WO2017051334A1 - Apparatus and process for sea surface microalgae cultivation - Google Patents

Abstract

A method and apparatus for providing the cultivation of microalgae is provided. The container has a wall material, an impeller, and at least one aperture. The process starts with seeding a feed stock, circulating a growth medium through a container, providing nutrients to the growth medium, providing a gas exchange, and extracting the growth once the process is completed.

Description

"APPARATUS AND PROCESS FOR SEA SURFACE MICROALGAE CULTIVATION"

INVENTOR:

Thomas J. Digby

CLAIM OF PRIORITY

This application claims priority to U.S. Application Serial Number 62/221,906 filed September 22, 2015, the contents of which are hereby fully incorporated by reference in its entirety.

FIELD OF THE INVENTION The invention relates to a method and apparatus for the cultivation of microalg; preferably for the cultivation of microalgae at the sea surface.

BACKGROUND OF THE INVENTION Microalgae is the general term used for a variety of microscopic photosynthetic organisms, often called phytoplankton, which can be single-celled or simple multi-cellular structures, and which includes eukaryotes (e.g. diatoms, green algae) and prokaryotes (e.g. cyanobacteria). Their small size distinguishes them from macroalgae such as kelp and sea grasses.

Large scale cultivation of microalgae originated with research in the US, Japan, Germany and other countries for food production (Burlew, J.S. (1953) Algal culture from laboratory to pilot plant. Carnegie Institute of Washington, Washington, D.C., Publication 600, pp. 357). Early research led to initiation of industrial scale production of microalgae (Chlorella) in large open ponds, in Japan in the early 1960s. Another microalgae species, Spirulina, was recognized during the 1960s to be a traditional food of people living around the alkaline Lake Chad in Africa. Spirulina has been produced at large scale in circulating raceway ponds for human consumption since the 1980s by companies such as Earthrise Nutritionals, LLC, (having a first production plant in the early 1980s near the Salton Sea, Calif.), followed by Cyanotech Corp. in Kona, Hawaii. Large scale production of microalgae also takes place in municipal wastewater treatment ponds. Several thousand small (< 10 hectare) and a few large scale (>100 hectare) open pond systems are currently operated for such purposes in the US. In this case the microalgae is rarely harvested, its purpose being mainly for oxygen production to assist aerobic digestion of wastewater by other bacteria. Further background on large scale cultivation of microalgae may be found at Lundquist et al. (2010) "A Realistic Technology and Engineering Assessment of Algae Biofuel Production" online publication of Energy Biosciences Institute, University of California, Berkeley, California, October 2010.

A smaller scale method of cultivating microalgae is the photobioreactor. These cultivation systems comprise clear tubes or glass panels, use artificial light sources, optionally supported by sunlight, and thereby provide highly controlled production of selected microalgae species. Background on photobioreactors may be found in T. Wencker's 2011 publication at http://www.submariner-project.eu/images/stories/events/algae- trelleborg/presentations/wencker.pdf (accessed 30-July-2015). Typically the growth volumes are in the range of 0.1 to 1 m3, with maximum volume per unit about 15 m3. Since the 1980s photobioreactors have been used to generate microalgae feedstock for research, for high value specialty chemical products, and for source feedstocks for larger open pond systems. Existing microalgae cultivation systems are not well-suited for large scale production of low-value high-volume products such as biofuels because production cost is high, and they suffer from inherent limitations on scalability. Photobioreactors, for example, are energy intensive and require substantially more energy to operate than can be obtained from the product. On the other hand, open ponds and raceway ponds demand large flat land areas, and high quantities of water. Given the need for sunlight, such ponds are often situated in deserts, presenting water resource challenges. Where such ponds are situated in water-rich agricultural areas, they compete with resources for food products. This problem is known as the "food versus fuel" debate. The limits on existing production technology is demonstrated by the modesty of current worldwide commercial microalgae production, estimated at 20,000 tonnes dry weight in 2013. This deficit is a great disappointment to those who perceive the potential for microalgae to provide feedstocks for human and animal nutrition, for specialty chemicals, for biofuel production, and for other valuable products.

With this background we turn to the present invention for a large-scale microalgae production process and apparatus which overcomes numerous difficulties, recognized and unrecognized, in the prior art.

SUMMARY OF THE INVENTION

The present invention provides for a container for cultivation of microalgae comprising: a wall material having transparency to at least some photosynthetic wavelengths on a solar facing surface, an impeller to circulate microalgae growth medium in the container, at least one aperture to periodically add nutrients to modulate the growth of microalgae in the microalgae growth medium, a mechanism for gas exchange between interior and exterior aspects of the container, wherein the container is sealed to contain the microalgae growth medium and is disposed to float with approximately neutral buoyancy at the sea surface.

The present invention also provides for a process for cultivation of microalgae comprising: seeding a feedstock of microalgae into a container comprising wall material having transparency to at least some photosynthetic wavelengths, the container being sealed to contain microalgae growth medium, and disposed to float with approximately neutral buoyancy at the sea surface, circulating the microalgae growth medium within the container, providing nutrients to the microalgae growth medium to modulate the growth of microalgae during the course of a growth cycle, providing gas exchange between interior and exterior aspects of the container; and extracting the microalgae growth medium from the container when desired microalgae growth has been achieved.

The present invention also provides for a method of manufacturing the longitudinal channels in parallel arrangement comprising, providing a first sheet of membrane material, overlaying a second sheet of membrane material, sealing the second sheet to the first sheet by heat, chemical or radiative means along a plurality of seams, each seam of width l-20cm wide, and each seam separated by lanes of a desired width (20cm to 200 cm), thus forming a series of parallel or semi-parallel lanes separated by seams.

The present invention also provides for a bioreactor for growth of microalgae, said bioreactor being sealable to contain microalgae growth medium wherein at least one portion of the solar facing surface is permeable to oxygen and carbon dioxide but substantially

impermeable to water, and the solar facing wall is transparent to at least some photosynthetic wavelengths but substantially blocking to UVB wavelengths 295-305 nm. The present invention further provides for a computer-implemented method for managing the growth conditions of a microalgae cultivation platform disposed on the sea surface comprising: monitoring in a microalgae cultivation platform disposed on the sea surface one or more conditions of the growth medium selected from among pH, levels of dissolved O2, CO2, inorganic carbon, organic nitrogen (including any form of ammonium, nitrate and urea), solar insolation, solar flux, temperature, and optical density to generate data; transmitting the data by automated device to a receiver, analyzing the data received at a computer to determine if the conditions of the growth medium correspond to optimized growth conditions; wherein if a condition of the growth medium does not correspond to optimized growth conditions, the computer sends a further signal to adjust one or more of impeller speed, nutrient flow and gas flow in the microalgae cultivation platform.

Therefore, the present invention succeeds in conferring the following, and others not mentioned, desirable and useful benefits and objectives.

It is an object of the present invention to provide an apparatus for the cultivation of microalgae.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a breakout perspective view of a container of the invention which is a fluidly sealed loop. Two longitudinal channels are linked by directional U-joint at each end, creating a sealed circular raceway. Examples of attachments for supplying feedstock and nutrients, and for removing biomass for harvesting, are shown.

Fig. 2 shows a cross-section of a longitudinal channel, with exemplary dimensions. Fig. 3 shows an aerial view of an example of a nutrient adjustment segment of a fluidly sealed loop, including components for gassing/degassing, for nutrient addition, and an impeller.

Fig. 4 provides a horizontal cross-sectional view of an arrangement for receiving and modifying the growth medium. A first component adjusts the dissolved gasses while a second component adds nutrients.

Fig. 5 sets out an example of a Field array comprising six fluidly sealed loops, and supported by a catenary system of tension cables moored between mooring posts.

Fig. 6 shows the field array of Figure 5 providing only the tension cables and mooring posts (e.g. without the container system).

Fig. 7 provides a perspective view of a field array in a catenary mooring system disposed to float at the sea surface with approximately neutral buoyancy.

Fig. 8 provides an embodiment of the invention comprising two field arrays, each covering 1 Ha sea surface, and linked by a dock giving convenient worker access to pumping mechanisms of each individual fluidly sealed loop, all of which is supported by a catenary mooring system.

Fig. 9 provides an embodiment of a floating field array where the field array is supported by an exterior frame, optionally via tension cables fixed to the field array. Anchoring by catenary lines below the surface to the sea floor is depicted.

Fig. 10 provides one embodiment of a manufacturing process for a container of the invention. A four channel (two circular closed loop) field array is depicted.

Fig. 11 is a cut-away view illustrating one method by which tension cables can be fixed to the field array to distribute stress. Tension cable is fixed with a plurality of cross securing members made from material compatible to fix securely to the thin film membrane of the field array, without causing damage or tearing when subject to the stresses encountered at the sea surface. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention herein has seized upon the possibility that truly large scale production of microalgae could be achieved by a striking solution: Moving production off of land, and into the sea. The numerous advantages of the invention, as exemplified in the embodiments and description herein, will quickly become apparent. Chiefly, the use of sea surface eliminates the land resource limitation, providing access to flat surface area with good solar exposure, particularly in equatorial latitudes. Sea water provides temperature stability that will reduce the day/night temperature variation experienced with land based systems, and eliminate the need for additional cooling systems. And, in one embodiment, the use of sea water itself as the basis for the growth medium takes advantage of an ample resource that can be reliably recycled, without competing for fresh water. High productivity with low demand for energy encourages scalability, making feasible very large scale cultivation of microalgae biomass. Other advantages will also become apparent to the reader.

The invention thus provides an improved process and apparatus for cultivation of microalgae, suitable for operation on the sea surface. The features of the invention, being used individually or in combination include: a sealable container comprising a fluidly sealed loop enclosing microalgae growth media, horizontally disposed to float at the sea surface. Affixed with the container: an impeller, a nutrient supply, and a gas exchange mechanism; one or more apertures for inoculating microalgae feedstock and fresh growth medium into the container, and one or more apertures for removal of the growth medium when the cultivation cycle is completed. The container further comprising: A solar facing surface, which among other things enhances light absorbance by the growth medium, The solar facing surface being transparent to at least some photosynthetic wavelengths, and optionally being substantially UV blocking in at least the wavelengths 290nm-320 m. A lower ocean facing container surface suitable for heat exchange between growth medium and the ocean. Surface (or "wall") materials of the container being a material flexible in 3-dimensions with a tear strength and tensile strength suitable for sea surface conditions. The ratio of the lower surface area to the upper surface area being from about 1.1 to about 2.0. The volume of growth medium liquid to gas headspace in the container having a ratio of at least 3: 1.

To enhance the growth rate and productivity of the process and apparatus, and to lower the overall cost of production, nutrients are supplied to the container, constantly monitored and adjusted to optimal levels, thus providing a fed-batch or continuous process, optionally controlled by computer. Impeller speed and nutrient addition is adjusted depending on time of day or night, atmospheric conditions such as solar insolation and temperature, and stage of the growth cycle of the microalgae under cultivation.

In one embodiment, the gas-exchange mechanism is provided by a sparging tank. In a preferred embodiment the gas-exchange mechanism is provided by an upper surface container wall, which is a thin film membrane permeable to oxygen and carbon dioxide gas, but having limited permeability to water.

The container is optionally flexibly connected to a plurality of similar containers, creating a field array which is held in place on the surface of the sea by an exterior support structure comprised of mooring posts and catenary cables, or by an anchoring system. The invention provides a substantial advantage over raceway pond technology by, among other things, preventing exposure of the growth medium to uncontrolled environmental factors including organisms, dust, sand, rain or hail.

These and other intriguing features of the invention are further described below.

Definitions

Special terms used herein are defined as follows unless the context clearly requires an adjusted meaning. Words and phrases not specifically defined herein are assumed to have the common meaning used by a person skilled in the art.

"Contains" means substantially containing, such that the container prevents release or escape of the contained substance, and that the contained substance is not exposed to external contaminants or factors external to the contained volume such as those driven by weather. Minor leaking, or leaking due to unintended or temporary damage is nonetheless contained. In this invention, where gas exchange occurs between interior and exterior of the container, as long as substantially all of the liquid phase is retained, the container including the growth media, the microalgae, and the liquid phase is still considered "contained".

