Bioreactors for Algae Growth

Abstract

[0003] A photobioreactor (PBR) cell comprising a riser portion in the form of a tube; a downcomer portion in the form of a tube; a means for connecting the riser portion and the downcomer portion to allow liquid flow; and an aeration means. There is a need for new methods and apparatus for growing algae; or at least methods and apparatus to complement previously known methods and apparatus for growing algae. The present invention seeks to provide one or more of improved or alternative methods and apparatus for growing algae, and improved or alternative algae cultures and compositions.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of Australian Provisional Patent Application No. 2022903067, filed October 18, 2022, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present invention relates to modularizable photobioreactor cells for producing algae, methods for growing algae, and algae cultures and compositions. [Background technology]

[0003] Background technology Livestock production, particularly ruminants, contributes to global anthropogenic greenhouse gas (GHG) emissions. The majority of GHG emissions from livestock production are in the form of methane (CH4), which is produced mostly through enteric fermentation and, to a lesser extent, organic manure decomposition. Enteric CH4 emissions not only contribute to total GHG emissions from agriculture, but also represent an energy loss totaling 11% of dietary energy consumption. Therefore, reducing enteric CH4 emissions could reduce agriculture's overall contribution to climate change and improve productivity through feed energy conservation.

[0004] Mitigation of intestinal CH4 emissions through organic feed supplements derived from the red algae Asparagopsis taxiformis (A. taxiformis) and Asparagopsis armata (A. armata) alters the rumen environment and directly inhibits methane production, resulting in lower intestinal CH4 production (>80% reduction) in ruminant livestock. Asparagopsis spp. synthesize and store halogenated CH4 analogs (e.g., bromoform and dibromochloromethane) in specialized glandular cells as a natural defense mechanism.

[0005] Asparagopsis spp. were found to reduce CH more effectively in vitro compared to similar inclusions of pure bromoform, likely due to multiple antimethanogenic CH analogs (e.g., bromomethane and iodomethane, and bromoethane and iodoethane) functioning synergistically, and methanogenic species differed in their sensitivity to CH inhibitors.

[0006] Approaches for commercial-scale production of Asparagopsis spp. should enable the large-scale use of seaweed in ruminant livestock feed. Therefore, low-capital-cost, scalable production systems are needed to generate feedstocks that can be used as organic feedstocks to reduce GHG emissions and improve ruminant livestock productivity. Improved cultivation methods and improved feedstocks are also desirable. Typical open-ocean aquaculture of Asparagopsis produces annual seaweed yields of between 1 and 3.8 tons of dry weight per hectare using current practices (Agrifutures 2022). However, many oceans are not suitable for growing Asparagopsis seaweed. One sustainable alternative is to use coastal, non-agricultural land to grow seaweed. Coastal areas can provide thousands to tens of thousands of hectares for land-based seaweed farming. However, land-based seaweed farming is very different from open-ocean farming and poses different challenges. For example, attempts have been made to use land-based tanks, ponds, or closed indoor photobioreactor modules, but these methods have not been found to be cost-effective on a large scale.

[0007] There is a need for new methods and apparatus for growing algae; or at least methods and apparatus to complement previously known methods and apparatus for growing algae. The present invention seeks to provide one or more of improved or alternative methods and apparatus for growing algae, and improved or alternative algae cultures and compositions.

[0008] The preceding discussion of the background art is intended merely to facilitate understanding of the present invention, and is not intended as an admission or acknowledgement that any of the material referred to is or was part of the general knowledge at the priority date of this application. Summary of the Invention [Means for solving the problem]

[0009] Summary of disclosure The present invention provides a) a riser section in the form of a tube; b) Downcomer section in the form of a tube; c) means for connecting said riser section and said downcomer section to permit liquid flow; d) ventilation means; A photobioreactor (PBR) cell comprising:

[0010] Preferably, the PBR cell is used or adapted for use in growing algae species, more preferably macroalgae. Preferably, the macroalgae is Asparagopsis spp.

[0011] Preferably, the PBR cell tubes are made from UV-resistant low-density plastic film and have a diameter of 75 to 300 mm and / or a length of 500 to 3000 mm. Preferably, the total working volume of each PBR cell is 1.5 to 215 liters.

[0012] Preferably, the temperature of the PBR cell is maintained at 12°C to 28°C. Preferably, the air permeation rate of the PBR cell is 1 to 70 L / min. Preferably, the PBR cell of the present invention has a permeation rate of 100 to 1500 μmol m -2 .s -1 The animals are exposed to a light intensity of 1000 and a photoperiod of 8:16 to 14:10 light:dark cycle.

[0013] The present invention provides a method for growing algae, the method comprising: i. inoculating a photobioreactor (PBR) cell with algal cells, wherein the PBR cell comprises: a. A rising pipe section in the form of a tube; b. Downcomer section in the form of a tube; c. means for connecting said riser section and said downcomer section to permit liquid flow; d. Ventilation means; a process comprising: ii. maintaining the PBR cells in an aqueous culture medium under light and aeration conditions suitable for growth of the algal cells; Further provided is a method comprising:

[0014] The present invention provides a method for growing algae, the method comprising: i. providing algal cells in an aqueous culture medium having a biomass density and average fragment diameter greater than 0.1 g / L; ii. removing the algal biomass from the culture medium; a. a biomass density of at least 0.1 g / L; and b. an average fragment diameter less than the average fragment diameter of the algal cells in the aqueous culture medium before removing the algal biomass; generating a culture of retained algal cells having iii. maintaining said culture of algal cells maintained in an aqueous culture medium under light and aeration conditions suitable for growth of said algal cells; Further provided is a method comprising:

[0015] The present invention provides a culture of algal cells produced using the method of growing algae using a PBR cell as described herein. Preferably, the culture of algal cells has an average fragment diameter of

[0016] The present invention further provides a culture of retained algal cells produced using the method of growing algae using a PBR cell as described herein. The culture of retained algal cells is a culture of algal cells grown using a PBR cell as described above, from which an algal biomass has been removed. Preferably, the culture of retained algal cells has an average fragment diameter that is less than the average fragment diameter of the culture of algal cells immediately prior to removing the algal biomass.

[0017] The present invention further provides a composition comprising an algal biomass, the algal biomass being biomass produced in a culture of algal cells by the method of growing algae using a PBR cell as described herein. Preferably, the algal biomass has an average fragment diameter of 100 μm.

[0018] The present invention further provides a composition comprising a removed algal biomass. The removed algal biomass is biomass removed from the algal biomass in culture as described above. Preferably, the removed algal biomass has an average fragment diameter that is greater than the average fragment diameter of the algal biomass from which it was removed. Preferably, at least 60%, preferably 70%, 80%, or 90% of the removed algal biomass has a fragment diameter of at least 2 mm.

[0019] The present invention further provides a composition comprising retained algal biomass. The retained algal biomass is biomass retained in a culture of algal cells as described above after removal of the removed algal biomass. Preferably, the retained algal biomass has an average fragment diameter that is less than the average fragment diameter of the algal biomass from which it was removed. Preferably, the retained algal biomass has at least 60%, preferably 70%, 80%, or 90% of the algal biomass having a fragment diameter less than 2 mm. BRIEF DESCRIPTION OF THE DRAWINGS Further features of the present invention will be more fully described in the following description of some non-limiting embodiments thereof. This description is included merely for purposes of illustrating the present invention. It should not be understood as a limitation on the broad summary, disclosure, or description as set forth above. The description is made with reference to the accompanying drawings. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram of an exemplary photobioreactor cell according to the present invention. [Figure 2A] FIG. 2 is a diagram of an example photobioreactor cell (FIG. 2A) and photobioreactor cells connected in series according to the present invention (FIG. 2B). [Figure 2B] FIG. 2 is a diagram of an example photobioreactor cell (FIG. 2A) and photobioreactor cells connected in series according to the present invention (FIG. 2B). [Figure 3] Figure 3 shows images of Asparagopsis taxiformis tetrasporophyte cultures, demonstrating that tetrasporophytes can self-fragment and grow continuously, requiring a continuous harvesting process to maintain culture density. Figure 2(a) shows a dense culture (5 g / L) with a range of tetrasporophyte sizes reaching a harvestable size of >8 mm diameter. Removal of the airlift reveals that the culture also consists of medium (3-7 mm) fragments (Figure 2(b)) and small (<3 mm) fragments (Figure 2(c)). [Figure 4] Figure 4 shows images of Asparagopsis taxiformis tetrasporophytes cultured under optimized operation in interconnected photobioreactor cells (10 cells) showing a 30-day production cycle to harvestable size. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description In order to provide a new approach to the problems associated with the terrestrial growth of algae, the present invention aims in one aspect to provide a system for cost-effective large-scale terrestrial algae cultivation, and in another aspect to provide improved algae growth methods, cultures and compositions.

