Abstract:
A bioreactor for culturing cells in a liquid environment is provided that is designed to reduce the chance of contamination, contain the contamination should it occur, and readily clean and sterilize all or part of the bioreactor in response to contamination, or on a schedule. A processor-controlled method of promoting sterility in a bioreactor is also provided.

Description:
FIELD 
     The present technology relates to a system for scale up and steady state production of liquid cultures under sterile conditions. More specifically, the technology relates to a safe bioreactor system for growing aquatic biological materials including salt water zooplankton and phytoplankton and combinations thereof. 
     BACKGROUND 
     Bioreactors have been used for many years for cell culture, most notably for fermentation and more recently for the growth of bacteria. These cultures are usually contained in stainless steel vessels where gas exchange, temperature, pH, dissolved oxygen levels, and circulation are closely monitored and controlled. 
     Photobioreactors are reactors for material that requires light. There are many designs, ranging from open-air races, to tubes, to transparent vessels. The vessels may have banks of lights around the periphery or a central core of lights. The level of control ranges from essentially none, to strict monitoring of the growth conditions. Where there is no control over the growth conditions, sterility and maintenance of cell culture purity are not considered. This may be adequate for growth of algae for biofuel production, but is not for the growth of algae as a food source. In this instance, sensors and controls, as disclosed in US Publication No. 20110136225, are employed. A bioreactor module can be connected to one or more functional modules such as a pump module, a stimulation signal generation module, a motor module, a mechanical transmission module, a gas exchange module, a temperature module, a humidity module and/or a CO2 module, among others. The bioreactor and functional modules can include standard or universal connectors to facilitate connection and movement of modules. The bioreactor system can be controlled and/or monitored by a controller that can individually identify and control each connected module and that can be adapted to collect signal data from sensors embedded in any of the modules. 
     The use of sensors may require special adaptations. As disclosed in US Publication No. 20110111489, a sensor adapter comprises an accommodating channel, in which the sensor can be positioned and the one end region of which is closed off by a semipermeable membrane. Moreover, the sensor adapter comprises a hollow cylindrical sealing structure, which is disposed within the accommodating channel coaxially with the longitudinal axis of the latter and with which the sensor can be disposed gas tight adjacent to the semipermeable membrane. 
     Processors and programmes can be used to monitor outputs from sensors and run the various controllers. As disclosed in US Publication No. 20050208473, decision making software can be used that utilizes detected changes in the course of fermentation. Decisions are aimed at determining the optima for cellular growth, optimizing for production or degradation of metabolites or substrates, or determining the limits of growth under various combinations of conditions. The invention determines optima or limits in a manner more quickly and at less cost than traditional methods. The basis for the computer generated decisions may be first or second derivative changes observed such as inflection points, limits on allowable rates of change, or the like. The most common measured parameter controlling the decision making process is the optically observed growth of the cells (e.g. microbial, animal, or plant cell cultures) under study. Any other measurable parameter (e.g. pH, temperature, pigment production) may be used to control the process (i.e., the independent variable). This process and variations of this process on a laboratory scale are valuable for research and development, education, pilot plant models, and bio-manufacturing optimization, including scale up to production volumes. 
     SUMMARY 
     The present technology is an integrated bioreactor comprising air, carbon dioxide, nutrient, sterilizant and neutralizer sources, lines from the sources to at least one culture vessel, a culture line for delivering seed culture to the vessel, a manifold to direct flow to and from the culture vessel, lights, sensors and a processor to control the functions of the bioreactor. 
     In one embodiment, the bioreactor has an integrated sterilization system for in situ sterilization. The technology allows for regular automated cleaning and sterilizing of a bioreactor with minimal interruption in production. Downtime can be less than 1 hour each week. From one to a plurality of culture vessels make up the bioreactor. The bioreactor provides controlled, closed scale up. 
     Specifically, the bioreactor, which is for culturing cells in a liquid environment, comprises:
         culture lines, culture medium lines, and a combined gas and sterilizant manifold, the lines and manifold comprising valves to control flow direction and flow rates, optionally, pressure relief valves to relieve pressure and optionally, pumps to maintain pressure;   a source of pressurized carbon dioxide, a source of pressurized air and a sterilizant source each in communication with the manifold;   a culture medium source in liquid communication with the culture medium lines;   at least one vessel, the vessel comprising a side wall, a lid, a bottom, sensors for reporting culture conditions, a sparger, a sprayer, an inlet and an outlet;   a transfer system for accepting a seed culture container, the transfer system in communication with a first vessel;
 
and
   a processor programmed to control culture conditions, execution of sterilization schedules, and incremental increases of volume of a culture on a schedule.       

     For use with phototrophic or mixotrophic cultures, at least the side wall is light transmitting and the vessels are provided with lighting proximate the side wall. 
     The bioreactor may further comprise a base, wherein the side wall comprises substantially vertical contours and the base is contoured to mate with the side wall. 
     The vessels may further be provided with reflectors proximate the lighting. 
     The bioreactor may further comprise at least one cleaner, the cleaner comprising a blade, an arm and a drive, the blade located within the at least one vessel and magnetically coupled to the arm, or directly driven, the arm configured to rotate around the vessel, and the drive for driving rotation of the arm, such that in use, the blade wipes the side walls within the vessel. 
     The sterilizant source may be a steam boiler or a liquid sterilizant pack. 
     The processor may be programmed to increase culture volume on a cell density based schedule. 
     The bioreactor may comprise at least two vessels, wherein the processor is programmed to transfer the culture from a first vessel to a second vessel to increase culture volume. 
     The bioreactor may comprise one vessel, wherein the processor is programmed to add culture medium to the vessel to increase culture volume. 
     In another embodiment, a bioreactor is provided, the bioreactor comprising:
         culture lines, culture medium lines, and gas lines, the lines comprising valves and optionally, pumps;   gas sources in gaseous communication with the gas lines;   a culture medium source in liquid communication with the culture medium lines;   at least one culture vessel comprising a side wall, a lid, a bottom, sensors for reporting culture conditions, a gas sparger in communication with the gas line, a culture medium sprayer in communication with the culture medium line, a culture inlet and a culture outlet;   a pressure driven transfer system for transferring a culture from a seed culture container to the culture vessel;
 
and
   a processor programmed to control culture conditions, incremental increases in culture volume and execution of sterilization cycles,
 
the improvement being an integrated sterilization system for in situ sterilization of the bioreactor.
       

     The integrated sterilization system may comprise the gas lines, a sterilizant source in communication with the gas lines, and sterilization cycle protocols programmed in the processor. 
     The sterilizant source may be a steam boiler. 
     The sterilizant source may be a sterilizing fluid pack. 
     The bioreactor may further comprise a cleaner, the cleaner comprising a blade, an arm and a drive, the blade located within the vessel and coupled to the arm, the arm configured to rotate and the drive for driving rotation of the arm, such that in use, the blade wipes the side walls within the vessel. 
     At least the side wall may be light transmitting, and the vessels may be provided with lighting proximate the side wall. 
     A bioreactor vessel is also provided, the vessel comprising a side wall, a lid, a bottom, a base, the base contoured to mate with the side wall, sensors for reporting culture conditions, a gas sparger for communication with a gas line, a culture medium sprayer for communication with a culture medium line, a culture inlet and a culture outlet, wherein the side wall is light-transmitting and comprises substantially vertical contours of peaks and valleys. 
     The bioreactor vessel may further comprise a layer proximate the lighting, the vertical contours and layer defining air channels. 
     The bioreactor vessel may further comprise a combined stand and cooling system, the combined stand and cooling system comprising a framework of conduits and at least one blower, the blower in gaseous communication with a conduit inlet, the frame work of conduits having a series of outlets aligned with the air channels, such that in use, air is blown into a lower end of the channels and rises to the top of the channels thereby cooling the bioreactor vessel. 
     A processor-controlled method of promoting sterility in a bioreactor is also provided, the bioreactor comprising at least two culture vessels, sensors, culture lines, culture medium lines, a combined gas and sterilizant manifold, a sterilizant source, and inline filters between the ambient environment and the bioreactor, and a processor, the method comprising:
         the processor signaling a start of the sterilizing cycle;   delivering sterilizant through the manifold to the bioreactor, at least downstream of the inline filters;
 
and
   signaling an end of the sterilizing cycle, thereby promoting sterility in the bioreactor.       

