Abstract:
A pneumatic bioreactor includes a vessel containing a fluid to be mixed and at least one mixing device driven by gas pressure. A first embodiment includes a floating impeller that rises and falls in the fluid as gas bubbles carry it upward to the surface where the gas is then vented, permitting the impeller to sink in the fluid. The floating impeller may be tethered to a second impeller with a flexible member and pulley. The mixing speed is controlled with electromagnets in the vessel acting upon magnetic material in the impeller or its guides. In another embodiment, floating pistons mix the fluid, pushing it through a mixing plate with one or more apertures. In a third embodiment, the mixing device is a rotating drum with bubble-catching blades and rotating mixing plates with apertures. The top of the vessel for these mixers may include a closed top and sterile filters.

Description:
RELATED APPLICATIONS 
       [0001]    This application is a continuation application claiming priority under 35 U.S.C. §120 of U.S. patent application Ser. No. 14/444,695 filed Jul. 28, 2014, now issued as U.S. Pat. No. 9,453,194, which is a continuation application claiming priority under 35 U.S.C. §120 of U.S. patent application Ser. No. 12/884,431 filed Sep. 17, 2010, now issued as U.S. Pat. No. 8,790,913, which is a continuation-in-part application claiming priority under 35 U.S.C. §120 of U.S. patent application Ser. No. 11/739,089 filed Apr. 23, 2007, now abandoned. 
     
    
     TECHNICAL FIELD 
       [0002]    The invention pertains to apparatus for mixing solutions. More particularly, the invention relates to methods for using pneumatically operated mixers for use in closed, sterile environments. 
       BACKGROUND OF THE INVENTION 
       [0003]    Efforts of biopharmaceutical companies to discover new biological drugs have increased exponentially during the past decade-and-a-half. Bioreactors have been used for cultivation of microbial organisms for production of various biological or chemical products in the pharmaceutical, beverage, and biotechnological industry. Most biological drugs are produced by cell culture or microbial fermentation processes which require sterile bioreactors and an aseptic culture environment. An increasing number of biological drug candidates are in development. Stringent testing, validation, and thorough documentation of process for each drug candidate are required by FDA to ensure consistency of the drug quality used for clinical trials to the market. However, shortages of global biomanufacturing capacity are anticipated in the foreseeable future, particularly as production needs will increase as such new drugs are introduced to the market. 
         [0004]    A production bioreactor contains culture medium in a sterile environment that provides various nutrients required to support growth of the biological agents of interest. Stainless steel stir tanks have been the only option for large scale production of biological products in suspension culture. Manufacturing facilities with conventional stainless bioreactors, however, require large capital investments for construction, high maintenance costs, long lead times, and inflexibilities for changes in manufacturing schedules and production capacities. Conventional bioreactors use mechanically driven impellors to mix the liquid medium during cultivation. The bioreactors can be reused for the next batch of biological agents after cleaning and sterilization of the vessel. The procedure of cleaning and sterilization requires a significant amount of time and resources, especially to monitor and to validate each cleaning step prior to reuse for production of biopharmaceutical products. Due to the high cost of construction, maintenance and operation of the conventional bioreactors, single use bioreactor systems made of disposable plastic material have become an attractive alternative. 
         [0005]    While several mixing methods of liquid in disposable bioreactors have been proposed in recent years, none of them provides efficient mixing for large scale (greater than 1000 liters) without expensive operating machinery. For this reason, a number of non-invasive and/or disposable mixing systems that do not require an external mechanical operation have been developed. Many of these systems work well within certain size ranges, however, problems sometimes arise as larger mixing systems are attempted. 
         [0006]    Single use disposable bioreactor systems have been introduced to market as an alternative choice for biological product production. Such devices provide more flexibility on biological product manufacturing capacity and scheduling, avoid risking major upfront capital investment, and simplify the regulatory compliance requirements by eliminating the cleaning steps between batches. However, the mixing technology of the current disposable bioreactor system has limitations in terms of scalability to sizes beyond 200 liters and the expense of large scale units. Therefore, a disposable single use bioreactor system which is scalable beyond 1000 liters, simple to operate, and cost effective will be needed as a substitute for conventional stainless steel bioreactors for biopharmaceutical research, development, and manufacturing. 
         [0007]    It is an objective of the present invention to provide a pneumatic bioreactor that is capable of efficiently and thoroughly mixing solutions without contamination. It is a further objective to such a reactor that can be scaled to relatively large sizes using the same technology. It is a still further objective of the invention to a bioreactor that can be produced in a disposable form. It is yet a further objective of the invention to provide a bioreactor that can be accurately controlled by internal pneumatic force, as to speed and mixing force applied to the solution without creating a foaming problem. Finally, it is an objective to provide a bioreactor that is simple and inexpensive to produce and to operate while fulfilling all of the described performance criteria. 
       SUMMARY OF THE INVENTION 
       [0008]    A pneumatic bioreactor providing all of the desired features can be constructed from the following components. A containment vessel is provided. The vessel has a top, a closed bottom, a surrounding wall and is of sufficient size to contain a fluid to be mixed and a mixing apparatus. The mixing apparatus includes at least one gas supply line. The supply line terminates at an orifice adjacent the bottom of the vessel. At least one buoyancy-driven mixing device is provided. The mixing device moves in the fluid as gas from the supply line is introduced into and vented from the mixing device. When gas is introduced into the gas supply line the gas will enter the mixing device and cause the device to mix the fluid. 
