METHOD FOR THE ADAPTIVE EVOLUTION OF LIVING CELLS BY CONTINUOUS CELL CULTURE

Abstract

The present application relates to a method for adaptive evolution of living cells by continuous culture of said living cells.

Claims

1. A method for adaptive evolution of living cells, excluding human embryonic stem cells, by continuously culturing said living cells, wherein n culture vessels (RCi) are used, i ranging from 1 to n, where n≥2, characterized in that said method comprises the following steps consisting of: a) introducing at least one liquid culture medium and living cells into each of the n culture vessels, b) in each of the n culture vessels, culturing said living cells according to a given selective regime, using predefined culture parameters, until a determined growth stage is reached in at least one of the n culture vessels, so as to obtain, in each of the n culture vessels, a suspension of living cells in said liquid culture medium, c) combining at least a portion of the suspensions of living cells from at least two culture vessels (RCi) obtained in step b) to obtain a mixed suspension of living cells, d) homogenizing the mixed suspension of living cells obtained in step c) to obtain a homogenized suspension of mixed living cells, e) distributing, into at least two culture vessels (RCi), at least part of the homogenized suspension of mixed living cells obtained in step d), f) repeating steps b) to e), g) collecting after several culture cycles living cells that have acquired a phenotype of interest in at least one of the n culture vessels.

2. The method according to claim 1, characterized in that the living cells are selected from human, animal, or plant eukaryotic or prokaryotic cells.

3. The method according to any one of claims 1 to 2, characterized in that the selective regime of step b) is selected from: chemostat, turbidostat, medium swap, and iterated batch.

4. The method according to any one of claims 1 to 3, characterized in that the predefined culture parameters of step b) are selected from: temperature, pH, cell density, culture medium composition, gas composition, exposure to electromagnetic radiation of a particular wavelength, exposure to a mutagenic agent, or a combination thereof.

5. The method according to any one of claims 1 to 4, characterized in that step c) consisting of combining at least part of the suspensions of living cells from at least two culture vessels (Ri) obtained in step b) is carried out either by using one of said at least two culture vessels as a mixing vessel, or in a mixing vessel independent of the at least two culture vessels and making it possible to accommodate all or some of the contents of said at least two culture vessels.

6. The method according to any one of claims 1 to 5, characterized in that step c) is carried out using a mixing vessel, and in that at least part of the suspension obtained in step b) is transferred from at least two culture vessels to at least one mixing vessel.

7. The method according to any one of claims 1 to 6, characterized in that the homogenization step d) is carried out in whole or in part by an agitation means selected from a mechanical agitator and an injection of a gas stream.

8. The method according to any one of claims 1 to 7, characterized in that step e) consists of transferring at least part of the homogenized suspension of mixed living cells obtained in step d) to at least two culture vessels (RCi).

9. The method according to claim 8, characterized in that the at least part of the suspension transferred in step e) corresponds to a fraction between 1 and 100% of the volume of said homogenized suspension of mixed living cells.

10. The method according to any one of claims 1 to 9, characterized in that when repeating step b), the selective regime and/or the culture parameters used during a culture cycle may be the same or different from those used during the preceding culture cycle.

11. The method according to any one of claims 1 to 10, characterized in that n culture vessels (RCi) are used, i ranging from 1 to n, n being at least equal to 2, and at least n−1 mixing vessels (RMj), j ranging from 1 to n−1, the at least n−1 mixing vessels being respectively a culture vessel (RCi) arranged to receive the contents of at least two culture vessels, said method being further characterized in that steps c) to e) are carried out as follows: i) transferring all or part of the suspension obtained in step b) from a culture vessel (RCi), known as the starting culture vessel, to a mixing vessel (RMj), known as the destination vessel, so as to perform a destination transfer, ii) homogenizing the suspension from the starting culture vessel (RCi) with that of the destination vessel (RCj) in the destination vessel (RMj), to obtain a homogenized suspension of mixed living cells, iii) transferring at least part of the suspension obtained in step ii) from the destination vessel (RMj), to the starting culture vessel (RCi), so as to perform a return transfer, iv) repeating the preceding steps i) to iii) while varying RCi and RMj so that all suspensions have been combined 2-by-2 at least once.

12. The method according to any one of claims 5 to 10, characterized in that the at least one mixing vessel is a single vessel, independent of the set of culture vessels, and is arranged to receive the contents of the n culture vessels.

13. The method according to any one of claims 1 to 10, characterized in that n culture vessels (RCi) are used, i ranging from 1 to n, n being at least equal to 2, and at least one mixing vessel (RM), the at least one mixing vessel being a single vessel independent of the set of culture vessels, and being arranged to receive the contents of the n culture vessels, characterized in that steps c) to e) are carried out as follows: c) transferring all or part of the suspension of living cells obtained in step b) from at least two culture vessels (RCi) to the at least one mixing vessel (RM) to obtain a suspension of mixed living cells, d) homogenizing the mixed suspension of living cells obtained in step c) in the at least one mixing vessel (RM), to obtain a homogenized suspension of mixed living cells, e) transferring at least part of the suspension obtained in step d) from the at least one mixing vessel (RM) to each of the at least two culture vessels (RCi).

Description

FIGURES

[0194] FIG. 1a represents a device for continuous culture of living cells according to a particular embodiment of the invention, the device comprising a sterile fluid supply unit and three culture vessels, all of the culture vessels being arranged to be respectively and successively a mixing vessel, one mixing vessel being arranged to receive the contents of at least two culture vessels.

[0195] FIG. 1b represents a device according to FIG. 1a and shows step c) of the method according to a particular embodiment of the invention wherein the entire suspension from a first culture vessel is transferred to a second culture vessel which becomes a mixing vessel.

[0196] FIG. 1c represents a device according to FIG. 1a and shows step e) of the method according to a particular embodiment of the invention wherein at least a fraction of the suspension obtained in step d) in the second culture vessel is transferred to the first culture vessel.

[0197] FIG. 2a represents a device for continuous culture of living cells according to a second embodiment, wherein the device comprises a single mixing vessel, independent of the set of n culture vessels, and is arranged to receive the contents of the set of n culture vessels.

[0198] FIG. 2b represents a device according to FIG. 2a and shows step c) of the method according to a particular embodiment of the invention wherein the entire suspension from the n culture vessels is transferred to the mixing vessel.

[0199] FIG. 2c represents a device according to FIG. 2a and shows step e) of the method according to a particular embodiment of the invention wherein at least a fraction of the suspension obtained in step d) from the mixing vessel is transferred to each of the n culture vessels.

