WO2024115096A1

WO2024115096A1 - Method for treating an aqueous-phase mixture of compounds comprising sugars having 5 and 6 carbon atoms

Info

Publication number: WO2024115096A1
Application number: PCT/EP2023/081734
Authority: WO
Inventors: Matthieu ARAGONES; Sophie COUDERC; Gilles Ferschneider
Original assignee: IFP Energies Nouvelles
Priority date: 2022-11-28
Filing date: 2023-11-14
Publication date: 2024-06-06
Also published as: FR3142492A1

Abstract

The invention relates to a method for treating an aqueous-phase mixture (J0) of compounds comprising C5 sugars (51) having 5 carbons and C6 sugars (52) having 6 carbons, in which mixture the content by weight of C5 sugars is greater than the content by weight of C6 sugars. Said aqueous-phase mixture is brought into contact with a microorganism (2) that essentially consumes only the C6 sugars from the sugars in the mixture, in order to obtain a mixture depleted in C6 sugars.

Description

METHOD FOR TREATING AN AQUEOUS PHASE MIXTURE OF COMPOUNDS COMPRISING SUGARS WITH 5 AND 6 CARBON ATOMS.

Technical area

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

These sweet juices can be used to produce other products chemically or biochemically (e.g. alcohols such as ethanol, butanol, or other molecules, e.g. solvents such as acetone, intermediate products used in chemical industry, etc.), particularly as substitutes for petrochemical derivatives.

Prior art

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 industry, etc.

Different types of processes exist to convert lignocellulosic biomass into sweet juices depending on the type of biomass. If these are sugar-producing plants (sugar beet, sugar cane), starchy plants (corn and wheat), so-called first generation sweet juices (1 G) are obtained, for example by operations extraction.

If it is biomass such as agricultural, forestry or paper residue, so-called second generation (2G) sweet juices are obtained 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 an acidic or basic or oxidizing liquor, then cooking the impregnated biomass which is possibly accompanied by a steam explosion.

The sweet juices resulting from hydrolysis can then be further treated, for example by fermentation to convert them into alcohol, and the process also includes separation and/or purification steps.

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 essentially made up of hexoses; hemicellulose (20 to 30% by weight), which is a polysaccharide most often essentially made up of pentoses; and lignin (15 to 25 wt%), which is a polymer of 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 in a complex tangle.

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

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

Numerous technologies of interest to the invention exist for carrying out this pretreatment, which will be grouped below under the generic term "cooking": acid cooking, alkaline cooking, cooking by auto-hydrolysis, steam explosion, so-called "organosolv pulping" processes. » 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.

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 (201 1) 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.

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

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 biomass impregnated, 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 acidic liquor, then cooking and steam explosion of the impregnated biomass, with an adjustment of the acidity of the acid liquor and recycling thereof. Patent FR 3 075 201 also describes a process for pre-treating biomass by acid impregnation then steam explosion, with further washing of the reactor supply means and recycling of the washing water in the process.

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 sugars are found called C5 (i.e. 5 carbons), such as xylose and arabinose, and so-called C6 sugars (i.e. 6 carbons), such as glucose, mannose and galactose .

Particularly in the case of type 2G sweet juices resulting from the pretreatment of lignocellulosic biomass, we observe for the majority of lignocellulosic biomasses, in the sweet juice, a proportion of C5 sugars significantly higher than that of C6 sugars. By sweet juice, we mean that sugars are released by the pretreatment, and that they can be solubilized by mixing the pretreated biomass with water, for example. However, there are outlets for juices sweetened with C5 specifically (for example to convert xylose into xylitol). To a lesser extent, these sweet juices can also contain furfural or furfural derivatives, sugar degradation products, carboxylic alcohols, furan compounds, which are fermentation inhibitors, which is detrimental when we want to continue the conversion of sweet juices into alcohols by fermentation using microorganisms such as yeast or bacteria. For example, the publication “Removal of the Fermentation Inhibitor, Furfural, Using Activated Carbon in Cellulosic-Ethanol Production” by Kuang Zhang and al. (Industrial & Engineering Chemistry Research 201 1, 50, 14055-14060) demonstrates the inhibitory nature of furfural at a content of 4 g/L for the bacterium Zymomonas mobiliz A3 during fermentations.

There is therefore a need to separate or purify C5 sugars from other sugars, in particular C6 sugars, and possibly other undesirable compounds, in order to obtain pure C5 sugar juices, or at least with an increased sugar content. C5 compared to other sugars in sweet juice.

Different purification/separation techniques have already been considered.

Thus, patent application WO 2022/023686 describes a biomass treatment comprising a pretreatment then an enzymatic hydrolysis to obtain a sweet juice comprising glucose, which is then clarified and then purified by filtration on activated carbon, in order to eliminate suspended matter and capture certain soluble contaminants. It is a simple technique, but it does not allow C5 sugars to be separated from C6 sugars. The publication “Removal of furfural and HMF from monosaccharides by nanofiltration and reverse osmosis membranes” by Tielin Wang and al. (Journal of the Energy Institute 91 (2018) 473-480) proposes removing furfural and its derivative from a sweet juice to increase the fermentation yield of the sweet juice, by nanofiltration and reverse osmotic membrane techniques. It is a complex and expensive technique, and which does not make it possible to separate C5 sugars from both C6 sugars and furfural derivatives, unless the number of membrane filtration operations is increased.