         [0009]    In a variant of the invention, the buoyancy-driven mixing device further includes at least one floating plunger. The plunger has a central, gas-holding chamber and a plurality of mixing elements located about the central chamber. The mixing elements are shaped to cause the plunger to agitate the fluid as the plunger rises in the fluid in the containment vessel. In a variant, the mixing elements are generally in the shape of a disc. 
         [0010]    In yet another variant, the buoyancy-driven mixing device further includes at least one floating impeller, which is also provided as a mixing element. The impeller has the central, gas-containing chamber and a plurality of impeller blades arcurately located about the central chamber. The impeller blades are shaped to cause the impeller to revolve about a vertical axis as the impeller rises in fluid in the containment vessel. 
         [0011]    The central chamber has a gas-venting valve. The valve permits escape of gas as the central chamber reaches a surface of the fluid. A constraining member is provided. The constraining member limits horizontal movement of the floating plunger and/or impeller (“plunger/impeller”) as it rises or sinks in the fluid. When gas is introduced into the gas supply line, the gas will enter the gas-holding chamber and cause the floating plunger/impeller to rise by buoyancy in the fluid while agitating the fluid. When the gas-venting valve of the central chamber reaches the surface of the fluid, the gas will be released and the floating plunger/impeller will sink toward the bottom of the containment vessel where the central chamber will again be filled with gas, causing the floating plunger/impeller to rise. 
         [0012]    In a further variant, a mixing partition is provided. The partition is located in the containment vessel adjacent the floating plunger/impeller and has at least one aperture to augment a mixing action of the floating plunger/impeller. 
         [0013]    In another variant, means are provided for controlling a rate of assent of the floating plunger/impeller. 
         [0014]    In still another variant, the means for controlling the rate of assent of the floating plunger/impeller includes a ferromagnetic substance attached to either of the floating plunger/impeller, the constraining member, or the outside housing, and a controllable electromagnet located adjacent the bottom of the containment vessel. The gas flow is interrupted by an on/off switch which is controlled by interactions of two magnetic substances. Therefore, the volume of gas supplied into the gas-holding chamber is determined by the strength of the electromagnetic power since the gas flow stops as the floating plunger/impeller starts to rise when the buoyancy becomes greater than the magnetic holding force. 
         [0015]    In yet another variant, the central, gas-holding chamber further includes an opening. The opening is located at an upper end of the chamber. A vent cap is provided. The vent cap is sized and shaped to seal the opening when moved upwardly against it by buoyancy from gas from the supply line. A support bracket is provided. The support bracket is located within the chamber to support the vent cap when it is lowered after release of gas from the chamber. When the chamber rises to the surface of the fluid the vent cap will descend from its weight and the opening will permit the gas to escape, the chamber will then sink in the fluid and the vent cap will again rise due to buoyancy from a small amount of gas permanently enclosed in the vent cap, thereby sealing the opening. 
         [0016]    In a further variant, a second floating plunger/impeller is provided. A second constraining member is provided, limiting horizontal movement of the second plunger/impeller as it rises in the fluid. At least one additional gas supply line is provided. The additional supply line terminates at an orifice adjacent the bottom of the vessel. At least one pulley is provided. The pulley is attached to the bottom of the containment vessel. A flexible member is provided. The flexible member attaches the chamber of the floating plunger to a chamber of the second floating plunger/impeller. The flexible member is of a length permitting the gas venting valve of the chamber of the floating plunger/impeller to reach the surface of the fluid while the chamber of the second floating plunger/impeller is spaced from the bottom of the containment vessel. When the floating plunger/impeller is propelled upwardly by buoyancy from the gas from the supply line the second floating plunger/impeller is pulled downwardly by the flexible member until the gas is released from the chamber of the floating plunger/impeller as its gas venting valve reaches the surface of the fluid. The chamber will then sink in the fluid as the second floating plunger/impeller rises by buoyancy from gas introduced from the second supply line. 
         [0017]    In yet a further variant, the containment vessel is formed of resilient material, the material is sterilizable by gamma irradiation methods. 
         [0018]    In still another variant, the pneumatic bioreactor further includes a cylindrical chamber. The chamber has an inner surface, an outer surface, a first end, a second end and a central axis. At least one mixing plate is provided. The mixing plate is attached to the inner surface of the chamber. First and second flanges are provided. The flanges are mounted to the cylindrical chamber at the first and second ends, respectively. First and second pivot points are provided. The pivot points are attached to the first and second flanges, respectively and to the containment vessel, thereby permitting the cylindrical chamber to rotate about the central axis. A plurality of gas-holding members are provided. The members extend from the first flange to the second flange along the outer surface of the cylindrical chamber and are sized and shaped to entrap gas bubbles from the at least one gas supply line. The gas supply line terminates adjacent the cylindrical chamber on a first side of the chamber below the gas-holding members. When gas is introduced into the containment vessel through the supply line it will rise in the fluid and gas bubbles will be entrapped by the gas-holding members. This will cause the cylindrical chamber to rotate on the pivot points in a first direction and the at least one mixing plate to agitate the fluid. 
