PHOTOAUTOTROPHIC GROWTH OF MICROALGAE FOR OMEGA-3 FATTY ACID
PRODUCTION
FIELD OF INVENTION
The present invention relates to a method of cultivating microalgae to prepare concentrated microalgae products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) docosahexaenoic acid. This invention also relates to a concentrated microalgae composition and purified lipid products from microalgae comprising EPA and DHA and their use in human or animal feed.
BACKGROUNDOF THE INVENTION
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are Long Chain Poly-Unsaturated Fatty Acids (LCPUFA) and belong to the omega-3 family. These polyunsaturated fats play a very important role in the function of our bodies and have been shown to be important in maintaining brain, retina and cardiovascular health (1-9). These fatty acids also play an important role in inflammation and thus they are useful for fighting diseases linked to inflammation, which include cardiovascular disease and arthritis (10-12).
The nutritional importance of EPA and DHA began emerging in the mid 1980s. The paleolithic diet contained small and roughly equal amounts on Omega-6 and Omega-3 PUFAs (ratio of 1 - 2:1) (13). An imbalance of this ratio can cause many age related health problems and neurodegenerative diseases (14).
In the 1980s, the major source of these LCPUFAs in the human diet was from fish or from fish oil capsules. But fish stocks are declining throughout the world due to overfishing. In addition, fish accumulate methyl mercury, PCB's and other toxins in their fat tissue, and these thus contaminate fish oils.
Fish do not synthesize EPA and DHA; they accumulate them from eating phytoplankton or eating animals that eat phytoplankton. It is the phytoplankton and other microbes that are the primary producers of EPA and DHA. Thus, an alternative source of EPA and DHA is microorganisms, and particularly phytoplankton.

A search is on to find suitable organisms to produce EPA- and DHA-containing oils (15-19).
The main difference in fish oils and algal oils is their structure. Fish oils are storage lipids and are in the form of triacylglycerides. The algal lipids are a mixture of storage lipids and membrane lipids. The EPA and DHA present in algae is mostly in the form of glycolipids and a small percentage is in the form of phospholipids. Glycolipids are mostly part of chloroplast membranes and phospholipids are part of cell membranes. Since glycolipids and phospholipids comprise a maximum of approximately 10-15% of the dry weight of algae, EPA and DHA production in this form is not considered economically viable. It has been suggested that cost effective production of EPA and DHA from algae (or any other microbes) would require the use of microbial strains that could produce large amounts of triacyglycerides (21).
Marine algae rich in EPA or DHA are produced by hatcheries in greenhouses or indoors in large tanks or transparent cylinders. But such methods are expensive and commercially not viable.
Economical ways of raising microorganisms that accumulate EPA and DHA are lacking. Microalgae have the potential to be raised photoautotrophically - by photosynthesis without a reduced carbon source, using CO2 as their carbon source. Since sunlight is free and land is inexpensive in some areas, it would be advantageous to raise microalgae outdoors photoautotrophically with sunlight in a way that results in accumulation of EPA and/or DHA. But culturing microalgae outdoors photoautotrophically is challenging because the cultures grow slowly and are prone to become contaminated when cultured outdoors. In addition, strains that may accumulate significant quantities of EPA or DHA under carefully controlled conditions may not accumulate as much under outdoor photoautotrophic conditions.
New sources of EPA and DHA for human nutritional supplements and as animal feed and aquaculture feed are needed. New improved methods of culturing microalgae to serve as a source of EPA or DHA are needed. Identification of microorganisms that are suitable sources of DHA and EPA for humans, seafood, and livestock is needed.

SUMMARY OF INVENTION
The invention relates to a method of culturing microalgae photoautotrophically outdoors to accumulate EPA and/or DHA and thus serve as a source of EPA or DHA supplementation in human nutritional supplements or in animal feed or aquaculture feed.
