Method for injection of co2 and removal of o2 from a bioreactor

Abstract

The present invention relates to a method for culturing microorganisms in a bioreactor, the culture medium in the bioreactor comprising carbon dioxide (CO2) as a carbon source. The CO2 feed in the bioreactor is controlled by a membrane contactor.

Description

Technical Field

[0001] The present invention relates to a method for cultivating microorganisms in a bioreactor, wherein the culture medium contains carbon dioxide (CO2) as a carbon source. A membrane contactor is used to regulate the CO2 supply in the bioreactor. Background Art

[0002] Methods for cultivating microorganisms are well known to those skilled in the art. Cultivation can be carried out in a laboratory in a petri dish or in a bioreactor at an industrial scale. Cultivation of microorganisms requires the reproduction of the conditions of the original environment to achieve optimal growth. Therefore, the presence of an organic substrate is necessary. Other parameters such as light, temperature, pressure, humidity, pH and gas concentration must also be reproduced.

[0003] Many microorganisms use carbon dioxide as a carbon source. In particular, autotrophic cultures do not include any carbon source other than the CO2 contained in the air. CO2 is converted into molecular oxygen (O2) and eliminated in the atmosphere. However, it has been found that excessive concentrations of CO2 lead to acidification of the culture medium and a decrease in pH. At the same time, excess O2 in the culture medium leads to reduced microbial growth and promotes the growth of bacteria. Therefore, the regulation of CO2 and O2 concentrations in microbial culture media is essential for their healthy growth.

[0004] There are many devices for regulating the flow of CO2 and O2, such as membranes (YSLai et al., Algal Research 2020, 52, 102098; US2021 / 053012), membrane bubblers and bubble towers, ceramic bubblers, and even bubble diffusers. Despite the fact that these methods present good transfer rates, material losses are still observed. Moreover, these devices often lead to the phenomenon of agglomeration on the walls of the bioreactor, blades, lighting devices or bubblers, which significantly leads to the obstruction of the air / gas inlet and the reduction of the light supply.

[0005] Furthermore, when desorption of O2 is performed via staged agitation (sequence of large bubbles, gentle mechanical agitation with a banana-shaped blade), it is necessary to send a strong gas flow (98% air) with a small amount of CO2 to obtain an appropriate dissolved CO2 concentration, which exacerbates the shear constraints in the culture medium.

[0006] It is desirable to avoid these phenomena which damage the growth of the strains.Therefore, there is a need to develop a method for cultivating microorganisms in which the flows of CO2 and O2 are optimally regulated and in which the problems associated with the use of existing devices are minimal or even absent.

[0007] In view of the environmental problems associated with the production of CO2 released into the atmosphere, in particular with the production of CO2-rich gases such as gaseous industrial effluents, it has been envisaged to use these gases to supply bioreactors for the growth of microorganisms that can grow with CO2 as the sole carbon source. The decontamination of such gases (also known as decarbonation) is described in particular in patent applications WO 2018 / 060197, WO 2019 / 101899 and WO 2021 / 165621. Although these methods show good levels of efficacy in terms of consuming the CO2 provided by the gas to be decarbonated, they still need to be improved to avoid some of the unconsumed CO2 being released into the atmosphere.

[0008] Therefore, the inventors have developed a method for culturing microorganisms comprising CO 2 as a carbon source, the method comprising the step of culturing the microorganisms in a bioreactor, thereby making it possible to improve the CO 2 supply, in particular for the decarbonation of industrial gases. Summary of the invention

[0009] The present invention relates to a method for culturing microorganisms containing CO2 as a carbon source, the method comprising the step of culturing the microorganisms in a bioreactor comprising a CO2 supply device, and is characterized in that the CO2 supply device comprises a membrane contactor to regulate the flow of CO2 and O2 in the culture medium, wherein the flow of CO2 is directed to the culture medium and the flow of O2 is directed to the outside of the culture medium.

[0010] Within the framework of the present invention, the microorganisms are primarily bacteria, yeasts and protists.

[0011] The culture method advantageously comprises the following steps:

[0012] (a) cultivating the microorganism; and

[0013] (b) recovering the biomass produced by the microorganism.

[0014] The present invention also relates to the use of a membrane contactor for regulating the flow of CO2 and O2 in a method for culturing microorganisms, wherein the CO2 is a carbon source in the culture medium.

[0015] The cultivation method according to the invention is particularly suitable for the decarbonation of CO2-rich gases.

[0016] The present invention also relates to a method for decarbonating a CO2-rich gas, the method comprising injecting the gas into a bioreactor, the bioreactor comprising a culture medium and microorganisms capable of growing in the culture medium using CO2 as the sole carbon source, and a CO2 supply device, characterized in that the CO2 supply device comprises a membrane contactor to regulate the flow of CO2 and O2 in the culture medium, wherein the flow of CO2 is directed to the culture medium and the flow of O2 is directed to the outside of the culture medium.

[0017] Finally, the present invention relates to a microalgae fermentation device, which includes a fermentation reactor, a CO2 supply device and a lighting device when applicable, characterized in that the CO2 supply device includes a membrane contactor for regulating the flow of CO2 and O2, wherein the flow of CO2 is directed to the culture medium and the flow of O2 is directed to the outside of the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A method of culturing microorganisms in the presence of a membrane contactor.

[0019] Figure 2 The membrane contactor according to the present invention ( Figure 2 A) and the cross section of the transfer membrane of the membrane contactor ( Figure 2 B).

[0020] Figure 3A and Figure 3B represents the phenomena occurring in the transfer membrane of the membrane contactor according to the present invention, especially diffusion ( Figure 3A ) and the flow of carbon dioxide (CO2), molecular oxygen (O2) and culture medium ( Figure 3B ) phenomenon.

[0021] Figure 4 Indicates the biomass productivity based on the measurement of 0.2kg / m 3 / J, velocity of culture medium in membrane contactor.

[0022] Figure 5 The measured biomass productivity is 0.79 kg / m 3 / J, velocity of culture medium in membrane contactor.

[0023] Figure 6 Represents the change in CO2 pressure in the transfer membrane of the membrane contactor depending on the amount of CO2 in the culture medium.

[0024] Figure 7 Represents the amount of CO2 to be injected into the culture medium depending on the temperature of the culture medium and the pressure in the transfer membrane of the membrane contactor.

[0025] Figure 8is a schematic diagram of a membrane contactor according to one embodiment of the present invention.

[0026] Fig. 9 is a graph showing the change in O2 pressure (PO2) in the culture medium over time.

[0027] Fig.10 is a graph presenting optical density (OD) results and daily productivity (based on OD) of Chlorella sorokiniana cultured in a bioreactor equipped with a membrane contactor in accordance with the present invention compared to the results obtained with Chlorella sorokiniana cultured in a control bioreactor that did not include a membrane contactor. DETAILED DESCRIPTION

[0028] The present invention relates to a method for culturing microorganisms in a culture medium containing carbon dioxide (CO2) as a carbon source, the method comprising the step of culturing the microorganisms in a bioreactor comprising a CO2 supply device, characterized in that the CO2 supply device comprises a membrane contactor for regulating the flow of CO2 and molecular oxygen (O2) in the culture medium, and wherein the flow of CO2 is directed to the culture medium and the flow of O2 is directed to the outside of the culture medium.

[0029] Methods of culturing microorganisms, whether heterotrophic, autotrophic or mixotrophic, are well known to those skilled in the art.

[0030] The cultivation of microorganisms involves many parameters, such as the properties and composition of the culture medium itself, as well as external factors that must be controlled and adjusted throughout the process, such as light, temperature, pressure, humidity, pH, gas concentration in the reactor, etc.

[0031] The culture media essential for the growth of microorganisms are well known to those skilled in the art, who will be able to determine and adjust the amount and nature of substrates used for microbial culture, such as sources of carbon, nitrogen, oxygen, phosphorus, etc.; minerals such as calcium, iron, magnesium, potassium, sodium, sulfur, etc.; and trace elements (boron, zinc, copper, cobalt, etc.) according to the one or more microorganisms to be cultured, the size of the reactor, the amount of biomass required, etc.

[0032] The present invention is particularly suitable for methods of cultivation in bioreactors. In the framework of the present invention, a bioreactor is understood to mean a reactor in which biological phenomena develop in highly variable aspects (such as the treatment of effluents or the production of biomass containing molecules of interest), such as the growth of pure cultures or microorganisms or microbial consortia.

[0033] Bioreactors are well known to those skilled in the art and generally comprise a closed tank in which a stirring or agitation element is provided to promote homogenization of the contents of the tank. The contents of the tank may also be stirred or agitated via a liquid pump which causes a circulation flow of the culture medium within the tank.

[0034] The present invention is particularly suitable for cultivation in autotrophic mode, said cultivation comprising carbon dioxide (CO2) as a carbon source and a light source.The cultivation method then also includes an illumination phase.

[0035] The bioreactor may also include lighting means, wherein a light supply may be provided continuously or cyclically (WO 2019 / 034792, WO 2021 / 160776). It should be understood that within the framework of the present invention, a person skilled in the art knows how to adjust the agitation speed and the amount of light to obtain optimal culture conditions.

[0036] Bioreactors also typically include air inlets and outlets to enable gas exchange between the exterior and interior of the reactor.

[0037] Thus, the air inlet can be coupled to a device such as a pump so that gas is injected into the culture medium. The injected gas is selected from gases necessary for the growth of the microorganisms cultured in the bioreactor. They are especially carbon dioxide (CO2), carbon monoxide (CO), molecular oxygen (O2) or molecular nitrogen (N2) or mixtures thereof. In a preferred embodiment, the gas injected into the culture medium is carbon dioxide.

[0038] The carbon dioxide injected into the reactor is supplied via the ambient gas stream or via an external source such as industrial gas, fermentation process or gas cylinders.

[0039] The CO2 is thus dissolved in the culture medium and consumed by the microorganisms, producing O2. This O2 is eliminated via the air outlet of the bioreactor.

