Method for converting lignocellulosic biomass

Abstract

The present invention relates to a method for converting lignocellulosic biomass by bringing, in the aqueous phase, pretreated lignocellulosic biomass into contact with at least one biocatalyst (3) in a first reactor (1) containing a reaction medium comprising the pretreated lignocellulosic biomass (2) in the aqueous phase and the biocatalyst, the method comprising: - (a) a first step of liquefaction by adding the pretreated lignocellulosic biomass and at least one biocatalyst to the reactor without removing any or all of the reaction medium from the reactor; - followed by (b) a second step of continuous liquefaction with the continuous removal of some of the reaction medium from the first reactor, the addition of at least one biocatalyst, and the continuous addition of pretreated lignocellulosic biomass.

Description

[0001] LIGNOCELLULOSIC BIOMASS CONVERSION PROCESS

[0002] Technical field

[0003] The invention relates to a process for converting lignocellulosic biomass to produce so-called second generation (2G) sugars (or sweet juices).

[0004] These sugars can be used to produce other products biochemically and / or catalytically (for example alcohols such as ethanol, butanol, or other molecules, for example xylitol, solvents such as acetone, etc.).

[0005] This process generally involves pre-treatment of the biomass, which may include, for example, impregnation with a liquor containing a chemical catalyst, such as an acid, a base, or an oxidizing compound, followed by cooking of the impregnated biomass, which cooking may be coupled with a steam explosion. Once pre-treated, the biomass is then converted into sugars by enzymatic hydrolysis, generally under the action of an enzymatic cocktail comprising at least one cellulolytic enzyme. The sugars thus formed may be fermented into alcohol under the action of yeasts or bacteria, either in a fermentation step separate from the enzymatic hydrolysis step, or simultaneously with the enzymatic hydrolysis.In the latter case, we speak of SSCF, the Anglo-Saxon acronym for "Simultaneous Saccharification and Co-Fermentation" when the fermented sugars are a mixture of C5 and C6 sugars obtained by enzymatic hydrolysis (i.e. with 5 or 6 carbons), or of SSF for "Simultaneous Saccharification and Fermentation" when only C6 sugars are fermented.

[0006] Prior art

[0007] The invention is more particularly concerned with the enzymatic hydrolysis of pretreated biomass, possibly associated with SSF or SSCF type fermentation. The pretreated biomass is the substrate for the enzymatic hydrolysis reaction. The residence time of the substrate is defined as the average residence time of the substrate under the reaction conditions. The cycle time is considered here as the time between two cleanings. The enzymatic hydrolysis can be carried out in different ways. Conventionally, enzymatic hydrolysis can be carried out in batch, fed-batch or continuous.

[0008] Batch operation can be summarized as follows: the substrate is added to the conversion reactor at the beginning of the cycle, then left in contact with the biocatalysts for a reaction time necessary to achieve the targeted conversion, before completely emptying the reaction medium. In this configuration, the substrate residence time is homogeneous, since all the substrate is added and removed at the same time. The reactor cycle time consists of the reactor preparation time, the reaction time (the substrate residence time) and the emptying and cleaning time.

[0009] A continuous operation can be summarized as follows: the substrate is added and a fraction of the reaction medium is withdrawn over time. These additions and withdrawals can be done in true continuous mode, or in pseudo-continuous mode: that is to say, the time between withdrawals / additions is much faster than the average residence time of the substrate. In this configuration, the reactor cycle time can be significantly extended. The average residence time of the substrate can be defined as the ratio between the volume of the reaction medium in the reactor and the average hourly flow rate of added substrate.This configuration allows better use of the available reactor volume, however, it has the disadvantage of having heterogeneity in the residence time of the substrate and biocatalysts in the reactor: in fact, the withdrawal carried out is a withdrawal of the reaction medium and therefore a part of the substrate and / or the added biocatalysts will be removed during the withdrawal without having achieved the average residence time. Conversely, another part will remain longer than the average residence time in the reactor. Therefore, in the case of reactions involving biocatalysts, i.e. microorganisms such as yeasts or bacteria, there is a greater risk of performance drifts linked either to the appearance of contamination or to an evolution of the biocatalyst (for example loss of a genetic modification of interest) due to the extended residence time for part of the inventory.

[0010] Fed-batch operation is quite similar to batch operation: part of the substrate is gradually added to the reactor while the desired reaction(s) have started in the reactor, then the entire reactor inventory is emptied at the end of the cycle. This type of operation is classic for bioprocesses and makes it possible to circumvent the typical limitations of bioconversions: fed-batch operation is, for example, carried out when the medium has too high a content of toxic molecules, or when the initial rheology of the medium is difficult.

[0011] It is thus possible, as described in patent EP-3 461 902, to carry out enzymatic hydrolysis in batch mode with a sequential feed of pretreated biomass (called "fed-batch" feed according to the English term), where the sequential addition to the hydrolysis reactor is carried out at increasingly longer intervals over time, so as to obtain a predetermined final dry matter content, without withdrawal during the hydrolysis.This type of feed is interesting in that it allows for an improved sugar conversion yield and allows for working at a high dry matter content, which leads to high concentrations of the product of interest in the medium, and also because it allows for better control of the viscosity variations of the reaction medium: as the hydrolysis reaction progresses, the reaction medium becomes less and less viscous, and pre-treated biomass can thus be added to increase the dry matter content of the reaction medium. However, this solution has limitations, similar to the limitations of batch operation, with draining and cleaning times that are regularly necessary. These times cannot be compressed and reduce the utilization rate of the tank, particularly when the reaction time is less than 24 hours, which penalizes productivity.

[0012] As described, for example, in patent WO 2013 / 088001, it is also known to decompose the enzymatic hydrolysis into two stages, each carried out in a specific reactor:

[0013] - a so-called liquefaction stage, which corresponds to the start of hydrolysis, during which the reaction medium is viscous and requires a complex stirring system and significant stirring energy for the reactor. On the other hand, the residence time of the biomass is generally short, which makes it possible to limit the volume of the reactors to be installed with such a stirring system.

[0014] - a next step, which corresponds to saccharification in the case where hydrolysis is only prolonged, or to SSF or SSCF in the case where microorganisms are introduced to ferment the sugars while hydrolyzing or hydrolyzing and fermenting, in another reactor. This step requires simpler stirring, less energy, but a longer residence time and therefore a larger reactor volume. According to the teaching of this patent, a rheological characteristic of the reaction medium is monitored during the first liquefaction step, so as to adjust the feed rates of the reactor in pretreated biomass / water / enzymes / other inputs (chemical products, acid or base for example), and thus optimize the liquefaction.This solution is interesting in that it allows for efficient management of at least the first liquefaction stage, regardless of the nature of the biomass, without having to characterize it. On the other hand, in industrial production, it requires a significant number of reactors over-equipped with stirring equipment and of relatively small volume to carry out the liquefaction. Batch mode operation also penalizes productivity due to the frequency of emptying, cleaning and filling operations.

