BIOREACTOR

Abstract

A temporary immersion bioreactor (10) for in vitro production of differentiated plant biomass including a growth chamber (12) having one or more transparent side walls (14) and a mesh bottom (16), the mesh bottom (16) defining a plurality of pores (18) to receive plant material. The bioreactor (10) includes a flexible bag (20) formed from a transparent material, the flexible bag (20) having a sealable opening and being dimensioned to receive the growth chamber (12) together with a liquid medium (22). The bioreactor (10) also includes an outer chamber (24) having one or more transparent side walls (26). the outer chamber (24) is formed to correspond in shape to the growth chamber (12) and dimensioned to receive the growth chamber (12) within the flexible bag (20) so that the mesh bottom (16) of the growth chamber (12) faces a bottom (28) of the outer chamber (24) that in use is intended to rest on a support surface. Movement of the growth chamber (12) within the outer chamber (24) is restrained to movement along a single axis such that the mesh bottom (16) of the growth chamber (12) moves towards and away from the bottom (28) of the outer chamber (24).

Claims

1. A temporary immersion bioreactor for in vitro production of differentiated plant biomass comprising: a growth chamber having one or more transparent side walls and a mesh bottom, the mesh bottom defining a plurality of pores to receive plant material; a flexible bag formed from a transparent material, the flexible bag having a sealable opening and being dimensioned to receive the growth chamber together with a liquid medium; an outer chamber having one or more transparent side walls, the outer chamber being formed to correspond in shape to the growth chamber and dimensioned to receive the growth chamber within the flexible bag so that the mesh bottom of the growth chamber faces a bottom of the outer chamber that in use is intended to rest on a support surface and movement of the growth chamber within the outer chamber is restrained to movement along a single axis such that the mesh bottom of the growth chamber moves towards and away from the bottom of the outer chamber; and a driving mechanism arranged to selectively drive movement of the growth chamber along a single axis, when the growth chamber and flexible bag are received in the outer chamber, between a first position in which the mesh bottom of the growth chamber is located at or towards the bottom of the outer chamber and a second position in which the mesh bottom of the growth chamber is spaced from the bottom of the outer chamber.

2. The temporary immersion bioreactor according to claim 1 wherein the flexible bag further includes a sealable port to receive liquid medium and/or inoculum therethrough.

3. The temporary immersion bioreactor according to claim 1 further including an air pump for connection to the flexible bag so as to allow a gas to be conveyed through the flexible bag.

4. The temporary immersion bioreactor according to claim 3 wherein the flexible bag includes at least three ports, first and second ports being configured for connection to the air pump to allow the flow of gas into and out of the flexible bag and a third port configured to receive liquid medium and/or inoculum therethrough.

5. The temporary immersion bioreactor according to claim 1 wherein the driving mechanism includes a drive arm located within the outer chamber so as to engage an underside of the mesh bottom of the growth chamber, via the flexible bag, when the growth chamber and flexible bag are received in the outer chamber, the drive arm being movable in a first direction along the single axis upwards from the bottom of the outer chamber to an extended position so as to push the growth chamber from the first position to the second position, and the drive arm being movable from the extended position in a second, opposite direction along the single axis so as to guide movement of the growth chamber downwards from the second position to the first position.

6. The temporary immersion bioreactor according to claim 1 wherein the driving mechanism includes a float element mounted so as to extend around the mesh bottom of the growth chamber and thereby allow the growth chamber to float on a liquid medium contained in the flexible bag when the growth chamber and flexible bag are received in the outer chamber and thereby locate the growth chamber in the second position, and the driving mechanism further including one or more drive elements provided in the outer chamber to selectively engage the growth chamber and drive movement of the growth chamber from the second position to the first position.

7. The temporary immersion bioreactor according to claim 6 wherein the float element has a depth of at least 5 cm so that, when the growth chamber floats on a liquid medium, the mesh bottom of the growth chamber is spaced at least 5 cm from the liquid medium.

8. The temporary immersion bioreactor according to claim 7 wherein the floatation element has a depth of 10 cm.

9. The temporary immersion bioreactor according to claim 1 wherein the driving mechanism includes at least two electro-magnets located within the outer chamber, towards the bottom of the outer chamber, and at least two electro-magnets located on an external surface of the growth chamber, in the vicinity of the mesh bottom of the growth chamber, the electro-magnets being selectively operable so as to create opposing magnetic fields and thereby drive movement of the growth chamber in a first direction along the single axis, from the first position to the second position, and the driving mechanism further including one or more drive elements provided in the outer chamber to selectively engage the growth chamber and drive movement of the growth chamber in a second, opposite direction along the single axis from the second position to the first position.

