AU2008261616A1 - Apparatus and method for the culture of photosynthetic microorganisms - Google Patents

Description

WO 2008/151376 PCT/AU2008/000848 "Apparatus and Method for the Culture of Photosynthetic Microorganisms" Field of the Invention [0001] The present invention is directed to an apparatus and method for culturing photosynthetic microorganisms in the presence of artificial light. 5 Background Art [0002] Photosynthesis based on plant metabolism produces cells of photosynthetic microorganisms which are rich in a variety of useful products including proteins, photosynthetic pigments, vitamins, and natural oils, amongst others, that are increasingly being used in different applications from 10 supplementing diets, especially diets of domesticated animals, producing "health foods", through to medical applications and the use of organic molecules such as ethanol and natural oils for the production of biofuels. [0003] With regard to the production of biofuels, photosynthetic microorganisms such as algae are currently being experimented with and algae 15 have emerged as one of the most promising sources for biodiesel (and ethanol) production. This is as a result of the widespread availability and higher oil yields (in orders of magnitude) from these microorganisms than those from traditional oilseeds such as soybean. In addition, they can grow in places away from the farmlands and forests, thus minimising the damages caused to the eco- and food 20 chain systems as well as be grown in sewages and next to power-plant smokestacks where they digest the pollutants and produce these biofuels. Though research into photosynthetic microorganisms such as algae as a source for biodiesel is not new, the current oil crises and fast depleting fossil oil reserves have made it more imperative for organisations and countries to invest more time 25 and efforts into research on suitable renewable feedstock such as algae. [0004] Thus, development of a cultivation method for these photosynthetic' microorganisms including natural oil producing algae, wildly growing in the WO 2008/151376 PCT/AU2008/000848 -2 natural environment, is needed for stable, high yield production of these useful products and high efficiency of these products requires efficient photosynthesis by these microorganisms. While use of these microorganisms appears promising, in practice, there are many questions to be answered and multiple 5 issues to be resolved before products such as biofuels can be produced sustainably and affordably on a large-scale from algae. [0005] There are number of requirements which need to be addressed for the cultivation of photosynthetic microorganisms. Some of these are comparable with requirements for cultivation of non-photosynthetic microorganisms and some 10 are specific to the requirements for the process of photosynthesis. [0006] The general requirements and culture conditions for cultivation of microorganisms are known by those skilled in the art and include a cultivation medium which contains solutes and the nutrient requirements of the microorganisms. Variables for the culture environment which need to be 15 substantially constant and controlled comprise pH, temperature, oxygen concentration, pressure and the turbidity or degree of mixing; all of which are specific for the microorganism being cultivated. [0007] Photosynthesis requires a nitrogen source, carbon dioxide and sunlight or equivalent electromagnetic radiation. Photosynthetic microorganisms such as 20 algae perform biosynthesis of a useful substance such as natural oils by photosynthesis through absorption of carbon dioxide. [0008] The current cultivation methods comprise "open" and "closed" cultivation systems. "Open" cultivation systems are very popular for large scale cultivation of photosynthetic microorganisms and generally comprise a cultivation 25 pond which may be in a form of, for instance a "raceway" or a salt lake in an outside environment. [0009] One of the first difficulties associated with large scale "open" culture in a cultivation pond is consistent light which is usually in the form of sunlight. For example, cultivation of microalgae using a cultivation pond causes a high WO 2008/151376 PCT/AU2008/000848 -3 concentration of algal cells in the culture environment resulting in colouration of the culture solution which prevents sunlight reaching the bottom of the pond. This reduced light results in reduced algal growth and thus a lower cell density is achieved, subsequently reducing the total algal photosynthetic efficiency and 5 therefore the biosynthesis of the product. [0010] .Consequently, the depth of the pond must be set to approximately 30 cm and the area required for large scale cultivation of algal cells is vast. In addition, the light intensity of sunlight is variable throughout the day, in particular at night where there is no sunlight and the seasons and thus no consistency is 10 provided for the growth of the photosynthetic microorganisms, reducing the efficiency of growth and biosynthesis of products. [0011] Although it is relatively easy to stir cultivation ponds of this type, large amounts of energy are required to mix the huge volumes of culture medium. Other problems are encountered such as inefficient mixing resulting in nutrient 15 clouding, and clouding of high concentrations of microorganisms reducing photosynthetic efficiency. Further, exogenous matters such as dust, waste, pollutants, and other airborne microorganisms and cells of other species can contaminate the cultivation ponds. Furthermore, the mechanical mixing required causes high shear stress on the microorganisms resulting in high levels of cell 20 death. Thus, a culture of a high purity and a high quality cannot be maintained. [0012] Aside from inconsistent light, there are also inconsistencies faced with these "open" cultivation ponds such as temperature and air pressure which also affect the dissolution of carbon dioxide required for photosynthesis and removal of oxygen. Thus as a result of the above factors, cultivation of photosynthetic 25 microorganisms using the cultivation ponds cannot be practically applicable to many microorganisms. (00131 With regard to the "closed" cultivation systems in operation, they are commonly expensive, with bioreactors often constructed from steel and glass to handle the pressures and weight of water some are expected to contain. 30 Irradiation is via a few incandescent bulbs in the sides of the bioreactors which WO 2008/151376 PCT/AU2008/000848 -4 prevent consistent irradiation of all microorganisms in the system and the excess heat from these bulbs inhibits maintenance of a consistent temperature and thus efficient photosynthesis. Furthermore, as the propagation of the microorganisms increases, the access to the preferred levels of radiation for efficient 5 photosynthesis decreases. These bioreactors are limited beyond a certain volume due to cost, materials, weight, cleaning and further issues of inconsistent irradiation. Thus, there has been limited success to date in the large scale cultivation of photosynthetic microorganisms by these methods. [0014] Those skilled in the art will appreciate that the invention described 10 herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. 15 [0015] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. 20 [0016] 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 herein by reference, and may be employed in the practice of the invention. [0017] The present invention is not to be limited in scope by any of the specific 25 embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein. [00181 The invention described herein may include one or more range of values (e.g. size, concentration etc). A range of values will be understood to WO 2008/151376 PCT/AU2008/000848 -5 include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. 5 [0019] Throughout this specification, unless the context requires otherwise, the word "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. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as 10 "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are 15 found in the prior art or that affect a basic or novel characteristic of the invention. [0020] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the 20 invention belongs. Summary of the Invention [0021] In accordance with the present invention, there is provided an apparatus for culturing photosynthetic microorganisms comprising at least one bioreactor and at least one source of electromagnetic radiation wherein the at 25 least one source of electromagnetic radiation is a light emitting diode comprising an organic or polymeric emissive layer. [0022] Preferably, the apparatus is retained within a structure substantially impenetrable by sunlight. Advatageously, the use of artificial electromagnetic WO 2008/151376 PCT/AU2008/000848 radiation in the present invention obviates the problems associated with the use of sunlight. [0023] Preferably, the at least one electromagnetic radiation source is an organic light emitting diode (OLED) which is known to include small molecule 5 OLEDs, polymer OLEDs (POLEDs), passive-matrix OLEDs, active-matrix OLEDs, transparent OLEDs, top-emitting OLEDs, foldable OLEDs and white OLEDs. [0024] In a highly specific form of the invention, the electromagnetic radiation source is a polymer OLED. 10 (00251 Advantageously, OLEDs emit less heat than incandescent light. Further, the power consumption of OLEDs is significantly less than other sources of electromagentic radiation. Further still, the heat/light ratio of OLEDs is in the order of < 1:10, whilst for incandescent light, the ratio is > 10:1. Advantageously, the use of light sources with low heat output results in significant advantages 15 over the use of incandescent light. [0026] Advatageously, OLEDS may be prepared in large emitting sheets unlike conventional LEDs which occur only as point sources of light. [0027] Where there are provided at least two electromagnetic radiation sources, their spatial arrangement may be such that when in use, a portion of 20 each beam of electromagnetic radiation overlaps with an adjacent beam of electromagnetic radiation. [0028] Preferably, it is possible to alter the spatial arrangement of the electromagnetic radiation sources to accommodate differing conditions in the apparatus. 25 [0029] Advantageously, the spatial arrangement of the electromagnetic radiation sources increases the exposure of the culture in the culture vessel to electromagnetic radiation.