[0040] It should be noted that the reactor may be equipped with other devices or equipment for controlling and regulating the reactor and the growth of the microorganisms. These may in particular be sensors or probes for measuring different cultivation parameters, such as temperature, pH, pressure when entering and leaving the reactor, etc.

[0041] According to the present invention, in order to regulate the gas flow rate, especially the flow of CO2 within the bioreactor, one or more membrane contactors are used.

[0042] The membrane contactor is a device comprising at least two fluid circulation loops separated by a transfer membrane. The fluid circulating in the membrane contactor is also referred to as a circulating fluid. Therefore, the membrane contactor comprises a first circulation loop of a first circulating fluid (referred to as a CO2 supply fluid) supplying carbon dioxide (CO2) in contact with a first side of the transfer membrane, and a second loop, wherein the second loop is used for the circulation of a second circulating fluid (referred to as a CO2 receiving fluid) in contact with a second side of the transfer membrane for receiving CO2. Each fluid circulation loop is independent of each other and can be closed or open.

[0043] In one embodiment, the circulation loop of the CO2 receiving fluid is a closed loop. In another embodiment, the circulation loop of the CO2 receiving fluid and the circulation loop of the CO2 supply fluid are closed loops to ensure optimal conditions for the growth of microorganisms. Figure 1 , the CO2 receiving fluid loops (3) and (4) and the CO2 supply fluid loops (6) and (7) are closed loops, in which the exchange between the two fluids occurs in the membrane contactor (2).

[0044] The CO2 supply fluid is in the form of a gas, preferably an air / CO2 mixture containing at least 5% CO2, even at least 25% CO2, further preferably at least 50% CO2, and even further preferably at least 75% CO2. Advantageously, the supply fluid has a gaseous CO2 content of 100% CO2.

[0045] The CO2 receiving fluid is preferably in liquid form according to the invention. This is more specifically a culture medium. In this case, the CO2 receiving fluid contains the microorganisms and the nutrients necessary for their growth.

[0046] Thus, according to the invention, a membrane contactor makes it possible to ensure the exchange of CO 2 molecules between a CO 2 supply fluid, preferably in gaseous form, and a CO 2 receiving fluid, which is a culture medium, via a membrane, known as a transfer membrane.

[0047] Within the membrane contactor, two fluids, namely, CO2 supply fluid and CO2 receiving fluid, are in circulation.

[0048] The circulation of the CO2 supply fluid can be ensured by all means known to the person skilled in the art, in particular by using a pressurized CO2 source or by using a gas compressor capable of ensuring a regular flow in the membrane contactor. At the same time, the circulation of the CO2 receiving fluid, i.e. the culture medium, is allowed by the rotation of the blades in the bioreactor or by pumping the culture medium which is then re-injected into the bioreactor using a pump. Preferably a vacuum pump is used.

[0049] Advantageously, the use of a membrane contactor according to the invention enables a method of injecting CO 2 without creating detrimental shear constraints in the reactor, which avoids the formation of foam in the culture.

[0050] In one embodiment, the flow of the CO2 supply fluid and the CO2 receiving fluid is parallel. In another embodiment, the flow is vertical. In yet another embodiment, the flow is crossed. Preferably, the flow of the CO2 supply fluid and the CO2 receiving fluid is crossed, i.e., the CO2 supply fluid and the CO2 receiving fluid circulate in opposite directions.

[0051] refer to Figure 2 The membrane contactor (1) then comprises an inlet (20) or (21) and an outlet (20) or (21) for a first circulating fluid, and an inlet (22) or (23) and an outlet (22) or (23) for a second circulating fluid. The two fluids are separated by a transfer membrane (30). The membrane contactor then also comprises two circulation loops: a first circulation loop of the first circulating fluid in contact with the first side of the membrane, and a second circulation loop of the second circulating fluid in contact with the second side of the membrane.

[0052] The membrane thus makes it possible to promote contact between two circulating fluids without the two circulating fluids having direct physical contact.The properties of the membrane determine the nature and characteristics of the exchange between the two circulating fluids.

[0053] Membranes can be porous or non-porous (also called dense membranes or pervaporation membranes). Dense membranes are membranes that have no porosity created. They are divided into two categories: hydrophilic membranes and organophilic membranes.

[0054] Hydrophilic membranes allow water to pass through, and therefore, for example, achieve solvent dehydration. Typically, these membranes are made of polymeric materials (polyvinyl alcohol) deposited on a porous medium. For example, their geometrical characteristics are identical to those of ultrafiltration membranes. These membranes are mainly implemented in flat modules, with the "permeate" compartment in contact with a partial vacuum chamber to maintain agitation and discharge the separated product.

[0055] Organophilic membranes are mainly silicone-based (PDMS polydimethylsiloxane), and in this case it is the affinity of the silicone material for one or more compounds of the mixture that enables the selective transfer across the membrane. Very high selectivities are thus obtained, while the transfer flux generally remains weak.

[0056] Membrane contactors are characterized by the transfer membranes that comprise them, and more specifically, by the geometry and structure of those membranes.

[0057] The membranes may be flat, spiral, tubular, or in a hollow fiber geometry (ie, pipe or tube).

[0058] According to a preferred embodiment, the membrane (s) has a hollow fiber geometry. Advantageously, the membrane contactor comprises several fibers dispersed in a tube bundle. The outer diameter of the membrane is greater than 50 μm, preferably greater than 60 μm, and even more preferably greater than 70 μm. In another embodiment, the outer diameter of the membrane with a hollow fiber configuration is at least 100 μm. In another embodiment, the outer diameter of the membrane is less than 2 mm, preferably less than 1.5 mm, and further preferably less than 1 mm. Advantageously, the outer diameter of the membrane with a hollow fiber configuration is between 100 μm and 1 mm. The inner diameter of the membrane according to the present invention is preferably less than 900 μm, or even less than 850 μm. In a preferred embodiment, the outer diameter of the membrane with a hollow fiber configuration is between 100 μm and 800 μm.

[0059] The internal diameter of the membrane with hollow fiber configuration is generally less than 1mm, preferably less than 900μm, less than 800μm, less than 700μm, less than 600μm, less than 500μm, less than 400μm, less than 300μm, less than 200μm, even less than 100μm. In one embodiment, the internal diameter of the membrane with hollow fiber configuration according to the present invention is between 1μm and 100μm. In another embodiment, the internal diameter is between 5μm and 100μm, preferably between 7μm and 100μm, even further preferably between 10μm and 100μm. Advantageously, the internal diameter of the membrane with hollow fiber configuration is between 20μm and 90μm.

[0060] refer to Figure 2 A and Figure 8 , and according to one embodiment, the membrane contactor comprises a transfer membrane in a hollow fiber configuration, the hollow fibers being dispersed in the form of a bundle. Figure 2 B, the membrane (30) is a hollow fiber (or tube) configuration having an inner diameter and an outer diameter. Therefore, there is an inner side (31) and an outer side (32) of the membrane, corresponding to the inner side of the fiber (or tube) and the outer side of the fiber (or tube), respectively.

[0061] In one embodiment, the CO2 supply fluid circulates inside the fibers constituting the membrane and is therefore in contact with the inner side of the membrane, while the CO2 receiving fluid is in contact with the outer side of the membrane. In a second preferred embodiment, the CO2 supply fluid is in contact with the outer side of the membrane and the CO2 receiving fluid is inside the fibers and in contact with the inner side of the membrane.

[0062] Within the framework of the present application, the transfer membrane having a hollow fiber configuration is a microporous membrane or a nonporous membrane.

[0063] In one embodiment, the transfer membrane of the membrane contactor is a porous membrane and includes pores with a diameter of less than 10 μm. Advantageously, the porous membrane is microporous and exhibits pores with a diameter of less than 8 μm, preferably less than 5 μm, even less than 3 μm, and further preferably less than 2 μm. In another embodiment, the microporous membrane includes pores with a diameter exceeding 0.001 μm, 0.002 μm, 0.003 μm, 0.004 μm, or even exceeding 0.005 μm. Advantageously, the diameter of the pores of the membrane is between 0.005 μm and 2 μm. In one embodiment, the diameter of the pores is less than 1 μm, even less than 0.5 μm, and preferably less than 0.2 μm, or even less than 0.1 μm. In another embodiment, the diameter of the pores of the membrane is between 0.005 μm and 0.1 μm. Furthermore, in another embodiment, the diameter of the pores is between 0.008 μm and 0.09 μm, preferably between 0.009 μm and 0.07 μm, even preferably between 0.01 μm and 0.06 μm, and further preferably between 0.01 μm and 0.05 μm. Advantageously, the diameter of the pores is about 0.01 μm, 0.02 μm, 0.03 μm, 0.04 μm or 0.05 μm.

[0064] In a preferred embodiment, the transfer membrane of the membrane contactor is a dense membrane. Then, the molecular diffusion is characterized by the phenomenon of diffusion across the membrane via adsorption of molecules with a strong affinity to the membrane. Relative to the porous membrane with potential adhesion areas in its structure, this embodiment presents the advantage of reducing or even preventing the membrane of the membrane contactor from being blocked by different elements (such as cell debris) carried in the liquid culture medium, and thus makes it possible to maintain the same performance of gas exchange through the membrane throughout the duration of the culture method.

[0065] According to one embodiment, the transfer membrane is a non-porous membrane having an inner diameter of 150 μm to 450 μm and an outer diameter of 250 μm to 550 μm.

[0066] According to one embodiment, the transfer membrane is a porous membrane having an inner diameter of 150 to 250 μm and an outer diameter of 250 to 350 μm and comprising pores having a diameter of 20 to 40 μm.

[0067] According to another embodiment, the transfer membrane is a dense membrane having an inner diameter of 350 to 450 μm and an outer diameter of 5 to 100 mm.

[0068] Advantageously, the porous or dense, in particular dense, transfer film has a thickness of less than 1,000 μm, preferably from 100 μm to 1,000 μm.

[0069] The use of a membrane contactor according to the invention makes it possible in particular to ensure all or some exchanges between the two fluids in circulation via the presence of a transfer membrane as defined above.The membrane contactor may also feature an exchange surface between the two fluids in circulation.