[0200] FIG. 3 represents a device for continuous culture of living cells according to a third embodiment, wherein the device comprises four culture vessels, each of the culture vessels being connected individually, and independently of each other, to a sterile fluid supply unit, and wherein the four culture vessels are arranged so that they can be respectively and successively a mixing vessel, one mixing vessel being arranged to receive the contents of at least two culture vessels.

[0201] FIG. 4 represents a device for continuous culture of living cells according to a fourth embodiment, wherein the device comprises four culture vessels, each of the culture vessels being connected individually, and independently of each other, to a sterile fluid supply unit, and a single mixing vessel, independent of the set of 4 culture vessels, said mixing vessel being arranged to receive the contents of the set of 4 culture vessels.

[0202] FIG. 5 represents the evolution of a bacterial strain of the Pseudomonadacea family. In this figure, the total number of dilutions triggered each day is plotted against the number of days

[0203] FIG. 6 represents the adaptation of the bacterial strain of the Pseudomonadacea family at 30° C. by plotting the change in the temperature in each of the culture vessels RC1 and RC2 against the number of culture cycles.

[0204] FIG. 7 represents the adaptation of the bacterial strain of the Pseudomonadacea family at 30° C. by plotting the change in the temperature in each of the culture vessels RC1 and RC2 against the number of days of the experiment.

[0205] FIG. 8 represents the dilution rate adaptation of a bacterial strain of the Pseudomonadacea family grown at 25° C. either in a single 15 mL culture vessel (solid squares) or in a single 80 mL culture vessel (solid rounds) according to a method not in accordance with the invention. In this figure the dilution rate in hour.sup.−1 is a function of the number of days.

[0206] FIG. 9 shows the adaptive evolution at a temperature of 25° C. with a forced dilution rate of 0.2 hours.sup.−1 of a bacterial strain of the Pseudomonadacea family cultured with successive combination and separation of the suspensions between 2 15 mL culture vessels (RC1: solid diamonds; RC2: solid triangles) according to the method in accordance with the invention. In this figure the temperature (° C.) is a function of the number of days.

DETAILED DESCRIPTION

[0207] The design and functionality of the device for continuous culture of living cells for the evolutionary adaptation of said living cells are described in FIGS. 1a to 4.

[0208] The device for continuous cell culture of living cells, as shown in FIGS. 1a to 2c, comprises three culture vessels, a first culture vessel RC1, a second culture vessel RC2 and a third culture vessel RC3. The first culture vessel RC1 is adjacent to the second culture vessel RC2, which is adjacent to the third culture vessel RC3. The culture vessels are arranged to contain living cells and culture media, and allow the culture of said cells.

[0209] With reference to FIG. 1a, the culture device includes a sterile fluid supply unit 10 comprising an external gas source GS, a sterilizing solution reservoir AS, a cleaning solution reservoir AC, a rinsing solution reservoir AR, and three culture medium reservoirs M1, M2 and M3. The circulation of the fluids in the sterile fluid supply unit 10, namely the circulation of the gas, the circulation of the sterilizing, cleaning and rinsing solutions, and the circulation of the culture media, is achieved through the use of pumps and valves. The pumps and valves can be operated mechanically, for example, and can be controlled electrically and/or electronically, advantageously automatically using control means which are not shown. The sterile fluid supply unit 10 further comprises a harvesting means, advantageously a syringe 11, for introducing cells into the culture vessels and for sampling living cells that have acquired a phenotype of interest. For simplicity's sake, in FIGS. 1b, 1c, 2a, 2b, 2c, 3, and 4 the sterile fluid supply unit 10 is shown as a block or square.

[0210] With reference to FIGS. 1a, 1b, 1c, 2a, 2b, 2c, the culture device comprises a main supply line C10 and supply valves 1, 2, 3, Va1, Va2 and Va3 connected to the main supply line C10. Said supply line C10 and said valves are located at the bottom of the culture vessels. The sterile fluid supply unit 10 is connected to the three culture vessels via said supply line C10 and said valves. They allow the filling of gas, sterilizing solution, cleaning and rinsing solution, culture media and living cells into the culture vessels. They also allow the transfer of the contents of the culture vessels, for example when transferring all or part of the suspension from the mixing vessel to a culture vessel and allow the emptying of the culture vessel when transferring all or part of the suspension from the culture vessel to the mixing vessel. The supply valve Va1 allows the culture vessel RC1 to be filled or emptied. The supply valve Va2 allows the culture vessel RC2 to be filled or emptied. The supply valve Va3 allows the culture vessel RC3 to be filled or emptied. Valves 1, 2, 3, Va1, Va2 and Va3 are normally closed in the inactive state. When the supply valve Vai is in the open position, the culture vessel RCi can be filled or emptied. When the supply valve Vai is in the closed position, it is not possible to fill or empty the culture vessel RCi.

[0211] The culture device comprises three gas supply devices G1, G2 and G3. Each culture vessel RC1, RC2 and RC3 is connected to a gas supply device G1, G2 or G3 which makes it possible to inject a pressurized gas stream into the culture vessel, inject gas into the suspension, homogenize said suspension (bubbling agitation), and pressurize the culture vessel as needed. Each gas supply device G1, G2 or G3 is connected to the culture vessel from its lower part by means of a gas supply line CG opening into the culture vessel at a height of about one-quarter of the total height of the vessel from the bottom of said vessel.

[0212] The culture device comprises three discharge devices W1, W2 and W3. Each culture vessel RC1, RC2 and RC3 is connected to a discharge device W1, W2 or W3 making it possible to evacuate the gases injected into the suspension during the culture, as well as the evacuation of the gases, from the culture medium/media and the sterilization, cleaning and rinsing solutions during the filling operations of the culture vessel. The discharge device is located in the upper part of the culture vessel. The culture device comprises three discharge valves Vd1, Vd2 and Vd3. Each culture vessel RC1, RC2 and RC3 is connected to a discharge valve Vd1, Vd2 and Vd3, respectively, so as to control the discharge of the gases injected during the culture, as well as the discharge of the gases, from the culture medium/media and the sterilization solution AS, cleaning solution AC and rinsing solution AR during the filling operations of the culture vessel. The discharge valves Vd1, Vd2 and Vd3 are normally in the open position in the inactive state. When the discharge valve is in the open position, the culture vessel can be filled. When the discharge valve is in the closed position, the transfer of all or part of the suspension contained in the culture vessel to the mixing vessel can be done by pressurizing said culture vessel.

