Abstract:
A self-contained breathing closure for flasks and other containers that require gas exchange. An illustrative embodiment of the closure is comprised of a splashguard, an adaptor for attaching the closure to the container, a bellows element and a gas-permeable barrier element. The splashguard is intended to keep liquid contents under vigorous agitation in the container without wetting the gas-permeable barrier. The adaptor couples the closure to the container in a secure fashion. The bellows element allows for repeated changes in the internal volume of the container-closure system. The gas-permeable barrier allows desired gases to enter and leave the container while excluding small particles and/or microorganisms. The technology may be used with existing glass flask technology, or coupled to a plastic flask that may be configured for either single-use, or multiple-use.

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This application is the national stage of International Patent Application No. PCT/US2010/002916, filed Nov. 5, 2010, which claims the benefit of U.S. Provisional Patent Application No. 61/280,554, filed Nov. 5, 2009, the disclosures of which patent applications are incorporated by reference as if fully set forth herein. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     Not Applicable 
     THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT 
     Not Applicable 
     INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC 
     Not Applicable 
     BACKGROUND OF THE INVENTION 
     This invention relates to closures and containers for use in chemistry, biology and biotechnology as well as any other fields which require gas exchange during mixing processes. In particular, the invention relates to closure devices for microbiological and chemical containers such as flasks and test tubes. 
     In the fields of chemistry, biology and biotechnology, container closures are used to prevent contamination of the chemical reaction or microorganism solution being reacted, cultivated or stored, from airborne particulates or other contaminants. In addition, closures are used to prevent the escape of the chemicals, particles or microorganisms from the container into the atmosphere, where they could be potentially harmful. Furthermore, such closures preferably allow easy access to the container&#39;s contents for the purpose of sampling, exchanging or adding media/reagents, etc. 
     A further requirement for closures in some applications is the need for gas exchange into the container of interest. For aerobic fermentation, for example, oxygen is required for the growth of microorganisms in a nutrient medium. At the same time, waste gasses such as carbon dioxide are often produced and must be eliminated from the container. Therefore, such closures must allow the passage of oxygen molecules into, and carbon dioxide molecules out through, the closure while maintaining appropriate conditions inside the container. 
     Growth rates and rates of subsequent formation of desired metabolites and products, by aerobic microorganisms are often governed by the available supply of dissolved oxygen in the nutrient medium. Since the solubility of oxygen in water is very low, dissolved oxygen often represents the limiting species for the growth rate of microorganisms. The negative consequences of not maintaining an adequate dissolved oxygen level in the nutrient range from mild to severe (Buchs, J., 2001, Introduction to advantages and problems of shaken cultures,  Biochemical Engineering Journal,  7, 91-98.) The first potential consequence is a slowdown of metabolism. While culture experiments may yield some useful results, repeatability is difficult because small differences in flask geometry or operating conditions often have greater effects than the experimental variable under study. 
     A second potential consequence is a changeover to partial anaerobic metabolism. This results in undesirable by-products that are excreted that change pH and inhibit cell growth. Product formation may, or may not, be affected. A third potential consequence may result if product formation is highly sensitive to oxygen supply. For example, glucoamylase production from  Saccharomycopsis fibuligera  has been shown to exhibit a narrow oxygenation optimum, even though growth of the organism is not as sensitive to oxygen levels. A fourth potential consequence is a complete change of metabolism mechanisms. Several examples of organisms that completely change their metabolism, secreting new secondary products in response to oxygen limitations have been noted (Katzer, W., Blackburn, M., Charman, K., Martin, S., Penn, J., &amp; Wrigley, S., 2001, Scale-up of filamentous organisms from tubes and shake-flasks into stirred vessels.  Biochemical Engineering Journal,  7, 127-134). These changes completely obscure the goals of the original culture experiment. A fifth potential consequence occurs for fermentations that require the organism of interest to grow in the presence of a toxic compound. Sufficient energy production (via oxidative respiration) is required to continuously excrete the toxin from the interior of the cell. In this case, oxygen transfer rate limitations lead to significant cell death of the organism being cultured. 
     Classically, a flask&#39;s or container&#39;s closure consists of a gauze or cotton plug inserted into the neck, or opening which acts to allow the diffusion-based exchange of gas molecules between the inside and outside of the container, while also preventing contamination of the container&#39;s contents from outside particles or microorganisms. Such cotton plug closures are deficient in many respects, including the tendency to fall apart, difficulty in maintaining homogeneous gas exchange between closures and difficulties in re-sterilization. In addition, cotton plug closures offer substantial resistance to gas transfer, thus causing severe limitations to the level of oxygen, or other desired gasses, diffusing into the container of interest. 
     The background art is characterized by U.S. Pat. Nos. 920,791; 2,287,746; 2,754,931; 2,849,147; 2,918,192; 3,128,899; 3,326,401; 4,027,427; 4,148,619; 4,665,035; 4,797,367; 4,971,219; 5,037,754; 5,180,073; 5,269,431; 5,395,006; 5,578,491; 5,649,639; 5,783,440; 6,170,684; 6,190,913; 6,536,938 and 7,381,559; the disclosures of which patents are incorporated by reference as if fully set forth herein. 
     BRIEF SUMMARY OF THE INVENTION 
     An object of illustrative embodiments of the present invention is to provide improved and novel means and methods for providing enhanced gas exchange to flasks and other containers in the fields of chemistry, biology, and biotechnology. Improved gas exchange in a simple format provides for the enhanced aerobic culture of microorganisms, improved chemical reaction kinetics, and general improvement of any process requiring substantial gas exchange while simultaneously excluding contaminating particles and microorganisms. These features, objects, and advantages of the present invention will be seen from the following description, illustrations, and examples. 
     One aspect of illustrative embodiments of the invention is that it provides a closure device for containers used in the biological, chemical, and biotechnological fields. This embodiment actively enhances gas exchange with the container without the necessity of a pressurized gas supply. The need for such a closure is especially pronounced in the biological arena for the cultivation of microorganisms. The need arises from the dual requirements of maintaining asepsis at all times combined with the necessity for large amounts of oxygen transfer to the container in order to support rapid growth of microorganisms. The passive filter membrane barrier that is most commonly used in the field presents a substantial resistance to the transfer of oxygen molecules into the container. By activating an illustrative embodiment of the present invention though the use of a vertical displacement mechanism, either for the entire container plus closure, or for just the closure itself, the interior volume of the chamber can be rapidly oscillated in real time, creating a substantial driving force pumping gas molecules into and out of the container. This substantial increase in gas transfer across the filter membrane has the effect of greatly enhancing the rate and efficiency of processes requiring gas exchange, such as the aerobic fermentation of microorganisms. 