[0070] According to one embodiment of the present invention, the membrane contactor exhibits a 2 / l, preferably at least 0.006m 2 / l of CO2 receiving fluid. At the same time, the exchange surface between the two fluids in the cycle is less than or equal to 0.020m per liter of CO2 receiving fluid. 2 / l, preferably less than or equal to 0.017, even less than or equal to 0.015, and preferably less than or equal to 0.012m 2 / l CO2 receiving fluid. In another embodiment, the membrane contactor exhibits a 2 / l and 0.012m 2 / l CO2 receiving fluid, preferably at 0.006m 2 / l and 0.012m 2 / l exchange surface between CO2 receiving fluid.

[0071] The materials used to produce the transfer membrane are generally polymers, such as polyethylene (PE), polypropylene (PP), polysulfone (PSu), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyimide (PI), polyamide (PA), polycarbonate (PC), polyacrylonitrile (PAN) and cellulose acetate (CA). The transfer membrane can also be made of silicone or silicone derivatives. Within the framework of the present application, the term silicone includes inorganic compounds formed by chains of silicon atoms and oxygen atoms and silicon atoms that may carry groups. Particular mention should be made of polymers belonging to the siloxane family, and more specifically polydimethylsiloxane (PDMS) or compounds comprising polydimethylsiloxane (PDMS), especially a ternary system consisting of PDMS, water and tetrahydrofuran (THF). Advantageously, the transfer membrane of the present application is made of silicone, preferably PDMS, and in particular, the membrane is a thin film of PDMS in sheet or roll form with a thickness between 0.01 cm and 1.00 cm. Examples of commercial transfer membranes are from SSP-M823 thin silicone membrane or Saint-Gobain PDMS. Such membrane contactors and their operating principles are known to those skilled in the art for other applications. They are commercially available, especially under reference Pervélys and 3M TM liqui-Cel.

[0072] A membrane contactor is thus defined as a system comprising one or more transfer membranes and whose purpose is to facilitate contact between two fluids in circulation without direct contact between the two fluids in circulation.

[0073] The use of a membrane contactor according to the invention makes it possible in particular to ensure all or some of the ion or gas exchanges necessary for optimal growth of the organisms present in the culture medium in the bioreactor.

[0074] refer to Figure 3A and Figure 3B At the transfer membrane (30) of the membrane contactor, substances are transferred according to the pressure or concentration gradient between the two circulating fluids, the CO2 supply fluid and the CO2 receiving fluid. CO2 molecules present in the CO2 supply fluid pass through the transfer membrane of the membrane contactor and are consumed by the microorganisms and converted into O2 molecules in the CO2 receiving fluid. The characteristics of the membrane and the pressure and concentration differences between the two sides of the membrane mean that O2 molecules pass through the membrane and are found in the CO2 supply fluid and are then eliminated from the CO2 supply fluid by techniques well known to those skilled in the art.

[0075] The use of a membrane contactor according to the invention makes it possible in particular to ensure all or some of the ion or gas exchanges necessary for optimal growth of the organisms present in the culture medium in the bioreactor; the membrane contactor is said to be a device coupled to the bioreactor and separate therefrom. Thus, the growth of the organisms takes place in the bioreactor and not in the membrane contactor to which it is coupled and in which the culture medium circulates.

[0076] Preferably, the membrane contactor exhibits dimensions that are smaller than the dimensions of the bioreactor to which it is coupled. The coupling of a membrane contactor to a bioreactor has several advantages, in particular an improvement in gas exchange. In fact, when using a membrane contactor, CO2 is supplied by osmosis and is therefore completely dissolved and evenly distributed in the culture medium, while supplying CO2 by bubbling results in the transfer of gas only at the surface of the bubbles when the gas enters the culture medium.

[0077] In addition, unlike CO2 bubbling, when using a membrane contactor, there is no limitation on the desorption of CO2 or the gas / liquid exchange surface due to the effect of bubbles agglomerating together.

[0078] Finally, the use of membrane contactors makes it possible to improve ion and gas exchange and, therefore, CO2 consumption in the culture medium due to the circulation of the culture medium. This circulation is achieved by the rotation of blades in the bioreactor or by pumping the culture medium from the bioreactor to the membrane contactor. The culture medium is then re-injected from the membrane contactor into the bioreactor.

[0079] exist Figure 2 A and Figure 8In one embodiment shown in , the membrane contactor comprises a transfer membrane in a hollow fiber configuration dispersed in the form of a fiber (or tube) bundle. The membrane contactor is thus divided into two spaces where a CO2 supply fluid and a CO2 receiving fluid circulate, the two spaces being separated by a transfer membrane (in this case, a membrane in a hollow fiber configuration).

[0080] exist Figure 2 In one embodiment shown in A, the CO2 supply fluid circulates in a first space (311) corresponding to the interior of the hollow fibers, wherein the fluid circulates according to the flow between the inlet (20) or (21) and the outlet (20) or (21) of the hollow fiber bundle. According to this embodiment, the CO2 receiving fluid circulates in a second space (321) defined by the waterproof wall of the membrane contactor, wherein the fluid circulates according to the flow between the inlet (22) or (23) and the outlet (22) or (23) of the membrane contactor.

[0081] exist Figure 8 In the second preferred embodiment shown in , the membrane contactor includes a transfer membrane in a hollow fiber configuration dispersed in the form of a fiber (or tube) bundle. According to this preferred embodiment, the CO2 supply fluid circulates in a first space (321) defined by a waterproof wall of the membrane contactor, wherein the fluid circulates according to a flow applied between an inlet (22) or (23) and an outlet (22) or (23) of the membrane contactor. At the same time, the CO2 receiving fluid is present in a second space (311) defined by the membrane (inside the hollow fiber) and circulates according to a flow between an inlet (20) or (21) and an outlet (20) or (21) of the hollow fiber bundle.

[0082] The layout of this embodiment has the advantage of limiting clogging phenomena which may occur when a CO2 receiving fluid circulates outside the hollow fibers and a CO2 supply fluid circulates inside the fibers, in particular when the CO2 receiving fluid is culture medium and the CO2 supply fluid is gaseous CO2.

[0083] Preferably, the membrane contactor is placed outside the bioreactor. The membrane contactor can also be placed inside the bioreactor. If necessary, the membrane contactor can be placed in a waterproof box, which can then be placed inside or outside the bioreactor.

[0084] Membrane contactors enable efficient and uniform supply of CO2 to all microorganisms in a cultivation process, due to the phenomenon of diffusion across the transfer membrane. These create a physical separation between the circulating fluids, especially between the gas phase of the CO2 supply and the liquid culture medium CO2 receiver, allowing only some molecules to pass through the membrane.

[0085] In one embodiment, the gas molecule pressure or concentration gradient is caused by an overpressure of CO2 concentration. A person skilled in the art will know how to determine the CO2 flow at the inlet of the membrane contactor to be applied to generate this overpressure, especially depending on the bioreactor and the amount of biomass required, without affecting the cultivation of the microorganisms.

[0086] In another embodiment, CO2 is injected at a regular flow rate according to the CO2 consumption required for the growth of the microorganisms. The consumed CO2 is then converted into O2 molecules that diffuse across the transfer membrane and cause an increase in pressure in the gas phase of the bioreactor. The O2 concentration threshold can be determined by the operator to arrange for the release of O2 molecules to the outside of the membrane contactor and / or bioreactor via the air outlet.

[0087] In another embodiment, the CO2 concentration threshold value may be determined by the operator to arrange that the CO2 supply fluid is released to the outside of the membrane contactor only when the CO2 concentration of the supply fluid drops below the threshold value, so that the CO2 supply fluid source can be replaced with a CO2 supply fluid source having a CO2 concentration greater than the threshold value. In another embodiment, the concentration gradient is caused by the lack of O2 molecules in the gas phase. The absence or low concentration of O2 molecules can be achieved in particular using a pump, preferably a vacuum pump, which eliminates O2 molecules from the membrane contactor and / or from the bioreactor.

[0088] Within the framework of the present application, it is understood that the membrane contactor and / or the bioreactor may comprise other devices and equipment that ensure the safety of the microbial culture and the operator.

[0089] Thus, the CO 2 concentration is regulated by at least one of the aforementioned means, namely via a pressure or concentration gradient caused by an overpressure of CO 2 and / or O 2 in the membrane contactor, or even a low O 2 concentration in the membrane contactor.

[0090] Preferably, the CO 2 concentration is regulated by an overpressure of CO 2 in the membrane contactor.

[0091] The pressure in the membrane contactor is monitored using a pressure gauge and regulated with a control valve.

[0092] It should be noted that the concentration of dissolved gas in the culture medium depends on the temperature of the culture medium. Therefore, the lower the temperature, the higher the concentration of dissolved gas, and vice versa.

[0093] In one embodiment, the concentration of dissolved CO2 in the culture medium is therefore between 0 and 3,500 mg / l. In another embodiment, the concentration of dissolved CO2 is at least 100 mg / l, even at least 200 mg / l, preferably at least 400 mg / l, further preferably at least 600 mg / l. In another embodiment, the concentration of dissolved CO2 in the culture medium is less than 3,400 mg / l, even less than 3,200 mg / l, preferably less than 3,000 mg / l. In another embodiment, the concentration of dissolved CO2 in the culture medium is between 600 mg / l and 3,000 mg / l, preferably between 600 mg / l and 2,500 mg / l, further preferably between 600 mg / l and 2,000 mg / l. Advantageously, the concentration of dissolved CO2 in the culture medium is between 600 mg / l and 1,500 mg / l, even between 800 mg / l and 1,500 mg / l. In a preferred embodiment, the concentration of dissolved CO2 is at least 1,000 mg / l, even at least 1,200 mg / l. In a further preferred embodiment, the concentration of dissolved CO2 is between 1,000 mg / l and 1,500 mg / l, even between 1,200 mg / l and 1,500 mg / l. In this latter embodiment, the temperature of the culture medium is preferably ambient temperature (i.e., 18°C ​​to 25°C).