In one aspect, the invention provides a method of preparing a concentrated microalgae composition involving: (a) cultivating microalgae photoautotrophically outdoors in open ponds under filtered sunlight in continuous or batch mode at a dilution rate of less than 35% per day; (b) harvesting the microalgae in exponential phase when cell number is increasing at a rate of at least 20% of maximal rate; and (c) concentrating the microalgae; wherein at least 40% of lipids in the microalgae are in the form of glycodiacylglycerides, phosphodiacylglycerides, or a combination thereof and at least 5% (preferably at least 10%) by weight of fatty acids are DHA, EPA, or a combination thereof.
In another aspect, the invention provides a concentrated microalgae composition prepared by a process involving: (a) cultivating microalgae photoautotrophically outdoors in open ponds under filtered sunlight in continuous or batch mode at a dilution rate of less than 35% per day; (b) harvesting the microalgae in exponential phase when cell number is increasing at a rate of at least 20% of maximal rate; and (c) concentrating the microalgae; wherein at least 40% by weight of lipids in the microalgae are in the form of glycodiacylglycerides, phosphodiacylglycerides, or a combination thereof and at least 5% (preferably at least 10%) by weight of fatty acids are DHA, EPA, or a combination thereof.
In yet another aspect, the invention relates to a food grade dietary supplement for human consumption comprising a concentrated microalgae composition of the invention.
In yet another aspect, the invention relates to an aquaculture or animal feed comprising a concentrated microalgae composition of the invention.
In still another aspect, the invention discloses a purified lipid composition prepared by a process involving purifying lipids from a concentrated microalgae composition of the invention, wherein the purified lipids comprise at least 5% (preferably at least 10%) by weight EPA or DHA or a combination thereof.
In still another aspect, the invention relates to a lipid composition comprising at least 5% by weight of the EPA or DHA or a combination thereof, wherein said lipid composition is prepared from a concentrated microalgae of the invention.

The invention provides using a net over the cultures of EPA-accumulating and DHA-accumulating microalgal strains to filter sunlight to a reduced intensity. The invention also involves diluting the photoautotrophic cultures at a rate of about 15% to 30% per day. This is significantly less than the maximal doubling rate of the cultures, but allows stable maintenance of the cultures in outdoor photoautotrophic conditions and good accumulation of EPA and DHA. The invention also involves harvesting cultures in the exponential phase of cell growth, instead of stationary phase, and harvesting cultures with EPA and DHA predominantly in the form of membrane lipids instead of storage lipids. Cells in stationary phase accumulate more lipids in triacylglycerides as storage lipids. During exponential growth the cells have less storage lipid and most lipids is in membranes in the form of phospho- and glyco-diacylglycerides. The inventors have found that the photoautotrophic cultures accumulate large amounts of EPA and DHA as phospho- and glyco-diacylglycerides when harvested in exponential growth.
The invention also provides for the successful cultivation of microalgae photoautotrophically for EPA and DHA accumulation at the relatively high temperatures of about 20°C to about 40°C. This is important because others have reported better EPA and DHA accumulation at low temperatures, but microalgae grow faster at high temperatures (15, 22). Furthermore, microalgae can best be cultured outdoors year round in the tropics, but this requires culturing them at high temperatures unless expensive refrigeration systems are used.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "microalgae" as used herein refers to photosynthetic organisms that are native to aquatic or marine habitats and are too small to be seen easily as individual organisms with the naked eye.
The term "photoautotrophic" as used herein refers to growth with light as the primary source of energy and carbon dioxide as the primary source of carbon.
As used herein, the term "a dilution rate of [e.g.] 30% per day" means that 30 ml of medium is added to 100 ml of culture each day, either continuously over the course of a day or in a single batch addition each day. The term "a dilution rate of less than X% per day" means that

The average dilution rate over a period of days is less than X% per day and that no individual dilution during culturing is greater than X% in a single day.
As used herein, the term "maximal rate" of cell number increase refers to the maximal rate achieved at any stage during the outdoor photoautotrophic growth of the particular harvested culture being referenced.