WO 2008/151376 PCT/AU2008/000848 -7 [0030] It will be appreciated that the intensity of the electromagnetic radiation sources will depend on many factors, including the type, number and spatial arrangement of the electromagnetic radiation sources. Without being limited by theory, it is expected that the preferred intentisty will be about 10-15 % of that on 5 a bright sunny day, expected to be in the region of 3000 - 6000 candela. [0031) Preferably, adjacent bioreactors are substantially coplanar. [0032] Preferably the at least one electromagnetic radiation source is substantially planar. Within the context of the present specificaiton, the temi substantially planar will be taken to include electromagnetic radiation source 10 substantially larger in two dimensions than a third dimension. [0033] Preferably, the at least one electromagnetic radiation source is provided in the form of a single sheet capable of emitting electromagnetic radiation. [0034] It will be appreciated that the electromagnetic radiation sources may be 15 substantially rigid or flexible. [0035] Preferably, the single sheet is approximately the same shape and size as the bioreactors. [0036] The substantially planar electromagnetic radiation sources comprise first and second surfaces. The electromagentic radiation may be emitted from 20 the first surface, the second surface or both surfaces. [0037] In one form of the invention, the electromagnetic radiation source is provided on a substrate. [0038] Preferably, the substrate Is approximately the same shape and size as the bioreactors.

WO 2008/151376 PCT/AU2008/000848 [0039] Preferably, the at least one electromangnetic radiation source is tailored to emit electromagnetic radiation over a preferred wavelength or wavelength range. Without being limited by therory, it is believed that microorganisms absorb electromagnetic radiation of certain wavelengths in 5 preference to electromagnetic radiation of other wavelengths. With the ability to control the wavelength of emitted radiation, the efficiency of microorganism growth may be increased. [00401 In one forn of the invention, the electromagnetic radiation is substantially monochromatic. 10 [0041] In a second form of the invention, the electromagnetic radiation has a wavelength in air of between about 380 nm and 780 nm. Preferably, the electromagnetic radiation has a wavelength in air of between about 600 nm and 700 nm. More preferably, the electromagnetic radiation has a wavelength in air of between about 630 nm and 690 nm. The skilled addressee will appreciate 15 that the choice of wavelength or wavelengths of the electromagenetic radiation may be affected by the type of microorganism present. [0042] It will be appreciated that the electromagnetic radiation sources may emit monochromatic radiation of various wavelengths. [0043] In one form of the invention, the electromagnetic radiation sources emit 20 electromagnetic radiation intermittently. [0044] In one form of the invention, the or each bioreactor is transparent or translucent. Where the bioreactor is transparent or translucent, the electromagnetic radiation source may be located extemal to the bioreactor. It will be appreciated that the bioreactor must be at least partially permeable to the 25 electromagnetic radiation. [0045] Where the or each bioreactor is transparent or translucent, the or each bioreactor is preferably prepared from a polymeric material such as polyethylene, polypropylene, polyurethane, polycarbonate, polyvinylpyrrolidone, WO 2008/151376 PCT/AU2008/000848 -9 polyvinylchlorid.e, polystyrene, poly(ethylene terephthalate), poly(ethylene naphthalate), poly(1,4-cyclohexane dimethylene terephthalate), polyolefin, polybutylene, polyacrylate and polyvinlyidene chloride. [0046] Preferably, the or each biorector defines a tortuous fluid flow path for 5 the flow of culture medium therein. [0047] In one form of the invention, the or each bioreactor comprises a translucent or transparent conduit. Preferably, the conduit is configured to define straight sections and turn sections, wherein the straight sections are disposed one adjacent another. Preferably, the conduit is so configured by being folded 10 upon itself at intervals along its length. [0048] In a second form of the invention, the or each bioreactor comprises a plurality of baffles arranged so as to define the tortuous flow path within each bioreactor. [0049] In a preferred form of the invention, each substantially planar bioreactor 15 is exposed to electromagnetic radiation on both sides of the bioreactor. [0050] It will be appreciated that the electromagnetic radiation penetrates the bioreactors and irradiates the culture medium. [0051] Where the or each bioreactor is transparent or translucent, there is preferably provided an electromagnetic radiation source between each 20 bioreactor. Where the bioreactors are substantially planar, it is preferable that the or each electromagnetic radiation source is substantially planar. Preferably, the or each substantially planar electromagnetic radiation source is substantially coplanar with adjacent bioreactors. [0052] Where the substantially planar electromagnetic radiation sources and 25 the bioreactors are substantially coplanar, the sizes of the substantially planar electromagnetic radiation sources and the bioreactors are preferably about the same.