     The method may further comprise sensing contamination, and the processor signaling emptying of a culture vessels prior to signaling the start of the sterilization cycle. 
     The method may further comprise a cleaning step prior to signaling the start of the sterilization cycle. 
     A processor controlled method of culturing plant cells in a bioreactor is also provided, the bioreactor comprising a processor, a sterilizable transfer valve for accepting a seed culture container, at least one culture vessel with a culture line inlet and a culture line outlet, sensors for the culture vessel, lights, culture lines between the transfer valve and the at least one culture vessel, culture medium lines, a combined gas and sterilizant manifold, a sterilizant source, and inline filters between the ambient environment and the bioreactor, the method comprising:
     i) attaching the seed culture container to the transfer valve;   ii) the processor signaling a start of the sterilizing cycle, controlling delivering sterilizant through the manifold to the bioreactor, at least downstream of the inline filters, then signaling a stop of the sterilizing cycle;   iii) the processor signaling opening of the transfer valve and signaling opening of the culture medium lines, thereby controlling delivering culture medium and culture to a first vessel;   iv) the sensors sending culture condition data to the processor, the processor controlling culture conditions; and   v) the processor terminating culturing and signaling emptying of the first culture vessel.   

     The method may further comprise:
     vi) the processor signaling cleaning of the at least one culture vessel.   

     The method may further comprise:
     vii) the processor controlling transferring the emptied culture to at second culture vessel and signaling opening of the culture medium lines, thereby filling the second culture vessel.   

     The method may further comprise:
     viii) the processor signaling cleaning of the culture vessels.   

     In another embodiment, a bioreactor for culturing cells in a liquid environment is provided, the bioreactor comprising:
         culture lines, culture medium lines, and a combined gas and sterilizant manifold, the lines and manifold comprising valves to control flow direction and flow rates, optional pressure release valves to relieve pressure and optionally, pumps to maintain pressure;   a culture vessel, the vessel comprising a transparent side wall, wherein the side wall comprises substantially vertical contours, a base, the base contoured to mate with the side wall, a lid, sensors for reporting culture conditions, a sparger, a sprayer, an inlet and an outlet;   a light source disposed around the side wall;
 
and
   a processor programmed to control culture conditions and execution of sterilization schedules.       

     The side wall contours may be ridges and valleys, the peak to valley height about 1/16th of an inch to about 12 inches and the distance between the peaks about 1/16th of an inch to about 12 inches. 
     The peak to valley height may be about 1 inch to about 6 inches and the distance between the peaks may be about 1 inch to about 6 inches. 
     The bioreactor may further comprise a cooling system, the cooling system comprising at least one fan and a distribution plate in communication with the at least one fan, the distribution plate having a network for directing air flow into each valley. 
     The bioreactor may further comprise a cooling plate or a cooling water jacket disposed beneath the distribution plate and for communication with a refrigeration source. 
     The bioreactor may further comprise a source of pressurized carbon dioxide, a source of pressurized air and a sterilizant source each in gaseous communication with the manifold. 
     A processor-controlled method of promoting sterility in a bioreactor is also provided, the bioreactor comprising:
         culture lines, culture medium lines, and gas lines, the lines comprising valves and optionally, pumps;   gas sources in gaseous communication with the gas lines;   a culture medium source in liquid communication with the culture medium lines;   at least one culture vessel comprising a side wall, a lid, a bottom, sensors for reporting culture conditions, a gas sparger in communication with the gas line, a culture medium sprayer in communication with the culture medium line, a culture inlet and a culture outlet;   a pressure driven transfer system for transferring a culture from a seed culture container to the culture vessel;   a processor; and   an integrated sterilization system for in situ sterilization of the bioreactor,
 
the method comprising:
   the processor signaling a start of the sterilizing cycle;   delivering sterilizant through the integrated sterilization system of the bioreactor, at least downstream of the inline filters; and   the processor signaling an end of the sterilizing cycle, thereby promoting sterility in the bioreactor.       

     The method may further comprise the sensors reporting data to the processor, the processor determining contamination, and the processor signaling emptying of a culture vessels prior to signaling the start of the sterilization cycle. 
     The method may further comprise a cleaning step prior to signaling the start of the sterilization cycle. 
     A processor-controlled method of promoting sterility in a bioreactor is also provided, the bioreactor comprising:
         culture lines, culture medium lines, and a combined gas and sterilizant manifold, the lines and manifold comprising valves to control flow direction and flow rates, optional pressure release valves to relieve pressure and optionally, pumps to maintain pressure;   a culture vessel, the vessel comprising a transparent side wall, wherein the side wall comprises substantially vertical contours, a base, the base contoured to mate with the side wall, a lid, sensors for reporting culture conditions, a sparger, a sprayer, an inlet and an outlet;   a light source disposed around the side wall;
 
and
   a processor programmed to control culture conditions and execution of sterilization schedules.
 
the method comprising:
   the processor signaling a start of the sterilizing cycle;   delivering sterilizant through the combined gas and sterilizant manifold, at least downstream of the inline filters; and   signaling an end of the sterilizing cycle, thereby promoting sterility in the bioreactor.       

     The method may further comprising the sensors reporting data to the processor, the processor determining contamination, and the processor signaling emptying of a culture vessels prior to signaling the start of the sterilization cycle. 
     The method may further comprise a cleaning step prior to signaling the start of the sterilization cycle. 
     A processor controlled method of culturing plant cells in a bioreactor is also provided, the bioreactor comprising:
         culture lines, culture medium lines, and gas lines, the lines comprising valves and optionally, pumps;   gas sources in gaseous communication with the gas lines;   a culture medium source in liquid communication with the culture medium lines;   at least one culture vessel comprising a side wall, a lid, a bottom, sensors for reporting culture conditions, a gas sparger in communication with the gas line, a culture medium sprayer in communication with the culture medium line, a culture inlet and a culture outlet;   a pressure driven transfer system for transferring a culture from a seed culture container to the culture vessel;   a processor programmed to control culture conditions, incremental increases of culture volume and execution of sterilization cycles; and   an integrated sterilization system for in situ sterilization of the bioreactor,
 
the method comprising:
       i) attaching the seed culture container to a first culture line;   ii) the processor signaling pressurizing the seed culture container to deliver culture to the culture vessel;   iii) the processor signaling opening of the culture medium lines, thereby controlling delivering culture medium to the culture vessel;   iv) the sensors sending culture condition data to the processor, the processor controlling culture conditions and controlling incremental increases in culture volume in the culture vessel; and   v) the processor terminating culturing and signaling emptying of the culture vessel.   

     The method may further comprise:
     vi) the processor signaling cleaning of the culture vessel.   

     The method may further comprise:
     vii) the processor signaling execution of the sterilization cycle.   

