Lignocellulosic biomass conversion process

Abstract

The present invention relates to a process for converting lignocellulosic biomass by contacting, in aqueous phase, pretreated lignocellulosic biomass 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, and (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. Figure for the abstract: Fig. 2

Description

Title of the invention: Process for converting lignocellulosic biomass technical field

[0001] The invention relates to a process for converting lignocellulosic biomass to produce so-called second generation (2G) sugars (or sugar juices).

[0002] These sugars can be used to produce other products by biochemical and / or catalytic means (for example alcohols such as ethanol, butanol, or other molecules, for example xylitol, solvents such as acetone etc.).

[0003] This process generally involves a pretreatment 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 the impregnated biomass, possibly coupled with a steam explosion. Once pretreated, the biomass is then converted into sugars by enzymatic hydrolysis, generally using an enzyme cocktail comprising at least one cellulolytic enzyme. The sugars thus formed can be fermented into alcohol by 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, an 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. Previous technique

[0004] The invention relates more particularly to the enzymatic hydrolysis of pretreated biomass, possibly combined with SSF or SSCF fermentation. The pretreated biomass is the substrate for the enzymatic hydrolysis reaction. The substrate residence time is defined as the average time it remains in the reaction conditions. The cycle time is considered here to be the time between two cleanings. Enzymatic hydrolysis can be carried out in different ways. Classically, enzymatic hydrolysis can be conducted in batch, fed-batch or continuous mode.

[0005] 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 Perform a complete emptying of the reaction medium. In this configuration, the substrate residence time is homogeneous, since all the substrate is added and removed simultaneously. The reactor cycle time consists of the reactor preparation time, the reaction time (the substrate residence time), and the emptying and cleaning time.

[0006] 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 carried out in true continuous operation, or in pseudo-continuous operation: that is, the time between withdrawals / additions is much shorter than the average residence time of the substrate. In this configuration, the reactor cycle time can be significantly increased. 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 substrate added.This configuration allows for better use of the available reactor volume; however, it has the disadvantage of heterogeneity in the residence time of the substrate and biocatalysts in the reactor. Indeed, the withdrawal process involves the withdrawal of the reaction medium, and therefore some of the substrate and / or added biocatalysts will be removed during the withdrawal without having completed the average residence time. Conversely, another portion will remain in the reactor longer than the average residence time. Consequently, in the case of reactions involving biocatalysts, i.e., microorganisms such as yeasts or bacteria, there is a greater risk of performance drift due to either the appearance of contamination or an evolution of the biocatalyst (for example, the loss of a genetic modification of interest) because of the extended residence time for some of the components.

[0007] Fed-batch (or fed-batch) operation is quite similar to batch operation: a portion of the substrate is gradually added to the reactor while the desired reaction(s) have started in the reactor, and then the entire reactor inventory is drained at the end of the cycle. This type of operation is common in bioprocesses and makes it possible to overcome the typical limitations of bioconversions: fed-batch operation is used, for example, when the medium has too high a concentration of toxic molecules, or when the initial rheology of the medium is difficult.

[0008] It is thus possible, as described in patent EP-3 461 902, to operate enzymatic hydrolysis in batch mode with a sequential feed of pre-treated biomass (called "fed-batch" feed according to the Anglo-Saxon term), where the sequential addition in the hydrolysis reactor is carried out in an increasingly spaced manner in time, so as to obtain a predetermined final dry matter rate, without withdrawal during the hydrolysis. This type of power supply is interesting because it allows for a yield of Conversion to improved sugar allows for processing at high dry matter content, leading to high concentrations of the desired product in the medium. Furthermore, it allows for better control of viscosity variations in the reaction medium: as the hydrolysis reaction progresses, the reaction medium becomes less viscous, allowing pre-treated biomass to be added to increase the dry matter content. However, this solution has limitations, similar to those of batch processing, requiring regular emptying and cleaning. These times are unavoidable and reduce tank utilization, particularly when the reaction time is less than 24 hours, thus negatively impacting productivity.

[0009] As described, for example, in patent WO 2013 / 088001, it is also known to decompose enzymatic hydrolysis into two steps, each carried out in a specific reactor: - a so-called liquefaction stage, which corresponds to the beginning of hydrolysis, during which the reaction medium is viscous and requires a complex stirring system and significant stirring energy from the reactor. On the other hand, the residence time of biomass is generally short, which makes it possible to limit the volume of reactors required with such a stirring system.

[0010] - a subsequent step, which corresponds to saccharification in the case where one prolongs Hydrolysis alone, or SSF or SSCF in the case where microorganisms are introduced to ferment the sugars while hydrolyzing, or hydrolyzing and fermenting, in a separate reactor. This step requires simpler agitation, 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 stage, in order to adjust accordingly the feed rates of the reactor in pre-treated biomass / in water / in enzymes / in other inputs (chemicals, acid or base for example), and thus optimize the liquefaction. This solution is attractive because it allows for efficient control of at least the first liquefaction stage, regardless of the type of biomass, without requiring prior characterization. However, in industrial production, it necessitates a significant number of reactors, each equipped with advanced agitation systems and relatively small in volume, to perform the liquefaction. Batch operation also reduces productivity due to the frequency of emptying, cleaning, and refilling.

