WO2008076998A1 - Closed system bioreactor - Google Patents

Abstract

A closed system bioreactor system includes at least one flexible tube capable of containing an aqueous medium. A thermal barrier is preferably within the tube and used to regulate the temperature of the aqueous medium. Rollers and movable septum clamps and actuators can be used to alternatively direct the aqueous medium to an upper chamber or a lower chamber of the tube, the chambers being separated by the thermal barrier, to thermally isolate or thermally expose the aqueous medium to the environment. Various parts in the system and the methods of their use, such as the thermal barrier, the rollers, the septum clamp that is used to raise and lower the thermal barrier, and an oxygen capture system of gas vents, are used to optimize the operation of the system.

Description

CLOSED SYSTEM BIOREACTOR

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional No. 60/875,294, filed December 15, 2006, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION 1. The Field of the Invention

The present invention relates to a closed system bioreactor apparatus, suitable for growing and/or harvesting a variety of aquatic organisms. 2. The Relevant Technology

Attempts have been made to culture a wide variety of aquatic organisms. Typically, the systems or apparatus used for aquaculture have been open systems, such as open ponds, pools or tanks. Such open systems have been used to culture everything from lobsters to oysters to algae. An advantage of open systems is that, in many cases, the organisms are cultured in a semi- natural environment, reducing operational costs such as feeding. For example, exposure of cultured marine organisms to seawater provides a ready source of food and/or other nutrients. For the same reason, open systems are subject to numerous problems. The presence of pathogenic micro-organisms or species that feed on the cultured organism may be difficult to control. Opportunistic species from the outside may compete with the cultured organisms for space, food or other resources. Open systems are difficult to insulate from environmental changes in temperature, salinity, turbidity, pH and other factors that may kill or reduce the growth or reproduction of the cultured organisms.

One group of organisms of recent interest for aquaculture efforts has been algae. The National Renewable Energy Laboratory (NREL) in Golden, Colo, operated a 10 year, $25 million Aquatic Species Program that focused on extracting biodiesel from high oil-producing species of algae. Before losing funding in 1996, the NREL scientists had demonstrated oil production rates 200 times greater per acre than achievable with fuel production from soybean farming (see, e.g., Sheehan et al., "A Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae," NREL Close-Out Report, NREL/TP-580-24190, 1998).

However, three fundamental problems limited the commercialization potential of aquaculture. First, petroleum-based oil prices were low in 1996 and hard to compete against. Second, the oil rich algae were difficult to protect from consumption or displacement by invading organisms as they were grown in ponds open to the environment. Third, algae best

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#233432 vl produce oil within a narrow temperature band, yet night sky radiation and low temperatures and high temperature days and excessive solar IR radiation interfered with NREL's open system pond experiments by wildly varying the cultivation temperature. While recent changes in petroleum prices have reduced or eliminated the first barrier, the latter two issues remain a concern with open bioreactor systems.

A need thus exists in the field for technologies and methods to address these issues and provide a competitively priced, biologically closed system, with better control of predators, competing species, temperature and other environmental factors than the open pond model.

BRIEF SUMMARY OF THE INVENTION The present invention is a closed system photo-bioreactor design that may be used to culture aquatic organisms that rely in whole or in part on exposure to sunlight, such as photosynthetic algae. It is designed to be preferably installed and operated in an outdoor environment where it is exposed to environmental light, temperature and weather. The herein disclosed bioreactor provides for improved thermal regulation and is therefore designed to maintain temperature within the range compatible with optimal productivity of aquatic organisms. Various features of the photo-bioreactor provide for improved capture of oxygen produced by the aquatic organisms and improved performance of the apparatus to efficiently keep aqueous material, and hence the aquatic organisms, where they are desired. In this way the apparatus also reduces problems from competing organisms, predatory organisms, pathogenic organisms and/or other extraneous species.

Another advantage of the apparatus is that it may be constructed and operated on land that is marginal or useless for cultivation of standard agricultural crops like corn, wheat, rice, canola or soybeans. The closed system bioreactor apparatus is scalable to any level of production desired, from a small backyard operation to one covering square miles of surface. The disclosed bioreactor technology stabilizes aquaculture medium temperature with low energy usage, practical on any scale. By solving the problems of temperature and invading species at an affordable cost and adding a few other technologies, the invention is useful as a system for creating a host of high value products from aquatic organisms that are largely fed by industrial, agricultural, and municipal waste products. In some embodiments, the apparatus may be used to produce an animal or human food source, for example by culturing edible algae such as Spirulina. In other embodiments, culture of photosynthetic algae may be used to support growth of a secondary food source, such as shrimp or other aquatic species that feed on algae. Methods of shrimp farming and aquaculture of other edible species are known in the art and may utilize well-characterized species such as Penaeus japonicus, Penaeus duorarum, Penaeus

#233432 vl aztecus, Penaeus setiferus, Penaeus occidentalis, Penaeus vannamei or other peneid shrimp. The skilled artisan will realize that this disclosure is not limiting and other edible aquatic species may be grown and harvested, utilizing photosynthetic algae to support growth of algae-consuming species. Embodiments of the invention can generally be implemented within a closed system photo-bioreactor that includes one or more or more flexible tubes capable of holding an aqueous medium and culturing the growth of aquatic organisms therein. The tubes haves open ends that connect to one or more control housings where nutrients are added, aquatic organisms are added or removed, and so forth. An aqueous medium is circulated through the tubes and to and from the control housings, preferably with the use of peristaltic rollers that substantially collapse the flexible tubes and force the aqueous medium in advance of its movement. Thermal control to optimize the growth environment is preferably accomplished at least in part with a thermal barrier inside the tubes that can be raised or lowered so that the aqueous medium is either exposed to or protected from the environment. Accordingly, a first example embodiment of the invention is a closed system bioreactor system. The system generally includes: at least one flexible tube capable of containing an aqueous medium, a thermal barrier within the at least one flexible tube to regulate the temperature of the aqueous medium, a first control housing operably coupled to the first open end of the at least one flexible tube and configured to circulate the aqueous medium to or from the upper chamber of the at least one flexible tube and to or from the lower chamber of the at least one flexible tube, and a flow control mechanism in mechanical association with the first control housing to direct the medium above or below the thermal barrier of the at least one flexible tube. The thermal barrier divides the at least one flexible tube into an upper chamber above the thermal barrier and a lower chamber below the thermal barrier, wherein the medium may be alternatively directed to the upper chamber or the lower chamber to thermally isolate or thermally expose the aqueous medium to the environment.

In this example the first control housing is operably coupled to the first open end of the at least one flexible tube and configured to help circulate the aqueous medium to or from the upper chamber of the at least one flexible tube and to or from the lower chamber of the at least one flexible tube. The flow control mechanism is in mechanical association with the first control housing and serves to direct the a flow of the aqueous medium above or below the thermal barrier. The flow control mechanism generally includes: a septum clamp connected to the first end of the thermal barrier; at least one actuator connected to the septum clamp and operable to move the septum clamp between an up position to facilitate a flow of the aqueous medium below

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#233432 vl the septum clamp and the thermal barrier and thus between the control housing and the lower chamber of the tube, or a down position to facilitate the flow of the aqueous medium above the septum clamp and the thermal barrier and thus between the control housing and the upper chamber of the tube; an upper valve member connected to the septum clamp for restricting the flow of the aqueous medium into the upper chamber of the tube when the septum clamp is in the up position but permitting a flow of the aqueous medium out of the upper chamber of the tube when the septum clamp is in the up position; and a lower valve member connected to the septum clamp for restricting the flow of the aqueous medium into the lower chamber of the tube when the septum clamp is in the down position but permitting a flow of the aqueous medium out of the lower chamber of the tube when the septum clamp is in the down position.

Another example embodiment of the invention is, in an aqueous bioreactor system using flexible tubes to contain aquatic organisms in an aqueous medium, a method for cleaning the flexible tubes. This method generally includes first providing a bioreactor that includes: first and second sections of flexible tube capable of containing an aqueous medium, each section of tube having a first open end and a section open end and an interior surface; a first control housing coupled to the first open end of the first section of flexible tube and the first open end of the second section of flexible tube. The first section of flexible tube, the second section of flexible tube, and the first control housing define an aqueous medium chamber; an aquatic organism containing aqueous medium in the aqueous medium chamber; a first peristaltic roller operably coupled to the first section of flexible tube to circulate the aqueous medium through the first section of flexible tube and scrub the surface of the tube to reduce biofilm on the tube surfaces; and a second peristaltic roller operably coupled to the second section of flexible tube to circulate the aqueous medium through the second section of flexible tube and scrub the surface of the tube to reduce biofilm on the tube surfaces. The method next involves moving the first peristaltic roller to a position on the first section of flexible tube distal the first control housing and moving the second peristaltic roller to a position on the second section of flexible tube distal the first control housing. Next, the rollers are simultaneously moved towards the first control housing to create aqueous medium pressure in the portion of the aqueous medium chamber between the peristaltic rollers, the increased pressure causing increased interior surface scrubbing of the first and second sections of flexible tube and increased particle re- suspension of the aquatic organisms within the first and second sections of flexible tubes.

Another example embodiment of the invention is a closed system bioreactor system that generally includes: a flexible tube capable of holding an aqueous medium, the at least one flexible tube having a first open end, a second open end, and an upper layer; a first control

#233432 vl housing operably coupled to the first open end of the at least one flexible tube; and a peristaltic roller configured for movement along the flexible tube towards and away from the first control housing to circulate the aqueous medium through the flexible tube and to remove gas bubbles from the flexible tube. The upper layer of the flexible tube has a gas venting section adjacent the first control housing, the gas venting section having at least one tube gas vent for release of gasses that accumulate as the peristaltic roller moves along the flexible tube towards the first control housing.

