Process for converting lignocellulosic biomass

Abstract

The invention relates to a method for converting lignocellulosic biomass by contacting pretreated lignocellulosic biomass with at least one biocatalyst (3) in an aqueous phase in a first reactor (1) containing a reaction medium, the reaction medium comprises the pretreated lignocellulosic biomass (2) and the biocatalyst in an aqueous phase, the process comprises: (a) a first liquefaction step without removing all or part of the reaction medium from the reactor by adding the pretreated lignocellulosic biomass and at least one biocatalyst to the reactor, followed by (b) a second continuous liquefaction step, wherein a portion of the reaction medium is continuously withdrawn from the first reactor, at least one biocatalyst is added, and pretreated lignocellulosic biomass is continuously added.

Description

Technical Field

[0001] The present invention relates to a method for converting lignocellulosic biomass to produce "second generation" (2G) sugars (or sugar liquors).

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

[0003] This method generally includes pretreatment of the biomass, which may include, for example, impregnation with a liquid containing a chemical catalyst such as an acid, base or oxidant compound, followed by cooking the impregnated biomass, which may be optionally combined with steam explosion. Once pretreated, the biomass is then converted into sugars by enzymatic hydrolysis, usually under the action of an enzyme mixture comprising at least one cellulolytic enzyme. The sugars thus formed can be fermented into alcohols under the action of yeast or bacteria - in a fermentation step separate from the enzymatic hydrolysis step or simultaneously with the enzymatic hydrolysis. In the latter case, when the fermented sugar is a mixture of C5 and C6 sugars (i.e., having 5 or 6 carbons) obtained by enzymatic hydrolysis, it is referred to as SSCF, an acronym for "simultaneous saccharification and co-fermentation", or when only C6 sugars are fermented, it is referred to as SSF, i.e., "simultaneous saccharification and fermentation". Prior art

[0004] The invention more particularly relates to the enzymatic hydrolysis of pretreated biomass, optionally in combination 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 here considered to be the time between two cleaning operations.

[0005] Enzymatic hydrolysis can be performed in various ways. Conventionally, enzymatic hydrolysis can be performed in batch mode, fed-batch mode or continuously.

[0006] Operation in batch mode can be summarized as follows: substrate is added to the conversion reactor at the beginning of the cycle, then remains in contact with the biocatalyst for the reaction time required to achieve the target conversion rate, and then the reaction medium is completely emptied. In this configuration, the residence time of the substrate is uniform because all substrate is added and removed simultaneously. The cycle time of the reactor is composed of the reactor preparation time, the reaction time (residence time of the substrate), and the emptying and cleaning time.

[0007] The operation of the continuous mode can be summarized as follows: substrate is added and a portion of the reaction medium is removed over time. These additions and removals can be performed in a real-continuous mode or a pseudo-continuous mode: that is, the time between removal / addition is much faster than the average residence time of the substrate. In this configuration, the cycle time of the reactor can be greatly 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 the added substrate. This configuration enables better utilization of the available reactor volume; however, its disadvantage is that it exhibits the heterogeneity of the residence time of the substrate and the biocatalyst in the reactor: this is because the removal performed is the removal of the reaction medium, so a portion of the added substrate and / or biocatalyst will be removed during the removal process without achieving the average residence time. On the contrary, the residence time of another part in the reactor is longer than the average residence time. Therefore, in the case of a reaction involving a biocatalyst, i.e., a microorganism such as yeast or bacteria, due to the extended residence time of a portion of the inventory, there is a greater risk of performance drift associated with the occurrence of contamination or changes in the biocatalyst (e.g., loss of genetic modification of interest).

[0008] The operation in fed-batch mode is quite similar to that in batch mode: a portion of the substrate is gradually added to the reactor when the desired reaction has already started in the reactor, and then the entire inventory of the reactor is emptied at the end of the cycle. This type of operation is routine for bioprocesses and makes it possible to circumvent typical limitations of bioconversions: for example, fed-batch mode operation is performed when the medium has an excessively high content of toxic molecules or when the initial rheology of the medium is problematic.

[0009] It is thus possible to carry out the enzymatic hydrolysis in batch mode with sequential feeding of pretreated biomass (called "fed-batch" feeding) as described in patent EP-3461902, in which sequential additions to the hydrolysis reactor are carried out at increasingly large intervals in order to obtain a predetermined final solids content, without withdrawal during the hydrolysis process.

[0010] This type of feeding is interesting because it makes it possible to obtain improved yields of conversion to sugars, and it makes it possible to work at high solid contents, which brings high concentrations of the product of interest in the medium, and also because it makes it possible to better control the viscosity changes of the reaction medium: as the hydrolysis reaction proceeds, the viscosity of the reaction medium becomes lower and lower, so pretreated biomass can be added again to increase the solid content of the reaction medium. However, similar to the limitations of batch mode operation, this solution has limitations, wherein emptying and cleaning times are regularly required. These times cannot be shortened and reduce the utilization of the container, particularly when the reaction time is less than 24 hours, which adversely affects productivity.

[0011] It is also known to split the enzymatic hydrolysis into two steps, each carried out in a specific reactor, as described, for example, in patent WO 2013 / 088001:

[0012] - a step known as the liquefaction step, which corresponds to the beginning of the hydrolysis, during which the reaction medium is viscous and the reactor requires a complex stirring system and a large amount of stirring energy. On the other hand, the residence time of the biomass is generally short, which makes it possible to limit the volume of the reactor to which such a stirring system is to be installed.

[0013] - A subsequent step in another reactor, which corresponds to saccharification in the case of continued hydrolysis alone, or to SSF or SSCF in the case of hydrolysis or hydrolysis and fermentation with the introduction of microorganisms to ferment the sugars. This step requires simpler stirring and less energy, but longer residence times and therefore larger reactor volumes.

[0014] According to the teaching of this patent, the rheological properties of the reaction medium are monitored during the first liquefaction step, with the rate of supplying pretreated biomass / water / enzyme / other inputs (such as chemical products, acids or alkalis) to the reactor accordingly adjusted, and therefore optimized liquefaction. This solution is interesting because it makes it possible to efficiently carry out at least the first liquefaction step, regardless of the nature of the biomass, without having to characterize it. On the other hand, in industrial production, a large number of super-equipped stirring devices and relatively small reactors of volume are required to liquefy. The operation of batch mode also adversely affects productivity due to the frequency of emptying, cleaning and filling operations.

