Process for the production of cellulolytic and / or hemicellulytic enzymes

Abstract

The present invention relates to a process for producing cellulolytic and / or hemicellulytic enzymes by a cellulolytic and / or hemicellulolytic microorganism, said process comprising at least: a) a growth phase of the microorganism in the presence of at least one carbon substrate, then b) a production phase of the enzymes in the presence of at least one inducing substrate, and also: c) a preparation phase of a carbon substrate comprising glucose and / or fructose, carbon substrate which is used in one or both of the growth and production phases a) and b) of production, said preparation phase comprising a step c1) of hydrolysis in acidic aqueous medium of sucrose to glucose and fructose. Figure for the abstract: Fig 1

Description

Title of the invention: Process for the production of cellulolytic and / or hemicellulytic enzymes Technical field

[0001] The present invention relates to the production of cellulolytic and / or hemicellulolytic enzymes, in particular in the context of the production of sugars from cellulosic or lignocellulosic materials involving enzymatic hydrolysis of these materials. The sugars can be used / recovered as such or continue their conversion into alcohol, in particular ethanol, by fermentation. Prior art

[0002] Since the 1970s, the transformation of lignocellulosic materials into ethanol, after hydrolysis of the constituent polysaccharides into fermentable sugars, has been the subject of numerous studies. Examples include the reference work of the National Renewable Energy Laboratory (Process Design and Economies for Bio-chemical Conversion of Lignocellulosic Biomass to Ethanol, Humbird et al., NREL / TP-5100-57764, May 2011).

[0003] Lignocellulosic materials are cellulosic materials, i.e. materials made up of more than 90% by weight of cellulose, and / or lignocellulosic materials, i.e. materials made up of cellulose and hemicelluloses, which are polysaccharides essentially made up of pentoses and hexoses, as well as lignin, which is a macromolecule of complex structure and high molecular weight, based on phenolic compounds. For the sake of brevity, they may be grouped together in this text under the generic term biomass.

[0004] Wood, straw, corn cobs are the most used lignocellulosic materials, but other resources, dedicated forest crops, residues of al-cooligenic, sugar and cereal plants, products and residues of the paper industry and products of transformation of lignocellulosic materials are usable. They are mostly made up of about 35 to 50% cellulose, 20 to 30% hemicellulose and 15 to 25% lignin.

[0005] The process for the biochemical transformation of lignocellulosic materials into sugars and then possibly into ethanol comprises a physicochemical pretreatment step, followed by an enzymatic hydrolysis step using an enzymatic cocktail. It can continue with a step of ethanolic fermentation of the released sugars, the ethanolic fermentation and the enzymatic hydrolysis being able to be carried out simultaneously, and a step of purification of the ethanol. An example of such a process converting biomass into ethanol is described in patent EP 3 484 945, to which we can refer to it for more details.

[0006] The enzyme cocktail used for hydrolysis is a mixture of cellulolytic enzymes (also called cellulases) and / or hemicellulolytic enzymes. Cellulolytic enzymes have three main types of activities: endoglucanases, exoglucanases and cellobiases, the latter also being called [3 glucosidases. Hemicellulolytic enzymes have xylanase activities in particular.

[0007] Enzymatic hydrolysis is efficient and is carried out under mild conditions. On the other hand, the cost of producing enzymes remains very high, representing at least 20% of the cost of converting biomass into ethanol, for example. As a result, much work has been carried out to reduce this cost: firstly, optimizing the production of enzymes, by selecting hyperproducing microorganisms and improving the processes for producing said enzymes, then reducing the quantity of enzymes in hydrolysis, by optimizing the pretreatment step, by improving the specific activity of these enzymes, and by optimizing the implementation of the enzymatic hydrolysis step.

[0008] The cellulolytic microorganism most used for the industrial production of the enzyme cocktail is the fungus Trichoderma reesei. Wild strains have the ability to secrete, in the presence of an inducing carbon substrate, cellulose for example, the enzyme cocktail considered to be best suited to the hydrolysis of cellulose. Other proteins possessing properties essential for the hydrolysis of lignocellulosic materials are also produced by Trichoderma reesei, xylanases for example. The presence of an inducing carbon substrate is essential for the expression of cellulolytic and / or hemicellulolytic enzymes. The nature of the carbon substrate has a strong influence on the composition of the enzyme cocktail. This is the case of xylose, which, associated with an inducing carbon substrate such as cellulose or lactose, makes it possible to significantly improve the so-called xylanase activity. Recombinant strains were obtained from Trichoderma reesei strains Qm9414 (Mandels M. (1975). Microbial sources of cellulase. Biotechnol. Bioeng. 5, 81-105), RutC30 (Montenecourt, BS and Eveleigh, DE, Appl. Environ. Microbiol. 1977, 34, 777-782), CL847 (Durand et al, 1984, Proc. Colloque SFM "Génétique des microorganismes industriels". Paris. H. HESLOT Ed, pp 39-50) by cloning heterologous genes, invertase (TAspergillus niger for example, to diversify the carbon source necessary for the production of cellulases and / or overexpressing [3-glucosidase in order to improve the yield of enzymatic hydrolysis, [S-glucosidases being considered as the limiting enzymes in the reaction. These strains have retained their hyperproductivity and their ability to be cultivated in fermenters.