Yet another example embodiment of the invention is a closed system bioreactor system that includes a flexible tube having an upper layer and a lower layer, a first open end, and a second open end and being capable of holding an aqueous medium, the flexible tube having a portion extending from the first open end to the second open end that defines a length of the flexible tube. The system also includes a peristaltic roller having a roller axis perpendicular to the length of the flexible tube and being configured for movement along the length of the flexible tube between the first open end and the second open end to circulate the aqueous medium. A thermal barrier is within and extends along the length of the flexible tube, the thermal barrier having a substantially flat upper surface oriented towards the upper layer of the flexible tube, the thermal barrier dividing the flexible tube into an upper chamber located between the upper layer of the flexible tube and the thermal barrier and a lower chamber below the lower layer of the flexible tube and the thermal barrier. The aqueous medium may be routed to the upper chamber to thermally expose the aqueous medium to the environment or the lower chamber to thermally isolate the aqueous medium from the environment, the thermal barrier having a non-uniform structure along the length of the thermal barrier such that the thermal barrier has a flexibility that enables the flat upper surface of the thermal barrier to conform to the upper layer of the flexible tube as the peristaltic roller moves thereover. Another example embodiment of the invention is a closed system bioreactor system including: a flexible tube having an upper layer, a lower layer, a first open end, and a second open end and being capable of holding an aqueous medium, the portion of the flexible tube extending from the first open end to the second open end defining a length of the flexible tube; a peristaltic roller configured for movement along the length of the flexible tube to circulate the aqueous medium in the flexible tube; and a thermal barrier within the flexible tube, the thermal barrier dividing the flexible tube into an upper chamber located between upper layer of the flexible tube and the thermal barrier and a lower chamber below the lower layer of the flexible tube and the thermal barrier so the aqueous medium may be routed to the upper chamber or the lower chamber to thermally isolate or thermally expose the aqueous medium to the environment.

#233432 vl In this embodiment the thermal barrier includes: a substrate having a top surface and a bottom surface; and an insulative layer, the insulative layer having a plurality of discontinuities that increase the flexibility of the insulative layer.

These and other objects and features of the present invention will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

FIG. 1 illustrates a bioreactor system in which the invention may be practiced; FIG. 2 illustrates another bioreactor system in which the invention may be practiced;

FIG. 3 is a top view of a closed system bioreactor apparatus according to one example embodiment of the invention;

FIG. 4A is a side cross sectional view of a closed system bioreactor apparatus according to one embodiment of the invention; FIG. 4B is a side cross sectional view of part of the closed system bioreactor apparatus of

FIG. 4A according to one embodiment the invention;

FIG. 4C is another cross sectional side view of part of the closed system bioreactor apparatus of FIG. 4A according to one embodiment the invention;

FIG. 5 illustrates an end cross sectional view of a closed system bioreactor apparatus according to one embodiment the invention

FIG. 6 illustrates an end cross sectional view of a closed system bioreactor apparatus according to one embodiment the invention that includes thermal control;

FIG. 7 also illustrates in an end cross sectional view of a closed system bioreactor apparatus according to one embodiment the invention that includes thermal control; FIG. 8 illustrates a computer simulation of water temperature in a closed bioreactor with and without the use of a thermal barrier according to the invention;

FIG. 9 illustrates the exemplary transmittal profile of idealized material for the thermal barrier;

#233432 vl FIG. 10 illustrates the construction of a thermal barrier according to one embodiment of the invention;

FIG. 11 illustrates the construction of a thermal barrier according to another embodiment of the invention; FIG. 12 illustrates control of the flow of an aqueous medium in a closed system bioreactor apparatus in conjunction with the invention;

FIG. 13 illustrates control of the flow of an aqueous medium in a closed system bioreactor apparatus according to one embodiment of the invention;

FIG. 14 illustrates a one way valve for use in controlling the flow of an aqueous medium in a closed system bioreactor apparatus according to one embodiment of the invention;

FIG. 15 illustrates a Frenel pattern for use on the top surface of a tube for use with embodiments of the invention;

FIG. 16 illustrates a bioreactor apparatus controller system according to an example embodiment of the invention; FIG. 17 illustrates a top view of a bioreactor apparatus controller system according to an example embodiment of the invention;

FIG. 18 illustrates the movement of rollers in a bioreactor apparatus according to an example embodiment of the invention;

FIG. 19 also illustrates the movement of rollers in a bioreactor apparatus according to an example embodiment of the invention;

FIG. 20 also illustrates a gas bubble formed by the movement of rollers in a bioreactor apparatus according to an example embodiment of the invention;

FIG. 19 illustrates one method of removing gas bubbles from in a bioreactor apparatus according to an example embodiment of the invention; FIG. 20 illustrates a gas valve collar for use in a bioreactor apparatus according to an embodiment of the invention; and

FIG. 21 illustrates a gas vent for use in a bioreactor apparatus according to an example embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made to the drawings to describe various aspects of exemplary embodiments of the invention. It is to be understood that the drawings are diagrammatic and schematic representations of such exemplary embodiments, and are not limiting of the present invention, nor are they necessarily drawn to scale.

#233432 vl Terms that are not otherwise defined herein are used in accordance with their plain and ordinary meaning. As used herein, "a" or "an" may mean one or more than one of an item. As used herein, "about" means plus or minus ten percent. E.g., "about 100" refers to any number between 90 and 110. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be obvious, however, to one skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known aspects of closed system photo-bioreactors and of aquatic organisms such as algae have not been described in particular detail in order to avoid unnecessarily obscuring the present invention.

The methods, compositions, apparatus and system disclosed and claimed herein concern technology that supports large scale and low cost cultivation and harvesting of aquatic organisms. This technology may be used to support industrial manufacturing of the various products that different species of aquatic organisms can provide, such as biodiesel, methane, animal or human food, precursors for polymer production or other chemical products. This technology may be of use to economically support the massive cultivation and harvesting of aquatic organisms, such as algae. Unless specifically mentioned otherwise, it is understood that a reference to "algae" will include within its meaning other suitable aquatic organisms. The disclosed apparatus is generally referred to herein as a "bioreactor," "photo -bioreactor," "closed system bioreactor" and/or "bioreactor apparatus." Other machinery, apparatus and/or technologies of use with the bioreactor may include sterilization technology, CO₂ infusion technology, and/or extraction technology.

With reference now to FIG. 1, the bioreactor apparatus may be utilized as part of a comprehensive system for growing, collecting and utilizing aquatic organisms, such as algae. FIG. 1 illustrates an exemplary system schematic. Elements of the exemplary system include bioreactor technology, harvesting technology, sterilization technology, CO₂ infusion technology, extraction technology, and/or remote driven bioreactor technology. Further details regarding such technology is known to those skilled in the relevant art and their use in conjunction with embodiments of the present invention will be apparent in view of the disclosure herein. As illustrated in FIG. 1, the aquaculture operation may derive nutrients from animal feeding operations, such as pig manure. After processing and sterilization, such organic nutrients may be stored and/or added to the culture medium to support algal growth. Since photosynthetic aquatic organisms "fix" CO₂ for conversion into organic carbon compounds, a

#233432 vl CO₂ source, for example the gas exhaust from a power plant, may be utilized to add dissolved

CO₂ to the culture medium.

The CO₂ and nutrients may be utilized by algae to produce oil and other biological products. The algae may be harvested and the oil, protein, lipids, carbohydrates and other components extracted. Organic components not utilized for biodiesel production may be recycled into animal feed, fertilizer, nutrients for algal growth, as feedstock for methane generators, or other products. The extracted oil may be processed, for example by transesterification with low molecular weight alcohols, including but not limited to methanol, to produce glycerin, fatty acid esters and other products. The fatty acid esters may be utilized for production of biodiesel. As is well known in the art, transesterification may occur via batch or continuous flow processes and may utilize various catalysts, such as metal alcoholoates, metal hydrides, metal carbonates, metal acetates, various acids or alkalies, especially sodium alkoxide or hydroxide or potassium hydroxide.

The products of the closed bioreactor system are not limited, but may include Biodiesel, Jet fuels, Spark ignition fuels, Methane, Bio-polymers (plastic), Human food products, Animal feed, Pharmaceuticals products such as vitamins and medicines, Oxygen, Waste stream mitigation (product removal), Waste gas mitigation (e.g. sequestering CO₂).

FIG. 2 shows an aerial view in block diagram form of an exemplary closed bioreactor system for aquaculture. In this exemplary illustration, an algae crop is grown in substantially horizontal clear plastic tubes, laying flat on the ground, that have aqueous growing media moving through, thereby keeping the algae in suspension. In preferred embodiments, the tubes are thin- walled so as to be economical and are constrained by sidewalls to spread out on the ground until they are full of water about 8 to 12 inches thick. The width of the tubes may be nominally about 10 to 20 feet and the length approximately 100 to 600 feet. However, the skilled artisan will realize that such dimensions are not limiting and other lengths, widths and thicknesses may be utilized. In general, nutrients, proper salinity or mineral content, CO₂, and sunlight are present in the aqueous media. The media has been seeded with a desirable algae picked to provide a particular end product and grow well in the bioreactor and so it propagates and multiplies as long as the growing conditions are sufficient. The bioreactor illustrated in FIG. 2 is only one component of an overall system that feeds the bioreactor and harvests the aquatic organisms from it. FIG. 2 the Figure illustrates an exemplary layout of a relatively small farm, capable of producing 6000 gallons of biodiesel a day. The view shows 1400 individual bioreactors that are connected, like leaves on a fern, to central servicing rails. Note that various elements of the bioreactors and the overall system are

#233432 vl not illustrated for clarity in depicting this embodiment. Also note that the skilled artisan will realize that other configurations are possible, although in preferred embodiments a more or less linear tube arrangement containing the growing aquatic organisms is utilized.