[0213] With reference to FIGS. 1a, 1b, 1c, the culture device comprises three leveling valves Vt1, Vt2 and Vt3. Each culture vessel RC1, RC2 and RC3 is connected to a leveling valve Vt1, Vt2 and Vt3 respectively via a leveling line CT. Each leveling line CT opens into a culture vessel at a height less than or equal to half the total height of the culture vessel from the lower part of said vessel. Each leveling valve Vt1, Vt2 and Vt3 is also connected to the main line C10. Each leveling valve controls the volume contained in each of the culture vessels, so that the volume remains constant when culture medium is added. The leveling valves Vt1, Vt2 and Vt3 are normally in the closed position in the inactive state. When the leveling valve is in the open position, a volume of suspension in excess of the suspension volume defined by the position of the leveling line is discharged from the culture vessel to the supply line C10.

[0214] FIGS. 1a, 1b and 1c represent a first particular embodiment wherein the three culture vessels are arranged to respectively and successively become a mixing vessel during the cell culture method.

[0215] The method for continuous cell culture of living cells associated with the culture device shown in FIGS. 1a, 1b and 1c will now be described.

[0216] According to FIG. 1a, each of the three culture vessels RC1, RC2, and RC3 comprises living cells in a culture medium. Each culture vessel RC1, RC2 and RC3 is filled to about half its capacity. The living cells are cultured according to a given selective regime, using defined culture parameters, until they reach a given growth stage, in order to obtain a suspension of living cells in each of the three culture vessels. For this purpose, a pressurized gas stream is injected into each of the three culture vessels via the gas supply device G1, G2 or G3 respectively, allowing the injection of gas and the homogenization of the suspension (bubbling agitation) inside each of the three culture vessels. Valves 1, 2, 3, 4, the leveling valves Vt1, Vt2 and Vt3, and the supply valves Va1, Va2 and Va3 are in the closed position. Only the discharge valves Vd1, Vd2 and Vd3 are in the open position.

[0217] Next, FIG. 1b represents step c) of the method according to the invention consisting of transferring the entire suspension obtained in step b) from at least one culture vessel (RCi) to the at least one mixing vessel and step d) consisting of mixing the suspension in step c) in the at least one mixing vessel.

[0218] According to FIG. 1b, all of the suspension in culture vessel RC1 is transferred to culture vessel RC2, as shown by arrow f12. The supply valves Va1, 2, Va2 are in the active position (in black in FIG. 1b) and therefore open, so that the suspension from the first vessel RC1 passes through said valves to the second culture vessel RC2. The discharge valve Vd1 of the first vessel is in the active position (black in FIG. 1b) and therefore closed, so as to pressurize the first culture vessel. The discharge valve Vd2 of the second vessel remains in the inactive and thus open position, so as to allow filling into the second culture vessel RC2. In order to allow the culture vessel RC1 to be emptied, the gas supply device G1 injects a pressurized gas stream, known as the transfer stream, via the gas supply line CG, which makes it possible to increase the pressure in the culture vessel RC1 and thus pushes the suspension like a syringe plunger. The culture vessel RC2 then becomes the mixing vessel and comprises both the suspension initially contained in the second culture vessel RC2 and the suspension from the first culture vessel RC1.

[0219] In a manner not illustrated, a pressurized gas stream is injected into the second culture vessel RC2 allowing, via the gas supply device G2, the gas injection of the suspension and the homogenization of the cell suspension (bubbling agitation) inside the culture vessel RC2.

[0220] Regarding the third culture vessel RC3, the culture step is maintained by injecting pressurized gas stream into the culture vessel RC3 and by keeping the valve Vd3 open.

[0221] Regarding culture vessel RC1, the sterilization, cleaning and rinsing steps are carried out by opening the valves 1, Va1. First, the sterilization step is started by adding a sterilizing solution from the sterilizing solution reservoir AS to the culture vessel RC1. The emptying of the sterilizing solution is done by the valve Va1. A cleaning solution is then applied from the cleaning solution reservoir AC to the culture vessel RC1. The emptying of the cleaning solution is done by the valve Va1. A rinsing solution is then applied from the rinsing solution reservoir AR to the culture vessel RC1. The emptying of the rinsing solution is done by valve Va1. These steps are not represented.

[0222] According to FIG. 1c, part of the suspension in culture vessel RC2 is transferred to culture vessel RC1, as shown by arrow f21. Leveling valve Vt2 and supply valves Va1 and 2 are in the active position (in black in FIG. 1c) and therefore open, so that part of the suspension from the second vessel RC2 passes through said valves to the first culture vessel RC1. The discharge valve Vd2 of the second vessel is in the active position (in black in FIG. 1c) and therefore closed, so as to allow the pressurization of RC2 and allow the transfer of half of the suspension to RC1. Discharge valve Vd1 of the first vessel remains in the inactive and thus open position, so as to allow filling into the first culture vessel RC1. In order to allow culture vessel RC2 to be emptied, gas supply device G2 injects a pressurized gas stream, known as the transfer stream, via the gas supply line CG, which increases the pressure in the second culture vessel RC2 and thus pushes the suspension like a syringe plunger via the transfer line.

[0223] In a manner not illustrated, a pressurized gas stream is injected into both culture vessels RC1 and RC2 respectively, via the gas supply device G1 and G2, allowing gas to be injected into the suspension and homogenization of the suspension (bubbling agitation) inside culture vessels RC1 and RC2.

[0224] According to FIG. 1b, all of the suspension in culture vessel RC3 is transferred to culture vessel RC1, as suggested by arrow f31. Supply valves Va3, 3, 2, Va1 are in the active position and therefore open, so that the suspension from the first vessel RC3 passes through said valves to the second culture vessel RC1. Discharge valve Vd3 of the third vessel is in the active position and thus closed, so as to pressurize the third culture vessel. Discharge valve Vd1 of the first vessel remains in the inactive and thus open position, so as to allow filling into the first culture vessel RC1. In order to allow culture vessel RC3 to be emptied, gas supply device G3 injects a pressurized gas stream, known as the transfer stream, via gas supply line CG, which makes it possible to increase the pressure in culture vessel RC3 and thus pushes the suspension like a syringe plunger. Culture vessel RC1 then becomes the mixing vessel and comprises both the suspension initially contained in the first culture vessel RC1 and the suspension from the third culture vessel RC3.

[0225] In a manner not illustrated, a pressurized gas stream is injected into the first culture vessel RC1 allowing gas to be injected, via gas supply device G1, into the suspension and the homogenization of the suspension (bubbling agitation) inside culture vessel RC1.