"Sealed to contain" means sealed to a sufficient degree that substantially all the liquid volume remains inside the container under normal operating conditions. Minor leaking, or leaking due to unintended or temporary damage is nonetheless "sealed".

"Fluidly sealed" means that the seal is sufficient such that a liquid substance will not normally or readily exchange with a liquid substance on the exterior of the container.

"Fluidly sealed loop" means a closed loop circulation system which is fluidly sealed.

"Sealed tube" means a fluidly sealed tube. "Sealed volume" is the total volume contained within a fluidly sealed loop, including gas and liquid volumes.

"Fluidly connecting", or in the nominative form a "fluidic connection", means that two conduits are aligned such that the flow of a liquid in a first conduit is directed to, and captured, by a second conduit which provides a continuous coherent flow path for the flow of the liquid.

Conduits are fluidly connecting even if there exists a valve between the two conduits that can be opened or closed by the user.

References in this specification to the container "surface", "wall", "wall material", "membrane", "film" or "thin film membrane" and the like, are more specifically referring to a material which can contain a liquid when fluidly sealed, while exhibiting some or all of the flexibility and design features set out herein. It does not need to be rigid.

"Photosynthetic wavelengths" means electromagnetic radiation between about 400-700 nm which may be absorbed by a living organism in a photosynthetic process.

"Transparency" and "transparent" refer to the capacity of an object or a wall material to permit the transmission of electromagnetic radiation in the ultraviolet, light or short-infrared range (i.e. wavelength between about 200-1000 nm).

The phrase "at least partially transparent" refers to a wall material or component which permits enough photosynthetic wavelength light energy to pass through to drive photosynthesis within a phototrophic organism.

"Solar facing surface" means the portion of the container which is oriented upwards, i.e. towards the atmosphere, when floating on the sea surface. It is the surface which is principally responsible to transmit sunlight during daylight hours to the growth medium contained in the container. It is opposed to the lower or "ocean facing surface" which is oriented towards the marine environment and which is principally responsible for heat exchange of the growth medium with the external sea volume. As the container is fluidly sealed, the exact dividing line between solar facing and ocean facing surfaces (e.g. in vertical cross section) is a functional one, and depends on which surface segment performs which of the noted functions. The ratio of cross-sectional lengths of the two surfaces is calculated based on the principal function of each segment. In Figure 2, the upper surface from one seam to the next defines the solar facing surface, and the lower surface from one seam to the next defines the ocean facing surface.

"Solar insolation" is a measure of solar radiation energy received on a given surface area in a given time. It is a function of solar flux (the spectrum of energy transmitted by the sun) as reduced by cloud cover and atmospheric conditions between the sun and the surface being measured.

"Blocking" as in "blocking to UVB wavelengths" means that transmission of such wavelengths are substantially reduced or eliminated, either by reflection or absorbance of the wavelengths by the blocking substance. "Substantially blocking" means transmission is at least 30% blocked, more preferably 40%, 50% or 60% blocked, and most preferably at least 75% blocked.

"Impeller" means any kind of pump, impeller system or propeller system which is used to impart pressure onto a fluid such as a liquid or gas.

"Aperture" means an opening in a wall or vessel which optionally may be adjustable in size and optionally include a closeable valve.

"Growth cycle" means the time from initiation of a crop of microalgae by inoculation into growth medium to the time the crop is harvested from the growth medium. It may include all of the standard sigmoidal growth process of a closed container, or it may include only part of the sigmoidal growth curve, if for example the crop is initiated in log phase growth and is harvested before plateau phase is reached. It may include one or multiple divisions of the inoculated parent cells. "Growth cycle" and "cultivation cycle" are interchangeable.

"Crop" means the biomass of preferred microalgae and all associated co-species, viruses and other organisms (including both heterotrophs and autotrophs) that are recoverable from the growth medium at the end of a growth cycle. Sometimes crop applies to the growing biomass, it may also apply to the biomass after harvesting, depending on context.

The phrase "mechanism for gas exchange" means the use of one or more techniques or devices, described herein or elsewhere, which in application permit control over the level of dissolved gases in the growth medium of the container.

"Nutrient" means a mineral and/or a salt, in inorganic or organic form, and includes dissolved gases, except when the context clearly distinguishes dissolved gasses as a separate category.

"Neutral buoyancy" means the condition in which a physical body's average density is equal to the density of the fluid in which it is immersed. As used herein, "approximately neutral buoyancy" includes slightly positive buoyancy and slightly negative buoyancy, as demonstrated by the cultivation platform of the invention which, when filled with growth media and disposed in sea-water, floats with its solar facing surface at the sea- surface and the majority of its mass and volume just below the surface. Approximately neutral buoyancy means average density of the physical body taken as a whole is within +/- 5% of the immersion fluid or +/- ("plus or minus") 3.5% or 3.0% or 2.5% or 2% or 1.5% or 1% or 0.5% or 0.05% or 0.005% of the immersion fluid. In the claims (as well as in the specification), all transitional phrases or phrases of inclusion, such as "comprising," "including," "carrying," "having," "containing," "composed of," "made of," "formed of," "involving" and the like shall be interpreted to be open-ended, i.e. to mean "including but not limited to" and, therefore, encompassing the items listed thereafter and equivalents thereof as well as additional items. The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

Certain other definitions are provided elsewhere in this disclosure.

All references cited in this specification are hereby incorporated by reference.

One embodiment of the invention is set out in Figure 1. Figure 1 illustrates an apparatus for cultivation of microalgae at the sea surface comprising two longitudinal channels 11 flexibly connected in a parallel arrangement, two directional joints (herein also sometimes called a U- tube), exemplified by 12 where one end of the directional joint is fluidly sealed to a first channel and the other end of the directional joint is fluidly sealed to a second channel such that liquid flow through the interior volume of one channel is directed back into the interior volume another channel, at least one impeller 13 fluidly connected to a channel, a gas exchange apparatus 14 disposed to exchange gases in the interior volume as liquid flows past the apparatus, at least one portal or aperture for nutrient addition 15 disposed to add liquid nutrient to the interior volume as it flows past the portal, an aperture for loading and extracting the liquid microalgae growth medium volume 16, wherein the apparatus comprises an interior volume sealed in a closed loop so as to substantially contain a liquid growth medium which may be circulated by the impeller situated therein. The apparatus is disposed to float on the sea surface thus presenting a solar- facing (or upper) surface, and an ocean-facing (or lower) surface (lower surface not shown). The material employed to form the longitudinal channel may be a flexible thin film membrane. The solar-facing surface is transparent to at least some photosynthetic wavelengths. To minimize power loss during fluid directional changes, the apparatus, which may be considered a photobioreactor, has a unit length which exceeds its width. The ratio of length to width is generally greater than 5: 1, and may exceed 100: 1. A growth medium supply tube is illustrated at 17; while the harvesting ("off-take") flow tube is shown at 18.

Figure 2 provides a cross sectional image of one of the plurality of channels. The channel walls have a solar-facing surface 21 and ocean-facing surface 22 and are made from a flexible thin film membrane. Dimension X is 10 cm to lm, preferably 20cm-80cm, most preferably 40-60cm; Dimension Y is 30-300 cm, preferably 100-200 cm, most preferably 140cm; length Z is 0-20 cm but preferably 10 cm. As shown in the Figure, the cross-sectional length of the lower surface is greater than the upper surface. Therefore the ratio of the lower surface area to the upper surface area is from about 1.1 to about 9.0.

Figure 2 also illustrates the circular turbulence 23 that results as liquid is driven by the impeller through the channel. The turbulence may be increased or decreased by fins or directional elements (not shown). Turbulence provides many advantages in the design, including heat transfer of heat gained from the atmosphere and by solar flux at the upper surface, to the lower surface, which due to its greater surface area will effectively transfer heat through the lower surface to the deeper ocean layers.

Pressure resulting from loading the liquid medium (growth medium) into the closed loop causes the container to fill and adopt a bulging (convex) upper face. Due to buoyancy of the entire apparatus the upper face may optionally project slightly above the mean sea surface level 24, though the majority of the volume of the container will be at or below the sea surface level. The convex upper surface may optionally be supported by ribs or other mechanical members fixed on the exterior or interior of the channel (not shown). Apart from bubbles and limited froth, the channel illustrated is predominantly fluid filled and largely devoid of gaseous headspace. In one embodiment, the sealed tube volume comprises at least half liquid phase and less than half gas phase preferably at least about 75% liquid phase and less than about 25% gas phase, more preferably at least about 90% liquid phase and less than about 10% gas phase, and most preferably the sealed tube volume comprises at least about 95% liquid phase and less than about 5% gas phase.

Solar radiation is (during daylight hours) incident on the upper surface 21, and at least some photosynthetic wavelengths are transmitted to the growth medium contained in the interior. Lower surface 22 may optionally further transmit any or all wavelengths to which it may be exposed so as to minimize light reduction in the sea water column and sea floor under the apparatus. In some embodiments the lower surface may include regions of light absorbing pigments to induce heat driven convection inside the container.

Figure 3 illustrates an aerial view of an embodiment of a gas exchange apparatus 31 , nutrient input portal 32 and liquid pump impeller 33. The direction of flow of the growth medium is indicated. In this embodiment, gas exchange and nutrient addition takes place at the end of a channel, shortly before the direction change of the U-tube 34 and impeller 33 act upon the medium. This arrangement assists with mixing of the growth medium.

Figure 4 illustrates a cut-away view of a gas exchange apparatus and nutrient input portal, which may be called the "receiving" channel segment. This embodiment of the gas exchange mechanism 41 is illustrated with an aeration pump 411 connected to a feed line 412 which leads to a sparger 413 embedded in the container volume. Gas 414 is pumped into the growth medium 415 and dissolves into the liquid phase or bubbles through and exits the liquid phase into a collecting headspace 416. In this process, over-saturated gases, for example 0₂, are released from the liquid, while gas which is under-represented such as CO2, is absorbed by the liquid medium. Exiting gases are collected in the headspace 416 and released by a release valve (not shown) or returned to the aeration pump via a return line 417 for storage 418 and re-cycling to the system.

The nutrient input unit 42 comprises a nutrient source 421, a liquid pump 422 and a feed line 423 inserted into the container interior volume via an aperture for nutrient addition 424 with at least one opening in the feed line disposed to release the liquid nutrient to the passing growth medium 415.

In Figure 3, the liquid pump impeller 33 is disposed to circulate the liquid medium in the interior volume of the container. Preferably the mechanisms in Figure 3 and Figure 4 are co- located at one end of the sealed closed loop to simplify access to and maintenance of these mechanical parts. Approximately neutral buoyance is also desired for the mechanical elements of the container illustrated in Figure 3 and Figure 4 (exclusive of the gas headspace 416 of the gas exchange unit). The user will adjust the entire unit with weight or flotation devices to ensure the mechanical elements sit just below the water surface, in horizontal line with the main volume of the container.

Having thus described certain visible features of the container of the invention, several less visible features and details which generally apply to the apparatus and process of the invention, are set out below.

The invention provides a large solar facing surface area relative to the volume of the closed loop system. The interior volume is designed to contain a liquid medium in which microalgae grows, herein referred to as the microalgae growth medium, or simply growth medium. The preferred growth medium is substantially seawater, or brackish water, which may be nutritionally adjusted by means known in the art or as set out below. Wastewater from human or animal sewage treatment plants is a potential source which can form the base growth medium. Whatever the source of the base growth medium, it is further adjusted by fed-batch or continuous flow addition of nutrients during the course of cultivation.

The impeller, in one embodiment a liquid pump, operates over a power range suitable to circulate the interior volume of the closed, sealed loop at a preferred rate of flow. In a preferred embodiment, the interior volume would be in the range of 90 M³ for a two channel system of 100 M each direction (including U-tubes), 140cm width and 50cm depth. Smaller volumes are possible by employing narrower channels containing as little as 1.5 M³ (e.g. a closed loop of width 30 cm, depth 10 cm, and total loop length 50 M). A larger system comprising multiple parallel channels linked by multiple U-tubes would contain correspondingly larger volumes. A closed loop system covering a full hectare at 60 cm depth in the design proposed contains up to about 5000 M³ of liquid medium. Based on the invention disclosed herein, one skilled in the art can create a wide range of sealed loop volumes, which may be circulated from one or more impellers fluidly connected to the interior volume of the channel.