[0094] At the same time, the amount of dissolved O2 in the culture medium is kept at less than 35 g / m 3 In one embodiment, the concentration of dissolved CO2 in the culture medium is less than 34 g / m 3 , even 33g / m 3 , preferably less than 32g / m 3 , even more preferably less than 31g / m 3 In another embodiment, the amount of dissolved O2 in the culture medium is at least 8 g / m 3 In a preferred embodiment, the concentration of dissolved O2 in the culture medium is 8 g / m 3 With 30g / m 3 between.

[0095] Advantageously, the CO2 concentration in the culture medium is regulated to provide the necessary and sufficient amount for the cultivation of the microorganisms, so that all the CO2 is consumed without being lost to the atmosphere. The bioreactor is equipped with a control system for this regulation.

[0096] The target value is determined and entered into the control system or each parameter that must be controlled. The control system measures the parameters that must be controlled in the culture medium. Notably, the control system measures the pH, temperature and the concentration of dissolved CO2 in the culture medium. The measurement of these parameters can be performed continuously or discontinuously.

[0097] The parameter value thus measured is then processed and compared with the target value. As the case may be, the control system indicates that there is a difference between the measured value and the target value. This indication makes it necessary to adjust the relevant parameter manually or automatically, for example by adding acid or alkaline solution to the culture medium, heating or cooling the culture medium, and / or increasing or decreasing the CO2 supply in the culture medium.

[0098] In particular, the only gas exchange between the reactor and the external environment takes place via the membrane contactor.

[0099] According to a particular embodiment of the invention, the injected CO2 originates from a gas rich in CO2 which should be treated to eliminate CO2 without releasing it into the atmosphere. In particular, the CO2 is a gaseous industrial effluent rich in CO2. In particular, the gaseous industrial effluent rich in CO2 has been pretreated to eliminate particulates and / or toxic components that may affect the cultivation of microorganisms.

[0100] It should be noted that:

[0101] - the factors that have the greatest influence on the variation of the concentration of dissolved O2 are the initial concentration of O2 in the liquid and the flow of the liquid in the membrane contactor (for a model of the cultivation of Chlorella vulgaris in a photobioreactor, see Example 1);

[0102] -The factors that most influence the change in the concentration of dissolved CO2 in the liquid are the pressure of CO2 in the CO2 supply fluid in the membrane contactor, the content of CO2 in the CO2 supply fluid and the initial concentration of dissolved CO2 in the culture medium; the temperature of the liquid, the gas flow rate, the liquid flow rate and the initial concentration of O2 in the liquid have no significant effect on the transfer rate of CO2 (for a model of Chlorella vulgaris cultivation in a photobioreactor, see Example 2).

[0103] FIG. 3 shows the flow of CO 2 and O 2 at the transfer membrane of a membrane contactor.

[0104] Referring to Figure 3, at the transfer membrane (30), an exchange phenomenon between CO2 and O2 molecules occurs. When CO2 diffuses from the inside of the membrane to the outside, O2 molecules pass through the membrane from the culture medium and are eliminated simultaneously with the excess CO2 molecules. Figure 3B Represents an embodiment in which the circulating fluids are crossed. The CO2 supplying fluid and the CO2 receiving fluid circulate in opposite directions.

[0105] Microorganisms and cultured microorganisms are well known to those skilled in the art. They are in particular bacteria, yeasts or protists, and more specifically algae and microalgae. According to a preferred embodiment, the cultured microorganism is a protist.

[0106] The term protist covers all unicellular eukaryotic microorganisms. Microalgae (green algae such as Chlorella, Senedesmus, Tetraselmis, Haematococcus; Charophyte, chrysophyte are diatoms; Nannochloropsis; Euglenophyte such as Euglena, Phacus; Rhodophyte (including Galdieria), etc.), unicellular fungi (Thraustochytrid, such as Schizochytrium, Aurantiochytrium, etc.), cyanobacteria (Anabaena, Nostoc, Microcystis, Arthrospira, Spirulina, etc.) and heterotrophic flagellates (Crypthecodinium, etc.) belong to the protist group.

[0107] In one embodiment, the microorganisms according to the invention are algae and microalgae, which preferably belong to the classes: Chlorophyceae, Rhodophyceae, Thraustochytrids and Bacillariophyceae.

[0108] If the microalgae belong to the genus Amphidinium, they may be selected from the species Amphidinium acutum, Amphidinium aloxalocium, Amphidinium asymmetricum, Amphidinium bipes, Amphidinium carbunculus, Amphidinium carterae, Amphidinium carteri, Amphidinium celestinum, Amphidinium coeruleum, Amphidinium conradi, Amphidinium corallinum, Amphidinium crassum, Amphidinium cyaneoturba, Amphidinium dubium, Amphidinium elegans, Amphidinium extensum, Amphidinium flagellans, Amphidinium flexus flexum), Amphidinium fusiforme, Amphidinium glaucum, Amphidinium globosum, Amphidiniumhoefleri, Amphidinium hyalinum, Amphidiniumincoloratum, Amphidiniuminflatum, Amphidinium kesslitzi, Amphidinium klebsii, Amphidinium lacustre, Amphidinium lanceolatum, Amphidinium latum, Amphidinium lilloense, Amphidinium longum, Amphidinium luteum, Amphidinium machapungarum, Amphidiniummacrocephalum, Amphidiniummammillatum), Amphidinium massartii, Amphidinium microcephalum, Amphidinium operculatum, Amphidinium ornithocephalum, Amphidinium ovoideum, Amphidinium ovum, Amphidinium pellucidum, Amphidinium phthartum Skuja, Amphidinium psammophilum, Amphidinium pseudogalbanum, Amphidinium purpureum, Amphidinium salinum Ruinen, Amphidinium schroederi, Amphidinium scissoides, Amphidinium sphenoides), Amphidinium steini, Amphidinium stellatum, Amphidinium subsalsum, Amphidinium tortum, Amphidinium trochodinoides, Amphidinium turbo, Amphidinium vasculum, Amphidinium vittatum, Amphidinium wislouchii.

[0109] If the microalgae belong to the genus Chlorella, they may be selected from the species C. acuminata, C. angustoellipsoidea, C. anitrata, C. antarctica, C. aureoviridis, C. autotrophica, C. botryoides, C. caldaria, C. candida, C. capsulata, C. chlorelloides, C. caldaria, C. C.cladoniae), C.coelastroides, C.colonialis, C.communis, C.conductrix, C.conglomerata, C.desiccata, C.ellipsoidea, C.elongata, C.emersonii, C.Faginea, C.fusca, C.glucotropha, C.homosphaera, C.infusionum, C.kessleri, C.Koettlitzii, C.lacustris, C.lewinii, C.lichina, C.lobophora, C.luteo-viridis, C.marina, C.miniata, C.minor, C.minutissima, C.mirabilis, C.mucosa, C.minutissima C. mutabilis, C. nocturna, C. nordstedtii, C. oblonga, C. oocystoides, C. ovalis, C. paramecii, C. parasitica, C. parva, C. peruviana, C. photophila, C. pituita, C. pringsheimii, C. protozoa.protothecoides), C.pulchelloides, C.pyrenoidosa, C.Regularis, C.reisiglii, C.reniformis, C.rotunda, C.rubescens, C.rugosa, C.saccharophila, C.salina, C.simplex, C.singularis, C.sorokin sorokiniana), C. spaerckii, C. sphaerica, C. Stigmatophora, C. subsphaerica, C. terricola, C. trebouxioides, C. vannielii, C. variabilis, C. viscosa, C. volutis, C. vulgaris, C. zopfingiensis. Advantageously, according to the invention, the algae of the genus Chlorella may be an algae chosen from the species Chlorella sorokiniana or Chlorella vulgaris. .

[0110] If the microalgae belong to the genus Euglena, they may especially be chosen from the species E. viridis, E. gracilis, E. limosa, E. globosa, E. prowsei, E. polomorpha.

[0111] If the microalgae belong to the genus Scenedesmus, they may be selected from the species S. abundans, S. aciculus, S. aculolatus, S. aculeotatus, S. acuminatus, S. acutiformis, S. acutus, S. aldavei, S. alternans, S. ambuehlii, S. anhuiensis, S. anomalus, S. antennatus, S. antillaru m, S.apicaudatus, S.Apiculatus, S.arcuatus, S.aristatus, S.armatus, S.arthrodesmiformis, S.arvernensis, S.asymmetricus, S.bacillaris, S.baculiformis, S.bajacalifornicus, S.balatonicus, S.basiliensis, Bernard S. Bernardii, S. bicaudatus, S. bicellularis, S. bidentatus, S. bijuga, S. bijugatus, S. bijugus, S. brasiliensis, S. breviaculeatus, S. breviaculespina, S. caribeanus, S. caudato-aculeolatus, S. caudatus , S.chlorelloides, S.circumfusus, S.coalitus, S.costatogranulatus, S.crassidentatus, S.curvatus, S.decorus, S.denticulatus, S.deserticola, S.diagonalis, S.dileticus, S.dimorphus, S.disciformis, S.dispar), S. distentus, S. ecornis, S. ellipsoideus, S. ellipticus, S. falcatus, S. fenestratus, S. flavescens, S. flexuosus, S. furcosus, S. fuscus, S. fusiformis, S. gracilis, S. graevenitzii, S. graneliensis S. grahneisii, S. granulatus, S. gujaratensis, S. gutwinskii, S. hanleyi, S. helveticus, S. heteroacanthus, S. hindakii, S. hirsutus, S. hortobagyi, S. houlensis, S. huangshanensis, S. hystrix, S. incrassat ulus), S. indianensis, S. indicus, S. inermis, S. insignis, S. Intermedius, S. Javanensis, S. jovais, S. jugalis, S. kerguelensis, S. kissii, S. komarekii, S. lefevrei, S. linearis, S. littoralis, S. longispinus S. longispina, S. longus, S. luna, S. lunatus, S. magnus, S. maximus, S. microspina, S. minutus, S. mirus, S. morzinensis, S. multicauda, ​​S. multiformis, S. multispina, S. multistriatus, S. naegelii, S. nanus, S.notatus, S. nygaardii, S. oahuensis, S. obliquus, S. obtusiusculus, S. obtusus, S. olvalternus, S. oocystiformis, S. opoliensis, S. ornatus, S. ovalternus, S. pannonicus, S. papillosum, S. parisiensis, S. sphenotype S.parvus, S.pecsensis, S.pectinatus, S.perforatus, S.planctonicus, S.plarydiscus, S.platydiscus, S.pleiomorphus, S.polessicus, S.polydenticulatus, S.polyglobulus, S.polyspinosus, S.praetervisus, S. prismaticus, S. producto-capitatus, S. protuberans, S. pseudoarmatus, S. pseudobernardii, S. pseudodenticulatus, S. pseudogranulatus, S. pseudohystrix, S. pyrus, S. quadrialatus, S. quadricauda, ​​S. .quadricaudata), S.quadricaudus, S.quadrispina, S.raciborskii, S.ralfsii, S.reginae, S.regularis, S.reniformis, S.rostrato-spinosus, S.rotundus, S.rubescens, S.scenedesmoides, S.schnepfii, S.schroeteri), S.securiformis, S.semicristatus, S.semipulcher, S.sempervirens, S.senilis, S.serrato-perforatus, S.serratus, S.serrulatus, S.setiferus, S.sihensis, S.smithii, S.soli, S.sooi, S.spicatus, S.spinoso-aculeolatus, S.spinosus, S.spinulatus, S.striatus., S.su bspicatus), S. tenuispina, S. terrestris, S. tetradesmiformis, S. transilvanicus, S. tricostatus, S. tropicus, S. tschudyi, S. vacuolatus, S. variabilis, S. velitaris, S. verrucosus, S. vesiculosus, S. westii, S. weberi, S. wisconsinensis, S. wuhanensis, S. wuhuensis. Advantageously, according to the invention, the algae of the genus Scenedesmus may be an algae selected from the species Scenedesmus obliquus or Scenedesmus affluensis. .