[0015] The object of the present invention is therefore to overcome the disadvantages of existing solutions. It aims to improve the enzymatic hydrolysis process, in particular to reduce and simplify the necessary equipment and / or reduce the energy consumption of said equipment, without reducing or even also increasing the biomass conversion yield. SUMMARY OF THE INVENTION

[0017] A first subject of the present invention is a method for converting lignocellulosic biomass by contacting a pretreated lignocellulosic biomass with at least one biocatalyst in an aqueous phase in a first reactor containing a reaction medium comprising said biomass and said biocatalyst in an aqueous phase, said method comprising:

[0018] -(a) a first liquefaction step by adding the pretreated lignocellulosic biomass and at least one biocatalyst to the reactor without withdrawing all or part of the reaction medium from the reactor, - and then (b) a second continuous liquefaction step, wherein a part of the reaction medium is continuously withdrawn from the first reactor, at least one biocatalyst is added and pretreated lignocellulosic biomass is continuously added.

[0019] (This continuous addition makes it possible to keep the reaction volume in the reactor at a given level in the reactor).

[0020] The biomass targeted by the present invention is of lignocellulosic nature and has a very diverse matrix, including woody matrices, such as various woods (hardwood and softwood), by-products from agriculture (wheat straw, corn cobs, etc.) or by-products from other agricultural food or paper industries, lignocellulosic waste, etc.

[0021] The term "pretreated" with respect to biomass is understood in its usual sense in the field of lignocellulosic biomass treatment. This generally involves impregnation with an acidic, alkaline or oxidizing liquid or simply with water, followed by optional cooking, in particular in combination with steam explosion. For more details on this prior operation, reference may be made, for example, to patents FR 3054141, FR 3075202 and FR 3075203.

[0022] The reaction medium is in the aqueous phase: the water may originate from the biomass itself (which naturally contains water and / or has been impregnated with an aqueous solution prior to the treatment according to the invention). The water may also originate from a specific water supply.

[0023] As described in detail below, the term "biocatalyst" is understood to mean an enzyme or a mixture of enzymes and / or one type of microorganism or multiple types of microorganisms, in particular of bacterial or yeast type. The biocatalyst can be added continuously or discontinuously or all at once as with the biomass.

[0024] For the present invention, the term "continuous" taking out and "continuous" feeding includes continuous taking out and feeding in the strict sense, or pseudo-continuous taking out and feeding. The term "pseudo-continuous (pseudo-continuous)" is understood to encompass the fact that the taking out and / or feeding of the substrate can be carried out in sequence. As an example of this transfer mode: x kg of medium y minutes of duration can be taken out, then x kg of substrate and other inputs required for the reaction (such as biocatalysts, chemicals, acids or bases) z minutes of duration are added, and the like is inferred to continue the entire duration of the step, such as 20 to 24 hours. The duration of taking out and adding can be different or similar. The amount of the substrate added and all other inputs is the same as those taken out, so that the volume in the reactor remains the same.

[0025] The invention therefore proposes to carry out the conversion of pretreated biomass by splitting the conversion into two stages, the first of which, liquefaction, has been modified compared to the known operating modes for liquefaction - by adding to the liquefaction step a subsequent step carried out continuously, preferably by sequential addition ("fed batch" mode). In this continuous step, the withdrawal from the reactor and the feed of pretreated biomass are continuous or pseudo-continuous. The withdrawal flow and the feed of pretreated biomass are adjusted throughout this step, in particular to adjust the rheology of the reaction medium in the reactor.

[0026] The method according to the invention thus provides a liquefaction step (a) in which the components of the reaction medium are added to / contacted in the reactor (biomass, water, biocatalyst) to initiate liquefaction without withdrawal. The liquefaction then continues with step (b) with continuous withdrawal.

[0027] By virtue of this additional continuous liquefaction step, the present invention provides a huge industrial advantage for the entire conversion process (including the post-liquefaction enzymatic hydrolysis step itself): it makes it possible to maximize the utilization of the reactor dedicated to liquefaction.

[0028] By limiting the frequency of non-productive feeding, emptying and cleaning phases, the invention therefore makes it possible to limit the number of liquefaction reactors in use for a given yield of converted biomass (or to increase the yield of converted biomass for a given number of liquefaction reactors in use).

[0029] This is all the more advantageous in terms of industrial return on investment, since, as already mentioned, liquefaction reactors usually have to be equipped with complex stirring systems, the operation of which is very energy-intensive.

[0030] It has also been found that with this liquefaction mode, the frequency of cleaning of the liquefaction reactor can be reduced without negative effects.

[0031] It has additionally been found that with the liquefaction according to the invention followed by the conversion stage itself in a further reactor (enzymatic hydrolysis or enzymatic hydrolysis and simultaneous fermentation SSF or SSCF), the same conversion yields to sugars or alcohols are achieved.

[0032] It should also be noted that with the liquefaction carried out according to the invention, no problems were encountered with regard to viscosity control of the reaction medium and no difficulties were encountered in removing / emptying the liquefaction reactor or filling the next reactor to allow the conversion reaction to continue.

[0033] Advantageously, the pH of the aqueous phase can be adjusted by controlled addition of at least one acidic and / or basic compound to the first reactor in at least one of the two liquefaction steps (a) and (b). Thus, generally, if the biomass is pretreated with an acid solution, the pH tends to be adjusted to a set value by controlled addition of a base and then maintained at the set value, and if the biomass is pretreated with an alkaline solution, the pH tends to be adjusted to a set value by controlled addition of an acid. The pH can also be adjusted prior to liquefaction.

[0034] In the process of the first liquefaction step (a), the pretreated lignocellulosic biomass can be added to the first reactor according to a fixed or variable frequency and with a fixed or variable amount. Biomass can also be added to the reactor at one time. This also applies to biocatalysts and water (when adding make-up water when the water content of biomass is insufficient). Water can be added separately from biomass, or biomass can be contacted with all or part of water before adding to the reactor. According to one embodiment, all components of the reaction medium, therefore water, pretreated biomass and biocatalyst are added at one time, optionally added simultaneously, and preferably added at the very beginning of step (a).

[0035] During the first liquefaction step (a), the pretreated lignocellulosic biomass may be added to the first reactor according to increasingly larger time intervals as described, for example, in the aforementioned patent EP 3461902, and preferably a fixed amount of biomass is used.

[0036] During the first liquefaction step (a), the biocatalyst or at least one biocatalyst may also be added sequentially to the reaction medium - at the same frequency and at the same intervals as the pretreated biomass or at a different frequency and intervals.

[0037] Alternatively, the biocatalyst may be added once during the liquefaction step (a), in particular at the very beginning of the liquefaction step (a).

[0038] During the first liquefaction step (a) the pretreated lignocellulosic biomass may be added to the first reactor continuously or sequentially.