[0010] The incorporation of invertase into Trichoderma gives it the property of being able to consume sucrose, and therefore effluents including sugary juices from washing beets or sugar cane, and / or sugar molasses, which are less expensive and / or more available. This has been verified, for example, in patent EP 2 222 865.

[0011] Lactose and glucose remain, in an industrial production process of enzyme cocktail, among the most suitable carbon substrates. Their cost, particularly that of glucose, is high and is likely to vary significantly. The production process of enzyme cocktail is also dependent on external carbon sources.

[0012] Therefore, the use of carbon substrates resulting from the process of biochemical conversion of lignocellulosic materials is an interesting approach, and patent WO 2013 / 190214 has thus proposed a process for producing enzymes comprising a phase of growth of the microorganisms in the presence of a carbon growth substrate, then a phase of production of the enzymes with an inducing carbon substrate in the form of a liquid residue resulting from a pretreatment of lignocellulosic material comprising C5 sugars.

[0013] Current economic logic dictates that second-generation biofuel production sites can be the same as first-generation production sites, the whole constituting a "biorefinery" where all the plant material is recovered. Thus, starting with a sugar plant, we seek to recover sugar cane and lignocellulosic cane residues, and effluents containing sucrose can be available on site.

[0014] This is not, however, the only possible way of improving enzyme production, and it may have certain drawbacks, or at least limitations in its application. Thus, it generally requires, in fact, that the production of enzymes takes place in the biomass conversion installation, which is not always the case encountered / envisaged. Then, it takes a portion of the sugars, intended, in the biomass conversion process, to be used / converted elsewhere. Finally, this sugary liquid may not be sufficient to ensure on its own both the growth of the fungus and its production of enzymes: these sugars must be supplemented, in particular by an inducing substrate such as lactose, in the enzyme production phase.

[0015] The aim of the invention is then to improve a process for producing enzymes, in particular by remedying the aforementioned drawbacks, by seeking in particular to simplify the process, to reduce its costs, to make it more flexible in its industrial implementation. Summary of the invention

[0016] The invention firstly relates to a method for producing cellulolytic and / or hemicellulytic enzymes by a cellulolytic and / or hemicellulytic microorganism, said method comprising at least - a) a growth phase of the microorganism in the presence of at least one carbon substrate, then - b) a phase of production of the enzymes in the presence of at least one inducing substrate, said method also comprising: - c) a phase of preparation of a carbon substrate comprising glucose and / or fructose, carbon substrate which is used in one and / or the other of the phases a) of growth and b) of production, said preparation phase comprising a step cl) of hydrolysis in an acidic aqueous medium of sucrose into glucose and fructose.

[0017] The invention sought to exploit a source of carbon substrate, sucrose, to replace glucose, because sucrose is a less expensive compound and has a more stable price than glucose, and is similar in its sweetening properties. Indeed, sucrose is a disaccharide composed of glucose and fructose molecules combined via a glycosidic bond. But a simple replacement of glucose by sucrose is not enough: in fact, sucrose cannot be assimilated as such by microorganisms of the filamentous fungi type such as Trichoderma, because these microorganisms are not naturally equipped with the invertase gene.

[0018] A first solution may consist of genetically modifying microorganisms to endow them with this gene. The publication by Lucas Miranda Fonseca et al. “Rational engineering of the Trichoderma reesei RUT-C30 strain into an industrially relevant platform for cellulase production” published in the journal Biotechnology for biofuels (2020) thus proposes making several genetic modifications to the RUT-C30 strain of T. reesei, in particular allowing the expression of the invertase gene, and therefore being able to use at least in part sucrose as a carbon substrate in the process of producing enzymes from this modified strain. If this targeted genetic modification technique is effective, it is complex to implement, and must be repeated as soon as one wants to change strain.

[0019] The invention therefore consisted not of modifying the strains, but of modifying the sucrose to make it assimilable. This modification is an acid hydrolysis, which makes it possible to cut the glycosidic bond of the molecule so that it breaks down into glucose and fructose, both of which can be assimilated by the microorganisms targeted by the invention.

[0020] Advantageously, the cellulolytic and / or hemicellulolytic microorganism of interest for the invention is chosen from microorganisms lacking the invertase gene and capable of consuming glucose and / or fructose: it is therefore possible to use in the invention unmodified strains, or, more precisely, non-modified strains. modified to make them capable of assimilating sucrose. The microorganisms targeted by the invention are preferably microorganisms of the fungal type, in particular of the filamentous fungal type.