[0022] The devices of the present invention generally include photobioreactor cells (PBR cells) that can be used in a series assembly to create a low capital cost, modular photobioreactor system (PBR system). The PBR system can also provide advantages such as the ability to control the photoperiod of light provided to the algae as needed, which can be used to induce sporulation and / or allow for continuous harvesting to control biomass.

[0023] The PBR cells and systems of the present invention are designed as modular, linearly scalable, and resilient systems that can operate with a low land and water footprint, which can provide increased renewable algae-based feedstock production compared to conventional marine-based and other land-based tank-based algae growth systems.

[0024] The methods of the present invention generally involve growing algae in an aqueous culture medium using PBR cells. The methods may include removing an algal biomass from the aqueous culture medium. Preferably, the removed biomass has an average fragment diameter greater than the average fragment diameter of the culture prior to removing the algal biomass. Removing the biomass preferably results in the production of a culture having an average fragment diameter less than the average fragment diameter of the culture prior to removing the algal biomass.

[0025] The methods of the present invention are designed to potentially allow for continuous cultivation of algae, which may allow for the continuous harvest of algal biomass for downstream uses, particularly as a feedstock for ruminant feed.

[0026] Thus, the cultures and compositions of the present invention generally comprise: - a culture of algal cells with an average fragment diameter; a culture from which an algal biomass has been removed, wherein such retained algal cell culture has an average fragment diameter that is less than the average fragment diameter of said algal culture prior to removal of the algal biomass; a composition comprising an algal biomass having an average algal fragment diameter; a composition comprising a removed algal biomass removed from a culture of algal cells, wherein the removed algal biomass composition has an average fragment diameter that is greater than the average fragment diameter of the algal culture prior to removing the algal biomass; and a composition comprising a retained algal biomass, wherein the retained algal biomass is the algal biomass retained after removal of the removed algal biomass, and wherein the retained algal biomass composition has an average fragment diameter that is less than an average fragment diameter of the algal culture prior to removal of the algal biomass.

[0027] algae In the present invention, the terms "seaweed" and "algae" are used interchangeably. Algae are simple, non-flowering, typically eukaryotic, photosynthetic aquatic organisms. Algae contain chlorophyll but lack true stems, roots, leaves, or vascular tissue. The algae may be macroalgae or microalgae.

[0028] Microalgae are unicellular algae throughout their life cycle, while macroalgae have at least one multicellular stage during their life cycle. When associated with an aqueous culture medium, the algae of the present invention may be referred to herein as "algal cells." When not associated with an aqueous culture medium, the algae of the present invention may be referred to as "algal biomass."

[0029] Algae suitable for use in the present invention are preferably macroalgae. These macroalgae include green algae, brown algae, and red algae. Brown algae and red algae are preferred because they typically require lower light intensities to grow than green algae, which can reduce electricity costs for land-based aquaculture using artificial lighting. Preferably, the macroalgae is red algae. Red algae are preferred because they tend to produce extracellular material, including cell wall polysaccharides, which can result in improved ruminant feed.

[0030] Algal cells in aqueous culture media can form multicellular clusters, particularly under growth conditions. These clusters can be referred to as "biomass fragments" or simply "fragments." Fragments can range in size from less than 2 mm to more than 8 mm in diameter. Biomass fragments with a diameter of less than 2 mm can be referred to as "very small," biomass fragments with a diameter of 2 mm to 4 mm can be referred to as "small," biomass fragments with a diameter of 4 mm to 6 mm can be referred to as "medium," while biomass fragments with a diameter greater than 6 mm can be referred to as "large." The term "diameter" in the context of biomass fragments does not limit the shape of the fragment but refers to the largest axial dimension. Biomass fragments can form during the gametophyte or sporophyte phase. The algal sporophyte can be, and preferably is, a tetrasporophyte.

[0031] Preferably, the algae is of the class Florideophyceae. Florideophyceae are multicellular red algae that form biomass fragments. Preferably, the algae is of the order Bonnemaisoniales within the class Florideophyceae. Bonnemaisonialea forms biomass fragments in the sporophyte phase, which includes tetrasporophytes. Preferably, the algae is Asparagopsis spp., as described below. Asparagopsis spp. may be Asparagopsis taxiformis (A. taxiformis) and / or Asparagopsis armata (A. armata), preferably Asparagopsis taxiformis (A. taxiformis) and / or Asparagopsis armata (A. armata). Although Asparagopsis spp. are macroalgae, the algae may be in the form of very small (microscopic) fragments, for example, after maceration prior to inoculation or during early growth. Fragments of the macroalgae Asparagopsis may initially be as small as one or a few cells and may be microscopic; however, the cells undergo substantial cell division and after a day or longer of growth form clearly visible fragments of macroalgae.

[0032] Asparagopsis A. taxiformis has a wide climatic range but is typically grown in temperate to tropical climates, while A. armata is typically grown in cool temperate climates. Both species have a diplo-haplontic life cycle with three morphologically distinct stages (two macroscopic and one microscopic): the gametophyte (macroscopic), the carposporophyte (microscopic), and the tetrasporophyte (macroscopic). During the gametophyte stage, both species share similar characteristics but are morphologically distinct from each other. Both species have rhizoids that give rise to several erect, polysiphonous stems. These repeatedly branch into trisiphonous ramuli, defining a thallus. The gametophyte produces male gametes on the antheridium and female gametes on the artefactium. In contrast, A. armata has spinous branches (harpoon-like, serrated appendages), very slender, upright branches, and a habit of spreading by entangling itself around other benthic organisms and artificial structures. A. taxiformis has a more compact rhizoid system, lacks spinous branches, and forms more patchy tufts.

[0033] The carposporophyte is a microscopic life stage that remains attached to the female gametophyte. The carposporophyte produces carpospores, which are released into the water column and, upon settlement, develop into tetrasporophytes, referred to as "Falkenbergia stages" (A. taxiformis-F. hillebrandii; A. armata-F. rufolanosa). These develop by branching trisiphonal, branching filaments, giving them a red, pompom-like appearance. The Falkenbergia stages were thought to be morphologically identical, but were later found to have terminal cell sizes that differ between the two species when maintained in culture. The tetrasporophytes can produce tetraspores through asexual reproduction (meiosis). The tetraspores can also be released into the water column, settle to the substratum, and develop into gametophytes.

[0034] The gametophyte and tetrasporophyte life stages of both species are sources of halogenated compounds with significant antifungal and antibiotic activity. The tetrasporophyte stage tends to have more halogenated compounds per unit biomass than the gametophyte stage due to its lower structural biomass. The tetrasporophyte life stage is the focus life stage for growth using the PBR cells of the present invention.

[0035] The commercial demand for these two species stems from their unique ability not only to produce biologically active metabolites (e.g., bromoform, as well as smaller amounts of other bromine-, chlorine-, and iodine-containing methane, ethane, ethanol, acetaldehyde, acetone, 2-acetoxypropane, propene, epoxypropane, acrolein, and butenone), but also to partition and store these compounds in specialized storage or glandular cells to prevent autotoxicity. In addition to exhibiting potent antimethanogenic applications, terrestrial cultures of Asparagopsis may represent an important source of other bioactive compounds responsible for antioxidant and cytotoxic activity in pharmaceutical and veterinary settings.

[0036] The present invention provides the ability to commercially produce and harvest tetrasporophytes of A. taxiformis and / or A. armata using the PBR cells, PBR systems, and methods described herein. The present invention further provides access to organically produced metabolic products (e.g., cell wall polysaccharides) that can result in improved ruminant feed.