WO 2008/151376 PCT/AU2008/000848 - 10 [0053] It will be appreciated that the preferred distance between adjacent bioreactors is such that when in use, a portion of a beam of electromagnetic radiation from a first electromagnetic radiation source on one side of a bioreactor overlaps with a beam of electromagnetic radiation from a second 5 electromagnetic radiation source on the second side of the same bioreactor. In this manner, all of the culture medium in the bioreactor is subjected to electromagnetic radiation. [0054] Advantageously, it is possible to adjust the distance between adjacent bioreactors to alter the coverage of electromagnetic radiation within each 10 bioreactor. [0055] It will be appreciated that the size of the bioreactor will be influenced by many factors including the weight of water, the flow rate and residence time of the culture medium. Preferably, each bioreactor is about 12 m high and about 3 m wide. More preferably, each bioreactor is about 4 m high and about 2 m 15 wide It will be appreciated that the larger the bioreactor and the greater the amount of culture medium contained within the bioreactor, the greater the requirement for stronger polymeric material to reduce the likelihood of leakage of culture medium from the bioreactor. [0056] The skilled addressee will appreciate that the bioreactor will need to 20 withstand pressures exerted by the culture medium and will need to make allowances when preparing the bioreactor from a polymeric material. It will be appreciated that greater pressures will be present in larger bioreactors. [0057] In one form of the invention, the or each bioreactor comprises a rectangular base with two opposing side walls and two opposing end walls. It will 25 be appreciated that the final dimension of the or each bioreactor will be dependant on many factors including the materials from which the or each bioreactor is constructed and the ability of said materials to withstand the pressure imparted by the volume of water necessary to fill the or each bioreactor. Preferably, the length of the or each bioreactor is about 2 m to 20 m, the width of 30 the or each bioreactor is about 0.75 m to 3 m and the height of the or each WO 2008/151376 PCT/AU2008/000848 -11 bioreactor is about 1 m to 2 m. In one embodiment of the invention, the or each bioreactor is about 10 m long, 2 m wide and 1 m high. In a preferred form of the invention, the or each bioreactor further comprises a lid. Preferably, the bioreactor is slightly tapered to form a trapezoid in cross section to permit 5 bioreactors to be stacked inside each other for to reduce volume during trasnportation and storage. [0058] Where there are provided more than one bioreactor, they may be arranged in series or parallel. In one embodiemnt of the invention, 10 x 2 m bioreactors are aligned lengthways, to provide, for example an apparatus about 10 2 m wide of elongated length, for example, about 100 m. Such an apparatus may be housed in a strucutre about 3 m wide, 4 m high and over 100 m in length. [0059] In an alternate form of the invention, the or each bioreactor comprises a substantially circular base with a side wall. [0060] It will be appreciated that where the or each bioreactor is not 15 transparent or translucent, the or each electromagnetic radiation source will reside at least partially within the or each bioreactor. It will be appreciated that the configuration of the or each electromagnetic radiation source and substrates where provided, should be such that they do not reduce the volume of the- or each bioreactor to a significant extent. 20 [0061] In a preferred form of the invention, the electromagnetic radiation source substrates are substantially planar. Preferably, the dimensions of the or each bioreactor and the electromagnetic radiation source substrates are complimentary. [0062] The substantially planar electromagnetic radiation source substrates 25 may be substantially flat or may be corrugated. Where the substantially planar electromagnetic radiation source substrates are corrugated, the apparatus may comrise supports for supporting the substantially planar electromagnetic radiation source substrates. Said supports may be provided in the form of rods, WO 2008/151376 PCT/AU2008/000848 - 12 wherein the rods are adjacent alternating grooves and ridges in each substantially planar electromagnetic radiation source substrates. [0063] The plurality of electromagnetic radiation sources are preferably arranged in the or each bioreactor in a substantially coplanar arrangement. In 5 one form of the invention, the substantially planar electromagnetic radiation sources are arranged substantially vertically. In a second form of the invention, the substantially planar electromagnetic radiation sources are arranged substantially horizontally. [0064] It will be appreciated that the preferred distance between adjacent 10 substantially planar electromagnetic radiation sources is such that when in use, a portion of a beam of electromagnetic radiation from a first electromagnetic radiation source overlaps with a beam of electromagnetic radiation from an adjacent electromagnetic radiation source. In highly preferred forms of the invention, adjacent substantially planar electromagnetic radiation sources are 15 about 3 cm to 5 cm apart. [0065] Preferably, the electromagnetic radiation sources are removable from the bioreactor. [0066] It will be appreciated that the preferred distance between adjacent electromagnetic radiation sources within a bioreactor is such that when in use, a 20 portion of a beam of electromagnetic radiation from a first electromagnetic radiation source overlaps with a beam of electromagnetic radiation from an adjacent electromagnetic radiation source. In this manner, all of the culture medium in the bioreactor is subjected to electromagnetic radiation. [0067] Advantageously, it is possible to adjust the distance between adjacent 25 electromagentic radiation sources within a bioreactor to alter the coverage of electromagnetic radiation within said bioreactor. [0068] Preferably, the or each bioreactor comprises an inlet for the ingress of water and/or culture medium and an outlet for the egress of culture medium.