Patent application US 2013/0149761 proposes to cause the precipitation of compounds which are not sugars using barium or calcium hydroxide, in order to obtain a juice which only contains sugars of the glucose type. , xylose and other sugars. This technique therefore does not make it possible to selectively separate the sugars, but simply to remove from the sugary juice minority compounds which are by-products of the pretreatment of the biomass.

Patent EP3990464 describes the separation of glucose, from a mixture of C5 sugars and C6 sugars, by adsorption of glucose on an FAU type zeolite adsorbent. It is an interesting method, which however does not allow optimal separation of glucose, which presents a selectivity which can be improved.

Patent EP3990668 describes the separation of xylose from a mixture of C5 sugars and C6 sugars, also by adsorption, here of xylose, on a zeolite adsorbent of the FAU type, with the same limit in terms of selectivity in the separation carried out . However, in one case (separation of glucose) as in the other (separation of xylose), it may be necessary, depending on the targeted applications, to obtain extremely pure separated sugars.

The invention therefore aims to remedy the aforementioned drawbacks. Its aim is to improve the techniques for selective separation of sugars in a sweet juice comprising different sugars, in particular C5 sugars and C6 sugars, aiming in particular to obtain juice enriched with C5 sugars. By “enriched” juice we mean that the sweet juice obtained by the invention has a ratio of C5/C6 sugars greater than the ratio of the sweet juice obtained without the invention.

Summary of the invention

The invention firstly relates to a process for treating a mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons, and such that said mixture is brought into contact in the aqueous phase. aqueous with a microorganism consuming, among the sugars in the mixture, essentially only the C6 sugars, in order to obtain a mixture depleted in C6 sugars.

In this text, we understand by “essentially only C6 sugars” the fact that the microorganism does not consume the other sugar(s) present in the mixture, or in such a small proportion that it is negligible/not measurable.

Preferably, the initial sweet juice comes mainly from the depolymerization of the hemicelluloses present in the lignocellulosic biomass. Preferably, sweetened juice contains more C5 sugars than C6 sugars by weight. Preferably, the sweet juice is obtained without the addition of enzymes. Finally, preferably, the initial sweet juice is obtained following the pretreatment of a lignocellulosic biomass.

Here we understand by “mostly derived” the fact that at least half of the initial sweet juice in question comes from the depolymerization in question (notably all of it).

The invention has thus developed a new technique for separating C5 and C6 sugars in a sweet juice, by selecting a microorganism which will selectively consume the sugar, here C6 sugar, which we want to remove from the sweet juice. , in order to obtain a juice said to be purified in C5 sugars, which can be used in the chemical industry in particular. It is a separation by biochemical route, rather than by physical or chemical route, which is very interesting: it is both simpler to implement than known filtration techniques, and significantly more efficient and selective.

For the purposes of the invention, the microorganism can be a single type of microorganism or an association of different microorganisms.

For the purposes of the invention, the term "mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons" could also be designated, for the sake of conciseness, under the term "sweet juice initial "

For the purposes of the invention, the term “mixture depleted in C6 sugars” could also be designated, for the sake of brevity, under the term “purified juice” or even “depleted juice”. For the purposes of the invention, “sugars” means sugars in their monomeric form (and not in an oligomeric/polymeric form). For the purposes of the invention, “juice” is equivalent to a mixture of compounds in the aqueous phase: the juice is aqueous.

The microorganism will consume the C6 sugar at least for its own maintenance or growth: the C6 sugar thus disappears. It can also, depending on the quantity of C6 sugars present in the sweet juice and depending on the quantity and type of microorganism chosen, convert the sugar into another valuable compound. (Note, however, that the microorganism can also consume a very small quantity of C5 sugar such as xylose, but it remains essentially a consumer of C6 sugars).

Thus, advantageously, the microorganism converts at least a part, in particular the majority, of the C6 sugars into alcohol, in particular into ethanol. The conversion to alcohol is not complete because the microorganism can use some sugars for non-alcohol by-products or for its metabolism. Here we understand by “the majority” the fact that the microorganism can convert at least half of the C6 sugars into ethanol (in particular more than half, for example at least two thirds of said C6 sugars, or even almost all of them).

However, ethanol is a highly valuable product, which can be more easily separated from sweet juice. For example, ethanol can be separated from an aqueous mixture by distillation or stripping. The microorganism can be separated by a solid/liquid separation operation known to those skilled in the art such as centrifugation for example.

The mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons (the initial sweet juice) can also include furfural, particularly when the sweet juice comes from the pretreatment of lignocellulosic biomass under acidic conditions. In this case, the microorganism can be chosen so that it also consumes furfural, in particular by converting at least part of the furfural into alcohol, in particular into furfuryl alcohol. Here again, this consumption is very interesting, to the extent that furfural is known to be an inhibitor of fermentation reactions: we thus improve the productivity and yields of possible biochemical conversions planned for the purified juice.

The aqueous phase mixture of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons (the initial sweet juice) can also include 5-hydroxymethyl furfural (5-HMF), particularly when the sweet juice comes from the pretreatment of lignocellulosic biomass in acidic conditions. In this case, the microorganism can be chosen so that it also consumes 5-hydroxymethyl furfural, in particular by converting at least part of the 5-hydroxymethyl furfural in particular into 5-hydroxymethyl furfuryl alcohol. Here again, this consumption is very interesting, to the extent that 5-HMF is also known to be an inhibitor of fermentation reactions: we thus improve the yields of possible biochemical conversions planned for the purified juice. The aqueous phase mixture of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons (the initial sweet juice) can also include other compounds for degrading C5 or C6 sugars, in particular furan compounds, acids organic, compounds with high molecular weight, particularly when the sweet juice comes from the pretreatment of lignocellulosic biomass at high temperature or by solvent. What is advantageous, and surprising, in the treatment process of the invention is that this treatment remains effective even when the initial sweet juice contains this type of compounds, whereas one could have feared that they would not play a role. potentially a role of inhibitor(s) with respect to the conversion of C6 sugars according to the invention.

The aqueous phase mixture of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons may also include mineral salts.

The aqueous phase mixture of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons may also include sugars in oligomeric form.

The microorganism can advantageously be chosen from yeasts, bacteria, fungi.

It can for example be chosen from yeasts in the genus Saccharomyces, in particular the species Saccharomyces cerevisae or, from bacteria, in the genus Corynebacterium, in particular the species Corynebacterium, Zymomonas mobilis. The choice of Saccharomyces cerevisae, in particular, is interesting, because the wild strain naturally consumes C6 sugars, but not C5 sugars, and work has been carried out to genetically modify it so that it is able to consume both types of sugars. : the invention thus recommends returning to the non-genetically modified version of this yeast/the native version, precisely to exploit its selectivity with respect to sugars, hitherto perceived as a disadvantage.

Preferably, the treatment of the mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons with the microorganism is carried out at a temperature between 20 and 60°C, in particular between 25 and 40°C. C, especially between 30 and 35°C. The treatment temperature must be adapted according to the chosen microorganism, each microorganism having a temperature range where its activity is maximum.

Advantageously, the treatment of the mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons with the microorganism has a duration of between 5 minutes and 15 hours, in particular between 10 minutes and 8 hours. Processing can be carried out in batches, in a fed-batch of sweet juice, or continuously.

The selected microorganism which will selectively consume the C6 sugar according to the invention can be added all at once directly with the sweet juice, or with sequential feeding, independently or not of the feeding of the sweet juice.

Here again, the duration of the treatment must be adjusted depending on the type and quantity of microorganism used, depending on the quantity of C6 sugars in the sweetened juice, depending on the quantity of C6 sugars that we wish to reduce in the sweet juice, depending on the quantity of compounds known as inhibitors of microorganisms (furan compounds such as furfural or 5-hydroxymethyl furfural, carboxylic acids such as acetic acid or formic acid, or phenolic compounds) for Saccharomyces Cerevisae yeast and depending on the final acceptable C6 sugar content in the purified juice.

Preferably, the treatment of the mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons with the microorganism is carried out at a pH of between 2 and 10, in particular between 4 and 6, in particular between 4.5 and 5.5.

The treatment of the mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons with the microorganism can be carried out under aerobic or anaerobic conditions, depending on the microorganism chosen and the desired product from the consumption of the sugars. .

Advantageously, the concentration of microorganisms in the aqueous phase mixture of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons is between 1 and 200 g/kg of C6 sugars, in particular between 10 and 30 g/kg. kg of C6 sugars.

The mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons preferably comes from the treatment of biomass, in particular lignocellulosic, originating in particular from forestry and/or agricultural and/or paper residues, and/or sacchariferous plants and/or starchy plants and/or Fermentable Fractions of Household Waste (FFOM).

The aqueous phase mixture of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons can come from a biomass washing operation, or from a hydrolysis or pretreatment of the biomass including in particular an impregnation of the biomass with an acidic or basic or oxidizing liquor followed by cooking of the impregnated biomass, in particular cooking with steam explosion. The aqueous phase mixture of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons can come from a solid/liquid separation step using a biomass pretreated by steam explosion plumped with a solvent (from water for example).

The invention also relates to the purified juice obtained according to the treatment of the invention: it thus also relates to a mixture of compounds in aqueous solution, which comprises C5 sugars and conversion products of C6 sugars and optionally other minority compounds present in the juice to be purified.