[0021] The cellulolytic and / or hemicellulolytic microorganism can be chosen for implement the invention among strains of fungi belonging to the genera Trichoderma, Aspergillus, Penicillium or Schizophyllum, and preferably belonging to the species Trichoderma reesei.

[0022] Advantageously, at the end of preparation phase c), an aqueous solution comprising glucose and fructose is obtained which can be used directly or after possible operations such as concentration adjustment (or other treatment such as filtration, etc.) as a carbon substrate in growth step a) and / or production step b).

[0023] The hydrolysis can be total or partial: in this case, a (small) quantity of sucrose can still be present in the solution obtained at the end of hydrolysis.

[0024] According to one embodiment, at least one inducing substrate, in particular lactose, can be added to the aqueous solution comprising glucose and fructose obtained at the end of preparation phase c), to use said solution as a substrate in production step b). This provides a ready-to-use carbon substrate solution for production step b), with a mixture of inducing and non-inducing substrates. It can also be used for step a), lactose can also be used as a carbon growth substrate. In this case, there is only one solution feeding the bioreactor for both steps, which simplifies its preparation, storage and method of introduction into the bioreactor.

[0025] It should be noted that the method according to the invention can implement the growth steps a) and production steps b) in the same bioreactor. Alternatively, it can be implemented with a first bioreactor dedicated to the growth step a), and a second bioreactor dedicated to the production step b).

[0026] According to another embodiment, at least one inducing substrate, in particular lactose, can be added to the acidic aqueous medium during step c1) of hydrolysis of sucrose. The advantage is that lactose is a disaccharide, like sucrose, but which is the combination of a glucose and a galactose via a glycosidic bond, which is assimilable by the fungal strains targeted by the invention. However, the inventors have shown that lactose is significantly more difficult to hydrolyze under acidic conditions than sucrose. Even if a small portion of lactose is hydrolyzed, the glucose and galactose resulting from its hydrolysis are also assimilable by the strains.

[0027] This embodiment can be combined with the previous one, by adding the inducing substrate partly during the hydrolysis, partly at the end of the hydrolysis.

[0028] It should also be noted that at the end of hydrolysis, the aqueous solution obtained can be used immediately, in the enzyme production facility. It can also be stored for later use, and hydrolysis can advantageously continue during storage if required, generally at room temperature, the solution preferably being maintained at an acidic pH until use to limit the risk of contamination.

[0029] According to a first variant, step cl) of hydrolysis of sucrose can be carried out in an acidic aqueous medium at a pH less than or equal to 5, in particular less than or equal to 4, in particular less than or equal to 3.5 or 3, preferably between 1 and 3.

[0030] To obtain a hydrolysis medium at acid pH, a strong acid, such as hydrochloric acid or sulfuric acid, is added, for example, to an aqueous solution in sufficient quantity to adjust the pH of the solution to the chosen value.

[0031] Alternatively, a weak acid of the organic acid type can be added.

[0032] Yet another alternative may be to acidify the aqueous solution by introducing carbon dioxide CO2 in gaseous form (bubbling): this is in fact a (weak) acid and it comes from the fermentation of sugars into alcohol in the process of converting biomass into alcohol. It is therefore available if the production of enzymes takes place on the biomass conversion site.

[0033] It is also possible to combine at least two of these different alternatives: addition to an aqueous solution of at least one strong acid, of at least one weak acid, introduction of CO2.

[0034] Advantageously, in particular with this first variant, step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium at a temperature of at least 110°C, in particular between 110 and 125°C, in particular between 110 and 120°C, in order to also ensure the sterilization of said medium. Indeed, the hydrolysis is accelerated if the medium in which the hydrolysis is carried out is heated, but not necessarily to such high temperatures. However, carrying out the hydrolysis at at least 110°C makes it possible to carry out the sterilization of the solution at the same time, which is necessary for its introduction into the enzyme production process. It is then advantageous, as mentioned above, to add one or more inducing substrates, such as lactose, to this medium before sterilization, which makes it possible to introduce the sterile solution comprising all the necessary substrates into the bioreactor (and which therefore avoids having to carry out separate sterilization of the inducing substrate(s).

[0035] In a second variant, step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium comprising at least in part, in particular only, an acidic hemicellulosic hydrolysate.

[0036] Advantageously, this hemicellulosic hydrolysate comes from an acid pretreatment step of a lignocellulosic biomass, and it comprises monomers and oligomers of sugars mainly in C5. Unlike patent WO 2013 / 1900214 cited above, this hydrolyzate is not used here directly as a carbon substrate, but first as providing a (strongly) acidic medium for the preliminary hydrolysis of sucrose. It should be noted that this hydrolyzate is acidic, because the pretreatment of biomass from which it is derived preferably comprises an acid impregnation step, as an example is described in the aforementioned patent EP 3 484 945.

