Method for treating an aqueous-phase mixture of compounds comprising sugars containing 5 and 6 carbon atoms

Abstract

The invention relates to a method for treating an initial mixture M in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons, said C5 sugars and / or said C6 sugars being partly in monomeric form and partly in oligomeric form, such that said treatment includes an enzymatic hydrolysis step, so as to obtain a treated mixture M' enriched in C5 sugars in monomeric form and / or in C6 sugars in monomeric form and depleted in C5 sugars in oligomeric form and / or in C6 sugars in oligomeric form.

Description

[0001] PROCESS FOR TREATING AN AQUEOUS PHASE MIXTURE OF COMPOUNDS COMPRISING SUGARS WITH 5 AND 6 CARBON ATOMS.

[0002] Technical field

[0003] The present invention relates to the treatment of sweet juices, in particular those known as second generation (2G), which can be obtained from lignocellulosic biomass.

[0004] These sugary juices can be used to produce other products by chemical or biochemical means (for example, alcohols such as ethanol, butanol, or other molecules, for example, solvents such as acetone, intermediate products used in the chemical industry, etc.), particularly as substitutes for petrochemical derivatives. These are called bio-sourced compounds.

[0005] Prior art

[0006] Lignocellulosic biomass represents one of the most abundant renewable resources on Earth. The substrates considered are very varied, they concern both woody substrates such as different woods (hardwoods and softwoods), co-products from agriculture (wheat straw, corn cobs, etc.) or other industries, such as the food industry, paper, etc.

[0007] Different types of processes exist for converting lignocellulosic biomass into sugary juices depending on the type of biomass. In the case of sacchariferous plants (sugar beet, sugar cane) or starchy plants (corn and wheat), so-called first-generation (1G) sugary juices are obtained, for example through extraction operations.

[0008] If it is biomass of the agricultural, forestry or paper residue type, we obtain so-called second generation (2G) sweet juices by a biochemical transformation process which generally includes a pretreatment step and an enzymatic hydrolysis step by an enzymatic cocktail. The pretreatment most often includes an impregnation step with a liquor, in particular acidic or basic or oxidizing, then cooking of the impregnated biomass which is possibly accompanied by a steam explosion.

[0009] The sugary juices resulting from hydrolysis can then be further processed, for example by fermentation to convert them into alcohol, and the conversion process also includes separation and / or purification steps.

[0010] These sweet juices can also come from a mixture of sweet juices from different types of biomass. Lignocellulosic biomass is composed of three main polymers: cellulose (35 to 50% by weight), which is a polysaccharide consisting mainly of hexoses; hemicellulose (20 to 30% by weight), which is a polysaccharide most often consisting mainly of pentoses; and lignin (15 to 25% by weight), which is a polymer with a complex structure and high molecular weight, composed of aromatic alcohols linked by ether bonds. These different molecules are responsible for the intrinsic properties of the plant wall and are organized into a complex tangle.

[0011] Among the three basic polymers that integrate lignocellulosic biomass, cellulose and hemicellulose are those that allow the production of 2G sweet juices.

[0012] Most often, hemicellulose is mainly broken down into sugars during pretreatment, and the cellulose is converted into glucose by enzymatic hydrolysis. However, access to raw cellulose remains difficult for enzymes, hence the need for the pretreatment mentioned above. This pretreatment makes it possible to modify the physicochemical properties of lignocellulosic biomass in order to improve the accessibility of cellulose to enzymes and its reactivity to enzymatic hydrolysis.

[0013] Many technologies of interest to the invention for carrying out this pretreatment exist, which will be grouped hereinafter under the generic term of "cooking": acid cooking, alkaline cooking, cooking by auto-hydrolysis, steam explosion, processes called "organosolv pulping" according to the known English term (or treatment with organo-solvent in French). This last process concerns a pretreatment in the presence of one or more organic solvents and generally water. The solvent can be an alcohol (ethanol), an acid such as acetic acid, formic acid, or even acetone.

[0014] Different configurations are reported for example in the document “Production of bioethanol from lignocellulosic materials via the biochemical pathway: A review”, M. Balat, Energy Conversion and Management 52 (2011) 858-875, or in the document “Bioethanol production from agricultural wastes: an overview”, N. Sarkar, S. Kumar Ghosh, S. Bannerjee, K. Aikat, Renewable Energy 37 (2012) 19-27.

[0015] One of the most effective pretreatments is steam explosion, especially under acidic conditions, which allows almost complete hydrolysis of hemicellulose and a significant improvement in the accessibility and reactivity of cellulose to enzymes. This pretreatment may be preceded by other treatment(s).

[0016] Thus, the pretreatment can generally comprise three stages which are the preparation of liquor, the impregnation of the biomass with this liquor and the pretreatment of the impregnated biomass, for example by cooking possibly coupled with a steam explosion: Patent FR 3 075 203 describes a process with impregnation of the biomass with an acid liquor, then cooking and steam explosion of the impregnated biomass, with an adjustment of the acidity of the acid liquor and a recycling of the latter. Patent FR 3 075 201 also describes a process for pretreatment of the biomass by acid impregnation then steam explosion, with in addition a washing of the means of feeding the reactors and recycling of the washing water in the process.