For example, these conversion products of C6 sugars and other minority compounds are alcohols such as ethanol, 5-hydroxymethylfurfuryl alcohol and furfuryl alcohol, particularly in the following concentrations; - C5 sugars: between 10 and 100 g/kg of solution, in particular between 20 and 60 g/kg of solution- ethanol: between 1 and 12 g/kg of solution, in particular between 2 and 7 g/kg of solution- alcohol 5 -hydroxymethylfurfuryl: between 0.1 and 9 g/kg of solution, in particular between 0.2 and 4 g/kg of solution - furfuryl alcohol: between 0.05 and 9 g/kg of solution, in particular between 0.1 and 3 .8 g/kg, notably between 0.1 and 1.8 g/kg of solution

The purified juice according to the invention can thus contain alcohols, in particular an alcohol which results from the conversion of C6 sugars under the action of the microorganism, for example ethanol, butanol or isopropanol, and alcohols resulting in particular of the conversion of the two inhibitors (furfural and 5-HMF), also under the action of the microorganism.

The conversion products of C6 sugars can also be microorganisms themselves, in particular yeasts, in the case where the treatment according to the invention is carried out under aerobic conditions. C6 sugars are consumed by the microorganism for its own growth or maintenance

Advantageously, the purified juice according to the invention contains less than 0.5 g/kg of C6 sugar solution, and preferably no C6 sugar, less than 0.1 g/kg of furfural solution, in particular no furfural, and less of 0.1 g/kg of 5-hydroxymethyl furfural solution, in particular no 5-hydroxymethyl furfural. With the invention, we can therefore go so far as to completely remove (by consumption or conversion) all the C6 sugar from the initial sweet juice, and the same is true for the two furan derivatives: depending on the constraints of the degree of purity of the juice purified in C5 sugars, we can therefore eliminate the C6 sugar, or at least maintain it at a very low content, at the level of an acceptable content of impurities for example. Advantageously, the purified juice contains the following compounds:

- glucose: between 0 and 0.3 g/kg of solution xylose: between 9 and 91 g/kg of solution

- arabinose: between 1 and 9 g/kg of solution

- galactose: between 0 and 0.1 g/kg of solution

- mannose: between 0 and 0.1 g/kg of solution

- 5-hydroxymethylfurfuryl alcohol: between 0.1 and 9 g/kg of solution

- furfuryl alcohol: between 0.05 and 3.8 g/kg of solution

This purified juice is preferably obtained from a mixture in the aqueous phase of compounds comprising C5 sugars with 5 carbons and C6 sugars with 6 carbons according to the process of the invention.

The purified juice according to the invention may also contain carboxylic alcohols, such as acetic acid, formic acid or levullinic acid. Advantageously, the purified juice according to the invention contains less than 8 g/kg of acetic acid acid, preferably less than 5 g/kg acetic acid. The purified juice according to the invention may also contain sugars in oligomeric form, for example compounds of the arabinoxylan type, pentose and hexose oligomers. Advantageously, the purified juice according to the invention contains between 0% and 100% of oligomeric sugars relative to the monomeric sugars, preferably between 0.1% and 50%, in particular between 0.1% and 20% of oligomeric sugars relative to the monomeric sugars. monomeric sugars.

The invention also relates to the use of the mixture treated according to the invention/described above (purified juice), which is therefore a sweet juice with a very high content of C5 sugars (compared to C6 sugars), to convert C5 sugars by chemical means, in particular to convert xylose into xylitol, or by biochemical means, in particular to serve as a carbonaceous substrate for the propagation of yeasts or for the induction of fungi for the production of enzymes. Xylose is considered a platform molecule, and many valorization routes are possible (xylitol, monoethylene glycol, propanol, butanol, lactic acid, succinic acid, etc.).

The invention will be described below in more detail, using non-limiting examples and the following figures: List of Figures

Figure 1 is a very schematic representation of a reactor implementing the treatment according to the invention, and representing the compounds of interest entering and leaving the reactor.

Figure 2 is a graph representing on the abscissa the duration of the treatment according to the invention (in hours) and on the ordinate the evolution of the concentrations of compounds in a mixture of sugars treated according to the invention, in g/kg of reaction medium .

The abbreviations in Figure 2 mean: glu: glucose xyl: xylose ara: arabinose gal: galactose man: mannose

Figure 3 is a very schematic representation of a reactor implementing the treatment according to the invention, and representing the compounds of interest entering and leaving the reactor under aerobic conditions, to propagate the microorganisms (yeasts).

Description of embodiments

The invention therefore seeks, starting from a sweet juice containing C5 sugars and C6 sugars (and preferably only these two types of sugars), to obtain a so-called purified juice where the C6 sugar content has been reduced. , or even eliminated, by biochemical treatment.