[0037] This ultimately produces a solution containing the C5 sugars of the hydrolysate, which can be assimilated by the strains (microorganisms), and glucose and fructose from the hydrolysis of sucrose (and possibly also non-hydrolysed sucrose). This variant is very interesting due to its flexibility: the proportion of C5 sugars and the proportion of glucose / fructose in the solution obtained ultimately can be measured by measuring the quantity of hydrolysate and the quantity of sucrose added to it, depending on the availability of both carbon sources.

[0038] There is another advantage to using a hydrolysate as an acidic medium for hydrolyzing sucrose: the hydrolysis can be carried out by heating the solution, for accelerate / amplify it, but at lower temperatures than in the case of the first variant: thus, the hydrolysis step cl) can be carried out at a temperature less than or equal to 100°C, in particular less than or equal to 80°C, in particular between 30 and 60°C, because it is no longer necessary to sterilize the solution obtained before using it.

[0039] Indeed, hemicellulosic hydrolysates are acidic media, which contain sugars, but also inhibitors generated during the acid pretreatment of the biomass, such as acetic acid, furfural, 5-HMF or even phenolic compounds, which greatly limits the risks of contamination.

[0040] It is also possible to combine the two variants, using as acid hydrolysis medium a hemicellulosic hydrolysate, possibly diluted, concentrated, etc., and adding an acid or CO2.

[0041] It can be seen that the rate of hydrolysis of sucrose can be best adjusted during step cl) by varying the operating conditions, namely by adjusting the pH of the medium to a more or less acidic pH (by adding acid and / or hydrolysate at variable concentration) and / or by adjusting the temperature at which the hydrolysis is carried out and / or by adjusting the duration of the hydrolysis.

[0042] According to the invention, different operating conditions can be selected, in particular depending on the variant chosen. The temperature at which the hydrolysis takes place, as well as the pH of the medium in which it takes place and the duration of the hydrolysis are the most significant operating conditions. Generally speaking, it has been found that the hydrolysis takes place more quickly when the temperature is high and the pH is low.

[0043] According to a first embodiment, step cl) of hydrolysis of sucrose can be carried out in an acidic aqueous medium at a temperature of at least 110°C, at a pH of at most 5 and for a duration preferably between 10 minutes and 10 hours. It is preferred for the implementation of the first variant, where it is necessary to sterilize the medium, or when a (very) rapid hydrolysis is desired. And we note that in this case, where the temperature is high, we can adopt a pH a little less acidic than with a lower temperature (when we are not trying to sterilize).

[0044] According to a second embodiment, step cl) of hydrolysis of sucrose can be carried out in an acidic aqueous medium at a temperature of at most 100°C, in particular between 30 and 60°C, at a pH of at most 4, and for a duration preferably between 1 hour and 24 hours. It is preferred for the implementation of the second variant, in particular in the case where it is desired to have a relatively rapid hydrolysis duration.

[0045] According to a third embodiment, step cl) of hydrolysis of sucrose can be carried out in an acidic aqueous medium at a temperature of between 10 and 30°C, at a pH of at most 4 and for a duration preferably of more than 24 hours. It is preferred for the implementation of the second variant, in particular in the case where the hydrolysis can be carried out over a long period. Here, it is therefore not necessary to heat the hydrolysis medium. This is an interesting embodiment when there is a sucrose storage tank in which the hydrolysis can be left to take place until use, at room temperature (by feeding the bioreactor where the enzyme production takes place). List of figures

[0046] [Fig.l]

[0047] [Fig.l] represents the evolution of the biomass concentration (in the following examples, these are microorganisms in the form of fungi) over time in an enzyme production process with different carbon substrate solutions, (on the abscissa, the duration in hours, on the ordinate the quantity of biomass in g / 1).

[0048] [Fig.2]

[0049] [Fig.2] represents the evolution of the enzyme concentration over time in an enzyme production process with different carbon substrate solutions (on the abscissa, the duration in hours, on the ordinate the quantity of enzymes in g / 1).

[0050] [Fig.3]

[0051] [Fig.3] represents the evolution of the sucrose concentration over time: on the abscissa, the duration in hours, on the ordinate to the left of the graph the estimate of the quantity of sucrose in the fed-batch solution in g / 1, and on the ordinate to the right of the graph the estimate of the quantity of sucrose in the bioreactor where both the microorganism growth stage and the enzyme production stage are carried out, in g / 1. Description of the embodiments

[0052] Sucrose hydrolysis tests in acidic medium

[0053] Preliminary hydrolysis tests of sucrose and lactose (for comparison) were first carried out in aqueous solutions of sulfuric acid at different pH and different temperatures, according to the first variant of the invention.

[0054] The procedure is as follows: 3 solutions at approximately 220 g / L are prepared with: - 100% sucrose: solution called “pure sucrose” in the following tables - 100% lactose: solution called “pure lactose” in the following tables - 75% sucrose + 25% lactose weight: solution called “mixture” in the following tables

[0055] The solutions are separated into four, and the pH is adjusted with sulfuric acid, aiming for the following four pHs: 1.5; 2.0; 2.5-; 3.0.