[0017] A solid / liquid separation can then be carried out on the pretreated biomass, so as to recover a liquid fraction which is a sweet juice in the aqueous phase, and a solid fraction, which is a pretreated marc also called cake. Patent FR 3 083 126 thus describes a liquid / solid separation operation on the pretreated biomass obtained at the end of the pretreatment step or at the end of an additional step of said pretreated biomass, this separation step comprising two successive sub-steps:

[0018] - an upstream sub-step b1 of contacting the solid / liquid mixture implemented by a continuous mixer using a mixing fluid,

[0019] - and a downstream extraction / washing sub-step b2 implemented by a continuous filter, in particular of the belt filter type, using a washing fluid, to obtain a solid phase enriched in solid and a plurality of liquid phases enriched in liquid, with operation of the filter preferably in counter-current between the circulation of the solid / liquid mixture to be separated and the extraction / washing fluid, and at least partial recycling of a liquid phase extracted from the belt filter at the inlet of the mixer as a mixing fluid.

[0020] The sweet juices thus obtained from lignocellulosic biomass, in particular after its pretreatment or after enzymatic hydrolysis in the case of so-called 2G sugars, are in the form of a mixture of sugars in the aqueous phase, where we find so-called C5 sugars (i.e. with 5 carbons), such as xylose and arabinose, and so-called C6 sugars (i.e. with 6 carbons), such as glucose, mannose and galactose.

[0021] Particularly in the case of type 2G sweet juices resulting from the pretreatment of lignocellulosic biomass, for the majority of lignocellulosic biomasses, a proportion of C5 sugars in the sweet juice is observed to be higher than that of C6 sugars. By sweet juice, we mean that sugars are released by a treatment carried out on the biomass, in particular a pretreatment in the known sense of the term in the field of biomass conversion, and that they can be solubilized by mixing the pretreated biomass with water for example.

[0022] The sugars in sweet juice, as seen above, are mainly, or even exclusively, C5 and C6 sugars. In fact, they can be found in three different forms: - in a polymeric form, with a high degree of polymerization, greater than 6, that is to say that they have not been converted, they are insoluble in water, this is the case of cellulose,

[0023] - in monomeric form, they are soluble in water

[0024] - in an oligomeric form, that is, the sugars are in a polymeric form, but with a low degree of polymerization, between 2 and 6, they are also soluble in water.

[0025] This means that, after pretreatment of the biomass, and after having carried out a solid / liquid separation of the biomass thus treated, the liquid fraction obtained is a sweet juice with sugars in monomeric and oligomeric form, the sugars in polymeric form, insoluble, being found in the solid fraction.

[0026] The proportion of C5 sugars compared to C6 sugars, and the proportion, for each of these types of sugars, of the oligomeric form compared to the monomeric form are variable, notably depending on the nature of the biomass (straw, wood, etc.), depending on the type and severity of the pretreatment, depending on the washing conditions when there is washing, etc. But in most cases, there is indeed a significant content of sugars in oligomeric form in the sweet juices.

[0027] However, the presence of these sugars in oligomeric form can be detrimental, regardless of the intended application for these sweet juices. Indeed, when the aim is to convert these sweet juices into other compounds, whether by chemical or biochemical conversion (fermentation for example), sugars in monomeric form are more reactive than sugars in oligomeric form. And while there are outlets for sweet juices as is, without further conversion, these must generally meet data specifications, including a high monomeric sugar content.

[0028] There is therefore a need to improve the quality of these sweet juices, and in particular to increase the sugar content in monomeric form or to reduce the sugar content in oligomeric form.

[0029] Summary of the invention

[0030] The invention firstly relates to a process for treating an initial mixture M in aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons, said C5 sugars and / or said C6 sugars being partly in monomeric form and partly in oligomeric form, characterized in that said treatment comprises an enzymatic hydrolysis step, in order to obtain a treated mixture M' enriched in C5 sugars in monomeric form and / or in C6 sugars in monomeric form and depleted in C5 sugars in oligomeric form and / or in C6 sugars in oligomeric form.

[0031] For the purposes of the invention, the term “mixture in aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons” may also be designated, for the sake of brevity, under the term “initial sugar juice” or “initial mixture M”.

[0032] For the purposes of the invention, the term "mixture M' enriched in C5 sugars in monomeric form and / or in C6 sugars in monomeric form and depleted in C5 sugars in oligomeric form and / or in C6 sugars in oligomeric form" may also be designated, for the sake of brevity, under the term "treated sweet juice" or "treated mixture".

[0033] For the purposes of the invention, "depleted" means that the concentration (or content) of the sugar in question in the mixture decreases due to the treatment and "enriched", conversely, means that the concentration of sugar in the mixture increases due to the treatment.

[0034] For the purposes of the invention, the term “sugars” means sugars in their monomeric form and in their oligomeric form, unless otherwise specified.