The biochemical treatment is a bringing into contact of the juice sweetened with C5 and C6 sugars, juice obtained by treatment of material based on lig nocellu losic biomass and comprising mainly C5 sugar, with a microorganism which selectively consumes the C6 sugars: the C5 sugars are not modified, remain almost intact in the juice, while the C6 sugars will gradually be consumed by the microorganism for its own growth and/or to be converted into another compound, in the form of alcohol in the case where the biochemical reaction is a fermentation. The operating conditions of the treatment are adapted function of the initial content of C6 sugars and the final content of C6 sugars which is acceptable (no more C6 sugars at all, or a content lower than a given value): temperature, pH, duration, aeration, choice and concentration of the microorganism .

Examples

Example 1 (comparative)

We start from an initial sweet juice JO from biomass pretreated by acid impregnation of the biomass then explosion with steam, in accordance with the teaching of the aforementioned patent FR 3 083 126, to which we will refer for more details.

Briefly, this pretreatment is an operation of impregnation of lignocellulosic biomass with an acidic liquor, followed by an operation of steam explosion of the impregnated biomass. At least part of the pretreated biomass undergoes a liquid/solid separation operation at the end of the pretreatment step, this liquid/solid separation step comprising a contact step between the pretreated lignocellulosic biomass and water, and a filtration and optionally washing step. To obtain JO juice, 550 kg of pretreated biomass are mixed with 1010 kg of water, then filtered and then pressed. After filtration and pressing, 885 kg of JO juice, also called C5 hydrolyzate, is obtained. As pretreatment takes place under acidic conditions, this juice has a pH of 2.

The concentration of compounds of interest in this OJ juice 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 attempted to analyze, for example of the salt (mineral) or acid type, in very low levels.

[Table 1]

We see that 82% of the sugars in JO juice are C5 sugars, that 18% of the sugars are C6 sugars, and that 5-HMF and furfural, known to inhibit biochemical reactions that can subsequently be considered to be carried out on the sugars (for example for ethanolic fermentations using yeasts).

Sweet juices from pretreated biomass generally include a much higher quantity of C5 sugars than C6 sugars, and there is a need, to valorize them, to separate the C5 sugars from the C6 sugars. However, the techniques currently available are unsatisfactory (membrane filtration in particular), because they are complex to implement and generally do not make it possible to achieve the desired level of C5 sugar purity in the juice separated from C5 sugars.

For example, the publication “Removal of furfural and HMF from monosaccharides by nanofiltration and reverse osmosis membranes” by Tielin Wang and al. (Journal of the Energy Institute 91 (2018) 473-480) concludes that the most efficient membranes for separating 5-HMF and furfural from a sweet juice are the Desal-5 DK and Alfa Laval-NF membranes (less than 2% retention for 5-HMF and furfural) but they lead to too significant a loss of sugars (2 to 8% for glucose and 10 to 20% for xylose).

The publication “Removal of the Fermentation Inhibitor, Furfural, Using Activated Carbon in Cellulosic-Ethanol Production” by Kuang Zhang and al. (Industrial & Engineering Chemistry Research 201 1, 50, 14055-14060) concludes that the commercial activated carbon Norit_1240 from Norit Company can selectively reduce the furfural concentration of a model sweet juice solution from 4 g/L to 0 g/L, but C6 sugars (here glucose) are not selectively separated from C5 sugars (here xylose).

Example 2 (according to the invention)

We start from the sweet juice J0 defined in example 1. A fermentation of the J0 juice is carried out with a microorganism, the yeast Saccharomyces cerevisae, in a native version, not genetically modified, and which has the particularity of selectively consuming C6 sugars, and not C5 sugars.

The yeast is inoculated at 0.5 g of yeast per kg of solution, or 60 g of yeast per kg of C6 sugars. The experiment was carried out in a bioreactor for 6 hours, at 33°C and pH 5.3. The treatment was carried out under anaerobic conditions to maximize the production of ethanol from C6 sugars by fermentation, and not the production of biomass. This reaction can be carried out in batch, fed-batch or continuously. They can then be separated to treat/valorize them separately.

The reactions that result from the action of the yeast are schematized in Figure 1, which represents bioreactor 1, with the following inputs:

2: yeast

3: a base (to regulate the pH and maintain the reaction medium in the conditions necessary for the activity of the chosen yeast), of the NH ₄ OH, KOH or NaOH type for example

4: nutrients for the yeast

5: JO juice, which contains in the aqueous phase C5 51 sugars, C6 52 sugars, 5-HMF 53 and furfural 54, and with the outgoing products:

6: emission of CO2 in the gas phase, evacuated in the upper part of the bioreactor, witness to the activity of the yeast,

5': withdrawal of a juice J1, resulting from the juice JO after action of the yeast on the juice JO, and which contains the yeast 2, C5 sugars 51, 5-hydroxymethylfurfuryl alcohol 55, furfuryl alcohol 56, ethanol 57 and yeasts 2.