As used herein, cultivating microalgae "outdoors in open ponds" means cultivating them exposed to unfiltered outdoor air. The ponds may be covered with a fabric cover that shades the pond or filters sunlight provided the pond is exposed to unfiltered outdoor air.
Description:
The invention provides various methods for culturing microalgae photoautotrophically outdoors to produce EPA and DHA. One method used is filtering sunlight to reduce the light intensity on the photoautotrophic culture. Shade cloth or netting can be used for this purpose. The optimal solar intensity for growth, maintaining a pure culture, and omega-3 fatty acid accumulation for most strains is about 40,000 to 50,000 lux, approximately half of the 110,000 lux of full sunlight. Shade cloth or netting is suitable for filtering the sunlight to the desired intensity.
Another method used to successfully culture microalgae photoautotrophically outdoors to produce EPA and DHA is to use small dilutions and a slow dilution rate of less than 40% per day, preferably less than 35% per day, more preferably from about 15% to about 30% per day. In other embodiments, the dilution rate is 15-40% per day or 15-35% per day. In other embodiments, the dilution rate is 10-30%, 10-35%, or 10-40% per day. These smaller dilutions and lower dilution rates than are typically used help prevent contamination in outdoor photoautotrophic cultures. It also promotes thick culture growth that gives good DHA or EPA yield.
Another method used to successfully culture microalgae photoautotrophically outdoors to produce EPA and DHA is to harvest the microalgae in exponential phase rather than stationary phase. Harvesting in exponential phase reduces the risk of contamination in outdoor photoautotrophic cultures and has surprisingly been found to give good yield of EPA and DHA. Typically, to drive fat accumulation in microbial cultures, the cultures are harvested in stationary phase, since cells in stationary phase tend to accumulate storage lipids. The present invention

Discloses that the EPA and DHA accumulate to large amounts as membrane lipids in cultures harvested in the exponential phase. The membrane lipids containing EPA and DHA are predominantly phosphodiacylglycerides and glycodiacylglycerides, rather thaa the triaclyglycerides found in storage lipids. The cultures are typically harvested when cell number is increasing at a rate at least 20% of the maximal rate, i.e., the maximal rate achieved at any stage during the outdoor photoautotrophic growth of the harvested culture. In specific embodiments, the cultures are harvested in exponential phase when cell number is increasing at a rate of at least 30%, at least 40%, or at least 50% of maximal rate.
With these techniques and the strains grown, the inventors have also achieved good DHA and EPA yields with culture outdoors at high temperatures. Others have reported that DHA and EPA accumulate better at low temperatures, and that a cold shock step, where a culture is grown at higher temperatures but then shifted to low temperatures, e.g., 12°C, for several days before harvest, is needed to accumulate omega-3 fatty acids (15, 22). The inventors have found that DHA and EPA accumulate to good levels in the strains used even with growth at 3O-35°C in the Indian summer. Thus, some embodiments of the invention involve outdoor photoautotrophic growth at at least 20°C, at least 25°C, or at least 30°C. As used herein, reference to growth at at least a given temperature means that the culture is maintained at at least that temperature for a majority of the outdoor photoautotrophic culture time and a majority of the last 72 hours, 48 hours, and 24 hours before the culture is harvested. In preferred embodiments of the methods and compositions of the invention, the microalgae are not genetically modified by recombinant DNA techniques.
In one embodiment, the present invention discloses a method of preparing a concentrated microalgae composition comprising cultivating microalgae photoautotrophically outdoors in open ponds under filtered sunlight in continuous or batch mode at a dilution rate of less than 35% per day; harvesting the microalgae in exponential phase when cell number is increasing at a rate of at least 20% of maximal rate; and concentrating the microalgae; wherein at least 40% of lipids in the microalgae are in the form of glycodiacylglycerides, phosphodiacylglycerides, or a combination thereof and at least 5% by weight of fatty acids are DHA, EPA, or a combination thereof.
