Bioreactor with reverse circulation of air and medium, and method based on such a bioreactor

Abstract

The invention relates to a bioreactor, in particular a photobioreactor, comprising: - an enclosure (E) that comprises a lower end (I) and an upper end (S) having an outlet (S1) and an inlet (D1), respectively, for liquids; - a recirculation circuit (RC) connected to the inlet (D1) and to the outlet (S1); - a recirculation pump (P1) connected to the enclosure (E) and to the recirculation circuit (RC), characterised in that the recirculation pump (P1) is configured to produce an essentially vertical downward flow of liquid from the outlet (S1) to the inlet (D1); and in that the enclosure (E) further comprises at least one gas injector (G) arranged in the lower end (I). The invention also relates to a method based on such a bioreactor.

Description

Reverse air-medium circulation bioreactor and process based on such a bioreactor

[0001] The invention relates to the field of bioreactor arrangements, in particular photobioreactors.

[0002] Bioreactors are used to grow cultures of microorganisms in a medium.

[0003] Some strains of microorganisms of interest tend to sediment in the reactor, causing the cells to clump together. This poses several disadvantages for culture development, while the volume of the reactor allows for spacing between the cells if they are better dispersed.

[0004] This is even more problematic in the case of photobioreactors where the strains need to be exposed to light, and therefore distributed throughout the volume of the reactor.

[0005] Thus, the prior art has proposed solutions for stirring the medium, by mechanical stirring of the culture medium container, or by stirring a magnetic part placed in the culture medium.

[0006] These solutions are complex to implement in a large volume bioreactor so that agitation is not effective throughout the volume.

[0007] Other solutions use cell pipes in which the cells move with the environment for several hundred meters.

[0008] Unfortunately, this solution involves a complex and cumbersome layout.

[0009] A first objective of the present invention is to propose a bioreactor solution with a simplified and efficient agitation system throughout the volume of the bioreactor.

[0010] A second objective is to propose a less cumbersome solution than in the prior art.

[0011] To achieve these objectives, the invention provides a bioreactor comprising:- an enclosure which comprises a lower end having at least one outlet for liquids; and an upper end having at least one inlet for liquids;- a recirculation circuit connected to said inlet and to said outlet;- a recirculation pump connected to the enclosure and to the recirculation circuit,characterized in that the recirculation pump is configured to produce a flow of liquid from said outlet to said inlet so that the flow of liquid in the enclosure has an essentially vertical downward direction;and in that the enclosure further comprises at least one gas injector arranged in the lower end.

[0012] Advantageously, the combination of upward aeration and downward movements of the medium makes it possible to homogenize cell production and increase yield by illuminating the cells more.

[0013] Furthermore, agitation through aeration and water extraction is significantly simplified and effective. Placing these elements in several locations allows for agitation throughout the entire volume of the bioreactor.

[0014] Furthermore, the bioreactor of the invention is compact compared to prior art pipeline systems which run for several hundred meters.

[0015] According to one variant, the enclosure comprises several gas injectors arranged at several points of the lower end.

[0016] This allows for several vertical homogenization zones to be created upwards. The gas injectors transmit bubbles, preferably microbubbles, into the medium. The term "gas" refers to all gases in the air.

[0017] Alternatively, the recirculation pump is connected to the enclosure outlet.

[0018] This allows for efficient draw for a very large volume enclosure compared to the recirculation circuit, while avoiding pressure losses.

[0019] According to a variant, said outlet has a central position relative to the gas injectors and / or a central position in the lower end.

[0020] This allows for a vertical homogenization zone downwards at the center of the gas injectors, in the center of the enclosure.

[0021] According to a variant, said gas injector has a peripheral position relative to said outlet and / or a peripheral position in the lower end.

[0022] This allows for a vertical homogenization zone upwards around the said outlet, around the enclosure.

[0023] In one embodiment, the enclosure has a cylindrical shape and the gas injectors are angularly spaced within the enclosure about the central axis of the cylindrical shape.

[0024] This allows for a cylindrical shape that is easy to manufacture. Furthermore, a cylindrical shape makes it easier to observe the cell culture from several directions in space, compared to a parallelepiped or cubic shape, for example.