     An additional feature of illustrative embodiments of the invention includes providing means on the enclosure for transfer of materials into and out of the flask. Another feature of illustrative embodiments of the invention includes the use of a single-use container that has the enclosure attached and is pre-sterilized. The single use container may also include optical instrumentation for the purpose of measuring process variables such as pH, dissolved oxygen, dissolved carbon dioxide, biomass levels, temperature, etc. 
     As used herein, the following terms and variations thereof have the meanings given below, unless a different meaning is clearly intended by the context in which such term is used. 
     “A,” “an” and “the” and similar referents used herein are to be construed to cover both the singular and the plural unless their usage in context indicates otherwise. 
     “About” means within 20 percent of a recited parameter or measurement, and preferably within 10 percent of such parameter or measurement. 
     “Comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. 
     “Exemplary,” “illustrative,” and “preferred” mean “another.” 
     An illustrative embodiment of the invention is an apparatus (and/or method) for enhancing gas exchange between a container and the immediate surrounding environment, while preventing the exchange of airborne particulates or droplets. In this embodiment, droplets and particulates are prevented from contaminating the container&#39;s contents and particulates and droplets are prevented from escaping from the container. 
     An illustrative embodiment of the apparatus comprises a bellows to provide a pumping action, a filter membrane to selectively retain particulates or droplets, while allowing gas exchange and a splashguard to keep contents of the container from fouling the filter membrane. An illustrative embodiment of the apparatus is comprised of a stopper, a splashguard and a filter cap. 
     In a preferred embodiment, the invention is a closure for a container comprising a body and a neck with a tubular opening and an outer surface, said body being adapted to hold a fluid (e.g., a liquid) during a mixing operation that involves movement of said container, said closure comprising: a sleeve comprising a flexible portion having a plurality of annular corrugations therein, a top end having a vent therein, a bottom end and a longitudinal axis, said flexible portion being operative to flex during the mixing operation; an adaptor that is operative to attach said bottom end to said tubular opening, said adaptor having an axially-oriented passageway there-through that is in communication with and open to said bottom end and the container, said axially-oriented passageway having an interior surface; a filter that is attachable to said top end and that is operative to cover said vent, allow gases to enter and leave said container and prevent microorganisms from entering and leaving said container; and a splashguard that is attached to said adaptor and that is operative to prevent the fluid from entering said adaptor during the mixing operation, said splashguard comprising a main body, an upper droplet shield that is attached to said main body and that is disposed above said axially-oriented passageway, a lower droplet shield that is attached to said main body and that is disposed below said axially-oriented passageway and a plurality of panels that are attached to said main body and that are disposed substantially within said axially-oriented passageway, each of said panels having an edge that abuts said interior surface; wherein the flexing of said flexible portion is operative to cause said gases to move through said filter and in and out of the container. 
     In another illustrative embodiment, the invention is a closure for a container comprising a body and a neck with a tubular opening and an outer surface, said body being adapted to hold a fluid during a mixing operation that involves movement of said container, said closure comprising: a sleeve comprising a flexible portion having annular corrugations therein, a top end having a vent therein and a tubular bottom end, said flexible portion being operative to flex during the mixing operation; an adaptor that is operative to attach said tubular bottom end to said tubular opening; a filter that is attachable to said top end and that is operative to cover said vent, allow gases to enter and leave said container and prevent microorganisms from entering and leaving said container; and a splashguard that is attached to said tubular bottom end and that is operative to prevent the fluid from entering said tubular bottom end during the mixing operation; wherein the flexing of said flexible portion is operative to cause said gases to move through said filter and in and out of the container. In another embodiment, said sleeve, adaptor, filter and said splashguard are autoclavable. In another embodiment, said adaptor comprises a deformable insert that has a frustoconical shape and that has a passageway through it. In another embodiment, said adaptor comprises a tubular sidewall having inwardly-projecting, circumferentially-spaced fingers that are adapted to grip the outer surface of the neck of the container. In another embodiment, said adaptor comprises a tubular sidewall having inwardly-projecting threads that are adapted to screw onto threads on the outer surface of the neck of the container. In another embodiment, said flexible portion is fabricated from a biocompatible material. In another embodiment, said biocompatible material is silicone rubber. In another embodiment, said flexible portion is operative to oscillate during the mixing operation. In another embodiment, said flexible portion comprises stiffeners and is operative to rock from side to side during the mixing operation. In another embodiment, said flexible portion comprises an embedded spring. In another embodiment, said filter is operative to prevent selected gases from entering or leaving said container. In another embodiment, said filter is a membrane filter. In another embodiment, said filter is a high efficiency particulate air filter. In another embodiment, the closure further comprises: a cap that attaches said filter to said top end and that prevents said filter from flexing. In another embodiment, said cap is rotatable with respect to said top end and has an opening in it that is operative to uncover at a least a portion of said filter when said cap is rotated to a desired position. In another embodiment, said splashguard has a shape that is selected from the group consisting of: a cone, an inverted cone, a frustum of a cone and a disc. In another embodiment, said splashguard has one or more drain holes. In another embodiment, said splashguard has a non-stick surface. In another embodiment, the closure further comprises a humidifier. In another embodiment, the closure further comprises a sampling port. In another embodiment, said splashguard comprises a main body, an upper droplet shield that is attached to said main body, a lower droplet shield that is attached to said main body and a plurality of panels that are attached to said main body. 
