METHOD FOR PRODUCING COMPOUNDS OF INTEREST BY FERMENTATION

Abstract

The present invention relates to a process for producing at least one compound of interest by solid-state fermentation, comprising the following steps: - culture of microorganisms on a solid substrate, said substrate being placed on a membrane permeable to liquids but not permeable to microorganisms or to the substrate, - elution of at least one compound of interest by distributing a liquid on the upper surface of said solid substrate, - collection of the liquid containing at least one compound of interest in a compartment located under the permeable membrane, characterized in that the process is carried out continuously.

Description

Title of the invention: PROCESS FOR THE PRODUCTION OF COMPOUNDS OF INTEREST BY FERMENTATION FIELD OF THE INVENTION

[0001] The present invention relates to a process for preparing compounds of interest by fermentation of a substrate in solid phase, and their uses.

[0002] Fermentation can be defined as the transformation of organic matter under the action of enzymes produced by microorganisms. Among the best known types of fermentation are alcoholic fermentation, where fruit sugars, particularly grapes, are transformed into alcohol by yeasts; and lactic fermentation, or lacto-fermentation, which transforms sugars into lactic acid under the action of lactic acid bacteria such as lactobacillus or bifidobacterium.

[0003] Generally speaking, fermentation allows the transformation of a carbon source into metabolites, CO2 and H2O, when carried out under aerobic conditions.

[0004] The fermentation process allows the production of various microbial metabolites such as antibiotics, or enzymes which serve as technological aids in various industries.

[0005] Although fermentation in liquid medium, with stirring, is the most used technique nowadays, fermentation in solid medium (FMS) is developing more and more. FMS technology indeed presents economic, technological and environmental advantages, thus offering a promising alternative for the production of various metabolites.

[0006] Solid state fermentation (SSF) is defined as a culture of microorganisms that takes place in a solid porous matrix, on the surface but also inside said matrix. The porous matrix can consist of a moist substrate or an inert support, loaded with a substrate providing the nutrients necessary for the microorganisms.

[0007] Many microorganisms grow naturally on biodegradable solid supports, provided that a minimum quantity of water is present.

[0008] The first solid-state fermentations were implemented in bread making. Other ancestral processes are those for the manufacture of koji, tofu and tempeh. Cheese-making techniques for seeding crusts with filamentous fungi also correspond to an implementation of FMS. STATE OF THE ART

[0009] Nowadays, FMS is generally carried out in bioreactors adapted to optimize the different parameters of microbial culture, such as temperature, oxygenation, humidity level and possibly agitation.

[0010] A process for producing carboxylic acids from agricultural co-products, particularly from corn, by solid-state fermentation, has been described by (Huang et al., 2002). In this process, fermentation takes place under anaerobic conditions. An inert, fibrous and porous matrix constitutes the support for the microbial culture; a nutrient liquid, containing a corn hydrolysate, circulates at a rate of 75 ml / minute using a pump.

[0011] The production of antibiotics by FMS has also been described. In particular, the production of meroparamycin by a strain of Streptomyces sp. is described in the article by (El-Naggar et al., 2009). The preferred substrate consists of wheat bran. This substrate is placed on sintered glass beads (with stirring), or in a static glass column, serving as an inert support for FMS. A nutrient medium is added to moisten the whole, then inoculated with the microbial strain. The incubation lasts from 5 to 7 days, then washing the support with water allows the antibiotic produced to be collected.

[0012] International application WO 00 / 29544 describes a bioreactor suitable for a solid-state fermentation process; this bioreactor contains several assemblable modules, comprising communication channels for circulating liquids and / or gases within the bioreactor. Each module contains a solid substrate, in particular an agricultural co-product. The metabolites produced by FMS are collected by an extraction liquid which is added from the top of the bioreactor and circulates to a collection tray, from which the liquid containing the metabolites and everything that has been entrained by the liquid is extracted: substrate residues, microorganisms.

[0013] The present invention aims to produce by FMS compounds of interest collected in a sterile medium, not containing microorganisms.