[0056] An aliquot is taken and each of the 4 solutions is separated into two to test two sterilization conditions: (sterilization here is done at the same time as the hydrolysis of the disaccharides). - conditions (1): 110°C for 35 minutes - conditions (2): 121°C for 20 minutes

[0057] The results are as follows:

[0058] After sterilization, a coloration of the solutions is visually observed for pH values ​​lower than 2.0, whether with sucrose or with lactose, and whatever the conditions (1) or (2) chosen.

[0059] Residual sugars are measured after sterilization / hydrolysis by high-performance liquid chromatography (HPLC)

[0060] These results are detailed in the two tables below: - Table 1 shows the percentage of sucrose hydrolysis for the different solutions, depending on whether they were sterilized under conditions (1) or (2) - Table 2 shows the percentage of lactose hydrolysis, similar to Table 1

[0061] [Tables 1] % hydrolysis sucrose Conditions (2) Conditions (1 ) pH Pure sucrose mixture Pure sucrose mixture 1.5 99% 98% 100% 95% 2 100% 98% 98% 100% 2.5 100% 100% 95% 95% 3 100% 97% 88% 88%

[0062] [Tables2] % lactose hydrolysis Conditions (2) Conditions (1) pH Pure lactose in mixture Pure lactose in mixture 1.5 72% 68% 26%. 31% 2 24% 22% 12% 15% 2.5 0% 6% 0% 7% 3 0% 11% 0% 4%

[0063] We note that: - at 121°C, with conditions (2), sucrose is completely hydrolyzed into glucose + fructose, whatever the pH among the four pH values ​​tested, - at 110°C, with conditions (1), the sucrose is completely hydrolyzed, as soon as the pH is less than or equal to 2.5.

[0064] These tests therefore demonstrate that it is possible to hydrolyze all or almost all of the sucrose in an acid medium, by choosing a strongly acidic pH (at most 5, preferably at most 4, preferably at most 3.5 or 3), a high temperature allowing sterilization to be carried out at the same time, and this within a period of 20 minutes, therefore quickly.

[0065] It is also noted that lactose hydrolysis does not occur, or only slightly, unless a very acidic pH is adopted, at most 2. Examples

[0066] The enzyme production process used to illustrate the invention is first described using the examples which follow.

[0067] These examples relate to the second variant of the invention with hydrolysis of sucrose by a hemicellulosic hydrolysate (also called “C5 sugar juice” or “C5 juice”).

[0068] The strain

[0069] The fungal strain used in the following examples is described in patent WO2015 / 193587: it is the variant referenced 130G9 (SEQ ID No. 7 in nucleic acid, SEQ ID No. 8 in polypeptide in Table 1 of this patent) of Tri-choderma reesei. It will be referred to hereinafter as “the strain”. It does not possess the invertase gene and does not have the capacity to assimilate sucrose.

[0070] The preculture phase

[0071] The preculture phase of the strain is carried out as follows: The strain is precultured in a Fembach for 72 h at 30°C and under agitation at a stirring speed of 150 rpm.

[0072] Composition of the preculture medium: In a Fernbach: - 62.5 mL of 4N medium (composition given below) - 1.25g of dipotassium phthalate - 1g of “cornsteep” (corn protein), commercially available from the Roquette company under the trade name Solulys. - sufficient quantity: 200 mL of distilled water The mineral medium (called “4N medium”) has the following composition: KOH 1.66 g / L, H3PO 4 85% 2 mL / L, (NH4)2SO4 2.8 g / L, MgSO4, 7 H2O 0.6 g / L, CaCl2 0.6 g / L, MnSO4 3.2 mg / L, ZnSO4, 7 H2O 2.8 mg / L, CoCl2 10 H2O 4.0 mg / L, FeSO4, 7 H2O 10 mg / L.

[0073] The pH is adjusted to 5 with soda before sterilization.

[0074] In a separate vial: - 8.5 g of glucose in 50 mL of distilled water Cultures in bioreactors: The growth phase

[0075] The preculture is transferred into 4 mechanically stirred 1.5 L CSTR type bioreactors (acronym for "continuous stirred-tank reactor"), with a seeding volume of 10% (v / v).

[0076] Composition of the culture medium: - 400 mL of 4N medium - 320 mL of glucose at 37.5 g / L, i.e. an initial glucose concentration of 15 g / L - 1g of cornsteep Arugula - 80 mL from preculture in Fernbach

[0077] The pH is maintained at 4.4 by the addition of a 5.5 N ammonia solution. The pO2 (dissolved O2 concentration expressed relative to the saturation concentration) is regulated at 40% by stirring. The temperature is stabilized at 27°C.

[0078] The production phase

[0079] After consumption of the glucose, the production phase starts with induction with the continuous addition of a sugar solution at approximately 400 g / kg, at a flow rate of 1.25 mL / h. The pH is regulated at 4 and the temperature at 25 °C.