     In another embodiment a bioreactor for culturing cells in a liquid environment is provided, the bioreactor comprising:
         culture lines, culture medium lines, and a combined gas and culture manifold, the lines and manifold comprising valves to control flow direction and flow rates, pressure relief valves to relieve pressure and pumps to maintain pressure;   a source of pressurized carbon dioxide and a source of pressurized air in communication with the manifold;   a culture medium source in liquid communication with the culture medium lines;   at least one vessel, the vessel comprising a side wall, a lid, a bottom, sensors for reporting culture conditions, a sparger, at least one inlet and an outlet;   a sterilizant source in communication with the vessel;   a transfer system for accepting a seed culture container, the transfer system in communication with a first vessel;
 
and
   a processor programmed to control culture conditions, execution of sterilization schedules, and incremental increases of volume of a culture on a schedule.       

     For phototrophic or mixotrophic cultures, at least the side wall may be light transmitting and the vessels may be provided with lighting proximate the side wall. 
     The bioreactor may further comprise a base, wherein the side wall comprises substantially vertical contours and the base is contoured to mate with the side wall. 
     The vessels may be further provided with reflectors proximate the lighting. 
     The bioreactor may further comprise at least one cleaner, the cleaner comprising a blade, an arm and a drive, the blade located within the at least one vessel and magnetically coupled to the arm, or directly driven, the arm configured to rotate around the vessel, and the drive for driving rotation of the arm, such that in use, the blade wipes the side walls within the vessel. 
     The sterilizant source may be a steam boiler or a liquid sterilizant pack. 
     The processor may be programmed to increase culture volume on a cell density based schedule. 
     The bioreactor may comprise at least two vessels, wherein the processor is programmed to transfer the culture from a first vessel to a second vessel to increase culture volume. 
     The bioreactor may comprise one vessel, wherein the processor is programmed to add culture medium to the vessel to increase culture volume. 
     The bioreactor may further comprise a heat exchanger or water jacket for cooling the culture vessel. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a plan view of the bioreactor of the present technology. 
         FIG. 2  is a schematic of the bioreactor of  FIG. 1 . 
         FIG. 3  is a longitudinal sectional view of the scale up vessel of the present technology. 
         FIG. 4  is a longitudinal sectional view of the feed vessel of the present technology. 
         FIGS. 5A and 5B  are longitudinal sectionals view of the cleaner and the alternative cleaner. 
         FIG. 6  is a schematic of a second embodiment. 
         FIG. 7  is a longitudinal sectional view of the feed culture vessel of the bioreactor of  FIG. 6 . 
         FIG. 8  shows the side wall of the feed culture vessel of  FIG. 7 . 
         FIG. 9  is a schematic of the third embodiment of a bioreactor. 
         FIG. 10  is a schematic of the fourth embodiment of a bioreactor. 
     
    
    
     DESCRIPTION 
     Except as otherwise expressly provided, the following rules of interpretation apply to this specification (written description, claims and drawings): (a) all words used herein shall be construed to be of such gender or number (singular or plural) as the circumstances require; (b) the singular terms “a”, “an”, and “the”, as used in the specification and the appended claims include plural references unless the context clearly dictates otherwise; (c) the antecedent term “about” applied to a recited range or value denotes an approximation within the deviation in the range or value known or expected in the art from the measurements method; (d) the words “herein”, “hereby”, “hereof”, “hereto”, “hereinbefore”, and “hereinafter”, and words of similar import, refer to this specification in its entirety and not to any particular paragraph, claim or other subdivision, unless otherwise specified; (e) descriptive headings are for convenience only and shall not control or affect the meaning or construction of any part of the specification; and (f) “or” and “any” are not exclusive and “include” and “including” are not limiting. Further, The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. 
     To the extent necessary to provide descriptive support, the subject matter and/or text of the appended claims is incorporated herein by reference in their entirety. 
     Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Where a specific range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. All smaller sub ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Although any methods and materials similar or equivalent to those described herein can also be used, the acceptable methods and materials are now described. 
     Definitions: 
     Aquatic—in the context of the present technology, aquaculture includes the culturing of biological material in fresh water, salt water, brackish water, brine and the like—essentially any liquid. 
     Culture—in the context of the present technology, culture, as in culture line or culture vessel, refers to a combination of biological material, culture medium and any additional chemicals produced by the biological material during the culturing process. Cultures require appropriate sources of food and energy, provided by the culture medium, and a suitable physical environment. Tissue cultures can themselves become a culture medium for viruses, which grow only with live cells. Cultures of only one kind of cells are known as pure cultures, as distinguished from mixed or contaminated cultures. 
     Cell—in the context of the present technology, cell means any cell or cells, as well as viruses or any other particles having a microscopic size, e.g. a size that is similar to that of a biological cell, and includes any prokaryotic or eukaryotic cell, for example, but not limited to bacteria, fungi, plant and animal cells. A cell may be living or dead. As used herein, a cell is generally living unless otherwise indicated. Cells may be a plurality of individual cells or may be cell clumps, aggregates or groupings. The cells may be undifferentiated or differentiated, but are not formed into tissues. 
     Tissue—in the context of the present technology, tissue means an aggregation of cells more or less similar morphologically and functionally. 
     Sensor—in the context of the present technology, sensor is defined as any device that can measure a measurable quantity. For examples, a sensor can be, but is not limited to a thermal detector, an electrical detector, a chemical detector, an optical detector, an ion detector, a biological detector, an electrochemical detector, a magnetic detector, a capacitive detector, a pressure detector, an ultrasonic detector, an infrared detector, a microwave motion detector, an electric eye, and an image sensor. 
     Culture medium—in the context of the present technology, culture medium refers to a liquid comprising chemicals needed to support growth and maintenance of cells. The chemicals may be nutrients, including but not limited to vitamins, minerals, micronutrients, amino acids. The chemicals may also comprise osmoticum, a carbon source, biological extracts, and buffers. A medium can be provided with one or more analytes to be consumed by one or more cells. In some instances, culture medium may simply be salt water, wherein salt water is defined as ocean water or brine pond water, or it may be brackish water. 
     Plant—in the context of the present technology, plant refers to any organism, cell or cells that photosynthesize. 
     Apparatus: 
     Itemized list of the main components:
     1. Sterilizant system;   2. Water treatment system;   3. Clean-in-place system (CIP);   4. Air and CO2 addition;   5. Control system—Programmable Logic Controller (PLC) Based;   6. Seed culture container;   7. Scale up vessel;   8. Feed vessel; and   9. Cooling system (as described in “Second embodiment”)   