[0226] Regarding the second culture vessel RC2, the culture step is maintained by injecting pressurized gas stream into culture vessel RC2 and by keeping valve Vd2 open.

[0227] Regarding culture vessel RC3, the sterilization, cleaning and rinsing steps are carried out by opening valves 1, 2, 3, and Va3. First, the sterilization step is started by adding a sterilizing solution from the sterilizing solution reservoir AS to culture vessel RC3. The emptying of the sterilizing solution is done by valve Va3. A cleaning solution is then applied from the cleaning solution reservoir AC to culture vessel RC3. The emptying of the cleaning solution is done by valve Va3. A rinsing solution is then applied from the rinsing solution reservoir AR to culture vessel RC3. The emptying of the rinsing solution is done by valve Va3. These steps are not represented.

[0228] According to FIG. 1c, part of the suspension in culture vessel RC1 is transferred to culture vessel RC3, as suggested by arrow f13. Leveling valve Vt1 and supply valves 2, 3 and Va3 are in the active position and therefore open, so that part of the suspension from the first vessel RC1 passes through said valves to reach the third culture vessel RC3. Discharge valve Vd1 of the first vessel is in the active position and therefore closed, so as to allow the pressurization of RC1 and allow the transfer of half of the suspension to RC3. Discharge valve Vd3 of the third vessel remains in the inactive position and thus open, so as to allow the filling of the third culture vessel RC3. In order to allow culture vessel RC1 to be emptied, the gas supply device G1 injects a pressurized gas stream, known as the transfer stream, via the gas supply line CG, which increases the pressure in the first culture vessel RC1 and thus pushes the suspension like a syringe plunger via the transfer line.

[0229] In a manner not illustrated, a pressurized gas stream is injected into both culture vessels RC1 and RC3 respectively, allowing gas to be injected, via the gas supply device G1 and G3, into the suspension and homogenization of the cell suspension (bubbling agitation) inside culture vessels RC1 and RC3.

[0230] According to FIG. 1b, all of the suspension in culture vessel RC2 is transferred to culture vessel RC3, as suggested by arrow f23. Supply valves Va2, 3, Va3 are in the active position and therefore open, so that the suspension from the second vessel RC2 passes through said valves to the third culture vessel RC3. Discharge valve Vd2 of the second vessel is in the active position and thus closed, so as to pressurize the second culture vessel. Discharge valve Vd3 of the third vessel remains in the inactive position and thus open, so as to allow filling into the third culture vessel RC3. In order to allow culture vessel RC1 to be emptied, the gas supply device G1 injects a pressurized gas stream, known as the transfer stream, via the gas supply line CG, which makes it possible to increase the pressure in culture vessel RC1 and thus pushes the suspension like a syringe plunger. Culture vessel RC3 then becomes the mixing vessel and comprises both the suspension initially contained in the third culture vessel RC3 and the suspension from the second culture vessel RC2.

[0231] In a manner not illustrated, a pressurized gas stream is injected into the second culture vessel RC3 allowing, via the gas supply device G3, the gas injection of the suspension and the homogenization of the cell suspension (bubbling agitation) inside culture vessel RC3.

[0232] Regarding the first culture vessel RC1, the culture step is maintained by injecting pressurized gas stream into culture vessel RC1 and by keeping valve Vd1 open.

[0233] Regarding culture vessel RC2, the sterilization, cleaning and rinsing steps are carried out by opening valves 1, 2, and Va2. First, the sterilization step is started by adding a sterilizing solution from the sterilizing solution reservoir AS to culture vessel RC2. The emptying of the sterilizing solution is done by valve Va2. A cleaning solution is then applied from the cleaning solution reservoir AC to culture vessel RC2. The emptying of the cleaning solution is done by valve Va2. A rinsing solution is then applied from the rinsing solution reservoir AR to culture vessel RC2. The emptying of the rinsing solution is done by valve Va2. These steps are not represented.

[0234] According to FIG. 1c, part of the suspension in culture vessel RC3 is transferred to culture vessel RC2, as suggested by arrow f32. Leveling valve Vt3 and supply valves Va2 and 3 are in the active position and therefore open, so that part of the suspension from the third vessel RC3 passes through said valves to reach the second culture vessel RC2. Discharge valve Vd3 of the third vessel is in the active position and therefore closed, so as to allow the pressurization of RC3 and allow the transfer of half of the suspension to RC2. Discharge valve Vd2 of the second vessel remains in the inactive and thus open position, so as to allow the filling of the second culture vessel RC2. In order to allow culture vessel RC3 to be emptied, the gas supply device G3 injects a pressurized gas stream, known as the transfer stream, via the gas supply line CG, which increases the pressure in the third culture vessel RC3 and thus pushes the suspension like a syringe plunger via the transfer line.

[0235] In a manner not illustrated, a pressurized gas stream is injected into both culture vessels RC2 and RC3 respectively, via the gas supply device G2 and G3, allowing gas to be injected into the suspension and homogenization of the cell suspension (bubbling agitation) inside culture vessels RC2 and RC3.

[0236] A pressurized gas stream is again injected into each of the three culture vessels via the gas supply device G1, G2 or G3 respectively, allowing the injection of gas into the suspension and the homogenization of the cell suspension (bubbling agitation) inside each of the three culture vessels. Supply valves 1, 2, 3, leveling valves Vt1, Vt2 and Vt3, and supply valves Va1, Va2 and Va3 are in the closed position. Only discharge valves Vd1, Vd2 and Vd3 are in the open position.

[0237] The preceding steps are repeated as many times as necessary until cells having a phenotype of interest are obtained in the culture vessels.

[0238] When the cells have acquired a phenotype of interest, the collection step is performed; this step is not shown. The collection step can also be performed after several mixing cycles. Preferably, the collection step is performed using a harvesting means, in particular by syringe 11.

[0239] Once the collection step has been carried out, all the culture vessels are emptied, by closing discharge valves Vd1, Vd2 and Vd3, injecting pressurized gas stream from the external gas source GS, and opening supply valves Va1, va2, Va3 and 2, 3. The sterilization, cleaning, and rinsing steps are then carried out by opening valves 1, Va1, Vd1, 2, Va2, Vd2, 3, Va3, Vd3. First, the sterilization step is started by adding a sterilizing solution from the sterilizing solution reservoir AS to each of culture vessels RC1, RC2, and RC3. The emptying of the sterilizing solution is carried out by the respective valves Va1, Va2 and Va3. A cleaning solution is then applied from the cleaning solution reservoir AC to each of culture vessels RC1, RC2, and RC3. The emptying of the cleaning solution is carried out by the respective valves Va1, Va2 and Va3. A rinsing solution is then applied from the rinsing solution reservoir AR to each of culture vessels RC1, RC2, and RC3. The emptying of the rinsing solution is carried out by the respective valves Va1, Va2 and Va3. These steps are not shown in FIGS. 1a, 1b and 1c.