[0037] Preferably, tetrasporophyte cultures of Asparagopsis spp. are maintained at a culture density in PBR cells of 1.0 to 9.0 g / L, more preferably 2.0 to 7.0 g / L, as described below.

[0038] Photobioreactor device Each PBR cell consists of two connected vertical plastic film tubes, one serving as the riser portion of the PBR cell and a second serving as the downcomer portion of the PBR cell in turn.

[0039] Two or more PBR cells can be interconnected in series to create a PBR system.

[0040] The present invention therefore provides a) a riser section in the form of a tube; b) Downcomer section in the form of a tube; c) means for connecting said riser section and said downcomer section to permit liquid flow; d) Ventilation means A photobioreactor cell comprising:

[0041] Reactor Piping Preferably, the tube of the PBR cell is made from a plastic film. Thus, the PBR cell preferably includes a plastic film tube. Preferably, the plastic film tube is transparent or has minimal opacity. The percentage of transmitted light that is scattered by the plastic film tube is preferably between 1.3% and 27.5%. For example, the percentage of transmitted light that is scattered can be in a range having an upper limit of 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, 2.0%, or 1.5% and / or a lower limit of 1.3%, 1.5%, 2.0%, 2.5%, 5.0%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, or 25%.

[0042] For example, the advantage of plastic film tubes over glass tubes is that they are cheaper and less prone to breakage. They are also lighter, making them easier and cheaper to transport and assemble, especially when PBR systems are set up in more remote locations.

[0043] Preferably, the plastic film of the plastic film tube is a UV-resistant plastic film.

[0044] Preferably, the plastic film of the plastic film tube is a low-density plastic film, and preferably has a density of 50 to 150 microns, preferably 100 microns.

[0045] Preferably, the plastic film of the plastic film tube is a thin plastic film. Preferably, the plastic film has a thickness of 0.05 mm to 0.15 mm. More preferably, the plastic film has a thickness of 0.1 mm to 0.15 mm, for example, about 0.1 mm.

[0046] Preferably, the plastic film of the plastic film tube is a polyethylene (LDPE) plastic film. The tube can also be constructed from other amorphous plastics, including polyvinyl chloride (clear PVC), polycarbonate (PC), and acrylic.

[0047] Optionally, the tube can be made from glass (i.e., the term "plastic film tube" can include glass tubing) or high-density plastic, but these materials generally do not offer the favorable properties, such as light transmission, cost-effectiveness, ease of transportation and assembly, that low-density plastic film tubes offer.

[0048] Therefore, preferably, the plastic film tube of the PBR cell is made from a UV-resistant low-density plastic film (eg, a UV-resistant low-density polyethylene (LDPE) plastic film).

[0049] In some embodiments of the present disclosure, the diameter of the plastic film tube is 75 to 300 mm, 100 to 200 mm, or 125 to 175 mm. For example, the diameter of the plastic film tube can be 75 mm, 80 mm, 90 mm, 100 mm, 125 mm, 150 mm, 200 mm, 300 mm, or more preferably 150 mm. This diameter maximizes volume while still allowing light to pass through the entire tube.

[0050] In some embodiments of the present disclosure, the length of the plastic film tube is 500 to 3,000 mm, 1,000 to 3,000 mm, or 2,000 to 3,000 mm. For example, the length of the plastic film tube can be 500 mm, 750 mm, 1,000 mm, 1,200 mm, 1,500 mm, 2,000 mm, 2,500 mm, or 3,000 mm, more preferably 2,500 mm. This length allows aeration to be provided at a rate such that tetrasporophytes can reach medium to large sizes and disperse throughout the entire volume of the airlift cells of the PBR cell. When the PBR cell is assembled vertically, the length of the tube is approximately the height of the PBR cell.

[0051] In some embodiments of the present disclosure, the plastic film tubes are interconnected with U-connectors and H-connectors (see, for example, Figures 1 and 2). Preferably, the plastic film tubes joined by connectors to form a PBR cell are arranged in a vertical assembly. A vertical assembly minimizes the amount of area occupied by the PBR cell or PBS system. In this embodiment, the U-connector is a bottom U-connector and the H-connector is a top H-connector. Alternatively, the PBR cell may be provided in a horizontal assembly, with the U-connector and H-connector provided on opposite sides of the plastic film tube. A horizontal assembly maximizes the area exposed to natural light from above.

[0052] Preferably, the U-connectors and H-connectors are made from polyvinyl chloride, acrylic, or low-density polyethylene (LDPE). In some embodiments of the present disclosure, the U-connectors and H-connectors are constructed from a clear acrylic material.

[0053] In some embodiments of the present disclosure, the plastic film tubes of the riser section and downcomer section are discharged in a single unit with a U-shaped bend that serves the same purpose as the U-connector.

[0054] Preferably, the U-connectors and H-connectors are secured to the plastic film tube via fittings (e.g., rubber fittings) and ring clamps (e.g., stainless steel ring clamps, preferably 316 stainless steel ring clamps), which ensure no slippage of the plastic film tube from the U-connectors or H-connectors and provide a watertight seal. In some embodiments of the present disclosure, the U-connectors and H-connectors are constructed from a clear acrylic material. Alternatively, the U-connectors and H-connectors can be attached to the plastic film tube via turn screw fasteners (e.g., plastic turn screw fasteners).

[0055] In some embodiments, the H-connector is open at the top to allow off-gassing from the air in the vent means, hi some embodiments, the H-connector has a valve that prevents evaporative loss of water but allows off-gassing.

[0056] The alternating riser and downcomer sections may form a single PBR cell or may be interconnected as a PBR system. In a PBR system, the riser and downcomer sections of each PBR cell are spaced 50 mm to 300 mm apart (preferably about 150 mm) and 0.075 m apart. 2 ~0.27m 2 forming a vertical loop occupying the entire land footprint of

[0057] Preferably, the total working volume of each PBR cell is between 1.5 L and 215 L. For example, the total working volume of each PBR cell can be between 10 L and 200 L, or between 50 L and 150 L. In one example, the total working volume of the PBR cell can be about 100 L.

[0058] When two or more PBR cells are interconnected to form a PBR system (see, for example, FIG. 1b), the configuration is preferably such that there is an even number of plastic film tubes at the front and back of the system, so that the last plastic film tube in a series of PBR cells can be looped back and connected to the first plastic film tube in the series to complete the cycle of the PBR system.

[0059] When two or more PBR cells are interconnected, the rear plastic film tube is preferably positioned either directly behind the front plastic film tube or at an offset angle relative to the front plastic film tube. Preferably, the plastic film tubes are positioned such that the rear plastic film tube is 50 mm to 300 mm away from the front plastic film tube.

[0060] Temperature control The PBR can be thermally regulated either through external means such as a temperature-controlled greenhouse, shade sails or other means to block the sun's natural heat, blankets to retain heat within the PBR cell during cool weather, or heating and / or cooling means located within the tubes of the PBR cell or surrounding the PBR cell. If thermal regulation is internal, it preferably includes element(s) located within the vertical tubes from the top and connected to a central temperature-controlled heating and cooling unit to maintain the culture temperature within the PBR. The heating and / or cooling elements may be fabricated from stainless steel.

[0061] Preferably, the temperature of the PBR cell is maintained between 12°C and 28°C. The exact temperature can be determined by one skilled in the art with reference to the algae being grown. For example, if the algae being grown is A. taxiformis, the preferred temperature is between 15°C and 28°C, e.g., about 20°C. If the algae being grown is A. armata, the preferred temperature is between 12°C and 23°C, e.g., about 17°C.

[0062] light source Preferably, the PBR cells of the present invention have a pH of 100 μmol m -2 .s -1 ~1500 μmol m -2 .s -1 , more preferably 150 μmol m -2 .s -1 ~1000 μmol m -2 .s -1 is exposed to a light intensity of

[0063] The light source may be natural light or may be provided by artificial electric lighting, which, if used, preferably provides wavelengths between 400 nm and 700 nm to maximize algae growth.

[0064] When two or more PBR cells are interconnected, the rear plastic tube is preferably positioned either directly behind the front tube or at an offset angle relative to the front tube, with offsetting the tubes being preferred to increase light availability to all tubes and maximize growth.