[0080] Fed-batch solutions are prepared by dissolving lactose and sucrose or glucose in a solution of hemicellulosic hydrolyzate from straw pretreated by steam explosion under acidic conditions. This hemicellulosic hydrolyzate, called "BHS 7.3" juice, is obtained after washing with water a straw pretreated at 185°C for 5 minutes after impregnation with sulfuric acid (acidity of 1.8%).

[0081] The composition of BHS 7.3 juice is given in Table 3 below.

[0082] The acronym MS for Dry Matter corresponds to the solid part of a liquid / solid mixture which is soluble in the liquid + the solid part insoluble in the liquid in question

[0083] The acronym MSI, for Insoluble Dry Matter, only includes the insoluble solid part in the liquid / solid mixture.

[0084] [Tables3] MS (%) MSI (%) Glucose (g / L) Xylose (g / L) Acetic Acid (g / L) HMF (g / L) Furfural (g / L) 9.04 0.08 10.05 35.07 1.81 0.58 0.19

[0085] The compositions of the solutions are detailed below:

[0086] Example 1: Control with glucose + lactose (unsterilized)

[0087] A 1 kg solution was prepared by dissolving 330 g of glucose monohydrate, 120 g of water and 100 g of lactose (lactose as the inducing substrate) in 450 g of BHS 7.3 juice. The dissolution is carried out in a beaker at room temperature using a magnetic bar. The solution obtained is not sterilized. This is the positive control.

[0088] Throughout this text, the term “ambient temperature” is understood to mean a temperature usually between 10 and 30°C, generally between 15 and 25°C, and in this case around 20°C.

[0089] Example 2: Use of sucrose + lactose (without sterilization)

[0090] A 1 kg solution was prepared by dissolving 300 g of sucrose, 150 g of water and 100 g of lactose in 450 g of BHS 7.3 juice. There is no heating step to hydrolyze the sucrose at acidic pH. The fed-batch solution is kept at room temperature. It will partially and slowly hydrolyze throughout the 215 hours of experiment.

[0091] Example 3: Heating sucrose-i- C5 juice to hydrolyze it then adding lactose to the solution (without sterilization)

[0092] A 900 g solution was prepared by dissolving 300 g of sucrose + 150 g of water in 450 g of BHS 7.3 juice, then heating the solution to 110°C for 20 minutes, then adding 100 g of lactose under non-sterile conditions.

[0093] Example 4: Use of Sucrose and Lactose with Sterilization

[0094] A 1 kg solution was prepared by dissolving 300 g of sucrose + 150 g of water + 100 g of lactose in 450 g of BHS 7.3 juice, then heating (autoclaving) the solution at 120°C for 20 minutes. The solution is sterile (limits the risk of contamination) and the sucrose is hydrolyzed into glucose and fructose.

[0095] Example 5 (comparative): Use of sucrose + lactose without acid hydrolysis of sucrose

[0096] A 1 kg solution was prepared by dissolving, in 450 g of water, 300 g of sucrose, 150 g of water and 100 g of lactose, then heating (autoclaving) at 120°C and 20 minutes of the solution. The solution is sterile. There is therefore no contact of the sucrose with an acidic medium prior to its introduction into the bioreactor. The fed-batch solution is kept at room temperature.

[0097] The follow-up

[0098] On the samples taken daily, the following measurements were carried out: - Determination of biomass by membrane filtration - Determination of residual sugar by HPLC - Determination of extracellular enzymes by the Lowry method after precipitation with TCA and BSA standard, after separation of the microorganism by filtration or centrifugation

[0099] Cellulolytic activities are determined by: - Filter paper activity (UPF: filter paper unit), which allows the overall activity of the enzymatic cocktail to be measured: endoglucanases and exoglucanases - Aryl [3-glucosidase] activity, for specific activities.

[0100] UPF activity is measured on Whatman No. 1 paper (Procedure recommended by the IUP AC biotechnology commission) at the initial concentration of 50 g / L: the test portion of the enzymatic solution to be analyzed is determined, which releases the equivalent of 2 g / L of glucose (colorimetric assay) in 60 minutes. The principle of filter paper activity is to determine by DNS (dinitrosalicylic acid) assay the quantity of reduced sugars from Whatman No. 1 paper.

[0101] The substrate used to determine aryl [3-glucosidase] activity is p-nitrophenyl-BD-glucopyranoside (PNPG). It is cleaved by B-glucosidase which releases p-nitrophenol. One unit of aryl [3-glucosidase] activity is defined as the amount of enzyme required to produce 1 pmol of p-nitrophenol from PNPG per minute and is expressed as 1U / mL.

[0102] Specific activities are obtained by dividing the activities expressed in lU / mL by the protein concentration. They are expressed in lU / mg. The cultures proceeded well until the end of the experiments.

