Abstract:
System for modifying the chemical composition of atmosphere within an enclosed space and incubator system including such a system. The concentration of oxygen within the enclosed space may be either increased or decreased using an electrochemical device. The concentration of carbon dioxide within the enclosed space may be increased using an electrochemical or chemical device. As necessary, purging of the system with ambient air can be a part of the process of controlling the chemical composition of the atmosphere. The present invention obviates the need to use pressurized gas cylinders to supply atmospheric gases to the enclosed space.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 60/923,296, filed Apr. 13, 2007, the disclosure of which is incorporated herein by reference. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Contract Nos. 1 R43EB001511-01A1 and 1 R44EB001511-02 awarded by the National Institutes of Health. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    The present invention relates generally to systems for modifying the atmosphere within an enclosed space and relates more particularly to a new system for modifying the atmosphere within an enclosed space and to an incubator system including the same. 
         [0004]    There are many different types of situations in which it may be desirable to provide an enclosed space having a controlled environment. One such example involves cell culture, particularly mammalian cell culture. Mammalian cell culture currently plays an important role in a number of areas of great interest including, but not limited to, basic medical research, transplantation, tissue engineering, stem cell production, vaccine production, antibody production and cellular assays. Mammalian cell culture is typically performed in an incubator, an enclosed chamber in which various environmental conditions, e.g., atmospheric composition, temperature, etc., are carefully controlled. Control over the composition of gases within an incubator is typically provided using gas supplied from one or more pressurized gas cylinders. For example, conventional incubators for mammalian cell culture are typically operated at 142 mm Hg (18.6%) oxygen and 38 mm Hg (5%) carbon dioxide. Such oxygen and carbon dioxide concentrations are typically achieved using pre-prepared O 2 /CO 2  gas mixtures stored in pressurized gas cylinders. 
         [0005]    However, as the role of oxygen concentration in cellular physiology becomes better understood, the need for adjustable oxygen concentrations for both research and production is becoming more apparent. For example, lower concentrations of oxygen are often required for research on stem cells, embryonic cells, and tumor cells; higher concentrations of oxygen may be advantageous for multilayer tissues, such as cultures of skin, pancreatic islets or tissue engineering products. Such diversity in the requirements for oxygen means that an adjustable concentration of incubator oxygen could enhance the research and production of many cell lines and primary cells. Some commercially-available low O 2 -concentration incubator systems include a dual gas system, wherein one gas cylinder typically contains an O 2 /CO 2  mixture and another gas cylinder typically contains N 2 , the N 2  being used to achieve a desired low oxygen level by purging, i.e., supplanting some of the oxygen and carbon dioxide with nitrogen. Alternatively, there are “tri-gas” systems, in which CO 2 , O 2 , and N 2  are introduced from three different cylinders at varying ratios to provide adjustable oxygen and carbon dioxide levels. 
         [0006]    Current commercially-available incubators using gas cylinders are capable of achieving the following environmental specifications: CO 2  ranges of 0-20%, O 2  ranges of 0-90%, relative humidity up to 95%, and temperature ranges of 5° C. above ambient to 60° C. above ambient. Special auxiliary equipment can provide temperatures below ambient. The recovery time (after opening the incubator) to regain these atmospheric set points can range from 8 minutes for CO 2  to 10 minutes for temperature and humidity recovery. The temperature of the incubator atmosphere can be controlled through the use of heat exchangers which can increase or decrease the temperature of the gas stream before it enters the incubator. Similarly, the humidity of the incubator atmosphere can be conditioned through the use of humidifiers and dehumidifiers, which can increase or decrease the humidity of the gas stream before it enters the incubator. The sizes and weights of these commercially available incubation systems are variable, with weights varying from 187 lbs to 490 lbs (excluding gas cylinders) and incubator spaces ranging from 5 ft 3  to 29 ft 3 , respectively. 
         [0007]    Unfortunately, the utilization of gas cylinders is cumbersome and inconvenient, in part, because of the size and the weight of the cylinders and, in many cases, because of the need for frequent cylinder changes. Gas cylinders can also be dangerous due to the possibility of uncontrolled release of the highly compressed gases contained within the cylinders. As a result, facilities using gas cylinders typically have to establish and to enforce safety rules on where gas cylinders can be stored and in what quantity, which loading docks and elevators can be utilized for the cylinders, which personnel can handle such gas cylinders, and the methods of securing the cylinders to walls. Also, where specialty gas mixtures are involved, the use of gas cylinders may result in a lag time between experiments, in the possibility of running out of a desired gas type, and in the expense involved in ordering specialty gas mixtures. Gas cylinders also run the risk of introducing chemical contaminants to an incubator due to previous gases stored in the cylinders and due to oils present in valves. 
       SUMMARY OF THE INVENTION 
       [0008]    It is an object of the present invention to provide a new system for modifying the atmosphere within an enclosed space, said enclosed space being, for example, an incubation chamber of a cell culture incubator. 
         [0009]    It is another object of the present invention to provide a system as described above that overcomes at least some of the shortcomings associated with existing methods and systems for controlling the atmosphere within an enclosed space. 
         [0010]    It is still another object of the present invention to provide a system as described above that obviates the need for atmospheric gases to be provided to the enclosed space using pressurized gas cylinders. 
         [0011]    According to one aspect of the invention, the concentration of gaseous oxygen within an enclosed space may be selectively increased or decreased using an electrochemical oxygen concentrator. 