The quantities of inputs (in addition to the concentrations indicated above) are specified below: In a stirred tank, 985 kg of JO juice are mixed with:

- 0.4 kg of urea (for nitrogen needs)

- 0.5 kg of dry yeast S. Cerevisae

- 5 kg of yeast extract (nutrients)

- 9.1 kg of a 50% KOH solution to adjust the pH to 5.3

The concentration of compounds of interest in the mixture in the initial state is given in Table 2 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 attempted to analyze, such as salt or acid, in very low levels. [Table 2]

At the start of the reaction, the reaction medium contains:

- 8.3 kg of C6 sugars (glucose + galactose + mannose)

- 37.3 kg of C5 sugars (xylose + arabinose)

- 0.4 kg of 5-HMF

- 0.2 kg of furfural

The fermentation process is detailed below:

The fermentation lasted 6 hours and shows that the glucose, galactose and mannose (C6 sugars, dotted lines) are completely consumed. Arabinose and xylose (C5 sugars) are not consumed. Furfural and 5-HMF were consumed by the yeast which detoxifies its environment, to produce furfuryl alcohol and 5-hydroxymethyl furfuryl alcohol (also called 2,5-Bis(hydroxy methyl)furan) which do not inhibit yeast.

We therefore found that the initial JO juice, composed of 45.6 kg of monomer sugars (18% C6 sugars/82% C5 sugars) is purified in a few hours into a J1 juice composed of 37.3 kg of monomer sugars (0 % C6 sugars and 100% C5 sugars).

Under anaerobic conditions, at 33°C and pH 5.3, the yeasts consume the C6 sugars (mainly to produce ethanol) and the inhibitors (5-HMF and furfural) in six hours.

At the end of the reaction, the medium contains a juice J1 whose composition is given in Table 2 below, with concentrations expressed in g per kg of medium: [Table 3]

We therefore have after 6 hours of reaction:

- 0 kg of C6 sugars (glucose + galactose + mannose), i.e. consumption of 100% of C6 sugars

- 37.3 kg of C5 sugars (xylose + arabinose), i.e. a consumption of only 1.6% of C5 sugars

- 0 kg of 5-HMF, i.e. 100% consumption of 5-HMF

- 0 kg of furfural, i.e. consumption of 100% of furfural - 0.4 kg of 5-hydroxymethylfurfurlic alcohol

- 0.2 kg of furfuryl alcohol

2.9 kg of ethanol, i.e. an ethanol/C6 sugar yield of 0.35 kg of ethanol/kg _SU cres ce

Figure 2 represents the evolution of the contents of the compounds in JO juice throughout the fermentation period, by sampling and analysis.

The data in Table 2 and the graph in Figure 2 confirm that yeast has no action on C5 sugars, the content of which remains constant. In contrast, the yeast completely consumed both all the C6 sugars and the two inhibitors, converting them at least in part into alcohols (another part that can be consumed by the yeast for its own growth). Treatment with yeast therefore gives spectacular results, with a juice purified in C5 sugars which no longer contains C6 sugars at all and whose inhibitors have, simultaneously, also been completely eliminated.

The fact that at least part of the C6 sugars has been transformed into ethanol is also very advantageous, because ethanol is a highly valuable product and can be produced in processes dealing with lignocellulosic biomass.

We also observe from the data in the graph in Figure 2 that the fermentation duration for the C6 sugars to be entirely consumed is 6 hours, which is a reasonable duration, which can be further lowered by playing, in particular, on the quantity of added yeast, or if lowered but not zero levels of C6 sugars are acceptable. Experiments have thus been carried out successfully with much shorter fermentation times, in particular from 30 minutes to 2 hours, for example a duration of around 1 hour.

In conclusion, the invention manages to purify mixtures of sugars to obtain juices containing the type of sugars that we want to preserve, by choosing a selective microorganism, which can furthermore, optionally, also consume/eliminate in the sugar mixtures of undesirable compounds/impurities, including inhibitors of biochemical reactions such as furan derivatives. Its implementation is simple, in one fermentation step, and its effectiveness is formidable, with possibly complete elimination of the compounds that we want to remove from the sweet juices.

Example 3 (according to the invention) We start from the JO sweet juice defined in Example 1. An aerobic fermentation of the JO juice is carried out with a microorganism, the yeast Saccharomyces cerevisae, in a native version, not genetically modified, and which has the particularity of selectively consuming C6 sugars. , and not C5 sugars.

The experiment was carried out in a bioreactor for 30 hours, at 30°C and pH 5. The treatment was carried out under aerobic aerobic conditions (at 0.1 vvm, volume of air per volume of medium per minute) to maximize the production of biomass and not the production of ethanol from C6 sugars. This reaction can be carried out in batch, fed-batch or continuously. In this example, the reaction is carried out in a fed-batch of sweet juice for 24 hours then an additional mixing time of 6 hours without fed-batch.

The reactions which result from the action of the yeast are schematized in Figure 3, which represents bioreactor 1, with the following inputs:

2: yeast

4: nutrients for the yeast

5: JO juice, which contains in the aqueous phase C5 51 sugars, C6 52 sugars, 5-HMF 53, furfural 54, and acetic acid 60 and with the output products:

5': withdrawal of a juice J1, resulting from the juice JO after action of the yeast on the juice JO, and which contains the propagated yeast 2, C5 sugars 51, 5-hydroxymethylfurfuryl alcohol 55 and alcohol furfuryl 56.