The circulation speed is determined by the user based on calculations and empirical testing. A plug flow speed of 1.0 m/sec would aggressively circulate the medium. A preferred speed is less than this, preferably less than 0.2 m/sec, more preferably less than 0.1 m/sec, less than 0.05 m/sec, and most preferably 0.01 m/sec. Circulation speed may be modulated during the course of a 24 hour period. Factors which influence impeller speed are night/day, solar insolation, temperature, growth culture density and other factors detailed herein. The lowest impeller speed is desired to reduce the overall energy demand of cultivation.

An important further criteria on the impeller and its speed is the nature of the species selected for growth in the container. The impeller must minimize forces and shear stress which damage the organism and inhibits growth. Most authors agree that shearings and accelerations have the most influence. Michael W. Volk, Pump Characteristics and Applications. 3rd Ed. CRC Press 2014. Shearings generate tensions which may alter the cell integrity with tearing of the wall of the microorganisms and effusion of the cytosol. Accelerations alter the structure of the cell by increasing the gravitational force. One of the objects of the present invention is to reduce the mechanical effects imposed on the microorganisms, notably the effects of the shearing and acceleration type, in order to extend the number of cultivable species inside the reactor to those which are the most sensitive to these damageable mechanical effects, in other words provide a reactor allowing the cultivation of fragile microorganisms, such as for example fragile microalgae such as those forming chains and/or they having appendices such as bristles, flagella, and spicules. Certain microalgae have higher stress resistance, such as for example the algae of the Haematococcus pluvialis type, which in contrast to Chlorella vulgaris or Nannochloropsis oculata do not have any appendage and have a relatively thick cell wall.

Impellers for liquid medium in the invention are selected from designs well known to those skilled in the art. This includes pumps and propellers. In a particular embodiment, the circulation means is a mechanical propulsion means positioned at the leading end of a longitudinal channel. Preferentially, the impeller comprises a propeller driven into rotation by a motor, and the longitudinal channel has a housing with a widened cross-section inside which said propeller is mobile in rotation. Advantageously, the housing of the propeller is positioned shortly after the gas-exchange and or nutrient addition members of the apparatus, in order to enhance mixing of the growth medium. Advantageously, the circulation means comprises a propeller driven into rotation by a motor and in which the speed of rotation of the propeller is less than about 100 revolutions per minute, so as to limit mechanical stresses on the organisms within the liquid culture medium.

Gas Exchange

As with all microalgae cultivation processes, the invention disclosed herein requires gas exchange between interior and exterior aspects of the container. Photosynthetic production of microalgae is accompanied by evolution of oxygen and consumption of carbon dioxide. Oxygen levels in liquid growth medium above that provided by steady-state air saturation (0.2247 mol O2/M³ at 20°C) can inhibit photosynthesis in many algal species and can become prohibitive over 300% of normal air saturation. On the other hand, carbon is in high demand since carbon constitutes about 50% of dried algal biomass. The local dissolved inorganic carbon

concentration at any point in the container should not fall below a critical value, or the availability of the carbon source will limit photosynthesis.

The equilibrium concentrations of oxygen and carbon dioxide gases in the liquid can be calculated using Henry's law. The relevant Henry's constants for oxygen and carbon dioxide are 1.07 mol 02/M³/atm and 38.36 mol C02/M³/atm, respectively, in sea water at 20°C (Doran, 1995). Warm low-latitude sea surface water generally holds less CO2 (-10 μmol kg-1) and∑ CO₂ (-2000 μmol kg-1) than cold high-latitude surface water (CO₂ -15 μmol kg-1 and∑ CO₂ -2100 μmol kg-1 ), because of the enhanced solubility at low temperature. Steady state CO2 level in sea surface waters, at atmospheric pressure, in the absence of photosynthesis, is in the range of 38.36 mol CO2/M³, in sea water at 20°C (Doran, 1995). Weissman et al. (1988) reported that CO2 concentration in bulk liquid of the relatively low amount of 65 mM and pH 8.5 were satisfactory for optimal productivity of some marine- and saline-water diatoms. Similarly, Markl and Mather (1985) observed a low critical CO2 for Chlorella vulgaris: Concentrations as low as 60 mM enabled unlimited photosynthesis. In principle, CO2 limitation can be easily avoided by supplying it in excess, but use of carbon dioxide represents a major operational expense of microalgal culture; hence, means of accessing CO2 and/or preventing loss of residual CO2 in the exhaust gas should be considered.

The instant invention provides gas exchange in several technically different

embodiments. Where the embodiments describe gas flushing, they are considered "sparging" herein. The embodiments of sparging specifically recited are not intended to be limiting and further combinations and alternatives are available to achieve the function of gas exchange.

One mechanism for gas exchange includes embodiments such as those illustrated by a sparging tank in Figure 4. Gas may be sparged into the liquid growth medium via a gas passageway such as a hollow tube with a sparging valve at the end immersed in the growth medium. Sparging creates gas bubbles providing a gas-liquid interface which permits exchange of dissolved gases in the growth medium with the supplied gas as the bubble migrates towards lower pressure (generally upwards). Gas will exchange from liquid to gas or from gas to liquid according to its individual rate constant, its relative solubility in the liquid, and its partial pressure in the gas phase. Sparging is a well-known technique for exchange of gas to liquid, employed in many existing microalgae production systems and disclosed in some detail in Lundquist et al "A Realistic Technology and Engineering Assessment of Algae Biofuel

Production" online publication of Energy Biosciences Institute, University of California, Berkeley, California, October 2010 at page 111 ; and WO2012107544 to LGem SA and Georg Fischer AG. In the sparging model, the gas flow is selected to provide sufficient gas-liquid contact time to provide a desired level of mass transfer between the gas and the liquid medium. Contact time of bubble to liquid depends upon a variety of factors, especially the bubble size, gas pressure, the depth/pressure of release, and the liquid medium density. Feed gas may be selected from atmospheric gas, or from carbon dioxide enriched gas, for example, as may be obtained from flue gas (e.g. from burning fossil fuels, cement production, or the like) or from another carbon dioxide capture technique, including Direct Air Capture.

Another mechanism for gas exchange, not illustrated herein, is to insert flexible tubing for gas delivery along the bottom of a channel, and to have sparging outlets attached periodically along the tubing, thereby having a steady release of gas at multiple points along the bottom of a flowing channel. In this embodiment the gas tubing is weighted or anchored to the bottom of the channel so that it does not float, and it is fixed in place relative to the channel to limit movement. One or multiple standard gas release sparger valves may be present at each gas release site. The user can adjust the number and distribution of sparger valves along the bottom of the channel to obtain optimized gas exchange results. As is well known, smaller bubble size is preferred to increase the gas to liquid ratio, and enhance gas exchange as the bubble migrates upwards to the surface.

In this embodiment, gas will accumulate above the upper surface of the growth medium, under the solar facing surface of the container. It is necessary to release this gas. Such may be achieved by vents spaced periodically along the channel. Alternatively, an upper air channel chamber can be laid along the top of the flow channel, and accumulated gas can be swept by suction through the upper air channel and released at a central vent. The invention recognizes it is preferred to design the gas release to achieve the lowest reasonable gas headspace in the flow channel.

In another embodiment, the invention provides excellent gas exchange by means of a selective gas permeable membrane forming at least part of the upper (solar facing) surface of the container. The membrane is selected to permit exchange of O2 and CO2 between the liquid growth medium and the atmosphere through the membrane, while limiting or prohibiting the exchange of liquids. With such a membrane it is possible to reduce or even eliminate the need for a sparging tank, such as that illustrated in Figure 4.

For principles, see Camacho Rubio, F., F.G. Acien Fernandez, J.A. Sanchez Perez, F. Garcia Camacho, and E. Molina Grima. 1999. Prediction of dissolved oxygen and carbon dioxide concentration profiles in tubular photobioreactors for microalgal culture. Biotechnology and Bioengineering 62(l):71-86.

Composition of growth media and growth dynamics

The dynamics of growth in the container are understood to be extremely complex at a theoretical level. Comprehensive multivariate analysis would require knowledge of the microalgae colony growth curve (normally a sigmoidal curve with phases of initiation, exponential growth and plateau), the nutrient demand, the inhibitory effect of O2 generated and other by-products of growth, fluid dynamics of the circulating growth media (impacting light exposure and photo-inhibition of individual cells), all placed in the context of variable pH, solar flux and temperature. Though efforts to design and model the growth dynamics are encouraged (see e.g. Papacek et al. 2015 "Modeling and Optimization of Microalgae growth in

Photobioreactors: A Multidisciplinary Problem" in ISCS 2014: Interdisciplinary Symposium on Complex Systems Emergence, Complexity and Computation Volume 14, 2015, pp 277-286; Wu, X.; Merchuck, J.C. (2001). A model integrating fluid dynamics in photosynthesis and photoinhibition processes. Chemical Engineering Science, 56, 3527-3538.; Camacho Rubio, F.; Garcia Camacho, F.; Fernandez Sevilla, J.M.; Chisti, Y.; Molina Grima, E. (2003). A

mechanistic model of photosynthesis in microalgae. Biotechnology Bioenergy, 81, 459-473; Sforza, E.; Simionato, D.; Giacometti, G.M.; Bertucco, A.; Morosinotto, T. (2012). Adjusted light and dark cycles can optimize photosynthetic efficiency in algae growing in

photobioreactors. PLoS ONE, 7, art. no. e38975.), it is understood that theoretical calculations are only a starting point, and that a user of the apparatus and process of the invention will empirically determine the preferred conditions for growth using a variety of test conditions well within the competence of one skilled in the art.

Given the complex multi-organism environment, and daily changes to the environmental conditions, the user will test impeller designs and speeds within the ranges disclosed herein to select the preferred range for the stage and condition of the cultivated microalgae.

The liquid nutrient input device feeds nutrients needed to obtain the desired growth of the microalgae in a fed-batch or continuous process. Key nutrients to stimulate growth are inorganic carbon (CO2, bicarbonate and carbonate being known collectively as Dissolved Inorganic Carbon, or DIC), organic nitrogen, phosphorous, silicon, potassium, iron and others know to those in the art. Forms of organic nitrogen most valuable for growth are selected from among urea, ammonium and nitrate.

In a preferred embodiment, seawater forms the basis of the growth medium, and key nutrients are added thereto. Seawater is a solution of salts of nearly constant composition. There are over 70 elements dissolved in seawater but only 6 make up >99% of all the dissolved salts:

Since the growth medium is normally re-cycled from previous use (further described below), it is important to maintain levels of all salts to ensure sufficient micronutrients are present to permit vigorous growth. The user must be adept at measuring salts in the re-cycled growth medium and adjusting if necessary.

The pH of seawater is normally 8.1-8.3, but this can be substantially effected by the growing microalgae culture due to the consumption of nutrients and production of by-products.

For practical purposes, it can be assumed that in sea water based media, pH is regulated substantially by the partial pressure of CO2 in the growth medium, e.g. the seawater pH buffer is mainly a result of the capacity of carbonate and bicarbonate ions to accept protons. Other drivers of pH can be generally ignored in the present context. The dissolved carbonate species react with water, hydrogen and hydroxyl ions and are related by the equilibria:

of the dissolved CO₂ is in the form of HC0₃ and not in the form of CO₂. The sum of the dissolved carbonate species denotes the total dissolved inorganic carbon.

Careful consideration is given by one skilled in the art to the path and intensity of solar radiation on the growth medium because sunlight provides the energy driving photosynthesis and its efficient conversion of nutrients into biomass. Algae grown in large scale cultivation systems, such as PBRs, are exposed to a complex light environment. First of all sunlight is not constant but its intensity continuously changes during the day and the seasons. Illumination intensity has an important influence on alga productivity, for the case of Nannochloropsis salina: up to 150 μmol of photons m^-2 s^-1 an increase in illumination stimulates growth, showing that, in this range of intensities, available light is the limiting factor. Once this limit is surpassed, however, growth is not stimulated anymore by an increase in light intensity but, on the contrary, it has an inhibitory effect, causing reduction in duplication rate, a concept known as photoinhibition (see E. Sforza, et al (2012) Adjusted light and dark cycles can optimize photosynthetic efficiency in algae growing in photobioreactors, PLoS One 7 (2012) e38975. Simionato et al. 2013

Optimization of light use efficiency for biofuel production in algae. Biophys. Chem. 182 71-78.) Photoinhibition is a well-known phenomenon common to most photosynthetic organisms.