In another embodiment, the invention relates to cultivating microalgae in the range of
about 20°C to about 40°C.

In a particular embodiment, CO2 is added to the open ponds during cultivation. This helps to neutralize pH and to enhance photoautotrophic growth.
In one embodiment, the cultures are maintained outdoors photoautotrophically for a period of 7-14 days. In another embodiment, the cultures are maintained outdoors photoautotrophically for a period of 7 -10 days. In a preferred d embodiment the cultures are maintained outdoors photoautotrophically for atleast 7 days.
In another embodiment, the microalgae are diatoms. In another embodiment, the diatoms are Thalassiosira sp. or Chaetoceros sp. Further the EPA yield is at least 5 mg/liter culture and at least 10% by weight of the fatty acids are EPA.
In another embodiment, the diatoms are cultivated photoautotrophically outdoors in open ponds in batch system under filtered sunlight.
In yet another embodiment, the microalgae are Chlorophyta. In some embodiments, the Chlorophyta are Tetraselmis sp. Preferably the EPA yield is at least 5 mg/liter culture.
In another embodiment, the Chlorophyta are cultivated photoautotrophically outdoors in open ponds in batch system under filtered sunlight.
In one preferred embodiment, the microalgae suitable for DHA production include Prymnesiophyta, more specifically those of class Prymnesiophyceae, more specifically those of order Isochrysales, more specifically Isochrysis sp. or Pavlova sp.
In another embodiment, the DHA yield is at least 3 mg/liter culture. Further the DHA is at least 5% by weight of total fatty acids. In another embodiment, the DHA yield is at least 5 mg/liter culture and DHA is at least 10% by weight of fatty acids in the microalgae.
In a particular embodiment, the Prymnesiophyta are cultivated photoautotrophically outdoors in open ponds in batch system under filtered sunlight.
In another preferred embodiment, the present invention relates to a concentrated microalgae composition prepared by the process comprising cultivating microalgae photoautotrophically outdoors in open ponds under filtered sunlight in continuous or batch mode at a dilution rate of less than 35% per day; harvesting the microalgae in exponential phase when cell number is increasing at a rate of at least 20% of maximal rate; and concentrating the microalgae; wherein at least 40% of lipids in the microalgae are in the form of glycodiacylglycerides, phosphodiacylglycerides, or a combination thereof and at least 5% by weight of fatty acids are DHA, EPA, or a combination thereof.

In another embodiment, the microalgae is selected from a group consisting of diatoms, Chlorophyta, Prymnesiopyta and a combination thereof.
In another embodiment, the diatoms are Thalassiosira sp. or Chaetoceros sp, the Chlorophyta are Tetraselmis sp and the Prymnesiophyta are Isochrysis sp. or Pavlova sp. Preferably at least 5% of fatty acids in the lipid composition are EPA and at least 3% of fatty acids in the lipid composition are DHA.
In another embodiment, the present invention discloses a food grade dietary supplement for human consumption comprising the concentrated microalgae composition of the invention, wherein the lipids will be extracted from algal cells.
In another embodiment, the invention relates to an aquaculture or animal feed comprising the concentrated microalgae composition of the invention, wherein whole algal dried cells is used for feed purpose.
In another embodiment, the present invention relates to a lipid composition prepared by a process comprising: extracting lipids from said concentrated microalgae composition, wherein the lipids comprise at least 5% by weight EPA or DHA or a combination thereof. In a particular embodiment at least 5% of fatty acids in the lipid composition are EPA. In another embodiment at least 5% of fatty acids in the lipid composition are DHA.
In another embodiment, at least 30%, at least 40%, at least 50%, or at least 60% of the EPA or DHA or both in the composition are in phosphodiacylglycerides or glycodiacylglycerides or a combination thereof.