10. The temporary immersion bioreactor according to claim 6 wherein the driving mechanism includes one or more drive elements in the form of a plunger located within the outer chamber, the or each plunger having an engagement member to engage an upper edge of the or at least one side wall of the growth chamber, via the flexible bag, when the growth chamber and the flexible bag are received in the outer chamber, the or each plunger being movable to drive movement of the respective engagement member from a rest position at the top of the outer chamber, in the first direction towards the extended position, and thereby drive movement of the growth chamber from the second position to the first position, and the or each plunger being movable to drive movement of the respective engagement member from the extended position in a second, opposite direction along the single axis so as to guide movement of the growth chamber from the first position to the second position under the action of the float member or opposing electro-magnets.

11. The temporary immersion bioreactor according to claim 10 wherein the driving mechanism includes at least three such plungers located within the outer chamber at equidistantly spaced locations about an inner circumference of the outer chamber.

12. The temporary immersion bioreactor according to claim 6 wherein the driving mechanism includes at least two drive elements in the form of at least two electro-magnets located within the outer chamber, towards the bottom of the outer chamber, and at least two electro-magnets located on an outer surface of the growth chamber, in the vicinity of the mesh bottom of the growth chamber, the electro-magnets being selectively operable so as to create attractive magnetic fields and thereby drive movement of the growth chamber in the second direction along the single axis, from the second position to the first position.

13. The temporary immersion bioreactor according to claim 1 wherein the driving mechanism includes a timer to control operation of the driving mechanism and thereby, in use, control movement of the growth chamber between the first and second positions.

14. The temporary immersion bioreactor according to claim 1 wherein the flexible bag is formed from a flexible thermoplastic polymer.

15. The temporary immersion bioreactor according to claim 14 wherein the flexible bag is formed from polypropylene, polyethylene or polyurethane.

16. The temporary immersion bioreactor according to claim 1 wherein the cross-sectional shapes of the growth chamber and the outer chamber, in a plane generally perpendicular to the single axis, are the same, the cross-sectional shape being selected from circular, elliptical, square, triangular or rectangular.

17. The temporary immersion bioreactor according to claim 1 wherein the mesh bottom of the growth chamber is formed to define pores having a size in the range of 50-500 μm.

18. The temporary immersion bioreactor according to claim 17 wherein the mesh bottom of the growth chamber is formed to define pores having a size in the range of 100-200 μm.

19. The temporary immersion bioreactor according to claim 1 wherein the growth chamber includes a mesh section located in the or one side wall, at or towards an opposite edge from the mesh bottom, the mesh bottom and the mesh section being formed from the same material and having the same pore size as the mesh bottom of the growth chamber.

20. The temporary immersion bioreactor according to claim 1 wherein the growth chamber is formed so as to define an inner volume in the range of 10-1,000 liters.

21. The temporary immersion bioreactor according to claim 20 wherein the growth chamber is formed so as to define an inner volume in the range of 30-150 liters.

Description

[0056] Preferred embodiments of the invention will now be described, by way of non-limiting examples, with reference to the accompanying drawings in which:

[0057] FIGS. 1 and 2 show schematic illustrations of a temporary immersion bioreactor according to a first embodiment of the invention;

[0058] FIG. 3 shows the drive arm of the bioreactor of FIGS. 1 and 2;

[0059] FIGS. 4a and 4b show a growth chamber of the bioreactor of FIGS. 1 and 2;

[0060] FIGS. 5a and 5b show the growth chamber and a flexible bag of the bioreactor of FIGS. 1 and 2;

[0061] FIGS. 6a and 6b show the growth chamber, flexible bag and an outer chamber of the bioreactor of FIGS. 1 and 2;

[0062] FIGS. 7 and 8 show schematic illustrations of a temporary immersion bioreactor according to a second embodiment of the invention;

[0063] FIG. 9 illustrates the rate of growth of leafy biomass using the bioreactor of FIGS. 1 and 2; and

[0064] FIG. 10 shows leafy biomass of Nicotiana tabacum produced using the bioreactor of FIGS. 1 and 2.

[0065] A temporary immersion bioreactor 10 for in vitro production of differentiated plant biomass, according to an embodiment of the invention, is shown in FIGS. 1 and 2.

[0066] The bioreactor 10 includes a growth chamber 12 (FIGS. 4a and 4b) having a plurality of transparent side walls 14 and a mesh bottom 16 defining a plurality of pores 18 to receive plant material or inoculum. It is envisaged that the plant material or inoculum may include leaves or root pieces, cell suspensions or callus.