[0015] The invention then aims to overcome the drawbacks of previous solutions. It aims to improve enzymatic hydrolysis processes, in particular to reduce and simplify the equipment required and / or to reduce the energy consumption of said equipment, without degrading - or by increasing - biomass conversion yields. Summary of the invention

[0016] The invention firstly relates to a process for converting lignocellulosic biomass by bringing pretreated lignocellulosic biomass into contact, in aqueous phase, with at least one biocatalyst in a first reactor containing a reaction medium comprising said biomass in aqueous phase and said biocatalyst, said process comprising: - (a) a first liquefaction step by adding said pretreated lignocellulosic biomass and at least one biocatalyst to said reactor without withdrawing all or part of said reaction medium from said reactor, - then (b) a second continuous liquefaction step, with continuous withdrawal from said first reactor of part of said reaction medium, addition of at least one biocatalyst, and continuous addition of pretreated lignocellulosic biomass.

[0017] (This continuous addition allows the reaction volume in the reactor to be maintained at a given level in the reactor).

[0018] The biomass targeted by the invention is of a lignocellulosic nature with very varied substrates including woody substrates such as different woods (hardwoods and softwoods), co-products from agriculture (wheat straw, corn cobs, etc.) or other agri-food industries, paper, lignocellulosic waste, etc.

[0019] The term "pretreated" is understood in relation to biomass in its usual meaning in the field of lignocellulosic biomass treatment. This generally involves impregnation with an acidic, basic, oxidizing liquor or simply with water, followed by possible cooking, in particular associated with a steam explosion. For more details on this preliminary operation, please refer, for example, to patents FR 3054141, FR 3075202 and FR 3075203.

[0020] The reaction medium is in aqueous phase: The water can come from the biomass itself (which contains water natively and / or which has been impregnated with aqueous solution(s) prior to the treatment of the invention. The water can also come from a specific water supply.

[0021] The term "biocatalyst", as detailed below, means an enzyme or a mixture of enzymes and / or a type of microorganism or several types of microorganisms, in particular of the bacteria or yeast type. The biocatalyst(s) can be added like biomass continuously or discontinuously, or all at once.

[0022] For the purposes of the present invention, the term "continuous" withdrawal and "continuous" feeding includes continuous withdrawal and feeding in the strict sense, or pseudo-continuous withdrawal and feeding. The term "pseudo-continuous" means that the withdrawal and / or feeding of substrate can be done sequentially. As an illustration of this transfer method: x kg of medium can be withdrawn - for a period of y minutes, then x kg of substrate and other inputs necessary for the reaction (biocatalysts, chemicals, acid or base for example) are added for a period of z minutes, and so on for the total duration of the step, for 20 to 24 hours for example. The duration of withdrawal and addition can be different or similar. The quantities of substrate and all other inputs added are the same as those withdrawn to be at iso-volume in the reactor.

[0023] The invention therefore proposes to carry out the conversion of pretreated biomass by breaking down the conversion into two stages, the first of which, liquefaction, has been modified compared to the operating modes already known for liquefaction, by adding to a liquefaction stage, preferably by sequential addition, ("fed-batch" mode according to English terminology) a following stage operating continuously. In this continuous stage, the withdrawal from the reactor and the supply of pretreated biomass are continuous, or pseudo-continuous. The withdrawal flow rate and the supply of pretreated biomass will be adapted throughout this stage to regulate, in particular, the rheology of the reaction medium in the reactor.

[0024] The process according to the invention therefore provides a liquefaction step (a), where the components of the reaction medium are added to the reactor / brought into contact (biomass, water, biocatalyst) to start the liquefaction, without withdrawal. Then the liquefaction continues with step (b) with continuous withdrawal.

[0025] And with this additional step of continuous liquefaction, the invention brings to the conversion process as a whole (including a step of enzymatic hydrolysis itself after liquefaction) an enormous industrial advantage: it makes it possible to maximize the utilization rate of the reactors dedicated to liquefaction.

[0026] The invention thus makes it possible, for a given production of converted biomass, to limit the number of liquefaction reactors to be mobilized (or to increase the production of converted biomass for a given number of liquefaction reactors to be mobilized) by limiting the frequency of the non-productive phases of feeding, emptying and cleaning.

[0027] This point is all the more advantageous, in terms of saving on industrial investments, since liquefaction reactors must generally, as already mentioned, be equipped with complex stirring systems whose operation is energy-intensive.

[0028] It was also found that with this liquefaction method, the cleaning frequency of the liquefaction reactors could be reduced, without any negative impact. And it was also found that with the liquefaction according to the invention, continued by the actual conversion stage in another reactor (enzymatic hydrolysis or simultaneous enzymatic hydrolysis and fermentation SSF or SSCF), the same conversion yields into sugar or alcohol were obtained.

[0029] It should also be noted that with the liquefaction carried out according to the invention, there is no problem in controlling the viscosity of the reaction medium, and there is no difficulty in withdrawing / emptying the liquefaction reactor or in filling the next reactor allowing the conversion reaction to continue.

[0030] Advantageously, the pH of the aqueous phase can be regulated by controlled addition to the first reactor of at least one acid and / or basic compound in at least one of the two liquefaction stages (a) and (b). Thus, generally if the biomass has been pretreated with an acid liquor, the pH will tend to be adjusted and then maintained at a set value by controlled addition of base, and if it has been pretreated with a basic liquor, the pH will tend to be adjusted to the set value by controlled addition of acid. The pH regulation can also be done before liquefaction.

[0031] During the first liquefaction step (a), the additions of said pretreated lignocellulosic biomass to the first reactor can be carried out according to a fixed or variable frequency, and according to fixed or variable quantities. The biomass can also be added to the reactor all at once. The same applies to the biocatalyst(s), and to the water (when additional water is added, if the biomass does not contain sufficient water). The water can be added separately from the biomass, or the biomass may have already been brought into contact with all or part of the water before addition to the reactor. According to one embodiment, all of the components of the reaction medium, therefore the water, the pretreated biomass and the biocatalyst(s) are added all at once, at the same time or not, and preferably at the very beginning of step (a).

[0032] During the first liquefaction step (a), the additions of said pretreated lignocellulosic biomass to the first reactor can be carried out at increasingly spaced time intervals, as described for example in the aforementioned patent EP 3 461 902, and preferably with fixed quantities of biomass.

[0033] During the first liquefaction step (a), the or at least one of the biocatalysts may also be added sequentially to the reaction medium, either with the same frequency or spacing as the pretreated biomass, or with a different frequency or spacing. Alternatively, the biocatalyst(s) may be added all at once, during, in particular at the very beginning of, the liquefaction step (a). During the first liquefaction step (a), the additions of the pretreated lignocellulosic biomass to the first reactor may be carried out continuously or sequentially.

[0034] The second liquefaction step (b) is preferably carried out at a constant volume of the reaction medium contained in the first reactor. Since a portion of the reaction medium is continuously withdrawn, pretreated biomass and the necessary inputs are gradually added so that the volume of the reaction medium remains imperceptibly unchanged and so that “fresh” pretreated biomass can be liquefied throughout this step (b). These additions are continuous at a given frequency, or controlled by monitoring a given physicochemical, rheological, or operating characteristic of the reaction medium.