The invention herein addresses light issues in several ways. In one feature, as illustrated in Figure 2, the solar facing surface has a convex (bulging) disposition, supported by interior water pressure or structural members on the surface of the container. The bulging surface allows morning and evening sunlight to be better channeled into the growth medium by increasing the angle of incidence of sunlight with its bulging shape. The curvature is selected so that a net increase in collected sunlight in the up-rising face of the surface is greater than the light lost by having partial shading of the declining side of the surface, thus leading to an overall enhanced uptake of morning and evening sunlight and extending the effective photosynthetic period. It is noted that collection of sunlight by the uprising face also directs the sunlight into the main body of the channel, as opposed to the declining side, which if it catches low incidence sunlight, would tend to direct it by short-light path to the outer wall of the lower facing surface. The bulging surface is not anticipated to reduce solar collection when the sun is overhead, as the angle of incidence will be high enough to result in minimal reflection. It is noted that the refractive index of the wall material selected for the upper surface is preferably selected so as to be equal or better than sea water, thus tending to increase light channeling into the growth medium, rather than reflecting to space.

Another feature of the invention which enhances light management is the turbulence and depth of the longitudinal channel. It is understood that sunlight at the immediate surface may be too intense for microalgae, leading to photoinhibition, whereas cells sheltered 5-25 cm below the surface, preferably 10-20cm below the surface may receive optimal light exposure for growth. The turbulent design of the longitudinal channel of the invention is selected to cycle the growing cells through the optimal light zone (photic zone), through the photoinhibition zone and back to the dark zone, overall providing enhanced light absorption, including time for recovery from photoinhibition. By minimizing exposure to photoinhibiting levels of sunlight, and providing extended dark photorecovery, a greater net growth per volume is obtained. See

http://www.photobiology.info/Hader.html (accessed 13-July 2015); Nath et al. 2013. Towards a critical understanding of the photosystem II repair mechanism and its regulation during stress conditions. Febs 587 (2013) 3372-3381; Han et al. (2000) Effect of photoinhibition on algal photosynthesis: a dynamic model. Journal of Plankton Research Vol.22 no.5 pp.865-885, 2000. The user will understand the need for dynamic change of turbulence according to solar conditions, e.g. by changing impeller speed if growth reduction due to photo-inhibition is too high.

Container Wall - Thin Film Membrane

As acknowledged previously, the container walls must be carefully selected to achieve the objects of the invention. References in this specification to the container "surface", "wall" (particularly a flexible portion of the wall), "membrane", "film" or "thin film membrane" are more specifically referring to a material (herein sometimes "wall material") which maintains its capacity to contain a liquid (when fluidly sealed) while exhibiting some or all of the features set out herein.

The material for the upper surface may be the same or different from the lower surface, but the materials must be amenable to durable sealing between them for the formation of seams and thereby establishing channels, lanes and the flexible connections between the channels. They may be mono-crystalline or may be composite materials or formed from layers. Key functional characteristics that may be analyzed for proper selection of membranes include:

Both upper and lower membranes

High tear resistance, tensile strength and puncture resistance: Standard tests are known to those skilled in the art (including ASTM D1004 - Standard Test Method for Tear Resistance (Graves Tear) of Plastic Film and Sheeting); Substantial three dimensional flexibility to permit transfer of sea surface energy through the material without breaking; Repairability in situ:

Inevitable tears and punctures can be patched or otherwise sealed by adhesive, chemical, thermal treatments or by radiation. This criterium applies to both atmospheric face (solar surface) and the ocean face (water surface); Resistance to sea-water degradation: Able to withstand chemical and physical stress existing at the sea surface for intended effective lifespan of up to 10 or more years; Biofouling resistance: chemical or surface treatments which prevent accumulation of biofilms, other sea-life, or sediment.

Lower membrane:

Permitting rapid heat transfer between interior volume and sea-water; Optionally transparent to avoid interference of light transfer to the deeper ocean; and to avoid unwanted heating; Optionally pigmented to absorb sunlight and by heating generate internal convection currents to increase vertical circulation; Optionally reflective to return solar radiation back through the growth medium to permit further capture by microalgae.

Upper membrane

Optically clear to at least some photosynthetic wavelengths. The invention recognizes that the solar facing membrane must transmit some, but not necessarily all, photosynthetic wavelengths. Microalgae photosynthesis saturates around instantaneous light levels of 500 - 750 within the 400 to 700 nm waveband. When adapted to high-light environments, the

rate of photosynthesis saturates at a much higher irradiance, even as high as

The membrane selected preferably transmits high levels of light transmission in wavelengths suitable for the growth of the species selected; Optionally blocking to UVB wavelengths from 275-320nm, from 285-3 lOnm or especially from 295-305 nm. The invention recognizes that microalgae use considerable energy recovering from damaging UVB wavelengths. Some algae species either grow much more slowly or die when exposed to ultraviolet light. Also, algae in log-phase growth is more sensitive to UVB than when in other growth phases. If the specific algae species being utilized in the photobioreactor, or growth stage, is sensitive to ultraviolet light, then, for example, certain portions of the cover, or alternatively, the entire cover outer and/or inner surface, could be prepared with, or coated or covered with one or more UV blocking or reflecting substances that can reduce transmission of the undesired radiation. Alternatively a film or foil having UV blocking capacity in the selected wavelength range could be applied to the solar facing surface with adhesive. Light filters as described can readily be designed to permit entry into the photobioreactor system of wavelengths of the light spectrum that the algae need for growth while barring or reducing entry of the harmful portions of the radiation spectrum. Such optical filter technology is already commercially available for other purposes (e.g., for coatings on sunglasses, car and home windows). A suitable optical filter for this purpose could comprise a transparent polymer film optical filter such as SOLUS™

(manufactured by Corporate Energy, Conshohocken, Pa.). A wide variety of other optical filters and light blocking/filtering mechanisms suitable for use in the above context will be readily apparent to those of ordinary skill in the art; Optionally permeable to O2/CO2 gas exchange but not permeable to liquid H2O exchange. In one embodiment, the gas exchange apparatus is provided by a gas permeable membrane on at least a portion of the solar facing surface of the container. The principles of the gas exchange by circulating the growth medium against the solar facing surface, which is permeable to O2/CO2 gas exchange but not permeable to liquid H2O exchange are set out above.

Further desirable characteristics for the wall material employed in the invention (e.g. for extended use at the sea-surface) include optionally cleanability (to maintain optical

transparency); limited elasticity; low cost; low carbon footprint.

Moving then to the particular choice of wall material, herein sometimes called the "thin- film membrane" or simply "membrane", typical membranes may be a plastic sheet, e.g. a polyethylene sheet, or any other suitable liner. The upper surface may be constructed from a wide variety of transparent or translucent materials that are suitable for use in constructing a bioreactor. Some examples include, but are not limited to, a variety of transparent or translucent polymeric materials, such as polyethylenes, polypropylenes, polyethylene terephthalates, polyacrylates, polyvinylchlorides, polystyrenes, polycarbonates, etc. The material, in certain embodiments in combination with support elements may be designed to support external loads such as rain and hail. A wide range of plastics that are used in agriculture can be employed, including, polyolefin, polyethylene (PE), Polypropylene (PP), Ethylene- Vinyl Acetate

Copolymer (EVA), Poly- vinyl chloride (PVC) and, in less frequently, Polycarbonate (PC) and poly-methyl-methacrylate (PMMA). Permeable membranes such as Gore-Tex permits gas transfer, though the expense of large scale use suitable for the invention may be prohibitive. LDPE, HDPE, Polypropylene are examples of Polyolefins (PO). Flexibility suggests use of a low-density polyethylene (LDPE), if it has satisfactory tear strength. Biobased plastics can be considered if they do not biodegrade at the ocean surface.

The thickness selected is designed to maintain integrity under the anticipated deployment conditions on the sea surface. For plastic sheeting or film, the common unit for expressing thickness is the mil, which is one thousandth of an inch. (1000 gauge = 10 mil = 254

micrometers = 0.254 mm.). In preferred embodiments the wall material is between about 1 to 200 mil, preferably 10-100 mil in thickness, and most preferably 40-100 mil, depending on the material. The desired flexibility of thin film membranes will be found at 120 mil (3.048 mm) or below.

Also satisfactory are: EVOH (Ethylene- Vinyl Alcohol copolymer, used in coextruded plastic films to improve oxygen barrier properties. Its oxygen transfer rate (OTR) depends on its VOH (vinyl alcohol) content; LDPE (Low density, (0.92-0.934) polyethylene: Used mainly for heatsealability and bulk in packaging); LLDPE (Linear low density polyethylene): Tougher than LDPE and has better heatseal strength, but higher haze); MDPE (Medium density, (0.934- 0.95) polyethylene): Has higher stiffness, higher melting point and better water vapor barrier properties; PP (Polypropylene): Has much higher melting point, thus better temperature resistance than PE. Two types of PP films are used for packaging: cast and oriented (see OPP); PE (Polyethylene): depending on its density, it may be low density (see LDPE), medium density (see MDPE), or high density, (see HDPE), though HDPE, due to its stiffness is unlikely to be suitable as a flexible wall material. Also preferred are fluoropolymers (PTFE or FEP), polyamides, and LLDPE (metallocene linear low density PEs), polyamides, HOPE, Topas CDC, OPP, O-PET, 0-PA6, EVOH-F, EVOH-E, 0-MXD6, PEN Homo-polymer, (see Massey LK, Permeability Properties of Plastics and Elastomers, 2^nd Ed: A guide to ) It is noted that packaging for fresh meat often has similar desired characteristics of low vapour transmission with high oxygen (gas) permeation. A Modified Atmosphere Packaging (MAP) can control transmission of oxygen, carbon dioxide and water vapor. These suggestions are intended to guide one skilled in the art to a desirable selection of flexible container wall material.

Flexible thermoplastics can be used such as Acrylonitrile butadiene styrene - ABS

Polycarbonate - PC Polyethylene - PE Polyethylene terephthalate - PET Poly(vinyl chloride) -

PVC Poly(methyl methacrylate) - PMMA Polypropylene - PP Polystyrene - PS Expanded

Polystyrene - EPS. If higher quality plastics are desired, Engineered Thermoplastics such as UHMWPE (Ultra-high-molecular-weight polyethylene, sometimes shortened to UH) may be considered.

Where LDPE is chosen, selection of a preferred LDPE variant film for use in the invention requires optimization of certain functional features. The lifespan of LDPE materials in common agricultural (e.g. greenhouse) use is generally 1 to 4 years. One substantial problem leading to reduced effective lifetime is photo-oxidation. Pure polyethylene, based on its basically inert chemical structure, should not in principle be affected by photo-oxidation, since it contains no groups (such as double bonds) capable of absorbing in the near UV spectrum.

However, commercial polyethylene films contain various internal or external impurities, usually photo- absorbing chromophores, which impart photosensitivity to the films (Rabek, 1995;

Gugumus, 1979). Initiators of photo-degradation are introduced to the polymer during manufacture (i.e. polymerisation) and during processing (i.e. extrusion). The impurities lead to enhancement of photodegradation by either absorbing energy of the UV spectrum, or by being initiators of photo-oxidation reactions (in the case of carbonyl and hydroperoxide groups). Of course, the presence of these impurities depends strongly on the manufacture and processing conditions, which can in principle be altered in order to minimize them (such as metalocene- based PE). However, the associated costs may make such alterations neither practical nor desirable for the industry. See Dilara et al J. agric. Engng Res. (2000) 76, 309-321.