[0025] According to one variant, said gas injector is a gas diffuser.

[0026] This allows very fine bubbles to be generated to limit flow turbulence, improve the visibility of the cells and their lighting.

[0027] According to one variant, the recirculation circuit includes a transparent control window.

[0028] This allows for specific visual control or measurements to be taken using optical devices.

[0029] In one variant, the bioreactor forms a photobioreactor and the enclosure is mostly transparent.

[0030] This allows the cultivation of photosynthetic microorganisms such as green, blue or red algae.

[0031] Another subject of the invention relates to a method for manufacturing a bioreactor according to the invention, comprising the following steps: - setting up the enclosure; - connecting the recirculation pump to the outlet of the enclosure and to the recirculation circuit; - connecting the recirculation circuit to the inlet of the enclosure.

[0032] The invention will be further detailed by the description of non-limiting embodiments, and on the basis of the appended figures illustrating preferred embodiments of the invention, in which: - schematically illustrates a view in space of a photobioreactor structure according to a preferred embodiment of the invention; - schematically illustrates a front view of a piece of furniture according to the invention comprising the photobioreactor structure of the ; - schematically illustrates a front view of a lower end piece of the photobioreactor structure of the ; - schematically illustrates a top view of the lower end piece of the.

[0033] The invention firstly concerns the general structure of the photobioreactor. The structure comprises a cylindrical enclosure E, the lower base and / or the upper base of which are open. The enclosure E is used in a vertical position so that the main axis of the cylinder is vertical.

[0034] The structure further comprises a lower end piece I configured to be connected to said lower base by means of a lower connecting piece RI.

[0035] The structure further comprises an upper end piece S configured to be connected to said upper base by means of an upper connecting piece RS.

[0036] The connection is made with an I / S end piece which can be slightly larger or smaller, or of the same size (here diameter) as the corresponding part of the enclosure E. The RI, RS connection piece is configured as appropriate for a watertight connection.

[0037] According to the invention, the lower end piece I comprises a proximal portion PP and a distal portion PD better visible in.

[0038] The proximal portion PP is cylindrical in shape. It comprises a connecting part cooperating with said connecting part RI / RS and the transparent enclosure E to close the corresponding opening of the enclosure E in a sealed manner.

[0039] The distal portion PD is conical in shape. The base of this cone is continuous with the cylindrical proximal portion PP. This cone has a distal end AP, in the form of an apex (best seen at).

[0040] The lower end piece I may be of metallic type, for example stainless steel. Other materials may of course be considered within the scope of the invention, such as in particular plastic materials or structures produced by 3D printing.

[0041] Likewise, the upper end piece S comprises a proximal portion PP and a distal portion PD, preferably with the same characteristics as those detailed above.

[0042] Furthermore, according to the invention, said distal end AP comprises at least one mouthpiece orifice E1, S1 at the apex. The mouthpiece orifice may be equipped with a mouthpiece structure. These are in particular the upper and lower distal ends AP.

[0043] Furthermore, the proximal portion PP comprises at least one bypass orifice D1, D2, D3, D4 in a lateral position, or even several angularly spaced bypass orifices. The bypass orifices D1, D2, D3, D4 are equipped with bypass structures, better visible in figures 3 and 4. Pumps P1, P2 can also be provided.

[0044] In particular, the lower end piece I comprises a first set of several angularly spaced bypass ports, configured to receive gas injectors G inserted into the end piece. In the illustrated variant, there are 4 gas injectors arranged around the photobioreactor structure.

[0045] Furthermore, the lower end piece I comprises at least one bypass orifice configured to receive a probe, for example a pH probe, inserted into the end piece I.

[0046] In addition, a D2 bypass can be provided, connected to a liquid injector, by a sterile circuit allowing liquid to be injected into the medium without opening the reactor, limiting the risk of contamination. The liquid injector can be connected via an EV solenoid valve.

[0047] The invention also relates to a lower end piece I, and an upper end piece S as described above, as well as a corresponding mounting kit.