[0039] The second liquefaction step (b) is preferably carried out at a constant volume of the reaction medium contained in the first reactor. As a portion of the reaction medium is continuously withdrawn, the pretreated biomass and the necessary inputs are gradually added so that the volume of the reaction medium remains essentially constant and "fresh" pretreated biomass can be liquefied throughout this step (b). These additions are carried out continuously according to a given frequency or are controlled by monitoring given physicochemical, rheological or operating characteristics of the reaction medium.

[0040] Thus, during the second liquefaction step (b), the pretreated lignocellulosic biomass and at least one other compound referred to as "input", at least one of which is selected from one of the following compounds: water, an acidic compound, an alkaline compound, a biocatalyst, are added over time, these compounds can be added together with the biomass, or added several times but not at the same time as the biomass, or added all at once at the beginning of this step. Preferably, the biocatalyst is added at the same time as / with the biomass.

[0041] These inputs, as well as those present at the beginning of liquefaction, may also contain other compounds, for example additives of the antifoam, antimicrobial type, or nutrients (particularly in the case of SSF or SSCF, for the microorganisms used for the fermentation).

[0042] The rheology of the reaction medium can be adjusted during the second liquefaction step (b) according to at least one of the following operating conditions: the residence time of the pretreated lignocellulosic biomass in the first reactor, the amount and / or frequency of addition of pretreated lignocellulosic biomass and inputs, wherein at least one input is selected from one of the following compounds: water, an acidic compound, an alkaline compound, a biocatalyst.

[0043] The rheology of the reaction medium can be adjusted during the second liquefaction step (b) so that it is the same as 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 the reaction medium is, the more particularly the reactors must be equipped with high-performance stirring devices and / or the more energy must be consumed to operate them.

[0044] It is therefore sought to maintain appropriate rheology within the liquefaction reactor in the continuous liquefaction according to the invention so that the viscosity conditions of the reaction medium are maintained in such a way as to allow it to be stirred in the reactor and withdrawn to another reactor in a manner compatible with industrial-scale production.

[0045] The rheology of the reaction medium can be monitored by monitoring the viscosity of the reaction medium or the mechanical torque on the shaft of the stirring system mounted to the first reactor or the electrical power consumed by the motor driving the stirring system.

[0046] The first liquefaction step (a) preferably has a duration of between 1 and 48 hours, more preferably between 2 and 24 hours and in particular between 5 and 12 hours.

[0047] The second liquefaction step (b) has a duration of preferably 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.

[0048] 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, such as between 5 hours and 14 hours.

[0049] During the second liquefaction step (b), a portion of the reaction medium can advantageously be continuously withdrawn from the first reactor and passed to a second reactor, wherein in the conversion step (c), the conversion of the biomass contained in the withdrawn reaction medium is continued in the presence of at least one biocatalyst.

[0050] The biocatalyst is already contained in the reaction medium transferred from one reactor to the other, but a biocatalyst different from or the same as the one already introduced into the first reactor may be added specifically to the second reactor.

[0051] Therefore, when the goal is to convert biomass to alcohols via SSF or SSCF:

[0052] - according to a first embodiment, it is possible to add all the biocatalyst (enzymes and microorganisms) in the first reactor from the start of liquefaction (so that no biocatalyst is added in step c) in the second reactor), - and according to another embodiment, it is possible to add the enzymes during liquefaction in the first reactor and then add the microorganisms and optionally additional enzymes in step c) in the second reactor.

[0053] At the end of the second liquefaction step (b), all the reaction medium is advantageously transferred from the first reactor to the second reactor, wherein the conversion of the biomass contained in the transferred reaction medium is continued in the presence of at least one biocatalyst in the conversion step (c).

[0054] Advantageously, the duration of the second liquefaction step (b) is less than or equal to the duration of the conversion step (c).

[0055] Thus, step (c) in the above-mentioned second reactor can be carried out 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 duration of operation in batch mode. The total duration of phase (c) carried out in fed-batch mode and then in batch mode is preferably between 10 and 170 hours, in particular between 70 and 140 hours. "Batch mode" should be understood in its usual sense, i.e. without removal from the reactor during the entire duration of the conversion carried out in this reactor.

[0056] The coupling of steps (a) and (b) in a dedicated liquefaction reactor and step (c) in another reactor makes it possible to benefit from the combined advantages of the previous batch, fed-batch and continuous embodiments: - the liquefaction reactor is operated according to steps (a) and then (b), which makes it possible to maximize the reactor for containing the reaction medium and reduce the time allocated for emptying and cleaning during a cycle, - the reactor carrying out step (c) ends in batch mode, which makes it possible to maximize the degree of conversion achieved and to control the maximum residence time of the biocatalyst, thereby avoiding the drift mentioned above.

[0057] Advantageously, the volume of the second reactor in which step (c) is carried out is greater than the volume of the first reactor in which steps (a) and (b) are carried out. Preferably, the volume of the second reactor is greater than 100%, preferably greater than 120%, preferably greater than 200%, and more preferably greater than 300% of the volume of the first reactor. The second reactor in which step (c) is carried out may be fed by multiple reactors in which steps (a) and (b) are carried out.

[0058] It is also possible to carry out step (c) using a plurality of reactors which are smaller in size and which are operated in particular in series.

[0059] The biocatalyst added for at least one, in particular all, of the first liquefaction (a), the second liquefaction (b) and the conversion (c) steps advantageously comprises at least one enzyme for at least partially converting the pretreated biomass into sugars by enzymatic hydrolysis, and optionally at least one microorganism of the yeast or bacterial type for converting all or part of this / these sugars into alcohol by fermentation.

[0060] According to one variation, the biocatalyst used in each of the first liquefaction step (a) and the second liquefaction step (b) may comprise at least one enzyme for at least partially converting the pretreated biomass into sugars by enzymatic hydrolysis, and the biocatalyst added in the conversion step (c) may comprise a mixture of enzymes and yeast (or other microorganisms) or only at least one yeast for converting all or part of the converted sugars into alcohols by fermentation.

[0061] According to one variant, the biocatalyst used for the total or partial fermentation of sugars into alcohol or products of interest is a bacterium, for example a Clostridium, such as Clostridium acetobutylicum. It can be added to the process in the same way as the yeast mentioned above.

[0062] Specifically, the present invention is intended to convert pretreated biomass to produce sugar by enzymatic hydrolysis, which generally uses an enzyme mixture comprising at least a cellulolytic enzyme. Cellulolytic enzymes are, for example, cellulases, endoglucanases, beta-glucosidases. The enzyme mixture can also include hemicellulolytic enzymes (hemicellulase). The enzyme can be produced by bacteria or fungi. Preferably, the enzyme is produced by fungi, such as Trichoderma reseii. Liquefaction according to the present invention therefore uses this type of mixture, as does the conversion reaction continued in a reactor different from the liquefaction reactor. The sugar involved can be utilized as is or after conversion.