[0035] For the purposes of the invention, “juice” is equivalent to a mixture of compounds in aqueous phase: the juice is aqueous, and the sugars it contains are soluble in water.

[0036] To give an order of magnitude, the initial sweet juice preferably has a sugar concentration of at least 20 g / kg. Its concentration can thus be between 20 and 80 g / kg, in particular between 30 and 70 g / kg, for example between 40 and 60 g / kg. And the dry matter content (DM) (defined below) of the initial sweet juice can typically be between 2 and 20%, in particular between 5 and 15%.

[0037] It can therefore be seen that, preferably, the treatment according to the invention is carried out on a sweet juice with a low dry matter content / with a low sugar concentration, even if the invention can also be applied to higher dry matter levels / concentrations.

[0038] The invention has therefore developed a treatment which will transform at least part of the sugars in oligomeric form into sugars in monomeric form, by enzymatic hydrolysis converting all or part of the oligomers into monomers. What is very advantageous is that it is thus possible to reduce / remove the undesirable compounds, namely the sugars in oligomeric form, to convert them into sugars in monomeric form, which are the desired products: it is not simply a matter of removing them from the initial sugary juice, but of converting them to the desired form. The conversion efficiency of oligomers into monomers obtained with the treatment according to the invention can in fact be up to 50% and more.

[0039] The operating conditions of this enzymatic hydrolysis, in particular, the nature and quantity of enzymes to be added are to be adjusted according to, in particular, the nature of the biomass, the type of pretreatment it has had when the sugar juice comes from a treatment / pretreatment of lignocellulosic biomass, the dry matter content MS of the juice (which can be adjusted by concentration or dilution operations), which determine the oligomer content of the mixture, or even the monomeric sugar purity specifications if it is used as is. It is thus possible to modulate the operating conditions of the treatment according to the sugar juice to be treated and the monomeric sugar purity to be achieved.

[0040] This treatment is very advantageous, because the treated sweet juice sees its content of sugars in monomeric form increase and its content of sugars in oligomeric form decrease or even disappear: it has been shown that it is thus possible to easily achieve the purity specifications required for the recovery of these juices. And when these juices are intended to be converted by chemical or biochemical processes, it has also been verified that their reactivity is improved, and therefore that their conversion yield is higher than that of untreated sweet juice according to the invention.

[0041] Advantageously, the temperature T of the treatment can be at most 80°C or at most 70°C, in particular at least 20°C, and is preferably between 40 and 60°C. Indeed, it is useful not to raise the temperature of the juice too much, on the one hand so that the enzymes retain / reach their full effectiveness, and on the other hand to avoid any parasitic reaction of the Maillard reaction type which would degrade the sugars.

[0042] Advantageously, the duration of the treatment may be at least 5 minutes, in particular at least 10 minutes, in particular at least 30 minutes, and preferably at most 3 hours, in particular at most 2 hours or at most 1 hour 30. It has been found that short treatment durations, of 1 hour or less, may be sufficient to obtain interesting results.

[0043] Advantageously, the treatment of the mixture M comprising an enzymatic hydrolysis step according to the invention is carried out at a pH of at least 3, in particular at least 3.5, in particular at least 4, and preferably at most 6. It is in fact in this pH range that the enzymes tend to be most effective. To obtain this pH, it may be necessary to adjust the pH of the initial mixture M by adding acid or base, in particular by adding base, preferably KOH or NaOH, when the pH of the initial mixture M is less than or equal to 3. This is particularly the case when the juice comes from a pretreatment with acid impregnation of biomass, and therefore strongly acid (with a pH which may be of the order of 2 or less).

[0044] The treatment according to the invention can be carried out continuously, or in batches, in particular in a stirred or piston-type reactor.

[0045] The enzymes used for the enzymatic hydrolysis step are preferably chosen from at least one of the following enzymes: cellulase(s) and hemicellulase(s). Preferably, a mixture of enzymes comprising these two types of enzyme is used. It may, for example, be an enzymatic cocktail comprising endoglucanases, cellobiohydrolases, beta-glucosidases, xylanases, in particular produced by bacteria or fungi of the genus Trichoderma or Aspergillus, in particular Trichoderma Reesei. This cocktail may be produced by a Trichoderma Reesei fungus, according to a method described, for example, in patent FR2555603 or in patent WO2020 / 052952 or in patent EP3461902. This fungus may have been genetically modified to be hyperproductive in enzymes, particularly cellulase, such as strains MCG77 (Gallo - US patent 4,275,167), MCG 80 (Allen, AL and Andreotti, RE, Biotechnol-Bioengi 1982, 12, 451-459 1982), RUT C30 (Montenecourt, BSand Eveleigh, DE, Appl. Environ. Microbiol. 1977, 34, 777-782) and CL847 (Durand et al, 1984, Proc. Colloque SFM "Genetics of industrial microorganisms". Paris. H. HESLOT Ed, pp 39-50).

[0046] Advantageously, the enzymatic hydrolysis step can be carried out by adding to the initial mixture (juice) M a quantity of enzymes corresponding to at least 0.05 g protein / kg of mixture, in particular at least 0.08 g protein / kg of juice for a concentration of 50 g of monomeric sugars / kg of juice, and preferably corresponding to at most 2 or 3 g protein / kg of mixture at the same concentration of monomeric sugars in the mixture.