FIG. 3 and FIGS. 4A-4C show a non-limiting example of a closed system bioreactor apparatus 100. An aqueous medium 102 including aquatic organisms is contained in substantially transparent flexible tubes 104. The liquid contents of the tube may be circulated by movable rollers 106 that roll across the surface of the tube and substantially collapse it, pushing liquid in front of them.

In this non-limiting example the rollers 106 track along a roller support rail 107 and are driven by cables attached to carriages that roll on the top of the rail. A roller drive system shown in FIG. 16 provides a motive force for roller movement. In an alternative embodiment not shown here, when the rollers reach the end of the tube, they may be rotated or lifted upwards to travel back to the starting point in a continuous oval path. However, in the preferred embodiment shown, bidirectional rollers 106 are used that travel from one end of a tube 104 to the other and then reverse direction to return to the starting point. The use of a roller system provides liquid circulation while generating low hydrodynamic shear force, in contrast to standard mechanical pumps for fluid movement.

FIG. 3 shows an exemplary two tube system, each tube operably coupled to a roller. The tubes are joined at the ends by control housings 108, 110, which can hold CO₂ bubblers, a whirlpool device, various sensors (e.g., pH, dissolved O₂, conductivity, temperature), actuators for moving the thermal barrier, and connections to pipes for transport of water, nutrients and/or harvested aquatic organisms, such as algae.

As indicated in the side view in FIG. 4A, in a bidirectional roller apparatus 100 the tubes 104 may be laid out along the ground 120, with the rollers 106 moving substantially parallel to the ground surface. However, at the ends of the tubes 104, the ground 120 under the tube 104 may be excavated to form a dip 122, which may be lined with a "belly pan." This arrangement allows water in the tubes 104 to flow under the rollers 106 when the rollers 106 reach the ends of the tubes 104 and position over the belly pans. After water flow has slowed sufficiently, the rollers 106 may reverse direction and travel back to their starting position, resulting in an alternating clockwise and counterclockwise flow of water through the apparatus 100.

The rollers 106 form a kind of peristaltic pump but differ in two respects. First, the peristaltic filling force is provided by the leveling action of gravity on the fluid rather than the elastic return that is seen in many pumps. Second, the rollers 106 preferably only squeeze the tubes down about 85% rather than completely. This means the fluid pressure differential from

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#233432 vl front to back of the roller causes a relatively high speed reverse flow right under the roller, as discussed below. In some embodiments, the roller speed (and accordingly the fluid velocity) may be approximately 1 foot/sec. More generally, preferred roller speeds range from about 0.5 feet/sec to about 2 feet/sec. In various embodiments, the aqueous medium may be used to culture photosynthetic organisms, such as algae. During photosynthesis, the algae absorb CO₂ and release oxygen gas. As the roller 106 moves along the upper surface of the tube 104, oxygen, other gases, fluid medium and algae are pushed ahead of the roller. This not only moves the algae through the tube but also provides a mixing action for the medium. The rollers 106 may push a gas pocket 130 in front of them. This is a combination of gases released from the water, un-absorbed CO₂, and oxygen generated by photosynthetic algae. The gas pocket 130 in front of the rollers 106 may be collected in end chambers and vented to the atmosphere or stored, to avoid oxygen inhibition of photosynthesis. In some embodiments stored oxygen may be reinjected into the apparatus at night to support algal metabolism during non-photosynthetic periods. Alternatively the collected oxygen may be piped to a power plant to increase the efficiency of its combustion processes. The rollers 106 may also cause optical turnover of algae, which is desired to modulate its light input. Otherwise algae either becomes over- saturated with light or starved of light causing the algae to not no thrive and the operation is slowed down or stopped altogether. As illustrated in FIG. 4A-C, the roller 106 preferably does not reach all the way to the bottom of the tube 104. This results in a high velocity backwash, immediately under the roller 106, where the force applied to the liquid in front of the roller results in fluid movement backwards under the roller 106. This backwash has several effects, including scrubbing the bottom surface of the tube to reduce biofouling and resuspending algae or other aquatic organisms that have settled to the bottom of the tube in the medium. A thermal barrier 124 may be included within the tube 104, separating the liquid into either upper chamber 126 or lower chamber 128 for thermal control. Depending on how fluid movement is regulated, the liquid may be diverted primarily into the upper chamber 126 of the tube above the thermal barrier (FIG. 4C) or into the lower chamber 128 of the tube below the thermal barrier (FIG. 4B). FIG. 4A shows the rollers 106 in two alternative positions to illustrate the thermal barrier control. When the liquid is in the upper chamber 126, the collected gas pocket 130 is forced against the upper layer 132 of the flexible tube 104 (FIG. 4C). The moving air- water interface in front of the roller then acts to scrub the inside surface of the upper layer 132, reducing biofouling and maintaining light transmission of the upper layer 132.

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#233432 vl When the liquid is in the lower chamber 128 (FIG. 4B) the underside of the thermal barrier 124 is scrubbed in the same manner to maintain light transmission through it.

This scrubbing action may be enhanced by the inclusion of slightly buoyant scrubber disks 1 inch diameter by 1/4 inch thick that are deliberately circulated in the fluid and that tend to be pushed ahead of the roller. Other solid shapes of similar size may be designed by those skilled in the art of scrubbing the inside of fluid systems. In practice, thousand of these disks or other solid shapes might be resident in the bioreactor but not so many as to reduce the light transmission appreciably. They would be separated from the algae mixture with screens before harvesting and would be of sufficiently low buoyancy that they could be washed into the air bubble space ahead of a roller by the prevailing fluid current caused by the previous roller. As shown in FIGS and 4A, control housings 108, 110 may be incorporated into the apparatus, for example at the ends of the tube 104, and include mechanisms to harvest aquatic organisms, add or remove gases, nutrients and/or waste products or for other purposes. In a preferred embodiment, the hydrodynamic fluid movement at the ends of the tubes 104 may be designed to promote formation of standing whirlpool circulation, as described in greater details in U.S. Patent Publication No. 2007/0048848, published Mar. 1, 2007, which is incorporated herein by reference in its entirety. A whirlpool may be utilized to improve efficiency of aquatic organism harvesting, gas and/or nutrient introduction, waste removal, or for other purposes. The control housing 110 in FIG. 4A houses an example whirlpool device for harvesting aquatic organisms.

The illustrative embodiment in FIGS. 3 and 4A-C shows a research model that is only 65 feet long, with individual bioreactor tubes that are 52 inches wide. In one preferred production scale embodiment each of the two tubes would be about from about 100 to 500 feet long, more preferably from about 200 to about 400 feet long, still more preferably 300 feet long and about 10 to 20 feet wide. For example, an apparatus having two 300 ft. tubes would have a total photosynthesis area of 0.15 to 0.30 acre per bioreactor assembly. Each such bioreactor should grow about 7 to 14 gallons of biodiesel per day or more.

In some embodiments, a single tube 104 may be formed to contain an upper chamber 126, an internal thermal barrier 124, and a lower chamber 128 as shown in FIGS. 4A to 4C. In alternative embodiments disclosed in FIG. 5, a dual top/bottom tube system 150 may be utilized with separate upper tube 152 and lower tube 154 and a thermal barrier 156 in between. In operation, such a system would behave similarly to the single tube system discussed above. The advantage of the dual tube system is that it potentially eliminates the need for sealed side seams, providing greater structural stability and decreasing costs. Further, since the high emissivity

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#233432 vl layer and insulator (discussed below) do not need to be waterproof, there are additional options for selection of materials. Also, since the thermal barrier layer 156 is not exposed to the aquatic organisms, it eliminates the possibility of biofouling of that material. Finally, the thermal barrier 156 and high emissivity layer 157 may be retained when the tubes are replaced, providing additional cost savings.

FIG. 5 also shows an additional optional feature of the invention which is a ground smoothing layer 158, such as fly ash, deposited between the lower tube 154 and the ground 160. The ground soothing layer 158 may be used with either a one-tube or two-tube system. Fly ash is a low cost material that may be obtained in the local of power plants and one that has a sufficient caustic nature as to retard the growth of plants under the bioreactor tubes. Other materials including salt may be placed under the tubes to retard growth. A netting 162 over the top tube is optional.

Technology for preventing or delaying biofouling of the inner tube plastic layers by adhering aquatic organisms is important. If the tubes need to be replaced too often then it becomes an economic drain on the operation. There are a number of approaches to preventing biofouling under development worldwide, although nano-textured hydrophobic surfaces that are very pointy on a nano scale are one possibility. One exemplary way to make a non-fouling inner surface for the bioreactors at very low cost is to use flocking technology to electrostatically embed the ends of polyethylene fibers that are approximately 1-2 microns diameter by 10-20 microns long into the soft, still cooling, polyethylene plastic blown film "bubble" just as it leaves the blown film annular nozzle. Alternatively, a tacky or curable adhesive coating may be applied to the inside of the tube 104 or to one side of a sheet of plastic film used for tube construction prior to the flocking of the fibers and exposure to fluorine gas.