[0063] The invention also aims at producing alcohol by fermentation from these sugars according to two main types of processes: - saccharification and fermentation are carried out simultaneously, which is called SSF or SSCF process. In this case, suitable microorganisms (yeast, bacteria, as indicated above) are added to the enzymes. The microorganisms can thus be added from the beginning of liquefaction, - or the fermentation is carried out after saccharification in a reactor dedicated to fermentation and supplied with suitable microorganisms. In this case, a single type of biocatalyst is added in each step (enzymes for hydrolysis and then microorganisms for fermentation).

[0064] It should be pointed out that, for carrying out fermentation, the biocatalyst is a microorganism which may be a yeast or a bacterium, even though in this text, for the sake of brevity, it is possible to refer only to yeast.

[0065] According to one embodiment, the method according to the invention may comprise the following steps: - an initial step (a0) of filling a first reactor with pretreated lignocellulosic biomass, a biocatalyst, water and optionally acidic and / or alkaline compounds supplied for the first time in said reactor, - a first liquefaction step (a) in the first reactor - by adding the pretreated lignocellulosic biomass to said reactor without taking it out, optionally also adding a biocatalyst, - then a second step (b) of continuous liquefaction in the first reactor, wherein a portion of the reaction medium is continuously taken out from the first reactor and the portion of the reaction medium taken out is transferred to the second reactor and pretreated lignocellulosic biomass and optionally water and / or biocatalyst, and / or acidic and / or alkaline compounds are continuously added, - then an optional step (b1) of homogenizing the reaction medium in the first reactor, - then a step (b2) of transferring all the reaction medium from the first reactor to the second reactor, wherein the conversion step (c) is preferably carried out in batch mode, - and a step (b3) of cleaning the first reactor, in particular with the aid of an aqueous solution, preferably an acidic or alkaline solution.

[0066] The addition of the pretreated lignocellulosic biomass to the first step a) may be sequential or non-sequential.

[0067] The addition of the biocatalyst to the first step a) may be sequential or non-sequential.

[0068] The operating conditions of the first liquefaction (a) in an enzymatic hydrolysis-only configuration (i.e. when the goal 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 more preferably between 45 and 55°C,

[0069] - a pH between 3 and 7, preferably between 4 and 6 and more preferably between 4.8 and 5.5.

[0070] The operating conditions of the first liquefaction (a) in a simultaneous enzymatic hydrolysis and fermentation configuration (SSF or SSCF) (i.e. when the goal 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 more preferably between 30°C and 35°C, - a pH between 3 and 7, preferably between 4 and 6, and more preferably between 5.0 and 5.5.

[0071] The operating conditions (temperature and pH) of the second liquefaction (b) are preferably the same as those of the first liquefaction (a).

[0072] The operating conditions (temperature and pH) of the conversion step (c) may be the same as or different from the operating conditions of the first liquefaction (a) and the second liquefaction (b). They are, for example, different if the biocatalyst introduced during this step is different from the biocatalyst introduced during the first liquefaction (a) and the second liquefaction (b).

[0073] Preferably, the solid content SC of the pretreated lignocellulosic biomass used in the method 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 method according to the invention contains at least 10 g of cellulose / 100 g of solids, in particular at least 20 g of cellulose / 100 g of solids.

[0074] List of Figures

[0075] Figure 1 The different stages of the liquefaction step of pretreated biomass according to the prior art are shown.

[0076] Figure 2 The different stages of the liquefaction step of pretreated biomass according to one embodiment of the invention are shown.

[0077] Figure 3 is a diagram showing the evolution of ethanol and xylose concentrations over time during a SSCF process according to prior art liquefaction and according to the invention. On the y-axis, the concentrations are in g / kg of reaction medium, while on the x-axis, the time is expressed in hours.

[0078] The accompanying drawings, more precisely, Figure 1 and Figure 2 , are very schematic and not drawn to scale. The same reference numerals from one figure to another denote the same flows / devices.

[0079] Description of the implementation plan

[0080] The object of the present invention is to improve the liquefaction procedure of pretreated lignocellulosic biomass. Liquefaction is understood to be a step that causes a biomass conversion under the action of a biocatalyst. This is a conversion by enzymatic hydrolysis (and / or then optionally fermentation). This liquefaction is sometimes referred to as "prehydrolysis".

[0081] One known protocol for carrying out either enzymatic hydrolysis alone or enzymatic hydrolysis and simultaneous fermentation of biomass or lignocellulosic waste is a first liquefaction step of the fed-batch type in a reactor specifically sized / designed for this purpose (sequential addition of biomass without blowdown throughout the step), followed by transfer to a more standard reactor to continue the enzymatic hydrolysis alone or simultaneous enzymatic hydrolysis and fermentation in batch mode in a second step.

[0082] For a description of this type of solution, reference may be made to, for example, the aforementioned patent WO 2013 / 088001.

[0083] The scheme according to a preferred embodiment of the present invention proposes a first step of liquefaction of the lignocellulosic biomass of the fed-batch type in a dedicated reactor (relatively limited effective volume, with high-performance stirring equipment), followed by continuous operation steps in this same reactor: - continuous or pseudo-continuous transfer to a more standard reactor (whose effective volume can be much larger and whose stirring equipment is simpler than the first reactor) to continue the separate enzymatic hydrolysis or simultaneous enzymatic hydrolysis and fermentation in batch mode, - and continuous or pseudo-continuous feeding of the lignocellulosic substrate and various inputs / biocatalysts to the first reactor.

[0084] With this new approach it has been shown that the utilization of the liquefaction reactors is maximized, so that it is possible to limit the number of liquefaction reactors in use at a given output, at the same productivity in enzymatic hydrolysis (or SSF or SSCF).

[0085] The present invention relates to the implementation of enzymatic hydrolysis for the production of sugars, or of enzymatic hydrolysis and simultaneous fermentation for the production of alcohols from lignocellulosic biomass / waste.

[0086] The raw material processed by the method according to the present invention is a pretreated lignocellulosic biomass. The pretreatment of the lignocellulosic biomass makes the cellulose accessible to and reactive to the enzyme, and comprises contacting the lignocellulosic biomass with a solvent and an optional catalyst (usually combined in a slurry) at a given temperature and pressure for a given residence time. Any type of pretreatment can be applied to obtain a pretreated lignocellulosic substrate.

[0087] The pretreated biomass may also be washed with water after its pretreatment and before the start of liquefaction according to the invention (resuspension of the pretreated biomass with water or a mixed fluid, solid / liquid filtration, washing of the solid part with water, and then solid / liquid filtration).