WO 2008/151376 PCT/AU2008/000848 -13 [0069] In one form of the invention, the bioreactors are interconnected in a parallel configuration- In such a configuration, culture medium flows into each bioreactor, undergoes photosynthesis and exits the bioreactor, wherein the microorganisms are harvested. In an alternate form of the invention, the 5 bioreactors are interconnected in a series configuration. In such a configuration, culture medium flows into a first bioreactor, undergoes photosyntheses and exits the bioreactor. The culture medium then enters a second bioreactor for further photosynthesis and so on. It will be appreciated that there may be provided a combination of parallel and series configurations in the same apparatus and that 10 it may be possible to switch configurations. [0070] The apparatus may be provided with an inlet manifold and an outlet manifold, the inlet manifold being in fluid communication with each bioreactor inlet and the outlet manifold being in fluid communication with each bioreactor outlet. 15 [0071] The inlet manifold preferably comprises at least one inlet and at least one outlet. Preferably, the number of inlet manifold outlets is the same as the number of bioreactors and are in fluid communication therewith. [0072] The outlet manifold preferably comprises at least one inlet and at least one outlet. Preferably, the number of outlet manifold inlets is the same as the 20 number of bioreactors and are in fluid communication therewith. [0073] Preferably, the inlet of the inlet manifold has provided therein a valve. Each of the outlets of the inlet manifold may have valves provided therein. [0074] Preferably, the outlet of the outlet manifold has provided therein a valve. Each of the inlets of the outlet manifold may have valves provided therein. 25 [0075] It will be appreciated that it may be necessary to provide the apparatus with means to deoxygenate the culture medium. It is known that oxygen is generated during the microorganism culture process and the presence of oxygen in the culture medium can inhibit microorganism photosynthesis.