         [0019]    In yet another variant, a rate of rotation of the cylindrical chamber is controlled by varying a rate of introduction of gas into the gas supply line. 
         [0020]    In a further variant, a second gas supply line is provided. The second supply line terminates adjacent the cylindrical chamber on a second, opposite side of the chamber below the gas holding members. Gas from the second supply line causes the cylindrical chamber to rotate on the pivot points in a second, opposite direction. 
         [0021]    In still a further variant, the at least one mixing plate has at least one aperture to augment mixing of the fluid in the containment vessel. 
         [0022]    In yet a further variant, the containment vessel further includes a closable top. The top has a vent, permitting the escape of gas from the gas supply line through a sterile filter. 
         [0023]    In another variant of the invention, a temperature control jacket is provided. The jacket surrounds the containment vessel. 
         [0024]    In a variant of the invention, an outside housing is provided. The housing is ring-shaped and surrounds the floating impeller and constrains its lateral movement. At least one supporting pole is provided. The pole extends from the bottom upwardly toward the top. The outside housing is slidably attached to the supporting pole. The floating impeller is rotatably attached to the outside housing. 
         [0025]    In still another variant, the impeller blades are rotatably mounted to the central chamber and the central chamber is fixedly attached to the outside housing. 
         [0026]    In a further variant, the impeller blades are fixedly mounted to the central chamber and rotatably mounted to the outside housing. 
         [0027]    In still a further variant, the outside housing further includes a horizontal interior groove located on an inner surface of the housing. The impeller blades include a projection, sized and shaped to fit slidably within the groove. 
         [0028]    In yet another variant, the vent cap further includes an enclosed gas cell. The cell causes the cap to float in the fluid and thereby to reseal the opening after the gas has been released when the chamber reached the surface of the fluid. 
         [0029]    In a further variant, wherein the pneumatic bioreactor further includes a second floating impeller, a second outside housing surrounding the second floating impeller is provided. At least one additional supporting pole is provided. At least one additional gas supply line is provided. The additional supply line terminates at an orifice at the bottom of the vessel. The second outside housing is slidably attached to the additional supporting pole. The second floating impeller is rotatably attached to the second outside housing. At least one pulley is provided. The pulley is attached to the bottom of the containment vessel. 
         [0030]    An appreciation of the other aims and objectives of the present invention and an understanding of it may be achieved by referring to the accompanying drawings and the detailed description of a preferred embodiment. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0031]      FIG. 1  is a perspective view of a first embodiment of the invention illustrating floating impellers and their control mechanisms. 
           [0032]      FIG. 2  is a top view of the  FIG. 1  embodiment illustrating the floating chamber affixed to the constraining member with the impeller blades rotating upon the chamber. 
           [0033]      FIG. 2A  is a top view of the  FIG. 1  embodiment illustrating the floating chamber rotating within the constraining member with the impeller blades fixed to the chamber. 
           [0034]      FIG. 3  is a side elevational view of the  FIG. 1  embodiment. 
           [0035]      FIG. 4  is a side elevational view of the  FIG. 2A  embodiment of the floating impeller. 
           [0036]      FIG. 4A  is a side elevational view of the  FIG. 2  embodiment of the floating impeller. 
           [0037]      FIG. 5  is a perspective view of a second embodiment of the invention illustrating floating plungers and their control mechanisms. 
           [0038]      FIG. 6  is a top view of the  FIG. 5  embodiment illustrating the floating plungers. 
           [0039]      FIG. 7  is a perspective view of the gas supply line and magnetic assent control mechanism. 
           [0040]      FIG. 8  is a cross-sectional side elevation of the floating chamber illustrating the vent cap in a closed position. 
           [0041]      FIG. 9  is a cross-sectional side elevation of the floating chamber illustrating the vent cap in an open position. 
           [0042]      FIG. 10  is a perspective view of a third embodiment of the invention illustrating a rotating drum mixer with gas supply line. 
           [0043]      FIG. 11  is an end view of the  FIG. 10  embodiment illustrating a single gas supply line. 
           [0044]      FIG. 12  is an end view of the  FIG. 10  embodiment illustrating a pair of gas supply lines. 
           [0045]      FIG. 13  is a side elevational view of the  FIG. 10  embodiment illustrating a containment vessel. 
           [0046]      FIG. 14  is a perspective view of the  FIG. 5  embodiment illustrating a closable top and sterile filters. 
           [0047]      FIG. 15  is a perspective view of the  FIG. 5  embodiment illustrating a temperature control jacket surrounding the vessel. 
           [0048]      FIG. 16  is a perspective view of a pneumatic bioreactor shown through a transparent housing and containment vessel for clarity. 
           [0049]      FIG. 17  is a front view of the pneumatic bioreactor of  FIG. 16 . 
           [0050]      FIG. 18  is top view of the pneumatic bioreactor of  FIG. 16 . 
           [0051]      FIG. 19  is a perspective view of the top and mixing apparatus of the pneumatic bioreactor of  FIG. 16 . 
           [0052]      FIG. 20  is a perspective view of one wheel of the pneumatic bioreactor of  FIG. 16 . 