     In another embodiment, the invention is a closure for a container comprising a body and a two necks, each neck having a tubular opening and an outer surface, said body being adapted to hold a fluid during a mixing operation that involves movement of said container, said closure comprising: a sleeve comprising a flexible portion having annular corrugations therein, a top end and a tubular bottom end, said flexible portion being operative to flex during the mixing operation; an adaptor that is operative to attach said tubular bottom end to one of the tubular openings; and a filter that is attachable to another of the tubular openings and that is operative to allow gases to enter and leave said container and prevent microorganisms from entering and leaving said container; wherein the flexing of said flexible portion is operative to causes said gases to move through said filter and in and out of the container. In another embodiment, the closure further comprises: a splashguard that is attached to said tubular bottom end and that is operative to prevent the fluid from entering said tubular bottom end during the mixing operation. In another embodiment, said top end has a vent therein. 
     In yet another illustrative embodiment, the invention is a method for enhancing gas movement into and out of an opening in a container having contents, said method comprising: attaching a bellows to said opening to produce a combination, said bellows having a volume, a splashguard and a vent that is covered by a filter through which particles of a selected size cannot pass; and moving said combination in an oscillating motion or an orbital motion to cause said volume to increase and then decrease in a cyclic manner. In another embodiment said oscillating motion comprises vertical displacements. In another embodiment, said moving step is accomplished with a small motor or a voice coil. In another embodiment, said moving step is accomplished by exposing said bellows to a magnetic field. In another embodiment, said moving step is accomplished by attaching said bellows to an externally activated mechanical member. In another embodiment, the method further comprises: monitoring a characteristic of said contents during the moving step. 
     In a further illustrative embodiment, the invention is a single-use mixing vessel comprising: a container comprising a body and a neck, said body being adapted to hold a fluid during an operation that involves movement of said container; a sleeve comprising a flexible portion, a top end having a vent therein and a bottom end that is attached to said neck, said flexible portion being operative to flex during the mixing operation; a filter that is attachable to said top end and that is operative to cover said vent, allow gases to enter and leave said container and prevent microorganisms from entering and leaving said container; and a splashguard that is attached to said bottom end and that is operative to prevent said fluid from entering said bottom end during said operation; wherein the flexing of said flexible portion is operative to cause said gases to move through said filter and in and out of the container. 
     In another illustrative embodiment, the invention is a closure for a container comprising a body and a neck with an opening and an outer surface, said body being adapted to hold a fluid during a mixing operation that involves movement of said container, said closure comprising: a sleeve comprising a flexible portion having at least one annular corrugation therein, a first end having a vent therein and a second end, said flexible portion being operative to flex during the mixing operation; an adaptor that is operative to attach said second end to said opening; a filter that is attachable to said first end and that is operative to cover said vent, allow gases to enter and leave said container and prevent microorganisms from entering and leaving said container; and a splashguard that is attached to said second end and that is operative to prevent the fluid from reaching said filter during the mixing operation; wherein the flexing of said flexible portion is operative to cause said gases to move through said filter and in and out of the container. In another embodiment, said sleeve is molded from fluoro liquid silicone rubber. In another embodiment, said flexible portion is operative to oscillate with a displacement of about one quarter inch during the mixing process. In another embodiment, said flexible portion has a wall thickness in the range from about 0.040 inches to about 0.060 inches. In another embodiment, said flexible portion has a wall having a durometer of about 40 Shore A to about 50 Shore A. In another embodiment, said splashguard comprises a main body, an upper droplet shield that is attached to said main body, a lower droplet shield that is attached to said main body and a plurality of panels that are attached to said main body. In another embodiment, said splashguard has a height to diameter ratio in the range of about 0.13 to 0.20. In another embodiment, each of said droplet shields has a cone angle of 90.5 to 104 degrees. In another embodiment, each of said droplet shields has a flat plate inclination angle of 80 degrees to 90 degrees. 
     Further aspects of the invention will become apparent from consideration of the drawings and the ensuing description of exemplary embodiments of the invention. A person skilled in the art will realize that other embodiments of the invention are possible and that the details of the invention can be modified in a number of respects, all without departing from the concept. Thus, the following drawings and description are to be regarded as illustrative in nature and not restrictive. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
       The features of the invention will be better understood by reference to the accompanying drawings which illustrate exemplary embodiments of the invention. In the drawings: 
         FIG. 1  is a perspective view of a container with a closure in accordance with an illustrative embodiment of the present invention. 
         FIG. 2A  is a perspective view of a closure with the port in accordance with an illustrative embodiment of the invention. 
         FIG. 2B  is a perspective view of a closure with a variable gas port in accordance with an illustrative embodiment of the invention. 
         FIG. 2C  is a perspective view of a closure for orbital motion in accordance with an illustrative embodiment of the invention. 
         FIG. 2D  is a perspective view of a bellows for a separate gas port in accordance with an illustrative embodiment of the invention. 
         FIG. 2E  is a perspective view of a closure with a helical bellows in accordance with an illustrative embodiment of the invention. 
         FIG. 3  is a cross sectional view of an illustrative embodiment of a closure assembly installed on a flask. 
         FIG. 4A  is a perspective cross sectional view of a bellows having a variable vent in accordance with an illustrative embodiment of the invention. 
         FIG. 4B  is a perspective view of the bellows of  FIG. 2B . 
         FIG. 5  is a perspective view of an illustrative embodiment of a helical bellows assembly installed on a container. 
         FIG. 6  is a perspective view of an illustrative embodiment of a screwed on closure having an additional port. 
         FIG. 7A  is a perspective view of an illustrative embodiment of a dual neck flask-one way valve closure assembly. 
         FIG. 7B  is a perspective view of an illustrative embodiment of a dual neck flask-ventless bellows assembly. 
         FIG. 8A  is a perspective view of a background art standard flask. 
         FIG. 8B  is a perspective view of a threaded top flask. 
         FIG. 8C  is a perspective view of a snap on top flask. 
         FIG. 8D  is a perspective view of a double necked flask. 
         FIG. 9A  is a perspective view of a flat perforate splashguard. 
         FIG. 9B  is a perspective view of a conical splashguard. 
         FIG. 10  is a perspective view of an illustrative embodiment of a container/closure system that illustrates the vertical/linear vessel motion used to actuate the system. 
         FIG. 11  illustrates the orbital motion that actuates the  FIG. 2   c  embodiment. 
         FIG. 12  is a schematic cross sectional view of a humidification device. 
         FIG. 13A  illustrates the linear vertical actuation caused by a base actuator. 
         FIG. 13B  illustrates the linear vertical actuation caused by an external electromagnetic actuator. 