[0035] During the second liquefaction step (b), pretreated lignocellulosic biomass and at least one other compound called an "input" will be added over time, including at least one chosen from one of the following compounds: water, acid compound, basic compound, biocatalyst(s), which can be added with the biomass, or in several times but not at the same time as the biomass, or in one go at the start of the step. Preferably, the biocatalyst(s) are added at the same time / with the biomass.

[0036] These inputs, as well as those present at the start of liquefaction, may also include other compounds, additives for example such as anti-foam agents, antibacterial agents, or even nutrients (in the case of an SSF or SSCF in particular, for the micro-organisms used for fermentation).

[0037] The rheology of the reaction medium can be adjusted during the second liquefaction step (b) as a function of at least one operating condition from among: the residence time of the pretreated lignocellulosic biomass in the first reactor, the quantity and / or frequency of additions of pretreated lignocellulosic biomass and input(s) including at least one chosen from one of the following compounds: water, acid compound, basic compound, biocatalyst(s).

[0038] The rheology of the reaction medium can be adjusted during the second liquefaction step (b) so that it is identical to or less severe than the rheology of the reaction medium at the end of the first liquefaction step (a). The more "severe" the rheology of a reaction medium, the more, in particular, the reactor will need to be equipped with efficient stirring means and / or the more energy will need to be consumed to operate them.

[0039] It is thus sought to maintain in the liquefaction reactor an appropriate rheology in the continuous liquefaction according to the invention, so that the reaction medium remains at viscosity conditions allowing its stirring in the reactor and its withdrawal to another reactor in a manner compatible with production on an industrial scale. The rheology of the reaction medium can be monitored by following the viscosity of the reaction medium or the mechanical torque of the shaft of a stirring system equipping the first reactor or the electrical power consumed by the motor driving said stirring system.

[0040] The first liquefaction step (a) preferably has a duration of between 1 and 48 hours, even more preferably between 2 and 24 hours, in particular between 5 and 12 hours.

[0041] The second liquefaction step (b) preferably has a duration of between 1 and 170 hours, in particular between 10 and 72 hours, in particular between 15 and 30 hours or between 20 and 28 hours.

[0042] During the second liquefaction step (b), the residence time of the pretreated biomass in the first reactor is preferably greater than or equal to 4 hours, in particular greater than or equal to 5 hours, for example between 5 hours and 14 hours.

[0043] During the second liquefaction step (b), it is advantageous to continuously withdraw part of the reaction medium from the first reactor to a second reactor where the conversion of the biomass contained in the withdrawn reaction medium is continued in a conversion step (c), in the presence of at least one biocatalyst.

[0044] Biocatalysts are already contained in the reaction medium transferred from one reactor to another, but different or identical biocatalysts to those already introduced into the first reactor can be specifically added to the second reactor.

[0045] Thus, when the aim is to convert biomass into alcohol by SSF or SSCF: - according to a first embodiment, all the biocatalysts (enzymes and micro-organisms) can be added as soon as the liquefaction occurs in the first reactor (and therefore no biocatalyst can be added in step c) in the second reactor), - and according to another embodiment, the enzymes can be added during the liquefaction in the first reactor, then the micro-organisms, and possibly additional enzymes, can be added in step c) in the second reactor.

[0046] At the end of the second liquefaction step (b), the entire reaction medium is advantageously transferred from the first reactor to a second reactor where, in a conversion step (c), the conversion of the biomass contained in the transferred reaction medium is continued, in the presence of at least one biocatalyst.

[0047] Advantageously, the duration of the second liquefaction step (b) is less than or equal to the duration of the conversion step (c). Step (c) in the second reactor mentioned above can therefore be operated in fed-batch mode, preferably with a feed duration of between 1 hour and 50 hours, and preferably between 10 hours and 40 hours, followed by a batch operation duration. The total duration of step (c), in fed-batch then in batch, is preferably between 10 and 170 hours, in particular between 70 and 140 hours. “Batch” is to be understood in its usual sense, namely that there is no withdrawal from the reactor throughout the duration of the conversion carried out in this reactor.

[0048] The coupling of steps (a) and (b) in a dedicated liquefaction reactor and (c) in another reactor makes it possible to benefit from the combined advantages of previous batch, fed-batch and continuous implementations: - the liquefaction reactor is operated according to steps (a) then (b) which make it possible to maximize the use of this reactor to contain the reaction medium, and to reduce the times allocated to emptying and cleaning during a cycle - the reactor operating step (c) ends in batch, which makes it possible to maximize the conversion levels achieved, and to control the maximum residence time of the biocatalysts, thus avoiding the drifts mentioned above.

[0049] Advantageously, the second reactor in which step (c) takes place has a volume greater than that of the first reactor in which steps (a) and (b) take place. Preferably, the volume of the second reactor is greater than 100% of the volume of the first reactor, preferably greater than 120%, preferably greater than 200% and even more preferably greater than 300%. The second reactor in which step (c) takes place may be fed by several reactors in which steps (a) and (b) take place.

[0050] It is also possible to use several reactors to carry out step (c), of smaller size and operating in series in particular.

[0051] The biocatalyst added for at least one of the first liquefaction (a), second liquefaction (b) and conversion (c) steps, and in particular all of these steps, advantageously comprises at least one enzyme for converting the pretreated biomass at least partially into sugar(s) by enzymatic hydrolysis, and optionally at least one microorganism, such as yeast(s) or bacteria(s) for converting all or part of this sugar(s) into alcohol(s) by fermentation.

[0052] According to a variant, the biocatalyst used for each of the first liquefaction (a) and second liquefaction (b) steps may comprise at least one enzyme for converting the pretreated biomass at least partially into sugar(s) by enzymatic hydrolysis, and the biocatalyst added in conversion step (c) may comprise a mixture of enzyme(s) and yeast(s) (or other microorganism) or only at least one yeast for converting all or part of the sugar(s) transformed into alcohol(s) by fermentation.

[0053] Alternatively, the biocatalyst used to ferment all or part of the sugar(s) into alcohol(s) or products of interest is a bacterium, for example Clostridium, such as Clostridium Acetobutylicum. It can be added to the process in the same way as the yeasts mentioned above.

[0054] Indeed, the present invention aims at the conversion of pretreated biomass to produce sugars by enzymatic hydrolysis, generally from a cocktail of enzymes comprising at least cellulolytic enzymes. The cellulolytic enzymes are for example cellulases, endoglucanases, beta-glucosidases. The enzymatic cocktail can also comprise hemicellulolytic enzymes (hemicellulases). The enzymes can be produced by bacteria or fungi. Preferably the enzymes are produced from a fungus, for example Trichoderma reseii. The liquefaction according to the invention will therefore use this type of cocktail, as well as the conversion reaction which will continue in a reactor other than the liquefaction reactor. The sugars in question can be recovered as they are, or after transformation.