           [0053]      FIG. 21  is a perspective view of the top and mixing apparatus of a modified bioreactor of  FIG. 16 . 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0054]    A pneumatic bioreactor  10 , as illustrated in  FIGS. 1-3 , providing all of the desired features can be constructed from the following components. A containment vessel  15  is provided. The vessel  15  has a top  20 , a closed bottom  25 , a surrounding wall  30  and is of sufficient size to contain a fluid  35  to be mixed and a mixing apparatus  40 . The mixing apparatus  40  includes at least one gas supply line  45 . The supply line  45  terminates at an orifice  50  adjacent the bottom  25  of the vessel  15 . At least one buoyancy-driven mixing device  55  is provided. The mixing device  55  moves in the fluid  35  as gas  60  from the supply line  45  is introduced into and vented from the mixing device  55 . When gas  60  is introduced into the gas supply line  45  the gas  60  will enter the mixing device  55  and cause the device to mix the fluid  35 . 
         [0055]    In a variant of the invention, the buoyancy-driven mixing device  55  further includes at least one floating mixer  65 . The mixer  65  has a central, gas-holding chamber  70  and a plurality of mixing elements  75  located about the central chamber  70 . The mixing elements  75  are shaped to cause the mixer  65  to agitate the fluid  35  as the mixer  65  rises in the fluid  35  in the containment vessel  15 . The central chamber  70 , as illustrated in  FIGS. 8 and 9 , has a gas-venting valve  80 . The valve  80  permits escape of gas  60  as the central chamber  70  reaches a surface  85  of the fluid  35 . A constraining member  90  is provided. The constraining member  90  limits horizontal movement of the floating mixer  65  as it rises or sinks in the fluid  35 . When gas  60  is introduced into the gas supply line  45 , the gas  60  will enter the gas holding chamber  70  and cause the floating mixer  65  to rise by buoyancy in the fluid  35  while agitating the fluid  35 . When the gas venting valve  80  of the central chamber  70  reaches the surface  85  of the fluid  35 , the gas  60  will be released and the floating mixer  65  will sink toward the bottom  25  of the containment vessel  15  where the central chamber  70  will again be filled with gas  60 , causing the floating mixer  65  to rise. 
         [0056]    In another variant, means  95 , as illustrated in  FIG. 7 , are provided for controlling a rate of assent of the floating mixer  65 . 
         [0057]    In still another variant, the means  95  for controlling the rate of assent of the floating mixer  65  includes a ferromagnetic substance  100  attached to either of the floating mixer  65  or the constraining member  90  and a controllable electromagnet  105  located adjacent the bottom  25  of the containment vessel  15 . 
         [0058]    In yet another variant, as illustrated in  FIGS. 8 and 9 , the central, gas-holding chamber  70  further includes an opening  110 . The opening  110  is located at an upper end  115  of the chamber  70 . A vent cap  117  is provided. The vent cap  117  is sized and shaped to seal the opening  110  when moved upwardly against it by buoyancy from gas  60  from the supply line  45 . A support bracket  120  is provided. The support bracket  120  is located within the chamber  70  to support the vent cap  115  when it is lowered after release of gas  60  from the chamber  70 . When the chamber  70  rises to the surface  85  of the fluid  35  the vent cap  115  will descend from its weight and the opening  110  will permit the gas  60  to escape, the chamber  70  will then sink in the fluid  35  and the vent cap  115  will again rise due to buoyancy from a small amount of gas  60  permanently enclosed in the vent cap  115 , thereby sealing the opening  110 . 
         [0059]    In a further variant, as illustrated in  FIGS. 1-3 , a second floating mixer  125  is provided. A second constraining member  130  is provided, limiting horizontal movement of the second mixer  125  as it rises in the fluid  35 . At least one additional gas supply line  135  is provided. The additional supply line  135  terminates at an orifice  143  adjacent the bottom  25  of the vessel  15 . At least one pulley  140  is provided. The pulley  140  is attached to the bottom  25  of the containment vessel  15 . A flexible member  145  is provided. The flexible member  145  attaches the chamber  70  of the floating mixer  65  to a chamber  150  of the second floating mixer  125 . The flexible member  145  is of a length permitting the gas venting valve  80  of the chamber  70  of the floating mixer  65  to reach the surface  85  of the fluid  35  while the chamber  70  of the second floating mixer  125  is spaced from the bottom  25  of the containment vessel  15 . When the floating mixer  65  is propelled upwardly by buoyancy from the gas  60  from the supply line  45  the second floating mixer  125  is pulled downwardly by the flexible member  145  until the gas  60  is released from the chamber  70  of the floating mixer  65  as its gas venting valve  80  reaches the surface  85  of the fluid  35 . The chamber  70  will then sink in the fluid  35  as the second floating mixer  125  rises by buoyancy from gas  60  introduced from the second supply line  135 . 
         [0060]    In yet a further variant, the containment vessel  15  is formed of resilient material  155 , the material is sterilizable by gamma irradiation methods. 