[0067] The growth chamber 12 is located in a flexible bag 20 (FIGS. 5a and 5b) formed from a transparent material. The flexible bag 20 includes a sealable opening (not shown) and is dimensioned to receive the growth chamber 12 together with a liquid medium 22.

[0068] The flexible bag 20, together with the growth chamber 12 and liquid medium 22, are located in an outer chamber 24 (FIGS. 6a and 6b). The outer chamber 24 has a plurality of transparent side walls 26 and a bottom 28 that, in use, is intended to rest on a support surface.

[0069] The bioreactor 10 further includes a driving mechanism in the form of a drive arm 30 (FIGS. 1 and 2) located towards the bottom 28 of the outer chamber 24. The drive arm 30 engages an underside of the mesh bottom 16 of the growth chamber 12 via the flexible bag 20.

[0070] Preferably the drive arm 30 is configured to present a relatively smooth engagement portion 31 (FIG. 3) that engages the underside of the mesh bottom 16 of the growth chamber 12 via the flexible bag 20. The provision of a relatively smooth engagement portion 31, having no sharp edges or protrusions, reduces the risk of the drive arm 30 tearing or otherwise rupturing the flexible bag 20.

[0071] As shown in FIGS. 1 and 2, the drive arm 30 does not engage the entire underside of the mesh bottom 16 of the growth chamber 12. The drive arm 30 engages a relatively small proportion of the underside of the mesh bottom 16 at which point the flexible bag 20 is trapped between the drive arm 30 and mesh bottom 16. As a result, two pockets 32,34 are formed on either side of the drive arm 30 into which the liquid medium 22 flows.

[0072] The growth chamber 12, the flexible bag 20 and the outer chamber 24 are each formed from a transparent material so as to allow light to penetrate and reach any plant material located in the pores 18 of the mesh bottom 16 of the growth chamber 12.

[0073] Preferably the flexible bag 20 is formed from a disposable material so as to allow disposal of the bag 20 after use.

[0074] In the embodiment shown in FIGS. 1 and 2, the flexible bag 20 is formed from polypropylene. In other embodiments, the flexible bag 20 may be formed from another flexible thermoplastic polymer such as polyethylene or polyurethane.

[0075] Locating the flexible bag 20 containing the growth chamber 12 and the liquid medium 22 in an outer chamber 24 allows the liquid medium 22 to settle within the flexible bag 20 at the bottom of the outer chamber 24, as shown in FIGS. 1 and 2. This arrangement eliminates voids forming in the liquid medium 22.

[0076] The growth chamber 12 and the outer chamber 24 are both moulded from a transparent polyethylene plastics material, so as to define a rectangular cross-section in a plane generally perpendicular to axis A.

[0077] In other embodiments the growth chamber 12 and the outer chamber 24 may be formed from plexi glass, glass or another transparent material that can withstand sterilisation by chemical, autoclaving or gamma radiation.

[0078] It is envisaged that in other embodiments the growth chamber 12 and the outer chamber 24 may be formed so as to define a different cross-sectional shape in a plane generally perpendicular to axis A. The cross-sectional shape could, for example, be circular, elliptical, triangular or square.

[0079] The growth chamber 12 of the bioreactor 10 shown in FIGS. 1 and 2 is sized so as to have a height Y.sub.a of 25 cm, a width X.sub.a of 60 cm and a depth Z.sub.a of 40 cm (the X-Y-Z axes being illustrated in FIG. 3a). This results in a growth chamber 12 having an internal volume of 60 liters.

[0080] It is envisaged that in other embodiments the width X and depth Z of the growth chamber 12 may be varied to alter the maximum internal volume of the growth chamber 12. The applicant has discovered however that the height Y of the growth chamber 12 should preferably not exceed 25 cm if sufficient light is to reach the plant material or inoculum.

[0081] The outer chamber 24 is sized so as to have a height Y.sub.b of 60 cm, a width X.sub.b of 70 cm and a depth X.sub.b of 50 cm (FIG. 6a). These dimensions allow a clearance of 5 cm on each side of the growth chamber 12 in the width X and depth Z directions when the growth chamber 12 and the flexible bag 20 are located in the outer chamber 24, as shown in FIGS. 1 and 2.

[0082] The corresponding shapes of the growth chamber 12 and the outer chamber 24, and the relative dimensions of the chambers 12,26 in the width X and depth Z directions means that the growth chamber 12 fits snugly within the side walls 28 of the outer chamber 24, thereby restraining movement of the growth chamber 12 within the outer chamber 24 to movement along a single axis A, which is essentially parallel to the height Y.sub.a,Y.sub.b direction of the two chambers 12,26.