[0088] During the enzymatic hydrolysis or SSF or SSCF step (which includes the liquefaction step according to the invention exemplified below), the pretreated lignocellulosic substrate is mixed with a liquid solution containing enzymes (and optionally microorganisms, such as yeast or bacteria). The goal is to obtain a high concentration of ethanol (or sugar if no fermentation is performed). If the product of interest needs to be concentrated, the fermentation / enzymatic hydrolysis step should be carried out at a relatively high concentration of the pretreated lignocellulosic substrate, i.e., at a high solids content, to reduce the economic and energy costs of the process.

[0089] The solid content (acronym "SC") refers to the solid content measured according to the standard ASTM E1756-08 (2015) "Standard Test Method for Determination of Total Solids in Biomass". (The concentration of the pretreated lignocellulosic substrate in the medium may be expressed as a weight percentage of solids).

[0090] When solid content is high, intimate mixing of pretreated lignocellulosic substrate with the liquid solution containing enzyme (and optionally yeast) proves to be difficult. In particular, the start of enzymatic hydrolysis at high solid content particularly causes problems of mixing and homogenization. The reaction medium is very pasty and viscous.

[0091] To solve this problem, existing solutions are: - to equip the fermentation (or enzymatic hydrolysis) reactor with specific complex agitators to ensure homogenization of the reaction medium. - to feed the substrate gradually into the reactor, known as "fed-batch" feeding, without removing the reaction medium. As the reaction proceeds, the mixture becomes less and less viscous, and fresh substrate can be added to increase the amount of substrate in the medium.

[0092] - The fermentation (or enzymatic hydrolysis) is carried out in two steps: the first step is called liquefaction, which makes it possible to reduce the viscosity of the medium. This step corresponds in fact to the first hours of the 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 that allows transfer to a tank equipped with a standard stirrer to continue the enzymatic hydrolysis. The second step corresponds to the continuation of the enzymatic hydrolysis (or SSF / SSCF): the liquefied biomass obtained from the liquefaction step is transferred to a fermentation (or hydrolysis) reactor, in which the conversion of cellulose and residual hemicellulose into sugars and then into ethanol continues.

[0093] The present invention is interested in the latter approach and aims to improve it.

[0094] Existing / Comparative Implementation

[0095] The liquefaction step of the pretreated substrate is carried out in "fed-batch" mode, i.e. the substrate is gradually added to the mixture of water + biocatalyst + base. (In this case the non-limiting example of pretreatment with acid impregnation is specifically considered, thus adding base to increase the pH of the reaction medium).

[0096] 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, and the pH and temperature are adjusted to the desired set points, and then a portion or all of the enzymes (and possible yeast) are added. The remaining pretreated substrate is then used in a fed-batch (sequential feeding) mode to increase the solid content. A fed-batch mode of enzymes (and yeast) can also be performed. The rheology of the solid suspension requires specific embodiments to ensure good stirring so that the reaction is carried out in a medium in which the pretreated lignocellulosic substrate is as concentrated as possible. In order to alleviate some of the limitations, the liquefaction reactor can be equipped with specific agitator technology.

[0097] Screw-type agitators are usually most suitable, even though they are complex and limited in size due to the mechanical design.

[0098] Various parameters make it possible to define the operating conditions and to establish a fed-batch strategy. The ranges shown below are examples:

[0099] - Solid content (SC) of the pretreated biomass (2 to 60 wt%)

[0100] - Cellulose content of the substrate

[0101] - Enzyme dosage relative to cellulose (5 to 100 mg / g cellulose)

[0102] - yeast inoculum rate (0.1 to 3 g / kg medium),

[0103] - Solid content (SC) of the initial mixture (2 wt% to 60 wt%)

[0104] - Solid content (SC) of the final mixture (2 wt% to 60 wt%)

[0105] -Total volume and / or mass of the final mixture in the reactor

[0106] - Number of times feed is added in batches

[0107] - Duration of fed-batch addition (0 to 48 hours)

[0108] - Duration of liquefaction (1 to 48 hours)

[0109] The prior art fed-batch liquefaction scheme is described as follows:

[0110] 1- Preparation of the initial mixture in the liquefaction reactor: A certain amount of pretreated biomass is mixed with a certain amount of water to achieve the desired solid content (SC). Stirring is started to homogenize the mixture and adjust the pH and temperature. An antimicrobial agent can be added to the reaction medium, for example, an antimicrobial agent of the chloramphenicol type or sold by the company BetaTec under the trade name VitaHop.

[0111] 2- If the pretreated substrate was produced under acidic conditions (e.g. impregnation with sulfuric acid followed by steam explosion), by adding an alkaline solution, e.g. NH 4 The pH is adjusted by adding 1H OH or KOH or NaOH, or if the pretreated substrate is produced under alkaline conditions, by adding an acidic solution. The pH adjustment can be maintained during the subsequent stages of the protocol.

[0112] 3-Injection of enzymes (and yeast) and then start of fed-batch liquefaction: Once the initial mixture is suitably homogeneous, a certain amount of enzymes (and yeast) is introduced into the reactor. This injection makes it possible to achieve the selected dosage of the biocatalyst. After the injection of the biocatalyst, the fed-batch liquefaction begins.

[0113] 4-fed batch-addition of pretreated biomass: After a certain predetermined time, the viscosity of the medium is much lower, and the first addition of pretreated biomass can be carried out. According to the strategy of adding enzymes, additional enzymes can be added at this time. The quality of the enzyme introduced corresponds to the addition of pretreated biomass. It is possible to choose to add a certain amount of enzyme according to the amount of pretreated biomass added when each biomass is added, or all or most of the required enzyme amount is added at the beginning of the fed batch operation. Addition is regularly carried out in succession until a mixture with a desired final quality is obtained. Usually, the quality and rate of each addition are constant and regular. As seen above, the addition interval of biomass can also be larger and larger. Usually, the goal is to reach the target enzyme dosage represented by grams / kilogram of cellulose.

[0114] 5- End of liquefaction: After the stage of adding the pretreated biomass, the reaction continues to a constant final volume. The reaction and viscosity reduction continue until the viscosity is considered low enough to be able to transfer the medium to an enzymatic hydrolysis (or SSF or SSCF) reactor equipped with a standard stirrer.

[0115] 6- After transferring the reaction medium to the enzymatic hydrolysis (or SSF or SSCF) reactor corresponding to a conventional stirred tank, cleaning of the liquefaction reactor is carried out to limit the risk of contamination.

[0116] The operating conditions for liquefaction in an enzymatic hydrolysis-only configuration (i.e., when the goal is to convert the biomass into oligomeric or monomeric sugars) are:

[0117] - a temperature between 25 and 80°C, preferably between 40 and 60°C and more preferably between 45°C and 55°C,

[0118] - a pH between 3 and 7, preferably between 4 and 6 and more preferably between 4.8 and 5.5.