         FIG. 13C  illustrates the linear vertical actuation caused by a device attached to the flask. 
         FIG. 13D  illustrates the linear vertical actuation caused by a device attached to the bellows. 
         FIG. 14  is an elevation (side) view of an illustrative embodiment of a self aerating closure on a single use flask fitted with an optical sensor. 
         FIG. 15  is a perspective (top) view of a filter cap with seal features. 
         FIG. 16  is a perspective (bottom) view of the vent cap with seal features. 
         FIG. 17  is a perspective cross sectional view of the closure assembly showing the filter cap installed in a spring coil bellows. 
         FIG. 18  is a perspective view of an alternate embodiment of a splashguard labyrinth. 
         FIG. 19  is a perspective view of another alternate embodiment of the splashguard labyrinth. 
         FIG. 20  is a perspective view of another alternate embodiment of the splashguard labyrinth. 
         FIG. 21  is a view of another alternate embodiment of the splashguard labyrinth. 
         FIG. 22  is a cross sectional perspective view of the closure assembly with an alternate embodiment of the splashguard installed. 
         FIG. 23  is a chart that presents a comparison of the oxygen transfer coefficients (kLa) for identical Pyrex® 4442 250 milliliter (ml) shake flasks containing 100 ml of water with a preferred embodiment of the invention and a vertical laboratory mixing system and with a background art closure and a background art orbital shaker mixing system. 
         FIGS. 24A-24D  are charts that present test data for  Escherichia coli  growth performance with a preferred embodiment of the invention and a vertical laboratory mixing system and with a background art closure and a background art orbital shaker mixing system. 
         FIGS. 25A-25C  are charts that present test data for  Bacillus subtilis  growth performance with a preferred embodiment of the invention and a vertical laboratory mixing system and with a background art closure and a background art orbital shaker mixing system. 
         FIGS. 26A-26D  are charts that presents test data for  Pseudomonas fluorescen  growth performance with a preferred embodiment of the invention and a vertical laboratory mixing system and with a background art closure and a background art orbital shaker mixing system. 
     
    
    
     The following reference numerals are used to indicate the parts and environment of an illustrative embodiment invention on the drawings: 
       1  bellows, bellows portion, flexible member, flexible portion 
       2  bidirectional top vent, top vent 
       3  single conical splashguard, single inverted cone 
       4  stopper top flask, shake flask, flask, container 
       5  spring coil bellows 
       6  bellows stiffeners 
       7  stopper adaptor 
       9  material-addition port 
       10  threaded top 
       11  threaded top flask 
       12  one way check valve and vent 
       13  double necked flask 
       15  variable vent cover, filter membrane cover, rotatable cap 
       16  bidirectional variable vent 
       17  flat perforated plate 
       18  snap top flask 
       19  orbital bellows 
       20  ventless bellows 
       21  bidirectional neck vent 
       22  rocking motion 
       23  orbital motion 
       24  vertical motion 
       25  fluid trap/humidifier, humidification device 
       26  air/gas duct 
       27  filter membrane, filter 
       28  fluid refill port 
       29  fluid 
       30  vent hole 
       31  seal surface 
       32  seal feature 
       33  splashguard labyrinth, labyrinth, splashguard 
       40  closure assembly, closure 
       42  upper droplet shield, upper cone 
       44  lower droplet shield, lower cone 
       50  main body 
       52  panels, vertical vanes 
       54  stopper portion 
       56  anchor tabs 
       58  alignment tabs 
       60  vertical resonant mixer 
       62  small motor, voice coil 
       64  stationary member 
       66  external device 
       68  external mechanical apparatus 
       70  filter cap, vent cap 
       80  single use flask 
       82  optical sensor 
       84  optical fiber 
     DETAILED DESCRIPTION OF THE INVENTION 
     According to the previously discussed advantages of the present invention, a first illustrative embodiment thereof is illustrated in  FIG. 1 . The primary unique advantage common to preferred embodiments of the invention is the ability to substantially enhance gas exchange to the container of interest without the necessity for pressurized gas lines being linked to the container. 
     Referring to  FIG. 1 , an illustrative embodiment of closure assembly  40  is presented. In this embodiment, closure assembly  40  comprises stopper adaptor  7  which provides an attachment point to the container (e.g., stopper top flask  4 ), single conical splashguard  3  to prevent wetting of closure components, a flexible member in the form of bellows  1 , filter membrane  27  to permit gas exchange and top vent  2 . In this embodiment, bellows  1  comprises a sleeve comprising a flexible portion having a plurality of annular corrugations therein. Filter membrane  27  may be bonded to bellows  1  by glue, mechanical compression, heat weld, ultrasonic weld or other means compatible with the construction materials of bellows  1 . 
     This embodiment of the invention was experimentally tested and found to be highly successful. This embodiment comprised a silicone rubber stopper outfitted with an inverted conical splashguard  3  manufactured from polypropylene that extended into shake flask  4 . A silicone bellows  1  was affixed to the top of the silicone stopper. The top of the silicone rubber bellows  1  was outfitted with a 0.2 micron pore diameter hydrophobic ultrafine glass microfiber filter  27 . Shake flask  4  outfitted with closure assembly  40  was placed on a vibratory vertical mixing system (LabRAM®) developed by Resodyn Acoustic Mixers, Inc. (Butte, M T). The inverted cone  3  acted as a splashguard to prevent droplets from splashing from the fluid onto the gas transfer membrane at the top of bellows  1 . When flask  4  with closure assembly  40  was placed on the vertical mixer and activated, the bellows/filter vibrated at a specified frequency, rapidly oscillating the gas volume in flask  4 . 
     Referring to  FIGS. 3 ,  5  and  6  several alternative embodiments of the attachment portion of the present invention are illustrated, each of which couples the closure to the container of interest. Alternate embodiments of the closure could be configured to insert inside the neck of a container of interest ( FIG. 5 ), or be slipped securely over the neck of the container of interest ( FIG. 6 ), or be screwed on the top of a threaded container of interest ( FIG. 3 ), or integrated into the structure of a single-use container of interest. 