[0047] Preferably, the initial sugar juice is mainly derived from the depolymerization of hemicelluloses present in the lignocellulosic biomass. Preferably, the initial sugar juice contains more C5 sugars than C6 sugars by weight. Preferably, the initial sugar juice is obtained without the addition of enzymes or microorganisms such as bacteria. Finally, preferably, the initial sugar juice is mainly derived from the pretreatment of a lignocellulosic biomass.

[0048] Here, the term "originating mainly" is understood to mean that at least half of the initial sugary juice in question comes from the depolymerization in question (in particular all of it). According to one embodiment of the invention, the mixture M comes from the treatment of biomass, in particular lignocellulosic, originating in particular from forestry and / or agricultural and / or paper residues, and / or from sacchariferous plants and / or from starchy plants and / or from Fermentable Fractions of Household Waste (FFOM).

[0049] In this case, the mixture M is preferably derived

[0050] - a biomass washing operation,

[0051] - or pretreatment of the biomass including in particular impregnation of the biomass with a liquor, of neutral, acidic or basic pH or of an oxidizing nature, preferably with an acidic liquor, followed by cooking of the impregnated biomass, in particular cooking with steam explosion, possibly followed by a washing operation of the pretreated biomass,

[0052] - then a solid / liquid separation of said washed or pretreated biomass.

[0053] The method according to the invention may, moreover, comprise at least one operation subsequent to the treatment by enzymatic hydrolysis of the mixture M. This may involve bringing said mixture in aqueous phase into contact with a microorganism consuming, among the sugars in the mixture, essentially only the C6 sugars, in order to obtain a mixture also depleted in C6 sugars (in monomeric and oligomeric form). The microorganism may be chosen from yeasts in the genus Saccharomyces, in particular the species Saccharomyces cerevisae or, among bacteria, in the genus Corynebacterium, and is capable of converting C6 sugars into alcohol, but also of converting certain undesirable impurities such as furfural or 5-HMF. For further details, reference may be made to patent application FR2212406 filed on November 28, 2022.

[0054] Another type of operation subsequent to the treatment according to the invention may be any purification operation in the broad sense, for example to separate a solid or soluble fraction of enzymes....

[0055] The method according to the invention may comprise, after the enzymatic hydrolysis step of the mixture M, a separation step aimed at separating at least a portion of the enzymes from the rest of the mixture, in particular with a view to recycling the enzymes present in the treated juice. Generally, a fraction of the enzymes may be in solid form or supported on a solid support in an aqueous medium: at least a portion of this solid fraction of enzymes may then be separated by any known appropriate separation technique. All or a portion of the enzymes may also be in soluble form in the treated juice: to separate at least a portion of this soluble fraction from the mixture, any known technique may also be used, for example the technique using ultrafiltration membranes.

[0056] Note that a solid / liquid separation operation (any known conventional technique) can also be planned on the treated juice, if the salts resulting from the addition of base are precipitated in order to adjust / raise the pH of the juice in order to treat it according to the invention.

[0057] This step of separating the enzymes (solid fraction and / or soluble fraction) is carried out for example by centrifugation, on a filter press, a plate filter, or by microfiltration or ultrafiltration membranes.

[0058] The solid fraction or retentate (comprising part of the still active enzymes) can be recycled in part or in full to the enzymatic hydrolysis reactor, and the liquid fraction or permeate (if the soluble fraction of the enzymes is also separated) constitutes the treated sugar juice.

[0059] This additional separation step is advantageous for recycling enzymes that retain enzymatic activity even after the treatment according to the invention. The inventory of enzymes to be added for the enzymatic hydrolysis step according to the invention is therefore reduced.

[0060] This separation step is also useful in cases where the enzymes used for the enzymatic hydrolysis step are added to the juice in an (aqueous) solution which in fact dilutes the sugary juice to be treated. This enzyme separation step (particularly by ultrafiltration) can also allow the treated juice to be concentrated, which makes it possible to compensate, at least in part, for the dilution brought about by the addition of enzymes in water.

[0061] This separation step is also interesting in the case of continuous implementation.

[0062] The invention also relates to a mixture M' of compounds in aqueous solution, comprising C5 sugars and / or C6 sugars, in particular treated according to the process described above, and which has the following characteristics:

[0063] - an S1 content of C5 sugars and / or C6 sugars of between 10 and 100 g / kg of solution

[0064] - including a ratio P1 of C5 sugars and / or C6 sugars in oligomeric form to monomeric C5 and / or C6 sugars of at most 40% by weight, in particular at most 30% by weight or at most 20% by weight. The invention also relates to a mixture M' of compounds in aqueous solution, comprising C5 sugars and / or C6 sugars, obtained from a mixture M in aqueous phase of compounds comprising C5 sugars with 5 carbons and / or C6 sugars with 6 carbons according to the process described above.