[0065] Day length (photoperiod) is a robust seasonal signal that eukaryotic algae can use to initiate or complete different developmental programs. Multicellular algae (e.g., the macroalgae Rhodophyta) have a triphasic (gametophyte, carposporophyte, and tetrasporophyte) life cycle.

[0066] The PBR cells of the present invention are preferably exposed to a photoperiod of 8:16 to 14:10 light:dark, more preferably a 10:14 light:dark, light:dark cycle, which maximizes the tetrasporophyte stage of the algal life cycle, and more preferably maximizes the tetrasporophyte stage of the Asparagopsis life cycle.

[0067] ventilation To grow, the algae in the PBR cells of the present invention must be supplied with oxygen and carbon dioxide in an aqueous (i.e., water-based) culture medium. Providing oxygen and carbon dioxide can be accomplished by aerating the riser portion of the PBR cell. Aeration provides the added benefit of lifting and mixing the water within the PBR cell, mixing and dispersing the algae growing within the PBR cell.

[0068] Preferably, the aeration flow rate is 1 L / min to 70 L / min. Aeration fragments the tetrasporophyte culture, at least within the riser portion of the PBR cell. If the aeration flow rate is too low, the tetrasporophytes may aggregate and form a single mass, rather than fragment, which may impair system performance. If the aeration flow rate is too high, excessive fragmentation may occur, and the tetrasporophytes may not reach a large size. More preferably, the aeration flow rate is 1 L / min to 70 L / min or 1 L / min to 30 L / min; for example, about 1 L / min to 10 L / min.

[0069] Four types of bubble regimes exist in aeration systems. The ratio or presence / absence of the four types depends on the flow rate and can be further influenced by the porous sparger type and pore size. At low air flow rates, the system operates in a "bubble flow regime" (the small bubbles are highly dependent on the sparger type and pore size). As the air flow rate increases, the bubbles coalesce to form larger bubbles, which push a "slug" of water to the top of the pipe in a "slug flow regime." As the air flow continues to increase, the large bubbles become unstable, creating a "churn flow regime." At high air flows, an "annular flow regime" occurs and the water flow decreases. The highest efficiency and maximum capacity of the air and water flow, resulting in maximum airlift, maximum algae growth, and optimal fragmentation, occurs near the slug / churn flow regime transition region.

[0070] Preferably, aeration is provided by injecting compressed air into the riser of the PBR cell or via a blower. Aeration can be achieved by using a porous sparger (e.g., air stone, perforated tubing, or perforated plastic) with a pore size of 10 to 200 μm. More preferably, the pore size is 50 μm to 150 μm, e.g., about 80 μm. These are inexpensive and easy to assemble, reducing the cost of assembling and maintaining the PBR cell and / or PBR system.

[0071] CO2-enriched air (up to 5% v / v) can be used if desired via a CO2 cylinder and flow meter connected to the compressed air supply line. The presence of CO2 can help maintain the pH of the system at a desired level.

[0072] water flow Generally, the water flow rate or velocity is determined and controlled by the aeration rate—moving air bubbles simultaneously move the water around them. However, if desired, a separate water pump can be attached to the PBR cell or system to assist in water flow control. Preferably, the water velocity is between 0.1 m / s and 0.7 m / s.

[0073] Nutrients Seaweed was treated with various forms of nitrogen (NO - and N.H. + and organic forms of nitrogen) and phosphates (including inorganic and organic forms of phosphorous acid), trace metals (including copper, zinc, manganese, molybdenum, iron, and cobalt), and vitamins (including vitamin B12, biotin, and thiamine).

[0074] Suitable nutrient media are known to those skilled in the art of algae growth. These include commercially available nutrient media (Cell-Hi TM Nutrient media developed from seawater nutrient medium recipes (including F / 2 medium, Provasoli Enriched Seawater, Enriched Seawater, Erdschreiber medium); or organic forms of nutrient media (fish farm effluent, sewerage effluent, animal wastewater).

[0075] pH Preferably, the pH of the PBR cell is maintained at pH 7 to pH 9. More preferably, the pH is maintained at pH 7.5 to pH 9.0. For example, the pH of the PBR cell can be maintained at approximately pH 8.1 to 8.8, which is the average pH of seawater.

[0076] Methods for growing algae The present invention provides a method for growing algae, the method comprising: i. inoculating a photobioreactor (PBR) cell with algal cells, wherein the PBR cell comprises: a. A rising pipe section in the form of a tube; b. Downcomer section in the form of a tube; c. means for connecting said riser section and said downcomer section to permit liquid flow; d. Ventilation means; a process comprising: ii. maintaining the PBR cells in an aqueous culture medium under light and aeration conditions suitable for growth of the algal cells; The present invention provides a method comprising:

[0077] Preferably, the algae is a macroalgae, more preferably, the macroalgae is an Asparagopsis spp.

[0078] Preferably, the PBR cell tubes are made from UV-resistant low-density plastic film and have a diameter of 75 to 300 mm and / or a length of 500 to 3000 mm. Preferably, the total working volume of each PBR cell is 1.5 to 215 liters.

[0079] Preferably, the temperature of the PBR cell is maintained at 12°C to 28°C. Preferably, the air permeation rate of the PBR cell is 1 L / min to 70 L / min. Preferably, the PBR cell of the present invention has a saturation rate of 100 μmol m -2 .s -1 ~1500 μmol m -2 .s -1 and a photoperiod of 8:16 to 14:10 light:dark cycle. Preferably, the velocity of the aqueous culture medium (water velocity) is 0.1 m / s to 0.7 m / s. Preferably, the pH of the aqueous culture medium is maintained at pH 7 to pH 9.

[0080] The method may include the further step of harvesting algae from the PBR cell, preferably in a continuous manner. This may be accomplished by the use of a sieve (e.g., a nylon mesh sieve) or other collection means inserted into either the riser or downcomer section, preferably the top of the downcomer section. Such collection means may harvest some or all of the biomass as the algae passes through the riser and downcomer sections and due to water flow, and hence algae flux, through the collection means. This is described further below.

[0081] The present invention provides a method for growing algae, the method comprising: i. growing algal cells in an aqueous culture medium; ii. removing the algal biomass from the culture medium to produce a culture of retained algal cells; and iii. maintaining said culture of algal cells maintained in an aqueous culture medium under light and aeration conditions suitable for growth of said algal cells; The present invention provides a method comprising:

[0082] The present invention provides a method for growing algae, the method comprising: i. growing the algal cells in an aqueous culture medium until the algal cells reach a biomass density of greater than 0.1 g / L; ii. removing the algal biomass from the culture medium; a. a biomass density of at least 0.1 g / L; and / or b. an average fragment diameter less than the average fragment diameter of the algal cells in the aqueous culture medium before removing the algal biomass; generating a culture of retained algal biomass having iii. maintaining a culture of algal biomass maintained in said aqueous culture medium under light and aeration conditions suitable for growth of said algal cells; Further provided is a method comprising:

[0083] Preferably, the algae is a macroalgae, more preferably, the macroalgae is an Asparagopsis spp.

[0084] Preferably, the method is carried out using a PBR cell as described herein.

[0085] Growing the algal cells in an aqueous culture medium can be accomplished by inoculating the algae into the aqueous culture medium and maintaining the inoculated algal cells in the aqueous culture medium under light and aeration conditions suitable for algal cell growth until they reach a selected biomass density. The algae that is initially inoculated into the aqueous culture medium can be collected from its natural aquatic environment and / or grown on a solid culture medium using methods known in the art.

[0086] The phrase "average fragment diameter less than the average fragment diameter of the algal cells in the aqueous culture medium prior to removing the algal biomass" means that the average diameter size of the fragments can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% smaller than the average diameter size of the fragments prior to removing the algal biomass. The phrase "average fragment diameter that is larger than the average fragment diameter of the algal cells in the aqueous culture medium prior to removing the algal biomass" means that the average diameter size of the fragments can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% larger than the average diameter size of the fragments prior to removing the algal biomass.