     Referring to  FIGS. 2A-2E , several different types of bellows designs and features are illustrated that may be incorporated into the bellows portion of the closure.  FIG. 2A  illustrates that a port for gas or liquid exchange may be built into the bellows portion of the closure in one alternative embodiment.  FIG. 2B  illustrates that a rotatable cap that partially blocks a portion of the filter on top of the closure may be used to regulate airflow in one alternative embodiment. In this embodiment, the position of rotatable cap  15  may be continuously varied to allow access to zero percent to 100 percent of the area of filter membrane  27 .  FIG. 2C  illustrates that stiff vertical elements called bellows stiffeners may be placed 180 degrees opposite of each other and attached to the bellows portion of the closure. In this alternative embodiment, the bellows may be used to provide air pumping on an orbital mixing device due to the change in volume that will occur along a line as the bellows is compressed from one side, then expands when passing through the center, and being compressed again at the far end of the line (see  FIG. 11 ).  FIG. 2D  illustrates an alternate embodiment of the bellows portion of the closure for which the top of the bellows is sealed. In this embodiment, the filter element essential to gas exchange would be located on a secondary exit port on the culture flask. The bellows would provide pumping action for gas to exchange through the filter located at a secondary location (see  FIG. 7B ) on the culture flask.  FIG. 2E  illustrates an alternative design for the bellows portion of the closure for which the bellows is designed to have a helical spring configuration. 
     Motion (e.g., vertical vibration and/or orbital motion) of closure  40  provides a positive gas flow into and out of container  4 . In an illustrative embodiment, closure  40  is made of an elastomeric material capable of displacements sufficient to create volumetric changes. A more preferred elastomeric material is Silastic® fluoro liquid silicone rubber manufactured by Dow Corning, Midland, Mich. Changes in the volume of bellows  1  are caused by the motion of the vessel and closure  40 . Displacement of a preferred embodiment of closure  40  of about one quarter inch (amplitude of bellows motion) achieve an optimum balance between high gas transfer and stability of the part under a vertical mixing load. If bellows  1  is too thin or too soft, it may collapse on itself during oscillatory motion and/or not maintain a consistent vertical motion. Accelerations of closure  40  cause the elastomeric material to compress and expand creating a pumping action. Volumetric changes in closure  40  are determined by its structural design, the material properties of the closure and the forces created by the motions imposed upon it. Increased levels of displacement may be obtained by decreasing the wall thickness of the bellows or by using a softer durometer of the material for the bellows wall. In a more preferred embodiment, a bellows wall thickness of about 0.040 inches to about 0.060 inches and a durometer of the material for the bellows wall about 40 Shore A to about 50 Shore A are used. In a more preferred embodiment, closure  40  is capable of a range of volumetric changes from 0.1 percent to the 100 percent of the at rest volume of the interior of closure  40 , thus creating the desired volumetric flows and associated pressure fluctuations in container  4 . 
     A person having ordinary skill in the art would understand that bellows  1  may be designed with different materials, thicknesses, heights, diameters, number of folds and alternative end styles in order to achieve different oscillation parameters depending on the displacement and frequency of the vibratory motion employed to activate it. In an alternative embodiment, a spring (not shown) is embedded in bellows  1  that maintains the structural properties of closure  40 . 
     An additional aspect of illustrative embodiments of the present invention is the incorporation of a splashguard to prevent the wetting of closure components in the presence of vigorous agitation of fluid that is disposed within the container of interest. In order for sterile filter  27  to maintain gas exchange, it is important that it remains unfouled by media or biomass. The splashguard allows gas to freely exchange between the upper portion of the pumper stopper and the flask, while preventing liquid from reaching filter  27 . The splashguard may not be necessary for embodiments that do not involve vigorous splashing of liquid contents inside a container of interest. In alternative embodiments, that splashguard that prevents wetting of filter  27  may have the shape of a cone, inverted cone, frustum, inverted frustum or any other substantially planar geometry, such as a round disc or square. In other alternative embodiments, the splashguard may be rendered permeable by the inclusion of one or more holes in order to allow improved drainage of liquid. The splashguard may be comprised of biocompatible polymers, metals or fabrics and may have a non-stick surface to inhibit the attachment of microorganisms or chemicals. 
     Referring to  FIG. 3 , another illustrative embodiment of closure  40  installed on threaded top flask  11  is presented. In this embodiment, closure  40  comprises bellows  1  with integral bi-directional variable vent  15  and variable vent cover  15 . Closure  40  is installed on threaded top flask  11  by screwing it on threaded top  10 . 
     Referring to  FIGS. 4A and 4B , an illustrative embodiment of the bellows  1  of  FIG. 3  is presented. In this embodiment, variable vent  15  is molded into (or otherwise integral with) bellows  1 . Variable vent cover  15  is rotatably attached to bellows  1  and is turned relative to bellows  1  to vary the size of variable vent  15 . 
     Referring to  FIG. 5 , another illustrative embodiment of the invention is presented. In this embodiment, closure  40  comprises spring coil bellows  5  which is helical in shape and has bidirectional vent  2  in its top. Closure  40  is releasably attached to container  4  by means of stopper adaptor  7 . Stopper adaptor  7  is a compliant annular insert that is slipped over the outer diameter of the lower portion of spring coil bellows  5  in order to increase its diameter to properly fit in flasks with larger diameter openings. 
     Referring to  FIG. 6 , another illustrative embodiment of the invention is presented. In this embodiment, closure  40  is screwed onto threaded top flask  11  which comprises threaded top  10 . Bellows  1  is provided with material-addition port  9 . 
     Referring to  FIG. 7A , another illustrative embodiment of the invention is presented. In this embodiment, closure  40  comprises bellows  1  having top vent  2  that is attached to one neck of double neck flask  13 . One way check valve and vent  12  is attached to the other neck of double neck flask  13 . As bellows  1  oscillates, air is drawn in either top vent  2  or one way check valve and vent  12  and expelled through either one way check valve and vent  12  or top vent  2 , depending on the configuration of the check valve (which direction it allows air to flow). 
     Referring to  FIG. 7B , another illustrative embodiment of the invention is presented. In this embodiment, closure  40  comprises ventless bellows  20  that is attached to one neck of double neck flask  13 . Bidirectional neck vent  21  is attached to the other neck of double neck flask  13 . As ventless bellows  20  oscillates, air is drawn into and expelled out of bidirectional neck vent  21 . 