         [0012]    According to another aspect of the invention, the concentration of gaseous carbon dioxide within an enclosed space may be selectively increased using a direct alcohol fuel cell, a sodium bicarbonate electrolyzer, or a chemical oxidation of an alcohol solution. 
         [0013]    According to still another aspect of the invention, the concentration of gaseous oxygen within an enclosed space may be selectively increased or decreased using an electrochemical oxygen concentrator, and the concentration of gaseous carbon dioxide within the same enclosed space may be selectively increased using a direct alcohol fuel cell, a sodium bicarbonate electrolyzer, or a chemical oxidizer of an alcohol solution. 
         [0014]    One advantage of the present invention is that there is no need to have gas supplied from pressurized gas cylinders in order to control the concentration of atmospheric gases within an enclosed space. Instead, an integrated electrochemical device may be used, as desired, to increase or to decrease the oxygen concentration within the enclosed space, and an integrated electrochemical or chemical device may be used, as desired, to increase the carbon dioxide concentration within the enclosed space. 
         [0015]    Additional objects, as well as aspects, features and advantages, of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the invention. In the description, reference is made to the accompanying drawings which form a part thereof and in which is shown by way of illustration various embodiments for practicing the invention. The embodiments will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]    The accompanying drawings, which are hereby incorporated into and constitute a part of this specification, illustrate various embodiments of the invention and, together with the description, serve to explain the principles of the invention. In the drawings wherein like reference numerals represent like parts: 
           [0017]      FIG. 1  is a block diagram of one embodiment of an incubator system incorporating the atmosphere control system of the present invention; 
           [0018]      FIG. 2  is a table indicating the operating modes of the atmosphere control system of the present invention as a function of the user-setpoints and the oxygen and carbon dioxide concentrations in the incubator atmosphere; 
           [0019]      FIG. 3  is a schematic diagram of one embodiment of the electrochemical oxygen concentrator (EOC) subsystem shown in  FIG. 1 , the EOC subsystem being shown together with the incubator of  FIG. 1 ; 
           [0020]      FIG. 4  is a schematic diagram of a first embodiment of the carbon dioxide generation subsystem shown in  FIG. 1 , the carbon dioxide generation subsystem being shown together with the incubator of  FIG. 1 ; 
           [0021]      FIG. 5  is a schematic diagram of an alternate embodiment of a carbon dioxide generation subsystem suitable for use in the incubator system of  FIG. 1 ; and 
           [0022]      FIG. 6  is a schematic diagram of a further alternate embodiment of a carbon dioxide generation subsystem suitable for use in the incubator system of  FIG. 1 . 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0023]    Referring now to  FIG. 1 , there is shown a block diagram of an incubator system incorporating the atmosphere control system of the present invention, the incubator system being represented generally by reference numeral  10 . 
         [0024]    System  10  comprises an incubator  100 , an electrochemical oxygen concentrator (EOC) subsystem  200 , a carbon dioxide generation subsystem  300 , and a process controller  400 . EOC subsystem  200 , carbon dioxide generation subsystem  300 , and process controller  400  collectively constitute an atmosphere control system. The atmosphere control system may be used to control the oxygen and carbon dioxide concentrations in the internal atmosphere of incubator  100 . (If desired, the atmosphere control system may be modified to control only oxygen concentration by elimination of carbon dioxide generation subsystem  300  and appropriate modifications to process controller  400 .) 
         [0025]    Incubator  100  comprises an oxygen gas sensor  110  for use in measuring the concentration of oxygen gas present in the internal atmosphere of incubator  100 , a carbon dioxide gas sensor  120  for use in measuring the concentration of carbon dioxide gas present in the internal atmosphere of incubator  100 , and a hydrogen gas sensor  130  for use in measuring the concentration of hydrogen gas present in the internal atmosphere of incubator  100 . In addition, incubator  100  further comprises a temperature sensor  140  for use in measuring the temperature of the internal atmosphere of incubator  100  and a humidity sensor  150  for use in measuring the relative humidity of the internal atmosphere of incubator  100 . The aforementioned sensors may be included in a commercial incubator as standard features and/or may be retrofitted to a commercial incubator lacking such sensors. Although the aforementioned sensors are shown in the present embodiment as being part of incubator  100 , one or more of these sensors may alternatively be located elsewhere in system  10 , such as in a gas recirculation loop. Incubator  100  further comprises a user interface (not shown) for selecting an oxygen concentration setpoint and a carbon dioxide concentration setpoint. Instead of being included in incubator  100 , the user interface may be a part of process controller  400 . 
         [0026]    To begin operation, the user selects the desired oxygen and carbon dioxide concentrations via the oxygen concentration setpoint and the carbon dioxide concentration setpoint. Next, process controller  400  compares the readings taken by oxygen concentration sensor  110  and carbon dioxide concentration sensor  120  with the selected setpoints. Next, assuming there is a differential between the readings and the setpoints, process controller  400 : (1) initiates operation of EOC subsystem  200  in oxygen-addition or oxygen-depletion mode, and/or (2) initiates operation of the carbon dioxide subsystem  300 , or (3) purges incubator  100  with ambient air using an air blower of EOC subsystem  200 . When the setpoint conditions are satisfied, the atmosphere control system operates in idle mode, during which process controller  400  monitors the oxygen and carbon dioxide concentrations. The logic used to determine the operating mode of the atmosphere control system based on the user-setpoints and the detected oxygen and carbon dioxide concentrations is shown in the table of  FIG. 2 . The upper portion of the table of  FIG. 2  describes the action of the atmosphere control system with respect to oxygen concentration. Col. 1 of the table shows whether the user-selected oxygen setpoint is greater than, equal to, or less than 21%—the nominal