7: an air supply to be in aerobic condition

The quantities of inputs (in addition to the concentrations indicated above) are specified below: In a stirred tank, 505.4 kg of water are mixed with:

- 12.1 kg of urea (for nitrogen needs)

- 2 kg of alkali at 23.5% by weight (nutrient for yeast)

- 0.5 kg of phosphoric acid (nutrient for yeast) - 4.5 kg of a mineral cocktail (nutrients for yeast)

- 1.8 kg of sulfuric acid at 37% by weight to reduce the pH to 5.0

- 1.8 kg of dry yeast S. Cerevisae

The sweet juice is then added for 24 hours at a linear flow rate of 19.54 kg/h. In total, 469 kg of sweet juice are added in fed batch. 2.8 kg of alkali at 23.5% by weight are also added to regulate the pH to 5.0.

The fed-batch of 469 kg of sweet juice added in the middle:

- 3.9 kg of C6 sugars (glucose + galactose + mannose)

- 17.5 kg of C5 sugars (xylose + arabinose)

- 0.2 kg of 5-HMF

- 0.1 kg of furfural

- 0.6 kg of acetic acid

- 1.8 kg of yeast

The fermentation process is detailed below:

The aerobic fermentation lasted 30 hours (24 hours of fed-batch of sweet juice and 6 hours of mixing) and shows that glucose, galactose and mannose are consumed at 91%. Arabinose and xylose (C5 sugars) are not consumed. Furfural and 5-HMF were 100% consumed by the yeast which detoxifies its environment, to produce furfuryl alcohol and 5-hydroxymethyl furfuryl alcohol (also called 2,5-Bis(hydroxy methyl)furan ) which do not inhibit yeast. Acetic acid was also 100% consumed.

We therefore found that the initial JO juice, composed of 21.4 kg of monomeric sugars (18% C6 sugars/82% C5 sugars) is purified in 30 hours into a J1 juice composed of 17.3 kg of monomeric sugars (2 % C6 sugars and 98% C5 sugars) while multiplying the yeasts by a multiplication factor of 2.1.

So after 30 hours we have:

- 0.3 kg of C6 sugars (glucose + galactose + mannose), i.e. a consumption of 91% of C6 sugars

- 17 kg of C5 sugars (xylose + arabinose), i.e. a consumption of only 2.6% of C5 sugars - 0 kg of 5-HMF, i.e. 100% consumption of 5-HMF

- 0 kg of furfural, i.e. consumption of 100% of furfural

- 0.2 kg of 5-hydroxymethylfurfurlic alcohol

- 0.1 kg of furfuryl alcohol

- 0 kg of acetic acid, i.e. 100% consumption of acetic acid

- 3.8 kg of yeast, i.e. a multiplication factor of 2.1.

Yeast therefore has no action on C5 sugars, the content of which remains constant. On the other hand, the yeast completely consumed both all the C6 sugars and the two inhibitors (5-HMF and furfural) as well as the acetic acid, to convert them at least in part into alcohols (another part can be consumed by yeast for its own growth). The treatment with yeast therefore gives spectacular results, with a juice purified in C5 sugars which no longer contains C6 sugars at all, the inhibitors of which have been, simultaneously, completely eliminated while also multiplying the yeast.

In conclusion, the invention manages to purify mixtures of sugars to obtain juices containing the type of sugars that we want to preserve, by choosing a selective microorganism, which can furthermore, optionally, also consume/eliminate in the sugar mixtures of undesirable compounds/impurities, including inhibitors of biochemical reactions such as furan derivatives. Its implementation is simple, in one aerobic fermentation step, and its effectiveness is formidable, with possibly complete elimination of the compounds that we want to remove from the sweet juices.

It makes it possible to obtain very pure C5 sugar juices, with no or a very low content of C6 sugars, with a purity which until now could not be achieved by separation methods, such as selective adsorption on zeolites. , as described in the patents cited in the preamble to this application.

It is also very interesting, in that it does not really lead to a separation between two types of sugar, but to obtain a very pure type of sugar, and to convert the other sugar (C6 sugar) into a product recoverable as ethanol. With the combination of C5 sugars and ethanol resulting from the conversion of C6 sugars obtained with the process according to the invention, several possibilities are available, in particular depending on the recovery routes envisaged and the proportion between the two types of sugars. in the initial sweet juice. Thus, we can then separate the C5 sugars from the ethanol, and obtain on the one hand very pure C5 sugars and on the other hand ethanol, which can be recovered separately. They can also be kept together to a common valuation. We can still look at a low ethanol content with C5 sugars, to benefit from the preservative effect of alcohol on sugars....