[0011] The invention aims to overcome the drawbacks of prior solutions. It seeks to improve enzymatic hydrolysis processes, in particular to reduce and to simplify the necessary equipment and / or to reduce the energy consumption of said equipment, without degrading - or by increasing - biomass conversion yields. Summary of the invention

[0012] The invention relates firstly to a process for converting lignocellulosic biomass by contacting, in aqueous phase, pretreated lignocellulosic biomass 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 pre-treated lignocellulosic biomass and at least one biocatalyst into 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.

[0013] (This continuous addition makes it possible to maintain the reaction volume in the reactor at a given level in the reactor).

[0014] 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 from other agri-food industries, paper mills, lignocellulosic waste, etc.

[0015] The term "pre-treated" in relation to biomass is understood in its usual sense in the field of lignocellulosic biomass processing. It generally refers to impregnation with an acidic, basic, or oxidizing solution, or simply with water, followed by possible cooking, particularly involving steam explosion. For more details on this preliminary operation, reference may be made, for example, to patents FR 3054141, FR 3075202, and FR 3075203.

[0016] 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.

[0017] The term "biocatalyst," as detailed below, refers to an enzyme or mixture of enzymes and / or one or more types of microorganisms, particularly bacteria and yeast. The biocatalyst(s) may be added like biomass, continuously, discontinuously, or all at once.

[0018] For the purposes of the present invention, the term "continuous" dispensing is included, and Continuous feeding, strictly continuous withdrawal and feeding, or pseudo-continuous withdrawal and feeding. The term "pseudo-continuous" refers to the fact that the withdrawal and / or feeding of substrate can be carried out sequentially. For example, x kg of medium can be withdrawn for y minutes, then x kg of substrate and other inputs necessary for the reaction (biocatalysts, chemicals, acid or base, for example) are added for z minutes, and so on for the total duration of the step, for example, 20 to 24 hours. The withdrawal and addition times can be different or the same. The quantities of substrate and all other inputs added are the same as those withdrawn to maintain the same volume in the reactor.

[0019] The invention therefore proposes to carry out the conversion of pretreated biomass by breaking down the conversion into two stages. The first stage, liquefaction, has been modified compared to existing liquefaction methods by adding a subsequent continuous stage to a liquefaction stage, preferably by sequential addition (a "fed-batch" method). In this continuous stage, the withdrawal from the reactor and the feeding of pretreated biomass are continuous or quasi-continuous. The withdrawal rate and the feeding of pretreated biomass are adjusted throughout this stage to regulate, in particular, the rheology of the reaction medium in the reactor.

[0020] The process according to the invention therefore includes a liquefaction step (a), where the components of the reaction medium are added to the reactor / brought into contact (biomass, water, biocatalyst) to initiate liquefaction, without withdrawal. Liquefaction then continues with step (b) with continuous withdrawal.

[0021] And with this additional step of continuous liquefaction, the invention brings to the conversion process as a whole (including a proper enzymatic hydrolysis step after liquefaction) a huge industrial advantage: it makes it possible to maximize the utilization rate of reactors dedicated to liquefaction.

[0022] The invention thus makes it possible, for a given converted biomass production, 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.

[0023] This point is all the more advantageous, in terms of gains on industrial investments, since liquefaction reactors generally have to be equipped with complex agitation systems, as already mentioned, and whose operation is energy-intensive.

[0024] It has also been found that with this liquefaction method, the frequency of cleaning liquefaction reactors could be reduced without negative impact.

[0025] And it has also been found that with the liquefaction according to the invention, followed by the actual conversion stage in another reactor (enzymatic hydrolysis or simultaneous enzymatic hydrolysis and fermentation SSF or SSCF), the same conversion yields in sugar or alcohol were obtained.

[0026] It should also be noted that with the liquefaction carried out according to the invention, there is no problem with the control of the viscosity of the reaction medium, and there are no difficulties in drawing off / emptying the liquefaction reactor or in filling the next reactor allowing the conversion reaction to continue.

[0027] Advantageously, the pH of the aqueous phase can be regulated by the controlled addition of at least one acidic and / or basic compound in the first reactor during at least one of the two liquefaction steps (a) and (b). Thus, generally, if the biomass has been pretreated with an acidic liquor, the pH will be adjusted and then maintained at a setpoint value by controlled addition of base, and if it has been pretreated with a basic liquor, the pH will be adjusted to the setpoint value by controlled addition of acid. pH regulation can also be performed before liquefaction.

[0028] During the first liquefaction step (a), the additions of said pre-treated lignocellulosic biomass in the first reactor can be carried out at a fixed or variable frequency, and in 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 water (when water is added as a supplement if the biomass does not contain enough). Water can be added separately from the biomass, or the biomass may have already been in contact with all or part of the water before being added to the reactor. According to one embodiment, all the components of the reaction medium, i.e. water, pre-treated biomass and the biocatalyst(s) are added at once, at the same time or not, and preferably at the very beginning of step (a).

[0029] During the first liquefaction step (a), the additions of said pre-treated lignocellulosic biomass in the first reactor can be carried out at increasingly longer time intervals, as described for example in the aforementioned patent EP 3 461 902, and preferably with fixed quantities of biomass.