[0087] Preferably, the algal cells in the aqueous culture medium have a biomass density greater than 1.0 g / L, 2.0 g / L, 3.0 g / L, 4.0 g / L, or 5.0 g / L. Preferably, the algal cells in the aqueous culture medium have a biomass density greater than 3.0 g / L. The algal cells in the aqueous culture medium may have a biomass density of up to about 13 g / L, 12 g / L, 11 g / L, 10 g / L, 9 g / L, or 8 g / L. When expressed as a range, the algal cells in the aqueous culture medium may have a biomass density of greater than 0.1 g / L to 12.0 g / L, greater than 1.0 g / L to 11.0 g / L, greater than 2.0 g / L to 10.0 g / L, greater than 3.0 g / L to 9.0 g / L, greater than 4.0 g / L to 8.0 g / L, or greater than 5.0 g / L to 7.0 g / L. However, any minimum and maximum may be combined without limitation; for example, the algal cells in the aqueous culture medium may have a biomass density of greater than 0.1 g / L to 8.0 g / L, or greater than 1.0 g / L to 9.0 g / L, or greater than 4.0 g / L to 7.0 g / L, etc. Preferably, the algal cells in the aqueous culture medium have a biomass density of greater than 4.0 g / L to 8.0 g / L.

[0088] The algal cells in the aqueous culture medium may be comprised of biomass fragments having a diameter of less than and / or at least 2.0 mm. The biomass fragments may have a diameter of up to about 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, or 7 mm. Expressed as a range, the algal cells in the aqueous culture medium may be comprised of biomass fragments having a diameter of less than 2.0 mm to 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, or 7 mm. Preferably, the provided algal cells in the aqueous culture medium comprise biomass fragments having a diameter greater than 2.0 mm. Preferably, about 30% to about 95%, about 40% to about 90%, about 50% to about 85%, or about 60% to about 80% of the algal cells in the aqueous culture medium comprise biomass fragments having a diameter greater than 2.0 mm. Preferably, the provided algal cells in the aqueous culture medium comprise biomass fragments having a diameter greater than 4 mm. Preferably, the provided algal cells in the aqueous culture medium comprise biomass fragments having a diameter of greater than 5 mm. Preferably, immediately prior to harvesting the algal biomass, the provided algal cells in the aqueous culture medium are comprised of biomass fragments having a diameter of up to about 7 mm.

[0089] The removal of algal biomass may be referred to herein as "harvesting."

[0090] A culture of algal cells from which algal biomass has been removed can generally be referred to as a "culture of retained algal cells." Immediately after the algal biomass has been removed, the culture of retained algal cells preferably has a biomass density of at least 1.0 g / L, 2.0 g / L, 3.0 g / L, or 4.0 g / L. The culture of retained algal cells can have a biomass density of up to about 9.0 g / L, 8.0 g / L, 7.0 g / L, or 6.0 g / L. When expressed as a range, the culture of retained algal cells can have a biomass density of 0.1 g / L to 9.0 g / L, 1.0 g / L to 8.0 g / L, 2.0 g / L to 7.0 g / L, or 3.0 g / L to 6.0 g / L. However, any minimum and maximum may be combined without limitation; for example, the culture of retained algal cells may have a biomass density of 0.1 g / L to 8.0 g / L, or 1.0 g / L to 6.0 g / L, or 3.0 g / L to 7.0 g / L, etc. In a preferred embodiment, the culture of retained algal cells has a biomass density of at least 2.0 g / L, and preferably 2.0 g / L to 5.0 g / L.

[0091] Preferably, the culture of retained algal cells comprises biomass fragments having a diameter of about 2 mm or less. Preferably, at least about 30%, 40%, 50%, 60%, 70%, or 80% of the culture of retained algal cells is composed of biomass fragments having a diameter of less than 2 mm. Up to about 95%, 90%, 85%, or 80% of the culture of retained algal cells may be composed of biomass fragments having a diameter of less than 2.0 mm. Expressed as a range, about 30% to about 95%, or about 40% to about 90%, or about 50% to about 85%, or about 60% to about 80% of the culture of retained algal cells may be composed of biomass fragments having a diameter of less than 2 mm. The culture of retained algal cells may also comprise biomass fragments having a diameter of 2 mm to 4 mm. Preferably, about 40%, 50%, 60%, 70%, or 80% to about 85%, 90%, 95%, or 100% of the culture of retained algal cells is composed of biomass fragments having a diameter of about 4 mm or less than 4 mm.

[0092] Methods for determining biomass density are known to those skilled in the art. One method involves taking a sample of algal biomass in a known amount of aqueous culture medium and drying and weighing the algal biomass to determine its dry weight per volume of aqueous culture medium.

[0093] Methods for determining the average fragment diameter are known to those skilled in the art. One method involves taking a whole sample of biomass and measuring the size of the biomass fragments using a measuring tool such as a caliper. This can be done under a microscope or other magnification. It is not necessary to measure every biomass fragment in the aqueous culture medium, or even every biomass fragment in the sample. An average may be determined by measuring 20 or more biomass fragments. However, it is not always necessary to determine the average fragment diameter. This is because the harvesting works by removing algal biomass having an average fragment diameter greater than the average fragment diameter of the algal cells in the aqueous culture medium before removing the algal biomass, in order to produce a culture of retained algal cells having an average fragment diameter less than the average fragment diameter of the algal cells in the aqueous culture medium before removing the algal biomass. This can be achieved by selectively removing biomass fragments or larger-sized biomass fragments that are larger than the average size. In other words, removing medium and large biomass fragments from algal cells in an aqueous culture medium containing very small, small, medium, and large biomass fragments results in a culture of retained algal cells having an average fragment diameter less than the average fragment diameter of the algal cells in the aqueous culture medium before removing the algal biomass. This can be achieved by using a sieve or other collection means that disproportionately removes larger-than-average sized biomass fragments. This can be achieved using a collection means with an appropriate pore size. For example, a 2 mm pore size can be used to remove biomass fragments larger than 2 mm in diameter while allowing biomass fragments larger than 2 mm in diameter to pass through and be retained in the culture of retained algal cells.

[0094] Preferably, the removed algal biomass comprises biomass fragments having a diameter of about 2.0 mm or greater (i.e., small, medium, and / or large). Preferably, the removed algal biomass comprises biomass fragments having a diameter of about 4.0 mm or greater (i.e., medium and / or large). The biomass fragments in the algal biomass can have a diameter of up to about 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, or 7 mm. Expressed as a range, the algal cells in the aqueous culture medium can comprise biomass fragments having a diameter of about 2.0 mm, 4.0 mm, or greater than 2.0 mm, or 4.0 mm to 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, or 7 mm. Preferably, at least about 60%, 70%, 80%, 90%, or 95% to 100% of the algal biomass is composed of biomass fragments having a diameter of about or greater than 2.0 mm or about or greater than 4.0 mm. Preferably, the algal biomass is composed of biomass fragments having a diameter of up to about 7 mm (i.e., large).

[0095] In a preferred combination of algal cells in an aqueous culture medium, the culture of maintained algal cells and the removed biomass comprise: i. the algal cells in the aqueous culture medium have a biomass density of greater than 3.0 g / L, preferably greater than 4.0 g / L to 8.0 g / L, and / or about 60% to about 80% of the provided algal cells in the aqueous culture medium are composed of biomass fragments having a diameter greater than 2.0 mm; ii. after biomass removal, the culture of retained algal cells has a biomass density of at least 2.0 g / L, and preferably between 2.0 g / L and 5.0 g / L, and / or about 60% to about 80% of the culture of retained algal cells is composed of biomass fragments having a diameter of less than 2 mm, and about 70% to about 100% of the culture of retained algal cells is composed of biomass fragments having a diameter of less than 4.0 mm; and iii. About 70% to about 95% of the removed algal biomass is composed of biomass fragments having a diameter of about 4.0 mm or greater, including biomass fragments having a diameter of up to about 7.0 mm.

[0096] After removal of the algal biomass, the culture of retained algal cells is maintained in an aqueous culture medium under light and aeration conditions suitable for growth of the algal cells. The culture of retained algal cells can then be grown into a culture similar to the algal cells in aqueous culture medium.