The inner flocked surface on the inside of the bubble may be made hydrophobic by having the inside of the bubble pressurized with fluorine gas (rather than air), which reacts with the polyethylene to create a thin skin of hydrophobic polyfluoroethylene (which is similar to polytetrafluoroethylene, PTFE) on both the flock fiber's surface as well as the plastic film between the fiber bases.

In certain embodiments, the tube may be made completely black on at least one side of the two tube system. When an aquatic organism goes into the darkness it consumes oxygen and when in the light it produces oxygen. There may be an oil productivity advantage if even during the day the algae mixture is channeled alternately through light and through darkness on some selectable duty cycle so as to consume some of the dissolved oxygen in the fluid and stimulate the energy converting photosynthesis reactions.

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#233432 vl Everything that goes into the bioreactors is preferably sterile except for the desired seed culture of the microorganism. In order to do this inexpensively on an industrial basis we may utilize a continuous flow autoclave. This may be done not only for the nutrients but also for any liquid returned to the bioreactors. Gases like air going into the bioreactors can be HEPA filtered and smokestack gases can be assumed to be sterile from the power plant heat. Return fluids which are optically clear may be sterilized using UV light technology.

As previously mentioned, in the exemplary embodiment of FIGS. 4A-4C, as well in the non-limiting example of FIGS. 6 and 7, the tube 104 in a preferred configuration has a construction that includes a insulating septum or thermal barrier 124 installed horizontally down the center. FIGS. 4A-4C show partial cross section side views while FIGS. 6 and 7 show a cross section of one flexible tube looking through it lengthwise. The thermal barrier 124 divides the tube 104 into an upper chamber 126 and the lower chamber 128 so that liquid in the upper chamber 126 is relatively exposed to the environment and liquid in the lower chamber 128 is relatively insulated from the environment. The purpose of thermal control is to keep aquatic organisms in the medium at their optimum temperature and prevent the tubes from freezing at sub-zero ambient temperatures, or from overheating during hot summer days. The thermal control aspects involve use of different tube components with selected optical and/or thermal transmittance properties.

In embodiments employing a thermal barrier within the tubes, the aqueous medium may be directed either above or below the thermal barrier. Under conditions of low temperature, the liquid may be directed above the thermal barrier, where it is exposed to increased solar irradiation including the infrared wavelengths, resulting in temperature increase. Under high temperature conditions, the liquid may be directed below the thermal barrier, where it is partially shielded from solar irradiation and simultaneously may lose heat by contact with the underlying ground layer. In still other embodiments, the ground underpaying the closed bioreactor may be used as a heat sink and/or heat source, storing heat during the day and releasing it at night.

When the thermal barrier is up (at the top of the tube), the liquid in the tubes is isolated from both radiative and conductive heat transfer to the outside environment. However, it is in intimate thermal contact with the ground underneath. When the thermal barrier is down the liquid may easily gain or lose heat to the environment via both radiation and conduction. In effect, the thermal barrier acts as a thermal switch that can be used to take advantage of opportune environmental conditions like night, day, rain, clouds, etc. to gain or shed heat to control the temperature of the fluid. The ground beneath the apparatus has thermal mass whose temperature can also be modulated by close thermal contact when the thermal barrier is in the up

14

#233432 vl position. The heat energy in this thermal mass may be used to further control the temperature of the fluid. If a cold night is anticipated, the fluid can be allowed to warm during the day with the thermal barrier in the down position to slightly above optimum temperature. Shift of the thermal barrier to the up position transfers this positive heat energy to the ground thermal mass. Several cycles of fluid warming and ground heating may occur. The heat transferred into the ground thermal mass may then be transferred back to the liquid during a cold night by keeping the thermal barrier is in the up position, to stabilize the water temperature in an optimal range.

Alternatively, when an excessively hot day is anticipated, the barrier may be placed in the down position at night until the mixture is slightly below the optimum temperature and then shifted to the upper position, where the cooled water is in contact with the ground, to pump down the temperature of the ground. This cycle may be repeated several times during the night. As the ensuing day heats up, the thermal barrier is raised, thereby connecting the fluid thermally to the ground to lengthen the time that the fluid stays at an acceptably low temperature. The thermal control mechanism discussed above is highly effective at maintaining temperatures in a range for optimal algal growth.

FIG. 8 shows computer modeled water temperature data, using the environmental conditions at Fort Collins, Colo, between January and June, 2006, with an R-4 (1 inch thick foam) thermal barrier and an ideal infrared absorption layer (see FIG. 9). The water temperature ranges are modeled with and without the presence of a thermal barrier as noted in the legend. It can be seen that Spring and Summer temperatures were largely stabilized in the range of 20° to 30° C with the thermal barrier, whereas in the absence of the thermal barrier the summer water temperature reaches 45⁰C or higher. The thermal barrier decreases maximum summer temperature by about 10° C.

The barrier is less effective at maintaining winter water temperature in the optimum range. Various alternatives are available for winter aquatic organism production, such as use of heat from supplemental sources (e.g., power plant exhaust), location of production units in warmer climates where winter temperature is not as cold, or use of cold-tolerant algal species such as Haematococcus sp. Thus, in alternative embodiments, active thermal control with power plant water may be utilized. Heated water from a power plant's cooling towers may be pumped to a plastic mat placed under part of the bioreactor tubing. When it is cold this additional heat source may be utilized to prevent freezing and/or below optimum algal growth temperatures. The skilled artisan will realize that a variety of heat sources may be utilized, such as power plant exhaust, geothermal heat, stored solar heat or other alternatives. Additionally in hot seasons or

15

#233432 vl locations of high solar flux, evaporative or other cooling systems that can be efficiently powered can be used to keep the algae from overheating.

The tube is preferably formed of a durable material that may allow light in and heat in or out. For example, a top sheet of 0.01 inch thick clear polyethylene allows light in and heat in or out. The tube also comprises a bottom sheet that is normally, but not necessarily, identical in composition to the top sheet. The tube may be formed by side sealing two sheets (upper and lower) or three sheets (upper, thermal barrier, and lower) of flexible plastic, although other mechanisms may be utilized, such as providing a seamless tube by continuous extrusion or blowing of a cylindrical sheet of plastic. A ground sheet that is resistant to physical/mechanical disruption but is heat conductive may be placed between the ground and the tube. The ground may be treated or prepared to be relatively flat, smooth, heat conductive and plant resistant. Side walls may be provided to physically support the fluid-filled tube and/or provide additional thermal insulation from the sides of the tube and additionally to support and guide the roller carriages. The tubes and part or all of the thermal barrier may be constructed of a variety of materials, such as polyethylene, polypropylene, polyurethane, polycarbonate, polyvinylpyrrolidone, polyvinylchloride, polystyrene, poly(ethylene terephthalate), poly(ethylene naphthalate), poly(l,4-cyclohexane dimethylene terephthalate), polyolefin, polybutylene, polyacrylate and polyvinlyidene chloride. In embodiments involving culture of photosynthetic algae or organisms that are fed on algae, the material(s) of the thermal barrier preferably exhibits a transmission of visible light in the red and blue wavelengths of at least 50%, preferably over 60%, more preferably over 75%, more preferably over 90%, more preferably over 90%, most preferably about 100%. In other preferred embodiments, the material used for the top surface of the tubes exhibits a transmission of visible light of at least 90%, more preferably over 95%, more preferably over 98%, most preferably about 100%.

In the most preferred embodiments, polyethylene is used for the tube. Polyethylene transmits both long- wave black body radiation and red and blue visible light, allowing the temperature control system to radiate the inner heat of the water to the night sky and allowing algae or other photosynthetic species to receive visible light, whether the medium is above or below the thermal barrier. Polyethylene exhibits increased transmittance of long wave infrared light associated with room temperature blackbody radiation, in comparison to certain alternative types of plastic. In various embodiments, thin layers of UV blocking materials may be applied to the surface of the tubes to reduce UV-degradation of the plastic. In other embodiments,

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#233432 vl fluorescent dyes that convert infrared (IR) or ultraviolet (UV) light to the visible (photosynthetic) light spectrum may be incorporated into the tube to increase efficiency of solar energy capture by photosynthetic organisms. Such dyes are known in the art, for example for coating the glass or plastic surfaces of greenhouses, or in fluorescent lighting systems that convert UV to visible light wavelengths. (See, e.g., Hemming et al., 2006, Eur. J. Hort. Sci. 71(3); Hemming et al., in International Conference on Sustainable Greenhouse Systems, (Straten et al., eds.) 2005.)

The internal thermal barrier may comprise a single flexible sheet that is designed to absorb infrared but pass visible light for photosynthesis that overlays a conductive insulator. In some embodiments, the thermal barrier may be a composite comprising different materials and constructions configured to match the desired result. For example, the thermal barrier may be a flexible insulator sheet bonded to an IR absorbing substrate. In this embodiment the flexible insulator may be, for example, a 1/2 inch or 1 inch thick layer of low density poly foam (e.g., foamed polyethylene). In lieu of a thicker substrate a thin (e.g., 0.0035 inch) facing can be added to the top and/or bottom surface to decrease algal attachment to the thermal barrier. The thermal barrier 124 preferably includes a high emissivity material. Polyethylene, however, has a low emissivity and so other materials are preferably used. In some embodiments, the emissivity properties of the thermal barrier may be adjusted by incorporation of other materials of selected optical characteristics. For example, quartz sand from specific sources may have desirable optical properties and could be embedded within the upper surface of the thermal barrier. Alternatively, doped glass or quartz beads or ceramic tiles of selected optical properties might be embedded within the upper surface of the thermal barrier. Unfortunately, the use of glass or quartz makes the thermal barrier more difficult to dispose of after use as it cannot be easily incinerated or recycled.