[0119] The operating conditions for liquefaction in a simultaneous enzymatic hydrolysis and fermentation configuration (SSF or SSCF) (i.e. when the goal is to convert biomass to alcohol by hydrolysis and fermentation) are:

[0120] - a temperature between 25 and 80°C, preferably between 30 and 50°C and more preferably between 30°C and 35°C,

[0121] - a pH between 3 and 7, preferably between 4 and 6 and more preferably between 5.0 and 5.5.

[0122] The duration of liquefaction is between 1 hour and 48 hours, in particular between 2 hours and 24 hours, in particular between 5 hours and 12 hours.

[0123] Figure 1 The different cycle phases / times of liquefaction A to E are shown, representing the liquefaction reactor 1 at each of these phases:

[0124] A: Filling the liquefaction reactor 1 with pretreated biomass 2, water 3' and biocatalyst 3 and alkaline compound 4, and adjusting the temperature and pH of the reaction medium in the reactor: duration between 2 hours and 4 hours

[0125] B: Fed-batch operation of pretreated biomass with sequential addition of pretreated biomass 2 and optionally alkaline compound 4 to adjust pH: duration between 2 hours and 10 hours

[0126] C: Homogenization (optional): 1 to 2 hours

[0127] D: Transfer of reaction medium 5 from reactor 1 to a downstream reactor (not shown): duration between 2 hours and 4 hours

[0128] E: Cleaning of liquefaction reactor 1: between 2 hours and 4 hours

[0129] The enzymatic hydrolysis (or SSF or SSCF) is carried out in batch mode. The operating conditions of pH and temperature are generally the same as for liquefaction. The duration of the enzymatic hydrolysis (or SSF or SSCF) is between 10 hours and 170 hours, preferably between 48 hours and 140 hours. After draining the enzymatic hydrolysis (or SSF or SSCF) reactor, the reactor is cleaned to limit the risk of contamination.

[0130] The volume limitation of the liquefaction reactor compared to the SSF or SSCF or enzymatic hydrolysis reactor (the effective volume ratio between the SSF or SSCF or enzymatic hydrolysis reactor and the liquefaction reactor is, for example, between 2 and 10) usually requires the establishment of a timing diagram to ensure the continuity of the different operating phases (including filling, emptying, cleaning phases) between the two steps of liquefaction and SSF or SSCF (or enzymatic hydrolysis). Such a timing diagram is also established to limit the number of liquefaction reactors and SSF or SSCF or enzymatic hydrolysis reactors by limiting the downtime.

[0131] Embodiments according to the present invention

[0132] Figure 2 Schematically showing a description of a scheme for fed-batch liquefaction with an additional continuous step according to the invention, using Figure 1 Same convention:

[0133] The durations of the phases shown are examples.

[0134] A- Initial stage of filling liquefaction reactor 1

[0135] Preparation of the initial mixture in the liquefaction reactor: A certain amount of pretreated biomass 2 is mixed with a certain amount of water 3' to achieve the desired solid content (SC). Stirring is started to homogenize the mixture and adjust the pH and temperature. If the pretreated substrate is produced under acidic conditions (e.g. impregnation with sulfuric acid followed by steam explosion), the pretreatment is carried out by adding an alkaline solution 4, such as NH 4 The pH is adjusted by adding OH or KOH or NaOH, or if the pretreated substrate was produced under alkaline conditions, by adding an acidic solution.

[0136] B - Start of fed-batch liquefaction - Addition of pretreated biomass 2: Once the initial mixture is suitably homogeneous, a certain amount of enzymes 3 (and yeast) is introduced into the reactor 1. This injection makes it possible to achieve the selected dosage of the biocatalyst. After the injection of the biocatalyst, the fed-batch liquefaction starts: - After a certain time, the viscosity of the medium is already much lower and the first addition of pretreated biomass can be carried out. Depending on the strategy for adding enzymes 3, additional enzymes can be added at this time. The mass of enzymes 3 introduced is proportional to the addition of pretreated biomass 2, aiming at a constant enzyme / cellulose addition. The additions are carried out successively until a mixture with the desired final mass is obtained. Usually, the mass and frequency of each addition are constant and regular.

[0137] F - Continuous stage of 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 input is added to the liquefaction reactor 1 to maintain a constant volume: pretreated biomass 2, biocatalyst 3, water 3' and acidic or alkaline solution 4 for pH adjustment. The continuous stage of liquefaction is performed so that the rheology of the withdrawn medium remains less constrained or the same as the rheology of the medium at the end of the fed-batch so as not to negatively affect the operation of the enzymatic hydrolysis (or SSF or SSCF) reactor.

[0138] The operating conditions for liquefaction in an enzymatic hydrolysis-only configuration (i.e., when the goal is to convert the biomass into oligomeric or monomeric sugars) are preferably:

[0139] - a temperature between 25 and 80°C, preferably between 40 and 60°C and more preferably between 45°C and 55°C,

[0140] - a pH between 3 and 7, preferably between 4 and 6 and more preferably between 4.8 and 5.5.

[0141] The operating conditions for liquefaction in a simultaneous enzymatic hydrolysis and fermentation configuration (SSF or SSCF) (i.e. when the goal is to convert biomass to alcohol by hydrolysis and fermentation) are preferably:

[0142] - a temperature between 25 and 80°C, preferably between 30 and 50°C and more preferably between 30°C and 35°C,

[0143] - a pH between 3 and 7, preferably between 4 and 6 and more preferably between 5.0 and 5.5.

[0144] The operating conditions (solids content, dosage of biocatalyst) are kept at target conditions making it possible to obtain the desired rheological parameters, therefore the amount and frequency of additions and the residence time of the residue in the liquefaction reactor during the continuous stage. The residence time of the pretreated biomass in the liquefaction reactor during the continuous stage is greater than or equal to 4 hours.

[0145] The duration of this continuous phase is, for example, between 1 hour and 170 hours, in particular between 10 hours and 72 hours.

[0146] C: Homogenization (optional): 1 to 2 hours

[0147] D: Transfer of reaction medium 5 from reactor 1 to a downstream reactor (not shown); End of liquefaction: After the continuous stage, transfer of the entire liquefied medium to an enzymatic hydrolysis (or SSF or SSCF) reactor preferably equipped with a standard stirrer: duration between 2 hours and 4 hours

[0148] E: Cleaning of the liquefaction reactor 1. After the transfer of the medium to the downstream reactor, the liquefaction reactor is cleaned to limit the risk of contamination: duration between 2 hours and 4 hours

[0149] Enzymatic hydrolysis (or SSF or SSCF) is always carried out in batch mode. The operating conditions of pH and temperature may be the same or different from those of liquefaction. This is the case, for example, for configurations in which the biocatalyst introduced in the liquefaction step is different from those introduced in the SSF or SSCF step.