oxygen concentration in ambient air. Col. 2 shows whether the user-selected oxygen setpoint is greater than, equal to, or less than the reading of the incubator oxygen sensor  110 . Similarly, Col. 3 compares the reading of the incubator oxygen sensor  110  to 21%. Col. 4 indicates the action of the atmosphere control system that occurs in accordance with the conditions of Col. 1, Col. 2 and Col. 3. Col. 5 and Col. 6 provide specific examples of oxygen concentration setpoints and oxygen sensor  110  readings that meet the criteria of Col. 1 through Col. 3 and that would result in the action listed in Col. 4. For example, in the fourth row down, the user has entered an oxygen setpoint of 30% and the incubator atmosphere, as measured by the oxygen concentration sensor  110 , contains 21% oxygen. In this case, the atmosphere control system would activate EOC subsystem  200  in oxygen-addition mode. When the user-selected oxygen setpoint is greater than 21% oxygen and the incubator oxygen sensor reading is less than 21%, the incubator may first be purged with atmospheric air to increase the oxygen concentration to 21%. Then, EOC subsystem  200  may be activated in O 2 -addition mode and operated until the oxygen setpoint is reached (Row 5). Similarly, when the user-selected oxygen setpoint is less than 21% oxygen and the incubator oxygen sensor reading is greater than 21%, the incubator may first be purged with atmospheric air to decrease the oxygen concentration to 21%. Then, EOC  200  may be activated in O 2 -depletion mode until the setpoint is reached (Row 9). Purging incubator  100  as described above to achieve the oxygen setpoint is desirable since it enables the system to reach the setpoint more rapidly. However, it should be noted that such purging may be omitted, with EOC  200  being run either in an oxygen-addition or oxygen-depletion mode until such time that the setpoint is reached. 
         [0027]    The logic used to control the action of the atmosphere control system with respect to carbon dioxide concentration is shown in the lower portion of the table of  FIG. 2 . As can be seen, if the user-selected carbon dioxide setpoint is greater than the reading of carbon dioxide sensor  120 , carbon dioxide generation subsystem  300  is activated. If the user-selected carbon dioxide setpoint is lower than the reading of the carbon dioxide sensor  120 , incubator  100  will be purged. 
         [0028]    EOC subsystem  200  and carbon dioxide generator subsystem  300  are controlled independently by process controller  400 . However, the purge mode takes precedence over oxygen addition and oxygen depletion by EOC subsystem  200  and over CO 2 -addition by carbon dioxide generator subsystem  300 . Thus, if the process logic requires purging of incubator  100  to meet the oxygen setpoint, incubator  100  will be purged, regardless of the carbon dioxide concentration. Similarly, if incubator  100  needs to be purged to meet the carbon dioxide setpoint, purging will occur, regardless of the oxygen concentration. During the purge mode, EOC subsystem  200  does not run in its oxygen-addition or oxygen-depletion modes, and carbon dioxide generator subsystem  300  does not run in its CO 2 -addition mode. 
         [0029]    Referring now to  FIG. 3 , additional details of EOC subsystem  200  are shown, EOC subsystem  200  being shown together with incubator  100 . 
         [0030]    EOC subsystem  200  comprises an EOC stack  210 . Stack  210  may contain a single oxygen-transfer cell or a plurality of series-connected oxygen-transfer cells, each of said cells comprising a cathode  212  and an anode  214 , the anode  212  and the cathode  214  of each cell being in intimate contact with and separated by an ionically-conductive separator  213 . In a preferred embodiment, ionically-conductive separator  213  is a solid proton-exchange membrane (PEM), such as NAFION® 117 membrane (E.I. du Pont de Nemours &amp; Company, Wilmington, Del.), anode  212  is a platinum/iridium particulate catalyst (50/50) with binder, applied as a decal to the solid PEM, and cathode  214  comprises a structural base of carbon paper as a porous carbon diffusion medium, onto which the side facing the PEM is sprayed platinum black particulate catalyst with binder. The opposite side of the carbon paper is preferably sprayed with TEFLON® polytertrafluoroethylene (PTFE) to provide wet proofing for the cathode. Anode  212  and cathode  214  are preferably in electrical contact with titanium bipolar plates (not shown) which have electrical leads to a power supply  216  and to process controller  400 . Stack  210  preferably additionally includes support screens (not shown) and frames (not shown) for holding anode  212  and cathode  214  in physical and electrical contact. 
         [0031]    EOC subsystem  200  additionally comprises a water reservoir/O 2  separator  220 . A fluid line  221  is connected at one end to an outlet from anode  214  and at the opposite end to an inlet of reservoir/separator  220  to conduct water and pure O 2  from anode  214  to reservoir/separator  220 . A fluid line  223  is connected at one end to an outlet located at the bottom of reservoir/separator  220  and at the opposite end to an inlet of a water pump  225  to conduct water from reservoir/separator  220  to water pump  225 . A fluid line  227  is connected at one end to an outlet from water pump  225  and at the opposite end to an inlet of anode  214  to conduct water from water pump  225  to anode  214 . 
         [0032]    EOC subsystem  200  additionally comprises a first 3-way, electrically-activatable valve  230 . Valve  230  may be, for example, a solenoid valve. A fluid line  231  is connected at one end to an outlet located at the top of reservoir/separator  220  and at the opposite end to a first port  232  of valve  230  to conduct pure O 2  from reservoir/separator  220  to valve  230 . A fluid line  234  is connected at one end to a second port  235  of valve  230  and at the opposite end to an inlet  102  of incubator  100 . Depending upon the operation of valve  230 , pure O 2  may be conducted from valve  230  to incubator  100  or may be vented through a third port  237  of valve  230 . 
         [0033]    EOC subsystem  200  additionally comprises a second 3-way, electrically-activatable valve  240 . Valve  240  may be, for example, a solenoid valve. A fluid line  241  is connected at one end to an outlet  103  from incubator  100  and at the opposite end to a first port  243  of valve  240  to conduct the atmosphere from incubator  100  to valve  240 . A fluid line  245  is connected at one end to a second port  247  of valve  240  and at the opposite end to the inlet of a blower  248 . A fluid line  249  connects the outlet of blower  248  to the inlet of cathode  212 . Depending upon the operation of valve  240 , blower  248  may receive the atmosphere from incubator  100  or may receive ambient air through a third port  250  of valve  240 . 