Unlike polyethylene, polyester has a relatively high emissivity. Polyester, however lacks the durability or flexibility that make polyethylene so desirable for use in the tube and thermal barrier construction. Similarly, thick foamed polyethylene layers also lack the flexibility desired in the thermal barrier. The importance of flexibility in the thermal barrier in large part relates to the its ability to conform to the upper tube layer and to the rollers. When the thermal barrier 124 is in the upper position, an inflexible thermal barrier would likely not conform to the shape of the rollers or upper tube layer, increasing the likelihood that some aqueous medium and associated aquatic organisms would be trapped in pockets between the upper surface of the thermal barrier and the lower surface of the tube's lower surface. In such cases the aquatic organisms would typically overheat during the day or freeze at night, in either casing dying and becoming undesired food that would promote the growth of excess bacteria in the system.

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#233432 vl In one non limiting embodiment preferred thermal barrier designs overcome the above challenges by using a non-uniform structure of one or more materials to increase flexibility. Because uniform thermal protection or high emissivity are not required for the thermal barrier, well insulating layers can be separated by less well insulating layers that are more flexible. Durability and high emissivity can be enhanced without losing flexibility with selective use of polyethylene and polyester. In a first example embodiment a thick insulation layer, for example foamed polyethylene, is segmented, stripped, or partially scored to increase its flexibility at the resultant thinner areas. The foamed polyethylene is preferably connected to a thin polyethylene and/or polyester substrate to increase its strength and/or emissivity, respectively, and to provide a good surface for smooth matching with the overlying tube upper layer.

With reference now to FIG. 10, a particularly preferred thermal barrier according to the invention includes a plurality of thermal islands 200 connected to a flexible substrate 202, for example a thin polyethylene layer. Each thermal island includes one or more layers, in the depicted embodiment the islands include a thin polyester layer 204, e.g. about .01" in thickness, and a thick insulative layer 206, e.g. from about .5" to about 1". Thus, the polyethylene layer maintains waterproofing and strength for the layer while remaining flexible, the less flexible polyester can be used to provide high emissivity by being formed as discontinuous sections and the foamed polyethylene can also have increased flexibility by being formed as discontinuous sections. The thermal islands can take various shapes, for example close fitting hexagonal or square blocks bonded to the substrate in a close pattern. In one embodiment the thermal islands preferably are about 4" wide by 4" long. In another example the thermal islands could be narrow, e.g. 2 to 6 inch wide strips that extend along the width of the tube, thus providing a discontinuity that runs parallel to the roller axis and thereby maximizing flexibility and conformity to the upper layer under the roller's pressure. In either case, the thermal islands are preferably separated by a gap of about .125" to about .25" between blocks. In another embodiment the thermal islands are in fact not separate structures, but are in fact a single structure that has had discontinuities cut or otherwise formed therein to separate many adjacent sections of the thermal barrier to increase flexibility. Non limiting examples of other high E (emissivity) materials that could be used in a thermal island include Teflon, ceramic and glass.

Referring to FIG. 11, another example of a varying thickness structure for the thermal barrier is a substrate 210 of polyester and/or polyethylene with a "bubble- wrap" style closed cell air insulative layer 212 adhered thereto as the insulative layer.

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#233432 vl With reference now to FIGS. 12-13, a preferred closed system bioreactor is constructed utilizing a roller design that allows for reversal of the roller direction and does not require a mechanism for lifting the roller above the housings at the ends of the tubes. In a preferred embodiment the ground or other surface is flat and level for almost the entire track length, while at the two ends immediately adjacent to the housings there is a small trenched dip that runs the width of the track. This trench is preferably lined with a metal "belly pan" which serves to define the shape of the trench and to prevent soil from entering the bypass area. The trench and belly pan are designed to allow the fluid medium in the tubes to flow under the level of the roller. Because of hydrostatic pressure, the flexible tubes conform to the ground level and belly pan surface. When the rollers reached the ends of the track, roller movement is stopped by the drive system. The liquid medium is allowed to flow under the rollers into the chambers without resistance from the roller, which is elevated above the liquid flow. This continued flow may be due to inertial momentum or due to the movement of the opposite roller. Due to frictional forces against the thermal barrier, sides of the tubes and components of the chambers, the fluid eventually slows and stops. When fluid flow has reached a sufficiently low velocity, the roller drive can be engaged again and the roller moves in the opposite direction. When the first roller stops over the area of the trench, the second roller engages the fluid in the tube again and pushes it in the opposite direction, reversing the flow of algae through the system.

FIGS. 12-14 also show the actuators for diverting water above or below the thermal barrier. As shown, the end of the thermal barrier 124 is connected to a bar or septum clamp 244 that is attached to one or more actuator rods. When the actuators are in the up position, the septum diverted water below the thermal barrier 124 and the barrier floated to the top of the tube. When the actuator is in the down position, fluid is diverted above the thermal barrier 124, which then sits at the bottom of the tube. In one embodiment the septum clamp 244 is constructed with a one-way valve, permitting fluid or gas flow out of the upper or lower tube even when the thermal barrier 124 is clamped to prevent fluid entry. This permits the roller 106 to squeeze out residual fluid or gas from a chamber regardless of septum valve position. For example, in FIG. 4B the left hand roller 106 appears to be rolling the fluid in the bottom of the tube, below the thermal barrier 124, out into the left hand chamber. After that fluid recirculates back around to the right side, where the septum claim and the thermal barrier 124 are in the down position, it is channeled above the thermal barrier 124, allowing the fluid to fill the upper chamber of the tube. This is an example of how the thermal barrier 124 position can cause the movement of fluid between the upper and

19

#233432 vl lower parts of the tube without much energy usage. The purpose of this movement is thermal control of the fluid.

The septum clamp 244 may be driven up or down by a 4-bar linkage driven by 2 position feedback electro-hydraulic actuators connected by wires to the system controller. Many other actuator systems including common pneumatic linear actuators are also suitable for moving the thermal barrier 124 up and down.

When the rollers and septum valves are set to sink or maintain sunk the buoyant thermal layer, then leakage, or incomplete initial purging, can result in excess un-purged water volume that can leak back under the rollers and allow the thermal barrier 124 to partially float up. Accordingly, in one embodiment the one way valve is a flexible sealing lip that serves to prevent backflow into the closed chamber. In another embodiment the septum clamp 244 includes flexible plastic members such as sheets 240 that are pulled in by the backflow suction of any fluid that tries to enter a closed chamber and thus close over the space between the septum clamp 244, or flexible sealing lip, should any back flow commence. By making the septum clamp valves one-way valves that only exit water from the sealed off chamber, then every back-and-forth movement of the roller will express out a little more water from under the thermal barrier 124.

In various embodiments, the top surface of the tube may be patterned to maximize light absorption for photosynthesis during the winter months, particularly at higher latitudes. An exemplary Frenel pattern is shown in FIG. 15, which illustrates a cross-section of the tube's top layer, with Frenel light gathering prisms that are oriented east- west with the angled face pointed towards the equator. The overall thickness is 0.025 inches and the Frenel pattern is created during the plastic blowing process or during a post rolling process.

FIG. 16 shows a preferred roller drive system. The rollers may be thin and lightweight tubes, for example of fiber glass and fiber construction. Alternatively, the rollers may be stainless steel or other heavy cylinders. In either case they must be heavy enough to compensate for the volume of water they displace underneath themselves. In most cases this will be achieved by manufacturing a thin light weight cylinder that can be inexpensively manufactured and transported and then filling it with sufficient water, or low friction other material to give it the proper weight after installation. The rollers may comprise a solid axle between two support roller assemblies or they may roll on bearings arranged on a tube axle running through them. In a preferred version the roller carriages are either independently driven on each side or there is a driven differential mechanism between the carriages holding each end of the roller. This is because the roller perpendicularity to the drive direction is critical to prevent bunching or

20

#233432 vl wrinkling of the tube assemblies. Sensors may detect when one side of a roller is getting ahead of the other or when cross track stress is being put on the tubes and adjust the phasing of the drive from one side to the other so that the rollers smoothly track over the tubes with out causing damage or incurring excess friction. The kinematic design of the roller carriage system in FIG. 16 permits it to compensate for large misalignments and temperature changes.

Ten to twenty foot long rollers must be accurately driven, against a background of reflected waves, misalignments, temperature differences, and varying friction in order to avoid skewing of the roller and diagonal wrinkling of the tube. In certain embodiments, the rollers may weigh thousands of pounds and may move along a track that can be 300 feet or greater in length. The exemplary system shown in FIG. 16 utilizes a steel drive cable system, which is low cost and has low driveline inertia because the cable transmits force through tensile strength, which is very mass efficient. In this embodiment, nested, high bandwith velocity servos are used to drive the drive pulleys and keep the rollers from skewing.