[0055] The present invention also aims at the production of alcohols by fermentation from these sugars, according to two main types of process: - either saccharification and fermentation are carried out at the same time, this is the so-called SSF or SSCF process. In this case, appropriate microorganisms (yeast, bacteria as seen above) are added to the enzymes. The microorganisms can thus be added as soon as the liquefaction begins, - or fermentation takes place after saccharification, in a reactor dedicated to fermentation and supplied with appropriate microorganisms. In this case, only one type of biocatalyst is added at each stage (enzymes for hydrolysis, then microorganism for fermentation).

[0056] Note that to ensure fermentation, the biocatalyst is a micro-organism, which can be a yeast or a bacterium, even if, in this text, we can only mention yeasts, for the sake of brevity.

[0057] According to one embodiment, the method according to the invention may comprise the following steps: - an initial step (aO) of filling a first reactor with a first supply into said reactor of pretreated lignocellulosic biomass, biocatalyst(s), water and optionally acid and / or basic compounds, - a first step (a) of liquefaction in the first reactor by adding said pretreated lignocellulosic biomass into said reactor without withdrawal, with optional addition also of biocatalyst(s), - then a second step (b) of continuous liquefaction in the first reactor, with continuous withdrawal from the first reactor of a portion of the reaction medium and transfer to a second reactor of said portion of the withdrawn reaction medium and continuous addition of pretreated lignocellulosic biomass, and optionally water and / or biocatalyst(s), and / or acid and / or basic compounds,- then an optional step (b1) of homogenization of the reaction medium in the first reactor, - then a step (b2) of transfer of all the reaction medium from the first reactor to the second reactor, where the conversion step (c) takes place, preferably in batch mode, - and a step (b3) of cleaning the first reactor, in particular using an aqueous solution, preferably acidic or basic.,

[0058] The additions of said pretreated lignocellulosic biomass in the first step a) may be sequential or not.

[0059] The additions of biocatalysts in the first step a) can be sequential or not.

[0060] The operating conditions of the first liquefaction (a) in enzymatic hydrolysis configuration alone (i.e. when the aim is to convert the biomass into oligomeric or monomeric sugars) are preferably: - a temperature between 25 and 80°C, preferably between 40 and 60°C, and even more preferably between 45°C and 55°C, - a pH between 3 and 7, preferably between 4 and 6 and even more preferably between 4.8 and 5.5.

[0061] The operating conditions of the first liquefaction (a) in the enzymatic hydrolysis and simultaneous fermentation (SSF or SSCF) configuration, i.e. when the aim is to convert the biomass into alcohol by hydrolysis and fermentation, are: - a temperature between 25 and 80°C, preferably between 30 and 50°C and even more preferably between 30°C and 35°C, - a pH between 3 and 7, preferably between 4 and 6 and even more preferably between 5.0 and 5.5.

[0062] The operating conditions (temperature and pH) of the second liquefaction (b) are preferably identical to those of the first liquefaction (a).

[0063] The operating conditions (temperature and pH) of conversion step (c) may be identical or different from those of the first liquefaction (a) and the second liquefaction (b). They will be different, for example, if the biocatalysts introduced during this step are different from those introduced during the first liquefaction (a) and the second liquefaction (b).

[0064] Preferably, the dry matter content MS of the pretreated lignocellulosic biomass used in the process according to the invention is at least 2% by weight, in particular at least 5% by weight, at least 10% by weight. The biomass used in the process according to the invention contains at least 10 g of cellulose per 100 g of dry matter, in particular at least 20 g of cellulose per 100 g of dry matter.

[0065] List of figures

[0066] Figure 1 represents the different stages of a liquefaction step of pretreated biomass according to the prior art.

[0067] Figure 2 represents the different stages of a pretreated biomass liquefaction step according to one embodiment of the invention.

[0068] Figure 3 is a graph representing the evolution of ethanol and xylose concentrations during an SSCF process with liquefaction according to the prior art and with liquefaction according to the invention as a function of time. On the ordinate, the concentrations are in g / kg reaction medium, and on the abscissa the time is expressed in hours.

[0069] The figures, and more specifically figures 1 and 2, are very schematic and do not respect the scale. The same references relate to the same flows / devices from one figure to another.

[0070] Description of the embodiments

[0071] The invention aims at improving the operating mode of liquefaction of pretreated lignocellulosic biomass. Liquefaction is to be understood as a step of starting the conversion of biomass under the effect of biocatalysts. This is a conversion by enzymatic hydrolysis (and / then optional fermentation). This liquefaction is sometimes referred to as "pre-hydrolysis".

[0072] A known protocol for carrying out enzymatic hydrolysis alone or enzymatic hydrolysis and simultaneous fermentation of biomass or ignocellulosic waste is a first fed-batch liquefaction step (sequential addition of biomass and no withdrawal during the entire step) in a reactor specially sized / designed for this purpose, then transfer to a more standard reactor to continue in a second step the enzymatic hydrolysis alone or the simultaneous enzymatic hydrolysis and fermentation in batch mode. Reference will be made, for example, to the aforementioned patent WO 2013 / 088001 for the description of this type of protocol.

[0073] The protocol according to a preferred embodiment of the invention proposes a liquefaction with a first fed-batch type step of lignocellulosic biomass in an ad hoc reactor (relatively small useful volume with high-performance stirring equipment), which is followed in this same reactor by a step in continuous operation, with: - a continuous or pseudo-continuous transfer to a more standard reactor (whose useful volume can be much larger and whose stirring equipment is simpler than the first reactor) to continue the enzymatic hydrolysis alone, or the simultaneous enzymatic hydrolysis and fermentation in batch mode, - and a continuous or pseudo-continuous supply of lignocellulosic substrate and various inputs / biocatalysts in the first reactor.

[0074] By this new protocol, it has been shown that the utilization rate of liquefaction reactors is maximized, allowing, for a given production, to limit the number of liquefaction reactors to be mobilized, at iso-productivity in enzymatic hydrolysis (or in SSF or SSCF).

[0075] The invention relates to the implementation of enzymatic hydrolysis for the production of sugars or enzymatic hydrolysis and simultaneous fermentation for the production of alcohol from lignocellulosic biomass / waste.

[0076] The feedstock treated by the process according to the invention is a pre-treated lignocellulosic biomass. The pre-treatment of the lignocellulosic biomass makes the cellulose accessible and reactive to enzymes, and consists of bringing the lignocellulosic biomass into contact with a solvent and possibly a catalyst (generally combined in a liquor) at a given temperature and pressure for a given residence time. Any type of pre-treatment can be applied to obtain the pre-treated lignocellulosic substrate.

[0077] The pretreated biomass may also undergo water washing (resuspension of the pretreated biomass with water or a mixing fluid, solid / liquid filtration, washing of the solid fraction with water then solid / liquid filtration) after its pretreatment and before the start of the liquefaction according to the invention.