Photostabilizers are a useful addition to the PE films, because untreated LDPE films are easily influenced by solar radiation, heat and oxygen and degrade in only a few months by the combined action of all three elements. UV stabilizers in plastics such as benzophenones, can be added in concentrations normally ranging from 0.05% to 2%, with some applications up to 5%. Most LDPE greenhouse films in the market today comprise co-extruded multi-layered films with 4-10% EVA. Other additions to the formulation include photostabilizers (UV absorbers, hindered amine light stabilizers (HALS), Ni quenchers) and special anti-fog materials. Three- layered structures are the norm, and five- to seven-layered films are produced for specific applications. Such advanced layered films possess several advantages over a single film. Design engineering of the LDPE films to prolong both their performance and service lifetime is possible. For example, in other attempts at improving the service life of the films (Sanchez Lopez et al., 1994), use was made of blends of LDPE with linear low-density PE (LLDPE) which possess superior mechanical properties.

For the invention, it is preferred that the thin-film membrane employed as the upper (solar facing) surface, is substantially blocking to UVB wavelengths 290-3 lOnm, and particularly 295nm-305nm. This initial objective is for the prevention of DNA damage to microalgae which results in this range. Prevention of DNA damage will improve overall productivity of the cultivation apparatus. A suitable UV photostabilizer can be selected that prevents transmission of UV in this range.

Further, as indicated above, it may be advisable to provide further UV photostabilizers to improve effective lifetime of the membrane depending on the impurities. These additional UV photostabilizers may increase the range of UV wavelengths blocked from transmission, such as 290-400 nm, and thereby prevent photo-oxidative damage to the membrane. It is noted that use of such photostabilizers will further protect the microalgae from harmful UV radiation that reduces their growth rate.

The inventor recognizes that the sealed closed loop container may have a shorter effective lifespan than the exterior stabilizing frame described herein (e.g. the tension cables, catenary cables and mooring posts). It is contemplated by the invention that the container portion may be replaced every 1-2 years, while the stabilizing frame might be effective for 5-10 years or more. It is anticipated that the tension cables and catenary cables (described further below) and U-tube directional joints can all be re -used, whereas the degraded container will be re-cycled for further use, and replaced by a new one. Coatings to prevent biofilm formation may also enhance productivity of the container. Biofilms commonly cover submersed surfaces and have the capacity

to modify the accumulation of substances such as nutrients and suspended particles on the exterior face or an interior face. Biofilm development typically starts with the formation of a conditioning film of bacteria, which allows for the settlement of larger microorganisms on the new substrate. To maintain optical transparency of the container and to prevent biofilm induced drag on the exterior, bacteria resistant coatings may be employed. It is also recognized that adding neutral buoyancy solid plastic chips to the growth medium (1-5% by volume) will reduce biofilm accumulation on the interior surfaces due to their scraping and sliding against the container walls as they circulate with turbulent flow. These plastic chips are easily filtered for re-use before the harvest process begins.

Combined assessment of permeability to O2, CO2 and moisture highlights that LDPE has optimal functional characteristics, including for use as a gas exchange membrane, but other types of plastic may be suitable for certain embodiments of the invention:

Oxygen Transmission Rate and Water Vapor Transmission Rate of commonly used plastic films

Oriented polypropylene (BOPP) 550 100 9

Polyvinyl chloride PVC (rigid) 150 56 40

Polyvinyl chloride PVC(soft) 320 80 —

Polystyrene(PS) 5500 880 110-160

Polyester (PET) 60 25 27

Polycarbonate (PC) 200 35 —

Further concepts which are useful for selection of the thin-film membrane for use in the invention include Heatseal Strength: Strength of heatseal measured after the seal is cooled. This is relevant to the strength of the lane-forming seam between upper and lower sheets of the container.

It is further understood that a desirable selection criteria is durability and the potential for re-use or recycling of the container materials once they have reached the end of their useful life cycle.

Other polymeric materials, in addition to the plastics above, such as certain silicon and rubber based materials, may be suitable for use in the invention, if they demonstrate the important functional characteristics set out herein. Composites of the materials described herein may also be suitable. For example, rip-stop or reinforced composites may be effective to increase tear strength of preferred materials. Multilayer films can take advantage of the mechanical properties of one polymer and the barrier properties of another. Tie layers of adhesive polymers may be used as glue between the different layers.

The user will be adept at testing the materials that might be used for the wall material of the invention. Preferred LDPE for use in the invention is described further below. U-Tube directional joint

The directional joint, also called herein a U-tube or a "U-tube fitting" may be formed by sealing the flexible wall material on the ends of adjacent parallel channels into a closed loop for re-directing flow between the channels, or from another material such as a glass or resin- supported fiberglass or a high-strength polymer, which may in certain embodiments sufficiently rigid to be self-supporting. In all cases, the directional joint must withstand typical expected forces experienced during operation without collapse or substantial deformation. The internal forces caused by change of direction of growth medium highlight that a solid or semi-solid material with less flexibility may be desirable. Additionally, all or a portion of the U-tube may be non-transparent in certain embodiments, if it improves strength and durability. Suitable examples include a glass material or a synthetic material of the plastic or acrylic polymer type such as Plexiglas™. Such U-tubes may be made by injection molding or otherwise. It is noted that the interior dimensions of the U-tube must accommodate the flow of growth medium driven by the impeller, and thus may have approximately the same diameter and dimensions as the longitudinal channel, although improved shapes for mixing and re-directing fluid flow (e.g. various gauge changes; or use of guide vanes) are conceivable for use in the invention. The U- tube may also optionally contain a window for a gas exchange membrane. The U-tube may also optionally contain the fittings for apertures for loading and extracting growth medium, for nutrient addition, and fittings for an impeller and or a gas-exchange sparging tank as illustrated in Figure 1 , Figure 3 and Figure 4.

An optional rainwater drainage may be positioned between channels. To accommodate rain water runoff, a drain system (not shown) may be incorporated into any of the above described photobioreactor systems. In one embodiment of a drainage system, a drainage is provided along the seam between longitudinal channels. The drainage is shaped to collect run-off from the convex solar facing surface and to encourage flow along the seam between

photobioreactor units that are positioned side -by-side. This creates a drainage trench which leads water toward the edge of the photobioreactor system where it returns to the sea. In some embodiments, the drainage trench may also be designed to accommodate mechanized cleaning devices which travel the length of the seam to eliminate detritus and build-up on the apparatus.

These embodiments of the invention provide overall a sealed tube, or a sealed closed loop container, containing growth medium and suitable for cultivation of microalgae. With the gas- exchange mechanism, such as a gas permeable membrane on the solar facing surface, the bioreactor according the invention is designed in some aspects to resemble a porous cell.

Further insight into the design of microalgae cultivation systems intended for sea surface operation may be found at US patent application publication no. 2011283608 by Patel, Brennan and Magan at Cranfield University; R. Tulip at

http://rtulip.iiei/ocean based algae production system provisional patent (accessed at 15-July- 2015); J. Trent of the US National Aeronautics and Space Administration (NASA) in the proposed design for re-using wastewater aboard a spacecraft, called the OMEGA system; and Algae Systems LLC of Mobile, Alabama, with patent publications WO2008134010 and US Pat No. 8110315.

Field Array

The sealed closed loop container described herein may optionally be linked in a plurality of such containers, herein called a field array. The field array may contain from two closed loops up to 100 closed loops linked together, depending on the designer's choice. Based on this invention, the designer will attempt to maximize use of the solar facing surface while accommodating stresses of the location, anticipating failure modes for the system, and mitigating impacts on ocean and marine life to the maximum extent possible.

Any single sealed closed loop container, and any field array, requires a supporting framework in order to maintain its preferred shape in the sea surface environment. Because the field array is a preferred embodiment, the supporting framework will be further described in this context.

Among other stresses the field array will be subject to mechanical forces of tides, diverse water currents, wave action, wind, and weather conditions. Figure 5 and Figure 9 provide embodiments of the invention which are general examples of a suitable supporting framework. The user will immediately understand based on the invention and these figures that numerous variations and combinations of aspects of these figures would also be suitable to satisfy the objects of the invention.

Figure 5 and 7 provide an aerial view and a bird's eye view, respectively, of what is herein termed a "catenary frame", in this case supporting a field array comprising six closed loop containers. In Figure 5, mooring posts 51 are secured in the sea floor, preferably 2 to 30 meters below the sea surface. (Mooring posts may be supported, if necessary, by undersea supporting cables). The mooring posts rise above the sea surface from 1 to 6 meters for visibility and easy access. Mooring posts are connected around an outer perimeter by an adjustable tension cable 52, herein sometimes called a catenary cable. The catenary cable is selected from a material having high tensile strength and that floats or has approximately neutral buoyancy at the sea surface. The tension on the catenary cable may be adjusted by a tension motor (also located at 51) at a first mooring post (or alternately in the line of the catenary, not shown) which can alternately increase or reduce the tension on the catenary connected to a second mooring post. The catenary thereby provides horizontal stability to resist mechanical forces in the horizontal plane and by its neutral buoyancy avoids dipping or submergence below the horizontal plane. In these figures, the four mooring posts are connected by catenaries to form an exterior stabilizing structure. The exterior stabilizing structure is floating (except for the mooring posts) and provides an exterior stabilizing frame.

The field array is connected to the catenary cables through a series of tension cables 53 emanating from the field array (fixing and securing the tension cables to the field array are further described in Figure 6). The tension cables of the field array are either fixed or slideably connected to the catenary. An additional catenary 54 may be attached directly from a mooring post to the nearest corner of the field array. During flat calm weather, in the absence of any current, the catenaries are relaxed to a degree that provides a bare minimum of tension across the field array. Passing waves are transmitted through the flexible portions of the field array and the catenaries serve to hold the general shape in anticipation of conditions which will grow more challenging.

As water currents, wind, wave or weather conditions increase, the catenaries are tightened in the direction from which the force comes, and maintained or loosened in the trailing direction. A limited stretching across the structure due to the materials and the pressurized channels allows the area of the field array to stretch or shrink slightly under the changing tension on the catenaries. The tension on the catenaries is adjusted according to the vector addition of all forces acting on the field array (which may be contradictory). The field array continues to transmit the surface waves and minimize absorption of the force of the waves. Pinching and constriction of the channels 55 is minimized to permit continued flow of the growth medium under preferred pressure through each closed loop system. Approximate dimensions of the field array shown in Figure 5 are 100 m long by 18 m wide (and up to 0.6 m deep, not visible), with the mooring posts and catenaries lying a distance of a few meters outside of the field array.

With proper selection of thin film membranes with high tear resistance, and tension cables of high strength, and minimization of drag components, the field array with its supporting exterior stabilizing structure is enabled to withstand currents of up to 5 m/s, winds of over 100 km/hr and waves of 2 meters height (crest to valley). It is noted that preferred locations for such a design will be in relatively sheltered coastal areas naturally or artificially protected from wind, waves, currents, large tides, storm conditions and weather.

In Figure 6, an aerial view is provided where the container material of the field array is stripped away to expose the underlying tension cables and catenary frame of the invention.

Figure 8 shows a field array covering 2 hectares, with pumping, nutrient supply and gas exchange apparatuses and portals oriented towards a centre mid-line for convenient maintenance.

Those skilled in the art are familiar with materials suitable for an exterior stabilizing structure. Mooring posts maybe made from any organic (e.g. wood), synthetic (e.g. reinforced plastic) or metallic material, and any combinations of the above. An inset for a tension motor for the catenary cables is optional. Catenary cables and tension cables are made from any high tensile material or composite which is of approximately neutral buoyancy at the sea surface. Such a material can be formed of a purified polymer such as Dyneema® cable provided by Royal DSM. Alternatively, a metallic cable wrapped with foam or flotation material so as to have overall neutral buoyancy would equally achieve the objects of the invention. In any event the catenary cable and tension cable needs to demonstrate good resistance to sea water and to avoid bio-fouling. Again, the floating or approximately neutral buoyancy of the tension cables and catenaries is needed to prevent sagging below the horizontal plane of the container. Figure 9 provides an exterior stabilizing structure designed to operate without mooring posts, thus optionally in deeper ocean conditions. The exterior frame 91 is provided by sealed floating members which are flexibly connected but maintain essentially linear arrangement when disposed on the sea surface. The floating members are constructed to allow for articulating movement and temporary deforming of the configuration in response to external forces exerted on the frame. A compression member 92 urging the outward displacement of the exterior frame may be optionally provided above or below the field array (or both).