[0103] [Fig.l] represents the monitoring of the concentration in g / l of fungi in the bioreactor as a function of time in hours. The time t=0 corresponds to the start of the process, comprising a first phase of growth of the fungi of 24 hours, with a feeding of the bioreactor in carbon substrate (only glucose as mentioned above) in one go, called batch feeding, then a second phase of production of the enzymes, with a feeding of the reactor in carbon substrate called n fed-batch feeding, according to the compositions defined above (progressive feeding according to a given frequency of additions). The curve with the diamonds corresponds in example 1 (control), the curve with squares in example 2, the curve with triangles corresponds to example 3, the curve with solid circles in example 4, and the curve with empty circles in example 5 (comparative).

[0104] [Fig.2] represents the monitoring of the concentration in g / 1 of enzymes, in the bioreactor as a function of time in hours, the time t=0 corresponding to the start of the process, with the same conventions as for [Fig.l] (24 hours of growth, then production) for examples 1 to 5.

[0105] For the five fermentations, as seen in Figures 1 and 2, the production of enzymes in Examples 3 and 4 with hydrolyzed sucrose before the experiment is identical to that of Example 1 which is the control (with glucose). As for Example 5, it is seen that it leads to a much lower production of fungi and enzymes than the other examples, which demonstrates that, in this case, the non-hydrolyzed sucrose is not assimilated by the fungi, and that the growth of the fungi and the production of enzymes is only due to the presence of lactose in the fed-batch solution.

[0106] Regarding Example 2, with sucrose hydrolyzed without heating: it produces fewer enzymes than the other examples, but nevertheless in comparable quantities with them, which is surprising. Looking at the final dosages of residual sugar, it is noted that there is an accumulation of sucrose during the unconsumed time, which explains the lower production performance. However, if the sucrose was not hydrolyzed at all in the fed-batch solution, the final sucrose concentration would have been theoretically equal to 86 g / L (instead of 27 g / L), because the strain tested does not possess invertase and is therefore not able to consume sucrose. The final dosage of sugars of the fed-batch solution maintained at room temperature throughout the experiment after 215 hours finally contains very little sucrose: 50 g / L instead of the 350 g / L added initially.

[0107] The acidic pH of the fed-batch solution allowed the hydrolysis of sucrose, just as when hydrolysis is carried out with heating (sterilization), but at a slower rate.

[0108] [Fig.3] represents, for this example 2, a graph, with the duration in hours on the abscissa, the concentration of sucrose in the fed-batch solution (on the left of the graph) in g / 1 on the ordinate, and in the bioreactor (on the right of the graph) in g / 1 also, the time t=0 corresponding to the start of the process, with the same conventions as for figures 1 and 2 (24 hours of growth, then production)

[0109] The curve with circles corresponds to the evolution of the sucrose concentration in the fed-batch solution measured in its storage tank (in fluidic connection with the bioreactor in order to feed it).

[0110] The curve with squares corresponds to the theoretical accumulation of sucrose in the bioreactor.

[0111] The curve with triangles corresponds to the accumulation of sucrose measured in the bioreactor.

[0112] Assuming that the sucrose only hydrolyzes in the tank where the fed-batch solution is, and not in the bioreactor which is at pH 4, and using the measurement of the accumulation of sucrose over time in the bioreactor, we can thus estimate the kinetics of hydrolysis of the sucrose in the fed-batch solution.

[0113] We can thus see that, under these conditions, it took approximately one week to completely hydrolyze the sucrose in its storage tank at room temperature.

[0114] The specific [3-glucosidase and UFP] activities are indicated in Table 4 below for Examples 1 to 5. They are all of the same order, whatever the fermentation, which indicates that the enzymes produced are of equivalent quality.

[0115] [T ableaux4] Examples Activity [3-glucosidase (lU / mg) UFP Activity (lU / mg) Example 1 - Control 26 0.9 Example 2 24 1.1 Example 3 25 0.9 Example 4 24 1.0 Example 5 - Comparative 23 0.9

[0116] Table 5 below groups together for examples 1 to 5 the specific enzyme production rates qp. This is calculated by dividing the protein production rate expressed in g / L / h by the microorganism concentration (as is known to those skilled in the art). They are of the same order for the fermentations of examples 1 to 4, but approximately 2 times lower for example 5, which indicates that the lack of assimilable sugar did not allow good production performances to be obtained.

[0117] [Tables5] Examples Specific production rate qp(mgp / gx / h) Example 1 - Witness 23 Example 2 26 Example 3 22 Example 4 24 Example 5 - Comparative 12

[0118] HPLC analysis of the fed-batch solutions at the end of fermentation confirms that the lactose of example 4 has not been degraded (concentration ~ 100 g / L), after autoclaving or without autoclaving, and that the sucrose is completely hydrolyzed into glucose and fructose after autoclaving, thanks to the strongly acidic pH of the solution, equal to 2.9.