[0078] During the enzymatic hydrolysis or SSF or SSCF step (which includes the liquefaction steps according to the invention exemplified later), the pretreated lignocellulosic substrate is mixed with a liquid solution containing the enzymes (and possibly microorganisms such as yeasts or bacteria). The objective is to obtain a high concentration of ethanol (or sugars if fermentation is not carried out). The fermentation / enzymatic hydrolysis step is to be carried out at relatively high concentrations of pretreated lignocellulosic substrate, i.e. with a high dry matter content, in order to reduce the economic and energy costs of the process if the product of interest must be concentrated.

[0079] This dry matter content (acronym "MS") refers to the dry matter rate which is measured according to the ASTM E1756 - 08 (2015) standard "Standard Test Method for Determination of Total Solids in Biomass". (The concentration of pretreated lignocellulosic substrate in the medium can be expressed as a percentage by weight of dry matter).

[0080] The intimate mixing of the pretreated lignocellulosic substrate with the said liquid solution containing the enzymes (and possibly the yeasts) proves difficult when the dry matter contents are high. Indeed, the start of enzymatic hydrolysis at a high dry matter content poses problems of mixing and homogenization. The reaction medium is very pasty and viscous.

[0081] To address this problem, existing solutions are: - equipping fermentation (or enzymatic hydrolysis) reactors with a specific complex stirrer to ensure homogenization of the reaction medium. - carrying out a progressive feed of substrate, known as fed-batch feeding in the reactor, without withdrawing the reaction medium. As the reaction progresses, the mixture becomes less and less viscous and it is possible to add fresh substrate in order to increase the quantity of substrate in the medium.

[0082] - conduct fermentation (or enzymatic hydrolysis) in two stages: A first stage called liquefaction, allowing the viscosity of the medium to be reduced. This stage actually corresponds to the first hours of enzymatic hydrolysis (or SSF / SSCF), the cellulose (insoluble in the medium) is converted into oligomeric or monomeric sugars soluble in the medium. It ends when the viscosity has been reduced to a value allowing transfer to a tank equipped with a standard stirrer for the continuation of the enzymatic hydrolysis. A second stage corresponding to the continuation of the enzymatic hydrolysis (or SSF / SSCF): the liquefied biomass from the liquefaction stage is transferred into fermentation (or hydrolysis) reactors in which the conversion of the cellulose and residual hemicelluloses into sugars and then the conversion of the sugars into ethanol continues.

[0083] The invention focuses on this latter approach, and aims to improve it.

[0084] Previous / Comparative Embodiment

[0085] The liquefaction step of the pretreated substrate is carried out in a "fed batch" or "fed-batch", i.e. with the substrate gradually added to the mixture of water + biocatalysts + base. (We are taking here the non-limiting example of a pretreatment with impregnation by an acid liquor, hence the addition of a base to increase the pH of the reaction medium).

[0086] Due to the rheology of the medium, the liquefaction reactor is first loaded with a portion of the substrate to be treated and all of the water, the pH and temperature are adjusted to the required settings, then some or all of the enzymes (and possibly yeasts) are added. A fed-batch (sequential feeding) is then carried out with the remainder of the pretreated substrate in order to increase the dry matter content. A fed-batch of the enzymes (and yeasts) can also be carried out. The rheology of the solid suspension requires special implementation to ensure good stirring to carry out the reaction in a medium as concentrated as possible in pretreated lignocellulosic substrate. To overcome some of this constraint, a special agitator technology can be fitted to the liquefaction reactor.

[0087] Helical type agitators are generally the most suitable, although they are complex and limited in size for mechanical design reasons.

[0088] Different parameters are used to define the operating conditions and establish the fed-batch strategy. The ranges indicated below are examples: - the dry matter (DM) content of the pretreated biomass (2 to 60% by weight) - the cellulose content of the substrate

[0089] - the dose of enzymes in relation to cellulose (5 to 100 mg / g of cellulose)

[0090] - the yeast seeding rate (0.1 to 3 g / kg medium),

[0091] - the dry matter (DM) content of the initial mixture (2 to 60% by weight)

[0092] - the dry matter (DM) content of the final mixture (2 to 60% by weight)

[0093] - the total mass and / or volume of the final mixture in the reactor

[0094] - the number of fed-batch additions

[0095] - the duration of fed-batch additions (0 to 48 h)

[0096] - the duration of liquefaction (1 to 48 hours)

[0097] The description of the previous fed-batch liquefaction protocol is as follows:

[0098] 1 - Preparation of the initial mixture in the liquefaction reactor: a certain quantity of pretreated biomass is mixed with a certain quantity of water to achieve the desired dry matter (DM) content. Stirring is started to homogenize the mixture and adjust the pH and temperature. An antibacterial agent, for example chloramphenicol or the one marketed under the trade name VitaHop by the company BetaTec, can be added to the reaction medium.

[0099] 2- pH regulation with the addition of a basic solution, for example NH4OH or KOH or NaOH if the pretreated substrate was produced under acidic conditions (for example impregnation with sulfuric acid followed by steam explosion) or with the addition of an acid solution if the pretreated substrate was produced under basic conditions. pH regulation can be maintained during the following phases of the protocol.

[0100] 3- Injection of enzymes (and yeasts) then start of fed-batch liquefaction: once the initial mixture is homogeneous, a certain quantity of enzymes (and yeasts) is introduced into the reactor. This injection allows the chosen dose of biocatalysts to be reached. After the injection of the biocatalysts, fed-batch liquefaction begins.

[0101] 4- Fed-batch - additions of pretreated biomass: after a certain pre-defined time, the medium is already much less viscous and the first addition of pretreated biomass can take place. Depending on the enzyme addition strategy, additional enzymes can be added at this time. The mass of enzymes introduced corresponds to the addition of pretreated biomass. One can choose to add a quantity of enzymes based on the quantity of pretreated biomass added at each biomass addition, or add all or most of the quantity of enzymes required from the beginning of the fed-batch. The additions follow one another regularly until a mixture with the desired final mass is reached. Generally, the mass and rate of each addition are constant and regular. As seen above, one can also space out the biomass additions more and more. In general, the objective is to aim for a target enzyme dose expressed in grams per kilogram of cellulose.

[0102] 5- End of liquefaction: after the pre-treated biomass addition phase, the reaction continues at a constant final volume. The reaction and viscosity drop continue until the viscosity is deemed low enough to transfer the medium to the enzymatic hydrolysis reactor (or SSF or SSCF) equipped with a standard stirrer.

[0103] 6- After transferring the reaction medium to an enzymatic hydrolysis reactor (or SSF or SSCF) corresponding to a conventional stirred tank, the liquefaction reactor is cleaned to limit the risk of contamination.

[0104] The operating conditions for liquefaction in the enzymatic hydrolysis configuration alone (i.e. when the aim is to convert the biomass into oligomeric or monomeric sugars) are:

[0105] - a temperature between 25 and 80°C, preferably between 40 and 60°C, and even more preferably between 45°C and 55°C,

[0106] - a pH between 3 and 7, preferably between 4 and 6 and even more preferably between 4.8 and 5.5.