[0083] The bioreactor 10 shown in FIGS. 1 and 2 includes a gas pump (not shown) to create, in use, a flow of gas through the internal volume of the flexible bag 20. To facilitate connection of the air pump, the flexible bag 20 includes first and second ports 38,40 configured to define an inlet and an outlet for connection to the air pump so as to allow the flow of gas into and out of the flexible bag 20.

[0084] In use, the air pump may be activated to blow air into the sealed flexible bag 20 at the start of the bioreactor process. The air pump may then be used to renew the air in the flexible bag 20 at time intervals ranging from every 1 hour to every 6 hours, depending on the plant specifies, and preferably every 1 to 2 hours.

[0085] The volume of air pumped into the flexible bag 20 is between 2 liters per minute and 20 liters per minute, and the air is pumped from anywhere between 1 to 60 minutes each time.

[0086] The flexible bag also includes a third port 42 configured to receive liquid medium, thereby providing means for introducing the liquid medium 22 into the flexible bag 20. The third port 42 may then be used to introduce plant material or inoculum into the flexible bag 20. Preferably the amount of inoculum introduced into the flexible bag 20 is in the range of 1 to 10 g per liter of the internal volume of the growth chamber 12.

[0087] The provision of the third port 42 is advantageous in that it means that it is not necessary to seal the growth chamber 12 in the flexible bag 20 together with the liquid medium 22 or the plant material or inoculum when constructing the bioreactor 10. Introduction of the liquid medium 22 and the plant material or inoculum may be delayed until the bioreactor is fully constructed and ready for use.

[0088] This in turn means that it is only necessary to locate the third port 42 within a sterile environment during the introduction of the liquid medium 22 and/or plant material or inoculum into the bioreactor 10. It is not necessary to locate the entire bioreactor 10 within the sterile environment and accordingly the size of the sterile environment does not limit the maximum size and capacity of the bioreactor 10.

[0089] The mesh bottom 16 of the growth chamber 12 is made from stainless steel to define pores 18 having a size in the range of 50-200 μm, preferably 100 μm so as to enable them to retain the smallest cells.

[0090] The growth chamber 12 also includes a mesh section 36 located along an upper edge of a side wall 14. The mesh section 36 is also made from stainless steel and formed to define pores (not shown) corresponding in size to the pores 18 formed in the mesh bottom 16 of the growth chamber 12.

[0091] In use, the drive arm 30 is movable between a rest position in which the movable arm lies adjacent the bottom 28 of the outer chamber 24 (FIG. 2), upward along an axis A, to an extended position in which the movable arm is spaced from the bottom 28 of the outer chamber 24 (FIG. 1). As a result, the drive arm 30 drives movement of the growth chamber 12 along axis A from a first position in which the mesh bottom 16 is located towards the bottom 28 of the outer chamber 24 (FIG. 2) towards a second position in which the mesh bottom 16 is spaced from the bottom 26 of the outer chamber 28 (FIG. 1).

[0092] During movement of the drive arm 30, the pockets 32,34 formed in the flexible bag 20 on either side of the drive arm 30 increase in depth and liquid medium 22 drains from the growth chamber 12 into those pockets 32,34.

[0093] The amount of liquid medium 22 provided in the flexible bag 20 is chosen such that, when the growth chamber 12 is located in the first position, any plant material located in the pores 18 of the mesh bottom 16 of the growth chamber 12 are immersed within the liquid medium 22.

[0094] The amount of liquid medium 22 provided in the flexible bag 20 and the extended position of the drive arm 30 are preferably chosen such that, when the growth chamber 12 is located in the second position, the mesh bottom 16 of the growth chamber 12 is spaced from an upper surface of the liquid medium 22 by a distance A (FIG. 1). This ensures that the liquid medium 22 drains from the growth chamber 12. It also ensures that any plant shoots or roots protruding from the underside of the mesh bottom do not remain immersed in the liquid medium 22.

[0095] In particularly preferred embodiments the amount of liquid medium 22 provided in the flexible bag 20, and the extended position of the drive arm 30 are chosen such that the mesh bottom 16 is spaced by a distance A (FIG. 1) of at least 5 cm and preferably 10 cm from the upper surface of the liquid medium 22.