[0097] Preferably, the culture of algal cells and / or the culture of maintained algal cells is maintained in an aqueous culture medium at a temperature between 12°C and 28°C. Preferably, the culture medium is aerated at a rate between 1 L / min and 70 L / min. Preferably, the algal cells are maintained at a concentration of 100 μmol m -2 .s -1 ~1500 μmol m -2 .s -1 The cells are exposed to a light intensity of 1000 kJ / s and a photoperiod of 8:16 to 14:10 light:dark cycle. Preferably, the velocity of the aqueous culture medium (water velocity) is between 0.1 m / s and 0.7 m / s. Preferably, the pH of the aqueous culture medium is maintained between pH 7 and pH 9.

[0098] The culture of retained algal cells may be maintained under growth conditions until algal growth produces algal cells in the aqueous culture medium having the above-described properties, and a second removal of algal biomass from the culture medium (having the properties as described above) may be performed to produce a second culture of retained algal cells having the properties as described above; that is, each of steps i.-iii. may be repeated using the culture of retained algal cells produced in the previous step ii. Each succession of steps i.-iii. may be referred to as a "growing cycle." That is, in a preferred embodiment, in each growth cycle, the algal cells in the aqueous culture medium have a biomass density preferably greater than 5.0 g / L to 7.0 g / L, and after removal of algal biomass, the culture of retained algal cells has a biomass density of 3.0 g / L to 5.0 g / L, and the removed algal biomass contains a disproportionate amount of small, medium, and / or large biomass fragments.

[0099] The method may be carried out for 1, 2, 5, 10, 15, 20, 25, 30, or more growth cycles, or any integer number in between, or may be carried out indefinitely. Repeated cycles may be referred to as "continuous harvesting." After several cycles, i.e., 1, 2, 5, 10, 15, 20, 25, 30, or more, an intermediate step may be carried out, whereby the aqueous culture medium is replaced. This may be accomplished by removing substantially all of the algal biomass, a portion of which may be inoculated into fresh aqueous culture medium. This intermediate step may take the place of steps ii. and iii. during continuous harvesting. This intermediate step may also be inserted between steps ii. and iii. This results in a harvested algal biomass having an average fragment diameter less than the average fragment diameter of the algal cells in the aqueous culture medium before the algal biomass was removed.

[0100] The growth rate of algae in an aqueous culture medium begins to slow when the biomass reaches a certain density and / or when the biomass fragments reach a certain size. It has been discovered that by removing larger-sized biomass fragments from the culture, this slowdown in growth rate can be avoided and the higher growth rate of the smaller fragments can be maintained, while achieving the combined benefits of producing an improved feedstock for ruminant feed that is composed of a significant portion of larger-sized biomass fragments. By producing a culture of retained algal cells having an average fragment diameter less than the average fragment diameter of the algal cells in the aqueous culture medium before removing the algal biomass, harvesting the culture of retained algal cells also enables the production of a feedstock that is composed of a significant portion of smaller-sized biomass fragments. The smaller-sized biomass fragments may contain a higher proportion of organically produced metabolic products, such as cell wall polysaccharides, which may also result in improved ruminant feed.

[0101] Cultures and Compositions The present invention therefore also provides a culture of retained algal cells, which is a culture of algal cells from which an algal biomass has been removed, wherein the culture of retained algal cells has an average fragment diameter that is less than the average fragment diameter of said culture of algal cells immediately prior to removing said algal biomass.

[0102] In producing a culture of retained algal cells having an average fragment diameter less than the average fragment diameter of said culture of algal cells in aqueous culture medium immediately prior to removing the algal biomass, removing biomass having an average fragment diameter greater than the average fragment diameter of said culture of algal cells in aqueous culture medium prior to removing the algal biomass. As noted above, this can be accomplished by use of a sieve or other collection means (e.g., a collection means with an appropriate pore size) that disproportionately removes biomass fragments of larger than average size.

[0103] Preferably, the culture of retained algal cells has a biomass density of at least 1.0 g / L, 2.0 g / L, 3.0 g / L, or 4.0 g / L. The culture of retained algal cells may have a biomass density of up to about 9.0 g / L, 8.0 g / L, 7.0 g / L, or 6.0 g / L. When expressed as a range, the culture of retained algal cells may have a biomass density of 0.1 g / L to 9.0 g / L, 1.0 g / L to 8.0 g / L, 2.0 g / L to 7.0 g / L, or 3.0 g / L to 6.0 g / L. Any minimum and maximum may be combined without limitation; for example, the culture of retained algal cells may have a biomass density of 0.1 g / L to 8.0 g / L, 1.0 g / L to 6.0 g / L, 3.0 g / L to 7.0 g / L, etc. In a preferred embodiment, the culture of maintained algal cells has a biomass density of at least 2.0 g / L, and preferably between 2.0 g / L and 5.0 g / L.

[0104] Preferably, the culture of retained algal cells comprises biomass fragments having a diameter of about 2 mm or less. Preferably, about 30% to about 95%, or about 40% to about 90%, or about 50% to about 85%, or about 60% to about 80% of the culture of retained algal cells is composed of biomass fragments having a diameter of less than 2 mm. The culture of retained algal cells may also comprise biomass fragments having a diameter of 2 mm to 4 mm. Preferably, about 40%, 50%, 60%, 70%, or 80% to about 90%, 95%, or 100% of the culture of retained algal cells is composed of biomass fragments having a diameter of less than 4 mm.

[0105] Preferably, the culture of retained algal cells has a biomass density of 2.0 g / L to 7 g / L, about 60% to about 80% of the culture of retained algal cells is composed of biomass fragments having a diameter of less than 2 mm, and about 70% to about 100% of the culture of retained algal cells is composed of biomass fragments having a diameter of less than 4 mm.

[0106] The present invention also provides a composition comprising an algal biomass removed from a culture of algal cells in an aqueous culture medium, wherein the removed algal biomass has an average fragment diameter that is greater than the average fragment diameter of the culture of algal cells in the aqueous culture medium from which the algal biomass was removed.

[0107] Producing a harvested algal biomass having an average fragment diameter greater than the average fragment diameter of the culture of algal cells in the aqueous culture medium from which the algal biomass was harvested can be achieved by use of a sieve or other collection means (e.g., a collection means with an appropriate pore size) that disproportionately removes larger-than-average sized biomass fragments, as described above.

[0108] Preferably, the removed algal biomass comprises biomass fragments having a diameter of about or greater than 2 mm and / or about or greater than 4 mm. Preferably, at least about 60%, 70%, 80%, 90%, or 95% to 100% of the removed algal biomass is composed of biomass fragments having a diameter of about or greater than 2.0 mm or about or greater than 4.0 mm. Preferably, the removed algal biomass comprises biomass fragments having a diameter of up to about 7 mm.

[0109] Preferably, about 70% to about 95% of the extracted algal biomass is composed of biomass fragments having a diameter of about 4 mm or greater than 4 mm, and is composed of biomass fragments having a diameter of up to about 7 mm.

[0110] The present invention also provides compositions comprising an algal biomass, wherein at least 60%, preferably 70%, 80%, or 90% of the algal biomass has a diameter of at least 2 mm. The present invention also provides compositions comprising an algal biomass, wherein about 60% to about 80% of the algal biomass is composed of biomass fragments having a diameter of less than 2 mm. Algal biomass so characterized can be obtained as described herein.

[0111] The culture of retained algal cells can also be harvested. Thus, the present invention also provides a composition comprising a retained algal biomass removed from a culture of retained algal cells, where the culture of retained algal cells is a culture of algal cells from which algal biomass has been removed, and the retained algal biomass has an average fragment diameter that is less than the average fragment diameter of the culture of algal cells from which the algal biomass was removed. Algal biomass harvested from a culture of retained algal cells can generally be referred to as "retained algal biomass."

[0112] Producing a retained algal biomass having an average fragment diameter less than the average fragment diameter of the culture of algal cells in the aqueous culture medium from which the removed biomass was removed can be achieved by use of a sieve or other collection means that disproportionately removes biomass fragments that are larger than the average size, as described above, and then collecting the retained biomass fragments that are smaller than the average size.