[0014] Indeed, conventional sterilization methods, particularly by heating, are likely to degrade fragile compounds at high temperatures.

[0015] French patent FR 2 960 551 proposes to sterilize the fermented substrate, obtained after an FMS step, by irradiation / ionization, before harvesting the metabolites produced. This FMS process is particularly suitable for the production of enzymes, the microorganisms having produced them being able to be destroyed by ionizing radiation, without the structure of the enzymes being affected.

[0016] However, this technique cannot be applied to all compounds of interest. In addition, this process has the disadvantage of being discontinuous, it having to be stopped to treat the substrate after fermentation for sterilization and collection of the compounds produced.

[0017] Generally speaking, the main difficulty of FMS is the final step of separating the microorganisms and the substrate from the molecules produced. Gene- Rally, this step is carried out at the end of fermentation, by filtration or centrifugation.

[0018] There is therefore a need to have a continuous process making it possible to produce by FMS various compounds of interest, collected directly in a sterile medium, free of microorganisms, without requiring separate steps of collecting the metabolites and / or sterilizing said metabolites.

[0019] The present invention aims to satisfy this need. Statement of the invention

[0020] The present invention relates to a process for producing at least one compound of interest by solid-state fermentation, comprising the following steps: - cultivation of microorganisms on a solid substrate, said substrate being placed on a membrane permeable to liquids but not permeable to microorganisms or to the substrate, - elution of at least one compound of interest by distribution of a liquid on the upper surface of said solid substrate, - collection of the liquid containing at least one compound of interest in a compartment located under the permeable membrane, characterized in that the process is carried out continuously.

[0021] According to a preferred implementation, the solid substrate is composed of one or more food, agricultural or industrial co-product(s).

[0022] According to one implementation of the method of the invention, the microorganisms are fungi, microalgae, yeasts, archaea or bacteria.

[0023] According to another preferred implementation of the method of the invention, the at least one compound of interest is collected in the compartment located under the membrane, then transferred to another compartment where it can be purified and / or concentrated. DESCRIPTION OF FIGURES

[0024] [Fig.l] [Fig.l] represents a schematic illustration of the process according to the invention allowing the production and collection of metabolites secreted by microorganisms. Schematically, the process is divided into 4 steps a, b, c and d.

[0025] [Fig.2A] [Fig.2A] illustrates the production of proteins by the method of the invention using the microorganism Aspergillus Niger, on 4 different solid substrates: potato peelings, corn cob, coffee grounds and bread, as a function of the culture time.

[0026] [Fig.2B] [Fig.2B] illustrates the production of acid by the process of the invention using the microorganism Aspergillus Niger, on 3 different solid substrates: potato peelings, corn cob, coffee grounds, after 5 days of culture.

[0027] [Fig.3A] [Fig.3A] represents the continuous recovery of proteins secreted by Aspergillus Niger during 5 days of culture, on a solid substrate consisting of coffee grounds.

[0028] [Fig.3B] [Fig.3B] represents the continuous recovery of proteins secreted by Aspergillus Niger during 5 days of culture, on a solid substrate consisting of hard bread.

[0029] [Fig.4] [Fig.4] represents the production of pigments with or without microor organisms naturally present on bread. Absorbance between 200nm and 600nm is shown.

[0030] [Fig.5A] [Fig.5A] represents the production of acids by the bacterium Escherichia coli. The quantity of acids produced was measured by comparing the pH of a culture without microorganism, with the pH of a culture in the presence of said microorganism. The difference in pH between the two conditions is represented.

[0031] [Fig.5B] [Fig.5B] represents the production of proteins by the bacterium Escherichia coli. The quantity of proteins produced was measured by comparing the absorbance at 280nm of a culture without microorganism, with the absorbance at 280nm of a culture in the presence of said microorganism. The difference in absorbance between the two conditions is represented. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to a process for producing at least one compound of interest by solid-state fermentation, comprising the following steps: - cultivation of microorganisms on a solid substrate, said substrate being placed on a membrane permeable to liquids but not permeable to microorganisms or to the substrate, - elution of at least one compound of interest by distribution of a liquid on the upper surface of said solid substrate, - collection of the liquid containing at least one compound of interest in a compartment located under the permeable membrane, characterized in that the process is carried out continuously.