[0030] During the first liquefaction step (a), the biocatalyst or at least one of the biocatalysts can also be added sequentially to the reaction medium, either with the same frequency and spacing as the pre-treated biomass, or with a different frequency and spacing. Alternatively, the biocatalyst(s) can be added all at once, during, in particular at the very beginning, of the liquefaction step (a).

[0031] During the first liquefaction step (a), the additions of the pre-treated lignocellulosic biomass in the first reactor can be carried out continuously or sequentially.

[0032] 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 progressively added so that the volume of the reaction medium remains virtually unchanged and 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 operational characteristic of the reaction medium.

[0033] During the second liquefaction step (b), pretreated lignocellulosic biomass and at least one other "input" compound will be added over time. At least one of these input compounds will be chosen from among the following: water, an acidic compound, a basic compound, or biocatalyst(s). This biocatalyst(s) may be added with the biomass, in several stages but not simultaneously with the biomass, or all at once at the beginning of the step. Preferably, the biocatalyst(s) are added simultaneously with the biomass.

[0034] These inputs, as well as those present at the start of the liquefaction, may also include other compounds, additives for example such as antifoaming agent, antibacterial agent, or nutrients (in the case of an SSF or SSCF in particular, for the microorganisms used for fermentation).

[0035] The rheology of the reaction medium can be adjusted during the second liquefaction step (b) according to at least one operating condition among: the residence time of the pre-treated lignocellulosic biomass in the first reactor, the quantity and / or frequency of additions of pre-treated lignocellulosic biomass and input(s) of which at least one is chosen from one of the following compounds: water, acidic compound, basic compound, biocatalyst(s).

[0036] 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 equipment and / or the more energy will be required to operate it.

[0037] We thus seek 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 agitation in the reactor and its withdrawal to another reactor in a manner compatible with industrial-scale production.

[0038] The rheology of the reaction medium can be monitored by monitoring the viscosity of the reaction medium. actional or the mechanical torque of the shaft of an agitation system equipping the first reactor or the electrical power consumed by the motor operating said agitation system.

[0039] 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.

[0040] 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.

[0041] During the second liquefaction step (b), the residence time of the pre-treated 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.

[0042] During the second liquefaction step (b), it is advantageous to continuously withdraw a portion of the reaction medium 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 carried out in the presence of at least one biocatalyst.

[0043] Biocatalysts are already contained in the reaction medium transferred from one reactor to the other, but different or identical biocatalysts can be specifically added in the second reactor to those already introduced in the first reactor.

[0044] Thus, when the goal is the conversion of biomass into alcohol by SSF or SSCF: - according to a first embodiment, all the biocatalysts (enzymes and microorganisms) can be added as soon as the liquefaction is complete in the first reactor (and no biocatalyst should be added in step c) in the second reactor). - and according to another embodiment, the enzymes can be added during liquefaction in the first reactor, then the microorganisms, and possibly a supplement of enzymes, can be added in step c) in the second reactor.

[0045] At the end of the second liquefaction step (b), advantageously all the reaction medium from the first reactor is transferred to a second reactor where, in a conversion step (c), the conversion of the biomass contained in the transferred reaction medium is carried out in the presence of at least one biocatalyst.

[0046] Advantageously, the duration of the second liquefaction step (b) is less than or equal to the duration of the conversion step (c).

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

[0048] Coupling steps (a) and (b) in a dedicated liquefaction reactor and (c) in another reactor allows for the combined benefits of previous batch, fed-batch and continuous implementations: - The liquefaction reactor is operated according to steps (a) and then (b), which maximize the use of this reactor to contain the reaction medium and reduce the time allocated to emptying and cleaning during a cycle - the reactor operating step (c) ends in batch, which allows maximizing the conversion levels achieved, and controlling 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 larger volume than 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 can be fed by several reactors in which steps (a) and (b) take place.

[0050] It is also possible to use several smaller reactors to operate step (c), operating in particular in series.

[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, for converting all or part of this sugar(s) into alcohol(s) by fermentation.

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

[0053] According to one embodiment, 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. in the same way as the yeasts mentioned above.

[0054] Indeed, the present invention relates to the conversion of pretreated biomass to produce sugars by enzymatic hydrolysis, generally using an enzyme cocktail comprising at least cellulolytic enzymes. Cellulolytic enzymes include, for example, cellulases, endoglucanases, and beta-glucosidases. The enzyme cocktail may also include hemicellulolytic enzymes (hemicellulases). The enzymes may 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 used as is, or after processing.

[0055] The present invention also relates to the production of alcohols by fermentation from these sugars, according to two main types of process: - either saccharification and fermentation are carried out simultaneously; 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 product is liquefied. - either 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 microorganism, which can be a yeast or a bacterium, although in the present text we may only refer to yeasts, for the sake of brevity.

[0057] According to one embodiment, the process according to the invention may comprise the following steps: - an initial step (aO) of filling a first reactor with an initial input into said reactor of pre-treated lignocellulosic biomass, biocatalyst(s), water and possibly acidic and / or basic compounds, - a first step (a) of liquefaction in the first reactor by adding said pre-treated lignocellulosic biomass into said reactor without withdrawal, with possible addition of biocatalyst(s) as well, - 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 pre-treated lignocellulosic biomass, and possibly water and / or biocatalyst(s), and / or acidic and / or basic compounds, - then an optional step (bl) of homogenizing the reaction medium in the first reactor, - then a step (b2) of transferring all the reaction mixture from the first reactor to the second reactor, where step (c) of conversion 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 pre-treated lignocellulosic biomass in the first step a) may be sequential or non-sequential.