[0113] Preferably, the retained algal biomass comprises biomass fragments having a diameter of less than 2 mm. Preferably, at least about 30%, 40%, 50%, 60%, 70%, or 80% of the retained algal biomass is composed of biomass fragments having a diameter of less than 2 mm. Preferably, about 40%, 50%, 60%, 70%, or 80% to about 85%, 90%, 95%, or 100% of the culture of retained algal cells is composed of biomass fragments having a diameter of about 4 mm or less. Preferably, about 60% to about 80% of the retained algal biomass is composed of biomass fragments having a diameter of less than 2 mm. Preferably, about 70% to about 100% of the retained algal biomass is composed of biomass fragments having a diameter of less than 4 mm.

[0114] The algal biomass described herein (algal biomass, retained algal biomass, removed algal biomass) finds particular utility in ruminant feed. The algal biomass can be mixed with other ingredients suitable for use in ruminant feed.

[0115] general Those skilled in the art will recognize that the present invention is susceptible to variations and modifications other than those specifically described. The present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, formulations, and compounds referred to or shown herein, individually or collectively, as well as any and all combinations of said steps or features, or any two or more of said steps or features.

[0116] Each document, reference, patent application, or patent cited in this text is expressly incorporated herein by reference in its entirety, meaning that it should be read and considered by the reader as part of this text. It is for reasons of brevity only that documents, references, patent applications, or patents cited in this text have not been repeated in this text.

[0117] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein are hereby incorporated by reference and may be employed in the practice of this disclosure.

[0118] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention.

[0119] The present invention may include one or more ranges of values ​​(e.g., sizes, weights, percentages, etc.). A range of values ​​is understood to include all values ​​within that range, including both the values ​​defining the range and adjacent values ​​within that range that produce the same or substantially the same result as the values ​​immediately adjacent to that value defining the boundaries of the range. Thus, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Thus, "about 80%" also means "about 80%" and "80%." At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0120] Throughout this specification, unless the context requires otherwise, the words "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0121] It is also noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have the meaning ascribed to them in U.S. patent law; for example, they may mean "includes," "included," "including," and the like; and terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law. For example, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the fundamental or novel characteristics of the disclosure.

[0122] Other definitions for selected terms used herein can be found in the detailed description of the present invention and can be applied throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The term "active agent" can mean one active agent or can include two or more active agents.

[0123] The following examples serve to more fully describe the manner of using the above disclosure and to set forth the best modes contemplated for carrying out various aspects of the disclosure, it being understood that these methods in no way limit the true scope of the disclosure, but rather are presented for illustrative purposes. [Example]

[0124] Example Further features of the present invention are more fully described in the following non-limiting examples, which are included solely for the purpose of illustrating the present invention and should not be understood as a limitation on the broad description of the present disclosure as set forth above.

[0125] Example 1 Thin-film photobioreactor cell (PBR cell) An example of a PBR cell of the present invention is provided in FIG.

[0126] In Figure 1, a photobioreactor (PBR) cell 0100 of the present invention includes a riser section 0110 and a downcomer section 0120. The riser section 0110 is interconnected with the downcomer section 0120 by a top H-connector 0130 and a bottom U-connector 0140.

[0127] A compressed air source (not shown) provides air vent to the PBR cell 100 through the vent tube 150 to induce an upward flow of air and water in the riser section 110, as evidenced by the air bubbles 160. This upward flow in the riser section 110 then expels the water and air through the H-connector 130 to the downcomer section 120. The water and air then flow back to the riser section 110 through the U-connector 140. The flow of air and water is indicated by the bold arrows.

[0128] Algae

[0160] grow in the riser section

[0110] and the downcomer section

[0120] .

[0129] Example 2 Algae growth in PBR systems method PBR cells, consisting of two connected vertical plastic film tubes, were interconnected in series, with one tube of each PBR cell acting as the riser section and the second tube of each PBR cell acting as the downcomer section, alternately, to form a PBR system.

[0130] The plastic film tubes were made from 100 micron density UV-resistant low-density polyethylene (LDPE) plastic tubing. Each plastic film tube had a diameter of 150 mm and a height of 2500 mm. The plastic film tubes were interconnected with polyvinyl chloride (PVC) bottom U-connectors and top H-connectors (Figure 2a). The U-connectors and H-connectors were secured to the plastic film via rubber fittings and stainless steel (316) ring clamps to ensure no slippage of the plastic film from the U-connectors or H-connectors and to provide a watertight seal.

[0131] The H-connector was open at the top to allow gas release through the vent.

[0132] In the PBR system, the riser and downcomer sections of each PBR cell formed a vertical loop, with the vertical tubes spaced 150 mm apart. The total working volume of each PBR cell was 100 L (each cell had a riser and downcomer section plus an 8 L U-connector volume and a 4 L H-connector volume). As shown in Figure 2b, multiple cells were interconnected so that there was an even number of tubes at the front of the system as well as at the rear of the system, and the last tube in a series of cells looped back and connected to the first tube in the series to complete the system cycle.

[0133] In the PBR system, the rear tube was positioned at an offset angle relative to the front tube at a distance of 90 mm, with the rear tube 150 mm from the front tube.

[0134] An aeration flow rate of 20-30 L / min was used, performed using a porous sparger (air stone) with a pore size of 80 μm and a bubble size between 3-5 mm in diameter.

[0135] The PBR was externally thermoregulated by assembling the PBR system in a temperature-controlled room to maintain 20°C within the PBR system.

[0136] The algae culture was treated with nitrogen (NO - and N.H. + and organic nitrogen) and phosphate (including inorganic and organic phosphates), trace metals (including copper, zinc, manganese, molybdenum, iron, and cobalt), and vitamins (including vitamin B12, biotin, and thiamine).

[0137] When a seaweed harvesting sieve made from nylon mesh was placed and immersed in the top of one of the downcomer tubes, biomass could be harvested. The nylon mesh harvesting sieve had a pore size of less than 0.5 mm and was able to completely harvest the biomass. Other seaweed harvesting sieves with different pore sizes were able to partially harvest the biomass depending on the pore size of the sieve. A sieve with a pore size of 3.55 mm was able to collect 95% or more of the large biomass with a diameter of >6 mm. A stainless steel mesh sieve with a pore size of 2.88 mm was able to collect 95% or more of the large biomass and 90% or more of the medium biomass with a diameter of 4-6 mm. A stainless steel mesh sieve with a pore size of 2.00 mm was able to collect 95% or more of the large biomass, 90% or more of the medium biomass, and 40% or more of the small biomass with a diameter of 2-4 mm. The sieve could also be made from stainless steel mesh.

[0138] result Using the PBR system described above, tetrasporophyte cultures of Asparagopsis spp. were maintained at culture densities of 3–7 g / L. The airlift mechanism, aeration rate, and bubble size allowed the tetrasporophyte cultures to naturally self-fragment and continue growing within the riser tube of the PBR cell; as a result, a continuous harvesting process was required to maintain the culture density. For example, within a 5 g / L high-density culture, the tetrasporophyte sizes varied, with harvestable sizes >8 mm in diameter. The cultures also consisted of medium (3–7 mm) and small (<3 mm) fragments (Figure 3). Optimized operation of the photobioreactor cell assembly was achieved with a yield of 150–1000 μmol m−1. -2 .s -1 When grown at a light intensity of 1000 and a photoperiod of 10:14 light:dark cycle, it exhibited a 30-day production cycle from 0.5 mm fragments to a harvestable size of 10 mm for tetrasporophytes (Figure 4).

[0139] Tetrasporophyte cultures of Asparagopsis spp. maintained at 5 g / L wet weight achieved an average growth rate of between 10 and 20% biomass per day. This means that 0.5 to 1 g / L wet weight biomass per consecutive harvest per day is required to maintain a culture density of 5 g / L.

[0140] By extrapolation, a 1000-liter PBR system (ten interconnected 2500 mm × 150 mm PBR cells) produces 150–1500 μmol m -2 .s -1 Under natural light intensity of 1000 m / day, it could provide 500-1000 g wet weight per day or 182.5-365 kg wet weight per year. A 1-hectare site housing 1100 x 1000 L PBR cell modules (with a 1 m perimeter to allow sufficient light access and maneuverability around each PBR system) could produce 200-400 tonnes of Asparagopsis biomass per year through sustained, continuous cultivation throughout the year.