[0048] In use, the photobioreactor structure is arranged in a specific piece of furniture M, for example a cabinet, equipped with lighting means L, such as light strips comprising LEDs. The piece of furniture M may comprise panels and preferably a door. Thus, the continuous lighting of the photobioreactor can take place in the piece of furniture M without disturbing the outside of the piece of furniture M.

[0049] The cabinet M comprises a speaker support SE allowing the speaker E to be easily held in the cabinet M; and an end support SI allowing the end piece, here lower I, to be held in the cabinet M.

[0050] For this purpose, the speaker stand SE comprises a first circular receiving structure, receiving the speaker E. The first receiving structure can be removable, into two semicircles for example, with the semicircular parts being fixed by a hinge. Thus, this first receiving structure is opened to place the speaker E within it before closing it.

[0051] Furthermore, the end support SI comprises a second circular receiving structure, receiving the lower end piece I. The lower end piece I can be inserted therein by the tip, and be held there by the weight of the photobioreactor structure and the strength of the second receiving structure.

[0052] This type of M furniture serves as a support for control devices, monitoring devices or others, to participate in the operation of the photobioreactor.

[0053] We can also consider a piece of furniture in the form of strand supports without panels or doors.

[0054] The assembly of the photobioreactor structure is done in two main steps.

[0055] In a first step, the lower end piece I must be connected to said lower base of the enclosure E, by means of said lower connecting piece RI.

[0056] In a second step, the upper end piece S must be connected to the upper base of the enclosure E, by means of the said upper connecting piece RS.

[0057] Once the photobioreactor structure is assembled, all that remains is to insert it into the cabinet M. For this purpose, the enclosure holder SE can be opened to place the enclosure E. Then, the apex of the lower end piece I can be placed in the end holder SI.

[0058] Now regarding the arrangement, it can be adapted for a photobioreactor described previously, or for a bioreactor in the broad sense.

[0059] Thus, the bioreactor includes enclosure E, a recirculation circuit RC, and a recirculation pump P1.

[0060] The enclosure E comprises the lower end I having at least one outlet S1 for liquids (namely here the mouth S1); and an upper end S having at least one inlet D1 for liquids (namely here at the bypass orifice D1).

[0061] In particular, said outlet S1 has a central position at the center of the apex of the lower end I, relative to the gas injectors G which have a peripheral position. The gas injectors G are preferably gas diffusers, diffusing for example bubbles of less than 500µm. We can speak of microbubbling in the context of this part of the invention.

[0062] In the illustrated variant, the mouth orifice E1 is used to evacuate the gas injected into the enclosure E, as well as the foam created by microbubbling. The mouth orifice E1 also allows for overflow collection thanks to the injection of medium via the bypass orifice D2 (continuous collection). The mouth orifice E1 overflows into a sterilizable container, the gas is evacuated from this container through a filter.

[0063] Since enclosure E is cylindrical in shape, the gas injectors G are angularly spaced in enclosure E around the central axis of the cylindrical shape. There are at least four gas injectors G. This allows diffusion across the entire bioreactor of, for example, 200 mm in diameter. Each diffuser diffuses over a quarter of the enclosure's surface area of ​​revolution.

[0064] An individual injector device may be considered that can be inserted into the enclosure container. The injector and / or the injector gas inlet may or may not pass through a wall of the enclosure. Such an injector device may be in the shape of a ring or an arc of a circle.

[0065] The recirculation circuit RC is connected to said inlet D1 and said outlet S1. It allows liquid to be recirculated in a pipe of small diameter compared to enclosure E. This pipe measures, for example, 20 mm in diameter.

[0066] The recirculation pump P1 is connected to the outlet S1 and to the lower part of the recirculation circuit RC.

[0067] The upper part of the RC recirculation circuit is connected to the D1 input at the top of enclosure E.

[0068] According to the invention, the recirculation pump P1 is configured to produce a flow of liquid FL from said outlet S1 to said inlet D1 so that the flow of liquid FL in the enclosure E has an essentially vertical direction from top to bottom.

[0069] In addition, the gas injectors G are arranged at several points of the lower end I. Thus, they allow a vertical gas flow FG to be achieved from bottom to top.