         [0034]    EOC subsystem  200  additionally comprises a separator  260 . A fluid line  261  is connected at one end to an outlet of cathode  212  and at the opposite end to an inlet of separator  260  to conduct sub-ambient air (i.e., O 2 -depleted air) and product water from cathode  212  to separator  260 . A fluid line  262  is connected at one end to an outlet at the bottom of separator  260  and at the opposite end to an inlet of separator  220  to conduct water from separator  260  to separator  220 . 
         [0035]    EOC subsystem  200  additionally comprises a third 3-way, electrically-activatable valve  270 . Valve  270  may be, for example, a solenoid valve. A fluid line  271  is connected at one end to an outlet from separator  260  and at the opposite end to a first port  273  of valve  270  to conduct the sub-ambient air from separator  260  to valve  270 . A fluid line  275  is connected at one end to a second port  277  of valve  270  and at the opposite end to an inlet  101  of incubator  100 . Depending upon the operation of valve  270 , the sub-ambient air from separator  260  may be conducted to incubator  100  through line  275  or may be vented through a third port  279  of valve  270 . 
         [0036]    It should be understood that EOC subsystem  200  may be modified to include certain components to improve operation or to configure the system for certain applications. These components include, but are not limited to: air filters, water deionizers or purifiers, gas purifiers, heat exchangers, additional valves, pumps or blowers, and process sensors and alarms. 
         [0037]    Additionally, it should be noted that, although  FIG. 3  does not show the interactions between process controller  400  and EOC subsystem  200 , process controller  400  controls all aspects of system operation, including, but not limited to: energizing/de-energizing solenoid valves, pump and blower speed, power supply voltage or current output, monitoring EOC stack and individual cell voltage, monitoring and alarming process variables, and automated shutdown in the event of a fault condition. 
       Operation of EOC Subsystem  200  During Oxygen Addition 
       [0038]    When process controller  400  initiates operation of EOC subsystem  200  in oxygen-addition mode, water from water reservoir/O 2  separator  220  is pumped by pump  225  over the anode  214  of EOC stack  210 . (An ion-exchange membrane or beads (not shown) may be positioned between pump  225  and anode  214  to ensure that the water fed to anode  214  is de-ionized water.) Simultaneously, ambient air is drawn through 3-way valve  240 , flowing from port  250  to port  247 , by air blower  248  and pumped across the cathode  212  of the EOC stack  210 . Power is supplied at a controlled voltage or current to EOC stack  210  by direct current (dc) power supply  216  to effect the following electrochemical reactions: 
         [0000]      (Anode): H 2 O→½O 2 +2H + +2 e   −   EQN. 1 
         [0000]      (Cathode): ½O 2  (from ambient air)+2H + +2 e   − →H 2 O  EQN. 2 
         [0000]      (Overall): ½O 2  (from ambient air)→½O 2   EQN. 3 
         [0039]    The gaseous oxygen produced at the anode  214 , together with any excess water, is conducted to the water reservoir/O 2  separator  220 . Within reservoir/separator  220 , the gaseous oxygen is separated from the water by gravity. The pure humidified gaseous oxygen then exits reservoir/separator  220  and flows through 3-way valve  230  from port  232  to port  235  and then to incubator  100  through port  102 . To prevent over-pressurization of incubator  100 , some of the incubator atmosphere is exhausted through a vent  104  as the pure oxygen is added to incubator  100 . 
         [0040]    The sub-ambient air leaving cathode  212 , which air has an oxygen concentration lower than that of ambient air due to the reaction of some of its oxygen, is then separated by gravity from the product water in separator  260 . The sub-ambient air then exits separator  260  and is vented from the system through 3-way valve  270  from port  273  through port  279 . The product water within separator  260  is returned to the water reservoir/O 2  separator  220 . 
         [0041]    Operation of EOC subsystem  200  in the oxygen-addition mode may continue until the incubator oxygen concentration sensor  110  detects an oxygen concentration within a predetermined range, for example, within 0.5% of the user-selected oxygen concentration setpoint. 
       Operation of EOC Subsystem  200  During Oxygen Depletion 
       [0042]    When process controller  400  initiates operation of EOC subsystem  200  in oxygen-depletion mode, atmosphere from incubator  100  is drawn through port  103  and through 3-way valve  240  from port  243  to  247 , is drawn past air blower  248 , and is pumped across the cathode  212  of EOC stack  210 . Simultaneously, water from the water reservoir/O 2  separator  220  is pumped by pump  225  over anode  214  of EOC stack  210 . Power is supplied at a controlled voltage or current to EOC stack  210  by dc power supply  216  to effect the electrochemical reactions shown in EQN. 1 and EQN. 2, except that, in the present case, the oxygen on the left-hand side of EQN. 2 is derived from the incubator atmosphere, as opposed to ambient air. 
         [0043]    The air leaving cathode  212 , which air has a decreased oxygen concentration following reaction of some of the oxygen, is separated from the product water in separator  260 . The saturated sub-ambient then air flows through 3-way valve  270  from port  273  to  277  to incubator  100  through port  101 . The product water collected in separator  260  is returned to the water reservoir/O 2  separator  220 . 
         [0044]    The oxygen produced at anode  214  is separated by gravity from the excess water in water reservoir/O 2  separator  220 . The pure humidified oxygen is then vented from system  200  through 3-way valve  230  from port  232  to port  237 . 
         [0045]    Operation of EOC subsystem  200  in the oxygen-depletion mode may continue until the incubator oxygen concentration sensor  110  detects an oxygen concentration within a predetermined range, for example, within 0.5% of the user-selected oxygen concentration setpoint. 
       General Considerations of EOC Stack Voltage and Current 