[0240] FIGS. 2a, 2b and 2c represent a second particular embodiment wherein the culture device comprises a single mixing vessel, independent of the culture vessels, and is arranged to receive the contents of all the culture vessels.

[0241] The culture device of FIG. 2a will be described only in terms of its differences from the culture device in FIG. 1a. The culture device of FIG. 2a further comprises a single mixing vessel RM, independent of culture vessels RC1, RC2 and RC3, and arranged to receive the contents of said culture vessels. The culture device comprises a gas supply device GM connected to the mixing vessel RM at the top of said mixing vessel. The gas supply device GM allows the mixing vessel to be pressurized by injecting a gas stream. The device includes a gas supply valve Vgm controlling the gas supply to the gas supply device GM. The culture device comprises a discharge device Wm connected to the mixing vessel RM in the upper part of said mixing vessel. The discharge device (Wm) allows the evacuation of gases during mixing. The device comprises a discharge valve Vdm controlling the discharge of gases during mixing. The discharge valve Vdm is normally in the closed position in the inactive state. When the discharge valve Vdm is in the open position, the filling of the mixing vessel and/or evacuation of gases during mixing can be carried out. The culture device comprises a supply valve Vam controlling the filling and emptying of the mixing vessel. It is located in the lower part of the mixing vessel and is connected to the main supply line C10, allowing the filling of the mixing vessel when transferring all or part of the suspension from the culture vessels to the mixing vessel and allowing the emptying of the mixing vessel when transferring all or part of the suspension from the mixing vessel to a culture vessel. The supply valve Vam is normally in the closed position in the inactive state. When the supply valve Vam is in the open position, the mixing vessel can be filled or emptied. When the supply valve Vam is in the closed position, it is not possible to fill or empty the mixing vessel.

[0242] The method for continuous cell culture of living cells associated with the culture device shown in FIGS. 2a, 2b and 2c will now be described.

[0243] According to FIG. 2a, each of the three culture vessels RC1, RC2, and RC3 comprises living cells in a culture medium. They are filled to about 75% of their total capacity. The mixing vessel RM is empty and valves Vdm, Vam and Vgm are in the closed position.

[0244] According to FIG. 2b, the suspensions contained in culture vessels RC1, RC2 and RC3 are transferred to mixing vessel RM, as represented by arrows f1m, f2m, f3 m and fmp. For this purpose, the transfer of the suspension from culture vessel RC1 to mixing vessel RM is carried out by opening supply valves Va1, 2, 3, Vam and discharge valve Vdm. The transfer of the suspension from culture vessel RC2 to mixing vessel RM is carried out by opening supply valves Va2, 3, Vam and discharge valve Vdm. The transfer of the suspension from culture vessel RC3 to mixing vessel RM is carried out by opening supply valves Va3, Vam and discharge valve Vdm. Opening discharge valve Vdm allows the mixing vessel RM to be filled. The transfer of the suspensions is achieved by means of gas supply devices G1, G2 and G3, as described above.

[0245] Once the mixing vessel has been filled with all the suspensions from culture vessels RC1, RC2 and RC3, supply valve Vam is placed in the closed position.

[0246] Regarding culture vessels RC1, RC2 and RC3, the sterilization, cleaning and rinsing steps are carried out by opening valves 1, Va1, Vd1, 2, Va2, Vd2, 3, Va3, Vd3. First, the sterilization step is started by adding a sterilizing solution from the sterilizing solution reservoir AS to each of culture vessels R1C, RC2, and RC3. The emptying of the sterilizing solution is carried out by the respective valves Va1, Va2 and Va3. A cleaning solution is then applied from the cleaning solution reservoir AC to each of culture vessels RC1, RC2, and RC3. The emptying of the cleaning solution is carried out by the respective valves Va1, Va2 and Va3. A rinsing solution is then applied from the rinsing solution reservoir AR to each of culture vessels RC1, RC2, and RC3. The emptying of the rinsing solution is carried out by the respective valves Va1, Va2 and Va3. These steps are not shown in FIG. 2b.

[0247] According to FIG. 2c, part of the suspension in mixing vessel RM is transferred to culture vessel RC1, as shown by arrows fms and fm1. For this purpose, the transfer is made by opening valves Vgm, Vam, 3, 2, Va1 and Vd1. Valve Vdm is in the closed position and a pressurized gas stream is injected into mixing vessel Rm, via the opening of gas supply valve Vgm, allowing mixing vessel RM to be emptied. Opening discharge valve Vd1 allows culture vessel RC1 to be filled. Valves Va2 and Va3 are in the closed position.

[0248] As suggested by FIG. 2c, a portion of the suspension in mixing vessel RM is then transferred to the second culture vessel RC2, as represented by dashed arrows fms and fm2. For this purpose, the transfer is made by opening valves Vgm, Vam, 3, Va2 and Vd2. Valve Vdm is in the closed position and a pressurized gas stream is injected into mixing vessel Rm, via the opening of gas supply valve Vgm, allowing mixing vessel RM to be emptied. Opening discharge valve Vd2 allows culture vessel RC2 to be filled. Valves Va1 and Va3 are in the closed position.

[0249] As suggested by FIG. 2c, a portion of the suspension in mixing vessel RM is then transferred to the third culture vessel RC3, as represented by dashed arrows fms and fm3. For this purpose, the transfer is made by opening valves Vgm, Vam, Va3, and Vd3. Valve Vdm is in the closed position and a pressurized gas stream is injected into mixing vessel Rm, via the opening of gas supply valve Vgm, allowing mixing vessel RM to be emptied. Opening discharge valve Vd3 allows culture vessel RC3 to be filled. Valves Va1 and Va2 are in the closed position.

[0250] Once the transfer has been completed and the mixing vessel has been totally emptied, the sterilization, cleaning and rinsing steps are carried out by opening valves 1, 2, 3, Vam and Vdm. First, the sterilization step is started by adding a sterilizing solution from the sterilizing solution reservoir AS to the culture vessel RM. The emptying of the sterilizing solution is done by valve Vam. A cleaning solution is then applied from the cleaning solution reservoir AC to mixing vessel RM. The emptying of the cleaning solution is done by valve Vam. A rinsing solution is then applied from the rinsing solution reservoir AR to the mixing vessel RM. The emptying of the rinsing solution is done by valve Vam. These steps are not shown in FIGS. 2a to 2c.