5

Claims

1. Process for treating a mixture (JO) in the aqueous phase of compounds comprising C5 sugars (51) with 5 carbons, C6 sugars (52) with 6 carbons and whose content by weight of C5 sugars is greater than the content of weight of C6 sugars, furfural (54), and 5-hydroxymethyl furfural 5-HMF (54), characterized in that said mixture is brought into contact in the aqueous phase with a microorganism (2) chosen from yeasts in the genus Saccharomyces, in particular the species Saccharomyces cerevisae, consuming, among the sugars in the mixture, only C6 sugars (52), in order to obtain a mixture depleted in C6 sugars, in that the microorganism consumes furfural , in particular by conversion of furfural into alcohol, in particular into furfuryl alcohol (56), and in that the microorganism also consumes 5-hydroxymethyl furfural, in particular by conversion of 5-hydroxymethyl furfural into 5-hydroxymethyl furfuryl alcohol (55), said process is carried out at a temperature of between 20 and 60°C, for a duration of between 5 minutes and 15 hours, in anaerobic conditions, and at a pH of between 2 and 10, characterized in that said microorganism (2 ) converts at least a part, in particular the majority, of the C6 sugars (52) into alcohol (57), in particular into ethanol.

2. Method according to one of the preceding claims, characterized in that the treatment of the mixture (JO) in the aqueous phase of compounds comprising C5 sugars (51) with 5 carbons and C6 sugars (52) with 6 carbons with the micro -organism (2) is carried out at a temperature between 25 and 40°C.

3. Method according to one of the preceding claims, characterized in that the treatment of the mixture (JO) in the aqueous phase of compounds comprising C5 sugars (51) with 5 carbons and C6 sugars (52) with 6 carbons with the micro -organism (2) has a duration of between 10 minutes and 8 hours.

4. Method according to one of the preceding claims, characterized in that the treatment of the mixture (JO) in the aqueous phase of compounds comprising C5 sugars (51) with 5 carbons and C6 sugars (52) with 6 carbons with the micro -organism (2) is operated at a pH between 4 and 6.

5. Method according to one of the preceding claims, characterized in that the concentration of microorganisms (2) in the mixture (JO) in the aqueous phase of compounds comprising C5 sugars (51) with 5 carbons and C6 sugars ( 52) with 6 carbons is between 1 and 200 g/kg of C6 sugars, in particular between 10 and 30 g/kg of C6 sugars.

6. Method according to one of the preceding claims, characterized in that the mixture (JO) in the aqueous phase of compounds comprising C5 sugars (51) with 5 carbons and C6 sugars (52) with 6 carbons comes from treatment of biomass, in particular lignocellulosic, coming in particular from forestry and/or agricultural and/or paper residues, and/or from sacchariferous plants and/or starchy plants and/or from Fermentable Fractions of Household Waste (FFOM).

7. Method according to claim 6, characterized in that the mixture (JO) in the aqueous phase of compounds comprising C5 sugars (51) with 5 carbons and C6 sugars (52) with 6 carbons comes from a washing operation of the biomass, or a hydrolysis or pretreatment of the biomass comprising in particular an impregnation of the biomass with an acidic or basic or oxidizing liquor followed by cooking of the impregnated biomass, in particular cooking with steam explosion.

8. Mixture (J1) of compounds in aqueous solution obtained according to the process according to any one of claims 1 to 7, characterized in that it is obtained from a mixture (JO) in aqueous phase of compounds comprising C5(51) sugars with 5 carbons and C6(52) sugars with 6 carbons.

9. Mixture (J1) of compounds in aqueous solution according to the preceding claim, characterized in that it comprises C5 sugars (51), ethanol (57), 5-hydroxymethylfurfuryl alcohol (55) and furfuryl alcohol (56), in particular in the following concentrations;

- C5 sugars: between 10 and 100 g/kg of solution

- ethanol: between 1 and 12 g/kg of solution

- 5-hydroxymethylfurfuryl alcohol: between 0.1 and 8.6 g/kg of solution

- furfuryl alcohol: between 0.05 and 3.6 g/kg of solution

10. Mixture (J1) according to claim 8 or 9, characterized in that it contains less than 0.3 g/kg of C6 sugar solution (52), and preferably no C6 sugar, less than 0.1 g /kg of furfural solution (54), in particular no furfural, and less than 0.1 g/kg of 5-hydroxymethyl furfural solution (53)l, in particular no 5-hydroxymethyl furfural.

11. Mixture (J1) according to one of claims 8 to 10, characterized in that it comprises: - glucose: between 0 and 0.3 g/kg of solution - xylose: between 9 and 91 g/kg of solution

- arabinose: between 1 and 9 g/kg of solution

- galactose: between 0 and 0.1 g/kg of solution

- mannose: between 0 and 0.1 g/kg of solution - 5-hydroxymethylfurfuryl alcohol: between 0.1 and 9 g/kg of solution

- furfuryl alcohol: between 0.05 and 3.8 g/kg of solution

12. Use of the mixture (J1) according to one of claims 8 to 1 1 to convert the C5 sugars (51) chemically, in particular to convert xylose into xylitol, or biochemically, in particular to serve as a carbonaceous substrate for the propagation of yeasts or for the induction of fungus, s for the production of enzymes.