       [0046]    In both the oxygen-addition and oxygen-depletion modes, EOC stack  210  may be operated in either voltage control or current control mode. The dc power supply  216  may be instructed by process controller  400  to maintain a given voltage or current across EOC stack  210 . Process controller  400  may vary the given voltage or current as the process requires. In a preferred embodiment, power supply  216  maintains a set voltage across EOC stack  210 . The current of EOC stack  210  and, thus, the oxygen-generation or oxygen-depletion rate varies with the process conditions, primarily the temperature of EOC stack  210  and the oxygen concentration of the feed to cathode  212 . The voltage of EOC stack  210  is set at a value that will prevent the entire stack from reaching the electrochemical potential at which hydrogen evolution on the cathode  212  will occur (i.e., the standard water electrolysis potential). For example, the operating voltage of a 10-cell EOC stack may be set at 12 V, an average of 1.2 V/cell, which is below the standard water electrolysis potential of 1.23 V. 
         [0047]    To further minimize the possibility of hydrogen generation in EOC stack  210 , process controller  400  may monitor the voltage of the individual cells in EOC stack  210  to ensure that an individual cell is not generating hydrogen, instead of reducing oxygen. If an individual cell voltage is higher than a predetermined value indicative of hydrogen evolution, for example 1.23 V, the current provided by power supply  216  to EOC stack  210  may be decreased or operation of EOC subsystem  200  may be automatically shut down. A hydrogen sensor  130  is included in incubator  100  as a further precaution. If hydrogen sensor  130  detects a hydrogen concentration greater than a predetermined low level, process controller  400  may be configured to shut down operation of EOC subsystem  200 . 
         [0048]    The current at a set voltage that can be sustained by EOC stack  210  during oxygen depletion decreases as the oxygen concentration in the incubator recirculation stream that feeds cathode  212  decreases. At sufficiently low oxygen concentrations, diffusion of oxygen to cathode  212  limits the current that can be drawn by the EOC stack  210 . In one embodiment of the invention, this limiting current is used as a measurement of the oxygen concentration in the recirculating stream, either as a replacement for oxygen sensor  110  or as an additional measurement at low oxygen concentrations. 
       Operation of EOC Subsystem  200  During Purge Mode 
       [0049]    When process controller  400  initiates a purge of incubator  100  to make the atmosphere inside incubator  100  more similar to the external ambient atmosphere, ambient air is drawn through 3-way valve  240  from port  250  to port  247  by air blower  248 . This air is pumped through cathode  212  of EOC stack  210 , which is not powered, through the separator  260 , and through 3-way valve  270  from port  273  to port  277  into incubator  100  through port  101 . The purge air replaces the atmosphere within incubator  100 , which is vented through port  104 . The purge may continue until the incubator oxygen concentration sensor  110  detects an oxygen concentration within a predetermined range, for example, 0.5% of the oxygen concentration in ambient air, namely, 21%. Purge mode for achieving a desired oxygen concentration is optional but is preferable since it enables the system to reach its setpoint more rapidly. (Purge mode may also be used to adjust the carbon dioxide concentration within incubator  100 .) 
         [0050]    Referring now to  FIG. 4 , additional details of carbon dioxide generation subsystem  300  are shown, carbon dioxide generation subsystem  300  being shown together with incubator  100 . 
         [0051]    Subsystem  300  comprises a direct alcohol fuel cell stack  310 , which in the present embodiment is preferably a direct methanol fuel cell (DMFC) stack. Stack  310  may contain a single cell or a plurality of series-connected cells, each cell comprising an anode  312  and a cathode  314  in intimate contact with and separated by an ionically-conductive separator  313 . In a preferred embodiment, the ionically-conductive separator comprises a proton-exchange membrane  313 . Anode  312  and cathode  314  are electrically coupled to a load  316  and to process controller  400 . 
         [0052]    Subsystem  300  additionally comprises a feed mixing tank  320 . A fluid line  322  is connected at one end to an outlet of anode  312  and at an opposite end to an inlet of tank  320  to conduct anolyte from anode  312  to mixing tank  320 . 
         [0053]    Subsystem  300  additionally comprises a methanol tank  323 . A fluid line  325  is connected at one end to an outlet of tank  323  and at an opposite end to an inlet of a methanol pump  327 . A fluid line  329  is connected at one end to an outlet of pump  327  and at an opposite end to an inlet of mixing tank  320 . In this manner, methanol may be pumped from tank  323  into mixing tank  320 . 
         [0054]    Subsystem  300  additionally comprises a feed pump  331 . A fluid line  333  is connected at one end to an outlet at the bottom of mixing tank  320  and at the opposite end to an inlet of feed pump  331 . A fluid line  335  is connected at one end to an outlet of feed pump  331  and at the opposite end to an inlet of a methanol sensor  337 . A fluid line  339  is connected at one end to an outlet of sensor  337  and at the opposite end to an inlet of anode  312 . In this manner, methanol may be pumped, as needed, from mixing tank  320  to anode  312 . 
         [0055]    Subsystem  300  additionally comprises a CO 2  purifier  341 . A fluid line  343  is connected at one end to an outlet of feed mixing tank  320  and at the opposite end to an inlet of purifier  341 . A fluid line  345  is connected at one end to an outlet of purifier  341  and at the opposite end to a carbon dioxide inlet port  105  in incubator  100 . 
         [0056]    Subsystem  300  additionally comprises an air/water separator  351 . A fluid line  353  is connected at one end to an outlet of cathode  314  and at the opposite end to an inlet of separator  351 . A fluid line  355  is connected at one end to an outlet at the bottom of separator  351  and at the opposite end to an inlet of mixing tank  320  to conduct water to tank  320 . An outlet is provided at the top of separator  351  to permit air to be exhausted from separator  351 . 
         [0057]    Subsystem  300  additionally comprises a blower  360 . A fluid line  362  is connected at one end to blower  360  and at the opposite end to the inlet of cathode  314  so that air may be supplied to cathode  314  by blower  360 . 
         [0058]    It should be understood that subsystem  300  may be modified to include certain components to improve operation or to configure the system for certain applications. These components include, but are not limited to: air filters, water deionizers or purifiers, heat exchangers, additional valves, pumps or blowers, and process sensors and alarms. 