[0059] The additions of biocatalysts in the first step a) may be sequential or non-sequential.

[0060] The operating conditions for the first liquefaction (a) in enzymatic hydrolysis-only configuration (i.e., when the aim is to convert 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 for the first liquefaction (a) in the enzymatic hydrolysis and simultaneous fermentation (SSF or SSCF) configuration, i.e. when the aim is the conversion of 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 the conversion step (c) may be identical or different from those of the first liquefaction (a) and the second liquefaction (b). For example, they will be different 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 (DM) content 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, or 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. List of figures [Fig. 1]

[0065] Fig. 1 represents the different stages of a liquefaction step of pre-treated biomass according to the prior art. [Fig. 2]

[0066] Fig. 2 represents the different stages of a liquefaction step of pre-treated biomass according to an embodiment of the invention. [Fig. 3]

[0067] Figure 3 is a graph showing 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. The concentrations are expressed in g / kg of reaction medium on the y-axis, and time is expressed in hours on the x-axis.

[0068] The figures, and more specifically figures 1 and 2, are very schematic and not to scale. The same references refer to the same flows / devices from one figure to the other. Description of the implementation methods

[0069] The invention aims to improve the operating method for the liquefaction of pretreated lignocellulosic biomass. Liquefaction is to be understood as a starting step in the conversion of biomass under the effect of biocatalysts. This involves conversion by enzymatic hydrolysis (and / or optional fermentation). This liquefaction is sometimes referred to as "pre-hydrolysis".

[0070] A protocol known for operating enzymatic hydrolysis alone or enzymatic hydrolysis and simultaneous fermentation of biomass or lignocellulosic 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 either enzymatic hydrolysis alone or enzymatic hydrolysis and simultaneous fermentation in batch mode. For example, reference should be made to the aforementioned patent WO 2013 / 088001 for a description of this type of protocol.

[0071] The protocol according to a preferred embodiment of the invention proposes a liquefaction with a first fed-batch stage of lignocellulosic biomass in an ad hoc reactor (relatively small usable volume with high-performance stirring equipment), which is followed in the same reactor by a continuous operation stage, with: - a continuous or pseudo-continuous transfer to a more standard reactor (whose usable volume can be much larger and whose stirring equipment is simpler than the first reactor) to continue enzymatic hydrolysis alone, or simultaneous enzymatic hydrolysis and fermentation in batch mode, - and continuous or pseudo-continuous feeding of lignocellulosic substrate and various inputs / biocatalysts in the first reactor.

[0072] 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).

[0073] The invention relates to the implementation of enzymatic hydrolysis for the production of sugars or of enzymatic hydrolysis and simultaneous fermentation for the production of alcohol from lignocellulosic biomass / waste.

[0074] The feedstock treated by the process according to the invention is pretreated lignocellulosic biomass. The pretreatment of the lignocellulosic biomass makes the cellulose accessible and reactive to enzymes, and consists of contacting the lignocellulosic biomass with a solvent and optionally a catalyst (generally combined in a liquid) at a given temperature and pressure for a given residence time. Any type of pretreatment can be applied to obtain the pretreated lignocellulosic substrate.

[0075] The pre-treated biomass can also undergo water washing (resuspension of the pre-treated biomass with water or a mixing fluid, solid / liquid filtration, washing of the solid fraction with water and then solid / liquid filtration) after its pre-treatment and before the start of the liquefaction according to the invention.

[0076] During the enzymatic hydrolysis or SSF or SSCF step (which includes the liquefaction steps according to the invention exemplified below), the pretreated lignocellulosic substrate is mixed with a liquid solution containing enzymes (and optionally 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 performed at relatively high concentrations of the pretreated lignocellulosic substrate, i.e., at a high dry matter content, in order to reduce the economic and energy costs of the process if the product of interest is to be concentrated.

[0077] This dry matter content (acronym "DM") refers to the dry matter content 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).

[0078] The intimate mixing of the pretreated lignocellulosic substrate with said liquid solution containing the enzymes (and possibly the yeasts) proves difficult when the dry matter content is high. Indeed, the onset of enzymatic hydrolysis at high dry matter content poses, in particular, mixing problems and of homogenization. The reaction medium is very pasty and viscous.

[0079] To address this problem, the existing solutions are: - equip the fermentation (or enzymatic hydrolysis) reactors with a specific complex agitator to ensure homogenization of the reaction medium. - Perform a gradual feeding of substrate, known as fed-batch feeding, into the reactor, without withdrawing the reaction medium. As the reaction progresses, the mixture becomes less viscous and it is possible to add fresh substrate to increase the amount of substrate in the medium. - Conduct the fermentation (or enzymatic hydrolysis) in two stages: A first stage called liquefaction, which reduces the viscosity of the medium. This stage corresponds to the first few hours of enzymatic hydrolysis (or SSF / SSCF), during which 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 that allows transfer to a tank equipped with a standard agitator for the continuation of enzymatic hydrolysis. A second step corresponding to the continuation of enzymatic hydrolysis (or SSF / SSCF): the liquefied biomass from the liquefaction step is transferred into fermentation (or hydrolysis) reactors in which the conversion of residual cellulose and hemicelluloses into sugars and then the conversion of sugars into ethanol continues.