[0150] The duration of the enzymatic hydrolysis (or SSF or SSCF) is between 10 hours and 170 hours, preferably between 48 hours and 140 hours.

[0151] According to one variant, during the initial filling phase A and during the continuous phase F, only enzymes are added to the liquefaction reactor 1 , and yeast is added to the SSF or SSCF reactor starting from the continuous phase of liquefaction.

[0152] Of course, it is also possible to carry out only enzymatic hydrolysis without yeast, or to utilize sugars without converting them into alcohols, or to convert them in another way. It is also possible to carry out fermentation of sugars alone in a dedicated fermentation reactor.

[0153] Compared with liquefaction according to the existing method ( Figure 1 Compared with the example in the example in the embodiment of the present invention, the liquefaction ( Figure 2 ) provides the following advantages:

[0154] - Increased productivity of the liquefaction through better utilization of each liquefaction reactor. The significance of the gain depends on the operating time of this continuous stage and also on the flow that can be processed during this stage (according to the residence time of the pretreated biomass during the continuous stage).

[0155] For the same amount of pretreated biomass to be liquefied, the number of liquefaction reactors is thus reduced (or for the same number of liquefaction reactors, the amount of liquefied biomass is greater): the liquefaction solution according to the invention thus allows for reduced investments.

[0156] - The cleaning phase of the liquefaction reactor is carried out after transferring several reactor volumes, which makes it possible to reduce the frequency of cleaning of the reactor, thus reducing the consumption of chemicals and increasing the productivity by increasing the reaction time.

[0157] - The ethanol yield and the conversion of cellulose and hemicellulose to sugars remain at the same or nearly the same level.

[0158] Example 1 (comparative)

[0159] Use the above Figure 1 Describe the scheme.

[0160] To illustrate the gain in utilization of the liquefaction reactor, two timing diagrams are compared: one for a known fed-batch liquefaction ( Figure 1 ), and another for fed-batch liquefaction with continuous stages ( Figure 2 ).

[0161] Liquefaction (+SSCF) was carried out at a solids content of 20% by weight SC, the enzyme dosage was 8 mg enzyme / g SC, the amount of yeast was 0.5 g / kg medium.

[0162] The linearized flow rate of the pretreated substrate fed to the liquefaction step was 15.6 tSC / h. The solids content of the pretreated substrate was 38% by weight.

[0163] Table 1 below details the timing diagram for known liquefaction:

[0164] [Table 1]

[0165]

[0166] Therefore, four 435m 3 The liquefied biomass was transferred to four SSCF reactors, each with a capacity of 4500 m3. 3 , running in batch mode.

[0167] The duration of the SSCF phase is shown in Table 2 below:

[0168] [Table 2]

[0169] operate Duration Filling with liquefied medium 37h reaction 113h Transfer to SSCF reactor 6h clean 4h Total cycle time 160h

[0170] Example 2 (according to the present invention)

[0171] Following the above liquefaction scheme according to the present invention and Figure 2 The following Table 3 details the timing diagram of liquefaction according to the present invention:

[0172] [Table 3]

[0173]

[0174] Therefore, two 500m 3 The liquefaction reactor with an effective volume of 1000 m2 liquefies the pretreated biomass at an operating efficiency of 72.5%. The residence time of the pretreated biomass in the liquefaction reactor is 8 hours. The liquefied biomass is transferred to four SSCF reactors, each with a capacity of 4500 m2. 3 , running in batch mode.

[0175] The duration of the SSCF phase was the same as that shown in Table 2 for Comparative Example 1: Example 3 (Comparative)

[0176] Analogously to Comparative Example 1, a known liquefaction protocol was used.

[0177] The liquefaction reactor is loaded with a portion of the pretreated biomass, all of the water, and an alkaline solution NH 4 OH to adjust the pH to 5.3, then add all the enzymes and yeast; this corresponds to the time t0 of liquefaction. The temperature is maintained at 33°C. The solid content in this initial mixture is 14% by weight. The pretreated biomass is then added 12 times over 6 hours for a fed-batch operation of the pretreated biomass: 5% SC in 3 hours, then 3% SC in 3 hours.

[0178] The solid content of the liquefied medium was 22% by weight, the enzyme dosage was 8 mg enzyme / g SC, and the amount of yeast was 0.5 g / kg medium.

[0179] The enzyme solution had a protein concentration of 35 g / L and a density of 1.02 g / cm3 at 20°C.

[0180] The solid content of the pretreated biomass was 42 wt%.

[0181] The liquefaction reactor is equipped with a spiral stirrer to achieve good homogenization of the reaction medium and optimal management of torque and viscosity.

[0182] The liquefied medium is then homogenized for 2 hours to achieve a reduction in the torque of the stirrer and then transferred to the SSCF reactor, where the conversion of cellulose and hemicellulose into sugars and the fermentation of sugars into ethanol continue. The SSCF reactor is equipped with a conventional stirrer; this is, for example, a stirring system comprising two "TT" type three-blade impellers (axial flow movement) and a bottom turbine (radial flow rotor) with two straight blades.

[0183] The duration of liquefaction (initial charge + fed-batch operation of pretreated biomass + homogenization) was 12 hours. The duration of SSCF was 132 hours (excluding liquefaction).

[0184] The operating parameters monitored were the torque of the stirrer in the liquefaction reactor, the contents of ethanol, glucose and xylose in the medium during the SSCF reaction (analyzed by HPLC).

[0185] The performance of the SSCF process was evaluated using the following yields:

[0186] - ethanol / SC yield, equal to the ratio between the amount of ethanol produced and the total amount of solids introduced into the liquefaction reactor 1

[0187] - Total enzymatic hydrolysis of samples at the end of fermentation: hydrolysis yields of cellulose and xylan and ethanol production yields, 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 given by the following stoichiometric equation.

[0188] C 6 H 12 O 6 (Glucose) -> 2C 2 H 5 OH(ethanol)+2CO 2

[0189] It produces 51.1 kg of ethanol from 100 kg of glucose. Pasteur later demonstrated the existence of 2 The production of byproducts related to the production of sugars (especially glycerol, succinic acid, heavy alcohols, microbial development) is reduced. The Pasteur yield takes into account these sugar losses, i.e. 48.4 kg of ethanol are produced from 100 kg of glucose. The Pasteur yield therefore corresponds to 94.7% of the theoretical yield. The results are shown in Table 4 below:

[0190] [Table 4]

[0191]

[0192] Example 4 (according to the present invention)

[0193] As in Example 2, the liquefaction scheme according to the present invention is followed and Figure 2 Shown in.