[0033] Advantageously, this method makes it possible to completely separate the microorganisms from the compounds produced by fermentation, and thus to obtain a liquid containing said compound of interest which is free of microorganisms, and therefore sterile. In addition, this collection liquid is free of solid substrate residues, which makes it easier to analyze by chromatography, without requiring a pre-purification step.

[0034] For the purposes of the invention, the term “solid state fermentation”, also designated by the abbreviations FMS or SSF, means a culture of microorganisms on a solid substrate providing them with the necessary nutrients, in which the microorganisms produce at least one compound of interest from a carbon source. present in the solid substrate.

[0035] The cultivation of the microorganisms will be carried out according to the general knowledge of the person skilled in the art, who will know how to adapt the cultivation conditions such as the temperature, the humidity of the substrate, the ratio of microorganisms / substrate, so that the growth of the microorganisms and especially the production of the compound of interest is optimal.

[0036] Fermentation may be carried out with or without stirring, depending on the microorganisms and / or solid substrates used.

[0037] According to a particular implementation of the method of the invention, the culture of the microorganisms is carried out with stirring.

[0038] The method according to the invention is carried out continuously. This means, within the meaning of the invention, that the process for producing compounds of interest is carried out without interruption, and that in particular the compounds produced are recovered as they are produced. A continuous process is distinguished from a discontinuous or “batch” process, where the recovery of the compounds of interest requires stopping the fermentation process, or even partial dismantling of the bioreactor containing the substrate and the microorganisms, in order to be able to collect the compounds of interest produced.

[0039] It is understood that in the context of a “continuous” process, there will nevertheless be a step of initiation of the process, corresponding to the inoculation of the microorganisms on the solid substrate, and a final step, corresponding to the stopping of the fermentation when the substrate is exhausted in nutrients, the microorganisms therefore no longer being able to produce compound(s) of interest. This final step may however be postponed by a regular addition of solid substrate in the system, if the latter is adapted for such an addition. Solid substrate

[0040] For the purposes of the present invention, the term “solid substrate” means the source of nutrients necessary for fermentation by microorganisms.

[0041] According to a preferred implementation, the solid substrate is composed of one or more food, agricultural or industrial co-product(s).

[0042] A food co-product will be, for example, coffee grounds, stale bread, fruit or vegetable peelings, or any by-product generated by the food industry.

[0043] An agricultural co-product is chosen from plant waste (wood, grass, leaves, straw) and co-products from the processing of agricultural raw materials for food, such as for example: • wheat bran or any other cereal, • corn cobs, • beet pulp, • sugarcane bagasse, • starch production waste (extraction of starch from corn, rice, wheat or potatoes), • brewery or distillery waste.

[0044] The solid substrate may also be an industrial co-product, such as, for example, paper mill waste, in particular the sludge generated during the production of paper.

[0045] According to a first implementation of the method, the solid substrate is a food co-product.

[0046] According to a second implementation of the method, the solid substrate is an agricultural co-product.

[0047] According to a third implementation of the method, the solid substrate is an industrial co-product.

[0048] According to a fourth implementation of the method, the solid substrate is a combination of at least two agricultural, food and / or industrial co-products.

[0049] Preferably, this solid substrate will be in the form of powder or particles.

[0050] The particle size should be greater than the pore size of the membrane, so that the substrate is not carried along by liquid when collecting the compounds of interest produced by fermentation.

[0051] Thus, the particle size will preferably be between approximately 1 pm2 and 2 cm2. Preferably, the particle size will be between 1 pm2 and 1 cm2, and most preferably between 50 pm2 and 0.5 cm2.

[0052] Said particles may in particular be in the form of chips.

[0053] According to a particular implementation of the method of the invention, the solid substrate is previously sterilized before being inoculated with the microorganism(s).