     A bioreactor, generally referred to as  10 , is shown in  FIG. 1 . A seed culture container  12  connects via a first culture line  14  to a scale up vessel  200 , which in turn connects via a second culture line  18  to a feed vessel  300 . The seed culture container  12  is transiently attached to the first culture line  14  via a sterilizable transfer valve  22 , or alternatively, is directly attached to the scale up vessel  200  via the transfer valve  22 , again transiently. There is an incremental volume increase in the vessels from the seed culture container  12  to the scale up vessel  200  to the feed vessel  300 . Each vessel has a second bottom  16  to define a water chamber for cooling the vessels  200 ,  300 . This functions as a heat exchanger. 
       FIG. 2  is a schematic of the bioreactor  10 . A steam generator  24  is used for sterilizing the bioreactor  10 . An air source  26 , which may be a tank or ambient air and a pressurized CO2 tank  28  are connected via gas lines  30  to the injectors  32  located in the interior  201  of the scale up vessel  200  and the interior  301  of the feeder vessel  300 . A processor  34  controls delivery of air and CO2 as needed. A regulator and digital pressure gauge  36  is located downstream from the CO2 tank  28  on the CO2 line  38  portion of the gas line  30 . A valve  31  is located downstream. Three way, 2 position solenoid valves  40  communicate with the processor  34  and are located on the gas lines  30 . An air pump  42  is on the air line  44  portion of the gas line  30  and is calibrated to produce a pressure between about 2 psi to about 15 psi. A check valve  46  is located between the air pump  42  and one of the three way, two position solenoid valves  40 . A 0.1 μm steam-in-place filter  48  is located upstream from the solenoid valve  40 . The solenoid valve  40  splits the air line  44  into an air dump line  50  and the air line  44 . The CO2 line  38  and the air line  44  connect at three way solenoid valves  40  to form the gas lines  30 . The CO2 line  38 , the air line  44 , and the gas lines  30  form a manifold. This manifold also distributes steam or more generally, sterilizant, allowing for easy steam sterilization of the lines. 
     The first culture line  14  enters the scale up vessel  200  at an inlet  203 . Downstream from the transfer valve  22 , the first culture line  14  has a three way valve  430  that can be manually operated and a two way valve  432  in line. The first culture line  14  optionally has an inline pump to pressurize the transfer mechanism. 
     The second culture line  18  leaves the scale up vessel through an outlet  70 . A first dump line splits  72  from the second culture line  18 . Both have two way valves— 74  on the dump line and  76  on the second culture line  18 . The second culture line  18  enters the feed vessel  300  at an inlet  301 . 
     A third culture line  80  leaves the feed vessel  300  through an outlet  82 . The third culture line  80  passes through an inline pump  84 , which is preferably a peristaltic pump or a shuttle pump, but may be a rotary pump, and a second dump line  86  splits off. Both have two way valves— 88  on the dump line  86  and  90  on the third culture line  80 . Additionally, the third culture line  80  has a one way check valve  92  downstream. An outlet  94  terminates the third culture line  80 . At this point the feed culture is supplied to the customer either as is, or in a concentrated form, by including a concentrator  96  either upstream or downstream from the outlet  94 . The concentrator  96  may be any suitable concentrator, for example, but not limited to a centrifuge or a filtration system. 
     A water line  390  for sea water has an inline 100 μm filter  392 , is joined by two nutrient lines  394  from nutrient packs  396  to become a culture medium line  398  and then passes an ultravoilet (UV) light source  399  located downstream. The culture medium line  398  enters a booster tank  400  that is supplied with a heater  402  and a pressure sensor  404 . The line  398  leaves the tank  400  through an outlet  406 , passes through an inline pump  408 , which is preferably a peristaltic pump or shuttle pump, but may be a rotary pump, and a one way check valve  410  to a three way diverter valve  412  that directs flow to the scale up vessel  200  or the feed vessel  300 . A first sprayer  414  sprays the contents of the line into the scale up vessel  200 . A second sprayer  416  sprays the contents of the line into the feed vessel  300 . The sprayers  414  and  416  are preferably rotary spray nozzles. The processor  34  controls the one way check valve  410  and the three way diverter valve  412 , which are solenoid valves, to control flow. 
     A fresh water supply  430  passes through a 50 μm filter  432  and enters a steam generator, for example, a boiler  434 . A first steam line  436  from the steam generator  434  enters the CO2 line  38  and the air line  44  at the solenoid valves  40 . A second steam line  438  enters the water line downstream from the nutrient lines  394  and upstream from the UV light source  399 . A third steam line  440  delivers steam to the transfer valve  22 . The steam lines, manifold and overall integration of the bioreactor allow for in situ sterilization of either the entire bioreactor, or select vessels and lines. 
     The scale up vessel, generally referred to as  200  is shown in  FIG. 3 . The scale up vessel is about 200 to about 2,000 liters, or about 500 to about 1500 liters or 1,000 liters and all ranges therebetween. If algae or other plant material is to be cultured, at least the side walls  202  are transparent or light transmitting. The lip  203  of the wall  202  is formed into a flange  204  and has openings  206  to accept bolts  208  for affixing an airtight lid  210 . As the vessel is steam-cleaned, both the vessel  200  and the lid  210  are made of steam-resistant material, for example, but not limited to fiberglass or a heat resistant polyethylene such as Tyvar®. The lid  210  has an access port  212  for accepting a clean in place system(CIP), generally referred to as  214 . Gaskets  216  are located between the lid  210  and flange  204  and between a CIP flange  218  of the CIP  414  and the lid  210 . 
     The scale up vessel  200  is equipped with a bottom access  230  on or in the vicinity of the bottom  231  connected to the gas lines  30  and the outlet  70  connected to the second culture line  18 . The gas line  30  terminates in a sparger  232 . The first culture line  14  enters into the scale up vessel  200  on a side wall  202 . An optional thin plastic polymer shell  234  surrounds the side wall  202  and is equipped with light emitting diode grow lights  236 . An optional reflective surface  238  is located on an outer side of the shell  234 . Lights  205  may additionally be provided on the lid  210 . As shown in  FIG. 2 , the scale up vessel  200  is provided with sensors for reporting culture conditions, for example, but not limited to each of a pH  240 , optical density  242 , temperature  244 , and pressure sensor  246 . Capacitance sensors  248  are located at a number of depths, for example, two located at ⅓ and ⅔ depth, three located at ¼, ½, ¾ depth or four located at ⅕, ⅖, ⅗ and ⅘ depth. 
     The feed culture vessel, generally referred to as  300 , is shown in  FIG. 4 . The feed culture vessel is about 100 to about 100,000 liters, or about 250 to about 75,000 liters or 50,000 liters and all ranges therebetween. If algae or other plant material is to be cultured, at least the side walls  302  are transparent or light transmitting. The lip of the wall  303  is formed into a flange  304  and has openings  306  to accept bolts  308  for affixing an airtight lid  310 . As the vessel is steam-cleaned, both the vessel  300  and the lid  310  are made of steam-resistant material, for example, but not limited to fiberglass or a heat resistant polyethylene such as Tyvar®. The lid  310  has an access port  312  for accepting a clean in place system (CIP), generally referred to as  416 . Gaskets  316  are located between the lid  310  and flange  304  and between a CIP flange  318  and the lid  310 . 
     The feed culture vessel  300  is equipped with a bottom access  330  on or in the vicinity of the bottom  331  connected to the gas lines  30  and an outlet  82  connected to the third culture line  80 . The gas line  30  terminates in a sparger  332 . The second culture line  18  enters the feed culture vessel  300  at a side wall  302 . An optional thin plastic polymer shell  334  surrounds the vessel  300  and is equipped with light emitting diode grow lights  336 . An optional reflective surface  338  is located on an outer side of the shell  334 . Lights  305  may additionally be provided on the lid  310 . As shown in  FIG. 2 , the feed culture vessel  300  is provided with sensors for reporting culture conditions, for example, but not limited to each of a pH  340 , optical density  342 , temperature  344 , and pressure sensor  346 . Capacitance sensors  348  are located at a number of depths, for example, two located at ⅓ and ⅔ depth, three located at ¼, ½, ¾ depth or four located at ⅕, ⅖, ⅗ and ⅘ depth. 
     The bioreactor is controlled by the processor  34 . It receives and process data from the various sensors (pH, optical density, temperature, pressure), and coordinates the activity of the solenoids, pumps, steam cleaning, lighting and heating. If desired, the processor  34  can be made to interface wirelessly to a computer to allow remote monitoring and control. 
     As shown in  FIG. 5A , a cleaner, generally referred to as  100  is for placing in the scale up and feed culture vessels  200 ,  300 . A blade  102  for locating inside the culture vessels  200 ,  300  is magnetically coupled to a rotating arm  104  which is configured to move around the outside of the vessels  200 ,  300 . As would be known to one skilled in the art, the magnet  106  and the magnetic material  108  can be interchangeably located on the rotating arm  104  and the blade  102 . Alternatively, the blade  102  may be directly driven. The cleaner  100  is preferably contoured to the inner surface  110  of the vessel  200 ,  300 , or can be flexible, for example, but not limited to iron filings encased in a long flexible plastic covering or brushes located on the blade  102 . In an alternative embodiment, as shown in  FIG. 5B , small free floating parts  112  are placed inside the culture vessels  200 ,  300 . These free floating parts  112  are carried by gas currents  114  in the culture medium  116  and keep the inner surface  110  clean through continuous small impacts. 
     Method: 
     The design of the bioreactor provides for a minimum downtime and maximum efficiency. As each vessel is emptied, both the vessel and the lines leading to it can be sterilized. Additionally, the entire bioreactor can be cleaned and sterilized. Once scale up has begun, the system is closed and remains closed until harvest, which is preferably in late log phase, but may be earlier or later. In this closed system (i.e. one that does not require open transfers), the volume of the vessels increases incrementally from the seed vessel to the scale up vessel to the feed vessel, on a schedule and under control of the processor, hence contamination can be contained to a relatively small volume, as compared to having one large culture vessel filled with culture medium. Also, the level of security increases as the number of valves, lines and vessels from the ambient environment increase, hence the larger the vessel, the further it is removed from ambient and therefore the less chance there is of contamination. Culture medium used for cleaning the vessels may be dumped or retained to scale up the culture volume. Should contamination occur in any one vessel the processor will detect the contamination, based on data from at least one sensor and will control emptying of the vessel. The vessel may additionally be cleaned, by the processor signaling a cleaning step before the sterilizing cycle begins. Gaseous sterilizant is fed through the bioreactor by means of the steam lines and manifold. All transfers are automated, thereby reducing the risk of contamination. 
     Second Embodiment 
     Itemized list of the main components:
     1. Sterilizant system;   2. Water treatment system;   3. Clean-in-place system (CIP);   4. Air and CO2 addition;   5. Control system—Programmable Logic Controller (PLC) Based;   6. Seed culture container;   7. Feed culture vessel; and   8. Cooling system.   