[0080] The invention focuses on this latter approach, and aims to improve it. Previous / comparative embodiment

[0081] The liquefaction step of the pretreated substrate is carried out in a "fed-batch" or "fed-batch" manner, i.e., with the substrate being added progressively to the mixture of water + biocatalysts + base. (We are taking here the non-limiting example of a pretreatment with impregnation by an acidic liquor, hence the addition of a base to increase the pH of the reaction medium).

[0082] 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, pH, and temperature are adjusted to the required specifications. Then, some or all of the enzymes (and possibly yeast) are added. A fed-batch (sequential feeding) is then carried out with the remaining pretreated substrate to increase the dry matter content. A fed-batch of the enzymes (and yeast) can also be performed. The rheology of the solid suspension necessitates specific implementation to ensure proper agitation for carrying out the reaction in a medium with the highest possible concentration of pretreated lignocellulosic substrate. To alleviate some of this constraint, a specific agitator technology can be used to equip the liquefaction reactor.

[0083] Helical type agitators are generally the most suitable, even though they are complex and limited in size for mechanical design reasons.

[0084] Various parameters allow the operating conditions to be defined and the fed-batch strategy to be established. The ranges indicated below are examples: - the dry matter (DM) content of the pre-treated biomass (2 to 60% by weight) - the cellulose content of the substrate - the dose of enzymes relative to cellulose (5 to 100 mg / g of cellulose) - the yeast inoculation rate (0.1 to 3 g / kg medium), - the dry matter (DM) content of the initial mixture (2 to 60% by weight) - the dry matter (DM) content of the final mixture (2 to 60% by weight) - the total mass and / or volume of the final mixture in the reactor - the number of fed-batch additions - the duration of fed-batch additions (0 to 48 hours) - the duration of liquefaction (1 to 48 hours)

[0085] The description of the prior fed-batch liquefaction protocol is as follows: 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. Agitation is started to homogenize the mixture and adjust the pH and temperature. An antibacterial agent, such as chloramphenicol or the one marketed under the trade name VitaHop by BetaTec, can be added to the reaction medium. 2- pH regulation by adding a basic solution, for example NH4O, KOH, or NaOH, if the pretreated substrate was produced under acidic conditions (e.g., impregnation with sulfuric acid followed by steam explosion), or by adding an acidic solution if the pretreated substrate was produced under basic conditions. pH regulation can be maintained during subsequent phases of the protocol. 3- Injection of enzymes (and yeast) followed by the start of fed-batch liquefaction: once the initial mixture is thoroughly homogenized, a specific quantity of enzymes (and yeast) is introduced into the reactor. This injection allows the chosen dose of biocatalysts to be reached. After the biocatalysts are injected, the fed-batch liquefaction process begins. 4- Fed-batch – additions of pre-treated biomass: After a predetermined time, the medium is much less viscous, and the first addition of pre-treated biomass can take place. Depending on the enzyme addition strategy, additional enzymes can be added at this point. The mass of enzymes introduced corresponds to the amount of pre-treated biomass added. It is possible to choose to add a quantity of enzymes based on the amount of pre-treated biomass added with each biomass addition, or to add all or most of the amount of enzymes. This is necessary from the start of the fed-batch process. Additions are made regularly until the mixture reaches the desired final mass. Generally, the mass and frequency of each addition are constant and regular. As mentioned above, the biomass additions can also be spaced further and further apart. The goal is generally to achieve a target enzyme dose expressed in grams per kilogram of cellulose. 5- End of liquefaction: After the pre-treated biomass addition phase, the reaction continues at a constant final volume. The reaction and viscosity decrease continue until the viscosity is deemed sufficiently low to transfer the medium to the enzymatic hydrolysis reactor (or SSF or SSCF) equipped with a standard agitator. 6- After transfer of 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 risks of contamination.

[0086] The operating conditions for liquefaction in enzymatic hydrolysis-only configuration (i.e., when the goal is conversion of biomass into oligomeric or monomeric sugars) are: - 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.

[0087] The operating conditions for liquefaction in a simultaneous enzymatic hydrolysis and fermentation (SSF or SSCF) configuration, i.e. when the goal is the conversion of 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.

[0088] The duration of the liquefaction is between 1 hour and 48 hours, in particular between 2 hours and 24 hours, in particular between 5 hours and 12 hours.

[0089] Figure 1 represents the different cycle durations / phases A to E of the liquefaction, showing the liquefaction reactor 1 at each of these phases: A: Filling of the liquefaction reactor 1 with pre-treated 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 and 4 hours B: Fed-batch of pre-treated biomass, with sequential addition of pre-treated biomass 2 and possibly basic compound 4 to regulate pH: duration from 2 hours to 10 hours C: Homogenization (optional): 1 to 2 hours D: Transfer of reaction medium 5 from reactor 1 to a downstream reactor (not shown): duration between 2 h and 4 h E: Cleaning of liquefaction reactor 1: between 2 and 4 hours

[0090] 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 and 170 hours, preferably between 48 and 140 hours. After draining the enzymatic hydrolysis (or SSF or SSCF) reactor, the reactor is cleaned to limit the risk of contamination.