[0054] According to another implementation of the method of the invention, the solid substrate is previously moistened before the start of the culture of microorganisms, and / or is moistened regularly during the culture, in particular thanks to the distribution of the liquid on the upper surface of said solid substrate.

[0055] The quantities of liquid added relative to the quantity of solid substrate will advantageously be of the order of 1 milliliter of liquid for a weight of solid substrate of between 0.1 g and 5 g, preferably between 0.3 g and 2 g, or even between 0.5 g and 1 g. Microorganisms

[0056] The initiation of the culture of microorganisms on a solid substrate can be carried out according to two distinct modes: a. the microorganism(s) already present on the substrate are placed in conditions allowing their development: see example 1 relating to the production of pigments by the microorganisms present in the bread; b. the microorganism(s) used are inoculated onto the solid substrate.

[0057] The term "inoculated" means, within the meaning of the invention, that the microorganisms have been introduced voluntarily and in a directed manner by the person implementing the method. In this implementation, the microorganism(s) used are chosen according to the compound of interest to be produced, and the solid substrate used.

[0058] According to a particular implementation, the microorganisms already present on the substrate or inoculated are fungi, microalgae, yeasts, archaea or bacteria.

[0059] The term "fungi" includes filamentous fungi and non-filamentous fungi. As examples of fungi, the following genera and species may be used in the method according to the invention: Aspergillus and in particular Aspergillus Niger, Thermothelomyces and in particular Thermothelomyces he-terothallica, Neurospora, Fusarium, Rhizopus, Chrysosporium, Penicillium, Myce-liophthora, Trichoderma, Rasamsonia, Corynascus, Talaromyces, Sporotrichum thermophile, Thielavia, Aureobasidium, Filibasidium, Cryptococciis, Acremonium, Scytalidium, Schizophylhim, Gibberella, Mucor, Humicola, Cryplococcus, Toly-pocladium, Fitsarium, Thermoascus, Ciyplococcics, Chrysosporhim, Thermomyces, Piromyces, Schizophyllum, Rhizomucor, Hansenula, Emericella, Hypocrea and Chaetomium.

[0060] The microalgae, or marine diatoms, will for example be chosen from the following species: Chlamydomonas reinhardtii and Phaeodactylum tricomutum.

[0061] The yeasts considered for the implementation of the process will be chosen in particular from Saccharomycetes, and in particular from the following species: Sac-charomyces cerevisiae, Pichia Pastoris, Pichia angusta and Candida boidinii.

[0062] Archaea or archaeobacteria are prokaryotic organisms living in particular, often hostile environments (salt-saturated water, very hot sulfurous springs, etc.). Among the archaea that can be used in the process of the invention, the following species will be mentioned in particular: Sulfolobus solfataricus, Thermococcus litoralis, Pyrococcus furiosus, Sulfolobus acidocaldarius, and Methanothermobacter thermautotrophicus.

[0063] Finally, the bacteria will for example be chosen from the following genera and species: Enterobacteriaceae and in particular Escherichia coli, Bacillaceae and in particular Bacillus subtilis, and Corynebacteriaceae and in particular Corynebacterium glutamicum.

[0064] According to a first implementation of the method, a single species of microorganism is present or inoculated on the solid substrate.

[0065] According to a second implementation of the method, a co-culture of at least two different microorganisms is present or inoculated on the solid substrate.

[0066] According to a particular implementation of the method of the invention, the microorganisms are genetically modified microorganisms to induce or optimize their capacity to produce the compound of interest.

[0067] According to this implementation, the genetically modified microorganisms are generally inoculated onto the substrate.

[0068] The person skilled in the art is able to choose the genetically modified microorganisms for the production of a particular compound of interest.

[0069] Membrane permeable to liquids but not permeable to microorganisms nor to the substrate

[0070] For the purposes of the invention, the term "permeable membrane" designates the ability of said membrane to allow a fluid to pass through it under the effect of gravity. This ability is closely linked to the porosity of the membrane, which can be expressed as a function of the size of the pores of the membrane.