[0096] Operation of the drive arm 30 is controlled by means of a timer connected to a drive motor 33 arranged to drive movement of the drive arm 30 (FIG. 3). to the timer controls movement, in use, of the growth chamber 12 between the first and second positions and thereby controls immersion of plant material or inoculum in the liquid medium 22. By appropriate setting of the timer, it is possible to set specific periods of immersion and aeration as may be required for a particular plant material located in the pores 18 of the mesh bottom 16 of the growth chamber 12.

[0097] A bioreactor 110 according to a second embodiment of the invention is shown in FIGS. 6 and 7.

[0098] The structure of the bioreactor 110 is very similar to the structure of the bioreactor 10 shown in FIGS. 1 and 2. Those features common to both embodiments are referred to using the same reference numerals and will not be described again in detail.

[0099] The bioreactor 110 shown in FIGS. 7 and 8 differs from the bioreactor 10 shown in FIGS. 1 and 2 in that it omits a drive arm 30 to drive movement of the growth chamber 12 from the first position to the second position.

[0100] In the embodiment shown in FIGS. 7 and 8, the growth chamber 12 includes a float element 112 mounted so as to extend around the mesh bottom 16 of the growth chamber 12.

[0101] The float element 112 permits the growth chamber 12 to float, in use, on the liquid medium contained in the flexible bag 20 and thereby locate the growth chamber 12 in the second position (FIG. 7) where the mesh base 16 is spaced from the bottom 28 of the outer chamber 24.

[0102] In the embodiment shown in FIGS. 7 and 8, the float element 112 has a depth of 10 cm so that, when the growth chamber 12 floats on the liquid medium 22, the mesh bottom 16 of the growth chamber 12 is spaced by a distance A of 10 cm from the surface of the liquid medium 22.

[0103] So as to immerse the growth chamber 12 in the liquid medium 22 when required, the bioreactor 110 includes four plungers 114 located in the outer chamber 24. The plungers 114 are arranged so that engagement members 116 provided on the plungers 114 engage upper edges of the growth chamber 12.

[0104] In use, each plunger 114 is operable to selectively drive the respective engagement member 116 downward along the axis A from a rest position to an extended position so as to drive movement of the growth chamber 12 from the second position to the first position where the mesh bottom of the growth chamber 12 is immersed in the liquid medium 22 (FIG. 8).

[0105] Following immersion of the mesh bottom 16 of the growth chamber 12 in the liquid medium 22, each of the plungers 114 is operable to selectively drive the respective engagement member 116 upward along the axis A from the extended position to the rest position and thereby guide movement of the growth chamber 12 upwards from the first position to the second position under the action of the float member 112.

[0106] Operation of the plungers 114 is controlled by means of a timer so as to control movement, in use, of the growth chamber 12 between the first and second positions and thereby control immersion of plant material or inoculum in the liquid medium 22. By appropriate setting of the timer, it is possible to set specific periods of immersion and aeration as may be required for a particular plant material located in the pores 18 of the mesh bottom 16 of the growth chamber 12.

[0107] In one particular method of operation of the bioreactor 10 shown in FIGS. 1 and 2 or the bioreactor 110 shown in FIGS. 7 and 8, the bioreactor 10,110 is located in a controlled environment at 25° C., with a 16 hour/8 hour day/night cycle and a light intensity of 50 μMolm.sup.−2s.sup.−1 at the bag level.

[0108] The inoculum is introduced into the flexible bag 20 via the third port 42. Thereafter 50 liters of liquid medium 22 are introduced into the flexible bag 20 such that the liquid medium 22 covers 80% of the growth chamber 12 when the growth chamber 12 is immersed in the liquid medium 22 within the flexible bag 20.

[0109] Starting the bioreactor process with the growth chamber 12 in the second position, spaced from the bottom 28 of the outer chamber 24, the liquid medium 22 flows through the mesh bottom 16 of the growth chamber 12 so as to collect at the bottom of the flexible bag 20 within the outer chamber 24.

[0110] The air pump is connected to the first port 38 to pump ambient air into the flexible bag 20 at a rate of 15 liters per minute over a period of 10 minutes each hour. In other embodiments, the air may be enriched with up to 1% of carbon dioxide.

[0111] The timer is set to immerse the growth chamber 12 in the liquid medium 22 for a period of 4 minutes every 6 hours.

[0112] Following this mode of operation, the growth of biomass becomes exponential after a lag period of 3 weeks (as illustrated in FIG. 9) until it reaches a plateau after 50 days. The maximal biomass yield is estimated at about 300 g f.w./L (fresh weight per liter).

[0113] Leafy biomass of Nicotiana tabacum is shown by way of illustrative example of the biomass 150 that may be produced through use of the bioreactor 10 in accordance with the method outlined above.