[0091] Limiting the volume of liquefaction reactors relative to SSF or SSCF or enzymatic hydrolysis reactors (the ratio of usable volume between the SSF or SSCF or enzymatic hydrolysis reactor and the liquefaction reactor is, for example, between 2 and 10) generally requires the implementation of a timing schedule to ensure the continuity of the various operating phases (including filling, emptying, and cleaning phases) between the two steps: liquefaction and SSF or SSCF (or enzymatic hydrolysis). This timing schedule is also established to limit the number of liquefaction and SSF or SSCF or enzymatic hydrolysis reactors by minimizing downtime. Embodiment according to the invention

[0092] The description of the protocol according to the invention for fed-batch liquefaction with an additional continuous step is schematically represented in [Fig.2], which uses the same conventions as [Fig.1]:

[0093] The durations of the phases indicated are examples.

[0094] A - Initial phase of filling liquefaction reactor 1 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. Agitation is started to homogenize the mixture, and the pH and temperature are adjusted. pH regulation is achieved by adding a basic solution 4, for example NH4OH, KOH, or NaOH if the pretreated substrate was produced under acidic conditions (e.g., impregnation with sulfuric acid followed by steam explosion), or by adding an acidic solution if the pretreated substrate was produced under basic conditions. B - Start of fed-batch liquefaction - addition of pre-treated biomass 2: Once the initial mixture is homogeneous, a certain quantity of enzymes 3 (and yeast) is introduced into reactor 1. This injection allows the chosen dose of biocatalysts to be reached. After the injection of the biocatalysts, the fed-batch liquefaction begins: - after a certain time, the medium is already much less viscous and the first addition of Pre-treated biomass can be added. Following the enzyme 3 addition strategy, additional enzymes can be added at this point. The mass of enzyme 3 added is proportional to the amount of pre-treated biomass 2 added to maintain a constant enzyme / cellulose ratio. Additions are repeated until the mixture reaches the desired final mass. Generally, the mass and frequency of each addition are constant and regular. F - Continuous phase of the liquefaction specific to the invention: a portion 5' 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 maintain a constant volume: pretreated biomass 2, biocatalyst(s) 3, water 3', and acidic or basic solution 4 for pH regulation. The continuous phase of the liquefaction is carried out 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, in order not to negatively impact the operation of the enzymatic hydrolysis reactor (or SSF or SSCF).

[0095] The operating conditions for liquefaction in enzymatic hydrolysis-only configuration (i.e., when the goal is conversion of 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.

[0096] The operating conditions for liquefaction in a simultaneous enzymatic hydrolysis and fermentation (SSF or SSCF) configuration, i.e., when the goal is the conversion of biomass into alcohol by hydrolysis and fermentation, are preferably: - 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.

[0097] The operating conditions (dry matter, biocatalyst dosage) are maintained at the target conditions; therefore, the parameters that allow obtaining the desired rheology are the quantity and frequency of additions, as well as the residence time of the pomace 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 hours.

[0098] The duration of the continuous phase is for example between 1 hour and 170 hours, in particular between 10 hours and 72 hours.

[0099] C - Homogenization (optional): 1 to 2 hours D - Transfer of reaction medium 5 from reactor 1 to a downstream reactor (not (represented); End of liquefaction: after the continuous phase, the entire liquefaction medium is transferred to the enzymatic hydrolysis reactor (or SSF or SSCF), preferably equipped with a standard stirrer: duration between 2 and 4 hours. 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 and 4 hours.

[0100] Enzymatic hydrolysis (or SSF or SSCF) is always carried out in batch mode. The operating conditions of pH and temperature are either identical to those of liquefaction or different. This is the case, for example, for a configuration where the biocatalysts introduced in the liquefaction step are different from those introduced in the SSF or SSCF step.

[0101] The duration of the enzymatic hydrolysis (or SSF or SSCF) is between 10 hours and 170 hours, in particular between 70 hours and 140 hours.

[0102] According to one 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 beginning of the continuous phase of liquefaction.

[0103] Naturally, it is also possible to perform only enzymatic hydrolysis, without yeast, either to valorize the sugars without transforming them into alcohol or by transforming them differently. It is also possible to carry out the fermentation of the sugars separately, in a dedicated fermentation reactor.

[0104] Compared to liquefaction according to the prior method (example in [Fig. 1]), liquefaction according to the invention (example in [Fig. 2]) offers the following advantages:

[0105] - Increased liquefaction productivity through improved utilization rate of each liquefaction reactor. The importance 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).

[0106] - For the same quantity of pre-treated biomass to be liquefied, the number of reactors Liquefaction 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 investment.

[0107] - The cleaning phase of the liquefaction reactor 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 reaction time.

[0108] - The ethanol production yields and the conversion yields of the Cellulose and hemicellulose sugars are maintained the same or almost the same same. Example 1 (comparative)

[0109] It uses the protocol described above with the help of [Fig. 1].

[0110] To illustrate the gain on the utilization rate of liquefaction reactors, we will compare two chronograms: one for a known fed-batch liquefaction ([Fig.1]), the other for a fed-batch liquefaction with a continuous phase ([Fig.2]).