       FIG. 7  is a schematic of a second embodiment of a bioreactor  510 . The seed culture container  512  connects via the first culture line  514  to a feed vessel  600 . The seed culture container  512  is transiently attached to the first culture line  514 , which directly feeds the feed vessel  600 . The first culture line  514  enters the feed vessel  600  at an inlet  603 . The first culture line  514  has a two way valve  48  in line. 
     The air line  516  and the first culture line  514  enter the seed culture container  512  through a bung  518 . The air line  516  is connected to a pump  520  for pumping air into the carboy  512 , thereby increasing the pressure, and forcing culture through the first culture line  514 . The air line  516 , first culture line  514 , bung  518  and pump  520  are collectively referred to as the pressure driven transfer system. A steam generator  24  is used for sterilizing the bioreactor  510 . An air source  26 , which may be a tank or ambient air and a pressurized CO2 tank  28  are attached via gas lines  30  to the injectors  32  located in the interior  601  of the feed vessel  600 . A processor  34  controls delivery of air and CO2 as needed. A regulator and digital pressure gauge  36  is located downstream from the CO2 tank  28  on the CO2 line  38  portion of the gas line  30 . A valve  31  is located downstream. A three way, 2 position solenoid valve  40  communicates with the processor  34  and is located on the gas lines  30 . An air pump  42  is on the air line  44  portion of the gas line  30  and is calibrated to produce a pressure of about 2 psi to about 15 psi. Check valves  46  are located on the air line  44  both on the air intake line and the air pump line. A 0.1 μm steam-in-place filter  48  is located upstream from the solenoid valve  40 . It splits the air line  44  into an air dump line  50  and the air line  44 . The CO2 line  38  and the air line  44  connect at three way solenoid valves  40  to form the gas lines  30 . The CO2 line  38 , the air line  44 , and the gas lines  30  form a manifold. This manifold also distributes steam or more generally, sterilizant, allowing for easy steam sterilization of the lines. 
     A water line  390  for sea water has an inline 100 μm filter  392 , and a valve  393 . It is joined by two nutrient lines  394  from nutrient packs  396  to become a culture medium line  398 . Each nutrient line  394  is equipped with a pump  408 , which is preferably a peristaltic pump or shuttle pump, but may be a rotary pump and a check valve  410 . The nutrient lines  394  upstream from the peristaltic pump  408  are preferably disposable. The culture medium line  398  passes through an inline pump  408 , which is preferably a peristaltic pump or shuttle pump, but may be a rotary pump. A sprayer  416  sprays the contents of the line into the feed vessel  600 . The sprayer  416  is preferably a rotary spray nozzle. This is the CIP. The feed vessel  600  has a pressure relief line  700  with a pressure relief valve  702  and an atmosphere dump  704 . 
     A fresh water supply  430  passes through a 50 μm filter  432  and enters a steam generator, for example, a boiler  434 . A first steam line  436  from the steam generator  434  enters the air line  44  between the filter  48  and the solenoid valves  40 . A second steam line  438  enters the water line upstream from the nutrient lines  394 . The steam lines, manifold and overall integration of the bioreactor allow for in situ sterilization of either the entire bioreactor, or select vessels and lines. The steam lines  436 ,  438  have a pressure release valve  439 . 
     A third culture line  80  leaves the feed culture vessel  600  through an outlet  82 . The third culture line  80  passes through an inline pump  84 , which is preferably a peristaltic pump or a shuttle pump, but may be a rotary pump, and a second dump line  86  splits off. Both have two way valves— 88  on the dump line  86  and  90  on the third culture line  80 . Additionally, the third culture line  80  has a one way check valve  92  downstream. An outlet  94  terminates the third culture line  80 . At this point the feed culture is supplied to the customer either as is, or in a concentrated form, by including a concentrator  96  either upstream or downstream from the outlet  94 . The concentrator  96  may be any suitable concentrator, for example, but not limited to a centrifuge or a filtration system. 
     The feed culture vessel, generally referred to as  600 , is shown in  FIG. 7 . The feed culture vessel is about 100 to about 100,000 liters, or about 250 to about 75,000 liters or 50,000 liters and all ranges therebetween. If algae or other plant material is to be cultured, at least the side walls  602  are transparent or light transmitting. The side wall  602  is preferably polycarbonate. The lip  603  of the wall  602  is formed into a flange  604  and has openings  606  to accept bolts  608  for affixing an airtight lid  610 . As the vessel is steam-cleaned, both the vessel  600  and the lid  610  are made of steam-resistant material, for example, but not limited to fiberglass or a heat resistant polyethylene such as Tyvar®. The lid  610  has an access port  612  for accepting a clean in place system (CIP), generally referred to as  416 . Gaskets  616  are located between the lid  610  and flange  604  and between a CIP flange  618  and the lid  610 . 
     The feed culture vessel  600  is equipped with a bottom access  630  on or in the vicinity of the bottom  631  connected to the gas lines  30  and an outlet  82  connected to the third culture line  80 . The gas line  30  terminates in a sparger  632 . The first culture line  514  enters the feed vessel  600  at an inlet  603 . An optional thin plastic polymer shell  634  surrounds the vessel  600  and is equipped with light emitting diode grow lights  636 . Lights  605  may additionally be provided on the lid  610 . An optional reflective surface  638  is located on an outer side of the shell  634 . As shown in  FIG. 6 , the feed culture vessel  600  is provided with sensors for reporting culture conditions, for example, but not limited to each of a pH  640 , optical density  642 , temperature  644 , and pressure sensor  646 . Capacitance sensors  648  are located at a number of depths, for example, two located at ⅓ and ⅔ depth, three located at ¼, ½, ¾ depth or four located at ⅕, ⅖, ⅗ and ⅘ depth. 
     As shown in  FIG. 8 , the side wall  602  is formed into vertically disposed ridges  650  and valleys  652 . They may be rounded or sharp edged and may be wavy  651  about their vertical axis  653 . The vertical contours  654  may be, but are not limited to waves, or ridges and valleys, or peaks and troughs or are accordion-shaped, and are substantially vertical, for example, the vertical axis is normal to the floor, about 85 degrees relative to the floor, about 80 degrees relative to the floor or about 75 degrees relative to the floor. The vertical contours  654  function to increase the surface area of the side wall  602  and thereby increase light penetration in the feed culture vessel  600 . The peak to valley height of the contours  654  is about 1/16 of an inch to about 1 foot, or about 1 inch to about 6 inches or about 3 inches and all ranges therebetween. The distance between the peaks is about 1/16 of an inch to about 1 foot, or about 1 inch to about 6 inches, or 3 inches and all ranges therebetween. Additionally, the contours  654  preferably have small corrugations  655  to further increase the surface area. A bottom plate  656  retains the side wall  602  and has plate contours  658  that correspond to the contours  654  of the side wall  602 . Alternatively, the bottom plate  656  may have a contoured groove  660  (shown in  FIG. 8 , inset) to accept the side wall  602 . 
     A cooling system provides air flow to the space between the feed culture vessel  600  and light emitting diode grow lights  636  (See  FIG. 8 ). This space is referred to as the air channel  662 . As shown in  FIG. 7 , blowers or fans  664  force air down through the air channels  662 , which then exits from the bottom  672  of the air channels  662 . Similarly, blowers or fans force air down through the air channels in the vessels of  FIG. 3  and  FIG. 4 . 
     The bioreactor is controlled by the processor  34 . It receives and process data from the various sensors (pH, optical density, temperature, pressure), and coordinates the activity of the solenoids, pumps, steam cleaning, lighting and heating. If desired, the processor  34  can be made to interface wirelessly to a computer to allow remote monitoring and control. 
     The cleaner and alternative cleaner are shown in  FIGS. 5 and 6 . 
     Method: 
     The design of the bioreactor provides for a minimum downtime and maximum efficiency. As the vessel is emptied, both the vessel and the lines leading to it can be sterilized. Additionally, the entire bioreactor can be cleaned and sterilized. Once scale up has begun, the system is closed and remains closed until harvest, which is preferably in late log phase, but may be earlier or later. Initially, the feed culture vessel contains a small amount of culture medium. In this closed system (i.e. one that does not require open transfers), the volume of culture medium increases incrementally on a schedule, under control of the processor, hence contamination has a smaller chance of establishing itself. Since less medium (a vector for contamination) is added at the beginning of the scale up, there is a smaller chance that contaminant organisms are added early on. This limits the amount of time that contaminants are multiplying in the system, and increases competition for resources, which on average will produce significantly less contaminated cultures. Culture medium used for cleaning the vessels may be dumped or retained to scale up the culture volume. Should contamination occur in the vessel the processor will detect the contamination, based on data from at least one sensor and will control emptying of the vessel. The vessel may additionally be cleaned by the processor signaling a cleaning step before the sterilizing cycle begins. Gaseous sterilizant is fed through the bioreactor by means of the steam lines and manifold. 
     Third Embodiment 
     Itemized List of the Main Components:
     1. Sterilizant system;   2. Water treatment system;   3. Clean-in-place system (CIP);   4. Air and CO2 addition;   5. Control system—Programmable Logic Controller (PLC) Based;   6. Seed culture container;   7. Culture vessel; and   8. Cooling system.   