[0107] The operating conditions for liquefaction in the enzymatic hydrolysis and simultaneous fermentation (SSF or SSCF) configuration, i.e. when the aim is to convert the biomass into alcohol by hydrolysis and fermentation, are: - a temperature between 25 and 80°C, preferably between 30 and 50°C and even more preferably between 30°C and 35°C,

[0108] - a pH between 3 and 7, preferably between 4 and 6 and even more preferably between 5.0 and 5.5.

[0109] The duration of liquefaction is between 1 hour and 48 hours, especially between 2 hours and 24 hours, especially between 5 hours and 12 hours.

[0110] Figure 1 represents the different durations / phases of cycle A to E of liquefaction, representing liquefaction reactor 1 at each of these phases:

[0111] A: Filling of the liquefaction reactor 1 with pretreated biomass 2, water 3' and biocatalyst(s) 3, and a basic compound 4 and regulation of the temperature and pH of the reaction medium in the reactor: duration between 2 hours and 4 hours

[0112] B: Fed-batch of pretreated biomass, with sequential addition of pretreated biomass 2 and possibly basic compound 4 to regulate the pH: duration from 2 hours to 10 hours

[0113] C: Homogenization (optional): from 1 hour to 2 hours

[0114] D: Transfer of reaction medium 5 from reactor 1 to a downstream reactor (not shown): duration between 2 h and 4 h

[0115] E: Cleaning of liquefaction reactor 1: between 2 and 4 hours

[0116] Enzymatic hydrolysis (or SSF or SSCF) is carried out in batch mode. The operating conditions of pH and temperature are generally identical to those for liquefaction. The duration of enzymatic hydrolysis (or SSF or SSCF) is between 10 hours and 170 hours, preferably between 48 hours and 140 hours. After emptying the enzymatic hydrolysis reactor (or SSF or SSCF), the reactor is cleaned to limit the risk of contamination.

[0117] Limiting the volume of liquefaction reactors compared to SSF or SSCF or enzymatic hydrolysis reactors (the useful volume ratio between the SSF or SSCF or enzymatic hydrolysis reactor and the liquefaction reactor is for example between 2 and 10) generally requires the establishment of a timetable to ensure the continuity of the different operating phases (including the filling, emptying, cleaning phases) between the two liquefaction and SSF or SSCF (or enzymatic hydrolysis) stages. This timetable is also established to limit the number of liquefaction and SSF or SSCF or enzymatic hydrolysis reactors by limiting downtime. Embodiment according to the invention

[0118] The description of the protocol according to the invention for fed-batch liquefaction with an additional continuous step is shown diagrammatically in Figure 2, which uses the same conventions as Figure 1:

[0119] The phase durations indicated are examples.

[0120] A - Initial phase of filling of liquefaction reactor 1

[0121] Preparation of the initial mixture in the liquefaction reactor: a certain quantity of pretreated biomass 2 is mixed with a certain quantity of water 3' to achieve the desired dry matter (DM) content. Stirring is started to homogenize the mixture and the pH and temperature are adjusted. The pH is regulated by adding a basic solution 4, for example NH4OH or KOH or NaOH if the pretreated substrate was produced under acidic conditions (for example impregnation with sulfuric acid followed by steam explosion) or by adding an acid solution if the pretreated substrate was produced under basic conditions.

[0122] B - start of fed-batch liquefaction - additions of pretreated biomass 2: once the initial mixture is homogeneous, a certain quantity of enzymes 3 (and yeasts) is introduced into reactor 1. This injection allows the chosen dose of biocatalysts to be reached. After the injection of the biocatalysts, fed-batch liquefaction starts: - after a certain time, the medium is already much less viscous and the first addition of pretreated biomass can take place. Depending on the strategy for adding enzymes 3, additional enzymes can be added at this time. The mass of enzyme 3 introduced is proportional to the addition of pretreated biomass 2 to aim for a constant quantity of enzymes / cellulose added. The additions follow one another until a mixture with the desired final mass is reached. Generally, the mass and frequency of each addition are constant and regular.

[0123] F - Continuous phase of the liquefaction specific to the invention: a 5' portion of the reaction medium is withdrawn and transferred to a downstream reactor (not shown) equipped with a standard stirrer. A portion of the inputs is added to the liquefaction reactor 1 to be at constant volume: pretreated biomass 2, biocatalyst(s) 3, water 3' and acid or basic solution 4 for pH regulation. The continuous phase of the liquefaction is operated so that the rheology of the withdrawn medium remains less restrictive or identical to that of the medium at the end of the fed-batch, so as not to negatively impact the operation of the enzymatic hydrolysis reactor (or SSF or SSCF). The operating conditions of the liquefaction in the enzymatic hydrolysis configuration alone (i.e. when the aim is to convert the biomass into oligomeric or monomeric sugars) are preferably:

[0124] - a temperature between 25 and 80°C, preferably between 40 and 60°C, and even more preferably between 45°C and 55°C,

[0125] - a pH between 3 and 7, preferably between 4 and 6 and even more preferably between 4.8 and 5.5.

[0126] The operating conditions for liquefaction in the enzymatic hydrolysis and simultaneous fermentation (SSF or SSCF) configuration, i.e. when the aim is to convert biomass into alcohol by hydrolysis and fermentation, are preferably:

[0127] - a temperature between 25 and 80°C, preferably between 30 and 50°C and even more preferably between 30°C and 35°C,

[0128] - a pH between 3 and 7, preferably between 4 and 6 and even more preferably between 5.0 and 5.5.

[0129] The operating conditions (dry matter, dose of biocatalysts) are maintained at the target conditions, the parameters which allow to obtain the desired rheology are therefore the quantity and frequency of the additions as well as the residence time of the marc in the liquefaction reactor during the continuous phase. The residence time of the pretreated biomass in the liquefaction reactor during the continuous phase is greater than or equal to 4 h.

[0130] The duration of the continuous phase is, for example, between 1 hour and 170 hours, in particular between 10 hours and 72 hours.

[0131] C - Homogenization (optional): from 1 hour to 2 hours

[0132] D - Transfer of the reaction medium 5 from reactor 1 to a downstream reactor (not shown); End of liquefaction: after the continuous phase, all of the liquefaction medium is transferred into the enzymatic hydrolysis reactor (or SSF or SSCF) preferably equipped with a standard stirrer: duration between 2 hours and 4 hours

[0133] E - Cleaning of liquefaction reactor 1. After transferring the medium to the downstream reactor, the liquefaction reactor is cleaned to limit the risk of contamination: duration between 2 hours and 4 hours.

[0134] Enzymatic hydrolysis (or SSF or SSCF) is always carried out in batch mode. The operating conditions of pH and temperature are identical to those of liquefaction or different. This is the case, for example, for a configuration where the biocatalysts introduced in the liquefaction stage are different from those introduced in the SSF or SSCF stage.

[0135] The duration of enzymatic hydrolysis (or SSF or SSCF) is between 10 hours and 170 hours, particularly between 70 hours and 140 hours.