[0065] The invention also relates to the use of the mixture M' described above as a sweet juice with a high sugar content in monomeric form, which can be recovered as such, or as an intermediate product for subsequent chemical or biochemical conversion. It is thus possible to target the production of biofuel (ethanol and other alcohols obtained by fermentation of sugars), of intermediates for chemistry known as biosourced compounds, or that of sweet juices (the concentration, water content or other minor compounds of which can be modified according to the imposed specifications).

[0066] It should also be noted that the initial sweet juices (and the juices treated according to the invention) may also contain other minor compounds, in addition to sugars, including, for example, mineral salts, sugar degradation products, carboxylic alcohols, or furan compounds. They may thus comprise furfural, and / or 5-hydroxymethyl furfural (5-HMF), in particular when the initial sweet juice comes from the pretreatment of lignocellulosic biomass under acidic conditions. It is then possible that these are also converted by the treatment according to the invention, for example into formic or levulinic acid.

[0067] The invention will be described below in more detail, using non-limiting examples and the following figures:

[0068] List of figures

[0069] Figure 1 is a very schematic representation of a process for treating lignocellulosic biomass to obtain the initial sugary juice which will be treated according to the invention.

[0070] Figure 2 is a graph which represents on the abscissa the time in hours, and on the ordinate the evolution of the glucose (curve C1) and xylose (curve C2) contents as the treatment by enzymatic hydrolysis according to the invention takes place, contents expressed in g / kg of medium (the juice after addition of the enzymes).

[0071] Description of the embodiments

[0072] The invention therefore seeks, starting from a sweet juice containing C5 sugars and / or C6 sugars (and preferably only these two types of sugars), to obtain a so-called treated sweet juice where the content of sugars in oligomeric form has been reduced, or even eliminated, in favor of the monomeric form of said sugars.

[0073] The invention can be applied to all kinds of sweet juices, as long as the juice contains at least one type of sugar in liquid phase, in particular aqueous phase, which is present both in monomeric form and in oligomeric form.

[0074] In the non-limiting example of implementation of the invention described below, the initial sweet juice is entirely derived from an acid pretreatment of a lignocellulosic biomass. This pretreatment is described below using Figure 1.

[0075] The references in the figure represent the following flows / devices / steps:

[0076] 1: Biomass

[0077] 2: Conditioning and pretreatment

[0078] 3: Pretreated biomass

[0079] 3a: Pretreated biomass towards the filtration tool

[0080] 3b: Pretreated biomass towards enzymatic hydrolysis (optional stream)

[0081] 4: Solid / liquid separation (a belt filter or a filter press for example) to extract a sweet juice, with an optional washing step

[0082] 5: Water as contact and / or washing fluid

[0083] 6: Sweet juice containing mainly C5 sugars (C5 and / or C6 sugars in monomeric and oligomeric form)

[0084] 6a: Sweet juice comprising mainly C5 sugars for the production of enzymes 6b: Sweet juice comprising mainly C5 sugars which will be treated according to the invention

[0085] 7: Pretreated and washed biomass

[0086] 8: Enzyme production reactor

[0087] 9: Mushroom inoculum (Trichoderma Reese / for example) for enzyme production

[0088] 10: Enzyme cocktail (mixture of one or more enzymes, including cellulases, beta glucosidases, xylanases, etc.)

[0089] 14: Enzymatic hydrolysis reactor.

[0090] 15: Hydrolyzate

[0091] 18: Solid / liquid separation with an optional step (not shown) of washing the solid residue with a solvent (e.g. water)

[0092] 19: Solid residue (composed mainly of lignin)

[0093] 20: Liquid product of filtration, here the filtered hydrolyzate (sugars mainly C6)

[0094] Throughout this text, and particularly in the description of the figure, the term reactor does not require that there be only one, and can be understood as a unit comprising at least one reactor. The same applies to all other devices, particularly filtration, solid / liquid separation, etc. (for the sake of brevity).

[0095] If we take the process sequence according to figure 1: Biomass 1 undergoes conditioning / pretreatment in unit 2 (possible mechanical crushing, dust removal, removal of any metal parts, then, here as an example, impregnation with an acid liquor and cooking / steam explosion of the pretreated biomass 3).

[0096] A portion of this pretreated biomass, the stream 3a, goes to a solid / liquid filtration and separation tool 4, said tool also being supplied with water 5 as contact water and / or washing water for the pretreated biomass. Firstly, the pretreated biomass is contacted with water to improve its filterability. The mixture is then filtered on a belt filter and washed with water.

[0097] Another part of the pretreated biomass, stream 3b, can go to the enzymatic hydrolysis reactor 14.

[0098] At the outlet of the separation tool 4, the liquid fraction 6 is an aqueous sugary juice comprising mainly C5 sugars, but also, to a lesser extent, C6 sugars, the C5 and / or C6 sugars being partly in monomeric form and partly in oligomeric form. All or part of this fraction, the flow 6a, can go to the enzyme production reactor 8, which produces an enzymatic cocktail 10 to feed the enzymatic hydrolysis reactor 14: this flow can thus constitute a growth and / or production substrate for the microorganisms of the Trichoderma reese type which produce this mixture of enzymes 10. The remainder of the flow 6, the flow 6b (or which constitutes the entire flow 6 if this flow is not used for the production of enzymes) is the sugary juice which will be treated according to the invention.