WO 2008/151376 PCT/AU2008/000848 -14 [0076] It is known that photosynthesis of microorganisms requires carbon dioxide and it may be necessary for the apparatus to further comprise carbon dioxide introduction means. In one form of the invention, said means introduce carbon dioxide directly into the culture medium in the or each bioreactor. In a 5 second form of the invention, carbon dioxide is introduced into an external water source which is subsequently introduced into the culture medium in the or each bioreactor. It will be appreciated that said external water source may be provided in the form of fresh culture medium. [0077] Where the carbon dioxide is introduced into an external water source, 10 the external water source is preferably saturated with carbon dioxide at that temperature. [0078] It will be appreciated that mixing of the culture medium is important to achieve substantially constant cultivation of photosynthetic microorganisms throughout the culture environment. This is because the light, air and carbon 15 dioxide should be distributed as evenly as possible throughout the culture medium. The culture medium is preferably mixed by way of inflow of water and/or fresh culture medium into the bioreactor and outflow of culture medium out of the bioreactor. It will be appreciated that the mixing should be sufficient to distribute the light, air and carbon dioxide to a desired level bearing in mind that 20 high levels of mixing may result in shearing of the photosynthetic microorganisms which can result in cell death. [0079] Whilst it is possible to provide mixing means, the culture medium is preferably mixed by way of inflow of water and/or fresh culture medium into the vessel and outflow of culture medium out of the vessel. It will be appreciated that 25 the mixing should be sufficient to distribute the light, air and carbon dioxide to a desired level bearing in mind that high levels of mixing may result in shearing of the photosynthetic microorganisms which can result in cell death. [0080] Depending on the structure of the or each bioreactors, the culture medium may flow through the or each bioreactor under the force of gravity. 30 Preferably, the plurality of substantially planar bioreactors in the apparatus are WO 2008/151376 PCT/AU2008/000848 - 15 oriented substantially vertically such that the culture medium flows through each bioreactor under the force of gravity from the top or near the top of the bioreactor to the bottom or near the bottom of the bioreactor. [0081] The photosynthetic microorganism of the invention may be varied and 5 can be any photosynthetic microorganism which can be cultured in the cell culture apparatus of the invention. Preferably, the photosynthetic microorganism is an algae. In one form of the invention, the photosynthetic microorganism is an algae from the family of blue green algae. In another form of the invention, the photosynthetic microorganism is a photosynthetic bacteria. 10 [0082] The present invention may be applied to a wide variety of algal and other species of photosynthetic microorganisms having the ability to produce a variety of products. [0083] In another form of the invention, green algae may be cultivated in the invention. In a further form green algae of the genus Chlorella may be cultivated 15 in the invention, comprising amongst others species; Chlorella pyrenoldosa. Products which can be recovered from Ch/orella comprise, amongst others, chelatory agents, for example to extract mercury from the body. [0084] In another form of the invention, the green algae of the genus Danaliella can be cultivated in the invention, comprising, amongst other species, 20 Dunafella salina. Products which can be recovered from species of Dunaliella comprise beta carotene, for example to be used in nutritional supplementation and "health foods". Beta carotene amongst other products can be recovered or produced from Dunaliella salina cultivated using the present invention. [0085] Products which can be produced by the photosynthetic microorganisms 25 cultured in the invention comprise, but are not limited to: compositions comprising docosahexaenoic acid [DHA, 22:6(n-3)] and eicosapentaeneoic acid [EPA, 20:5(n-3)] which are essential polyunsaturated fatty acids (PUFA) in the human diet and known as Omega 3 PUFA. These PUFA may reduce the risk of coronary heart disease and alleviate inflammatory diseases and are believed to WO 2008/151376 PCT/AU2008/000848 -16 be useful in the treatment of Alzheimer"s disease. Some microalgae and thraustochytrids are a rich source of these PUFA; Pharmaceutical leads: DHA and other novel bloactive PUFA can be chemically modified by adding or changing functional groups within different regions in the molecule (combinatorial 5 chemistry) in order to enhance/change the bioactivity and hence lead to the synthesis of new pharmaceuticals. Other unusual fatty acids are likely to be bloactive. Aquaculture in which microalgae are used as essential live feeds and supplements in the aquaculture of larval and juvenile animals including oyster spat, juvenile abalone, finfish larvae and rotifer; food supplements; "health" food 10 supplements; antimicrobials; antifungals: agrochemicals; cosmetics; photosynthetic pigments; polysaccharides; and natural oils for the production of biofuels. [0086] For the biosynthesis of natural oils for the production of biofuels, the following species and microalga belonging in the same class as these species, 15 amongst others, may be cultured by the invention: Neochloris o/eoabundans is a microalga belonging in the class Chlorophyceae; Scenedesmus dimorphus is a unicellular algae in the class Chlorophyceae; Euglena gracilis; Phaeodactylum tricomutum; Phaeodactylum tricornutum is a diatom; Pleurochrysis carterae is a unicellular coccolithophorid alga of the class Haptophyta (Prymnesiophyceae); 20 Prymnesium parvum; Tetraselmis chui; Tetraselmis suecica; Isochrysis galbana; Nannochloropsis safina (Nannochioris oculata); Nannochloris atomus; Nannochloris maculate; Nannochloropsis gaditana; Nannochloropsis oculata, Botryococcus braunii; Dunaliella tertiolecta; Nannochlors species; Spirulina species; Chlorophyceae (green algae); and Bacillarophy (diatom algae). 25 (00871 Microalgae species for the production of aquaculture feed can be cultivated in the invention comprising the following, but not limited to: Chaetoceros muelleri, Chaetoceros calcitrans, Chaetoceros simplex, Skeletonema costatum, Skeletonema sp., Thalassiosira pseudonana, Thalassiosira pseudonana, Navicula jeffreyi, Nitzschia closterium, Dunaliella 30 tertiolecta, Rhodomonas salina, Rhodomonas safina, Nannochloropsis oculata, WO 2008/151376 PCT/AU2008/000848 -17 Nannaochloropsis sp., Tetraselmis suecica, Tetraselmis chuii, Isochiysis sp., Pavlova lutheri, Paviova saina, and Pavlova pinguis. [0088] Other photosynthetic microorganisms which could be cultivated with the present invention are those which can produce products used as nutritional 5 supplements and "health foods". In a preferred form of the invention, cyanobacteria comprising, but not limited to, Arthrospira platensis, and Arthrospira maxim and other photosynthesizing bacteria belonging to the Arthrospira genus and Spirulina genus can be cultivated in the invention. The products which could be recovered from these cyanobacteria include proteins, all 10 essential amino acids, gamma-linolenic acid (GLA), and also provides alpha linolenic acid (ALA), linoleic acid (LA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA), vitamin B1 (thiamine), B2 (riboflavin), B3 (nicotinamide), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), vitamin C, vitamin D, and vitamin E. 15 [0089] In accordance with the present invention, there is provided a method for culturing photosynthetic microorganisms in the presence of artificial light,. the method comprising the steps of: culturing photosynthetic microorganisms in an apparatus comprising at least one bioreactor and at least one source of 20 electromagnetic radiation wherein the at least one source of electromagnetic radiation is a light emitting diode comprising an organic or polymeric emissive layer. [00901 Preferably, the method comprises the further step of: adding carbon dioxide to the culture medium in the or each 25 bioreactor. [00911 It will be appreciated that the carbon dioxide may be acquired from many sources, including power station flue gases and petroleum sources, natural gas residues and fermentation residues and cement plants.