The velocity command of the upper master servo is derived from the controller by determining the difference between where the roller is and where it should be. By limiting the first and second derivatives of the resultant velocity command, the unstable water filled bioreactor tubes are minimally excited. Wave action oscillation from any source is not magnified and does not induce out-of-phase feedback signals due to drivetrain compliance, because the velocity feedback sensors being directly attached to the drive motors are isolated from compliant elements. The bottom servo is slaved to match the same velocity as the upper main servo but with enhanced velocity following due to the dV/dt lead feed-forward network in its command. The slave velocity command is summed and offset by the skew strain sensor outputs on the kinematic carriage system. This actively drives the roller to a precise angular alignment referenced to the alignment rail. The exact angle of skew can be adjusted by the controller to compensate for roller directionally unique effects or to relieve detected wrinkle formation in the bioreactors. The controller can also use the fore-aft roller hydrostatic pressure difference sensed by the film (bioreactor tube) level sensors to control the roller velocity in order to maintain a specific pressure head. Battery or solar powered skew and level sensors with RF telemetry output require no power wires to be hooked to the roller. The carrage system is of kinematic mechanical design. This provides that changes in width between the roller rails or roller length changes due to expansion do not bind the carrage system. It also means that the roller perpendicularity is constrained by only one carriage end and therefore can accurately be measured by sensors on that end and the result used to differentially control the drive systems velocity on each end so as to zero out accumulated skew.

21

#233432 vl Collection systems, such as sippers, may be arranged to siphon concentrated suspensions of aquatic organisms out of the system. In a more preferred embodiment, the hydrodynamic flow through the bioreactor is designed to produce a "whirlpool" effect, for example in a chamber at one end of the tubes. The whirlpool may be used to concentrate aquatic organisms such as algae within the liquid medium, allowing more efficient harvesting, or to remove undesired byproducts of metabolism like dead cells and mucilage containing bacteria. Other mechanisms for adding nutrients and/or removing waste products from the closed bioreactor may also be provided. One or more sipper tubes may be operably coupled to the whirlpool system to increase efficiency of harvesting from and/or nutrient input to the apparatus. An exemplary harvesting whirlpool of alternative design is illustrated at the right side of

FIG. 3 and described in further detail in U.S. Patent Publication No. 2007/0048848, published Mar. 1, 2007, which is incorporated herein by reference in its entirety. Although preferred embodiments of a bioreactor include such a whirlpool device, the apparatus is not so limited and in alternative embodiments other methods and devices for harvesting aquatic organisms from the medium may be utilized. The primary purpose of the whirlpool is to permit extraction of fluid which is enhanced with algae (or other aquatic organisms) containing a desired product. A secondary purpose may be to extract components of the fluid that need to be removed from the medium, like mucilage or foam that may primarily consist of deleterious bacteria.

There are numerous potential uses for a density separating whirlpool, corresponding to the many different product types that may be grown in a photo-bioreactor. Algae of different species and in different environmental circumstances or life stages may be either heaver or lighter than the fluid medium, depending upon their concentration of oil, carbohydrates, and gas vacuoles, as well as the growing media that can have various densities depending on salt content and temperature. Aquatic organisms other than algae may also be separated from the liquid via density differences in this manner.

Another purpose of the whirlpool may be to serve as an alternative CO₂ injection mechanism. In the case of a whirlpool, this would preferably happen on the bottom of the whirlpool where the fluid is spinning outward after leaving the control orifice. Gases like pure CO₂, or alternatively CO₂ rich flue gases obtained from a power plant, factory or other source, may be injected to the whirlpool.

It may be possible for the bioreactor to acquire CO₂ directly from the air to the flexible tube portion of the apparatus as well.

Various alternatives exist to separate aquatic organisms from the medium and the claimed methods and apparatus are not limited to the exemplary whirlpool and sipper tubes

22

#233432 vl discussed above. In one alternative embodiment, industrial scale commercial centrifuges of large volume capacity may be used to supplement or in place of other separation methods. Such centrifuges may be obtained from known commercial sources (e.g., Cimbria Sket or IBG Monforts, Germany; Alfa Laval A/S, Denmark). Centrifugation, sedimentation and/or filtering may also be of use to purify oil from other algal components. Separation of algae from the aqueous medium may be facilitated by addition of flocculants, such as clay (e.g., particle size less than 2 microns), aluminum sulfate or polyacrylamide. In the presence of flocculants, algae may be separated by simple gravitational settling, or may be more easily separated by centrifugation. Flocculent-based separation of aquatic organisms is disclosed, for example, in U.S. Patent Appl. Publ. No. 20020079270, incorporated herein by reference.

The skilled artisan will realize that any method known in the art for separating cells, such as algae, from liquid medium may be utilized. For example, U.S. Patent Appl. Publ. No. 20040121447 and U.S. Pat. No. 6,524,486, each incorporated herein by reference, disclose a tangential flow filter device and apparatus for partially separating algae from an aqueous medium. Other methods for algal separation from medium have been disclosed in U.S. Pat.

Nos. 5,910,254 and 6,524,486, each incorporated herein by reference. Other published methods for algal separation and/or extraction may also be used. (See, e.g., Rose et al., Water Science and Technology 1992, 25:319-327; Smith et al., Northwest Science, 1968, 42:165-171; Moulton et al., Hydrobiologia 1990, 204/205:401-408; Borowitzka et al., Bulletin of Marine Science, 1990, 47:244-252; Honeycutt, Biotechnology and Bioengineering Symp. 1983, 13:567-575).

In certain embodiments, exhaust gases that are enriched in CO₂ may be utilized to support photosynthetic carbon fixation, while simultaneously scrubbing the exhaust gases of their CO₂ content to prevent further buildup of greenhouse gases. In this way huge amounts of, for example, power plant flue gases can be "mined" for their CO₂ and the resulting gas piped to the algae farm. A further description of this technology is disclosed in U.S. Patent Publication No. 2007/0048848, published Mar. 1, 2007, which is incorporated herein by reference.

Other embodiments may comprise devices and methods for circulation of liquid within and extraction of oxygen or other gases from the closed bioreactor. In a preferred embodiment, large rollers may be arranged to roll over the surface of the closed tubes, pushing liquid along the tube. The roller system is a preferred method to move fluid through the tubes while minimizing hydrodynamic shear that would inhibit aquatic organism growth and division. Another benefit of the roller system is that when fluid is being diverted from below to above the thermal barrier, the roller provides a low-energy mechanism for moving a buoyant thermal barrier to the bottom of the tube, as the roller semi-seals the barrier to the tube bottom as it rolls along the tube.

23

#233432 vl Because the roller compression does not extend all the way to the bottom of the tube, the roller movement creates a high-velocity localized "backwash" immediately under the roller that serves to scrub the lower tube surface to reduce attachment to and biofouling of the tube surface and to re-suspend organisms that have settled to the bottom of the tube. Similarly, the movement of the accumulated gas bubble and gas/water interface in front of the roller at the top of the tube also scrubs the upper tube surface, reducing biofilm formation and increasing light transmission through the top surface.

In addition to moving fluid, the rollers would function to collect gas bubbles, such as oxygen that is generated by photo synthetic organisms, which may be removed from the system to reduce oxygen inhibition of growth. However, with reference now to FIG. 20, as the solid rollers approach the end housing where the tube attaches, they can get only so close. As a result a bubble of O₂ forms that is difficult to push into the end housing because there is always a lip involved, even with the best tube joining techniques. Even when there is no lip, the O₂ forms a pocket under the plastic even when the roller gets very close (to the end housing). This pocket, not only inhibits the capture of valuable oxygen, it also creates a stress on the tube that will shorten its usable life.

Accordingly, with reference to FIGS. 20-23, in one embodiment of the invention gas vents are placed in the tube and thermal barrier at each end of a tube in the space where the roller does not reach. For example, such vents can be placed every 3 feet across the width of each end of the tube. The vents are also preferably located midway between the roller terminal position and the tube seal to the end housing. In a preferred embodiment a plastic collar is welded or adhered over a vent opening in the tube and another tube, e.g. a 4" diameter tube is clamped to the plastic collar. One way pressure cracking valves and manifolds are preferably used to prevent infection entry. One skilled in the art will be able to select suitable sterile valves and associated parts to form a sterile air collection mechanism in view of the disclosure herein.

The thermal barrier preferably has a cracking flapper valve that covers the vent that is aligned with the collar on the tube vent so that when it opens to release air there is no mechanical blockage of the vent opening.

High oil strains of algae may be cultured in the closed system bioreactor apparatus and harvested. Algae may be extracted and their oil product removed without complex chemical treatment. The simplest way for large algae is to crush the algae and centrifugally separate the components into oil, crushed algae bodies for feed or nutrient, and nutrient laden water. However, algae is slippery and may be difficult to crush by standard means.

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#233432 vl In some embodiments, oil extracted from algae may be converted into commercial products, such as biodiesel. A variety of methods for conversion of photosynthetic derived materials into biodiesel are known in the art and any such known method may be used. For example, algae may be harvested, separated from the liquid medium, lysed and the oil content separated. The algal-produced oil will be rich in triglycerides. Such oils may be converted into biodiesel using well-known methods, such as the Connemann process (see, e.g., U.S. Pat. No. 5,354,878, incorporated herein by reference). Standard transesterification processes involve an alkaline catalyzed transesterification reaction between the triglyceride and an alcohol, typically methanol. The fatty acids of the triglyceride are transferred to methanol, producing alkyl esters (biodiesel) and releasing glycerol. The glycerol is removed and may be used for other purposes. The Connemann process is well-established for production of biodiesel from plant sources such as rapeseed oil and as of 2003 was used in Germany for production of about 1 million tons of biodiesel per year (Bockey, "Biodiesel production and marketing in Germany," www.projectbiobus.com/IOPD_E_RZ.pdf). However, the skilled artisan will realize that any method known in the art for producing biodiesel from triglyceride containing oils may be utilized, for example as disclosed in U.S. Pat. Nos. 4,695,411; 5,338,471; 5,730,029; 6,538,146; 6,960,672, each incorporated herein by reference. Alternative methods that do not involve transesterification may also be used. For example, by pyrolysis, gasification, or thermochemical liquefaction (see, e.g., Dote, 1994, Fuel 73: 12; Ginzburg, 1993, Renewable Energy 3:249-52; Benemann and Oswald, 1996, DOE/PC/93204-T5).