[0099] At the outlet of the separation tool 4, the solid fraction 7 which contains unconverted cellulose, and also unconverted residual hemicelluloses, feeds the enzymatic hydrolysis reactor 14.

[0100] Please note that throughout this text, “solid” is to be understood in opposition to “liquid”, but that any solid fraction may still contain a certain proportion of liquid (and vice versa). This proportion of solid and soluble compounds in a sample of the product can be assessed by measuring its Dry Matter (acronym “DM”) content, which is measured according to ASTM E1756 - 08(2015) “Standard Test Method for Determination of Total Solids in Biomass”. The enzymatic hydrolysis reactor 14 is fed by stream 3b, and also by stream 7 which is the solid fraction resulting from filtration in unit 4, which is therefore the solid washed pretreated biomass.

[0101] It should also be noted that the enzymatic hydrolysis operation 14 is preferably carried out in 2 stages: liquefaction with a fed-batch of substrate then enzymatic hydrolysis in batch, (possibly with two reactors in series, or in the same reactor), to work at the highest possible DM content and to convert lignocellulosic substrates whose rheology can be complex. Neutralization of the medium can be carried out before the enzymatic hydrolysis, for example by adding a basic solution in the case where the pretreatment is carried out under acidic conditions.

[0102] At the outlet of reactor 14, there is a flow 15 of hydrolyzate (sugars), which then feeds a solid / liquid separation device 18 of the filter press type for example, with an optional step of washing the solid residue with a solvent (for example water). From this device 18 emerge a liquid fraction 19, in the form of a sugary juice with mainly C6 sugars, and a solid fraction 20 which is a woody solid residue.

[0103] It should also be noted that enzyme production can be done on the biomass processing site, as shown in Figure 1. But it can also be done ex situ: ready-to-use enzymes can thus be brought to the site. This explains why, depending on the choices made on this subject, the flow can be divided into two flows 6a, 6b (with a distribution between the two flows which can be equal or not depending on the needs), or be used only for the production of enzymes (flow 6a) or only to be used as such (flow 6b).

[0104] The treatment according to the invention can advantageously be applied to the flow of sweet juice 6 or 6b.

[0105] We recall the reaction path which leads, during the pretreatment carried out in steps 2-3-4, to obtaining a sweet juice (flow 6): we start from a lignocellulosic biomass which includes cellulose and hemicelluloses. After pretreatment, at the end of step 2, we obtain an acidic pretreated marc where:

[0106] - cellulose is at least partly converted into monomers and oligomers of C6 sugars, such as glucose,

[0107] - hemicelluloses, namely galactan, mannan, arabinan, xylan are at least partly converted into monomers and oligomers of C5 and C6 sugars, such as galactose, mannose, arabinose and xylose.

[0108] The recovered sugar juice 6 at the outlet of the separation device contains these C5 and C6 sugars which are in monomeric form, but also, to a smaller extent, in oligomeric form. It may also contain very minor compounds, as already mentioned, such as 5-HMF and furfural. It does not contain sugars in polymeric form, which are insoluble and which therefore remain in the solid fraction 7 at the outlet of the separation device 4.

[0109] The composition of sweet juice 6 may vary depending on the biomass, the type of pretreatment, the pretreatment conditions, the washing conditions of the pretreated biomass.

[0110] For example, the sweet juice to be treated according to the invention may have the following composition:

[0111] - between 0.5 and 30 g of glucose per kg of juice, in particular between 5 and 15 g / kg of glucose

[0112] - between 10 and 100 g of xylose per kg of juice, in particular between 20 and 60 g / kg of xylose

[0113] - between 0.5 and 36 g / kg of potential glucose, in particular between 5.5 and 20 g / kg of potential glucose, or between 0.1 and 35 g / kg of oligomeric glucose, in particular between 0.5 and 30 g / kg of oligomeric glucose, in particular between 0.5 and 10 g / kg of oligomeric glucose

[0114] - between 10 and 130 g / kg of potential xylose, in particular between 22 and 75 g / kg of potential xylose, or between 0.1 and 30 g / kg of oligomeric xylose, in particular between 2 and 20 g / kg of oligomeric xylose with the following conventions:

[0115] - Glucose concentration is understood in the sense of monomeric glucose.

[0116] - The xylose concentration is understood in the sense of monomeric xylose.

[0117] - These concentrations can be analyzed for example by high performance liquid chromatography (HPLC) methods

[0118] - The potential sugar concentration is the measurement of the concentration of monomeric sugars after acid hydrolysis on the initial sugar juice at 120°C for 1 hour in the presence of a large excess of dilute acid: to 0.3 grams of DM of the initial juice are added 4.92 grams of 72% sulfuric acid and water, the quantity of which is calculated to obtain a mixture of 90 g.

[0119] - The concentration of oligomeric sugars corresponds to the difference between the concentration of potential sugars and the concentration of monomeric sugars.