     A schematic of a third embodiment, generally referred to as  700  is shown in  FIG. 9 . The seed culture container  702  connects via the first culture line  704  to the culture vessel  706 . The seed culture container  702  is transiently attached to a first culture line  704 , which directly feeds the culture vessel  706 . The first culture line  704  enters the culture vessel  706  at an inlet  714 . 
     A first air line  710  has an air source  716  which may be a tank or ambient air. A pump  718  forces the air to a T-junction  720 , to a second air line  722  that branches from the first air line  710  at the T-junction  720 . The pump  718  is calibrated to produce a pressure of about 2 psi to about 15 psi. The second air line  722  has a two way manual valve  724  and a fitting  726  downstream from the valve  724  for a user to attach a third air line  730  with an air filter  732 . The third air line  730  enters the seed culture container  702  through a bung  734 . The first culture line  704  similarly has a fitting  736  for attaching a second culture line  738  that enters the seed culture container  702  through the bung  734 . When the valve  724  is open and the air lines  706 ,  722 ,  730  are pressurized by the pump  718 , culture  740  is forced from the seed culture container  702  to the first culture line  704  that leads to the culture vessel  706 . The air lines  706 ,  722 ,  730 , culture line  704 ,  738  and pump  718  are collectively referred to as the pressure driven transfer system. Alternatively, the transfer valve  22  described above could be employed. 
     A pressurized CO2 tank  744  provides CO2 to a CO2 line  746 . A regulator and digital pressure gauge  748  is located downstream from the CO2 tank  744  on the CO2 line  746  and a three way two position solenoid valve  749  is located downstream from the regulator and digital pressure gauge  748 . The CO2 line  746  joins the first air line  710  to form a gas line  750 , which delivers to the culture vessel  706  through injectors or spargers  752  located in the interior  754  of the culture vessel  706 . Upstream from the gas line  750 , a three way, two position solenoid valve  756  is located on the first air line  710 . A processor  758  controls delivery of air and CO2 as needed by communicating with the valves  746 ,  756 . A 0.1 μm filter  760  is located on the gas line  750 . 
     A water line  762  for sea water has a two position solenoid valve  764  and optionally, an inline ultraviolet filter. The water line  762  and the gas line  750  connect to form a common line  766  downstream of the valve  764 . A two position solenoid valve  768  is downstream from the connection  770 . The common line  766  enters the culture vessel  706  at a sprayer  772  that sprays the contents of the common line  766  (which is normally primarily liquid, but, by closing the valve  726  on the water line  762 , can become a gas line) into the culture vessel  706 . The sprayer  772  is preferably a rotary spray nozzle. 
     Two nutrient lines  774  from nutrient packs  776  are each equipped with a pump  778 , which is preferably a peristaltic pump or shuttle pump, but may be a rotary pump. The nutrient lines  774  upstream from the peristaltic pump  778  are preferably disposable. The nutrient lines  774  enter the culture vessel  706  at an upper end  780 . 
     A sterilizant line  782  from a sterilizer pack  784  is equipped with a pump  786 , which is preferably a peristaltic pump or shuttle pump, but may be a rotary pump. Similarly, a neutralizer or detoxifier line  788  from a neutralizer or detoxifier pack  790  is equipped with a pump  792 . The lines  782 ,  788  enter the culture vessel  706  at an upper end  794 . The gas line  750 , common line  766  and sterilizant line  782  form a manifold to provide an integrated sterilization system for in situ sterilization. 
     A 2-directional air filter  795  extends from the culture vessel  706  at an upper end  794  and functions as a pressure release valve. A third culture line  800  leaves the culture vessel  706  through an outlet  802 . The third culture line  800  passes through an inline pump  804 , which is preferably a peristaltic pump or a shuttle pump, but may be a rotary pump, and a dump line  806  splits off. Both have two way valves— 808  on the dump line  806  and  810  on the third culture line  800 . Additionally, the third culture line  800  has a one way check valve  812  downstream. An outlet  814  terminates the third culture line  800 . At this point the feed culture is supplied to the customer either as is, or in a concentrated form, by including a concentrator  816  either upstream or downstream from the outlet  802 . The concentrator  816  may be any suitable concentrator, for example, but not limited to a centrifuge or a filtration system. 
     A liquid sterilizer pack  784  contains sterilizant that is used for sterilizing the bioreactor  700 . The sterilizant may be a weak sodium hypochlorite solution, for example, 1% in water. The neutralizer or detoxifier may be a de-chlorinator. The path of the sterilizant is as follows: 
     Sterilizant leaves sterilizant pack  784  and travels through sterilizant line  782 , under pressure resulting from the pump  786  to the culture vessel  706  where it is sprayed into the culture vessel  706  with the sprayer  772  (the CIP system). The sterilizant leaves the culture vessel  706  through the injector  752  and travels through the gas line  750  to the connection  770 , into the common line  766 , through open valve  768 . It is stopped by the filter  760  and the valve  762 , which is closed. It then re-enters the culture vessel  706  through the sprayer  772 , forming an integrated sterilization system for in situ sterilization. Once sterilization is completed, the system is neutralized by the neutralizer. The neutralizer leaves the neutralizer pack  776  and travels through neutralizer line  788 , under pressure resulting from the pump  792  to the culture vessel  706  where it is sprayed into the culture vessel  706  with the sprayer  772  (the CIP system). The neutralizer leaves the culture vessel  706  through the injector  752  and travels through the gas line  750  to the connection  770 , into the common line  766 , through open valve  768 . It is stopped by the filter  760  and the valve  762 , which is closed. It then re-enters the culture vessel  706  through the sprayer  772 , forming a closed neutralization loop. 
     The culture vessel, generally referred to as  706 , is the same of that of  FIG. 7  (where the culture vessel is generally referred to as  600 ). The culture vessel is about 100 to about 100,000 liters, or about 250 to about 75,000 liters or 50,000 liters and all ranges therebetween. If algae or other plant material is to be cultured, at least the side walls  602  are transparent or light transmitting. The side wall  602  is preferably polycarbonate, but may be acrylic or glass. The lip of the wall  602  is formed into a flange  604  and has openings  606  to accept bolts  608  for affixing an airtight lid  610 . As the vessel is steam-cleaned, both the vessel  600  and the lid  610  are made of steam-resistant material, for example, but not limited to fiberglass or a heat resistant polyethylene such as Tyvar®. The lid  610  has an access port  612  for accepting a clean in place system (CIP), generally referred to as  416 . Gaskets  616  are located between the lid  610  and flange  604  and between a CIP flange  218  and the lid  610 . An optional thin plastic polymer shell  634  surrounds the vessel  600  and is equipped with light emitting diode grow lights  636 . An optional reflective surface  638  is located on an outer side of the shell  634 . The culture vessel  706  is provided with sensors for reporting culture conditions, for example, but not limited to each of a pH  640 , optical density  642 , temperature  644 , and pressure sensor  646 . Capacitance sensors  648  are located at a number of depths, for example, two located at ⅓ and ⅔ depth, three located at ¼, ½, ¾ depth or four located at ⅕, ⅖, ⅗ and ⅘ depth. 