[0141] Example 3 Algae growth and biomass removal in PBR systems Juvenile Asparagopsis taxiformis seaweed was clonally generated from fragmentation of its mature tetrasporophyte. Fragmentation was achieved by macerating the tetrasporophyte in 50 ml of seawater. This was achieved by using a milk frother, stick mixer, or blender, which chopped the tetrasporophyte into thousands of fragments within 20–30 seconds. The fragments ranged in size from 0.3 to 1.5 mm in maximum longitudinal length.

[0142] Fractions of 80 g fresh weight total biomass were inoculated into a PBR system similar to that described above, but scaled up in series to a 1000 L multi-tubular airlift photobioreactor system, which was placed in a suitable rack indoors under controlled environmental conditions and filled with filtered seawater.

[0143] The tubing of each riser section in the PBR system series was aerated at its base using compressed air supplied via an air stone (upwelling tube), while the tubing of every alternate downcomer section in the series acted as a downwelling tube. Aeration was set at an average of 25 L / min to provide sufficient lift to the sections and circulate water throughout the PBR system. After 30 days of cultivation, the culture density was more than 5 g / L.

[0144] result Over another three days, the average daily growth rate using the PBR system was estimated to be 15% ± 5%. This average daily growth rate of 15% was achieved using appropriate conditions of temperature, light, and nutrient supply. Light intensity was maintained at 1000 μmol m while maintaining the culture at 5 g / L. -2 .s -1 was set as.

[0145] Using the PBR system described above, tetrasporophyte cultures of Asparagopsis spp. were maintained at a culture density of 5 g / L. The culture density was maintained by continuous daily harvesting. The amount harvested each day was calculated based on an average daily growth rate of 15%.

[0146] The culture consisted of large (>6 mm), medium (4-6 mm), small (2-4 mm), and very small (<2 mm) biomass fragments. Stainless steel mesh sieves with 2.00 mm pore size were used to harvest the algal biomass by placing them on top of the downcomer tube. The sieves collect tetrasporophytes larger than the pore size while allowing tetrasporophytes smaller than the pore size to pass through. The 2.00 mm pore size stainless steel mesh sieve collects at least 60% of the small, medium, and large tetrasporophytes while leaving at least 70% of the retained very small biomass fragments in the PBR system.

[0147] After one week of maintenance, the total biomass was harvested and weighed, revealing that the maintenance was successful.

[0148] Thus, maintenance of culture densities of 3-7 g / L and daily growth rates greater than 10% were achieved by continuous daily harvesting of algal cultures in the PBR system of the present invention. The daily harvesting process was capable of producing a feedstock consisting of Asparagopsis tetrasporophytes, with greater than 85% of the medium and large fragments.

Claims

1. A photobioreactor (PBR) cell, the cell comprising: a) a riser section in the form of a tube; b) a downcomer section in the form of a tube; c) means for connecting said riser section and said downcomer section to permit liquid flow; d) ventilation means; A photobioreactor cell comprising:

2. The tube of the PBR cell is i. Made from UV resistant low density plastic film; ii. has a diameter of 75 to 300 mm; iii. has a length of 500 to 3000 mm; and / or iv. has a total working volume of 1.5 to 215 liters; The PBR cell of claim 1 .

3. 1. A method for growing algae, said method comprising: i. inoculating a photobioreactor (PBR) cell with algal cells, wherein said PBR cell comprises: a. a riser portion in the form of a tube; b. A downcomer section in the form of a tube; c. means connecting said riser section and said downcomer section to permit liquid flow; d. ventilation means; a process comprising: ii. maintaining the PBR cells in an aqueous culture medium under light and aeration conditions suitable for growth of the algal cells; A method that encompasses

4. 5. The method of claim 4, wherein the algae is Asparagopsis spp.

5. i. The temperature of the PBR cell is maintained between 12°C and 28°C; ii. The air flow rate of the PBR cell is 1 to 70 L / min; iii. The PBR cell has a capacitance of 150 to 1500 μmol m －２ .s －１ and / or iv. The photoperiod to which the PBR cells are exposed is an 8:16 to 14:10 light:dark cycle; The method according to claim 3 or 4.

6. 1. A method for growing algae, said method comprising: i. providing algal cells having a biomass density and average fragment diameter greater than 0.1 g / L in an aqueous culture medium; ii. Removing the algal biomass from the culture medium; a. a biomass density of at least 0.1 g / L; and b. an average fragment diameter that is less than the average fragment diameter of the algal cells in the aqueous culture medium prior to harvesting the algal biomass; generating a culture of retained algal cells having iii. Maintaining the culture of algal cells maintained in an aqueous culture medium under light and aeration conditions suitable for growth of the algal cells; A method that encompasses

7. 7. The method of claim 6, wherein the algal cells in the aqueous culture medium have a biomass density of greater than 1.0 g / L to 11.0 g / L, preferably greater than 4.0 g / L to 8.0 g / L.

8. 8. The method of claim 6 or 7, wherein the algal cells in the aqueous culture medium are composed of biomass fragments having a diameter of less than 2.0 mm to 12 mm, preferably 2.0 mm to 7 mm.

9. 9. The method of any one of claims 6 to 8, wherein about 60% to about 80% of the algal cells in the aqueous culture medium are comprised of biomass fragments having a diameter greater than 2.0 mm.

10. 10. The method of any one of claims 6 to 9, wherein the culture of maintained algal cells has a biomass density of 1.0 g / L to 8.0 g / L, preferably 2.0 g / L to 5.0 g / L.

11. 11. The method of any one of claims 6 to 10, wherein the culture of retained algal cells consists of biomass fragments having a diameter of about 2 mm or less.

12. 12. The method of any one of claims 6 to 11, wherein about 60% to about 80% of the culture of retained algal cells consists of biomass fragments having a diameter of less than 2 mm.

13. 13. The method of any one of claims 6 to 12, wherein the removed algal biomass consists of algal biomass having an average diameter of about 2 mm or larger, and preferably up to about 7 mm.

14. 14. The method according to any one of claims 6 to 13, wherein at least 60%, preferably 70%, 80% or 90% of the algal biomass has a diameter of at least 2 mm, and preferably up to about 7 mm.

15. 15. The method of any one of claims 6 to 14, wherein the algal cells in the aqueous culture medium have a biomass density of greater than 2.0 g / L, preferably greater than 4.0 g / L to 8.0 g / L, and wherein removing the algal biomass from the culture medium produces a culture of retained algal cells having a biomass density of greater than 2.0 g / L, preferably between 2.0 g / L and 5.0 g / L.

16. 16. A culture of algal cells produced using the PBR cell of claim 1 or claim 2, or a maintained culture of algal cells produced by the method of any one of claims 3 to 15.

17. 1. A culture of retained algal cells, which is a culture of algal cells in an aqueous culture medium from which an algal biomass has been removed, wherein the culture of retained algal cells has an average fragment diameter that is less than the average fragment diameter of the culture of algal cells immediately prior to removing the algal biomass.

18. 1. A composition comprising an algal biomass derived from a culture of algal cells in an aqueous culture medium, wherein the algal biomass has an average fragment diameter that is greater than the average fragment diameter of the culture of algal cells from which the algal biomass was derived.

19. 19. The composition of claim 18, wherein at least 60%, preferably 70%, 80% or 90% of the algal biomass has a diameter of at least 2 mm.

20. 1. A composition comprising an algal biomass, wherein at least 60%, preferably 70%, 80%, or 90% of said algal biomass has a diameter of at least 2 mm.

21. 20. A composition comprising an algal biomass removed from said culture of maintained algal cells of claim 17.

22. 1. A composition comprising retained algal biomass removed from a culture of retained algal cells, wherein the culture of retained algal cells is a culture of algal cells from which algal biomass has been removed, and the retained algal biomass has an average fragment diameter that is less than the average fragment diameter of the culture of algal cells from which the algal biomass was removed.

23. 23. The composition of claim 22, wherein about 60% to about 80% of the retained algal biomass is comprised of biomass fragments having a diameter of less than 2 mm.

24. 1. A composition comprising an algal biomass, wherein about 60% to about 80% of the algal biomass is composed of biomass fragments having a diameter of less than 2 mm.

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