In Figure 9, the field array is again connected to the exterior frame by tension cables fixed to the field array. In this case the tension cables may optionally be fixed to the exterior frame by fixed or slideable connection.

Figure 9 illustrates that corners and various points along the exterior frame are attached to underwater tension cables 93 which are directly or indirectly connected to a plurality of sea floor anchors 94. The sea floor anchors are positioned at a horizontal distance to provide resistance to motion of the exterior frame. An optional tension motor 95 at each connection point permits the adjustment of tension for each underwater tension cable so as to optimize the positioning of the field array depending on sea condition, wind, weather, tide and current.

Designs and mechanical parts for designs of Figure 9, and their anchoring or mooring systems, may conveniently be taken from aquaculture practices, for example fish cages used in the open sea. Examples may be found in Aqualine AS patent applications W091/17653 and WO 2014/189383. Materials selected and designs can be seen in many working embodiments, or as disclosed in e.g. US Patent Application Pub. No. 2010/0224136; US Pat No. 4957064 to Nippon Kokan KK; US Pat No. 5299530 to Occidental Research Corp; or US Pat No. 5412903 to Marine Industries and Investments Ltd. For use in locations with particularly heavy ocean-going conditions, it may be desirable to use an adjustable flotation system (not shown). In one embodiment, floodable buoyancy is provided, comprising one or more floodable frame members, such as a ballast tank, located at the undersea cable connection point on the exterior floating frame. During normal use, such a member is adjusted to neutral buoyancy. The adjustable flotation can be induced to increase buoyancy under heavy conditions thus permitting increased tension on the undersea anchor lines, without causing submergence of the floating frame beneath the ocean surface.

It is further noted that when open sea conditions get too extreme and present risk of system failure, the most convenient way to protect the field array is to have floodable floating members which can be simultaneously flooded to submerge the field array, and all associated pumps and equipment, at a depth below the risk area presented by the storm current and waves. The field array can be safely secured at such depth (from 2 to 25 meters) until the storm conditions pass, causing little or no damage to the cultivated microalgae. Upon return of fair weather and daylight the buoyancy of the flotation system is reactivated as water is blown out from the tanks using compressed air or an air-pump and the field array can return to the sea surface under controlled conditions. An example of such a submergible construction in the aquaculture industry is found in US Patent Application publication no. US2006130728.

Figures 5 and 9 highlight that the field array is somewhat independent of the exterior floating frame employed to support it.

Figure 10 provides one embodiment of a manufacturing process for a container of the invention. The lower surface thin film membrane 101 is laid across a seaming roller 102, each individual roller wheel 103 being separated by a desired channel distance, and each roller wheel providing a surface for forming a seam by sealing 104 the upper membrane to the lower one. In this example, the lower surface, being greater length, is induced to droop between the roller wheels to a desired depth 106. A vacuum apparatus may be employed to position the lower surface membrane. The upper membrane 105 is provided flat and is induced to contact the lower membrane at the roller wheel for seaming at the seaming surface 104. The seam is a sealed across a desired width (1-20 cm, with 10 cm preferred) by heat, chemicals or radiation, according to the suitability for the materials selected. The seams form the channel barrier and substantially prevent liquid exchange between the channels. The resulting seam sealed membranes have a lower surface length somewhat longer than the upper surface (also shown in Figure 2). The ratio of lower to upper surfaces is preferably from 1.1 : 1 to 2.0: 1. Each channel between seams is normally a constant shape, and of length selected by the maker, and open ended when first manufactured. The shape of seams may be adjusted at the ends to enhance the circulation patterns (i.e. prevent sedimentation) around the directional joint (not illustrated).

As noted previously, it is desirable to support the field array with tension cables which can connect the Field array to the exterior floating frame. Figure 11 illustrates one method by which tension cables can be fixed to the field array to distribute stress. Tension cable 111 is fixed with a plurality of cross securing members 112 made from material compatible to fix securely to the tension cable and to the thin film membrane of the field array. The tension cable so fixed is laid along seam 113. The securing members are sealed to field array along the seam. This step can be taken either at the time of seam formation, or afterwards when the seam has been formed.

It is further noted that in alternative embodiments, one or more of the longitudinal channels may be arcuate, serpentine, or otherwise non-linear, if desired. The key design parameter is effective use of a defined solar surface area for enhanced productivity of microalgae per unit. The figures present non-limiting examples of preferred geometrical arrangements. Many alternatives are possible within the scope of the claims hereof.

Microalgae species

The process of the invention for cultivation of microalgae requires selection of a suitable microalgae species and growth conditions to achieve the desired growth rate.

"Microalgae" as used herein refers to the diverse range of single cell and simple multi- cell organisms which are photosynthetic organisms and primarily photoautotrophs. The word microalgae encompasses phytoplankton of diverse classes as diatoms and green algae (both of which are eukaryotes) and the prokaryotic cyanobacteria (blue-green algae), and many other classes, genuses and species. Microalgae does not include macroalgae, which are generally visible to the naked eye and sometimes called seaweed or seagrass.

The term "photosynthetic organism", as used herein, includes all organisms capable of photosynthetic growth, such as plant cells and micro-organisms (including algae, euglena and lemna) in unicellular or multi-cellular form that are capable of growth in a liquid phase. These terms may also include organisms modified artificially or by gene manipulation. While certain photobioreactors disclosed in the context of the present invention are particularly suited for the cultivation of microalgae, it should be understood that, in other embodiments, other

photosynthetic organisms may be utilized in place of or in addition to microalgae.

Diverse microalgae species are known to be suitable for cultivation. Preferred microalgae are those which can withstand mechanical stresses or contaminations better than the other ones, such as for example the algae of the Chlorella, Scenedesmus, Skeletonema, Odontella or Nannochloropsis type. Also preferred are species with high lipid content at harvest such as those set out in the Table:

In one embodiment, an axenic culture of one microalgae species is selected for growth, it being understood that in the large production volumes of the invention, the selected microalgae will co-exist with diverse other organisms such as bacteria, predators (multicellular or single cell), viruses and possibly some multicellular contamination. In one embodiment, a substantial proportion of the biomass may be from non-photosynthetic organisms which compete with the algae for resources, in which case, though detrimental to the microalgae growth rate, does not fall outside of the claimed invention.

A mixed species of microalgae may also be employed in the container of the invention. One such embodiment utilizes more than one species of algae, selected from among Chlorella, Chlamydomonas, Chaetoceros, Spirolina, Dunaliella, Porphyridum, etc. Combinations of microalgae are selected based on empirical or theoretical combinations which are anticipated to enhance the overall accumulation of biomass and desired products such as lipids. Species may be selected based on differential demands for nutrients. A superior source for axenic microalgal culture is the Bigelow lab at NCMA which can provide many diverse species. See

https://nema^>igelow.org/ncma-new (last accessed

July 14, 2015).

Further microalgae which may be cultivated in mono-culture or in mixed species culture include:

Cyanobacteria: Divisions Cyanophyta and Procholorphyta, including Spirulina, Nostoc and Arthrospira platensis;

Chlorophyta: Chlorella, Scenedesmus, Ettlia, Nannochloris and Monoraphidium;

Heterokonts: diatoms, Eustigmatophytes and Chrysophytes, including Nannochloropsis,

Nitzschia, Navicula, Amphiprora, Amphora and Phaedodactylum; and others known in the art including Dunaliella tertiolecta, Euglena gracilis, Phaeodactylum tricornutum, Chrysotila carterae, Prymenesium parvum, Scenedesmus dimorphus, and Tetraselmis chui. A good source for further details is Biomass and Biofuels from Microalgae (N R Moheimani et al. Eds.) Springer (2015).

It is recognized that it is preferred that the cultivated microalgae species be selected from among those naturally found in the oceanographic region near the location of the container. This preference recognizes that some leakage and container failure is inevitable over time, and that it would be preferred that the leaked species not be foreign to the local environment. For the same reason it is preferred that the species used are not genetically modified from the natural variants present. This preference for local, unmodified microalgae does not preclude natural breeding and hybridization programs, or targeted mutagenesis approaches, to select the optimal and highest performing local species. Feedstock for a cultivation cycle may be provided from either or both of a fresh feedstock cultivated in a photobioreactor, or from a portion of the previously grown crop. It is noted that not all the mature crop needs to be harvested. A portion of the crop may be left behind to provide the feedstock for the next generation. In one embodiment, the desired percentage of harvestable growth medium is extracted (between 5% and 99%, preferably about 25-60% or about 75-90%) while fresh growth medium is pumped into the container. In another

embodiment, the entire crop is extracted, and a sub-loop outside the container diverts a desired proportion of the harvest stream (e.g. between about 5-50%, preferably 10-20%) into the incoming fresh growth medium, thus providing more consistent mixing for the incoming medium.

Alternatively, the fresh feedstock can be provided from a culture grown in a preparation photobioreactor. A combination of both sources may also be used (existing crop plus new feedstock from a separate photobioreactor). This latter combination is especially useful when two or more species are grown together, and one grows more quickly than the other. The fresh feedstock would contain increased levels of the slow growing species.

Growth medium and fed-batch/continuous cultivation

"Growth medium" as used herein refers the liquid medium which provides the environment for microalgae growth, circulation, and/or transportation by liquid flow. Prior to addition of microalgae feedstock, the growth medium may be sterile or it may contain some level of contaminating species, so long as the contaminating species do not reduce overall growth of microalgae to an unacceptable level.

It is advantageous to utilize a growth medium based on sea water, brackish water, fresh water, wastewater effluent, and/or other non-potable water obtained from a locality in which the photobioreactor system will be operated and from which the microalgae contained therein was derived or is adapted to. Particular liquid medium compositions, nutrients, etc. required or suitable for use in maintaining a growing algae or other phototrophic organism culture are well known in the art. Potentially, a wide variety of liquid media can be utilized in various forms for various embodiments of the present invention, as would be understood by those of ordinary skill in the art. Potentially appropriate liquid medium components and nutrients are, for example, discussed in detail in: Rogers, L. J. and Gallon J. R. "Biochemistry of the Algae and Cyanobacteria," Clarendon Press Oxford, 1988; Burlew 1961 ; and Round, F. E. The Biology of the Algae. St Martin's Press, New York, 1965; each incorporated herein by reference). Well known growth media include BG11 , ASNIII and seawater enrichment medium. Many acceptable culture media have been developed for individual strains to optimize growth or other desirable characteristics. Culture media can be obtained through biological supply companies or prepared in the laboratory (UTEX, 2010). Generally, media are composed of macronutrients, trace elements, and vitamins (Anderson, 2005, Algal culturing techniques. Elsevier Academic Press). A good example of culture initiation and scale up is available from the UN Food and Agriculture Organization at

July 2015).

Those skilled in the art are aware that seawater based medium has a density of at least 1020 g/1, and normally closer to 1035 g/1, whereas typical municipal wastewater has density of about 1002.2 g/1, including both suspended solids and biological waste. Where sewage effluent is highly concentrated, it can have density of up to 1012 g/1 (See: T.G. Ellis; "Chemistry of Wastewater" EOLSS.net retrieved 18-Aug-2016; UN Food and Agricultural Organization.

recognizes that when microalgae is cultivated in wastewater, the platform will have an average density at 1012 g/1 (at maximum) or less; whereas a seawater based growth medium will have an average density of 1020 g/1 (at minimum) or higher, including 1025, 1030, 1035 or 1040 g/1. When a container disposed on the sea surface is filled with a wastewater based growth medium it has a significantly higher positive buoyancy than when filled with a seawater based growth medium.

In a preferred embodiment, growth medium is taken from microalgae-depleted medium resulting from dewatering operations used at harvest. Microalgae is harvested at very low densities, generally less than 0.1% w/v, therefor over 95% of liquid medium is available for reuse immediately after dewatering. The re -used growth medium may if necessary be treated to improve its characteristics, such as by ultraviolet treatment or chemical treatment to limit viruses, bacteria and other contaminants. Such treatment must not inhibit the potential for future use of the medium. The re -used medium may also desirably be adjusted by gas exchange and nutrient addition to make it suitable for the initiation of a new crop. Optionally the re-used growth medium is mixed with fresh liquid medium.