WO 2008/151376 PCT/AU2008/000848 -18 [0092] Preferably, the method comprises the further step of: adding water to the or each bioreactor. [0093] Preferably, the method comprises the further step of: adding further culture medium to the or each bioreactor. 5 [0094] Preferably, the method comprises the further step of: adding nutrients to the culture medium. [0095] The skilled addressee will appreciate that the choice of nutrients will depend on many factors, including the type of microorganisms present in the culture medium. The nutrients may include potassium nitrate, magnesium 10 sulphate, calcium chloride, boric acid, iron sulphate, zinc sulphate, manganese chloride; molybdenum oxide, copper sulphate and cobalt nitrate. [0096] The skilled addressee will appreciate that the pH of the culture medium will depend on many factors, including the type of microorganisms present in the culture medium. In a preferred embodiment, the pH is alkaline. 15 [0097] The skilled addressee will appreciate that the residence time of the culture medium in the or each bioreactor will depend on many factors, including the type of microorganisms present in the culture medium. (0098] Preferably, the method comprises the further step of removing culture medium from the or each bioreactor. 20 [00991 Preferably, the method comprises the further step of: deoxygenating the culture medium in the or each bioreactor. 00100] Preferably, the method comprises the further step of: WO 2008/151376 PCT/AU2008/000848 -19 mixing the culture medium in the or each bioreactor. [00101] The skilled addressee will appreciate that the temperature of the culture medium in the or each bioreactor will depend on many factors, including the type of microorganisms present in the culture medium. Preferably, the temperature of 5 the culture medium is between about 15 *C and 30 *C. More preferably, the temperature of the cell culture medium is between about 20 'C and 25 *C. [00102] Preferably, the pressure of the culture medium is about atmospheric pressure. [00103] Other aspects and advantages of the invention will become apparent to 10 those skilled in the art from a review of the ensuing description, which proceeds with reference to the following illustrative drawings. Brief Description of the Drawings [00104] The present invention will now be described, by way of example only, with reference to five embodiments thereof and the accompanying drawings, in 15 which: Figure 1 is a cross-sectional side view of a bioreactor of the apparatus in accordance with a first embodiment of the present invention; Figure 2 is a cross-sectional side view of a bioreactor of the apparatus in accordance with a second embodiment of the present invention; 20 Figure 3 is a side view of a bioreactor of the apparatus in accordance with a second embodiment of the present invention; Figure 4 is a persective view of an apparatus in accordance with a second embodiment of the present invention; WO 2008/151376 PCT/AU2008/000848 - 20 Figure 5 is a perspective view of a culture vessel of the apparatus in accordance with a third embodiment of the present invention; Figure 6 is side view of an apparatus in accordance with a third embodiment of the present invention; 5 Figure 7 is top view of an apparatus in accordance with a fourth embodiment of the present invention; and Figure 8 is top view of an apparatus in accordance with a fifth embodiment of the present invention. Detailed Description of the Invention 10 [00105] According to the present invention, the inventors have revealed that the problems with prior art can be addressed at least in part by culturing photosynthetic microorganisms within the apparatus of the present invention, wherein the microorganisms have a substantially consistent and constant light source to provide optimum growth conditions. 15 [00106] Those skilled in the art will appreciate that the invention described herein is amenable to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, 20 individually or collectively and any and all combinations or any two or more of the steps or features. [00107] Other aspects and advantages of the invention will become apparent to those skilled in the art from a review-of the ensuing description, which proceeds with reference to the following illustrative drawings. 25 [00108] In Figures 1 to 4, there is shown an apparatus 10 for culturing photosynthetic microorganisms in the presence of artificial light in accordance with two embodiments of the present invention, the apparatus 10 comprising a WO 2008/151376 PCT/AU2008/000848 -21 plurality of polyethylene bioreactors 12 wherein each bioreactor 12 is transparent or translucent and defines a tortuous fluid flow path adapted to contain a culture medium and wherein there is provided a plurality of electromagnetic electroluminescent radiation sources 14 adapted to irradiate the culture medium 5 with electromagnetic radiation. [00109] In accordance with a first embodiment of the present invention, each bioreactor comprises a translucent or transparent conduit 16 best seen in Figure 1. The conduit 16 is configured to define straight sections. 18 and turn sections 20, wherein the straight sections 18 are disposed one adjacent another and is so 10 configured by being folded upon itself at intervals along its length. [00110] In accordance with a second embodiment of the present invention, each bioreactor 12 comprises a plurality of baffles 22 arranged so as to define the tortuous flow path within each bioreactor 12, best seen in Figure 3. [00111] The bioreactors 12 are substantially planar and are approximately 4 m 15 high and 1 m wide. Adjacent bioreactors 12 are substantially coplanar, adjacent bioreactors placed about 5 -10 cm apart fom each other, best seen in Figures 2 and 4. [00112] An electromagnetic radiation source 14 is provided between each substantially coplanar bioreactor 12 as shown in Figures 2 and 4. The 20 electromagnetic radiation sources 14 are substantially planar and are substantially coplanar with adjacent bioreactors 14. The sizes of the substantially planar electromagnetic radiation sources 14 and the bioreactors 12 are preferably complimentary. [00113] The electromagnetic radiation sources 14. are provided in the form of 25 OLEDs, preferably provided as a single sheet capable of emitting electromagntic radiation. The electromagnetic radiation sources 14 comprise first 22 and second 24 surfaces and the electromagentic radation is emitted from both surfaces.

WO 2008/151376 PCT/AU2008/000848 -22 [001141 The usual structure of an OLED consists of an anode (eg. indium tin oxide), deposited onto a transparent substrate, followed by two conducting layers, a hole injection layer and an electron transport layer which sandwich an emissive organic layer. The structure is topped with a reflective metal cathode. 5 Various materials have been used as the emissive organic layer, including small crystalline molecules (small molecule OLEDs) or a polymer (polymer OLEDs or POLEDs). Known small molecules include anthracene and, fused benzene ring compounds. POLEDs may comprise a two-layer polymer film sandwiched between the electrodes. The bilayer film consists of an emitting polymer such as 10 polyparaphenylene or polyfluorene atop a conducting polymer layer such as a combination of polyaniline and poly(styrenesulfonate) or poly(ethylenedioxythiophene) and poly(styrene sulfonate). Substitution of side chains onto the polymer backbone may determine the color of emitted light or the stability and solubility of the polymer for performance, and ease of processing. 15 POLEDs may advantageously be prepared by inkjet or roll priitng light emitting polyers onto a sheet of glass or plastic. [00115] Each bioreactor 12 is provided with a fluid inlet 26 at the top of the bioreactor and a fluid outlet 28 at the bottom of the bioreactor. [00116] The apparatus 10 is provided with an inlet manifold 30 and an outlet 20 manifold 32, the inlet manifold 30 being in. fluid communication with each bioreactor inlet 26 and the outlet manifold 32 being in fluid communication with each bioreactor outlet 28. [00117] The inlet manifold 30 comprises an one inlet 34 and a plurality of outlets 36, each outlet 36 in fluid communication with a bioreactor 12. The outlet 25 manifold 32 comprises at a plurality of inlets 38 and an outlet 40, each inlet 38 in fluid communication with a bioreactor 12. [00118] Preferably, the inlet 34 of the inlet manifold 30 has provided therein a valve (not shown). Each of the outlets 36 of the inlet manifold 30 may have valves (not shown) provided therein.