     Referring to  FIGS. 8A-8D , examples are presented of container types that the applicants envision closure  40  to be used with.  FIG. 8A  presents background art standard flask  4 .  FIG. 8B  presents threaded top flask  11 .  FIG. 8C  presents snap on top flask  18 .  FIG. 8D  presents double necked flask  13 . A person having ordinary skill in the art would understand that closure  40  may be used with many other types of containers. 
     Referring to  FIG. 10 , another illustrative embodiment of the invention is presented. In this embodiment, vertical motion  24  is caused by the operation of a vertical vibratory mixing system (not shown). The vertical motion  24  causes bellows  1  to expand and contract, resulting in the movement of air in and out of top vent  2 . 
     Referring to  FIG. 11 , another illustrative embodiment of the invention is presented. In this embodiment, orbital bellows  19  is preferably provided with one or more one dimensional bellows stiffeners  6 . Orbital motion  23  of closure  40  is caused by an orbital mixer (not shown) which causes closure  40  to move with rocking motion  22 . The rocking motion  22  causes orbital bellows  19  to expand and contract, resulting in the movement of air in and out of top vent  2 . 
     The development of a splashguard that is effective under the demanding conditions of constant and reversing airflow combined with rapid vibrations is non-trivial and the resulting more preferred embodiment of splashguard  33  is quite different from background art splashguards. Many background art splashguards have a low height to diameter ratio and rely on various types of inverted cones to deflect droplets back to the reservoir below. Examples of this type of splashguard have been described by Dolvet (U.S. Pat. No. 5,649,639), Sakata (U.S. Pat. No. 5,269,431), Dombeck (U.S. Pat. No. 4,971,219) and Thompson (U.S. Pat. No. 2,849,147). When subjected to constant airflow and continuous vibrations at a frequency of about 60 Hertz (Hz), these types of conical splashguards proved ineffective at keeping liquid away from the filter membrane  27  above when tested with vertical mixers. 
     Another common type of splashguard described in the background art are designs that rely on a series of flat plates that cover alternating portions of the cross-sectional area of the throat of closure  40 , thus preventing large intermittent splashes of liquid from reaching to top of the splashguard. Examples of this type of splashguard have been described by Runo (U.S. Pat. No. 3,128,899), Dedman (U.S. Pat. No. 2,918,192). When this type of splashguard was tested under the required conditions of constant airflow and vibrations, these splashguards also failed to prevent splashing of liquid contents onto filter  27 . 
     Through experimentation, splashguards with better performance were achieved. Examples of better performing splashguards are the embodiments illustrated in  FIGS. 19-21 . These designs resulted from dramatically increasing the height to diameter ratio of the cone-type splashguard, reducing the angle of the cone at the bottom, changing impinging plates from flat to angled, incorporating curved surfaces as impinging plates, and/or separating the curved surfaces into two distinct zones, upper and lower. In more preferred embodiments, a height to diameter ratio of cone type splashguards of about 0.13 to 0.20 is used, a cone angle (the angle between the surface of the cone and the vertical longitudinal axis of closure  40 ) of 90.5 degrees to 104 degrees is used, a flat plate inclination angle (the angle between the surface of the plate and a plane that is normal to the vertical longitudinal axis of closure  40 ) of 80 degrees to 90 degrees is used, impinging plate curved surfaces such as those shown in  FIGS. 19 and 21  are used, and two distinct zones of impinging plate curved surfaces such as those shown in  FIGS. 19 and 21  are used. 
     While such designs were improved over the background art, substantial further improvement was needed to make closure  40  useful at fluid volumes greater than a 25 percent fill ratio. Therefore, other design features were incorporated into more preferred embodiments of the splashguards. One of these features was to incorporate cones  42 ,  44  at both the top and the bottom of splashguard  33  as shown in  FIGS. 18 and 22 . Cone  44  (at the bottom) is inverted to promote drainage of liquid back into the flask. A second more preferred aspect of splashguard  33  was to remove as much surface area between the upper and lower cones  42 ,  44  as possible. This is preferably accomplished by configuring main body  50  to have the lowest amount of surface area as is structurally feasible. This was unexpected and counterintuitive, since in background art splashguards, the more surface upon which splashes can impinge, the better the performance. However, under constant vibrational force, the additional surface area was detrimental to splashguard performance since acoustic vibrational forces proved to be more important than gravitational forces, which is opposite from the situation that occurs with background art splashguards. Another element that proved highly useful and unexpected was the incorporation of vertical vanes or panels  52  into the throat or passageway in which splashguard  33  is disposed. The vertical vanes  52  serve to alter the airflow pathways inside the splashguard, creating more favorable conditions for droplets to drop back into container  4  as opposed to becoming entrained in the airstream and fouling filter  27  at the top of closure  40 . More than sixty different types of diverse designs were explored before discovering the unexpected combination of features that resulted in the outstanding splashguard performance of a more preferred embodiment illustrated in  FIGS. 18 and 22 . 
     Referring to  FIGS. 18 and 22 , splashguard labyrinth  33  has several features of note. At both the upper and lower ends are disk-like features (upper droplet shield  42  and lower droplet shield  44 ) that prevent fluid droplets from moving in a vertical direction. Main body  50  of labyrinth  33  has three panels  52  to restrict horizontal motion of droplets and also to help anchor labyrinth  33  in stopper portion  54  of closure assembly  40 . Anchor tabs  56  on edges of panels  52  further help to anchor labyrinth  33  in stopper portion  54 . At the lower end of panels  52  are alignment tabs  58 . Alignment tabs  58  prevent the over-insertion of labyrinth  33  into stopper portion  54  which would otherwise block gas flow. Referring to  FIGS. 19-21 , alternative embodiments of splashguard  33  are illustrated depicting alternative geometries and support methods. Examples of other alternative embodiments include supporting inverted cone  3  from the interior of a single-use container of interest (see  FIG. 9B ), the use of a frustum supported from the interior of a single-use container of interest and containing drain holes, and the use of a circular disc with drain holes (e.g., flat perforated plate  17 ) supported from the interior of a single-use container of interest (see  FIG. 9A ). In an alternative embodiment (not shown), filter  27  is positioned in an alternative orientation to the direction of fluid motion (e.g., parallel instead of normal). 