The temperature of the liquid medium during the growth cycle should be optimized for the species selected. Generally, the medium will be maintained between about 5 degrees C. and about 45 degrees C, more typically between about 15 degrees C. and about 37 degrees C, and most typically between about 15 degrees C. and about 30 degrees C. For example, a desirable operating temperature for a photobioreactor utilizing Chlorella could have a liquid medium temperature controlled at about 30 degrees C. during the daytime and about 20 degrees C. during nighttime. In one embodiment, the temperature of the photobioreactor is maintained at about 20 degrees C. The growth medium is a dynamic environment with constantly changing nutrient and dissolved gas levels depending on the state of growth and density of the culture therein. It is well known from the field of biotechnology that cell-culture can be enhanced by careful control of the nutrient conditions. Fed-batch production provides nutrient and dissolved gasses in a periodic bolus. Fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. Where the feed is constantly adjusted, it is known as constant feed or continuous feed. Exponential feed is a form of continuous feed that may be considered where useful to drive a preferred result. A preferred embodiment of the invention employs fed-batch or continuous feed of the growth medium, as opposed to simply providing an initial well-stocked growth medium (e.g. wastewater) and allowing consumption of nutrients over time. Fed-batch and continuous feed are preferred to develop the high productivity culturing methods that are desired to achieve economic feasibility of these systems.

The invention may achieve its objects by maintaining culture pH through the use of stoichiometrically-balanced growth medium. Elevated CO₂ may be provided for pH control in addition to providing enhanced CO2 availability. pH may also be controlled by a balance of organic nitrogen compounds so that the total nitrogen in the form of ammonium ions is close to achieving a stoichiometric balance, which would avoid excess proton secretion or uptake.

Incremental addition of ammonium and/or nitrate ions can be used to control pH as long as the carbon availability is not severely limited. Substantial improvement in biomass yield can be observed by modulating the type and amount of organic nitrogen. (Scherholz and Curtis BMC Biotechnology 2013, 13:39) Enhanced productivity will depend on the single or multi-species microalgae selected, and the correct modulation of the growth medium during the course of a growth cycle. Those skilled in the art are aware of basic growth requirements, and substantial improvements can be gained by simple testing well within the capability of those skilled in the art.

Under high growth conditions sought by the invention, microalgae typically replicate every 0.5 to 3 days, preferably around once per day. The culture density thus exhibits a standard sigmoidal growth curve with an initiation phase followed by log phase growth. A plateau phase is normally reached due to nutrient limitation, catabolite repression, or density effects which prevent further rapid growth. More details on typical growth profiles are found in Schuler and Kargi, 2002, Bioprocess Engineering: Basic Concepts (2nd ed.). Upper Sadie River, N.J.:

Prentice Hall, Inc. Commonly, populations of cells are quantified by direct cell enumeration, otherwise known as cell counting. Such growth measurements can be easily translated into kinetics models for batch culture. Cell kinetics is usually plotted as concentration of cells versus time.

It is noted that the initiation phase may be avoided if the crop is harvested during log phase, and a portion of less than 100%, for example 90%, 80%, 70%, 60%, 50% or between 1- 50%, preferably 5-35%, more preferably 10-20% is diverted from the harvest stream and retained in the growth medium, there being fed with fresh growth medium, thus initiating a new crop with a highly active feedstock. The decision to employ log phase feedstock from a previous crop may be based on optimization of the intended result. At a macro level, the intended result is productivity per unit area over time.

Finishing of cultivation It is known in the art that prior to harvest, it may be desirable to adjust growth conditions, such as the nutrient conditions, in order to drive the microalgae towards accumulation of a preferred constituent, such as lipid quantity. Nitrogen limitation is one technique that has been employed to trigger an accumulation of lipid in microalgae, which accumulation is possibly a biological stress response. Limitation of other nutrients may be found to drive accumulation of lipids or other desired products. Similarly addition of chemical inhibitors may drive lipid synthesis pathways. Use of such techniques are described as "finishing" in the instant specification.

Finishing is not required for using the present invention, but the decision to employ it may again be based on optimization of the intended result. At a macro level, the intended result is productivity per unit area over time. If the net gain in product, e.g. lipid, is enhanced by a short phase of finishing, then finishing may be employed. However, if increase in lipid per cell is less than would be achieved by total lipid accumulation resulting from rapid growth under log phase growth, then finishing is not useful. If finishing is desired, the consumption of nutrients in an individual sealed closed loop system is allowed to proceed, and all nutrients/gasses except the limiting nutrient are added, thus leading to the desired stress condition, for the desired time. The stressed crop is harvested when the peak lipid accumulation is achieved.

Monitoring Growth and Cultivation

In an embodiment of the invention, the condition of the growth medium is periodically or constantly monitored for one or more of pH, level of dissolved 0₂, CO2, inorganic carbon, organic nitrogen (including any form of ammonium, nitrate and urea), solar insolation, solar flux, temperature, optical density, and/or any other aspect identified as critical to the growth process. Monitoring may be by any standard automated device, and the data on the condition of the growth medium may be transmitted electronically to a receiver. Single or multiple monitors may be employed and placed at various points in a sealed closed loop or on the field array. Careful monitoring provides the opportunity for real-time adjustments to growth medium in the fed-batch process, including the nutrient being added, the gas exchange rate, and the impeller speed.

For computer aided control of the cultivation apparatus and process, a computer, e.g. a computer controlled system, may be used to control the operation of the various components of the photobioreactor sections, units and systems disclosed herein, including nutrient supply, gas exchange, valves, sensors, pumps, etc. Operational experience monitoring the growth medium will lead to the development of algorithms for adjustments to improve productivity. Recently this technique has become known as precision agriculture, or in the instant invention, will be known as precision aquaculture. Certain embodiments may employ computer systems and methods described in International Publication No. WO2006/020177. In addition to automating operation of aspects of the photobioreactor system, use of a computer-implemented system may facilitate optimizing or improving the efficiency of the system by determining suitable values for various control parameters. In some embodiments, flow may be controlled to provide a desired level of turbulence and light/dark exposure intervals for improved growth, and described and determined according to methods also described in International Publication No.

WO2006/020177. The computer-implemented method for managing the growth conditions of a microalgae cultivation platform disposed on the sea surface therefore comprise monitoring one or more conditions of the growth medium selected from among pH, levels of dissolved 0₂, CO2, inorganic carbon, organic nitrogen (including any form of ammonium, nitrate and urea), solar insolation, solar flux, temperature, and optical density to generate data; transmitting the data by automated device to a receiver, analyzing the data received at a computer to determine if the conditions of the growth medium correspond to optimized growth conditions; and adjusting one or more of impeller speed, nutrient flow and gas flow in the microalgae cultivation platform if the determined conditions are not optimized. The computer-implemented method may also integrate monitoring of ambient environmental conditions relevant to the cultivation platform including but not limited to local wind speed, wave height, precipitation, sea currents and tides, and strain and stress measures on the supporting cables of the platform.

Harvesting the crop and use of the Microalgae Biomass

Over the past several decades a large body of knowledge has developed regarding harvesting microalgae, then processing it for food and feed value, or for extracting valued components for biofuels and specialty chemicals. Harvesting and extracting technologies are well known (See e.g. Milledge and Heaven (2013) A review of the harvesting of micro-algae for biofuel production. Rev Environ Sci Biotechnol 12: 165-178), and will not be described in detail herein, except to note that extracting the microalgae growth medium from the container when desired microalgae growth has been achieved requires one or more valves connected to an outflow line, which valve(s) can be opened when outflow is desired. Such lines are illustrated in Figure 1. The fluid lines are connected to a harvesting/filtration facility that may be floating, fixed to the seabed, or located onshore, in either case preferably relatively nearby so as to minimize time between extraction and processing.

After harvesting, it has been noted that Algae-depleted growth medium resulting from dewatering operations may be returned to cultivation system (after optionally being mixed with fresh liquid medium), to return unused nutrients to the system. Alternatively, the left-over growth medium may be disposed of. Harvest density of microalgae is normally very low, being less than about 0.1% w/v (weight per volume). The first dewatering step increases the density to 1-20% w/v, preferably 5- 15% w/v, and most preferably about 10% w/v. Further processing steps are normally employed to generate commercial products such as food supplements for humans and animals, or for specialty chemical feedstocks. In certain embodiments, at least a portion of the biomass, either dried or before drying, can be utilized for the production of products comprising organic molecules, such as fuel-grade oil (e.g. biodiesel) and/or organic polymers. Methods of producing fuel grade oils and gases from such dewatered microalgal biomass are known (e.g., see, Dote, Yutaka, "Recovery of liquid fuel from hydrocarbon rich micro algae by thermo chemical liquefaction," Fuel. 73:Number 12. (1994); Ben-Zion Ginzburg, "Liquid Fuel (Oil)

From Halophilic Algae: A renewable Source of Non-Polluting Energy, Renewable Energy," Vol. 3, No 2/3. pp. 249-252, (1993); Benemann, John R. and Oswald, William J., "Final report to the DOE: System and Economic Analysis of Micro algae Ponds for Conversion of CO.sub.2 to Biomass." DOE/PC/93204-T5, March 1996; and Sheehan et al., 1998; each incorporated by reference). Algal biomass can be used directly, when dried, as a solid fuel for use in a combustion device or facility and/or could be converted into a fuel grade oil (e.g., biodiesel) and/or other fuel (e.g., ethanol, methane, hydrogen). The biomass can alternatively be processed by hydrothermal liquefaction to obtain a bio-crude suitable for diverse uses, including upgrading to drop-in fuels. A wide variety of techniques for biofuel production from microalgae feedstocks are set out in such references as Y. Chisti (2007) Biotechnology Advances 25 (2007) 294-306 and the 2012 publication of the US National Research Council of the National Academies "Sustainable Development of Algal Biofuels in the United States". 2012 International Standard Book Number 13: 978-0-309-26032-9. All references cited in this specification are hereby incorporated by reference.

Examples

Example 1

A container is made from two sheets of a 50 mil LDPE, optically clear with a measured visible light transmission of 95%, both 100 m long. The top one is 4.5 m wide, the lower one is 6.1 m wide. The lower sheet is spread along a mold of 4 convex depressions, each depression separated by a horizontally flat sealing ridge of 10cm thickness. The lower sheet is held in place by vacuum suction. The depressions have a circumference length of 1.4 M each. The top sheet is stretched flat and overlays the lower sheet, making contact with the lower sheet along each sealing ridge. The width of the upper sheet is 1.0 m between sealing ridges (1.1 m from mid- ridge to mid-ridge). The 10 cm wide seals are formed by a heated roller. When the seam is secured sufficiently such that it will not leak liquid across it (between channels), and so it can withstand tugging and stretching forces to be encountered on the sea surface, the container is released from the mold. The seam sealed container bag contains 4 independent channels, initially open-ended.

One end of an optically clear U-tube directional joint made from an approximately neutral buoyancy acrylic polymer (such as Plexiglas) is inserted in the distal open end of a first channel, while the other end of the U-tube is inserted into the distal open end of the adjacent channel. The U-tube is sealed to the container, thus fluidly connecting the two parallel channels with no leakage. The U-tube directional joint is prepared from a mold and shaped to insert into the container without providing avenue for leakage (thus corresponding to the inflated shape of the channel), and to carry the liquid volume delivered in the channel. At the proximal end of the container bag channels, another U-tube is inserted. Again, the U-tube fluidly connects the open proximal end of the first channel to the open proximal end of the second channel. At this proximal end, the U-tube is adjusted to accommodate an impeller screw for the growth medium, a gas exchange apparatus including a sparger bubbling CO2 enriched gas, and a liquid nutrient supply portal. Each mechanism is connected to a power source and pump, as necessary. The entire combination of mechanisms and the U-tube is sealed to prevent fluid leakage and thus establishing a fluidly sealed loop and a sealed volume.

The container bag with four channels thus forms two separate liquidly sealed containers, in a field array, according to the invention.