[0070] The combination of the downward flow of liquid FL and the upward flow of gas FG allows for reverse circulation of gas and medium, homogenizing the culture and distribution of cells in enclosure E.

[0071] In the preferred embodiment, the recirculation circuit RC includes a transparent control window F. At this location, the wall is preferably completely transparent over a few centimeters. This window is preferably located at eye level so that it can be checked without bending down. For example, it is in a range between 1.60m and 1.75m from the ground.

[0072] The invention also relates to the method of manufacturing a bioreactor as described above.

[0073] In this process, the recirculation pump P1 is connected to the output S1 of the enclosure E, as well as to the recirculation circuit RC.

[0074] Then the recirculation circuit RC is connected to the D1 input of enclosure E.

[0075] Pump P2 is a pump used to completely or partially drain the photobioreactor.

[0076] Regarding specifically the arrangement of the gas injectors G (diffusers), it can be adapted for a photobioreactor described previously, or for a bioreactor in the broad sense.

[0077] Thus, the bioreactor comprises the enclosure E which comprises an enclosure wall, and a lower end I (here in the form of a separate end piece).

[0078] The lower end I comprises a lateral wall (here the proximal portion PP) and a bottom of a given shape (here the distal portion PD of conical shape).

[0079] The bioreactor further comprises the gas injectors G arranged in the PP side wall of the lower end I.

[0080] According to the invention, the gas injectors are arranged therein so that their gas bubbles rise in the medium while being in contact with the enclosure wall of the enclosure E.

[0081] In order to cover the entire enclosure wall, the gas injectors G are arranged at several points on the PP side wall, here at points angularly spaced around the central axis of the cylindrical enclosure wall.

[0082] In the illustrated variant, the enclosure wall is arranged vertically. The gas bubbles are therefore preferably projected onto it.

[0083] The invention also relates to the method of manufacturing a bioreactor as described above.

[0084] In this method, the gas injectors G are placed in the side wall PP of the lower end I, so that their gas bubbles rise into the medium while being in contact with the enclosure wall of the enclosure E.

Claims

Bioreactor comprising:- an enclosure (E) which comprises a lower end (I) having at least one outlet (S1) for liquids; and an upper end (S) having at least one inlet (D1) for liquids;- a recirculation circuit (RC) connected to said inlet (D1) and to said outlet (S1);- a recirculation pump (P1) connected to the enclosure (E) and to the recirculation circuit (RC), wherein the recirculation pump (P1) is configured to effect a flow of liquid from said outlet (S1) to said inlet (D1) such that the flow of liquid in the enclosure (E) has an essentially vertical downward direction;and the enclosure (E) further comprises at least one gas injector (G) arranged in the lower end (I),characterized in that said outlet (S1) has a central position relative to the gas injectors (G) and a central position in the lower end (I),and in that said gas injector (G) has a peripheral position relative to said outlet (S1) and a peripheral position in the lower end (I).; Bioreactor according to the preceding claim, characterized in that the enclosure (E) comprises several gas injectors (G) arranged at several points of the lower end (I). Bioreactor according to one of the preceding claims, characterized in that the recirculation pump (P1) is connected to the outlet (S1) of the enclosure (E). Bioreactor according to one of the preceding claims, characterized in that the enclosure (E) has a cylindrical shape and the gas injectors (G) are angularly spaced in the enclosure (E) around the central axis of the cylindrical shape. Bioreactor according to one of the preceding claims, characterized in that said gas injector (G) is a gas diffuser. Bioreactor according to one of the preceding claims, characterized in that the recirculation circuit comprises a transparent control window (F). Bioreactor according to one of the preceding claims, characterized in that it forms a photobioreactor and the enclosure (E) is mainly transparent. Method for manufacturing a bioreactor according to one of the preceding claims, comprising the following steps:- setting up the enclosure (E);- connecting the recirculation pump (P1) to the outlet (S1) of the enclosure (E) and to the recirculation circuit (RC);- connecting the recirculation circuit (RC) to the inlet (D1) of the enclosure (E).

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