[0112] If the microalgae are diatoms, they may be selected from the following genera: Nitzschia, Navicula, Bryophyte, Phaeodactylum, Thalassiosira, and the like.

[0113] If the microalgae belong to the genus Nitzschia, they may be selected from the species N. abbreviata, N. abonuensis, N. abridia, N. accedens, N. accommodata, N. aciculariformis, N. acicularioides, N. acicularis (including all these variants), N. acidoclinata, N. actinastroides, N. actydrophila, N. acula, N. cuspula, N. acuminata) (including all such variants), N. acuta, N. adamata, N. adamatoides, N. adapta, N. adducta, N. adductoides, N. admissa, N. admissoides, N. aequalis, N. aequatorialis, N. aequora, N. aequorea, N. aerophila, N. aerophiloides, N. aestuar i), N. affinis, N. africana, N. agnii, N. agnita, N. alba, N. albicostalis, N. alexandrina, N. alicae, N. allanssonii, N. alpina, N. alpinobacillum, N. amabilis, N. ambigua, N. americana, N. .amisaensis, N.amphibia, N.amphibia (including all such variants), N.amphibioides, N.amphicephala, N.amphilepta, N.amphioxoides, N.amphioxys (including all such variants), N.amphiplectans, N.amphiprora, N.amplectens, N.amundonii, N.anassae, N.andicola, N.angularis (including all such variants), N.angulata, N.angustata (including all such variants), N.angustatula, N.angustiforaminata, N.aniae, N.antarctica, N.antillarum, N.apiceconica, N.apiculata, N.archibaldii, N.arcuata, N.arcula, N.arculata N.arcus, N.ardua, N.aremonica, N.arenosa, N.areolata, N.armoricana, N.asperula, N.astridiae, N.atomus, N.attenuata, N.aurantiaca, N.aurariae, N.aurica, N.auricula, N.australis, N.austriaca, N.bacata ( including all such variants), N. bacilliformis, N. bacilliformis, N. bacillum, N. balatonis, N. balcanica, N. baltica, N. barbieri (including all such variants), N. barkleyi, N. barronii, N. barroiana, N. bartholomei, N. bathurst rstensis), N. bavarica, N. behrei, N. bergii, N. beyeri, N. biacrula, N. bicapitata (including all such variants), N. bicuneata, N. bifurcata, N. bilobata (including all such variants), N. birostrata, N. bisculpta, N. bita, N. bizertensis, N.blankaartensis, bombiformis, borealis, bosumtwiensis, braarudii, brebissonii (including all such variants), bremensis (including all such variants), N. brevior, brevirostris, brevissima (including all such variants), brevistriata, Brightwell Nitzschia N. brightwellii, N. brittonii, N. brunoi, N. bryophila, N. buceros, N. bukensis, N. bulnheimiana, N. buschbeckii, N. calcicola, N. caledonensis, N. calida (including all such variants), N. californica, N. campechiana, N. capens is), N. capitata, N. capitallata (including all such variants), N. capuluspalae, N. carnicobarica, N. carnico-barica, N. challengeri, N. chalonii, N. chandolensis, N. chardezii, N. chasei, N. chauhanii, N. chunga ra), N. chutteri, N. circumsuta, N. clarissima, N. clausii, N. clementei, N. clementia, N. clevei, N. closterium (including all such variants), N. coarctata, N. cocconeiformis, N. communis (including all such variants), N. commutata, N.commutatoides, N.compacta, N.compressa (including all such variants), N.concordia, N.confinis, N.conformata, N.confusa, N.congolensis, N.constricta (including all such variants), N.consummata, N.corpulenta, N.costei, N.coutei, N.creticola, N. cucumis, N. cursoria, N. curta, N. curvata, N. curvilineata, N. curvipunctata, N. curvirostris (including all such variants), N. curvula (including all such variants), N. cuspidata, N. cylindriformis, N. cylindrus, N. dakar kariensis), N. davidsonii, N. dealpina, N. debilis, N. decipiens, N. delauneyi, N. delicatissima, N. delicatula, N. delognei, N. denticula (including all such variants), N. denticuloides, N. desertorum, N. dianae, N. diaphana, N. diducta, N. didyma, N. dietrichii, N. dilatata, N. diluviana, N. dippelii, N. directa, N. diserta, N. disputata, N. dissipata (including all variants), N. dissipatoides, N. distans (including all variants), N. distans (including all variants), N.distantoides), N. divaricata, N. divergens, N. diversa, N. diversecostata, N. doljensis, N. draveillensis, N. droebakensis, N. dubia (including all such variants), N. dubiformis, N. dubioides, N. ebroicensis, N.eglei, N.elegans, N.elegantula, N.elegens, N.elliptica, N.elongata, N.entomon, N.epiphytica, N.epiphyticoides, N.epithemiformis, N.epithemioides, N.epithemoides (including all these variants), N. N. epsilon, N. erlandssonii, N. erosa, N. etoshensis, N. examinanda, N. eximia, N. familiara, N. fasciculata, N. febigeri, N. ferox, N. ferrazae, N. fibula-fissa, N. filiformis (including all these variants), N. flexa, N. flexus N. flexoides, N. fluminensis, N. fluorescens, N. fluvialis, N. fogedii, N. fonticola (including all such variants), N. fonticoloides, N. fonticula, N. fontifuga, N. forfica, N. fossalis, N. fossilis, N. fragilariiformis, N.franconica, N.fraudulenta, N.frauenfeldii, N.frequens, N.frickei, N.frigida (including all such variants), N.frustuloides, N.frustulum (including all such variants), N.fruticosa, N.fundi, N.fusiformis, N.gaarderi, N. gaertnerae, N. gandersheimiensis, N. garrensis, N. gazellae, N. geitleri, N. geitlerii, N. gelida (including all these variants), N. geniculata, N. gessneri, N. gieskesii, N. gigantea, N. gisela, N. glabra ), N.glacialis (including all such variants), N.glandiformis, N.goetzeana (including all such variants), N.gotlandica, N.graciliformis, N.gracilis (including all such variants), N.gracillima, N.graciloides, N.gradifera, N.graeffii, N.grana, N.gr andis), N. granii (including all such variants), N. granulata (including all such variants), N. granulosa, N. groenlandica, N. grossestriata, N. grovei, N. gruendleri, N. grunowii, N. guadalupensis, N. guineensis, N. guttula, N.gyrosigma), N. habirshawii, N. habishawii, N. hadriatica, N. halteriformis, N. hamburgiensis, N. hantzschiana (including all such variants), N. harderi, N. harrissonii, N. hassiaca, N. heidenii, N. heimii ), N. hemistriata, N. heteropolica, N. heuflerania, N. heufleriana (including all of these variants), N. hiemalis, N. hiengheneana, N. hierosolymitana, N. hoehnkii, N. holastica, N. hollerupensis, N. holsatica, N. felt hat.

[0114] N. homburgiensis, N. hudsonii, N. hummii, N. hungarica (including all such variants), N. hustedti, N. hustedtiana, N. hyalina, N. hybrida (including all such variants), N. hybridaeformis, N. ignorata (including all such variants), N. iltisii, N. impressa, N. improvisa, N. indeterminate

[0115] N.incerta, N.incognita, N.inconspicua, N.incrustans, N.incurva (including all these variants), N.indica, N.indistincta, N.inducta, N.inflatula, N.ingenua, N.inimasta, N.innominata, N.insecta, N.insignis (including all these variants), N.intermedia (including all these variants), N.intermissa, N.intermedia

[0116] N.interrupta, N.interruptestriata, N.invicta (including all these variants), N.invisa, N.invisitata, N.iranica, N.irregularis, N.irremissa, N.irrepta, N.irresoluta, N.irritans, N.italica, N.janischii, N.jelineckii, N.johnmartins N. johnmartinii), N. juba, N. jucunda, N. jugata (including all of these variants), N. jugiformis, N. kahlii, N. kanakarum, N. kanayae, N. kavirondoensis, N. kerguelensis, N. kimberliensis, N. kittlii, N. kittonii, N. knysnensis, N. kolaczeckii, N.kotschyi, N.kowiensis, N.krachiensis, N.krenicola, N.kuetzingiana (including all such variants), N.kuetzingii, N.kuetzingioides, N.kurzeana, N.kurzii, N.Kützingiana (including all such variants), N.labella, N.labuensis, N.lacrima, N.lacunarum, N.lacunicola, N.lacus-karluki, N.lacustris, N.lacuum, N.laevis, N.laevissima, N.lagunae, N.lagunensis, N.lamprocampa (including all such variants), N.lanceola (including all such variants), N.lanceolata (including all such variants), N.lancettula, N.lancettula-likelancettuloides), N. lange-bertalotii, N. latens, N. latestriata, N. latiuscula, N. lauenbergiana, N. lauenburgiana, N. lecointei, N. leehyi, N. legleri, N. lehyi, N. leistikowii, N. lesbia, N. lesinensis, N. lesothensis, N. leucosigma, N. levidensis (including all these variants), N. lieb etruthii (including all such variants), N.ligowskii, N.limicola, N.limulus, N.linearis (including all such variants), N.lineata, N.lineola, N.linkei, N.lionella, N.Littoralis (including all such variants), N.littorea, N.longa, N.longicollum, N.longirostris, N.longissima (including all such variants), N.lorenziana.