The field array is disposed to float on the sea surface in a sheltered bay. Tension cables attached to the field array are connected to an exterior frame of catenary cables which are themselves connected to fixed mooring posts. Sea water based microalgae growth medium is loaded from a portal in the U-tube into the interior of each fluidly sealed container. Total volume of each two channel raceway is 92 m³, with total circuit length slightly over 200m.

Growth medium flow rate is initiated at an effective plug-flow rate of 0.05 m/sec during daylight hours. At steady state the impeller generates a head pressure of 0.5-2.0 m. Average cycle time for the growth medium is slightly over one hour during daylight. Throughout the daylight hours, the gas sparger evenly distributes bubbled atmospheric gas enriched to 5% CO2 at 1 litre/min. Flow-through gases are removed from the headspace of the gas exchange apparatus at the same volume rate as added by the sparger. Temperature of the growth medium is the same as the sea water contacting the ocean face of the container, generally in the range of 22-30 C, though it may be a few degrees higher during daylight hours. Example 2

In the field array of Example 1, the growth medium selected is sea water adjusted to comprise 0.075 g/1 NaN0₃ and 0.00565 g/1 NaH₂P04.2H₂0. A seed stock of algae species Nannochloris sp. in a log-phase initiation colony is injected through the portal into the growth medium. The growth medium is circulated with turbulent flow. Average circulation cycle time for the growth medium is slightly over one hour during daylight. Flow rate may be reduced after the growth medium concentration has equilibrated after dusk. Throughout the daylight hours, the gas sparger evenly distributes bubbled atmospheric gas enriched to 5% CO₂ at 1 litre/min. Gas flow is reduced after dusk when excess oxygen has been removed from the growth medium and photosynthesis has ceased in the darkness.

Monitors provide data on concentration of dissolved gases, pH and liquid nutrients during the course of the growth cycle. Solar insolation and temperature are also recorded. Nitrate and phosphate is added as required, and when required, to maintain their concentrations within +/- 20% of starting values. pH is adjusted by increasing the rate of sparger flow if below pH 7.5 and decreasing sparger gas flow if pH is above 8.5 (pH being governed substantially by dissolved inorganic carbon (DIC) in the seawater based growth medium). Nannochloris grows until it reaches a culture density of from 0.2 to 10 grams/liter, as desired by the user, whereupon it is harvested according to techniques known in the art. Growth rate over the course of 7 days is 10- 20 g dry weight microalgae per m² (sea surface area) per day.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many variations and equivalents to the specific embodiments of the invention described herein, all of which are intended to be encompassed by the claims of this invention.

Claims

CLAIMS What is claimed is:

1. A container for cultivation of microalgae comprising:

a) a wall material having transparency to at least some photosynthetic wavelengths on a solar facing surface,

b) an impeller to circulate microalgae in a microalgae growth medium in the container, c) an aperture for nutrient addition, and

d) a mechanism for gas exchange between interior and exterior aspects of the container, wherein the interior volume of the container is sealed to contain the microalgae, and the container floats with approximately neutral buoyancy when filled with microalgae growth medium and disposed in sea water.

2. The container of claim 1 wherein gas exchange is provided by sparging.

3. The container of claim 1 wherein a substantial portion of the wall material comprises a flexible thin film membrane.

4. The container of claim 3 wherein the flexible thin film membrane is substantially

impermeable to water.

5. The container of claim 3, wherein at least a portion of the container wall is selected from a low-density polyethylene (LDPE).

6. The container of claim 5 wherein the LDPE is a modified or composite LDPE having higher tensile strength and/or higher tear resistance than pure LDPE.

7. The container of claim 3, wherein the flexible thin film membrane has greater than 80% transparency to at least some wavelengths in the range 400-700nm.

8. The container of claim 7, wherein the flexible thin film membrane is substantially

blocking to wavelengths 290-320nm.

9. The container of claim 1 wherein the aperture for nutrient addition is adapted for fed- batch or continuous feed.

10. The container of claim 3, wherein when sealed, the sealed volume comprises at least half liquid phase and less than half gas phase.

11. The container of claim 10, wherein the sealed volume comprises at least about 95%

liquid phase and less than about 5% gas phase.

12. The container of claim 3, which comprises two or more longitudinally attached channels with the same number of directional joints fluidly connecting the volume of the channels.

13. The container of claim 12, wherein one or more of the directional joints is made from an at least partially transparent material which is less flexible than the longitudinal sections of the container, or is substantially solid.

14. The container of claim 12, wherein one or more of the directional joints is a U-tube

fitting inserted at the end of adjacent channels, thereby fluidly connecting a first channel with a second channel.

15. The container of claim 12, wherein a plurality of channels are connected by directional joints including one or more U-tube fittings.

16. The container of claim 15, wherein the U-tube fitting is transparent to at least some

photosynthetic wavelengths.

17. The container of claim 12, wherein the sealed container consists of 2 or more longitudinal channels and the same number of directional joints.

18. The container of claim 12 wherein the sealed container consists of 4 or more longitudinal channels and the same number of directional joints.

19. The container of claim 12 wherein the container is disposed in a plural array of 2 or more flexibly connected containers comprising a field array.

20. The container of claim 19, wherein the field array comprises 4 or more fluidly sealed loops.

21. The container of claim 19, wherein the field array covers at least about 0.2 Ha of sea surface.

22. The container of claim 19, wherein the field array covers at least about 0.5 Ha of sea surface.

23. The container of claim 19, wherein the field array covers at least about 1.0 Ha of sea surface.

24. A process for cultivation of microalgae comprising

a. seeding a feedstock of microalgae into a container comprising wall material

having transparency to at least some photosynthetic wavelengths, the container being sealed to contain microalgae, and disposed to float with approximately neutral buoyancy at the sea surface,

b. circulating the microalgae in a microalgae growth medium within the container, c. providing nutrients to the microalgae growth medium via an aperture at more than one time-point or continuously during the course of a growth cycle, d. providing gas exchange between interior and exterior aspects of the container; and e. extracting the microalgae growth medium from the container when desired

microalgae growth has been achieved.

25. The process of claim 24 wherein the gas exchange is provided by sparging.

26. The process of claim 24 wherein a substantial portion of the wall material comprises a flexible thin film membrane

27. The container of claim 26 wherein the flexible thin film membrane is substantially

impermeable to water.

28. The process of claim 27 wherein at least a portion of the container wall is selected from a low-density polyethylene (LDPE).

29. The process of claim 28 wherein the LDPE is a modified or composite LDPE having higher tensile strength and/or higher tear resistance than pure LDPE.

30. The process of claim 28 wherein at least a portion of the container wall is selected from an LDPE having greater than 80% transparency to at least some wavelengths in the range 400-700nm.

31. The process of claim 28 wherein the LDPE has less than 50% transparency to

wavelengths 290-320nm.

32. The process of claim 24 wherein the growth medium has density of about 1020 g/1 or greater.

33. The process of claim 24 wherein a sealed volume is formed from two or more flexibly connected longitudinal channels with the same number of directional joints fluidly connecting the longitudinal channels into a circuit.

34. The process of claim 33 wherein one or more of the directional joints is made from an at least partially transparent material which is less flexible than the longitudinal sections of the container, or substantially solid.

35. The process of claim 34 wherein one or more of the directional joints is a U-tube fitting inserted at the end of parallel channels, thereby fluidly connecting a first channel with a second channel.

36. The process of claim 35, wherein the U-tube fitting is transparent to at least some

photosynthetic wavelengths.

37. The process of claim 33 wherein the sealed volume comprises at least about 75% liquid phase and less than about 25% gas phase.

38. The process of claim 33 wherein the sealed volume comprises at least about 95% liquid phase and less than about 5% gas phase.

39. The process of claim 33 wherein the container is disposed in a plural array of 2 or more flexibly connected containers comprising a field array.

40. The process of claim 39 wherein the field array comprises 4 or more flexibly connected containers.

41. The process of claim 39 wherein the field array covers at least about 0.2 Ha of sea

surface.

42. The process of claim 24 wherein the nutrient is provided to the microalgae growth

medium in a fed-batch or continuous process.

43. The process of claim 42 wherein the nutrient provided changes according to the growth stage of the microalgae colony.

44. The process of claim 43 wherein the nutrient provided is different between day and night.

45. The process of claim 42 wherein the nutrient provided is selected according to anticipated growth rates in the coming cycle based on predicted weather conditions.

46. The process of claim 45 wherein predicted weather conditions include air temperature, sea temperature and solar insolation.

47. The process of claim 24 wherein the condition of the growth medium is periodically or constantly monitored for one or more of pH and levels of dissolved 0₂, CO2, inorganic carbon, organic nitrogen, solar insolation, temperature, and optical density.

48. The process of claim 47 wherein the monitoring is by automated device, and the data on the condition of the growth medium is transmitted to a receiver.

49. The process of claim 48 wherein adjustments to the nutrient provided and/or impeller speed are made according to data received from a monitoring device.

50. The container of claim 1 wherein the impeller force can be adjusted by an operator or automatically.

51. The container of claim 50 wherein the impeller force is automatically adjusted based on an algorithm comprising factors including current or predicted solar insolation, temperature and growth culture density.

52. The container of claim 1 supported on the sea surface by an exterior stabilizing structure.

53. The container of claim 52 wherein the exterior stabilizing structure comprises a plurality of mooring posts and a plurality of catenary cables interconnecting the mooring posts, which catenary cables connect to tension cables fixed to the container itself.

54. The container of claim 53 wherein the catenary cables and tension cables are made from material that floats on sea water.

55. The container of claim 53 wherein tension on each catenary cable may be individually adjusted.

56. The container of claim 53 wherein the tension on each catenary cable is adjusted

according to weather and/or sea current conditions.

57. The container of claim 52 comprising a floating exterior frame operably connected to an underwater anchoring system and which floating exterior frame connects to tension cables fixed to the container itself.

58. A bioreactor for growth of microalgae, said bioreactor being sealable to contain

microalgae in a microalgae growth medium, wherein

a) the bioreactor wall material is a flexible thin film membrane which is

substantially impermeable to water, and

b) a solar facing wall is transparent to at least some photosynthetic wavelengths but substantially blocking to UVB wavelengths 295-305 nm, and

c) the microalgae growth medium has density of 1020 g/1 or higher.

59. A computer-implemented method for managing the growth conditions of a microalgae cultivation platform disposed on the sea surface comprising:

a) monitoring in a microalgae cultivation platform disposed on the sea surface one or more conditions of the microalgae growth medium selected from among pH, levels of dissolved 0₂, CO2, inorganic carbon, organic nitrogen, solar insolation, temperature, and optical density to generate data;

b) transmitting the data by automated device to a receiver,

c) analyzing the data received at a computer to determine if the conditions of the growth medium correspond to optimized growth conditions;

d) wherein if a condition of the growth medium does not correspond to optimized growth conditions, the computer sends a further signal to adjust one or more of impeller speed, nutrient flow and gas flow in the microalgae cultivation platform.

60. The computer-implemented method of claim 59 further comprising monitoring one or more ambient environmental conditions including wind speed, wave height, precipitation, sea current and tide, and strain/stress on the platform and/or its supporting cables.

61. Claim 1 or 24, wherein the container comprises an upper (solar facing) surface, and a lower (ocean facing) surface, and the ratio of the area of the lower surface to the upper surface is about 1.1 to about 2.0.

62. A method of manufacturing a plurality of flexibly connected longitudinal channels for cultivation of microalgae comprising,

a) Providing a first sheet of membrane material,

b) Overlaying a second sheet of membrane material

c) Sealing the second sheet to the first sheet by heat, chemical or radiative means along a plurality of seams,

wherein each seam has a width from about 1cm to about 20cm wide, and each seam is separated by about 20cm to about 200 cm, and wherein the length of each seam is from about 20 meters to about 200 meters.

63. The method of claim 62 wherein at least 6 longitudinal channels are created in parallel array.

64. The method of claim 62, further comprising the addition of tension cables aligned and fixed with one or more of the sealing seams and disposed to maintain the form and shape of the container when such cables are connected to a stabilizing exterior structure surrounding the container.

65. The method of claim 62 wherein the ratio of the distance separating seams of the first sheet to the distance separating seams of the second sheet is from 1.1 to 2.0.