     Activation (operation) of closure assembly  40  involves the use of a vertical displacement mechanism. The vertical displacements may be applied to the entire container of interest with the closure installed, or alternatively vertical displacements may be applied only to the flexible member of the closure itself. In a preferred embodiment illustrated in  FIG. 13A , container  4  and closure  40  are coupled to vertical resonant mixer  60 , which in addition to agitating the liquid contents of container  4 , induces vertical oscillations in closure assembly  40 . 
     An alternative embodiment is illustrated in  FIG. 13B  which depicts a small motor or voice coil  62  attached to stationary member  64  which induces vertical oscillation of flexible member  1  of closure  40 . Another alternative embodiment is illustrated in  FIG. 13C  which depicts the incorporation of magnetically active materials in the flexible member  1  of closure  40  to which an oscillating magnetic field is applied by external device  66  with the effect of inducing vertical displacements of filter  27  of closure  40 . Another alternative embodiment is illustrated in  FIG. 13D  which depicts external mechanical apparatus  68  that is releasably attached to flexible member  1  of the closure  40 . Activating external mechanical apparatus  68  induces vertical displacements of flexible member  1  of closure  40 . 
     Referring to  FIG. 14 , another illustrative embodiment of the invention is presented. In this embodiment, closure  40  is attached to single use flask  80 . Optical sensor  82  is also attached to closure  40  and signals from optical sensor  82  are transmitted to an instrument (not shown) via optical fiber  84 . 
     In an illustrative embodiment, another means of affixing the membrane/filter paper of filter  27  to spring coil bellows  5  involves the use of filter cap  70 . This allows the replacement of membrane/filter papers without requiring replacement of the spring coil bellows  5 . Filter cap  70  must form an airtight seal with spring coil bellows  5  so that air can only pass through the membrane surface area of filter  27  and not around filter  27 . 
       FIGS. 15-21  show a preferred embodiment of filter cap  70 . This embodiment of filter cap  70  has at least one vent hole  30  and includes a seal feature  32  to force gas molecules to pass only through membrane filter  27 . Seal feature  32  comprises lip type seal that is formed by spring coil bellows  5  and seal surface  31  of filter cap  70 . The sealing member is integral to spring coil bellows  5  while the seal face (e.g., seal surface  31 ) is integral to filter cap  70 . 
     Another preferred feature of filter cap  70  is a means of preventing the membrane of filter  27  from flexing under the momentum of the vertically oscillating forces imposed upon it. Vertical flexing of the membrane may cause flexure failure modes of the membrane and may increase the noise level of the device to unacceptable levels. Various types and numbers of anchor points may be used to prevent flexure of the membrane. Examples of such features include cross bars, spokes, or circular elements that extend into the center point of filter cap  70 . The filter membrane is then attached to filter cap  70  at one or more locations. A center point attachment is a more preferred and effective anchor point. Additional anchor points radiating out towards the periphery of filter cap  70  are also of potential value for flexure reduction. Membrane/filter papers may be bonded to filter cap  70  using gluing, mechanical compression, heat welding, ultrasonic welding or other methods that are compatible with the construction materials of filter cap  70 . 
     Another component that may optionally be incorporated into closure  40  is a mechanism for modulating the level of gas exchange obtained at a particular vertical displacement and frequency.  FIGS. 2B ,  3 ,  4 A and  4 B illustrate the incorporation of filter membrane cover  15  with a partial-circle or half-circle shaped opening (e.g., bidirectional variable vent  16 ). The filter membrane of closure  40  is also preferably configured to have a partial-circle or half-circle geometry. By rotating filter membrane cover  15 , gas exchange can be modulated between zero percent and  100  percent of the maximum available gas exchange for a particular set of vertical mixing conditions. 
     Another component that can optionally be incorporated into the attachment portion of the closure described in the present invention is a one-way gas flow restrictor (not shown). The one-way gas flow restrictor has the effect of allowing gas molecules to enter, but not exit, through closure  40 . By coupling this additional component with a passive vent (not shown) located in another location on container  4  a defined pathway for gas flow within container  4  can be created. This defined pathway may have the effect of further increasing the efficiency of closure  40 . 
     A component that may optionally be incorporated into another illustrative embodiment of the invention is a material-addition port  9  as shown in  FIG. 2A  and  FIG. 6 . This component allows the user to add materials such as fluids to flask  4  in any manner during operation. 
     Another component that can optionally be incorporated is a humidification device  25  as shown in  FIG. 12 . This device allows for the humidification of incoming gasses. With a proper volume of fluid  29  in the device, when the device is subjected to a vigorous vertical motion, fluid  29  effectively creates a fluidized air space at 100 percent humidity. Air flow under the influence of bellows  1  passes in and out of flask  4  through duct  26 , keeping the air inside the flask fully humidified to prevent evaporation. 
     A further illustrative embodiment of the invention is shown in  FIG. 2C  and  FIG. 11 . The present invention is applicable to orbital motion using this embodiment.  FIG. 2C  illustrates that stiff vertical elements (e.g., bellows stiffeners  6 ) may be placed 180 degrees apart (opposite each other) and attached to bellows portion  1  of closure  40 . In this alternative embodiment, bellows  1  may be used to provide air pumping on an orbital mixing device due to the change in volume that occurs as bellows  1  is compressed as it rocks toward one side, then expands when passing through the center of rocking motion  22 , and is compressed again as it rocks toward the other side (see  FIG. 11 ). 
     WORKING EXAMPLE NO. 1 
     The rates of oxygen transfer into an agitated liquid were compared for a preferred embodiment of the present invention (the configuration in  FIG. 1  with splashguard from  FIG. 18 ) and a combination of a background art culture system and background art closure. The closure  40  of  FIG. 1  was affixed to the top of a 250 ml Pyrex® 4442 shake flask containing 100 ml of water and placed on a vertical vibratory mixing system (LabRAM®, Resodyn Acoustic Mixers, Inc). A background art vented closure (BugStopper™, Whatman) was affixed to an identical flask and placed on a background art orbital shaker (Innova 2100, New Brunswick). Oxygen transfer values in water were evaluated using an optical dissolved sensing patch (PreSens Precision Sensing) with a fast response time using the dynamic gassing out method. 