         [0061]    In still a further variant, as illustrated in  FIGS. 5 and 6 , the buoyancy-driven mixing device  10  further includes at least one floating plunger  160 . The plunger  160  has a central, gas-holding chamber  70  and at least one disk  165  located about the central chamber  70 . The disk  165  is shaped to cause the plunger  160  to agitate the fluid  35  as the plunger  160  rises in the fluid  35  in the containment vessel  15 . The central chamber  70  has a gas-venting valve  80 . The valve  80  permits escape of gas  60  as the central chamber  70  reaches a surface  85  of the fluid  35 . A mixing partition  170  is provided. The partition  170  is located in the containment vessel  15  adjacent the floating plunger  160  and has at least one aperture  175  to augment a mixing action of the floating plunger  160 . A constraining member  180  is provided. The constraining member  180  limits horizontal movement of the plunger  160  as it rises or sinks in the fluid  35 . When gas  60  is introduced into the gas supply line  45  the gas  60  will enter the gas holding chamber  70  and cause the floating plunger  160  to rise by buoyancy in the fluid  35  while agitating the fluid  35 . When the gas venting valve  80  of the central chamber  70  reaches the surface  85  of the fluid  35 , the gas  60  will be released and the floating plunger  160  will sink toward the bottom  25  of the containment vessel  15  where the central chamber  70  will again be filled with gas  60 , causing the floating plunger  160  to rise. 
         [0062]    In another variant of the invention, a second floating plunger  185  is provided. A second constraining member  190  is provided, limiting horizontal movement of the second plunger  185  as it rises in the fluid  35 . At least one additional gas supply line  135  is provided. The additional supply line  135  terminates at an orifice  143  adjacent the bottom  25  of the vessel  15 . At least one pulley  140  is provided. The pulley  140  is attached to the bottom  25  of the containment vessel  15 . A flexible member  145  is provided. The flexible member  145  attaches the chamber  70  of the floating plunger  160  to a chamber of the second floating plunger  185 . The flexible member  145  is of a length permitting the gas venting valve  80  of the chamber  70  of the floating plunger  160  to reach the surface  85  of the fluid  35  while the chamber  70  of the second floating plunger  185  is spaced from the bottom  25  of the containment vessel  15 . The mixing partition  170  is located between the floating plunger  160  and the second floating plunger  185 . When the floating plunger  160  is propelled upwardly by buoyancy from the gas  60  from the supply line  45  the second floating plunger  185  is pulled downwardly by the flexible member  145  until the gas  60  is released from the chamber  70  of the floating plunger  160  as its gas venting valve  80  reaches the surface  85  of the fluid  30 . The floating plunger  160  will then sink in the fluid  35  as the second floating plunger  185  rises by buoyancy from gas  60  introduced from the second supply line  135 . 
         [0063]    In still another variant, as illustrated in  FIGS. 10-13 , the pneumatic bioreactor  10  further includes a cylindrical chamber  195 . The chamber  195  has an inner surface  200 , an outer surface  205 , a first end  210 , a second end  215  and a central axis  220 . At least one mixing plate  225  is provided. The mixing plate  225  is attached to the inner surface  200  of the chamber  195 . First  230  and second  235  flanges are provided. The flanges  230 ,  235  are mounted to the cylindrical chamber  195  at the first  210  and second ends  215 , respectively. First  240  and second  245  pivot points are provided. The pivot points  240 ,  245  are attached to the first  230  and second  235  flanges, respectively and to the containment vessel  15 , thereby permitting the cylindrical chamber  195  to rotate about the central axis  220 . A plurality of gas holding members  250  are provided. The members  250  extend from the first flange  230  to the second flange  235  along the outer surface  205  of the cylindrical chamber  195  and are sized and shaped to entrap gas bubbles  255  from the at least one gas supply line  45 . The gas supply line  45  terminates adjacent the cylindrical chamber  195  on a first side  260  of the chamber  195  below the gas holding members  250 . When gas  60  is introduced into the containment vessel  15  through the supply line  45  it will rise in the fluid  35  and gas bubbles  255  will be entrapped by the gas holding members  250 . This will cause the cylindrical chamber  195  to rotate on the pivot points  240 ,  245  in a first direction  262  and the at least one mixing plate  225  to agitate the fluid  35 . 
         [0064]    In yet another variant, a rate of rotation of the cylindrical chamber  195  is controlled by varying a rate of introduction of gas  60  into the gas supply line  45 . 
         [0065]    In a further variant, as illustrated in  FIG. 12 , a second gas supply line  135  is provided. The second supply line  135  terminates adjacent the cylindrical chamber  195  on a second, opposite side  265  of the chamber  195  below the gas holding members  250 . Gas  60  from the second supply line  135  causes the cylindrical chamber  195  to rotate on the pivot points  240 ,  245  in a second, opposite direction  270 . 
         [0066]    In still a further variant, as illustrated in  FIGS. 10 and 13 , the at least one mixing plate  225  has at least one aperture  275  to augment mixing of the fluid  35  in the containment vessel  15 . 
         [0067]    In yet a further variant, as illustrated in  FIG. 14 , the containment vessel  15  further includes a closable top  280 . The top has a vent  285 , permitting the escape of gas  60  from the gas supply line  45  through a sterile filter  290 . 
         [0068]    In another variant of the invention, as illustrated in  FIG. 15 , a temperature control jacket  295  is provided. The jacket  295  surrounds the containment vessel  15 . 