         [0059]    Additionally, it should be noted that, although  FIG. 4  does not show the interactions between process controller  400  and subsystem  300 , process controller  400  controls all aspects of system operation, including, but not limited to: energizing/de-energizing solenoid valves, controlling pump and blower speed, controlling load voltage or current settings, monitoring DMFC stack and individual cell voltages, monitoring and alarming process variables, and automated shutdown in event of a fault condition. Furthermore, it should be noted that stack  310  may be scaled up to provide sufficient electricity to power some or all of EOC subsystem  200 , as well as other components of system  10 . 
         [0060]    In use, when process controller  400  initiates operation of subsystem  300 , ambient air is supplied by blower  360  to cathode  314  of stack  310 . Simultaneously, a dilute aqueous methanol solution (anolyte) having a methanol concentration in the range of 0.25 M to 3 M, preferably in the concentration range of 0.5 M to 1.5 M, is pumped from feed mixing tank  320  across anode  312  by feed pump  331 . A load  316  having a controlled current and/or voltage is placed across stack  310 , resulting in the reactions shown below in EQN. 4 through EQN. 6. 
         [0000]      (Anode): CH 3 OH+H 2 O→CO 2 +6H + +6 e   −   EQN. 4 
         [0000]      (Cathode): 3/2O 2 +6H + +6 e   − →3H 2 O  EQN. 5 
         [0000]      (Overall): CH 3 OH+3/2O 2 →2H 2 O+CO 2   EQN. 6 
         [0061]    The effluent from anode  312 , which contains gaseous and/or dissolved carbon dioxide, as well as water and unreacted methanol, is returned to mixing tank  320 . The gaseous carbon dioxide is separated from the methanol solution in tank  320  by gravity and is then treated in purifier  341  to remove any methanol liquid or vapor present in the stream. The purified carbon dioxide gas then flows to incubator  100  through port  105 . To prevent over-pressurization of incubator  100 , some of the atmosphere from incubator  100  is exhausted through vent  104  as carbon dioxide is added to incubator  100 . 
         [0062]    Methanol fuel is stored in tank  323 . Tank  323  may be a refillable container or a pre-filled cartridge containing neat (pure) methanol or a methanol-water solution that is more concentrated than the solution in mixing tank  320 . For example, a 24 volume percent methanol solution may be used as the fuel to minimize flammability concerns of methanol fuel. 
         [0063]    The methanol concentration of the anode feed solution may be measured by methanol concentration sensor  337 . When sensor  337  detects a methanol concentration below a predetermined value, process controller  400  energizes methanol pump  327  to dispense methanol fuel from tank  323  into mixing tank  320 . Addition of methanol fuel may continue until a predetermined methanol concentration is obtained, as measured by sensor  337 . 
         [0064]    The effluent from cathode  314  contains sub-ambient air, product water, and water that came across separator  313  from anode  312 . This effluent stream flows to an air/water separator  351 . The sub-ambient air in separator  351  is vented from the system while the water is returned to mixing tank  320 . 
         [0065]    Operation of subsystem  300  may continue until carbon dioxide concentration sensor  120  detects a carbon dioxide concentration within a predetermined range, for example, within 0.5% of the user-selected carbon dioxide concentration setpoint. 
         [0066]    Referring now to  FIG. 5 , there is shown a schematic diagram of an alternate embodiment of a carbon dioxide generation subsystem suitable for use in incubator system  10 , the alternate carbon dioxide generation subsystem being usable in place of carbon dioxide generation subsystem  300 , said alternate carbon dioxide generation subsystem being represented generally by reference numeral  500 . 
         [0067]    Subsystem  500  generates carbon dioxide by the catalytic combustion of an alcohol fuel, which, in the present embodiment, is methanol. Subsystem  500  comprises a catalytic combustor  542 , which may be a single cell or a plurality of cells, each cell containing a fuel chamber  544 , a pervaporation membrane  545 , a catalyzed substrate  546 , and an air chamber  548 . 
         [0068]    Subsystem  500  additionally comprises a fuel tank  550 , fuel tank  550  containing a volume of an aqueous methanol solution. A fluid line  551  is connected at one end to an outlet of fuel tank  550  and at the opposite end to an inlet of fuel chamber  544 . 
         [0069]    Subsystem  500  additionally comprises a spent fuel reservoir  552 . A fluid line  553  is connected at one end to an outlet of fuel chamber  544  and at the opposite end to an inlet of reservoir  552 . A valve  555  may be connected to an outlet of reservoir  552  to drain reservoir  552 . 
         [0070]    Subsystem  500  additionally comprises an air pump  564 . A fluid line  565  is connected at one end to the output of air pump  564  and at the opposite end to an inlet of air chamber  548 . A fluid line  570  is connected at an outlet of air chamber  548  to convey carbon dioxide. 
         [0071]    It should be understood that subsystem  500  may be modified to include certain components to improve operation or to configure the system for certain applications. These components include, but are not limited to: air filters, water deionizers or purifiers, gas purifiers, heat exchangers, additional valves, pumps or blowers, and process sensors and alarms. 
         [0072]    Additionally, it should be noted that, although  FIG. 5  does not show the interactions between process controller  400  and subsystem  500 , process controller  400  controls all aspects of the operation of subsystem  500 . 
         [0073]    In operation, methanol or a methanol solution is supplied from fuel tank  550  to fuel chamber  544  of the catalytic combustor  542 . In a preferred embodiment, the fuel is an aqueous methanol solution containing from 4 vol % to  50  vol % methanol. The fuel solution may flow by gravity or a pump may be used. Fuel chamber  544  contains flow channels or passages for the fuel solution. One side of fuel chamber  544  houses pervaporation membrane  545 , which has a high permeability for methanol vapor but does not pass liquid methanol. The methanol in the solution pervaporates to the opposite side of membrane  545 , which is adjacent to catalyzed substrate  546  containing a high surface area noble metal catalyst, such as platinum. The catalyst is treated or structured to provide hydrophobic channels to allow air access to the catalyst sites. Catalyzed substrate  546  is positioned on one side of air chamber  548 . Ambient air is supplied to air chamber  548  by air pump  564 . Oxygen in the air reacts with methanol on catalyzed substrate  546  to form carbon dioxide according to EQN. 7. 