[0251] A pressurized gas stream is again injected into each of the three culture vessels RC1, RC2 and RC3 via the gas supply device G1, G2 or G3 respectively, allowing the injection of gas and the homogenization of the suspension (bubbling agitation) inside each of the three culture vessels. Supply valves 1, 2, 3, leveling valves Vt1, Vt2 and Vt3, and supply valves Va1, Va2 and Va3 are in the closed position. Only discharge valves Vd1, Vd2 and Vd3 are in the open position.

[0252] The collection step, after several culture cycles of the living cells that have acquired a phenotype of interest in the culture vessels, is then performed, this step not being shown.

[0253] Once the collection step has been completed, all of the culture vessels are emptied in the same manner as in the first embodiment. These steps are not shown in FIGS. 2a to 2c.

[0254] The culture device of FIG. 3 will be described only in terms of its differences from the culture device in FIG. 1a.

[0255] The device for continuous cell culture of living cells, as shown in FIG. 3, comprises four culture vessels, a first culture vessel RC1, a second culture vessel RC2, a third culture vessel RC3 and a fourth vessel RC4. In this embodiment, the set of culture vessels is arranged to respectively and successively become a mixing vessel during the cell culture method, each mixing vessel being arranged to receive the contents of at least two culture vessels. Each of culture vessels RC1, RC2, RC3, and RC4 is connected individually, and independently of each other, to a sterile fluid supply unit 10 described above. The sterile fluid supply unit (10) is connected to its culture vessel via a supply valve 1.

[0256] According to FIG. 3, each of the four culture vessels comprises living cells in a culture medium. The living cells are put in culture to obtain a suspension. For this purpose, a pressurized gas stream is injected into each of the four culture vessels via the gas supply device G1, G2, G3, or G4 respectively, allowing the injection of gas and the homogenization of the suspension (bubbling agitation) inside each of the four culture vessels RC1, RC2, RC3 and RC4. The supply valves 1, leveling valves Vt1, Vt2, Vt3, and Vt4, and supply valves Va1, Va2, Va3, and Va4 are in the closed position. Only discharge valves Vd1, Vd2, Vd3, and Vd4 are in the open position.

[0257] The culture device comprises valves V10, V20, V30, and V40 allowing the interconnection of the four culture vessels, and are normally in the closed position in the inactive state.

[0258] The method of continuous cell culture of living cells associated with the culture device shown in FIG. 3 is similar to the culture method associated with FIGS. 1a, 1b and 1c.

[0259] First, all of the suspension in the first culture vessel RC1 is transferred to culture vessel RC2, as suggested by arrow f12. For this purpose, the transfer is made by opening valves Va1, V10, V20, Va2, and Vd2. Valve Vd1 is in the closed position, allowing culture vessel RC1 to be emptied by the gas supply device as described above. Opening discharge valve Vd2 allows culture vessel RC2 to be filled. Culture vessel RC2 then becomes the mixing vessel and comprises both the suspension initially contained in culture vessel RC2 and the suspension from culture vessel RC1. A pressurized gas stream is injected into culture vessel RC2 allowing, via gas supply device G2, the gas injection of the suspension and the homogenization of the cell suspension (bubbling agitation) inside culture vessel RC2. In culture vessels RC3 and RC4, the culture step is maintained by injecting pressurized gas stream into culture vessel RC3 and RC4 and by keeping valves Vd3 and Vd4 open.

[0260] In culture vessel RC1, the sterilization, cleaning and rinsing steps are then carried out by opening valves 1, Va1, Vd1. First, the sterilization step is started by adding a sterilizing solution from the sterilizing solution reservoir AS to culture vessel RC1. The emptying of the sterilizing solution is done by valve Va1. A cleaning solution is then applied from the cleaning solution reservoir AC to culture vessel RC1. The emptying of the cleaning solution is done by valve Va1. A rinsing solution is then applied from the rinsing solution reservoir AR to culture vessel RC1. The emptying of the rinsing solution is done by valve Va1. These steps are not shown in FIG. 3.

[0261] Next, part of the suspension in culture vessel RC2 is transferred to culture vessel RC1, as shown by arrow f21. For this purpose, the transfer is made by opening valves Vt2, V20, V10, Va1, and Vd1. Valve Vd2 is in the closed position, allowing the emptying of culture vessel RC2 by applying pressure on the suspension using the gas injected by GC2. Opening discharge valve Vd1 allows culture vessel RC1 to be filled. A pressurized gas stream is injected into both culture vessels RC1 and RC2 respectively, allowing gas to be injected, via the gas supply device G1 and G2, into the suspension and homogenization of the cell suspension (bubbling agitation) inside culture vessels RC1 and RC2. In the third culture vessels RC3 and RC4, the culture step is maintained by injecting pressurized gas stream into culture vessels RC3 and RC4 and by keeping valves Vd3 and Vd4 open.

[0262] Then, the entire suspension in culture vessel RC3 is transferred to the first culture vessel RC1, as suggested by dotted arrow f13. For this purpose, the transfer is made by opening valves Va1, V10, V30, Va3 and closing valve Vd3. Then, the sterilization, cleaning, and rinsing of vessel RC3 is performed as described above for vessel RC1. Next, after mixing, part of the suspension in culture vessel RC1 is transferred to culture vessel RC3, as shown by arrow f31. For this purpose, the transfer is made by opening valves Vt1, V30, V10, Va1 and closing valve Vd1.

[0263] The entire suspension in culture vessel RC1 is transferred to the first culture vessel RC4, as suggested by dotted arrow f14. For this purpose, the transfer is made by opening valves Va1, V10, V40, Va4 and closing valve Vd1. Next, after mixing, part of the suspension in culture vessel RC4 is transferred to culture vessel RC1, as shown by arrow f41. For this purpose, the transfer is made by opening valves Vt4, V40, V10, Va1 and closing valve Vd4.

[0264] Then, the entire suspension in culture vessel RC2 is transferred to the third culture vessel RC3, as suggested by dotted arrow f23. For this purpose, the transfer is made by opening valves Va2, V20, V30, Va3 and closing valve Vd2. Then, the sterilization, cleaning, and rinsing of vessel RC2 is performed as described above for vessel RC1. Next, after mixing, part of the suspension in culture vessel RC3 is transferred to culture vessel RC2, as suggested by arrow f32. For this purpose, the transfer is made by opening valves Vt3, V30, V20, Va2 and closing valve Vd3.