[0071] In the implementation of the method of the invention, the permeable membrane is of the “macroporous” type, that is to say that the diameter of the pores is greater than 50 nanometers.

[0072] According to a particular implementation, the pores of the membrane have a diameter of between 0.1 and 0.5 μm.

[0073] According to another implementation, the pores of the membrane have a diameter of approximately 0.2 pm, plus or minus 0.02 pm.

[0074] According to an advantageous aspect of the invention, the membrane is made of a material which can be sterilized by heat treatment (conventionally, by autoclaving).

[0075] More particularly, the permeable membrane is made of a material chosen from the following materials: polyethersulfone (PES), cellulose, nylon, polycarbonate, cellulose nitrate, polypropylene, polytetrafluoroethylene and polyamide.

[0076] According to a preferred implementation of the invention, the membrane is made of PES.

[0077] The method according to the invention is implemented within a device schematically represented in [Fig.l].

[0078] The solid substrate, inoculated with at least one microorganism, is placed on a membrane ("filter") permeable to the liquid, but not permeable to the microorganisms or to the substrate.

[0079] The flow of liquid over the solid substrate allows the compounds of interest produced by fermentation to be collected in a solution which is sterile due to the inability of the microorganisms to pass through the membrane. This solution is collected in a compartment located under the membrane.

[0080] In this system, there is an "upper compartment" which contains the solid substrate, and a "lower compartment", or "collection compartment", into which the liquid distributed on the solid substrate flows. The permeable membrane is placed between these two compartments and delimits them. Collection of the compound of interest

[0081] For the purposes of the invention, the term “compound of interest” means any compound produced by fermentation using microorganisms, from a carbon source. This includes in particular any product resulting from microbial metabolism, also referred to as a “metabolite”.

[0082] For the purposes of the present invention, the term “compound of interest” means, in particular: • substances with therapeutic activity and in particular antibiotics, • pigments, • proteins, in particular enzymes, and also recombinant proteins produced by genetically modified microorganisms, and among these in particular growth factors, cytokines or hormones, • acids, in particular organic acids, and particularly carboxylic acids, • alcohols, • vitamins, • polysaccharides or simple sugars, • lipids, and • antioxidants.

[0083] Preferably, the compound of interest will be chosen from: a pigment, a protein or an organic acid.

[0084] More specifically, the compound of interest will be chosen from: glucoamylase, FGF (Fibroblast Growth Factor), citric acid and at least one carotenoid.

[0085] According to an advantageous implementation of the method of the invention, the compound of interest produced is a metabolite secreted by the microorganisms.

[0086] For the purposes of the invention, a compound of interest is described as “secreted” when it is released by a microorganism into the extracellular medium. Thus, the metabolites produced by fermentation and then secreted do not remain within the microorganism that produced them, but are present on the membrane of said microorganisms and mix with the substrate surrounding them.

[0087] In the method according to the invention, the elution of the compound(s) of interest is carried out by distributing a liquid on the upper surface of the solid substrate. Said liquid, by being thus distributed on the solid substrate, carries away the secreted metabolites by flow.

[0088] The liquid used for this elution is preferably water, a buffer solution, a nutrient solution or an organic solvent.

[0089] The water will preferably be sterile water, possibly demineralized.

[0090] The buffer solution may in particular be PBS (Phosphate-Buffered Saline) buffer.

[0091] The nutrient solution may in particular be LB, Potato Dextrose Broth (PDB), YPD medium (Yeast extract Peptone Dextrose), or any solution containing peptones.

[0092] The organic solvent may in particular be methanol, ethanol, butanol, chloroform or acetonitrile.

[0093] Advantageously, the compound of interest is soluble in the elution liquid used.

[0094] The elution liquid will preferably be distributed over the upper surface of the solid substrate continuously, in order to collect the metabolites continuously in the compartment located under the membrane, also referred to as the "collection chamber".

[0095] The elution liquid will be distributed over the solid substrate at a moderate flow rate, so as not to disturb the equilibrium established in the FMS process.

[0096] According to a particular implementation, said liquid is distributed at a rate of 0.5 to 500 ml of liquid, for 1 to 100 grams of solid substrate, per 24 hours.