     As shown in  FIG. 8 , the side wall  602  is formed into vertically disposed ridges  650  and valleys  652 . They may be rounded or sharp edged and may be wavy  651  about their vertical axis  653 . The vertical contours  654  may be, but are not limited to waves, or ridges and valleys, or peaks and troughs or are accordion-shaped, and are substantially vertical, for example, the vertical axis is normal to the floor, about 85 degrees relative to the floor, about 80 degrees relative to the floor or about 75 degrees relative to the floor. The vertical contours  654  function to increase the surface area of the side wall  602  and thereby increase light penetration in the feed culture vessel  600 . The peak to valley height of the contours  654  is about 1/16 of an inch to about 1 foot, or about 1 inch to about 6 inches or about 3 inches and all ranges therebetween. The distance between the peaks is about 1/16 of an inch to about 1 foot, or about 1 inch to about 6 inches, or about 3 inches, and all ranges therebetween. Additionally, the contours  654  preferably have small corrugations  655  to further increase the surface area. As shown in  FIG. 7  a bottom plate  656  retains the side wall  602  and has plate contours  658  that correspond to the contours  654  of the side wall  602 . Alternatively, the bottom plate  656  may have a contoured groove to accept the side wall  602 . 
     A cooling system provides air flow to the space between the feed culture vessel  600  and light emitting diode grow lights  636  (See  FIG. 8 ). This space is referred to as the air channel  662 . As shown in  FIG. 7 , a series of blowers or fans  664  forces air through the air channels  662 , which then exits from the bottom  672  of the air channels  662  (see  FIG. 7 ). 
     The bioreactor is controlled by the processor  758 . It receives and process data from the various sensors (pH, optical density, temperature, pressure), and coordinates the activity of the solenoids, pumps, cleaning, sterilizing, neutralizing, lighting and heating. If desired, the processor  758  can be made to interface wirelessly to a computer to allow remote monitoring and control. 
     The cleaner and alternative cleaner are shown in  FIGS. 5A and 5B . 
     A schematic of a fourth embodiment, generally referred to as  800  is shown in  FIG. 10 . The seed culture container  702  connects via the first culture line  704  to the culture vessel  706 . The seed culture container  702  is transiently attached to a first culture line  704 , which directly feeds the culture vessel  706 . The first culture line  704  enters the culture vessel  706  at an inlet  714 . 
     A first air line  710  has an air source  716  which may be a tank or ambient air. A pump  718  forces the air to a three way valve  725 , to a second air line  722  that branches from the first air line  710  at the three way valve  725 . The pump  718  is calibrated to produce a pressure of about 2 psi to about 15 psi. The second air line  722  has a fitting  726  downstream from the valve  725  for a user to attach a third air line. An air filter  732  is downstream from this. The second air line  722  enters the seed culture container  702  through a bung  734 . The first culture line  704  similarly has a fitting  736  for attaching a second culture line  738  that enters the seed culture container  702  through the bung  734 . When the valve  725  is open and the air lines  706 ,  722  are pressurized by the pump  718 , culture  740  is forced from the seed culture container  702  to the first culture line  704  that leads to the culture vessel  706 . The air lines  706 ,  722 , culture line  704 ,  738  and pump  718  are collectively referred to as the pressure driven transfer system. Alternatively, the transfer valve  22  described above could be employed. 
     A pressurized CO2 tank  744  provides CO2 to a CO2 line  746 . A regulator and digital pressure gauge  748  is located downstream from the CO2 tank  744  on the CO2 line  746  and a three way two position solenoid valve  749  is located downstream from the regulator and digital pressure gauge  748 . A processor  758  controls delivery of air and CO2 as needed by communicating with the valve  749 . A one way valve  761  is upstream from a 0.1 μm filter  760  on the gas line  750 . The CO2 line  746  joins the first air line  710  to form a gas line  750 . The gas line  750  enters a manifold  900  from which a delivery line  902  passes through a pump  804  to a sprayer, sparger or injector  772  located in the interior  754  of the culture vessel  706 . The sprayer  772  is preferably a rotary spray nozzle. The pump  804  is preferably a peristaltic pump or a shuttle pump, but may be a rotary pump, 
     A water line  762  for sea water has a two position solenoid valve  764  and an inline ultraviolet filter  763 . The water line  762 , nutrient lines  774  and sterilizant line  782  connect to form a common line  767  downstream of the valve  764  and upstream of the ultraviolet filter  763 . The common line  766  enters the culture vessel  706 . 
     The two nutrient lines  774  from nutrient packs  776  are each equipped with a pump  778 , which is preferably a peristaltic pump or shuttle pump, but may be a rotary pump. The nutrient lines  774  upstream from the peristaltic pump  778  are preferably disposable. The nutrient lines  774  enter the culture vessel  706  at an upper end  780 . A stir motor  777  is located below the nutrient packs  776  to keep the nutrients stirred. 
     A sterilizant line  782  from a sterilizer pack  784  is equipped with a pump  786 , which is preferably a peristaltic pump or shuttle pump, but may be a rotary pump. Similarly, a neutralizer or detoxifier line  788  from a neutralizer or detoxifier pack  790  is equipped with a pump  792 . The lines  782 ,  788  enter the culture vessel  706  at an upper end  794 . The liquid sterilizer pack  784  contains sterilizant that is used for sterilizing the bioreactor  700 . The sterilizant may be a weak sodium hypochlorite solution, for example, 1% in water. The neutralizer or detoxifier may be a de-chlorinator. 
     A 2-directional air filter  795  extends from the culture vessel  706  at an upper end  794  and functions as a pressure release valve. A common line  902  leaves the culture vessel  706  through an outlet  752  located at an aperture  802 . The common line  902  passes through the manifold  900  and a dump line  806  and a third culture line  800  splits off. Both have two way valves— 808  on the dump line  806  and  810  on the third culture line  800 . An outlet  814  terminates the third culture line  800 . 