         [0069]    In yet another variant, as illustrated in  FIGS. 1-3 , a pneumatic bioreactor  10  includes a containment vessel  15 . The vessel  15  has a top  20 , a closed bottom  25 , a surrounding wall  30  and is of sufficient size to contain a fluid  35  to be mixed and a mixing apparatus  40 . The mixing apparatus  40  includes at least one gas supply line  45 . The supply line  45  terminates at an orifice  50  at the bottom  25  of the vessel  15 . At least one floating impeller  300  is provided. The impeller  300  has a central, gas-containing chamber  70  and a plurality of impeller blades  305  arcurately located about the central chamber  70 . The impeller blades  305  are shaped to cause the impeller  300  to revolve about a vertical axis  310  as the impeller  300  rises in fluid  35  in the containment vessel  15 . 
         [0070]    The central chamber  70  has a gas-venting valve  80 . The valve  80  permits escape of gas  60  as the central chamber  70  reaches a surface  85  of the fluid  35 . An outside housing  315  is provided. The housing  315  is ring-shaped and surrounds the floating impeller  300  and constrains its lateral movement. At least one supporting pole  320  is provided. The pole  320  extends from the bottom  25  upwardly toward the top  20 . The outside housing  315  is slidably attached to the supporting pole  320 . The floating impeller  300  is rotatably attached to the outside housing  315 . When gas  60  is introduced into the gas supply line  45  the gas  60  will enter the gas containing chamber  70  and cause the floating impeller  300  to rise in the fluid  35  while rotating and mixing the fluid  35 . When the gas venting valve  80  of the central chamber  70  reaches the surface  85  of the fluid  35 , the gas  60  will be released and the floating impeller  300  will sink toward the bottom  25  of the containment vessel  15  where the central chamber  70  will again be filled with gas  60 , causing the floating impeller  300  to rise. 
         [0071]    In still another variant, as illustrated in  FIGS. 2 and 4A , the impeller blades  305  are rotatably mounted to the central chamber  70  and the central chamber  70  is fixedly attached to the outside housing  315 . 
         [0072]    In a further variant, as illustrated in  FIGS. 2A and 4 , the impeller blades  305  are fixedly mounted to the central chamber  70  and rotatably mounted to the outside housing  315 . 
         [0073]    In still a further variant, the outside housing  315  further includes a horizontal interior groove  322  located on an inner surface  325  of the housing  315 . The impeller blades  305  include a projection  330 , sized and shaped to fit slidably within the groove  322 . 
         [0074]    In yet a further variant, as illustrated in  FIG. 7 , means  95  are provided for controlling a rate of assent of the floating impeller  300 . 
         [0075]    In another variant of the invention, the means  95  for controlling a rate of assent of the floating impeller  300  includes a ferromagnetic substance  100  attached to either the floating impeller  300  or the outside housing  315  and a controllable electromagnet  105  located adjacent the bottom  25  of the containment vessel  15 . 
         [0076]    In still another variant, as illustrated in  FIGS. 8 and 9 , the central, gas-containing chamber  70  further includes an opening  110  located at an upper end  115  of the chamber  70 . A vent cap  115  is provided. The vent cap  115  is sized and shaped to seal the opening  110  when moved upwardly against it by pressure from gas  60  from the supply line  45 . A support bracket  120  is provided. The support bracket  120  is located within the chamber  70  to support the vent cap  115  when it is lowered after release of gas  60  from the chamber  70 . When the chamber  70  rises to the surface of the fluid  35  the vent cap  115  will descend from its weight and the opening  110  will permit the gas  60  to escape. The floating impeller  300  will then sink in the fluid  35  and the vent cap  115  will again rise due to pressure from gas  60  introduced into the chamber  70  from the gas line  45 , thereby sealing the opening  110 . 
         [0077]    In yet another variant, the vent cap  115  further includes an enclosed gas cell  310 . The cell  310  causes the cap  115  to float in the fluid  35  and thereby to reseal the opening  110  after the gas  60  has been released when the chamber  70  reached the surface  85  of the fluid  35 . 
         [0078]    In a further variant, as illustrated in  FIGS. 1 and 3 , the pneumatic bioreactor  10  further includes a second floating impeller  317 . A second outside housing  324  surrounding the second floating impeller  317  is provided. At least one additional supporting pole  326  is provided. At least one additional gas supply line  135  is provided. The additional supply line  135  terminates at an orifice  143  at the bottom  25  of the vessel  15 . The second outside housing  324  is slidably attached to the additional supporting pole  325 . The second floating impeller  317  is rotatably attached to the second outside housing  324 . At least one pulley  140  is provided. The pulley  140  is attached to the bottom  25  of the containment vessel  15 . 
         [0079]    A flexible member  145  is provided. The flexible member  145  attaches the chamber  70  of the floating impeller  300  to a chamber  70  of the second floating impeller  317 . The flexible member  145  is of a length to permit the gas venting valve  80  of the chamber  70  of the floating impeller  300  to reach the surface  85  of the fluid  35  while the chamber  70  of the second floating impeller  317  is spaced from the bottom  25  of the containment vessel  15 . When the floating impeller  300  is propelled upwardly by pressure from the gas  60  from the supply line  45  the second floating impeller  315  will be pulled downwardly by the flexible member  145  until the gas  60  is released from the chamber  70  of the floating impeller  300  as its gas venting valve  80  reaches the surface  85  of the fluid  35 , the floating impeller  300  will then sink in the fluid  35  as the second floating impeller  315  rises under pressure from gas  60  introduced from the second supply line  135 . 