[0097] According to one implementation of the invention, the elution liquid is recycled in the process, that is to say it is collected in the compartment located under the membrane and redistributed on the upper surface of the solid substrate. This implementation makes it possible to reduce the quantity of liquid used in the process, but also to concentrate the collected compounds of interest.

[0098] The method according to the invention is particularly suitable for the production by FMS of a “fragile” compound of interest, i.e. one whose structure is rapidly degraded at room temperature, or is sensitive to variations in pH and / or temperature, or is sensitive to proteases secreted by microorganisms. Indeed, the elution liquid collected in the compartment located under the membrane can be treated directly, without waiting for “the end” of the fermentation. In particular, this collection liquid containing the compound of interest can be directly conveyed to an additional purification compartment, coupled to the FMS device as shown in [Fig.l]. Thus, the purification steps of the collected compound of interest can take place continuously, like the FMS method.

[0099] According to one implementation of the method of the invention, the compound of interest is collected in the compartment located under the membrane, then transferred to another compartment continuously.

[0100] This other compartment, attached to the system, may in particular contain means for purifying and / or concentrating the compound of interest. This compartment may advantageously be at a temperature lower than ambient temperature, for optimal conservation of fragile compounds (ideally, between 2°C and 8°C).

[0101] This process for producing at least one compound of interest by fermentation in the state solid-state (FMS) can be implemented for the production of a single compound or for the production of multiple compounds of interest during the same culture of microorganisms.

[0102] The main advantages of this method are as follows: i. after collecting the compound(s) of interest, they are dissolved in a solution (aqueous or not) which does not contain any microorganisms, and is therefore sterile, and which is also free from residues of the solid substrate used; ii. the compound(s) of interest are collected continuously, which allows them to undergo subsequent treatments (purification, concentration) directly after their collection, without latency. EXAMPLES

[0103] The examples presented below are for purely illustrative purposes, and in no way limit the subject of the claimed invention. Materials and Methods Substrate treatment:

[0104] Four substrates were used: coffee grounds, hard bread, corn cobs, and potato peelings. These substrates were ground into powder or chips before being autoclaved. The coffee grounds were not ground into powder. The corn cob was ground into coarse chips using a blender to approximately 0.1 and 0.5 cm2. The hard bread was ground into powder (comprising particles of approximately 50 pm2) using a blender. The potato peelings were ground into chips of approximately 1 cm2.

[0105] Method of the invention for the growth of microorganisms and the collection of secreted metabolites:

[0106] The microorganism and the solid substrate are deposited on a PES membrane with a pore diameter of approximately 0.20 pm + / - 0.02. This membrane does not allow the passage of the microorganism or the substrate into the lower compartment. The pore size of the filter also makes it possible to maintain a sterile environment in the lower compartment. The addition of liquid in the upper part will humidify the substrate in order to improve the growth of the microorganism, but will also allow the soluble molecules in this liquid to pass from the upper compartment to the lower compartment.

[0107] Depending on the needs of the microorganisms, the agitation of the entire device will make it possible to oxygenate the environment in which the microorganism grows, allowing it in certain cases better growth.

[0108] Recycling of the liquid is possible between the lower compartment and the com upper compartment, this allows to reduce the quantity of liquid used, but also to concentrate the secreted molecules.

[0109] However, it is preferable to avoid this step when the compound of interest is fragile. In the case of a compound sensitive to degradation, the collected liquid can be directly used for the following purification steps, thus the purification can take place continuously.

[0110] Example 1, Production of pigments by microorganisms present in bread:

[0111] 7.5g + / - 0.5g of bread were added onto a 50mm diameter membrane. The The bread was previously moistened with 20 mL of water, giving a bread / H2O ratio of between 0.35 and 0.4 g / mL. The addition of water accelerates the growth of microorganisms at the start of fermentation. The devices were placed at 30°C.

[0112] 5mL of water was added every 24h. Pigment production was evaluated by comparing the absorbance spectrum of the solution produced during 4 days of culture with sterilization and without sterilization.