WO 2008/151376 PCT/AU2008/000848 -23 [00119] Preferably, the outlet 40 of the outlet manifold 32 has provided therein a valve (not shown). Each of the inlets 38 of the outlet manifold 32 may have valves (not shown) provided therein. (00120] The apparatus further comprises a holding vessel (not shown) 5 comprising fresh culture medium to replace the culture medium in each bioreactor 12 on harvesting of the photosynthetic microorganisms. The holding vessel is provided with carbon dioxide introduction means (not shown) to saturate the fresh culture medium with carbon dioxide prior to introduction of the fresh culture medium into the bioreactor 12. The fresh culture medium may also 10 comprise nutrients. [00121] In use, each vertically suspended bioreactor 12 is filled or at least partically filled with the culture medium from the holding vessel and electromagnetic radiation is emitted from the electromagnetic radiation sources 14. Given the distance between each electromagnetic radiation source 14-and 15 each adjacent bioreactor 12, the electromagentic radiation emitted through the culture vessel should provide consistent coverage throughout the culture medium. [00122] Fresh culture medium saturated with carbon dioxide and containing nutrients is continuously fed into the inlet manifold 30 and via the inlet manifold 20 outlets 36, to each corresponding bioreactor 12. The used culture medium is continuously removed from each bioreactor 12 into the outlet manifold 32. The continuous flow of liquid between each biorector inlet 26 and biorector outlet 28 serves to mix the culture medium within each bioreactor 12 and results in consistent exposure of the culture medium to electromagnetic radiation. 25 [001231 The algae in the culture medium exiting the apparatus is harvested via flocculation or centrifuging. It will be appreciated that after harvesting the algae, it may be possible to reuse the water in the grwoth of further mocroorganisms.

WO 2008/151376 PCT/AU2008/000848 - 24 [001241 Advantageously, the apparatus operates at about atmospheric pressure which results in considerable advantages compared to high pressure apparatuses of the prior art. [00125] It may be possible to power the entire apparatus by solar power, which 5 may include the use of solar power storage batteries. Advantageously, electromagenetic radiation sources such as OLED's draw very small amounts of current and can permit the use of solar generated power. [00126] The apparatus 10 is retained within a structure substantially impenetrable by sunlight such as as shed (not shown). In order to have greater 10 control over temperature fluctuations, it is advantageous that the structure be insulated. [00127] In Figures 5 and 6, there is shown an apparatus 50 for culturing photosynthetic microorganisms in the presence of artificial light in accordance with a third embodiment of the present invention, the apparatus 50 comprising a 15 culture vessel 52 comprising a plurality of electromagnetic radiation sources (not shown) and a plurality of electromagnetic radiation source substrates 54, wherein each electromagnetic radiation source substrate 54 comprises at least one electromagnetic radiation source. [00128] The bioreactor 52 comprises a substantially rectangular base 56 about 20 3 m long and about 2 m wide with side walls, each side wall being about 1 m high. The bioreactor 52 further comprises a removable lid 58. (00129] The electromagnetic radiation source substrates 54 are substantially flat and planar and comprise a plurality of electromagnetic radiation sources. The substrates 54 are about 3 m long and 1 m wide and are adapted to reside 25 within the bioreactor 52 in a substanaitly vertical orientation, best seen in Figure 6, an end view of the bioreactor 52 of Figure 5. For clarity, the substrates 54 have been removed from Figure 5. The substrates 54 are each provided with at least one OLED, each OLED being spaced apart from adjacent OLED's such that when in use, a portion of each beam of electromagnetic radiation overlaps WO 2008/151376 PCT/AU2008/000848 -25 with an adjacent beam of electromagnetic radiation. Each substrate 54 is about 3-5 cm apart from an substrate 54. [00130] The apparatus 50 further comprises a holding vessel (not shown) comprising fresh culture medium to replace the culture medium in the bioreactor 5 52 on harvesting of the photosynthetic microorganisms. The holding vessel is provided with carbon dioxide introduction means (not shown) to saturate the fresh culture medium with carbon dioxide prior to introduction of the fresh culture medium into the bioreactor 52. The fresh culture medium may also comprise nutrients. 10 [00131] The bioreactor 52 is provided with fluid input means (not shown) at one end of the culture vessel and fluid outlet means (not shown) at an opposing end of the culture vessel. Said fluid inlet means and fluid outlet menas are provided in the form of pipes and valves. [00132] The apparatus may further comprise means for removing culture 15 medium from the bioreactor 52. [00133] In use, the bioreactor 52 containing the substantially flat, planar frames 20 is filled with the culture medium and electromagnetic radiation is emitted from the electromagnetic radiation sources. Given the distance between the substrates 54 and the electromagnetic radiation sources on each substrate 54, 20 the electromagentic radiation emitted through the culture vessel is substantially consistent. [00134] Fresh culture medium saturated with carbon dioxide and further containing nutrients is continuously fed into the bioreactor 52 at the input means and used culture medium is continuously removed from the bioreactor 52 at the 25 output means. The continuous flow of liquid between the input means and the output means serves to mix the culture medium within the bioreactor 52 and results in consistent exposure of the culture medium to electromagnetic radiation.