     The results of the oxygen transfer test are presented in  FIG. 23  and demonstrate a dramatic improvement in oxygen transfer when the present invention was used. The present invention demonstrated oxygen transfer rates up to fourteen-fold higher than those obtained using the background art closure and orbital agitation. An interesting finding was that the background art closure effectively caps (sets an upper limits to) the oxygen transfer rate obtainable with the background art orbital shaker. This was confirmed in control experiments by removing the background art closure, which resulted in the oxygen transfer rate increasing with agitation speed, as would be expected in an open system. 
     From a practical point of the view, a sterile barrier is essential for biological culture and must be provided. Whether the barrier is a cotton plug, a loose aluminum cap, or a filter membrane, such background art closures provide resistance to oxygen penetration. Since the orbital motion of background art mixers is normal to the desired direction of airflow, such orbital motion does not contribute to gas flow across the closure, which must occur via diffusion alone. In contrast, because the present invention causes the sterile membrane to move in a direction parallel to the desired direction of air flow, a convective airflow is created across the closure, substantially increasing the transfer of oxygen molecules into the interior of the flask. This effect was strong enough to be felt as an air pressure wave on the hand when placed just above the oscillating closure of the present invention. 
     WORKING EXAMPLE NO. 2 
     The performance of a preferred embodiment of the present invention ( FIG. 1 ) was compared to the performance of a combination of a background art culture system and a background art closure in the culturing of several microorganisms. The closure of  FIG. 1  was affixed to the top of a 250 ml Pyrex® 4442 shake flask containing 100 ml of water and placed on a vertical vibratory mixing system (LabRAM®, Resodyn Corporation). A background art closure (BugStopper™, Whatman) was affixed to an identical flask and placed on a background art orbital shaker (Innova 2100, New Brunswick). 
     In a first experiment,  Escherichia coli  HB101 transformed with a green florescent protein (GFP) containing plasmid (pGLO) was cultured in 62.5 ml of H15 medium at 37° Centigrade (C). Mean green florescence was monitored using a Guava® easyCyte flow cytometer. 
     The results for a  Escherichia coli  culture are presented in  FIGS. 24A-24D . The error bars indicate the standard error of the mean for duplicate cultures. The results show four-fold, two-fold, and 13-fold greater optical density (OD600), dry cell weight (DCW), and mean green fluorescence protein intensity (GFP expression), respectively, for identical cultures performed on the vertical vibratory mixing system (at 12 g) when compared to control cultures performed on orbital shakers (at 400 rpm). The assumption that the mean fluorescence signal is directly proportional to the quantity of GFP protein accumulated in the cytosol has been validated (Hedhammar, M., Stenvall, M., Lonneborg, R., Nord, O., Sjolin, O., Brismar, H., Uhlen, M., Ottosson, J., &amp; Hober, S., 2005,  Journal of Biotechnology,  119, 133-146). A novel flow cytometry-based method for analysis of expression levels in  Escherichia coli , giving information about precipitated and soluble protein. Cultures were extended significantly past the glucose depletion point because the GFP in the pGLO plasmid is under the control of an arabinose operon promoter that is repressed in the presence of high levels of glucose. These results indicate that the dramatically improved oxygen transfer rates that are possible with the present invention correspond directly to improved bacterial culture responses. 
     In a second experiment, the gram positive bacterium  Bacillus subtilis  (ATCC 9799) was cultured in 62.5 ml of medium at 37° C. The culture medium is described in the following reference: Martinez, A., Ramirez, O. T., &amp; Valle, F., 1997, Improvement of culture conditions to overproduce beta-galactosidase from  Escherichia coli  in  Bacillus subtilis, Applied Microbiology and Biotechnology,  47, 40-45. 
     The results for are presented in  FIGS. 25A-25C . The error bars indicate the standard error of the mean for duplicate cultures. These results are similarly positive, showing approximately 50 percent increases in OD600 and DCW for the present invention compared to the orbital controls. 
     In a third experiment,  Pseudomonas fluorescens  A506::gfp2 transformed with GFP were obtained from J. K. Jansson and cultured in 62.5 ml of Kim/H15 medium at 30° C. (Lowder, M., Unge, A., Maraha, N., Jansson, J. K., Swiggett, J., &amp; Oliver, J. D., 2000, Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged  Pseudomonas fluorescens  A506, Applied and Environmental Microbiology, 66, 3160-3165). The Kim/H15 culture medium is described in the following references: Kim, G. J., Lee, I. Y., Choi, D. K., Yoon, S. C., &amp; Park, Y. H., 1996, High cell density cultivation of  Pseudomonas putida  BM01 using glucose,  Journal of Microbiology and Biotechnology,  6, 221-224 and Danielson, P. B., Buchs, J., Stockmann, C., &amp; Fogleman, J. C., 2004, Maximizing cell densities in miniprep-scale cultures with H15 medium and improved oxygen transfer,  Biochemical Engineering Journal,  17, 175-180.) Mean green florescence was monitored using a Guava® EasyCyte flow cytometer. 
     As indicated in  FIGS. 26A-26D , growth of cultures of  Pseudomonas fluorescens  were similarly positive in favor of the present invention. The error bars indicate the standard error of the mean for duplicate cultures. Approximately twice the level of biomass and a 50 percent improvement in GFP expression levels were obtained in RBS cultures. 
     For bacterial cultures, the composition of the culture medium is critical to allowing the benefits of the present invention to be clearly evident. Because of the significantly increased oxygen transfer, substantially more nutrients are required, along with the concomitant requirements to properly balance pH change in the medium. The H15 medium for  E. coli  was designed specifically for supporting high densities in a batch culture format. The medium formulations for the  B. subtilis  and  P. fluorescens  strains were not optimized for high density culture. These results clearly imply that with improved medium design, the  B. subtilis  and  P. fluorescens  results might have even more dramatically favored the cultures utilizing the present invention. 
     Although some embodiments are shown to include certain features or steps, the applicant(s) specifically contemplate that any feature or step disclosed herein may be used together or in combination with any other feature or step on any embodiment of the invention. It is also contemplated that any feature or step may be specifically excluded from any embodiment of the invention.