[0113] The results are presented in [Fig.4].

[0114] Microorganisms present in bread are known to produce brown pigments. Absorbance is higher in samples where microorganisms are present, showing that water has drained the pigments into the lower compartment.

[0115] Example 2, Production of endogenous proteins and var AsoersUlus Ni acids bound on different substrates:

[0116] ImL of Aspergillus Niger spore solution was inoculated into a quantity of bread, coffee grounds, corn cob, or potato peelings of between 25g and 15g for a 75mm diameter PES membrane, and between 5 and 10g for a 50mm diameter PES membrane.

[0117] The devices were placed at 30°C.

[0118] A quantity of water between 10 and 5mL of H2O was added every 24h.

[0119] The amount of protein present in the collection compartment was assessed daily by taking 1 1 / 2 L of liquid from this compartment and comparing the absorbance at 280 nm of a system with Aspergillus Niger to a system without As-pergillus Niger.

[0120] The amount of acid present in the collection compartment was evaluated after 5 days of culture by measuring the pH of the compartment.

[0121] The permeability of the substrate will influence the recovery of proteins in the first days of culture. Indeed, certain substrates, and in particular bread, tend to absorb water, which will slow down its passage into the recovery chamber. This is why no protein could be collected during the first two days days of cultivation. However, it is observed that prior saturation of the substrate with water allows for faster recovery of liquid and improves the growth of the microorganism. Then the corn husks and potato peelings are cut more coarsely, into particles between 0.1 and 1 cm2 in size, which allows them to better allow the liquid to pass through.

[0122] A decrease in the amount of protein is observed when the microorganism grows on corn husk, this can be explained by the dilution of the proteins in the collection chamber. In fact, the liquid remained in the collection chamber throughout the experiment, so if the amount of protein produced decreases during growth, the volume of water increases, which dilutes the proteins and therefore reduces the absorbance. The growth of the microorganism can also be improved by supplementing the liquid with nutrients, trace elements or sugars.

[0123] The results are presented in [Fig.2A] and [Fig.2B].

[0124] Example 3, Secretion of endogenous proteins by Aspergillus niger on different substrates:

[0125] The method according to the invention makes it possible to recover the proteins that are secreted continuously. Since the lower compartment containing the secreted molecules is accessible, it is possible to collect the proteins for analysis while allowing the microorganism to grow and produce new proteins, or to modify the lower compartment by adding sensors to directly carry out measurements on the composition of the liquid in this compartment.

[0126] In order to evaluate the impact of continuous production, but especially of continuous protein recovery, all of the liquid present in the collection compartment was recovered every 24 hours and the quantity of protein produced was evaluated by measuring the absorbance at 280nm and taking into account the volume of liquid recovered, which is why the absorbance was multiplied by the volume of liquid recovered.

[0127] [Fig.3A] shows that the amount of protein produced by the microorganism on coffee grounds is quite similar across the different recovery days, while the amount produced by the microorganism on hard bread increases between day 4 and day 5, as illustrated in [Fig.3B].

[0128] Example 4, Production of acids and proteins by ZLco / / ':

[0129] An E.coli preculture was carried out overnight in 25mL of LB nutrient medium. 1mL of preculture was added to 10mg + / - 0.5g of potato peelings on a 50mm diameter membrane. 25mL of water was added and the sample was stirred at 120rpm at 37°C to facilitate the descent of the liquid into the lower compartment as well as the oxygenation of the microorganisms.

[0130] As soon as all the liquid reaches the lower compartment, the liquid from the com lower compartment can be moved into the upper compartment to concentrate the acids and proteins produced.

[0131] Acid production was assessed by measuring the pH of the liquid after 28 hours of culture and comparing this value to that of a culture without bacteria. Protein production was assessed by comparing the absorbance at 280nm of a culture without bacteria to that of a culture with bacteria. The results are shown in Figures 5A and 5B.