In another embodiment the present invention discloses a lipid composition comprising at least 5% by weight EPA or DHA or a combination thereof, wherein said lipid composition is prepared from the concentrated microalgae composition prepared by a process comprising cultivating microalgae photoautotrophically outdoors in open ponds under filtered sunlight in continuous or batch mode at a dilution rate of less than 35% per day; harvesting the microalgae in exponential phase when cell number is increasing at a rate of at least 20% of maximal rate; and concentrating the microalgae; wherein at least 40% of lipids in the microalgae are in the form of glycodiacylglycerides, phosphodiacylglycerides, or a combination thereof and at least 5% by weight of fatty acids are DHA, EPA, or a combination thereof.
The invention is illustrated by the following examples and should not be construed to limit the scope of the present invention. The present invention is described in term of its specific

Embodiments and any modifications and equivalents to a person skilled in the art should be included within the scope of the present invention.
EXAMPLE
Example 1
Strain: - Thalassiosira sp. Thalassiosira sp. is a diatom, and the strain used was isolated from Bay of Bengal. This strain dominates during summer months, and it was isolated from seawater collected near Chennai, India. This culture was maintained in open tubs. The strain was identified as Thalassiosira weissflogii. This strain was capable of growth at high temperatures (35-38°C). The fatty acid profile was good even when the alga was grown at high temperature -with 25-30% EPA (as percentage of fatty acids).
Culturing
The lab cultures were maintained in tubs in artificial seawater medium, under fluorescent lights (3000-4000 lux) and the temperature was maintained at 25°C.
Initial expansion of the culture was done under laboratory condition in tubs. The dilution rate was 15% to 30% of the total culture volume per day. Once the volume was 40-50 liters, it was transferred to an outdoor pond. The outdoor ponds were covered with netting to control the light (40000 to 50000 lux). The dilution continued until the culture reached 100,000 liters volume. The culture was held in 500 sq. m ponds at this time, with a culture depth of 20 cm. The culture was stirred with a paddle wheel and CO2 was mixed to keep the culture pH neutral. When the EPA levels in the pond reached a desirable level (10-15 mg/lit), the whole pond was harvested by filtration. The filtered biomass was washed with saltwater (15 parts per thousand concentration) and then spray dried. The mode of culturing was batch mode. The EPA productivity was 2-3 mg/lit/day.
The ponds can also be run continuously for several weeks by harvesting part of the culture, recycling the filtrate into the ponds and replenishing required nutrients.

Example 2
Strain: Tetraselmis sp. Tetraselmis sp. is in the division Chlorophyta and the class Prosinophyceae or Micromanadophyceae. This strain was obtained from the Central Marine Fisheries Research Institute, India. It was isolated from the local marine habitats in India. The culture was maintained in flasks in artificial seawater medium, and expanded as described for Thalassiosira. With culture outdoors in open ponds as described for Thalassiosira, the strain gave a good lipid yield (200-300 mg/liter) and an EPA content of 6-7% of fatty acids.
Example 3
Strain: Chaetoceros sp. This is another diatom strain obtained from the Central Marine Fisheries Research Institute, India, and isolated from local marine habitats in India. Chaetoceros sp. was maintained in flasks and cultivated in outdoor ponds photoautotrophically as described in Example 1. It gave similar EPA productivity and EPA content as Thalassiosira, described in Example 1.
Example 4
Strain: Isochrysis sp. Isochrysis is in the Prymnesiophyta, class Prymnesiophyceae; order Isochrysidales. It was obtained from the Central Marine Fisheries Research Institute, India, and isolated from local marine habitats in India. It was maintained and grown as described in Example 1. It was expanded from laboratory culture to a 50,000 liter outdoor pond culture in 14-15 days with a dilution rate of 15-30% per day. The lipid content at harvest was 100-150 mg lipids/liter. The rate of lipid production was 25-50 mg/liter/day. DHA was 10-12% of total fatty acids.