[0120] The invention consists of treating this type of sweet juice by enzymatic hydrolysis (same enzymes as cocktail 10 or other more specific cocktail), in order to hydrolyze the sugars in oligomeric form into sugars in monomeric form, while preserving the monomeric sugars already present in the initial sweet juice.

[0121] The following non-limiting examples illustrate its implementation and use a biomass pretreated under acidic conditions, which means that the sweet juices to be treated according to the invention and which are derived therefrom can be / are already under acidic conditions. The invention applies equally well to juices resulting from the treatment / pretreatment of biomass under non-acidic conditions (in particular neutral or basic conditions).

[0122] Examples

[0123] Example 1 (comparative)

[0124] We start with an initial sweet juice M from biomass pretreated by acid impregnation of the biomass followed by steam explosion, pretreated biomass which is then washed and separated into liquid fraction (the sweet juice) and solid fraction in accordance with the teaching of the aforementioned patent FR 3 083 126, to which we will refer for more details. This is the juice from stream 6 described above.

[0125] Briefly, this pretreatment is an operation of impregnation of lignocellulosic biomass (here wheat straw) with an acid liquor, followed by a steam explosion operation of the impregnated biomass. The pretreated biomass undergoes a liquid / solid separation operation at the end of the pretreatment step, this liquid / solid separation step comprising a contacting step between the pretreated lignocellulosic biomass and water, and a filtration and optionally washing step. To obtain the initial sweet juice M to be treated according to the invention, 550 kg of pretreated biomass are mixed with 1010 kg of water, then filtered and then pressed. After filtration and pressing, 885 kg of a juice M, also called C5 hydrolysate, is obtained. The pretreatment taking place under acidic conditions, this juice has a pH of 1.7.

[0126] The concentration of compounds of interest in this initial sweet juice M is given in Table 1 below, with concentrations expressed in g per kg of juice. Note that it is possible that the composition contains other impurities, which we have not sought to analyze, for example of the salt (mineral) or acid type, 5-HMF and furfural, in (very) low levels. The dry matter content in the initial sweet juice is 6.9% by weight. Juice M contains 38.4 g / kg of sugars glucose and xylose.

[0127] The concentrations of minority sugars, such as arabinose, galactose and mannose for this biomass, are not indicated.

[0128] [Table 1]

[0129] This juice is the control juice, which will be treated according to the invention in the following examples.

[0130] Example 2 (according to the invention)

[0131] We start from the initial sweet juice M defined in example 1. It is treated according to the invention with the following operating conditions:

[0132] The initial M juice is at pH 1.7. It feeds a stirred reactor, the reaction is carried out in batch. A sufficient quantity of aqueous KOH solution is added to increase the pH of the medium to a pH of approximately 4.8.

[0133] The juice is then treated by adding 0.1 g protein / kg of juice at 49.6 g of potential sugars per kilo of juice, by adding an enzyme cocktail produced by Trichoderma reesei and having an FPase activity of 0.8 lU / mg, a beta-glucosidase activity of 35.6 lU / mg, a protein concentration of 35.8 g / kg (proteins assayed by the Kjeldhal method), then stirring the mixture for 24 hours at 50°C.

[0134] The so-called filter paper or "FPase" activity allows the overall activity of the enzyme pool (endoglucanases and exoglucanases) to be measured and is measured on Whatman No. 1 paper (procedure recommended by the IUPAC biotechnology commission): the test portion of the enzyme solution which achieves 4% progress in the enzymatic reaction in 60 minutes is determined. The principle of the filter paper activity is to determine by DNS (dinitrosalicylic acid) dosage the quantity of reduced sugars from a Whatman No. 1 paper.

[0135] Figure 2 represents:

[0136] - curve C1: the evolution of the glucose content in the juice over time

[0137] - curve C2: the evolution of the xylose content in the juice over time

[0138] Time t=0 corresponds to the addition of the enzymatic cocktail to the juice.

[0139] This graph shows an increase in the concentration of glucose and xylose sugars in the juice after only one hour of reaction, then the reaching of a plateau. The conversion of oligomers into monomeric sugars is rapid. The process according to the invention is therefore efficient, even with a rather short treatment time, which is advantageous industrially: this additional treatment is interesting, without significantly lengthening the biomass conversion process as a whole.

[0140] Table 2 below groups together the sugar concentrations of the examples once hydrolyzed according to the invention, at t=0 (sugar contents at the start of treatment) and at t = t0 + 1 hour.

[0141] C is the concentration of glucose and xylose sugars analyzed by HPLC (high performance liquid chromatography) in g / kg, and R is the change in the yield of monomeric sugars. The maximum change in sugar yield is 23%.

[0142] [Table 2]

[0143] The data in Table 2 correspond to those in the graph in Figure 2: after one hour of treatment, there is a significant conversion of oligomeric sugars into monomeric sugars, since the yield of monomeric sugars is increased by 12%, or more than 50% of the theoretical possible increase (which is 23%).