[0136] According to a variant, only the enzymes are added to the liquefaction reactor 1, during the initial filling phase A and during the continuous phase F, and the yeasts are added to the SSF or SSCF reactor from the start of the continuous liquefaction phase.

[0137] Naturally, it is also possible to carry out only enzymatic hydrolysis, without yeast, either to recover the sugars without transforming them into alcohol or by transforming them differently. The fermentation of the sugars can also be carried out separately, in a dedicated fermentation reactor.

[0138] Compared to liquefaction according to the previous method (example in figure 1), liquefaction according to the invention (example in figure 2) provides the following advantages:

[0139] - Increased liquefaction productivity through a better utilization rate of each liquefaction reactor. The magnitude of the gain depends on the operating time of this continuous phase, but also on the flow rate that can be treated during this phase (depending on the residence time for the pre-treated biomass during the continuous phase).

[0140] - For the same quantity of pre-treated biomass to be liquefied, the number of liquefaction reactors is therefore reduced (or for the same number of liquefaction reactors, the quantity of liquefied biomass is greater): the liquefaction protocol according to the invention therefore leads to a reduction in investments.

[0141] - The liquefaction reactor cleaning phase is carried out after the transfer of several reactor volumes, which makes it possible to reduce the frequency of reactor cleaning, therefore reducing the consumption of chemicals, and increasing productivity by increasing the reaction time.

[0142] - Ethanol production yields and conversion yields of cellulose and hemicellulose into sugars are maintained the same or almost the same.

[0143] Example 1 (comparative)

[0144] It uses the protocol described above using Figure 1. To illustrate the gain in the utilization rate of liquefaction reactors, we will compare two timelines: one for a known fed-batch liquefaction (Figure 1), the other for a fed-batch liquefaction with a continuous phase (Figure 2).

[0145] Liquefaction (+SSCF) is carried out with a MS content of 20% by weight, the enzyme dose is 8 mg of enzymes / gMS, the quantity of yeasts is 0.5 g / kg medium.

[0146] The linearized flow rate of pretreated substrate feeding the liquefaction stage is 15.6 tMS / h. The dry matter content of the pretreated substrate is 38% by weight.

[0147] Table 1 below details the known liquefaction timeline:

[0148] [Table 1]

[0149] 4 liquefaction reactors of 435 m 3 (useful volume) are therefore necessary to liquefy the pretreated biomass with an operational utilization rate of 45%. The liquefied biomass is transferred into 4 SSCF reactors of 4,500 m 3 each one, working in batch.

[0150] The durations of the phases in SSCF are indicated in the following table 2: [Table 2]

[0151] Example 2 (according to the invention) It follows the liquefaction protocol according to the invention described above and illustrated in Figure 2. Table 3 below details the liquefaction schedule according to the invention:

[0152] [Table 3]

[0153] 2 500 m liquefaction reactors 3 (useful volume) are therefore required to liquefy the pretreated biomass with an operational utilization rate of 72.5%. The residence time of the pretreated biomass in the liquefaction reactor is 8 h. The liquefied biomass is transferred to 4 SSCF reactors of 4,500 m 3 each one, working in batch.

[0154] The durations of the phases in SSCF are the same as those indicated in Table 2 for comparative example 1.

[0155] Example 3 (comparative)

[0156] Like comparative example 1, it uses the known liquefaction protocol. The liquefaction reactor is loaded with a portion of the pretreated biomass, all of the water, the basic NH4OH solution for adjusting the pH to 5.3, then all of the enzymes and yeasts are added, which corresponds to the tO time of liquefaction. The temperature is maintained at 33°C. The dry matter content in this initial mixture is 14% by weight. The fed-batch of pretreated biomass is then carried out with 12 additions of pretreated biomass over 6 hours: 5% DM increase in 3 hours, then 3% DM increase in 3 hours.

[0157] The dry matter content of the liquefaction medium is 22% by weight, the enzyme dose is 8 mg of enzymes / gMS, the amount of yeast is 0.5 g / kg medium.

[0158] The enzyme solution has a protein concentration of 35 g / L and a density at 20°C of 1.02 g / cm3. The dry matter content of the pretreated biomass is 42% by weight.

[0159] The liquefaction reactor is equipped with a helical agitator allowing good homogenization of the reaction medium and optimal management of torque and viscosity.

[0160] The liquefaction medium is then homogenized for 2 hours to allow the agitator torque to decrease, and is then transferred to a SSCF reactor in which the conversion of cellulose and hemicelluloses into sugars and the fermentation of sugars into ethanol continue. The SSCF reactor is equipped with a conventional agitator; for example, it is a stirring system comprising two 3-blade TT-type propellers (axial flow rotor) and a straight two-blade bottom turbine (radial flow rotor).

[0161] The liquefaction time (initial fed-batch filling of pretreated biomass + homogenization) is 12 hours. The SSCF time is 132 hours (excluding liquefaction).

[0162] The operating parameters monitored are the stirrer torque in the liquefaction reactor, the ethanol, glucose and xylose contents in the medium (HPLC analysis) during the SSCF reaction.

[0163] The performance of the SSCF process is evaluated from the following efficiencies:

[0164] - The ethanol / MS yield, equal to the ratio between the quantity of ethanol produced and the total quantity of dry matter introduced into the liquefaction reactor 1

[0165] - From the total enzymatic hydrolysis on a sample at the end of fermentation: the hydrolysis yields of cellulose and xylan and the ethanol production yield, calculated in relation to the Pasteur yield (i.e. 94.7% of the Gay-Lussac yield). As a reminder, the so-called Gay-Lussac yield is equal to the theoretical yield from the stoichiometric equation

[0166] C6H12O6 (glucose) -> 2 C2H5OH (ethanol) + 2 CO2

[0167] This gives 51.1 kg of ethanol produced from 100 kg of glucose. Pasteur then demonstrated that there were co-products associated with the production of ethanol and CO2 (notably glycerol, succinic acid, heavy alcohols, micro-organism development). Pasteur's yield takes into account these sugar losses, i.e. 48.4 kg of ethanol produced from 100 kg of glucose. Pasteur's yield therefore corresponds to 94.7% of the theoretical yield. The results are shown in Table 4 below:

[0168] [Table 4]

[0169] Example 4 (according to the invention)

[0170] Like Example 2, it follows the liquefaction protocol according to the invention described above and illustrated in Figure 2.

[0171] The filling phase of the liquefaction reactor up to the fed-batch phase of the substrate is identical to that of Example 3. The continuous phase is then implemented: part of the medium is withdrawn and transferred to the SSCF reactor equipped with a standard vertical stirrer. Part of the inputs (see Table 5 below) is added to the liquefaction reactor to operate at constant volume.