Example 5
Harvesting and Drying:
The harvesting may be done by flocculation. The commonly used flocculants include Alum with polymer; FeCl3 with or without polymer and chitosan. The concentration of flocculent will depend on the cell number in the culture before harvest. The range may vary from 100 ppm to 500 ppm. Alternatively harvesting is done by filtration using appropriate meshes. Removal of adhered chemicals (other than salt) is effected by washing the cells in low salinity water.

The harvested slurry is then taken for spray drying. If required the slurry is sometimes encapsulated to prevent oxidation. The concentration of encapsulating agent may vary from 0.1 to 1.0% on dry weight basis. Modified starch is a suitable encapsulating agent. The spray dryer used is of atomizer or nozzle type. The inlet temperature ranges from 160 to 190°C and the outlet temperature ranges from 60 to 90°C. The spray dried powder is used immediately for extraction. If storage is needed, the powder is packed in aluminum laminated pouches and sealed after displacing the air by nitrogen. The packed powder is stored at ambient temperature until further use.
Example 6
Extraction:
Extraction of EPA/DHA is carried out using wet slurry or dry powder. Extraction is carried out using solvents. The solvents include hexane, ethanol, methanol, acetone, ethyl acetate, isopropanol and cyclohexane and water. The above solvents are used alone or a combination of two solvents. The solvent to biomass ratio depends on the starting material. If it is a slurry the ratio is 1:2 to 1:10. In the case of a spray dried powder, the ratio is 1:4 to 1:30. The extraction is carried out in an extraction vessel under inert atmosphere. Extraction temperature ranges from 25 to 60°C and the time varies from one hour to 10 hours. Solvent addition is made one time or in parts based on the lipid level in the cells. After extraction of crude lipid, the mixture is passed through a centrifuge or filtration system to remove the cell debris. The lipid in the filtrate is then concentrated by removing the solvent by distillation. The distillation process is carried out under vacuum. The resulting product is a crude lipid extract, which contains approximately 10% omega 3 fatty acid (EPA/DHA). This lipid extract can be used as such or purified further to enrich the omega 3 fatty acids. Further purification may involve removal of unsaponifiables such as pigments, sterols and their esters.
REFERENCES
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2. Lim, G.P, Calon, F., Morihara, T., Yang, F., Teter, B., Ubeda, O., Salem, N. Jr, Fraiitschy, S.A. and Cole, G.M. 2005. A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. JNeurosci 25: 3032-3040
3. Hoffman, D.R., Birch, E.E., Birch, D.G. and Uauy, R.D. 1993. Effects of supplementation with omega-3 long-chain polyunsaturated fatty acids on retinal and cortical development in premature infants. AmJClin Nutr 57 (Suppl): 807S-812S.
4. Hoffman, D.R., Theuer, R.C., Castaneda, Y.S., Wheaton, D.H., Bosworth, R.G., O'Connor, A.R., Morale, S.E., Wiedemann, L.E. and Birch, E.E. 2004. Maturation of Visual Acuity Is Accelerated in Breast-Fed Term Infants Fed Baby Food Containing DHA-Enriched Egg Yolk. J
Nutr 134: 2307-2313.
5. Mukherjee, P.K., Marcheselli, V.L., Serhan, C.N. and Bazan, N,G. 2004. Neuroprotectin Dl: A docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress. PNAS 101: 8491-8496.
6.Vanschoonbeek, K., de Maat, M.P.M., and Heemskerk, J.W,M. 2003. Fish oil consumption and reduction of arterial disease. J Nutr 133: 657-660.
7. Leaf, A., Xang, J.X., 1996. Prevention of cardiac sudden death by N-3 fatty acids: a review of the evidence. J Intern Med 240: 5-12.
8. Nestel, P., Shige, H., Pomeroy, S., Cehun, M., Abbey, M. and Raederstorff, D. 2002. The n-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid increase systemic arterial compliance in humans. Am J Clin Nutr 76: 326-330.