[0265] The entire suspension in culture vessel RC4 is transferred to the first culture vessel RC2, as suggested by dotted arrow f24. For this purpose, the transfer is made by opening valves Va2, V20, V40, Va4 and closing valve Vd4. Then, the sterilization, cleaning, and rinsing of vessel RC4 is performed as described above for vessel RC1. Next, after mixing, part of the suspension in culture vessel RC2 is transferred to culture vessel RC4, as suggested by dotted arrow f42. For this purpose, the transfer is made by opening valves Vt4, V40, V20, Va2 and closing valve Vd2.

[0266] Lastly, the entire suspension in culture vessel RC3 is transferred to the first culture vessel RC4, as suggested by dotted arrow f34. For this purpose, the transfer is made by opening valves Va3, V30, V40, Va4 and closing valve Vd3. Next, after mixing, part of the suspension in culture vessel RC4 is transferred to the third culture vessel RC3, as suggested by dotted arrow f43. For this purpose, the transfer is made by opening valves Vt4, V40, V30, Va3 and closing valve Vd4.

[0267] The culture device of FIG. 4 will be described only in terms of its differences from the culture device in FIG. 3. FIG. 4 represents a fourth particular embodiment comprising four culture vessels and a single mixing vessel RM, independent of the set of culture vessels, and which is arranged to receive the contents of the set of culture vessels RC1, RC2, RC3 and RC4. Each of culture vessels RC1, RC2, RC3, and RC4 is connected individually, and independently of each other, to a sterile fluid supply unit 10 described above. The sterile fluid supply unit (10) is connected to its culture vessel via a supply valve 1.

[0268] The method of continuous cell culture of living cells associated with the culture device shown in FIG. 4 is similar to the culture method associated with FIGS. 2a, 2b, and 2c.

[0269] Advantageously, the suspensions contained in culture vessels RC1, RC2, RC3 and RC4 are transferred to the mixing vessel RM.

[0270] The sterilization, cleaning, and rinsing operations of vessels RC1, RC2, RC3 and RC4 are then applied.

[0271] Once the mixing step is completed, part of the suspension contained in the mixing vessel RM is then transferred to the first culture vessel RC1. Part of the suspension in the mixing vessel RM is then transferred to the second culture vessel RC2. Part of the suspension in the mixing vessel RM is then transferred to the third culture vessel RC3. Part of the suspension in the mixing vessel RM is then transferred to the fourth culture vessel RC4.

[0272] Next, the operations of sterilization, cleaning and rinsing of the mixing vessel RM are applied.

Examples

[0273] In all the examples described hereafter, the growth regime is that of the turbidostat for which the dilution rate (in hours.sup.−1) is defined as the ratio between the flow rate of growth medium, to maintain a constant concentration of microorganisms in the culture chamber during the evolution, and the volume of the culture chamber. In these experiments, the turbidostat was implemented such that the dilution rate was equivalent to the population growth rate.

Example 1: Evolutionary Adaptation of a Bacterial Strain to a Temperature of 30° C.

[0274] At a suboptimal temperature, that is below the optimal temperature, the growth rate of an organism is lower than at the optimal temperature. When the organism is intended for use in a context where the temperature is lower than the optimal temperature, it is relevant to adapt this organism to said temperature.

[0275] In experiments 1 and 2 described below, the strain used is a soil bacterium of the family Pseudomonadaceae. The specific growth rate of this strain on synthetic growth medium containing 20 g/L sucrose as a carbon source is 0.315 h.sup.−1 at the optimum temperature of 35° C.

[0276] For each of the two experiments, the objective is to adapt this strain to a temperature of 30° C., with the aim of achieving a growth rate at 30° C. comparable to that of the starting strain at 35° C.

[0277] To achieve this objective, two adaptive evolution experiments were conducted using the same strain described above, in the same setup and using the same reference medium described above.

1/ Experiment 1: Comparative Method not Part of the Invention: Turbidostat at the Target Temperature of 30° C.

[0278] Experiment 1 consisted of the evolutionary adaptation of the aforementioned bacterium according to a simple evolutionary protocol not in accordance with the present invention, implementing only the turbidostat selective regime in a single culture vessel.

[0279] At the beginning of the experiment, the culture vessel is inoculated with the strain described above.

[0280] During the experiment, the temperature is kept fixed and equal to 30° C.

[0281] The selective regime is implemented in a discontinuous way as follows: every ten minutes, the transparency measured by optical measurement is compared to a threshold arbitrarily set at 80:

[0282] If the measured value is above the threshold, no action is triggered,

[0283] If the measured value is below the threshold, a dilution of the suspension is performed by adding a volume of 4 mL of the growth medium described above in the culture vessel, keeping the volume of the suspension constant at 13.5 mL in the culture vessel, withdrawing the same volume V of suspension present in the culture vessel.

Results Obtained:

[0284] The results obtained are presented in FIG. 5.

[0285] The number of 29 daily dilutions, equivalent to a theoretical dilution rate or growth rate of 0.358 h.sup.−1, is reached after 20 days of adaptive evolution in turbidostat at 30° C.

2/ Experiment 2: Method According to the Invention Using Two Culture Vessels

[0286] Experiment 2 consisted of the evolutionary adaptation of the same bacterium mentioned above according to the method of the invention and implementing two culture vessels RC1 and RC2 at distinct temperatures. This protocol is implemented in the same device as the one used for experiment 1.

[0287] At the beginning of the experiment, each culture vessel RC1 and RC2 is inoculated with the same strain described above that was used for inoculation at the beginning of experiment 1 and the initial temperature is 35° C. in both RC1 and RC2.

[0288] In each of the two culture vessels RC1 and RC2, the selective regime is the turbidostat, implemented in a discontinuous manner as described in experiment 1, with the same parameters as used in experiment 1:

[0289] Growth medium as defined above

[0290] Transparency threshold=80,

[0291] Dilution of the suspension by adding a volume of 4 mL of the growth medium in the culture vessel, keeping the volume of the suspension constant at 13.5 mL in the culture vessel, withdrawing the same volume V of suspension present in the culture vessel.

[0292] The growth stage is defined by a duration arbitrarily set at 12 hours, at the end of which step c) is carried out by transferring the entire contents of the culture vessel RC1 to the culture vessel RC2, which becomes the mixing vessel. Each cycle therefore has a duration of 12 hours and there are two cycles per day.