WO 2008/151376 PCT/AU2008/000848 - 26 (00135] Advantageously, the apparatus operates at about atmospheric pressure which results in considerable advantages compared to high pressure apparatuses of the prior art. [00136] It may be possible to power the entire by solar power, which may 5 include the use of solar power storage batteries. Advantageously, electromagenetic radiation sources such as OLED's draw very small amounts of current and can permit the use of solar generated power. [00137] In Figure 7, there is shown an apparatus 60 for culturing photosynthetic microorganisms in the presence of artificial light in accordance with a fourth 10 embodiment of the present invention, the apparatus 60 comprising a bioreactor 62 comprising a plurality of electromagnetic radiation sources (not shown) and a plurality of electromagnetic radiation source substrates 64, wherein each substrate 64 comprises one electromagnetic radiation source. [00138] The electromagnetic radiation sources are OLEDs and are 15 approximately the same size as the substrates 64. The substrates 64 are flexible and are arranged in the bioreactor 62 in a corrugated fashion, best seen in Figure 7. [00139] The apparatus 60 may comprise rods (not shown) running vertically through the bioreactor 62 wherein the rods are adjacent alternating grooves and 20 ridges in each substrate 64 to support the substrate 64. [00140] In Figure 8, there is shown an apparatus 70 for culturing photosynthetic microorganisms in the presence of artificial light in accordance with a fifth embodiment of the present invention, the apparatus 70 comprising a bioreactor 72 comprising a plurality of electromagnetic radiation sources 74. 25 (00141] Without being limited by theory, it is anticipated that the present invention may result in microorganism yields of 2000 - 20000 dry tonnes per hectare.

WO 2008/151376 PCT/AU2008/000848 -27 [00142] Modifications of the above-described modes of carrying out the various embodiments of this invention will be apparent to those skilled in the art based on the above teachings related to the disclosed invention. The above embodiments of the invention are merely exemplary and should not be construed 5 to be in any way limiting.

Claims (18)

1. An apparatus for culturing photosynthetic microorganisms comprising at least one bioreactor and at least one source of electromagnetic radiation wherein the at least one source of electromagnetic radiation is a light 5 emitting diode comprising an organic or polymeric emissive layer.

2. An apparatus for culturing photosynthetic microorganisms according to claim 1, wherein the apparatus is retained within a structure substantially impenetrable by sunlight.

3. An apparatus for culturing photosynthetic microorganisms according to 10 claim 1 or claim 2, wherein the at least one electromagnetic radiation source is an OLED.

4. An apparatus for culturing photosynthetic microorganisms according to claim 3, wherein the electromagnetic radiation source is a polymer OLED.

5. An apparatus for culturing photosynthetic microorganisms according to any 15 one of the preceding claims, wherein the at least one electromagnetic radiation source is substantially planar.

6. An apparatus for culturing photosynthetic microorganisms according to any one of the preceding claims, wherein the at least one electromagnetic radiation source is provided in the form of a single sheet capable of 20 emitting electromagnetic radiation.

7. An apparatus for culturing photosynthetic microorganisms according to claim 6, wherein the substantially planar electromagnetic radiation sources emit electromagnetic radiation from both first and second surfaces.

8. An apparatus for culturing photosynthetic microorganisms according to any 25 one of the preceding claims, wherein the electromagnetic radiation source is provided on a substrate. WO 2008/151376 PCT/AU2008/000848 - 30-.

9. An apparatus for culturing photosynthetic microorganisms according to any one of the preceding claims, wherein the electromagnetic radiation has a wavelength in air of between about 630 nm and 690 nm. 1 0An apparatus for culturing photosynthetic microorganisms according to any 5 one of the preceding claims, wherein the or each bioreactor is transparent or translucent.

11.An apparatus for culturing photosynthetic microorganisms according to claim 10, wherein the or each bioreactor is prepared from a polymeric material such as polyethylene, polypropylene, polyurethane, 10 polycarbonate, polyvinylpyrrolidone, polyvinylchlorde, polystyrene, poly(ethylene terephthalate), poly(ethylene naphthalate), poly(1,4 cyclohexane dimethylene terephthalate), polyolefin, polybutylene, polyacrylate and polyvinlyidene chloride.

12.An apparatus for culturing photosynthetic microorganisms according to any 15 one of the preceding claims, wherein the or each biorector defines a tortuous fluid flow path for the flow of culture medium therein.

13.An apparatus for culturing photosynthetic microorganisms according to any one of the preceding claims, wherein, the or each bioreactor is substantially planar. 20 14.An apparatus for culturing photosynthetic microorganisms according to any one of claims 10 to 13, wherein the sizes of the substantially planar electromagnetic radiation sources and the bioreactors are preferably about the same.

15.An apparatus for culturing photosynthetic microorganisms according to any 25 one of claims I to 9, wherein the or each bioreactor comprises a rectangular base with two opposing side walls and two opposing end walls. WO 2008/151376 PCT/AU2008/000848 - 31

16.An apparatus for culturing photosynthetic microorganisms according to claim 15, wherein the or each electromagnetic radiation source resides at least partially within the or each bioreactor.

17.An apparatus for culturing photosynthetic microorganisms according to 5 claim 16, wherein the plurality of electromagnetic radiation source substrates are arranged in the or each bioreactor in a substantially coplanar arrangement.

18.An apparatus for culturing photosynthetic microorganisms according to claim 15 or claim 16, wherein, the or each electromagnetic radiation source 10 is removable from the bioreactor.

19.An apparatus for culturing photosynthetic microorganisms according to any one of the preceding claims, wherein the bloreactors are interconnected in a parallel or a series configuration or combinations thereof.

20.A method for culturing photosynthetic microorganisms in the presence of 15 artificial light, the method comprising the steps of culturing photosynthetic microorganisms in an apparatus comprising at least one bioreactor and at least one source of electromagnetic radiation wherein the at least one source of electromagnetic radiation is a light emitting diode comprising an organic or polymeric 20 emissive layer. 21 .An apparatus for culturing photosynthetic microorganisms substantially as hereinbefore described with reference to the figures.