[0117] (including all of these variants), N. lucisensibilis, N. lunaris, N. lunata, N. lurida, N. luzonensis, N. macaronesica, N. macedonica, N. macera, N. machardyae, N. macilenta (including all of these variants), N. magnacarina, N. mahihaensis, N. mahoodii ), Maillardii, N.major, N.Majuscula (including all such variants), N.makarovae, N.manca, N.mancoides, N.manguini, N.marginata, N.marginulata (including all such variants), N.marina, N.martiana, N.maxima, N.media, N.medioconstricta, N.medioconstricta N. mediocris, N. mediocris, N. mediterranea, N. metzeltinii, N. microcephala (including all these variants), N. migrans, N. minuta, N. minutissima, N. minutula, N. miramarensis, N. miserabilis, N. mitchelliana, N. modesta, N. moissacensi s (including all these variants), N. mollis, N. monachorum, N. monoensis, N. montanestris, N. morosa, N. multistriata, N. nana, N. natalensis, N. natans, N. nathorsti, N. navicularis, N. navis-varingica, N. navrongensis, N. neglecta, N. nelson's nitzschia (N.nelsonii), N.neocaledonica, N.neoconstricta, N.neofrigida, N.neogena, N.neotropica, N.nereidis, N.nicobarica, N.nienhuisii, N.normannii, N.notabilis, N.nova, N.novae-guinea N.eaensis), New Guinea Nitzschia (N.novae-guineensis), New Holland Nitzschia (N.novaehollandiae), New Zealand Nitzschia (N.nova-zealandia), N.nyassensis, N.oberheimiana, N.obesa, N.obliquecostata, N.obscura, N.obscurepunctata, N.obsidialis, N.obsoleta , N.obsoletiformis, N.obtusa (including all these variants), N.obtusangula, N.oceanica, N.ocellata, N.oliffi, N.omega, N.osmophila, N.ossiformis, N.ostenfeldii, N.ovalis, N.paaschei, N.pacifica N.pacifica), N.palacea, N.palea (including all of these variants), N.paleacea, N.paleaeformis, N.paleoides, N.palustris, N.pamirensis, N.panduriformis (including all of these variants), N.pantocsekii, N.paradoxa (including all of these variants), N.parallela), N. pararostrata, N. partita, N. parvula (including all these variants), N. parvuloides, N. paxillifer, N. peisonis, N. Pelagica, N. pellucida, N. pennata, N. peragallii, N. perindistincta, N. perminuta, N. narrow-leaved N. erpusilla) (including all such variants), N. perspicua, N. persuadens, N. pertica, N. perversa, N. petitiana, N. philippinarum, N. pilum, N. pinguescens, N. piscinarum, N. plana (including all such variants), N. planctonica, N. plicatula, N. plioveterana, N. polari s), N. polymorpha, N. ponciensis, N. praecurta, N. praefossilis, N. praereinholdii, N. princeps, N. procera, N. prolongata (including all such variants), N. prolongatoides, N. promare, N. propinqua, N. pseudoepiphytica, N. pseudoamphioxoid es), N. pseudoamphioxys, N. pseudoamphyoxys, N. pseudoatomus, N. pseudobacata, N. pseudocapitata, N. pseudocarinata, N. pseudocommunis, N. pseudocylindrica, N. pseudodelicatissima, N. pseudospringpseudofonticola), N. pseudohungarica, N. pseudohybrida, N. pseudomicroscopic nitzschia, N. pseudoseriata, N. pseudosigma, N. pseudosinuata, N. pseudostagnorum, N. pubens, N. pulcherrima, N. pumila, N. punctata (including all these variants), N. spiky N. pungens (including all such variants), N. pungiformis, N. pura, N. puriformis, N. pusilla (including all such variants), N. putrida, N. quadrangula, N. quickiana, N. rabenhorstii, N. radicula (including all such variants), N. rautenbachiae, N. recta (including all such variants), species), straight N.rectiformis, straight long N.rectilonga, straight and strong N.rectirobusta, straight N.rectissima, regular N.regula, N.reimeri, N.reimerii, N.reimersenii, N.retusa, N.reversa, N.rhombica, N.rhombiformis, N.rhopalodioides, N. .richterae), N.rigida (including all these variants), N.ritscheri, N.robusta, N.rochensis, N.rolandii, N.romana, N.romanoides, N.romanowiana, N.rorida, N.rosenstockii, N.rostellata, N.rostrata, N.ruda, N.rugosa, N.rockrupestris), N. rusingae, N. ruttneri, N. salinarum, N. salinicola, N. salpaespinosae, N. salvadoriana, N. sansimoni, N. sarcophagum, N. scabra, N. scalaris, N. scaligera, N. scalpelliformis, N. schoenfelder eldii), N. schwabei, N. schweikertii, N. scutellum, N. sellingii, N. semicosta, N. semirobusta, N. separanda, N. seriata (including all such variants), N. serpenticola, N. serpentiraphe, N. serrata, N. sibula (including all such variants), N. sigma (including all such variants), N. sigmata (including all such variants), N.sigmaformis, N.sigmatella, N.sigmoidea (including all such variants), N.silica, N.silicula (including all such variants), N.siliqua, N.similis, N.simplex, N.simpliciformis, N.sinensis, N.sinuata (including all such variants), N.smithii, N.sociabilis, N. socialis (including all such variants), N. solgensis, N. solida, N. solita, N. soratensis, N. sp., N. spathulata (including all such variants), N. speciosa, N. spectabilis (including all such variants), N. sphaerophora, N. spiculoides, N. spiculum, N. spinarum, N. spinifera, N.stagnorum), N. Steenbergensis, N. stellata, N. steynii, N. stimulus, N. stoliczkiana, N. stompsii (including all such variants), N. strelnikovae, N. stricta, N. strigillata, N. striolata, N. subaccommodata, N. subacicularis, N. subacuta, N. su bamphioxioides), N.subapiculata, N.subbacata, N.subcapitata, N.subcapitellata, N.subcohaerens (including all these variants), N.subcommunis, N.subconstricta, N.subcurvata, N.subdenticula, N.subfalcata, N.subpseudo-nitzschia N.subfraudulenta), N.subfrequens, N.subfrustulum, N.subgraciloides, N.subinflata, N.subinvicta, N.sublaevis, N.sublanceolata, N.sublica, N.sublinearis, N.sublongirostris, N.submarina, N.submarina, N.submarina submediocris), N.subodiosa, N.subpacifica, N.subpunctata, N.subromana, N.subrostrata, N.subrostratoides, N.subrostroides, N.subsalsa, N.subtilioides, N.subtilis (including all these variants), N.subtubicola, N.subvitrea), N.suchlandtii, N.sulcata, N.sundaensis, N.supralitorea, N.tabellaria, N.taenia, N.taeniiformis, N.tantata, N.tarda, N.taylorii, N.temperei, N.tenella, N.tenerifa, N.tenuiarcuata, N. tenuirostris), N. tenuis (including all of these variants), N. tenuissima, N. tergestina, N. terrestris, N. terricola, N. thermalis (including all of these variants), N. thermaloides, N. tirstrupensis, N. tonoensis, N. towutensis, N. translucida, N. tropica, N. try blionella) (including all these variants), N.tsarenkoi, N.tubicola, N.tumida, N.turgidula, N.turgiduloides, N.umaoiensis, N.umbilicata, N.umbonata, N.vacillata, N.vacua, N.valdecostata, N.valdestriata, N.valens, N.valga, N.valida (including including all such variants), N. vanheurckii, N. vanoyei, N. vasta, N. ventricosa, N. vermicularioides, N. vermicularis (including all such variants), N. vermicularoides, N. vexans, N. victoriae, N. vidovichii, N. vildaryana, N.N. villarealii), N. virgata, N. visurgis, N. vitrea (including all such variants), N. vivax (including all such variants), N. vixnegligenda, N. Vonhauseniae, N. vulga, N. weaveri, N. weissflogii, N. westii, N. williamsiii, N. wipplingeri, N. witkowskii, N. wodensis, N. Woltereckii, N. woltereckoides, N. wuellerstorfii, N. wunsamiae, N. yunchengensis, N. zebuana, N. zululandica. .

[0118] Advantageously, according to the invention, the algae of the genus Nitzschia may be an algae selected from the species N. sp.

[0119] If the microalgae belong to the genus Haematococcus, they can be selected from the following species: H. buetschlii, H. capensis, H. carocellus, H. droebakensis, H. grevilei, H. insignis, H. lacustris, H. murorum, H. pluvialis, H. salinus, H. sanguineis, H. thermalis, H. zimbabwiensis.