         [0080]      FIGS. 16 through 20  illustrate a bioreactor positioned in a housing, generally designated  410 . The housing  410  is structural and preferably made of stainless steel to include a housing front  412 , housing sides  414  and a housing back  416 . The housing back  416  does not extend fully to the floor or other support in order that access may be had to the underside of the bioreactor. The housing  410  includes a housing bottom  418  which extends from the housing sides  414  in a semi-cylindrical curve above the base of the housing  410 . One of the front  412  or back  416  may act as a door to facilitate access to the interior of the housing  410 . 
         [0081]    The bioreactor includes a containment vessel, generally designated  420 , defined by four vessel sides  422 ,  424 ,  426 ,  428 , a semi-cylindrical vessel bottom  430 , seen in  FIG. 17 , and a vessel top  432 . Two of the vessel sides  424 ,  428  which are opposed each include a semicircular end. The other two vessel sides  422 ,  426  join with the semi-cylindrical vessel bottom  430  to form a continuous cavity between the two vessel sides  424 ,  428 . All four vessel sides  422 ,  424 ,  426 ,  428  extend to and are sealed with the vessel top  432  to form a sealed enclosure. The vessel top  432  extends outwardly of the four vessel sides  422 ,  424 ,  426 ,  428  so as to rest on the upper edges of the structural housing front  412 , sides  414  and back  416 . Thus, the containment vessel  420  hangs from the top  432  in the housing  410 . The vessel, with the exception of the vessel top  432 , is of thin wall film which is not structural in nature. Therefore, the housing front  412 , sides  414 , back  416  and bottom  418  structurally support the containment vessel  420  depending from the vessel top  432  when filled with liquid. All joints of the containment vessel  420  are welded or otherwise sealed to provide the appropriate sealed enclosure which can be sterilized and closed ready for use. 
         [0082]    The vessel top  432  includes access ports  434  for receipt or extraction of liquids, gases and powders and grains of solid materials. The access ports  436  in the vessel top  432  provide for receipt of sensors to observe the process. Two orifices  438 ,  440  are shown at the vessel bottom  430  slightly offset from the centerline to receive propellant gas for driving the rotational mixer as will be discussed below. The semi-cylindrical vessel bottom  430  defining a semi-cylindrical concavity within the containment vessel  420  also includes a temperature control sheet  442  which may include a heater with heating elements, a cooler with cooling coils, or both as may be employed to raise or lower the temperature of the contents of the containment vessel  420  during use. Sealed within the enclosure defining the containment vessel  420 , struts  444  extend downwardly from the vessel top  432  to define a horizontal mounting axis at or close to the axis of curvature defined by the semi-cylindrical bottom  430 . 
         [0083]    A mixing apparatus includes a rotatably mounted rotational mixer, generally designated  448 . The rotational mixer  448  is a general assembly of a number of functional components. The structure of the rotational mixer  448  includes two parallel wheels  450 ,  452  which are displaced from one another. These wheels are tied to an axle  454  by spokes  456 . Additional stabilizing bars parallel to the axle  54  may be used to rigidify the rotational mixer  448 . 
         [0084]    Each wheel  450 ,  452  is defined by two parallel plates  460 ,  462 . These plates  460 ,  462  include buoyancy-driven mixing cavities  464  there between. These cavities  464  operate to entrap gas supplied from below the wheels  450 ,  452  through the gas supply at orifices  438 ,  440 . The orifices  438 ,  440  are offset from being directly aligned with the horizontal axis of rotation to insure that the buoyancy-driven cavities  464  are adequately filled with gas to power the rotational mixer  448  in rotation. In the embodiment of  FIGS. 16 through 20 , the buoyancy-driven cavity  464  in each one of the wheels  450 ,  452  are similarly oriented to receive gas from the orifices  438 ,  440  at the same time. 
         [0085]    Outer paddles  466  are equiangularly placed to extend axially outwardly from the outer parallel plates  460  where they are attached. These outer paddles  466  can mix the liquid between the rotational mixer  448  and either side  424 ,  428 . The outer paddles  466  are formed in this embodiment with a concavity toward the direction of rotation of the rotational mixer  448  and are inclined toward the direction of rotation as well such that they are disposed to induce flow entrained with constituents of the mix in the vessel inwardly toward the axis for flow through each wheel  450 ,  452  with the rotation of the rotational mixer  448 . The outer paddles  466  may exhibit an inclined orientation on each of the outer parallel plates  460  such that any induced axial flow through each wheel  450 ,  452  will flow toward the center of the rotational mixer  448  in opposite directions. The number of outer paddles  466  may be increased from the four shown, particularly when the constituents of the mix in the vessel are not easily maintained in suspension. The outer paddles  466  may extend close to the vessel bottom  430  to entrain constituents of the mix in the vessel which may otherwise accumulate on the bottom. Such extensions beyond the wheels  450 ,  452  preferably do not inhibit rotation of the rotational mixer  448  through actual or close interaction with the vessel wall. 