     The culture vessel, generally referred to as  754 , is the same of that of  FIG. 7  (where the culture vessel is generally referred to as  600 ). The vessel  754  is equipped with light emitting diode grow lights  637  and banks of fluorescent lights  636 . An optional reflective surface  638  is located on an outer side of the shell  634 . The culture vessel  706  is provided with sensors for reporting culture conditions, for example, but not limited to each of an optical density  642 , temperature  644 , and pressure sensor  646 . Fans  906  are used to cool the air surrounding the vessel  754 . A cooling heat exchanger  16  as shown in  FIG. 1 , is used to cool the vessel  754 . 
     As shown in  FIG. 8 , the side wall  602  is formed into vertically disposed ridges  650  and valleys  652 . They may be rounded or sharp edged and may be wavy  651  about their vertical axis  653 . The vertical contours  654  may be, but are not limited to waves, or ridges and valleys, or peaks and troughs or are accordion-shaped, and are substantially vertical, for example, the vertical axis is normal to the floor, about 85 degrees relative to the floor, about 80 degrees relative to the floor or about 75 degrees relative to the floor. The vertical contours  654  function to increase the surface area of the side wall  602  and thereby increase light penetration in the feed culture vessel  600 . The peak to valley height of the contours  654  is about 1/16 of an inch to about 1 foot, or about 1 inch to about 6 inches or about 3 inches and all ranges therebetween. The distance between the peaks is about 1/16 of an inch to about 1 foot, or about 1 inch to about 6 inches, or about 3 inches, and all ranges therebetween. Additionally, the contours  654  preferably have small corrugations  655  to further increase the surface area. As shown in  FIG. 7  a bottom plate  656  retains the side wall  602  and has plate contours  658  that correspond to the contours  654  of the side wall  602 . Alternatively, the bottom plate  656  may have a contoured groove to accept the side wall  602 . 
     A cooling system provides air flow to the space between the feed culture vessel  600  and light emitting diode grow lights  636  (See  FIG. 8 ). This space is referred to as the air channel  662 . As shown in  FIG. 7 , a series of blowers or fans  664  forces air through the air channels  662 , which then exits from the bottom  672  of the air channels  662  (see  FIG. 7 ). 
     The bioreactor is controlled by the processor  758 . It receives and process data from the various sensors (pH, optical density, temperature, pressure), and coordinates the activity of the solenoids, pumps, cleaning, sterilizing, neutralizing, lighting and heating. If desired, the processor  758  can be made to interface wirelessly to a computer to allow remote monitoring and control. 
     Method: 
     The design of the bioreactor provides for a minimum downtime and maximum efficiency. As the vessel is emptied, both the vessel and the lines leading to it can be sterilized. Additionally, the entire bioreactor can be cleaned and sterilized. Once scale up has begun, the system is closed and remains closed until harvest, which is preferably in late log phase, but may be earlier or later. Initially, the feed culture vessel contains a small amount of culture medium. In this closed system (i.e. one that does not require open transfers), the volume of culture medium increases incrementally on a schedule, under control of the processor, hence contamination can be contained to a relatively small volume. Since less medium (a vector for contamination) is added at the beginning of the scale up, there is a smaller chance that contaminant organisms are added early on. This limits the amount of time that contaminants are multiplying in the system, and increases competition for resources, which on average will produce significantly less contaminated cultures. Culture medium used for cleaning the vessels may be dumped or retained to scale up the culture volume. Should contamination occur in the vessel the processor will detect the contamination, based on data from at least one sensor and will control emptying of the vessel. The vessel may additionally be cleaned by the processor signaling a cleaning step before the sterilizing cycle begins. Liquid sterilizant is fed through the bioreactor by means of a closed loop recirculating system. 
     All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential. 
     Advantages of the exemplary embodiments described herein may be realized and attained by means of the instrumentalities and combinations particularly pointed out in this written description. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims below. While example embodiments have been described in detail, the foregoing description is in all aspects illustrative and not restrictive. It is understood that numerous other modifications and variations can be devised without departing from the scope of the example embodiment. For example, a heat exchanger could be integrated into the cooling system, filtering of the water entering the integrated system can be done using inline filters on each integrated system, or using a larger rotating drum or rotary screen micron filter to filter water for a number of integrated systems. The pore sizes of the filters are approximate sizes, for example, a 0.1 μm filter may be about 0.05 μm to about 0.15 μm, a 1 μm filter may be about 0.5 to about 1.5 μm, a 50 μm filter may be about 25 μm to about 75 μm, and a 100 μm filter may be about 75 μm to about 150 μm, or about 75 μm to about 125 μm and all ranges therebetween. Filtration may be combined with other known methods to remove or kill contaminants, whether algae, plankton, or bacteria or may be replaced with other methods. UV filtering can be done using one large filter for numerous integrated systems, or in our case integrating an individual UV filter with each integrated system. As would be known to one skilled in the art, sterilization may be effected by sterilizants other than steam and therefore the steam generator and various lines may be replaced with chemical tanks, for example, but not limited to tanks of ethylene oxide or ozone. The bioreactor may be used for fresh water, salt water, brine, brackish water and any other liquid that can be used as the fluid in bioreactor cultures. Algal cultures include isochrysis, nannochloropsis, pavlova, tetraselmis, or any of the variety of industry standard species. Mixed culture includes a nannochloropsis to rotifer production system, or a nannochloropsis and isochrysis to rotifer production system. It can also be used as a fermenter. The nutrient packs may contain a carbohydrate source, such as glucose. Should contamination be a recurring problem, an additional vessel can be added to the system having a volume that is larger than the preceding vessel and smaller than the next vessel, in other words, having an incremental volume increase. While the described embodiments have one or two permanent vessels, a series of vessels ranging from three, to four, to five or more vessels is contemplated. A number of spargers may be employed to ensure proper mixing. This is especially relevant in the alternative embodiment when the depth of the valleys increases. As would be known to one skilled in the art, components described in one embodiment may be used in the other embodiments. The processor may be programmed to incrementally increase culture volume on a cell density based schedule or on a time schedule.