[0132] Unlike fungi, E.coli is less known to grow on solid substrates, which is why this bacterium requires a greater quantity of water to allow its growth. Thus, by increasing the quantity of water used and allowing good oxygenation of the medium by stirring, we were able to produce acids and proteins by implementing the method according to the invention. Results:

[0133] Solid-state fermentation offers several advantages such as ease of operation, reduced energy requirements and high productivity (Silveira et al., 2006). However, the recovery of compounds secreted during solid-state fermentation is problematic. Indeed, the compounds remain in the substrate and must be extracted by adding a liquid in which the molecules are soluble. Then, the microorganisms and the substrate must be separated from the molecules produced. Most often, this step is carried out at the end of the culture by a filtration or centrifugation step which will leave particles in the substrate, making a second pre-purification step necessary before passing the liquid through a chromatography column.The method according to the invention makes it possible to recover the secreted molecules by completely separating the microorganism from the molecules it has produced, and to have a liquid free of particles which can be used in chromatography.

[0134] Furthermore, the fact that the compounds of interest produced are collected as they elut is advantageous, because this avoids possible degradation. For example, proteins produced and secreted by fungi are likely to be degraded by proteases produced by these same fungi; according to the method of the invention, the proteins are separated from the microorganisms having produced them and collected quickly after their production. BIBLIOGRAPHICAL REFERENCES PATENTS

[0135] WO 00 / 29544

[0136] FR 2 960 551 SCIENTIFIC ARTICLES

[0137] Huang YL, Wu Z, Zhang L, Cheung CM, Yang ST. Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor. Bioresour Technol. 2002 Mar;82(l):51-9. doi: 10.1016 / s0960-8524(01)00151-l.

[0138] El-Naggar MY, El-Assar SA, Abdul-Gawad SM. Solid-state fermentation for the production of meroparamycin by streptomyces sp. strain MAR01. J Microbiol Biotechnol. 2009 May;19(5):468-73. doi: 10.4014 / jmb.0807.457.

[0139] Silveira ST, Oliveira MS, Costa JA, Kalil SJ. Optimization of glucoamylase production by Aspergillus niger in solid-state fermentation. Appl Biochem Biotechnol. 2006 Feb; 128(2): 131-40. doi: 10.1385 / abab:128:2:131.

Claims

Claims

1. A method for producing at least one compound of interest by solid-state fermentation, comprising the following steps: - cultivation of microorganisms on a solid substrate, said substrate being placed on a membrane permeable to liquids but not permeable to the microorganisms or to the substrate, - elution of at least one compound of interest by distribution of a liquid on the upper surface of said solid substrate, - collection of the liquid containing at least one compound of interest in a compartment located under the permeable membrane, characterized in that the method is carried out continuously.

2. Production method according to claim 1, characterized in that the solid substrate is composed of one or more food, agricultural or industrial co-product(s).

3. Production method according to one of claims 1 to 2, characterized in that the permeable membrane is made of a material chosen from the following materials: polyethersulfone (PES), cellulose, nylon, polycarbonate, cellulose nitrate, polypropylene, polytetrafluoroethylene and polyamide.

4. Production method according to one of claims 1 to 3, characterized in that the microorganisms present on the substrate or inoculated are fungi, microalgae, yeasts, archaea or bacteria.

5. Production method according to claim 4, characterized in that the microorganisms are genetically modified microorganisms to induce or optimize their capacity to produce said compound of interest.

6. Production method according to one of claims 1 to 5, characterized in that the compound of interest produced is a metabolite secreted by the microorganisms.

7. Production method according to one of claims 1 to 6, characterized in that the culture of the microorganisms is carried out with stirring.

8. Production method according to one of claims 1 to 7, characterized in that the liquid is water, a buffer solution, a nutrient solution or an organic solvent.

9. Production method according to one of claims 1 to 8, characterized in that the liquid is distributed in a proportion of 0.5 to 500 ml of liquid, for 1 to 100 grams of solid substrate, per 24 hours.

10. Production method according to one of claims 1 to 9, characterized in that the at least one compound of interest is collected in the compartment located under the membrane, then transferred to another compartment continuously.

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