[0144] With the plateau effect shown in Figure 2, it is not necessary to continue the treatment for long periods, because the less significant improvement in the yield of monomeric sugars risks being less advantageous in terms of an extension of the reactor downtime, for example.

[0145] In conclusion, it can be seen that the enzymatic hydrolysis of juices according to the invention is favorable for obtaining juices with higher monomeric sugar contents and a lower oligomeric sugar content. The invention is flexible in terms of implementation, it can be operated at low temperature, the nature and quantity of enzymes added during the treatment can be adjusted, in particular according to the type of sweetened juice. And a significant increase in the monomeric sugar content is obtained with reasonable treatment times. If necessary, in particular if a very high purity of monomeric sugar is required, the treatment time can be extended or the parameters mentioned above can be adjusted.

Claims

Claims 1. Process for treating an initial mixture M in aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons, said C5 sugars and / or said C6 sugars being partly in monomeric form and partly in oligomeric form, characterized in that said treatment comprises an enzymatic hydrolysis step, in order to obtain a treated mixture M' enriched in C5 sugars in monomeric form and / or in C6 sugars in monomeric form and depleted in C5 sugars in oligomeric form and / or in C6 sugars in oligomeric form.

2. Method according to the preceding claim, characterized in that the temperature T of the treatment is at most 80°C or at most 70°C, in particular at least 20°C, and is preferably between 40 and 60°C.

3. Method according to one of the preceding claims, characterized in that the duration of the treatment is at least 5 minutes, and preferably at most 3 hours, in particular at most 2 hours or at most 1 hour 30.

4. Method according to one of the preceding claims, characterized in that the treatment of the initial mixture M is carried out at a pH of at least 3, in particular at least 3.5, in particular at least 4, and preferably at most 6.

5. Method according to the preceding claim, characterized in that the pH of the mixture is adjusted by adding acid or base, in particular by adding base, preferably KOH or NaOH, when the pH of the initial mixture M is less than or equal to 3.

6. Method according to one of the preceding claims, characterized in that the enzymes used for the enzymatic hydrolysis step are chosen from at least one of the following enzymes: cellulase(s) and hemicellulase(s), and preferably both cellulases and hemicellulases, in particular in the form of an enzymatic cocktail comprising endoglucanases, cellobiohydrolases, beta-glucosidases, xylanases in particular produced by bacteria or fungi of the genus Trichoderma or Aspergillus, in particular Trichoderma Reesei.

7. Method according to one of the preceding claims, characterized in that the enzymatic hydrolysis step is carried out by adding to the initial mixture M a quantity of enzymes corresponding to at least 0.05 g protein / kg juice, in particular to at least 0.08 g protein / kg of mixture, and preferably corresponding to at most 2 or 3 g protein / kg of mixture, for a concentration of 50 g of monomeric sugars / kg of mixture.

8. Method according to one of the preceding claims, characterized in that it comprises, after the step of enzymatic hydrolysis of the mixture M, at least one separation step to separate at least part of the enzymes from the rest of the mixture.

9. Method according to claim 8, characterized in that at least part of the enzymes separated in the enzymatic hydrolysis step of the mixture is recycled.

10. Method according to one of the preceding claims, characterized in that the initial mixture M comes from the treatment of biomass, in particular lignocellulosic, originating in particular from forestry and / or agricultural and / or paper residues, and / or from sacchariferous plants and / or from starchy plants and / or from Fermentable Fractions of Household Waste (FFOM).

11. Method according to the preceding claim, characterized in that the initial mixture M comes from - a biomass washing operation, - or pretreatment of the biomass including in particular impregnation of the biomass with a liquor, of neutral, acidic or basic pH or of an oxidizing nature, preferably with an acidic liquor, followed by cooking of the impregnated biomass, in particular cooking with steam explosion, possibly followed by a washing operation of the pretreated biomass, - then a solid / liquid separation of said washed or pretreated biomass.

12. Method according to one of the preceding claims, characterized in that it comprises a step subsequent to the step of enzymatic hydrolysis of the initial mixture M, said subsequent step comprising bringing said mixture in aqueous phase into contact with a microorganism consuming, among the sugars of the mixture, essentially only the C6 sugars, in order to obtain a mixture depleted in C6 sugars.

13. Mixture M' of compounds in aqueous solution, comprising C5 sugars and / or C6 sugars; - with an S1 content of C5 sugars and / or C6 sugars of between 10 and 100 g / kg of solution - including a ratio P1 of C5 sugars and / or C6 sugars in oligomeric form to monomeric C5 and / or C6 sugars of at most 40% by weight, in particular at most 30% by weight or at most 20% by weight.

14. Mixture M' of compounds in aqueous solution, comprising C5 sugars and / or C6 sugars, characterized in that it is obtained from an initial mixture M in phase aqueous solution of compounds comprising C5 sugars with 5 carbons and / or C6 sugars with 6 carbons according to the process according to one of claims 1 to 12.

15. Use of the mixture M' treated according to one of claims 13 or 14 as a sweet juice with a high sugar content in monomeric form, which can be recovered as is, or as an intermediate product for subsequent chemical or biochemical conversion.

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