[0111] Liquefaction (+SSCF) is carried out with a dry matter content of 20% by weight, the dose of enzymes is 8 mg of enzymes / gDM, the quantity of yeast is 0.5 g / kg medium.

[0112] The linearized flow rate of pretreated substrate feeding the liquefaction stage is 15.6 t DM / h. The dry matter content of the pretreated substrate is 38% by weight.

[0113] Table 1 below details the chronogram of the known liquefaction:

[0114] [Tables 1] Operation Duration Filling Water Ih Initial filling of pre-treated biomass in the liquefaction reactor 2h Addition of Biocatalysts Ih Addition of pre-treated biomass in fed-batch to the liquefaction reactor 9 h Homogenization 2h Transfer to SSCF reactor 2h Cleaning 3 h Total cycle time 20 h

[0115] 4 liquefaction reactors of 435 m3 (usable volume) are therefore required for The pre-treated biomass is liquefied with an operational utilization rate of 45%. The liquefied biomass is transferred into 4 SSCF reactors of 4,500 m3 each, operating in batch mode.

[0116] The durations of the phases in SSCF are indicated in the following table 2:

[0117] [Tables2] Operation Duration Filling Liquefaction Medium 37 h Reaction 113 h Transfer to SSCF Reactor 6 h Cleaning 4 h Total Cycle Time 160 h Example 2 (according to the invention)

[0118] It follows the liquefaction protocol according to the invention described above and illustrated in [Fig. 2]. Table 3 below details the liquefaction timing diagram according to the invention:

[0119] [Tables3] Operation Duration Water Filling 1 h Initial filling of pre-treated biomass in the liquefaction reactor 2 h Addition of Biocatalysts 1 h Addition of fed-batch pre-treated biomass to the liquefaction reactor 8 h Homogenization 1 h Continuous phase in the liquefaction reactor 20 h Homogenization 2 h Transfer to SSCF reactor 2 h Cleaning 3 h Total cycle time 40 h

[0120] Two liquefaction reactors of 500 m3 (useful volume) are therefore required to The pre-treated biomass is liquefied with an operational utilization rate of 72.5%. The residence time of the pre-treated biomass in the liquefaction reactor is 8 hours. The liquefied biomass is transferred to 4 SSCF reactors of 4,500 m³ each, operating in batch mode.

[0121] The durations of the phases in SSCF are the same as those indicated in Table 2 for comparative example 1. Example 3 (comparative)

[0122] 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, and the basic NH4O2 solution to adjust the pH to 5.3. Then, all the enzymes and yeast are added, corresponding to the liquefaction time t0. 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: a 5% dry matter increase over 3 hours, followed by a 3% dry matter increase over 3 hours.

[0123] The dry matter content of the liquefaction medium is 22% by weight, the dose of enzymes is 8 mg of enzymes / g DM, the quantity of yeast is 0.5 g / kg medium.

[0124] The enzyme solution has a protein concentration of 35 g / L and a density at 20°C of 1.02 g / cm3.

[0125] The dry matter content of the pre-treated biomass is 42% by weight.

[0126] The liquefaction reactor is equipped with a helical agitator allowing good homogenization of the reaction medium and optimal torque and viscosity management.

[0127] The liquefaction medium is then homogenized for 2 hours to allow the agitator torque to decrease, and is then transferred to an 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, an agitation system comprising two TT-type three-bladed propellers (axial flow impeller) and a two-bladed straight-bladed bottom turbine (radial flow impeller).

[0128] The liquefaction time (initial filling + fed-batch of pre-treated biomass + homogenization) is 12 hours. The SSCF time is 132 hours (excluding liquefaction).

[0129] The operating parameters monitored are the stirrer torque in the liquefaction reactor, the ethanol, glucose and xylose contents in the medium (analysis by HPLC) during the SSCF reaction.

[0130] The performance of the SSCF process is evaluated based on the following yields: - 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 - Based on the total enzymatic hydrolysis of a sample at the end of fermentation: the hydrolysis yields of cellulose and xylan, and the ethanol production yield, calculated relative to the Pasteur yield (i.e., 94.7% of the Gay-Lussac yield). As a reminder, the Gay-Lussac yield is equal to the theoretical yield derived from the stoichiometric equation. C6H12O6 (glucose) -> 2 C2H5OH (ethanol) + 2 CO2 This yields 51.1 kg of ethanol produced from 100 kg of glucose. Pasteur subsequently demonstrated that there were co-products associated with ethanol and CO2 production (notably glycerol, succinic acid, heavy alcohols, and microbial growth). Pasteur's yield takes these sugar losses into account, resulting in 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:

[0131] [Tables4] Glucose content at final t Ethanol content at final t EtOH / MS yield Cellulose hydrolysis yield Xylan hydrolysis yield EtOH / Potential EtOH g / kg g / kg g / 100g MS % wt % wt % wt SSCF A 1.15 56.0 25.4 81 90 79.7 Example 4 (according to the invention)

[0132] Like example 2, it follows the liquefaction protocol according to the invention described above and illustrated in [Fig.2].

[0133] The filling phase of the liquefaction reactor up to the fed-batch stage of the substrate is identical to that of Example 3. The continuous phase is then implemented: a portion of the medium is withdrawn and transferred to the SSCF reactor equipped with a standard vertical agitator. A portion of the inputs (see Table 5 below) is added to the liquefaction reactor to operate at a constant volume.