[0120] If the microalgae belong to the genus Tetraselmis, they can be selected from the following species: T. alacris, T. apiculata, T. arnoldii, T. ascus, T. astigmatica, T. bichlora, T. bilobata, T. bolosiana, T. chui, T. contracta, T. convolutae, T. cordiformis, T. desikacharyi, T. elliptica, T. fontiana, T. gracilis, T. hazenii, T. helgolandica, T.impellucida, T.incisa, T.Inconspicua, T.indica, T.levis, T.maculata, T.marina, T.mediterranea, T.micropapillata, T.rubens, T.striata, T.suecica, T.tetrabrachia, T.tetrathele, T.verrucosa, T.viridis, T.wettsteinii.

[0121] According to a particular embodiment of the invention, the protist is selected from the genera: Chlorella, Galdiella, Euglena, Cyanobacteria and Diatom.

[0122] The microorganism can be cultivated in the absence or presence of a carbon source and / or in the absence or presence of light. Therefore, the method according to the invention is suitable for cultivating microorganisms in heterotrophic, autotrophic or mixed nutritional modes. In a preferred embodiment, the microorganism is a microalgae cultivated according to an autotrophic or mixed nutritional mode. Advantageously, the microorganism cultivated according to the invention is an autotrophic microalgae.

[0123] The method of culturing microorganisms comprises culturing the microorganisms in a bioreactor coupled to a membrane contactor according to the present invention, and recovering the biomass produced by the microorganisms. The produced biomass can be used to prepare pharmaceutical compounds, cosmetic compounds, nutraceutical compounds or food compounds.

[0124] The present invention therefore also relates to the use of a membrane contactor to regulate the flow of CO2 and O2 in a method for culturing microorganisms, wherein CO2 is a carbon source in the culture medium. The membrane contactor can be placed inside or outside the bioreactor. It should be understood that the membrane contactor can be placed in a box, which can be inside or outside the bioreactor. Preferably, the membrane contactor is placed inside the bioreactor. Microorganisms and other culturing methods are as described above.

[0125] The present invention also relates to a method for decarbonating a CO2-rich gas, the method comprising injecting the gas into a bioreactor, the bioreactor comprising a culture medium and microorganisms capable of growing in the culture medium using CO2 as the sole carbon source, and a CO2 supply device, characterized in that the CO2 supply device comprises a membrane contactor to regulate the flow of CO2 and O2 in the culture medium, wherein the flow of CO2 is directed to the culture medium and the flow of O2 is directed to the outside of the culture medium.

[0126] Advantageously, the CO2 concentration in the culture medium is adjusted to provide the necessary and sufficient amount for culturing the microorganisms so that all the CO2 is consumed and none is lost to the atmosphere.

[0127] In particular, in the decarbonation process according to the invention, the only gas exchange between the reactor and the external environment takes place via the membrane contactor. The reactor is advantageously closed.

[0128] Finally, the invention relates to a fermentation device for microorganisms, which are microalgae according to the invention.

[0129] The fermentation equipment includes:

[0130] (a) a fermenter or bioreactor, and

[0131] (b) A CO 2 supply device comprising a membrane contactor according to the present invention, which enables the flow of CO 2 and O 2 to be regulated, wherein the flow of CO 2 is directed to the culture medium and the flow of O 2 is directed to the outside of the culture medium.

[0132] refer to Figure 1 , the device comprises a membrane contactor (2) coupled to a bioreactor (1). The circulation of the CO2 receiving fluid contained in the bioreactors (3) and (4) (the flow of the culture medium) is ensured by a pump (5) arranged between the bioreactor and the membrane contactor. Measuring sensors or probes (10) and (11) can be added to the device. These are in particular sensors for measuring CO2 and O2.

[0133] Furthermore, the membrane contactor is connected to a CO2 source, causing the flow of CO2 (CO2 supply fluid) in the membrane contactors (6) and (7). The device according to the present invention may also include a vacuum pump (8) to generate the flow of CO2. The device may also include a buffer tank (9) capable of storing CO2. Preferably, the buffer tank is placed between the CO2 supply source and the membrane contactor.

[0134] refer to Figure 8 , the membrane contactor is connected to a source of carbon dioxide (CO2) at the inlet of the CO2 supply fluid (22). The CO2 supply fluid leaves the membrane contactor (23) and returns to the CO2 supply tank (9) according to a closed loop. At the same time, the CO2 receiving fluid circulates in the membrane contactor from the inlet (20) connected to the bioreactor (1) to the outlet (21), which is also connected to the bioreactor (1). Therefore, there are two circulating flows in the device: the circulating flow of the CO2 supply fluids (6) and (7), and the circulating flow of the CO2 receiving fluids (3) and (4). These two flows circulate according to a closed loop and enter the membrane contactor, in which material exchange occurs between the two fluids.

[0135] According to the present invention, the membrane contactor comprises a transfer membrane, preferably a transfer membrane having a hollow fiber configuration. In this embodiment, the hollow fiber transfer membrane is selected from a microporous membrane and a dense membrane. Preferably, the transfer membrane having a hollow fiber configuration is a dense membrane.

[0136] In a fermentation device, the membrane contactor can be placed inside or outside the reactor. It should be understood that the membrane contactor can be placed in a box, which itself is placed inside or outside the bioreactor. In a preferred embodiment, the membrane contactor is placed outside the reactor.

[0137] According to a particular embodiment, the reactor is closed, isolated from the outside, the only gas exchange with the outside being carried out via a membrane contactor by supplying CO 2 and exhausting O 2 .

[0138] Example

[0139] Materials and methods

[0140] Cell growth assay

[0141] Optical density (OD)

[0142] OD measurements were performed in duplicate at 750 nm using a spectrophotometer (7315, JENWAY). Especially when the biomass concentration was too low to have an accurate dry mass value, the daily productivity was calculated from the OD and the correlation coefficient (CC) specific to the cultured microorganism according to the following formula.

[0143]

[0144] Dry mass (DM)

[0145] DM measurements were performed in duplicate using a vacuum filtration collector. For this purpose, the samples were placed on an AP40 glass fiber prefilter (pores of 0.7 μm, Merck Millipore). The filtered dry matter was then weighed after incubation at 100° C. for 24 hours. The final DM value was calculated according to the following formula.

[0146]

[0147] The following formula was used to calculate the daily productivity from DM.

[0148]

[0149] Example 1: Change in the amount of molecular oxygen (O2)

[0150] Purpose: To eliminate dissolved O2 by varying the flow rate of the medium in the contactor, depending on the geometrical and physicochemical characteristics of the reactor.

[0151] Experimental conditions: Biomass of Chlorella vulgaris was cultivated in a photobioreactor, in which the light emitting surface was kept constant (250 m 2 ). The culture medium used is described in Table 1 below.

[0152] Table 1

[0153] Nutrients Concentration g / l KNO3 8.888 NaH2PO4 1.238709677 CaCl2,2H2O 0.04672 MgSO4,7H2O 1.312 EDTA with Iron 0.577

[0154] The amount of photons emitted from the light surface ranges from 1,050 μmol photons / m 2 / s to 0μmol photons / m 2 There is no unlit area in the reactor (fd=0).

[0155] The speed that must be applied to the flow of the culture medium over the transfer membrane can be determined according to the configuration of the photobioreactor, according to the amount of O 2 present in the culture medium and the amount of O 2 produced by the microalgae.

[0156] The concentration of dissolved molecular oxygen must be controlled at 8g / m 3 With 30g / m 3 In fact, it has been found that less than 8g / m 3 The dissolved oxygen concentration may have a negative impact on the growth of microalgae, and concentrations greater than 30 g / m 3 The solubility of O2 may lead to a loss in biomass productivity on Chlorella vulgaris.

[0157] The amount of O2 produced via biomass generation was modeled using the following method: Where YO2 / x equals 1.5 kg O2 / kg biomass produced and VP is the volumetric productivity.

[0158] Therefore, the flow of the medium in the membrane contactor depends on the ΔO2 corresponding to the following equation:

[0159] ΔO2 (mg / l) = -0.79-0.66*initial O2 concentration (mg / l) + 0.29*flow rate of culture medium (l / min)

[0160] Situation A : Size the system to produce 0.2 kg / m 3 / day, and the production of O2 will remain between the desired minimum and maximum limits of dissolved O2. To achieve the minimum limit, a flow rate of 15.72 l / min O2 is required ( Figure 4 ).

[0161] The production of O2 is still reasonable, since it remains within the tolerable limits for good photosynthesis. However, it can be kept as close to the minimum limits as possible by adjusting the flow rate. Secondly, if the productivity increases, the production of O2 will be harmful to the microalgae, since it is above those limits. The flow in the transfer membrane must then be adjusted to reach the maximum desired limits of dissolved O2.

[0162] Situation B :The system is defined to produce 0.79kg / m 3 / day; the production of O2 will exceed the maximum limit. Therefore, the O2 concentration must be reduced by 22.25 mg / l ( Figure 5 ).

[0163] Table 1-bis summarizes the different parameters of systems A and B.

[0164] Table 1-2

[0165]

[0166] Example 2: Changes in the amount of carbon dioxide (CO2)

[0167] By changing the pressure conditions on the transfer membrane of the contactor, the amount of dissolved CO2 can be increased or decreased depending on the amount initially present in the culture medium. Therefore, the change in the amount of CO2 can be defined according to the following formula:

[0168] ΔCO2 (mg / l) = 664.96-0.46*initial CO2 concentration (mg / l) + 888.30*pressure (bar)

[0169] If the concentration of the culture medium includes a CO2 concentration of 100 mg / l and a constant concentration of 1,200 mg / l dissolved CO2 is desired, the CO2 delta is 1,100 mg / l at a pressure of 0.542 bar across the transfer membrane ( Figure 6 ). This CO2 retention must be corrected over time based on the amount of photon emitting surfaces sent into the tank.

[0170] Various parameters can be adjusted to calculate the productivity in a photobioreactor. They are considered in the following model of the surface output of a photobioreactor.

