ethanol from a raw material generating at least one pentose, in particular xylose and / or arabinose, and at least one hexose, in particular glucose, comprising: a) a step extracting said material, to obtain an extraction juice containing said pentose, in particular xylose and / or arabinose, and a marc, b) a liquefaction step of said pomace obtained in the preceding step, to obtain a liquéfiat, said liquéfiat being a source of the aforesaid hexose, in particular glucose, c) a step of proliferation of an inoculum of a microbial population with the aid of said extraction juice containing the aforesaid pentose, in particular xylose and / or arabinose, in the presence of air, to obtain a propagation must comprising said microbial population, said propagation wort does not contain ethanol, d) a first fermentation step of the aforesaid pentose, in particular of xylose and / or arabinose, contained in said extraction juice, by said microbial population, to obtain a first source of ethanol, e) a second fermentation step of the aforesaid hexose, in particular glucose, generated by said liquéfiat, by said microbial population to obtain a second source of ethanol, f) a step of isolating said ethanol from the first and / or second sources of ethanol, in particular by distillation of said first and / or second sources of ethanol, said step of liquefying said marc being carried out in the presence of all or part of said first source of ethanol obtained at the end of the first fermentation step of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice said first source of ethanol being, in particular, substantially free of microorganisms from said microbial population. A process for the preparation of ethanol according to claim 2, wherein said distillation, in addition to said ethanol, generates a residue, which contains substantially no ethanol, all or part of the soluble elements, water and elements. insoluble, is added to said raw material during said extraction of said raw material. 4. A process for the preparation of ethanol according to any one of claims 2 to 3, wherein said microbial population comprises yeasts of Candida shehatae, Pichia stipitis, Pichia guilliermondii, Candia tropicalis or Pachysolen tannophilus strain, said microbial population comprising in particular yeasts of Candida shehatae strain. A process for the preparation of ethanol according to any one of claims 2 to 4, comprising a microbial population for preparing ethanol from a raw material generating at least one pentose, in particular xylose and / or arabinose, and at least one hexose, in particular glucose, by fermentation of said pentose and hexose by said microbial population, said raw material being extracted to give an extraction juice (9) containing said pentose, in particular xylose and and / or arabinose, and a marc (10), said marc giving, after being liquefied, a liquefied (23), said liquefied being a source of the aforesaid hexose, in particular glucose, said wort of propagation (13) being obtained by proliferation of an inoculum of said microbial population with the aid of said extraction juice (11), in the presence of air, said propagation wort containing no ethanol, said propagation wort (13) being subject to a se solid / liquid paration to give a propagation juice (16) and a fresh microbial population (17), said propagation wort containing no ethanol, said fresh microbial population (17) being separated into two parts, to obtain a first part (17a) and a second part (17b) of said fresh microbial population (17), said first part of said fresh microbial population (17a) being used for the preparation of a fermenting must (18) by fermentation of the aforesaid pentose , in particular xylose and / or arabinose, contained in said extraction juice (12), by said fresh microbial population, said fermentation wort (18) giving after said liquid / solid separation said first source of ethanol ( 19) and all or part of the recyclable microbial population (20), said second source of ethanol (24) being obtained by fermentation of the aforesaid hexose, in particular glucose, generated by said liquéfiat (23), by all or part of said recyclable microbial population (20) and said second fresh microbial population portion (17b). 6. Process for preparing ethanol according to any one of claims 2 to 5, comprising: a) a step of extracting a lignocellulosic biomass and all or part of the soluble elements, water and insoluble elements; of the residue (26) obtained after the distillation step h), to obtain an extraction juice (9) containing the aforesaid pentose, in particular xylose and / or arabinose, and a marc (10), b) a step of liquefying said pomace (10) obtained in the preceding step in the presence of all or part of said first source of ethanol (19) obtained after the first fermentation step f) to obtain a liquéfiat (23), said liquéfiat being a source of the aforesaid hexose, in particular glucose, c) a proliferation stage of an inoculat comprising yeasts of Candida shehatae strain, Pichia stipitis, Pichia guilliermondii, Candia tropicalis or Pachysolen tannophilus, in particular Candida shehatae, using said extraction juice (11) containing the aforesaid pentose, in particular xylose and / or arabinose, in the presence of air, in order to obtain a propagation must (13) comprising a population of said yeasts, said propagation must not containing no ethanol, d) a solid / liquid separation step relative to said propagation wort (13), to give a propagation juice (16) and a population of said fresh yeasts (17), e) a step of separating said population of fresh yeasts into two parts, to obtain a first portion (17a) and a second portion (17b) of said population of fresh yeasts, f) a first fermentation step of the aforesaid pentose, in particular xylose and / or arabinose, contained in said extraction juice (12), by said first portion of the population of said fresh yeasts (17a), to obtain after liquid / solid separation a first source of ethanol (19) and a population said yeasts, recyclable ( 20), g) a second fermentation step of the aforesaid hexose, in particular glucose, generated by said liquéfiat (23), said population of said recyclable yeasts (20) and said second portion of said population of said fresh yeasts (17b), to obtain a second source of ethanol (24), h) a step of isolating said ethanol from the first (19) and / or second (24) sources of ethanol by distillation of said first and / or second sources of ethanol, said distillation generating, in addition to said ethanol, a residue (26), which contains substantially no ethanol. 7. Process for the preparation of ethanol according to any one of claims 2 to 6, wherein said raw material is, prior to said extraction, impregnated and pretreated, said raw material being particularly subject, after said impregnation and said pretreatment, additional hydrolysis treatment. 8. Process for the preparation of ethanol according to any one of claims 2 to 7, wherein the raw material is a lignocellulosic biomass, in particular chosen from: - cereal straw, said cereals being in particular chosen from wheat, barley, triticale, sorghum and maize, - cereal bran, said cereals being in particular chosen from wheat, barley, triticale, sorghum and maize, - energy crops, chosen in particular from switchgrass, miscanthus and arundo donax, and - forest crops, chosen in particular from wood, short rotation coppices, very short rotation coppices, and forest chips. A process for the preparation of ethanol according to any one of claims 2 to 8, wherein said extraction juice containing said pentose, in particular xylose and / or arabinose, is detoxified prior to said proliferation and the preparing said first source of ethanol. 10. Process for the preparation of ethanol according to any one of claims 2 to 9, wherein said liquefied is: - hydrolyzed before said fermentation of the aforesaid hexose, in particular glucose, generated by the hydrolysis of said liquefied, or - hydrolysed said hydrolysis and fermentation of said hexose, in particular glucose, generated by the hydrolysis of said liquéfiat being concomitant.