[0293] At each new cycle, the temperature in each of these two culture vessels RC1 and RC2 is automatically adjusted according to the calculation of the average dilution rate in vessel RC1, with the instruction to decrease the culture temperature as soon as the dilution rate in vessel RC1, averaged over the cycle just completed, is greater than a threshold arbitrarily set at the value of 0.358 h.sup.−1 obtained during experiment 1 after 20 days in a turbidostat at 30° C.

[0294] Table 1 below lists the temperatures applied in each of the two culture vessels RC1 and RC2 during successive cycles, taking into account that only one cycle took place on day 7 of the experiment.

TABLE-US-00001 TABLE 1 Temperature of Temperature of Duration of the Vessel 1 (° C.) at Vessel 2 (° C.) at experiment the start of the the start of the (days) Cycle cycle cycle 1 1 35 35 1 2 34.9 34.8 2 3 34.9 34.8 2 4 34.8 34.9 3 5 34.6 34.8 3 6 34.4 34.6 4 7 34.1 34.4 4 8 33.7 34.1 5 9 33.3 33.7 5 10 32.5 33.3 6 11 31.7 32.5 6 12 30.1 31.7 7 13 28.5 30.1 8 14 28.5 30.1 8 15 29.3 30.1 9 16 29.7 30.1 9 17 29.9 30.1 10 18 30 30.1 10 19 29.9 30 11 20 29.8 29.9 11 21 29.6 29.8

Results Obtained:

[0295] The results obtained are presented in FIGS. 6 and 7.

[0296] During the 18th culture cycle, i.e. after 10 days of adaptive evolution, the temperature in culture vessel RC1 is 30° C. During this cycle, the average dilution rate is 0.387 h.sup.−1, higher than the average dilution rate obtained after 20 days of adaptive evolution in turbidostat at 30° C. in the same device.

[0297] To compare growth rates, it is legitimate to compare the dilution rate over one cycle of experiment 2 to that measured in experiment 1 because, during each cycle, the suspension is exposed to the turbidostat selective regime implemented according to the same batch protocol, with the same parameters and in the same device (culture vessel RC1 in experiment 2 being precisely the same culture vessel as the one used in experiment 1).

[0298] Thus, it can be stated that the suspension grown in the 18th cycle, on day 10 of Experiment 2, displays a slightly higher growth rate at 30° C. than that obtained on day 20 of turbidostat Experiment 1 at 30° C.

Conclusion

[0299] Therefore, the protocol in Experiment 2, which implements parallel evolutionary adaptation of two subpopulations that are regularly mixed and redistributed into the two culture vessels according to the method of the invention, made it possible to adapt a bacterium to a suboptimal temperature twice as fast as with a conventional turbidostat regime where a single population is grown in a single vessel.

Example 2: Evolutionary Adaptation of a Bacterial Strain to a Temperature of 25° C.

[0300] For both experiments, the objective is to adapt this strain to a temperature of 25° C. while increasing the growth rate.

[0301] To achieve this objective, two adaptive evolution experiments were conducted using the same strain as used in Example 1, initially evolved at 35° C. with a dilution rate of 0.315 hours.sup.−1, within the same setup and using the same reference medium.

1/ Experiment 3: Comparative Method not Part of the Invention: Turbidostat at the Target Temperature of 25° C.

[0302] This experiment was carried out under the same conditions as shown above in Example 1 for Experiment 1/but at a temperature of 25° C.

[0303] Experiments were performed with single culture vessels of either 15 mL or 80 mL volume.

Results Obtained:

[0304] The results are shown in the attached FIG. 8. In this figure the dilution rate (hours.sup.−1) is a function of the number of days. The curve with the solid circles corresponds to the experiment performed in the 80 mL vessel and the curve with the solid squares corresponds to the experiment performed in the 15 mL vessel.

[0305] It is observed that the dilution rate or growth rate of 0.2 hours.sup.−1 at 25° C. is obtained in 18 days in both the 15 mL volume culture vessel and the 80 mL volume vessel, indicating that the volume of the culture vessel is not a critical parameter for the evolution of microorganisms.

2/ Experiment 4: Method According to the Invention Using Two Culture Vessels with a Prescribed Dilution Rate of 0.2 Hours.SUP.−1

[0306] Experiment 4 consisted of the evolutionary adaptation of the same bacterium according to the method of the invention using two culture vessels RC1 and RC2, each having a volume of 15 mL, according to the protocol described in Experiment 2 of Example 1, but varying the temperature from 35° C. to 25° C.

[0307] At the beginning of the experiment, each culture vessel RC1 and RC2 is inoculated with the same strain as in Example 1 and the initial temperature is 35° C. in both vessels RC1 and RC2.

[0308] At each new cycle, the temperature in each of these two culture vessels RC1 and RC2 is automatically adjusted according to the calculation of the average dilution rate in the vessel, with the instruction to decrease the culture temperature as soon as the dilution rate in culture vessel RC1, averaged over the cycle just completed, is greater than a threshold arbitrarily set at the value of 0.2 hours.sup.−1, value obtained during Experiment 3 after 18 days in a turbidostat at 25° C.

[0309] Table 2 below lists the temperatures applied in each of the two culture vessels RC1 and RC2 during successive cycles.

TABLE-US-00002 TABLE 2 Temperature of Temperature of Duration of the Vessel 1 (° C.) at Vessel 2 (° C.) at experiment the start of the the start of the (days) Cycle cycle cycle 0.0 1 35 35 0.5 2 34.9 34.6 1.0 3 34.6 34.4 1.5 4 34.4 34 2.0 5 34.4 34 2.5 6 34 33.6 3.0 7 33.6 32.8 3.5 8 32.8 31.9 4.0 9 31.9 30.9 4.5 10 30.9 29.9 5.0 11 29.9 28.9 5.5 12 28.9 27.9 6.0 13 27.9 26.9 6.5 14 26.9 25.9 7.0 15 25.9 24.9

[0310] The results obtained are shown in the attached FIG. 9.

[0311] In this figure the temperature (° C.) is a function of the number of days. The curve with the solid diamonds corresponds to the experiment performed in vessel RC1 and the curve with the solid squares corresponds to the experiment performed in vessel RC2.

[0312] These results show that the adaptation of the bacteria at 25° C. for a dilution rate or growth rate of 0.2 hours.sup.−1 is obtained in 7 days according to the method of the invention implementing the repeated mixing of the contents of 2 culture vessels, i.e. 2.6 times faster than according to the method of Experiment 3/not in accordance with the invention.

[0313] The benefit observed on the evolutionary adaptation method is indeed caused by the method according to the invention consisting of periodically combining and separating the suspensions coming from at least two culture vessels.