         [0086]    Inwardly of the two wheels  450 ,  452 , vanes  468  may be employed in some embodiments as can best be seen in  FIG. 20 . These vanes  468  extend axially inwardly from the inner parallel plates  462  to span the distance there between. The vanes  468  can also extend to induce flow radially outwardly from the rotational mixer  448  and beyond the rotational mixer  448  so as to impact and mix liquid outwardly of the rotational mixer. As with the outer paddles  466 , the vanes  468  can be used to entrain constituents that tend to fall and collect on the vessel bottom  430 . These vanes  468  may, in some instances not be preferred because of flow resistance or disruption of circulating flow. Empirical analysis is necessary in this regard depending on such things as rotational mixer speed, liquid viscosity, space to the vessel walls and the like. Four vanes  468  are illustrated on each wheel  450 ,  452  but the number can, as with the outer paddles  466 , be increased or decreased with the performance of the mix. 
         [0087]    Inner paddles  470  also extend axially inwardly from the inner parallel plates  462 . These inner paddles  470  are convex facing toward the rotational direction and are inclined to draw flow axially through the wheels  450 ,  452 . The inner paddles  470  can enhance radially outward flow with rotation of the rotational mixer  448  as well at the location shown inside of the wheels  450 ,  452 . There can be any practical number of inner paddles  470 , four being shown. Such paddles  470 , if configured to extend past the perimeter of the wheels  450 ,  452 , can urge flow off of the bottom as well and direct that flow axially outwardly to either side. 
         [0088]    Located inwardly of each wheel  450 ,  452  is an impeller having blades  472 . The two impellers provide principal axial thrust to the flow through the wheels  450 ,  452 . The thrust resulting from these blades  472  both flow inwardly toward one another in this embodiment. This is advantageous in creating toroidal flow about the wheels and balance forces which would otherwise be imposed on the mountings. The placement of the blades  472  may be at other axial locations such as at either of the plates  460 ,  462 . Where the impellers act alone, the blades  472  can be located anywhere from exterior of to interior to the rotational mixer with appropriate reconfiguration in keeping with slow speed impeller practice. 
         [0089]    The mixing apparatus defined principally by the rotating rotational mixer  448  is positioned in the containment vessel  420  such that it extends into the semi-cylindrical concavity defined by the vessel bottom  430  and is sized, with the outer paddles  466 , vanes  468  and inner paddles  470 , to fill the concavity but for sufficient space between the mixing apparatus and the vessel sides  424 ,  428  and bottom  430  to avoid inhibiting free rotation of the rotational mixer  448 . In one embodiment, the full extent of the mixing apparatus  426  is on the order of 10% smaller than the width of the cavity in the containment vessel  420  and about the same ratio for the diameter of the rotational mixer  448  to the semi-cylindrical vessel bottom  430 . This spacing is not critical so long as the mixing apparatus is close enough and with commensurate speed to effect mixing throughout the concavity. Obviously, empirical testing is again of value. The liquid preferably does not extend above the mixing apparatus and the volume above the rotational mixer  448  will naturally be mixed as well. 
         [0090]    In operation, the liquid, nutrients and active elements are introduced into the containment vessel  420  through the ports  434 ,  436 . The level of material in the vessel  420  is below the top of the rotational mixer  448  to avoid the release of driving gas under the liquid surface which may cause foam. Gas is injected through the orifices  438 ,  440  to become entrapped in the buoyancy-driven cavity  464  in the rotational mixer  448 . This action drives the rotational mixer  448  in a direction which is seen as clockwise in  FIG. 17 . 
         [0091]    The blades  472  act to circulate the liquid within the containment vessel  420  with toroidal flow in opposite directions through the wheels  450 ,  452 , radially outwardly from between the wheels  450 ,  452  and then radially inwardly on the outsides of the rotational mixer  448  to again be drawn into the interior of the rotational mixer  448 . Mixing with turbulence is desired and the outer paddles  466 , the vanes  468  and the inner paddles  470  contribute to the mixing and to the toroidal flow about each of the wheels  450 ,  452 . The target speed of rotation is on the order of up to the low tens of rpm to achieve the similar mixing results as prior devices at  50  to  300  rpm. The difference may reduce shear damage in more sensitive materials. Oxygen may be introduced in a conventional manner as well as part of the driving gas to be mixed fully throughout the vessel  420  under the influence of the mixing apparatus. 
         [0092]      FIG. 21  illustrates a variation on the embodiment of  FIGS. 16 through 20 . In this embodiment, the buoyancy-driven mixing cavities  464  are reversed in one of the wheels  450 ,  452  for driving in the opposite direction. Similarly, the orifices  438 ,  440  are offset to either side of the horizontal axis of rotation. The gas through the orifices  438 ,  440  is independently controlled to allow selection of rotation of the rotational mixer in either direction. 
         [0093]    Thus, an improved pneumatic bioreactor is disclosed. While embodiments and applications of this invention have been shown and described, it would be apparent to those skilled in the art that many more modifications are possible without departing from the inventive concepts herein. The invention, therefore, is not to be restricted except in the spirit of the appended claims. 
         [0094]    An appreciation of the other aims and objectives of the present invention and an understanding of it may be achieved by referring to the accompanying drawings and the detailed description of a preferred embodiment.