         [0000]      CH 3 OH+3/2O 2 →CO 2 +2H 2 O  EQN. 7 
         [0074]    The effluent from air chamber  548  contains up to 14 vol % carbon dioxide in a subambient air stream. This stream may then be supplied to incubator  100  using line  570 . The spent methanol fuel leaving fuel chamber  544  is collected in spent fuel reservoir  552 . Although not shown in the present embodiment, the spent methanol fuel may be recirculated from reservoir  552  to fuel tank  550 . 
         [0075]    Referring now to  FIG. 6 , there is shown a schematic diagram of a further alternate embodiment of a carbon dioxide generation subsystem suitable for use in incubator system  10 , the alternate carbon dioxide generation subsystem being usable in place of carbon dioxide generation subsystem  300 , said alternate carbon dioxide generation subsystem being represented generally by reference numeral  600 . 
         [0076]    Subsystem  600 , which is based on the electrolysis of an aqueous sodium bicarbonate solution, comprises a sodium bicarbonate electrolyzer stack  661 . Stack  661  comprises a single cell or a plurality of cells connected in series or parallel. Each cell is divided into three chambers, an anode chamber  662 , a center chamber  665 , and a cathode chamber  668 . An anode electrode  663  containing Pt catalyst is in intimate contact with a perfluorocarbon proton-exchange membrane (PEM)  664  in the sulfonic acid form. Membrane  664  separates anode chamber  662  from center chamber  665 . A second PEM  666  separates center chamber  665  from cathode chamber  668 . PEM  666  may be in the sulfonic acid form, or more preferably, in the carboxylic acid form. A cathode electrode  667  containing Pt catalyst is in intimate contact with PEM  666 . 
         [0077]    In operation, hydrogen gas produced at cathode  667  is re-circulated to anode chamber  662  through a port  674 . The hydrogen is oxidized on anode electrode  663  to form H +  ions and electrons. The H +  ions are transported through PEM  664  to center chamber  665 . Center chamber  665  contains an aqueous saturated bicarbonate solution, which is supplied from a tank  670  by a pump  672  through a port  675 . Sodium sulfate may be added to the sodium bicarbonate solution to serve as a supporting electrolyte. In center chamber  665 , the H +  ions chemically react with the sodium bicarbonate to form carbon dioxide, water and sodium ions (Na + ). The carbon dioxide is vented through a port  677  and is supplied to incubator  100 . Simultaneous with the H +  ion transport, the Na +  ions generated in center chamber  665  are transported through second PEM  666  to cathode chamber  668 . Sodium bicarbonate solution is also supplied to cathode chamber  668  from tank  670  by pump  672  through a port  676 . Water from the sodium bicarbonate solution is electrochemically reduced at cathode electrode  667  to form OH −  ion and hydrogen gas. The OH-combines with the transported Na +  ions to form sodium hydroxide (NaOH). The NaOH reacts with the sodium bicarbonate in cathode chamber  667  to form sodium carbonate (Na 2 CO 3 ). The sodium carbonate solution, mixed with hydrogen gas, flows from cathode compartment  668  through a port  678  to a separator  680 . Hydrogen is released from separator  680  through a port  682  and is re-circulated to anode chamber  662 . The sodium carbonate flows from separator  680  through a port  684  and is collected for disposal. 
         [0078]    Power for the electrolyzer is provided by a power supply  690 . The voltage per cell is approximately 1.0 V. This voltage is lower than that of a standard water electrolyzer because the hydrogen re-circulated from the cathode to the anode depolarizes the anode, resulting in the hydrogen oxidation reaction, rather than the oxygen evolution reaction at the anode and, thus, a lower cell voltage. The reactions occurring in subsystem  600  are as follows: 
         [0000]      (Anode): H 2 →2H + +2 e   −   EQN. 8 
         [0000]      (Chemical—Center Chamber): 2H + +2NaHCO 3 →2H 2 O+2CO 2 +2Na +   EQN. 9 
         [0000]      (Cathode): 2H 2 O+2 e   − →H 2 +2OH −   EQN. 10 
         [0000]      (Chemical—Anode Chamber): 2NaOH+2NaHCO 3 →2Na 2 CO 3 +2H 2 O  EQN. 11 
         [0000]      (Overall): 4NaHCO 3 →2Na 2 CO 3 +2H 2 O+2CO 2   EQN. 12 
         [0079]    It should be understood that subsystem  600  may be modified to include certain components to improve operation or to configure the system for certain applications. These components include, but are not limited to: filters, heat exchangers, additional valves, pumps or blowers, and process sensors and alarms. 
         [0080]    Additionally, it should be noted that, although  FIG. 6  does not show the interactions between process controller  400  and subsystem  600 , process controller  400  controls all aspects of the operation of subsystem  600 . 
         [0081]    The following examples are illustrative only and do not limit the present invention. 
       Example 1 
       [0082]    An actual embodiment of system  10  was constructed. In this embodiment, EOC subsystem  200  included a 5-cell stack with an active cell area of 50 cm 2 /cell that was operated at a maximum current density of 100 mA/sq. cm. EOC subsystem  200  generated a maximum of 83 cm 3 /min of pure O 2  when in oxygen generation mode. When in oxygen removal mode, EOC subsystem  200  generated a gas stream of &lt;1% oxygen. Subsystem  300  included a 5-cell DMFC stack with an active area of 80 sq. cm./cell that was operated at current densities between 110-150 mA/sq. cm. Subsystem  300  generated 154 cm 3 /min of CO 2 . 
       Example 2 
       [0083]    The system of Example 1 was operated at current densities up to ˜100 mA/cm 2  using a 5-V, 5.0-amp variable power supply. At this current density, EOC subsystem  200  produced oxygen at the rate of 83 cm 3 /min and changed the concentration of oxygen in a 1-ft 3  incubator by 20 volume percent in a period of approximately 2 hours. 
       Example 3 
       [0084]    In another embodiment, a small on-board CO 2  control subsystem consisting of a 5-cell DMFC with a maximum power output of 12 watts could be integrated into the completed incubator and EOC subsystems. A larger DMFC unit (150 Watt DC-output, inverted to 120 VAC) could be utilized to provide power to the EOC subsystem as well as to power auxiliaries (electronics, blowers, valves, sensors). The integrated incubator system can operate directly from DMFC power by attaching the 150-W DMFC unit, or the integrated system can operate from 120-VAC grid power and still produce CO 2  with the small on-board CO 2  subsystem. 
       Example 4 
       [0085]    The following are certain specifications for an embodiment of system  10  actually reduced to practice: 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Gas Generation Unit 
               