In some embodiments, all aspects of bioreactor function may be controlled by a central processing unit, for example a computer controller. The controller may be operably coupled to various sensors and actuators on the bioreactor. The computer may integrate all functions of bioreactor operation, such as roller movement and alignment, fluid flow, whirlpool operation, harvesting of aquatic organisms, nutrient and fluid input into the apparatus, gas removal, and CO₂ injection. The computer may operate on a sensing and control program such as Lab View made by National Instruments Corporation and may use interface cards and circuits well known in the art to connect with the sensors and actuators of the bioreactor system. An exemplary operation cycle is illustrated in FIG. 17. The discussion refers to compass directions for clarity, however the skilled artisan will realize that the apparatus in actual use may be aligned in a variety of directions, depending on local geography, solar inclination, temperature, etc. As illustrated in FIG. 17, Rollers 314 and 316 are initially positioned over their belly pans at the ends of the tubes. Flapper valve 318 is in the up position so that water being

25

#233432 vl drawn south comes from the bottom deck of the whirlpool device and flapper valve 320 is in the down position so that water going north is channeled upward onto the top deck of the whirlpool device. The cycle begins as shown in FIG. 18A with roller 314 being directed by the controller to begin moving South at a constant speed of 1 foot/second. As it moves, pressure is built up in tube 334 ahead of roller 314 and algae growth media (water) begins moving South, westward through the CO2 housing 302 then north through tube 336, slipping under stationary roller 316 through the belly pan channel. As the water flows up flapper valve 320 onto the top deck 300 it begins whirling through the whirlpool 326 to the bottom deck and expands through flapper valve 318 to begin backfilling behind roller 314. FIGS. 17 and 18 show roller 314 having fully traversed tube 334 and having come to a stop at the whirlpool housing. Since both rollers are positioned over belly pans, the liquid is free to continue moving by inertia in the direction shown. With no delay, roller 316 is caused to begin moving north by the controller as is shown in FIG. 18C. This continues the clockwise flow of the liquid through the whirlpool and back through the CO₂ housing as it slips under roller 314 through the channel created by the belly pan. When roller 316 finally reaches the whirlpool housing all motion stops except for the fluid media that continues to move clockwise through stored momentum until friction slows the water movement to nearly zero.

At this point the circulation direction of the fluid is reversed. First flapper 318 is put in the down position so that counterclockwise water flow is directed first onto the top deck and flapper valve 320 is in the up position so that exiting lower deck water is expanded into the full height of the bioreactor tube. Roller 316 moving south in under control of the computer, pushing water ahead to start a counter-clockwise fluid movement. After it comes to rest at the end of tube 336 roller 314 immediately starts moving north, to keep the pressure head on the whirlpool and full flow moving. For a short time after roller 314 comes to rest at the end of tube 334 the fluid keeps moving under its own momentum until friction slows it down to near zero speed.

Once this is achieved, the controller commands the clockwise motion sequence shown in FIG. 18 to begin again in a constant reciprocating motion. This motion further has the advantages of being inexpensive to implement by not needing to lift the heavy rollers out of the water during turnaround and because of flow reversing is less likely to leave un-turbulent spots in the bioreactor where algae might settle.

The CO₂ injectors may be controlled so that only the bubble injector experiencing counter-current water flow is actuated to take advantage of the increased bubble dwell time and concurrent increased CO₂ absorption. The amount of CO₂ injected is not limiting and it is

26

#233432 vl anticipated that CO₂ injection will be intermittent, as determined by medium pH and other indicators.

The septum valves for tube 336 are septum valves 306, 308. The septum valves for tube 334 septum valves 304, 310. Each tube thermal barrier may be controlled independently of the other tube septum but each must be coordinated with its roller motion.

Before either roller leaves its rest position the controller must determine whether its associated septum should be placed in the up or down position. If the septum is decided to be in the up position, the septum valve at the roller start position must be in the up position such that water gets drawn under the septum during roller travel. The septum valve at the far end of the tube can be in either position during roller travel as long as the septum valve sealing method allows for expelling water from inside the tube regardless of position. When the roller has stopped however, the septum valve at the far end should be fixed into the upper position.

When the septum is desired to be in the down position, the septum valve at the roller start position must be in the down position so that water is drawn over the top of the septum by roller movement. The septum valve at the far end of the tube can be in either position as long as it is designed to allow the unimpeded expelling of water from either top or bottom tube chamber. When the roller stops however the septum must be fixed into the down position so that water is not allowed to seep under the septum which would allow it to float to the top.

A fluid temperature sensor 328 is interfaced to the computer, which compares the detected temperature with a set point of desired temperature for the algae. Depending on weather and time of day conditions, the computer decides to place the thermal septums in the up or down position and coordinates the actions of the septum valves with the roller movement accordingly. In some cases a sensor may be constructed to determine whether the fluid will gain or lose heat to the temperature and radiative environment. Such a sensor would be constructed by channeling a small amount of fluid (about 0.1 gallon per minute) through a plastic tube of about 3 feet square by 3 inches deep that is laying on ground substantially the same temperature as the ground the main bioreactors are sitting on. Differential temperature sensors with a resolution of 0.02 degree F. measure the temperature at both the intake and outlet of the sensor tube. If the temperature is calculated to be increasing as fluid passes through the tube then the computer positions the septums to expose the fluid to the environment if the fluid is too cold in the tubes or to insulate the tubes from the environment if the fluid is too warm. The converse logic would apply if the sensor tube indicates that environmental exposure would cool the fluid.

A pH sensor 330 is also interfaced to the computer. The value of the fluid pH is compared with a desirable pH set point that is indicative of the proper concentration of dissolved

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#233432 vl CO2 in the water to support optimum growth or harvesting. When the pH is too high the computer opens valves to the appropriate CO2 bubbler to allow pure CO2 or flue gas containing CO2 to bubble through the water making it more acid with the formation of carbonic acid and lowering the pH. With reference to FIG. 19, in a preferred embodiment a method of performing a deep scrub can include simultaneously moving a first peristaltic roller and a second peristaltic roller in the same direction toward a control housing to create an increased aqueous medium pressure in the portion of the aqueous medium chamber between the peristaltic rollers. The parallel movement creates a larger head in front of rollers as fluid must escape under the rollers rather than circulating to the next tube, thus causing the increased pressure This increased pressure will cause increased interior surface scrubbing of flexible tube and thermal barrier walls and increased particle re- suspension of the aquatic organisms and other particles as a high velocity backwash under the rollers occur from the higher pressure.

The scrubbing will occur in either the top or bottom chambers depending on the location of the thermal barrier.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

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Claims

CLAIMSWhat is claimed is:

1. A closed system bioreactor system comprising: at least one flexible tube capable of containing an aqueous medium, the at least one flexible tube having a first open end and a second open end; a thermal barrier within the at least one flexible tube to regulate the temperature of the aqueous medium, the thermal barrier dividing the at least one flexible tube into an upper chamber above the thermal barrier and a lower chamber below the thermal barrier, wherein the medium may be alternatively directed to the upper chamber or the lower chamber to thermally isolate or thermally expose the aqueous medium to the environment; a first control housing operably coupled to the first open end of the at least one flexible tube and configured to circulate the aqueous medium to or from the upper chamber of the at least one flexible tube and to or from the lower chamber of the at least one flexible tube; a flow control mechanism in mechanical association with the first control housing to direct the medium above or below the thermal barrier of the at least one flexible tube, the flow control mechanism comprising: a septum clamp connected to the first end of the thermal barrier; at least one actuator connected to the septum clamp and operable to move the septum clamp between an up position to facilitate a flow of the aqueous medium below the septum clamp and the thermal barrier and thus between the control housing and the lower chamber of the tube, or a down position to facilitate the flow of the aqueous medium above the septum clamp and the thermal barrier and thus between the control housing and the upper chamber of the tube; an upper valve member connected to the septum clamp for restricting the flow of the aqueous medium into the upper chamber of the tube when the septum clamp is in the up position but permitting a flow of the aqueous medium out of the upper chamber of the tube when the septum clamp is in the up position; and a lower valve member connected to the septum clamp for restricting the flow of the aqueous medium into the lower chamber of the tube when the septum clamp is in the down position but permitting a flow of the aqueous medium out of the lower chamber of the tube when the septum clamp is in the down position.

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2. The system of claim 1, further comprising at least one peristaltic roller operably coupled to the at least one flexible tube to circulate the aqueous medium through the at least one flexible tube and to remove gas bubbles from the at least one flexible tube.

3. The system of claim 2, wherein: movement of the peristaltic roller along the at least one flexible tube toward the first control housing pushes aqueous medium in the lower chamber through the lower valve member when the septum claim is in the down position, creating a backflow suction that tends to close the lower valve member against the septum clamp; and movement of the peristaltic roller along the at least one flexible tube toward the first control housing pushes the aqueous medium in the upper chamber through the upper valve member when the septum claim is in the up position, creating a backfill suction that tends to close the lower valve member against the septum clamp.