[0172] The residence time of the pretreated biomass in the liquefaction reactor during the continuous phase is set to:

[0173] - 12 hours in the case where the biomass has been pretreated under less acidic conditions (1.8% by weight of H2SO4 in the impregnation liquor)

[0174] - 6 hours in the case where the biomass has been pretreated in more acidic conditions (2.4% by weight of H2SO4 in the impregnation liquor)

[0175] This residence time allows a constant agitator torque to be maintained in the liquefaction reactor.

[0176] The duration of the continuous phase is 24 hours.

[0177] The quantities of medium to be withdrawn as well as the quantities of inputs to be added to the liquefaction reactor, whose useful volume is 3,000 kg, are indicated in Table 5 below. The nutrients are intended for the yeasts (source of nitrogen) and are here in the form of a solution of soluble corn proteins, in particular that marketed under the name Solulys by the Roquette company.

[0178] [Table 5]

[0179] The operating parameters monitored are the stirrer torque in the liquefaction reactor, the ethanol, glucose and xylose contents in the medium (analysis by HPLC, acronym for high performance liquid chromatography) during the SSCF reaction. The performance of the SSCFs is evaluated as for example 3 above, the results are grouped in table 6 below.

[0180] [Table 6]

[0181] We thus verify that the yields, in particular those of ethanol, are identical, whether we operate with liquefaction according to the known protocol (example 3) or according to the protocol of the invention (example 4).

[0182] The residence time allows to keep a constant agitator torque in the liquefaction reactor.

[0183] The kinetics of ethanol production and xylose consumption do not show any difference in reactivity between the SSCFs conducted with standard conventional fed-batch liquefaction and the SSCFs conducted with fed-batch then continuous liquefaction according to the invention. These good results in terms of performance are also evident from the graph in Figure 3. Indeed, curves C1 and C2 correspond respectively to the ethanol concentrations according to comparative example 3 and according to example 4 according to the invention: we see that the ethanol concentrations converge at 140 hours of SSCF. Similarly, curves C3 and C4 correspond respectively to the xylose concentrations according to comparative example 3 and according to example 4 according to the invention: here again, the drops in xylose concentration, due to their conversion to ethanol, reach very low and similar concentrations after 140 hours.We see that the invention also gives better results in terms of productivity in the first 48 hours.

Claims

Claims 1. Process for converting lignocellulosic biomass by bringing pretreated lignocellulosic biomass into contact, in aqueous phase, with at least one biocatalyst (3) in a first reactor (1) containing a reaction medium comprising said pretreated lignocellulosic biomass (2) in aqueous phase and said biocatalyst, said process comprising - (a) a first liquefaction step by adding said pretreated lignocellulosic biomass and at least one biocatalyst to said reactor without withdrawing all or part of said reaction medium from said reactor, - then (b) a second continuous liquefaction step, with continuous withdrawal from said first reactor of a portion of said reaction medium, addition of at least one biocatalyst, and continuous addition of pretreated lignocellulosic biomass.

2. Method according to the preceding claim, characterized in that, during the first liquefaction step (a), the additions of said pretreated lignocellulosic biomass (2) to the first reactor (1) are carried out according to a fixed or variable frequency, and according to fixed or variable quantities.

3. Method according to the preceding claim, characterized in that, during the first liquefaction step (a), the additions of said pretreated lignocellulosic biomass (2) to the first reactor (1) are carried out according to increasingly spaced time intervals, and preferably with fixed quantities of biomass.

4. Method according to one of the preceding claims, characterized in that the second liquefaction step (b) is carried out at a constant volume of the reaction medium contained in the first reactor (1).

5. Method according to one of the preceding claims, characterized in that, during the second liquefaction step (b), pretreated lignocellulosic biomass (2) and at least one other compound called "input" are added over time, at least one of which is chosen from one of the following compounds: water (3'), acid compound, basic compound (4), biocatalyst(s) (3).

6. Method according to one of the preceding claims, characterized in that the rheology of the reaction medium is adjusted during the second liquefaction step (b) as a function of at least one operating condition among the residence time of the pretreated lignocellulosic biomass (2) in the first reactor (1), the quantity and / or the frequency of the additions of pretreated lignocellulosic biomass and input(s) including at least one chosen from one of the following compounds: water (3'), acid compound, basic compound (4), biocatalyst(s) (3).

7. Method according to one of the preceding claims, characterized in that the rheology of the reaction medium is adjusted during the second liquefaction step (b) so that it is identical to or less severe than the rheology of the reaction medium at the end of the first liquefaction step (a).

8. Method according to one of claims 6 or 7, characterized in that the rheology of the reaction medium is monitored by monitoring the viscosity of the reaction medium or the mechanical torque of the shaft of a stirring system equipping the first reactor (1) or the electrical power consumed by the motor actuating said stirring system.

9. Method according to one of the preceding claims, characterized in that the first liquefaction step (a) has a duration of between 1 and 48 hours, in particular between 2 and 24 hours, and even more preferably between 5 and 12 hours.

10. Method according to one of the preceding claims, characterized in that the second liquefaction step (b) has a duration of between 1 and 170 hours, in particular between 10 and 72 hours, in particular between 15 and 30 hours or between 20 and 28 hours.

11. Method according to one of the preceding claims, characterized in that, during the second liquefaction step (b), a portion (5') of the reaction medium is continuously withdrawn from the first reactor to a second reactor where, in a conversion step (c), the conversion of the biomass contained in the withdrawn reaction medium is continued in the presence of at least one biocatalyst.

12. Method according to one of the preceding claims, characterized in that at the end of the second liquefaction step (b), the entire reaction medium (5) is transferred from the first reactor (1) to a second reactor where the conversion of the biomass contained in the transferred reaction medium is continued in a conversion step (c), in the presence of at least one biocatalyst.

13. Method according to one of claims 1 1 or 12, characterized in that step (c) in the second reactor is carried out in fed-batch then in batch, preferably for a duration of between 10 and 170 hours, in particular between 70 and 140 hours.

14. Method according to one of the preceding claims, characterized in that said method comprises - an initial step (aO) of filling the first reactor (1) with a first supply in said reactor of pretreated lignocellulosic biomass (2), biocatalyst(s) (3), water (3') and possibly acid and / or basic compounds (4), - a first stage (a) of liquefaction in the first reactor (1) by adding said pretreated lignocellulosic biomass (2) to said reactor without withdrawal, with possible addition also of biocatalyst(s) (3), - then a second step (b) of continuous liquefaction in the first reactor (1), with continuous withdrawal from the first reactor of a part (5') of the reaction medium and transfer to a second reactor of said part of the withdrawn reaction medium and addition over time of pretreated lignocellulosic biomass (2), - then an optional step (b1) of homogenization of the reaction medium in the first reactor (1), - then a step (b2) of transferring all of the reaction medium (5) from the first reactor (1) to the second reactor, where the conversion step (c) takes place, preferably in batch mode, - and a step (b3) of cleaning the first reactor, in particular using an aqueous solution, preferably acidic or basic.

15. Method according to one of the preceding claims, characterized in that the dry matter content MS of the pretreated lignocellulosic biomass used is at least 2% by weight and contains at least 10 g of cellulose per 100 g of dry matter.

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