               
                 Integrated Specs 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Power Requirements 
                 60 W (120 VAC, 0.5 A) - 
               
               
                   
                   
                 Max/15 W - Idle 
               
               
                   
                 Integrated 
                 87 lb (39.4 kg) 
               
               
                   
                 System Weight 
               
               
                   
                 Gas Generation/ 
                 30 lb (13.2 kg) 
               
               
                   
                 Control Weight 
               
               
                   
                 Incubator Volume 
                 1 ft 3  (28 L) 
               
               
                   
                 EOC 
                 5-cell, 50 cm 2 /cell 
               
               
                   
                 DMFC 
                 5-cell, 80 cm 2 /cell 
               
               
                   
                 O 2   
                 Concentration: 1-60% atmosphere: 
               
               
                   
                   
                 Rate: 83 cm 3 /min (Max) 
               
               
                   
                 CO 2   
                 Concentration: 0-5% atmosphere: 
               
               
                   
                   
                 Rate: 154 cm 3 /min (Max) 
               
               
                   
                 Reservoir 
                 ~500 mL 
               
               
                   
                 Capacity (Water) 
               
               
                   
                 Reservoir 
                 ~250 mL 
               
               
                   
                 Capacity (Methanol) 
               
               
                   
                   
               
             
          
         
       
     
         [0086]    The embodiments of the present invention described above are intended to be merely exemplary and those skilled in the art shall be able to make numerous variations and modifications to it without departing from the spirit of the present invention. All such variations and modifications are intended to be within the scope of the present invention as defined in the appended claims.