[0194] The stage of filling the liquefaction reactor until the fed-batch stage of the substrate was the same as that of Example 3. A continuous stage was then implemented: a portion of the medium was withdrawn and transferred to a SSCF reactor equipped with a standard vertical stirrer. A portion of the input (see Table 5 below) was added to the liquefaction reactor to operate at a constant volume.

[0195] The residence time of the pretreated biomass in the liquefaction reactor during the continuous phase is set to:

[0196] - Under the condition of low acidity of biomass (1.8 wt% H 2 SO 4 ) 12 hours for pretreatment

[0197] - When the biomass was exposed to more acidic conditions (2.4 wt% H 2 SO 4 ) 6 hours for pretreatment

[0198] This residence time makes it possible to maintain a constant agitator torque in the liquefaction reactor.

[0199] The duration of this continuous phase is 24 hours.

[0200] The amounts of medium to be removed and the amounts of inputs to be added to the liquefaction reactor having an effective volume of 3000 kg are shown below in Table 5. The nutrients are intended for the yeast (nitrogen source) and are here in the form of a soluble zein solution, in particular that sold under the name Solulys by the company Roquette.

[0201] [Table 5]

[0202]

[0203]

[0204] The operating parameters monitored were the torque of the stirrer in the liquefaction reactor, the contents of ethanol, glucose and xylose in the medium during the SSCF reaction (analyzed by HPLC (acronym for High Performance Liquid Chromatography)).

[0205] The performance of the SSCF was evaluated as in Example 3 above, and the results are summarized in Table 6 below.

[0206] [Table 6]

[0207]

[0208] It was thus demonstrated that the yields, in particular the yield of ethanol, are the same whether the operation is carried out under liquefaction according to the known protocol (Example 3) or according to the protocol of the invention (Example 4).

[0209] This residence time makes it possible to maintain a constant agitator torque in the liquefaction reactor.

[0210] The ethanol production and xylose consumption kinetics did not show any difference in reactivity between SSCF performed with standard conventional fed-batch liquefaction and SSCF performed with fed-batch and then continuous liquefaction according to the present invention.

[0211] These good results in terms of performance can also be seen from Figure 3As can be seen in the graph of. Specifically, curves C1 and C2 correspond to the ethanol concentration according to comparative example 3 and according to example 4 of the present invention, respectively: it can be seen that the ethanol concentration is consistent after 140 hours of SSCF. Similarly, curves C3 and C4 correspond to the xylose concentration according to comparative example 3 and according to example 4 of the present invention, respectively: again, the decrease in the xylose concentration due to its conversion into ethanol reaches a very low and similar concentration after 140 hours. It can be seen that the present invention also provides better results in terms of productivity in the first 48 hours.

Claims

1. A method for converting lignocellulosic biomass by contacting a pretreated lignocellulosic biomass with at least one biocatalyst (3) in an aqueous phase in a first reactor (1) containing a reaction medium, the reaction medium comprising the pretreated lignocellulosic biomass (2) and the biocatalyst in an aqueous phase, the method 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 then (b) a second continuous liquefaction step, wherein a portion of the reaction medium is continuously withdrawn from the first reactor, at least one biocatalyst is added, and pretreated lignocellulosic biomass is continuously added.

2. The method according to the preceding claim, characterized in that During the first liquefaction step (a) the pretreated lignocellulosic biomass (2) may be added to the first reactor (1) according to a fixed or variable frequency and in a fixed or variable amount.

3. The method according to the preceding claim, characterized in that During the first liquefaction step (a), the pretreated lignocellulosic biomass (2) is added to the first reactor (1) according to increasingly larger time intervals and preferably a fixed amount of biomass is used.

4. A method according to any one of the preceding claims, characterised 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, characterised in that During the second liquefaction step (b), pretreated lignocellulosic biomass (2) and at least one other compound referred to as "input", at least one of which is selected from one of the following compounds: water (3'), acidic compounds, alkaline compounds (4), biocatalyst (3), are added over time.

6. A method according to any one of the preceding claims, characterised in that The rheology of the reaction medium is adjusted during the second liquefaction step (b) according to at least one of the following operating conditions: the residence time of the pretreated lignocellulosic biomass (2) in the first reactor (1), the amount and / or frequency of addition of pretreated lignocellulosic biomass and inputs, wherein at least one input is selected from one of the following compounds: water (3'), acidic compounds, alkaline compounds (4), biocatalyst (3).

7. A method according to any one of the preceding claims, characterised in that The rheology of the reaction medium is adjusted during the second liquefaction step (b) so that it is the same as or less severe than the rheology of the reaction medium at the end of the first liquefaction step (a).

8. The method according to any 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 on the shaft of the stirring system mounted to the first reactor (1) or the electrical power consumed by the motor driving said stirring system.

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

10. A method according to any one of the preceding claims, characterised 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. A method according to any one of the preceding claims, characterised in that During the second liquefaction step (b), a portion (5') of the reaction medium is continuously withdrawn from the first reactor and sent to the second reactor, wherein in the 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. A method according to any one of the preceding claims, characterised in that At the end of the second liquefaction step (b), all of the reaction medium (5) is transferred from the first reactor (1) to the second reactor, wherein the conversion of the biomass contained in said transferred reaction medium is continued in the presence of at least one biocatalyst in the conversion step (c).

13. The method according to any one of claims 11 and 12, characterized in that Step (c) in the second reactor is carried out in fed-batch mode and then in batch mode for a duration preferably between 10 and 170 hours, in particular between 70 and 140 hours.

14. A method according to any one of the preceding claims, characterised in that The method comprises: - an initial step (a0) of filling a first reactor (1) with pretreated lignocellulosic biomass (2), biocatalyst (3), water (3') and optionally acidic and / or basic compounds (4) which are supplied for the first time in said reactor, - a first liquefaction step (a) in a first reactor (1) by adding the pretreated lignocellulosic biomass (2) to said reactor without withdrawal, optionally also adding a biocatalyst (3), - a second step (b) of continuous liquefaction in the first reactor (1), wherein a portion of the reaction medium is continuously withdrawn from the first reactor (5') and the withdrawn portion of the reaction medium is transferred to a second reactor and pretreated lignocellulosic biomass (2) is added over time, - then an optional step (b1) of homogenizing the reaction medium in the first reactor (1), - step (b2) of then transferring all the reaction medium (5) from the first reactor (1) to the second reactor, wherein the conversion step (c) is preferably carried out in batch mode, - and a step (b3) of cleaning the first reactor, in particular by means of an aqueous solution, preferably an acidic or alkaline solution.

15. A method according to any one of the preceding claims, characterised in that The pretreated lignocellulosic biomass used has a solids content SC of at least 2% by weight and contains at least 10 g of cellulose per 100 g of solids.

Images