[0134] The residence time of the pretreated biomass in the liquefaction reactor during the continuous phase is set at: - 12 hours in the case where the biomass has been pre-treated under less acidic conditions (1.8% wt H2SO4 in the impregnation liquor) - 6 hours in the case where the biomass has been pre-treated under more acidic conditions (2.4% wt H2SO4 in the impregnation liquor) This residence time allows a constant agitator torque to be maintained in the liquefaction reactor. The duration of the continuous phase is 24 hours. The quantities of medium to be withdrawn and the quantities of inputs to be added to the liquefaction reactor, which has a usable volume of 3,000 kg, are indicated in Table 5 below. The nutrients are intended for the yeast (nitrogen source) and are here in the form of a soluble maize protein solution, notably the one marketed under the name Solulys by the company Roquette.

[0135] [Tables5] Residence time in the liquefaction reactor: 6 hours (h) Quantity of medium to be withdrawn: 250 kg (125) Quantity of water to be added: 110 kg (55) Quantity of enzyme solution to be added: 13.2 kg (6.6) Quantity of yeast to be added: 0.125 kg (0.063) Quantity of nutrients to be added: 0.145 kg (0.073) Quantity of raw pretreated biomass to be added: 125 kg (63) Quantity of base solution (NH4O at 24% wt%) to be added: 1.91 kg (0.95)

[0136] 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 in the previous example 3, the results are grouped in table 6 below.

[0137] [Tableauxô] Glucose content at final t Ethanol content at final t EtOH / MS yield Cellulose hydrolysis yield Xylan hydrolysis yield EtOH / Potential EtOH g / kg g / kg g / 100g MS % wt % wt % wt SSCF B 0.96 58.0 26.4 85 100 82.1

[0138] It is thus verified that the yields, in particular those in ethanol, are identical, whether the liquefaction is carried out according to the known protocol (example 3) or according to the protocol of the invention (example 4).

[0139] The residence time allows a constant agitator torque to be kept in the liquefaction reactor.

[0140] The ethanol production and xylose consumption kinetics do not show any difference in reactivity between SSCFs conducted with standard conventional fed-batch liquefaction and SSCFs conducted with fed-batch liquefaction then continues according to the invention.

[0141] These good performance results are also evident from the graph in [Fig. 3]. Indeed, curves C1 and C2 correspond respectively to the ethanol concentrations according to comparative example 3 and example 4 of the invention: it can be seen that the ethanol concentrations converge after 140 hours of SSCF. Similarly, curves C3 and C4 correspond respectively to the xylose concentrations according to comparative example 3 and example 4 of the invention: here again, the drops in xylose concentration, due to their conversion to ethanol, reach very low and similar levels after 140 hours. It can be seen that the invention also gives better results in terms of productivity during the first 48 hours.

Claims

Demands

1. A process for converting lignocellulosic biomass by contacting, in aqueous phase, pretreated lignocellulosic biomass 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 into 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 part of said reaction medium, addition of at least one biocatalyst, and continuous addition of pretreated lignocellulosic biomass,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), and 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 carried out in the presence of at least one biocatalyst.

2. A process according to the preceding claim, characterized in that, during the first liquefaction step (a), the additions of said pre-treated lignocellulosic biomass (2) into the first reactor (1) are carried out at a fixed or variable frequency, and in fixed or variable quantities.

3. A process according to the preceding claim, characterized in that, during the first liquefaction step (a), the additions of said pre-treated lignocellulosic biomass (2) into the first reactor (1) are carried out at increasingly longer time intervals, and preferably with fixed quantities of biomass.

4. A process according to any 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. A method according to any one of the preceding claims, characterized in that that, during the second liquefaction step (b), pre-treated 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'), acidic compound, basic compound (4), bio-catalyst(s) (3).

6. A process according to any 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 frequency of additions of pretreated lignocellulosic biomass and input(s), at least one of which is selected from one of the following compounds: water (3'), acidic compound, basic compound (4), biocatalyst(s) (3).

7. A method according to any one of the preceding claims, characterized in that the rheology of the reaction medium is followed by following the viscosity of the reaction medium or the mechanical torque of the shaft of an agitation system equipping the first reactor (1) or the electrical power consumed by the motor operating said agitation system.

8. A process according to any 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.

9. A process according to any 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.

10. A process according to any 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 carried out in a conversion step (c), in the presence of at least one biocatalyst.

11. A process according to any one of the preceding claims, characterized in that step (c) in the second reactor is operated in fed-batch and then in batch, preferably over a period of between 10 and 170 hours, in particular between 70 and 140 hours.

12. A method according to any one of the preceding claims, characterized in that said method comprises: - an initial step (aO) of filling the first reactor (1) with a first input into said reactor of pre-treated lignocellulosic biomass (2), biocatalyst(s) (3), water (3') and possibly acidic and / or basic compounds (4), - a first step (a) of liquefaction in the first reactor (1) by adding said pre-treated lignocellulosic biomass (2) into 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 (bl) of homogenizing the reaction medium in the first reactor (1), - then a step (b2) of transferring all the reaction medium (5) from the first reactor (1) to the second reactor, where step (c) of conversion 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.

13. A process according to any one of the preceding claims, characterized in that the dry matter content DM 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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