4. The system of claim 1, wherein the at least one flexible tube is arranged horizontally along the ground.

5. The system of claim 1, wherein said at least one flexible tube comprises two flexible tubes, each flexible tube connected to the first control housing at the respective first open ends, the system further comprising: two peristaltic rollers, each flexible tube being operably coupled to a single peristaltic roller; and a second control housing operably coupled to the second open ends of the two flexible tubes to form a biologically closed system.

6. The system of claim 1, further comprising one or more aquatic organisms in the medium.

7. In an aqueous bioreactor system using flexible tubes to contain aquatic organisms in an aqueous medium, a method for cleaning the flexible tubes, the method comprising: providing a bioreactor comprising: a first section of flexible tube capable of containing an aqueous medium, the first section of flexible tube having a first open end and a section open end and an interior surface; a second section of flexible tube capable of containing an aqueous medium, the second section of flexible tube having a first open end and a second open end and an interior surface; a first control housing coupled to the first open end of the first section of flexible tube and the first open end of the second section of flexible tube, the first section of

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#233432 vl flexible tube, the second section of flexible tube, and the first control housing defining an aqueous medium chamber; an aquatic organism containing aqueous medium in the aqueous medium chamber; a first peristaltic roller operably coupled to the first section of flexible tube to circulate the aqueous medium through the first section of flexible tube and scrub the surface of the tube to reduce biofilm on the tube surfaces; and a second peristaltic roller operably coupled to the second section of flexible tube to circulate the aqueous medium through the second section of flexible tube and scrub the surface of the tube to reduce biofilm on the tube surfaces; moving the first peristaltic roller to a position on the first section of flexible tube distal the first control housing; moving the second peristaltic roller to a position on the second section of flexible tube distal the first control housing; and simultaneously moving the first peristaltic roller and the second peristaltic roller towards the first control housing to create an increased aqueous medium pressure in the portion of the aqueous medium chamber between the peristaltic rollers, the increased pressure causing increased interior surface scrubbing of the first and second sections of flexible tube and increased particle re-suspension of the aquatic organisms within the first and second sections of flexible tubes.

8. The method of claim 7, wherein the first section of flexible tube and the second section of flexible tube comprise adjoining sections of the same flexible tube.

9. The method of claim 7, wherein the first section of flexible tube is a first flexible tube and the second section of flexible tube is a second flexible tube.

10. The method of claim 7, wherein the first section of flexible tube is a first flexible tube and the second section of flexible tube is a second flexible tube.

11. A closed system bioreactor system comprising: a flexible tube capable of holding an aqueous medium, the at least one flexible tube having a first open end, a second open end, and an upper layer; a first control housing operably coupled to the first open end of the at least one flexible tube; a peristaltic roller configured for movement along the flexible tube towards and away from the first control housing to circulate the aqueous medium through the flexible tube and to remove gas bubbles from the flexible tube;

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#233432 vl wherein the upper layer of the flexible tube comprises a gas venting section adjacent the first control housing, the gas venting section having at least one tube gas vent for release of gasses that accumulate as the peristaltic roller moves along the flexible tube towards the first control housing.

12. The system of claim 11, tube gas vent comprises: an opening in the upper layer of the flexible tube; a collar connected to the upper layer of the flexible tube and forming a seal around the opening; a gas tube connected to the collar and configure to receive gas from the collar and transfer the gas away from the flexible tube; and a one way valve configured to permit the flow of air from the flexible tube to the gas tube but restrict the flow of air or contaminants in the opposite direction.

13. The system of claim 11, wherein the upper layer of the flexible tube comprises at least two tube gas vents.

14. The system of claim 13, wherein the system further comprises a gas collection manifold for receiving gas from the at least two tube gas vents.

15. The system of claim 11, wherein the tube gas vent comprises a section of the upper layer that is formed of a gas permeable water resistant material.

16. The system of claim 11, further comprising a thermal barrier within the flexible tube to regulate the temperature of the aqueous medium, the thermal barrier dividing the flexible tube into an upper chamber above the thermal barrier and a lower chamber below the thermal barrier, wherein the aqueous medium may be alternatively directed to the upper chamber or the lower chamber to thermally isolate or thermally expose the aqueous medium to the environment, the thermal barrier comprising: at least one thermal barrier gas vent to permit gasses that accumulate in the lower chamber to pass to the upper chamber through the at least one thermal barrier gas vent and then through the tube vent.

17. The system of claim 16, wherein the thermal barrier gas vent is adjacent a tube gas vent.

18. The system of claim 16, wherein the thermal barrier gas vent comprises a one way flapper valve configured to permit the flow of air from the lower chamber to the upper chamber but restrict the flow of gas or fluid from the upper chamber to the lower chamber.

19. The system of claim 16, wherein the thermal barrier gas vent comprises a section of the thermal barrier that is formed of a gas permeable water resistant material.

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20. A closed system bioreactor system comprising: a flexible tube having an upper layer and a lower layer, a first open end, and a second open end and being capable of holding an aqueous medium, the flexible tube having a portion extending from the first open end to the second open end that defines a length of the flexible tube; a peristaltic roller having a roller axis perpendicular to the length of the flexible tube and being configured for movement along the length of the flexible tube between the first open end and the second open end to circulate the aqueous medium; and a thermal barrier within and extending along the length of the flexible tube, the thermal barrier having a substantially flat upper surface oriented towards the upper layer of the flexible tube, the thermal barrier dividing the flexible tube into an upper chamber located between the upper layer of the flexible tube and the thermal barrier and a lower chamber below the lower layer of the flexible tube and the thermal barrier, wherein the aqueous medium may be routed to the upper chamber to thermally expose the aqueous medium to the environment or the lower chamber to thermally isolate the aqueous medium from the environment, the thermal barrier having a non-uniform structure along the length of the thermal barrier with regions of greater and lesser flexibility, whereby the thermal barrier has a flexibility that enables the flat upper surface of the thermal barrier to conform to the upper layer of the flexible tube as the peristaltic roller moves thereover.

21. The system of claim 20, wherein the thermal barrier has a varying thickness with thinner regions of the thermal barrier having a greater flexibility than thicker regions of the thermal barrier.

22. The system of claim 20, wherein the thermal barrier comprises: a substrate forming the top surface of the thermal barrier; and an insulative layer on the bottom surface of the substrate, the insulative layer having a plurality of discontinuities that increase the flexibility of the insulative layer.

23. The system of claim 20, wherein the thermal barrier comprises a substrate and an insulative layer connected to the substrate, the insulative layer comprising a material having a varying thickness.

24. The system of claim 20, wherein the thermal barrier comprises: a substrate having a first emissivity, an upper surface, and a lower surface; a high emissivity layer connected to the lower surface of the substrate and having a second emissivity that is greater than the first emissivity; and

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#233432 vl an insulative layer connected to the high emissivity layer, the insulative layer comprising a material having a varying thickness.

25. The system of claim 20, wherein the thermal barrier comprises: a polyester substrate having an emissivity of greater than about .6, the polyester substrate having a substantially uniform thickness, the polyester substrate having an upper surface oriented towards the upper layer of the flexible tube and a lower surface; and a thermally insulative layer connected to the lower surface of the substrate, the thermally insulative layer having a varying thickness.

26. The system of claim 20, wherein the thermal barrier comprises: a substrate having a substantially uniform thickness, the substrate having an upper surface oriented towards the upper layer of the flexible tube and a lower surface; and a plurality of separate thermally insulative members connected to the lower surface of the substrate, the thermally insulative members having substantially the same thickness and being separate from adjacent thermally insulative members by a gap.

27. The system of claim 20, wherein the thermal barrier comprises: a polyethylene substrate having a substantially uniform thickness, the substrate having an upper surface oriented towards the upper layer of the flexible tube and a lower surface; and a plurality of separate thermally insulative members connected to the lower surface of the substrate, the thermally insulative members comprising: a polyester substrate; and a foamed polyethylene insulative layer adhered to the polyester substrate.

28. A closed system bioreactor system comprising: a flexible tube having an upper layer, a lower layer, a first open end, and a second open end and being capable of holding an aqueous medium, the portion of the flexible tube extending from the first open end to the second open end defining a length of the flexible tube; a peristaltic roller configured for movement along the length of the flexible tube to circulate the aqueous medium in the flexible tube; and a thermal barrier within the flexible tube, the thermal barrier dividing the flexible tube into an upper chamber located between upper layer of the flexible tube and the thermal barrier and a lower chamber below the lower layer of the flexible tube and the thermal barrier so the aqueous medium may be routed to the upper chamber or the lower chamber to thermally isolate or thermally expose the aqueous medium to the environment, the thermal barrier comprising: a substrate having a top surface and a bottom surface; and

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#233432 vl an insulative layer, the insulative layer having a plurality of discontinuities that increase the flexibility of the insulative layer.

29. The system of claim 28, wherein the insulative layer comprises a plurality of completely separate insulative members.

30. The system of claim 28, wherein: the substrate has a first elasticity and a first emissivity; the insulative layer comprises a plurality of separate insulative members, each insulative member comprising: a high emissivity layer formed of a material having a second emissivity that is greater than the first emissivity and a second elasticity that is lower than the first elasticity; and an insulative material connected to the high emissivity layer.

31. The system of claim 28, wherein the insulative layer comprises a plastic material having an air filled cell construction.

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