[0119] A complementary experiment was carried out by preparing a fed-batch solution similar to that of Example 2. This solution was divided into two parts: one part incubated at 20°C for 215 hours, and one part incubated at 50°C for 215 hours. It appears that after 24 h at 50°C, all the sucrose is hydrolyzed, whereas 60% of the sucrose is hydrolyzed after 215 h at 20°C.

[0120] From these different examples and results, it is understood that the invention consisting of hydrolyzing sucrose in an acid medium makes it possible to make it assimilable by the fungal strain without having to genetically modify it to add the invertase gene to it. It is also understood that this hydrolysis is favored / accelerated by heating, either at a moderate temperature or at a higher temperature, in particular if it is also necessary to sterilize the substrate solution before putting it in contact with the strain.

[0121] The invention offers great flexibility of implementation, - using two possible types of acidic medium: made by adding acid, or obtained directly by a C5 sugar juice obtained during the conversion of the biomass, and which is in fact at a sufficiently acidic pH for this hydrolysis, - and using different operating conditions (notably the hydrolysis temperature) depending on requirements (high temperature if sterilization is required, lower temperature or even at room temperature if hydrolysis can be allowed to take place more slowly, pH which can be chosen to be more acidic the lower the temperature, etc.).

Claims

Claims

1. Process for the production of cellulolytic and / or hemicellulytic enzymes by a cellulolytic and / or hemicellulolytic microorganism, said process comprising at least - a) a phase of growth of the microorganism in the presence of at least one carbon substrate, then - b) a phase of production of the enzymes in the presence of at least one inducing substrate, characterized in that said process also comprises: - c) a phase of preparation of a carbon substrate comprising glucose and / or fructose, carbon substrate which is used in one and / or the other of the phases a) of growth and b) of production, said preparation phase comprising a step c1) of hydrolysis in an acidic aqueous medium of sucrose into glucose and fructose, - the cellulolytic and / or hemicellulolytic microorganism being chosen from microorganisms lacking the invertase gene and capable of consuming glucose and / or fructose, in particular strains of fungi,- and step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium comprising at least part of an acidic hemicellulosic hydrolyzate.,

2. Method according to one of the preceding claims, characterized in that the cellulolytic and / or hemicellulolytic microorganism is chosen from strains of fungi belonging to the genera Trichoderma, Aspergillus, Penicillium or Schizophyllum, and preferably belonging to the species Trichoderma reesei.

3. Method according to one of the preceding claims, characterized in that, at the end of preparation phase c), an aqueous solution comprising glucose and fructose is obtained, and in that said solution is used, directly or after possible concentration adjustment operations, as a carbon substrate in growth step a) and / or production step b).

4. Method according to the preceding claim, characterized in that at least one inducing substrate, in particular lactose, is added to the aqueous solution comprising glucose and fructose obtained at the end of preparation phase c), in particular to use said solution as a substrate in production step b).

5. Method according to one of the preceding claims, characterized in that at least one inducing substrate, in particular lactose, is added to the acidic aqueous medium during step cl) of hydrolysis of sucrose.

6. Method according to one of the preceding claims, characterized in that step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium at a pH less than or equal to 5, in particular less than or equal to 4, in particular less than or equal to 3.5 or 3, preferably between 1 and 3.

7. Process according to the preceding claim, characterized in that the acidic pH of the acidic aqueous medium of the hydrolysis step cl) is obtained by adding to an aqueous solution at least one strong acid and / or at least one weak acid and / or gaseous carbon dioxide, in particular from fermentation.

8. Method according to one of the preceding claims, characterized in that step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium at a temperature of at least 110°C, in particular between 110 and 125°C, in particular between 110 and 120°C, in order to also ensure the sterilization of said medium.

9. Method according to one of the preceding claims, characterized in that step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium comprising only an acidic hemicellulosic hydrolyzate.

10. Method according to the preceding claim, characterized in that the hemicellulosic hydrolyzate comes from an acid pretreatment step of a lignocellulosic biomass, and in that it comprises monomers and oligomers of sugars mainly in C5.

11. Process according to one of claims 1 to 7, characterized in that the hydrolysis step cl) is carried out at a temperature less than or equal to 100°C, in particular less than or equal to 80°C, in particular between 30 and 60°C, or between 10 and 30°C.

12. Process according to one of claims 1 to 10, characterized in that step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium at a temperature of at least 110°C, at a pH of at most 5 and for a duration preferably of between 10 minutes and 10 hours.

13. Process according to one of claims 1 to 7, characterized in that step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium at a temperature of at most 100°C, in particular between 30 and 60°C, at a pH of at most 4, and for a period preferably between 1 hour and 24 hours.

14. Process according to one of claims 1 to 11, characterized in that step cl) of hydrolysis of sucrose is carried out in an acidic aqueous medium at a temperature between 10 and 30°C, at a pH of at most 4 and for a duration preferably of more than 24 hours.

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