[0171]

[0172] in:

[0173] -(r' X ) max : Maximum surface output of the photobioreactor

[0174] -K: half-saturation constant of photosynthesis

[0175] -a 光 : corresponds to the ratio between the illuminated surface and the actual volume of the photobioreactor (S 光 / Vr)

[0176] -Vr: actual volume

[0177] -ň: average collimation of the incident radiation

[0178] -fd: dark fraction of the photobioreactor, or the ratio of the unilluminated volume to the actual volume

[0179] -A: Volume density of absorbed radiation power, in μmolhν·m-3·s-1

[0180] -ρ M : Maximum energy output from the conversion of light energy into physical and chemical energy

[0181] -Qn: Average photon flux density on the surface of the photobioreactor

[0182] -h: Planck constant

[0183] -v: frequency

[0184] -Sx: Surface yield

[0185] -Px Continuous: Volumetric Production

[0186] - Correction output = Px, correction factor is 20%

[0187] The yield of a photobioreactor will depend mainly on the following factors:

[0188] - Dark fraction (fd)

[0189] -Surface yield (Sx) caused by capturing surface photon flux

[0190] - Volumetric yield (Px) according to the surface photon flux relative to the total volume. In this case, it is preferred to reduce the non-illuminated part of the reactor as much as possible and to increase the surface receiving the photon flux.

[0191] The amount of CO2 supplied to achieve the target and surplus for microalgae growth was calculated according to the following formula:

[0192] ΔCO2*V 反应器 +(biomass productivity*V 反应器 *Assimilation of kgCO2 / kg biomass)

[0193] The parameters of Example 2 are summarized in Table 2 below.

[0194] Table 2

[0195]

[0196] Example 3: Amount of CO2 injected into the culture medium according to temperature and pressure

[0197] Purpose: To eliminate O2 produced by photosynthesis of microalgae by injecting concentrated CO2 (which will act as carrier gas).

[0198] Experimental conditions: The biomass of Chlorella vulgaris was cultured in a photobioreactor with the medium described in Example 1 (Table 1). 2 light surface and 1,050 μmol photons / m 2 / s surface emission. The biomass yield is estimated to be 1.03kg / m 3 / D, of which CO2 consumption is 2.12kgCO2 / kg biomass / m 3 / sky.

[0199] The CO2 flow was adjusted to eliminate at least 85% of the initial O2 produced. The parameters were then verified via pressure gauges and liquid and gas flow meters.

[0200] The O2 gas pressure of the incoming gas was maintained lower than the O2 gas pressure of the culture medium (PO2 of the incoming gas < PO2 of the culture medium). The CO2 gas pressure of the incoming gas was maintained higher than the CO2 gas pressure of the culture medium (PCO2 of the incoming gas > PCO2 of the culture medium).

[0201] The amount of injected CO2 depends on the temperature of the culture medium, the initial concentration of the culture medium, the pressure allowed in the transfer membrane carrying the CO2 and the CO2 concentration difference between the initial and target CO2 concentrations.

[0202] When the culture medium reaches saturation with CO2, additional amounts of CO2 must be added to act as a carrier of O2 and in anticipation of CO2 consumption by the biomass. This approach makes it possible to maintain the maximum availability of CO2 according to the strain associated with CO2 consumption.

[0203] Table 3 employs different parameters that allow the calculation of ΔCO2 and the CO2 pressure that must be reached (to obtain a given saturation of dissolved CO2) depending on the temperature of the culture medium. Therefore, these parameters also allow the calculation of the amount of CO2 added for the growth of the microalgae and the elimination of O2.

[0204] Table 3

[0205]

[0206] Based on the same principle as Example 2, the parameters of Example 3 are summarized in Table 4 below.

[0207] Table 4

[0208]

[0209] Example 4: Cultivation of Chlorella Sorokinensis with and without CO2 supply from a membrane contactor

[0210] The culture of Chlorella sorokinensis was started in two 5-liter STR bioreactors (Pierre Guérin, working volume 3 L). The system included a peristaltic pump ( Model 7518-00, Cole Parmer). The two reactors differ only in the means for supplying CO2 (the amount of CO2 supplied remains the same). One is equipped with a membrane contactor and a bubbling device according to the invention. The membrane contactor is a 200 mm x 550 mm structure comprising a transfer membrane of a dense gas permeable silicone fiber configuration with an inner diameter of about 200 μm and an outer diameter of about 300 μm, in which the culture medium is circulated at 400 rpm using a WG600S peristaltic pump.

[0211] A schematic diagram of the membrane contactor used is presented in Figure 8 middle.

[0212] The second (control) bioreactor was not equipped with a membrane contactor but was equipped with a CO2 sparging system.

[0213] The culture was kept at 500 μmol.m -2 .s -1 The samples were kept under light of the intensity of 1000K for 7 days.

[0214] The medium used was that described in Example 1 (Table 1) and each bioreactor was inoculated at 1.5% volume with biomass grown from cryovials (INOVA) with a target OD of 0.05.

[0215] The culture parameters were maintained as follows:

[0216] - pH: between 7 and 8:

[0217] - Temperature: 30°C;

[0218] - Agitation: 200 rpm;

[0219] - Pressure on the transfer membrane of the contactor: 0.1 bar, using a control valve and a pressure gauge;

[0220] During the first 48 hours, the culture was supplied with air (1m 3 .h -1 ), then CO2 (0.05 l / min). One to three times a day, 15 ml samples were taken for OD and DM analysis. Temperature, pH and CO2 and O2 pressure parameters were monitored.

[0221] After a few days, the color difference between the biomass of each bioreactor is visible to the naked eye. After 3 days of cultivation, in the case of a bioreactor according to the present invention, the following CO2 pressures are applied to the transfer membrane of the contactor using a stop valve: first 0.05 bar, then 0.20 bar, then 0.225 bar, then 0.25 bar. The O2 pressure in the reactor then tends to decrease. The target CO2 pressure applied has been determined according to the method described in Example 3 above, and enables gas exchange (O2 and CO2) across the transfer membrane to be promoted.

[0222] Fig. 9 A graph showing the evolution of the O2 pressure in the culture medium over time is presented in . It should be noted that a power outage occurred on day 8. Thus, in addition to the temporary absence of light and agitation, the pump that enables the culture medium to circulate in the contactor was also interrupted. This is visible in the graph: on day 8, there is a peak O2 pressure.

[0223] Growth of Chlorella sorokinensis was observed in both bioreactors. Results of OD and daily productivity (based on OD) are presented in Fig.10 However, starting from day 4, the daily production (based on OD) of the culture in the bioreactor according to the present invention was significantly higher compared to the control. This trend was maintained throughout the cultivation process. Considering that the only difference between the two bioreactors was the use or not of a membrane contactor according to the present invention, these differences in productivity indicate improved CO2 supply and efficient O2 elimination.

[0224] It should be noted that during the cultivation period, there was only moderate clogging of the membrane contactor and the culture medium could therefore be stirred uniformly. Furthermore, in the case of a bioreactor equipped with a membrane contactor according to the invention, the occurrence of foam appears to be limited.

[0225] For both cultures, the maximum theoretical productivity was achieved after 7 days (0.48 / g / L / day for the invention and 0.14 for the control).

Claims

1. A method for culturing microorganisms in a culture medium containing CO2 as a carbon source, the method comprising the step of culturing the microorganisms in a bioreactor containing a CO2 supply device, characterized in that The CO2 supply device includes a membrane contactor for regulating the flow of CO2 and O2 in the culture medium, and wherein the flow of CO2 is guided to the culture medium and the flow of O2 is guided to the outside of the culture medium through a transfer membrane of the membrane contactor.

2. The culture method according to claim 1, characterized in that The membrane contactor includes at least two fluid circulation loops, namely a first circulation loop of a CO 2 supply fluid and a second circulation loop of a CO 2 receiving fluid, and a transfer membrane separating the circulation loops.

3. The method according to claim 1 or 2, characterized in that: The membrane contactor includes a transfer membrane having a hollow fiber configuration.

4. The method according to claim 1, characterized in that The membrane contactor comprises a microporous transfer membrane or a nonporous transfer membrane, preferably a nonporous transfer membrane.

5. The method according to claim 3 , wherein: The outer diameter of the membrane(s) is between 100 μm and 800 μm.

6. The method according to claim 1, characterized in that The membrane contactor comprises a microporous transfer membrane, whereby the diameter of the pores is between 0.01 μm and 0.5 μm.

7. The method according to one of claims 2 to 6, characterized in that The CO2 supply fluid circulates outside the hollow fibers.

8. The method according to claim 2, characterized in that The CO2 receiving fluid circulates inside the hollow fibers.

9. The method according to claim 1, characterized in that The concentration of CO2 in the culture medium is at least 1,000 mg / l.

10. The method according to one of claims 1 to 9, characterized in that The amount of O2 in the reactor is less than 35 g / m 3 , preferably 8g / m 3 With 33g / m 3 between.

11. The method according to claim 1, characterized in that The concentration of CO2 in the culture medium is adjusted to supply the necessary and sufficient amount for culturing the microorganisms, wherein all of the CO2 is consumed and not lost to the atmosphere.

12. The method according to one of claims 1 to 11, characterized in that The only gas exchange between the reactor and the external environment takes place via the membrane contactor.

13. The method according to one of claims 1 to 12, characterized in that The microorganisms are bacteria, yeasts or protists, in particular microalgae.

14. The method according to one of claims 1 to 13, characterized in that The microorganisms are microalgae cultivated in an autotrophic mode.

15. Use of a membrane contactor as defined in one of claims 1 to 13 for regulating the flow of CO2 and O2 in a method for cultivating microorganisms, wherein the CO2 is the carbon source in the culture medium.

16. The use according to claim 15, characterized in that The culturing method comprises using a bioreactor containing a culture medium.

17. The use according to claim 15, characterized in that The membrane contactor is placed in the bioreactor.

18. A microalgae fermentation device, comprising a fermentation reactor, a CO2 supply device and, if applicable, a lighting device, characterized in that: The CO 2 supply device includes a membrane contactor for regulating the flow of CO 2 and O 2 , wherein the flow of CO 2 is guided to the culture medium and the flow of O 2 is guided to the outside of the culture medium.

19. The device according to claim 18, characterized in that The membrane contactor is defined according to one of claims 2 to 14 .

20. The device according to claim 18 or 19, characterized in that The membrane contactor is placed in the fermentation reactor.

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