9.Vanschoonbeek, K., Feijge, M.A.H., Paquay, M., Rosing, J., Saris, W., Kluft, C, L.A. Giesen, P. de Maat, M.P.M and Heemskerk, J.W.M. 2004. Variable hypocoagulant effect of fish oil

Intake in humans: Modulation of fibrinogen level and thrombin generation. Arterioscler Thromb Vase Biol 24: 1734-1740.
10. Lopez-Garcia, E., Schulze, MB., Manson, J.E., Meigs, J.B., Albert, CM., Rifai, M., Willett, W.C. and Hu, F.B. 2004. Consumption of (n-3) fatty acids is related to plasma biomarkers of inflammation and endothelial activation in women. JNutr 134: 1806-1811.
ll.Mishra, A., Chaudhary, A. and Sethi, S. 2004. Oxidized omega-3 fatty acids inhibit NF-nB activation via a PPARa-dependent pathway. Arterioscler Thromb Vase Biol 24: 1621-1627.
12.Mirnikjoo, B., Brown, S.E., Kim, H.F.S., Marangell, L.B., Sweatt, J.D. and. Weeber, EJ. 2001. Protein kinase inhibition by cu-3 fatty acids. J Biol Chem 276: 10888-10896.
13. Simopoulos, A.P. 2002. Omega-3 Fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr 21 (6): 495-505.
14. Song, C. and Horrobin, D. 2004. Omega-3 fatty acid ethyl-eicosapentaenoate, but not soyabean oil, attenuates memory impairment induced by central IL-1B administration. J Lipid Res45: 1112-1121.
15. Wen, Z. and Chen, F., 2005, Prospects for eicosapentaenoic acid production using microorganisms, in Cohen, Z. and Ratledge, C. (Ed), Single cell oils, ppl38-160, AOCS Press.
16. Kiy, T., Rtising, M. and Fabritius, D., Production of docosahexaenoic acid by the marine microalga, Ulkenia sp. in Cohen, Z. and Ratledge, C. (Ed), Single cell oils, pp99-106, AOCS Press.
17. Molina Grima, E., Garcia Camacho, G., Aden Fernandez, F.G., 1999, Production of EPA from Phaeodactylum tricornutum, in Cohen, Z.,(Ed.), Chemicals from microalgae pp57-92, London: Taylor & Francis Ltd.

18. Sukenik, A., 1999, Production of Eicosapentanoic acid by the marine eustigmatophyte Nannochloropsis, in Cohen, Z.,(Ed.), Chemicals from microalgae pp42-56, London: Taylor & Francis Ltd.
19. Boussiba, A., Sandbank, E., Shelef, G., Cohen, Z., Vonshak, A., Ben-Amotz, A., Arad, S. and Richmond, A., 1988, Outdoor cultivation of the marine microalga Isochrysis galabana in open reactors. Aquaculture 72: 247-253
20. Wynn, J., Behrens, P., Sundararajan, A., Hansen, J. and Apt, K., 2005, Production of single cell oils by dinoflagellates, in Cohen, Z. and Ratledge, C. (Ed), Single cell oils, pp86-98, AOCS Press.
21. Cohen, Z. and Khozin-Goldberg, I, 2005, Searching for PUFA-rich microalgae, in Cohen, Z. and Ratledge, C. (Ed), Single cell oils, pp. 53-72, AOCS Press.
22. Springer, S., Franke, H., and Pulz, O., 1994, Increase of the content of polyunsaturated fatty acids in Porphyridium cruentum by low-temperature stress and acetate supply. J. Plant Physiol. 143:534-537.
I/We claim:
1. A method of preparing a concentrated microalgae composition comprising: cultivating microalgae photoautotrophically outdoors in open ponds under filtered sunlight in continuous or batch mode at a dilution rate of less than 35% per day; harvesting the microalgae in exponential phase when cell number is increasing at a rate of at least 20% of maximal rate; and concentrating the microalgae; wherein at least 40% of lipids in the microalgae are-in the form of glycodiacylglycerides, phosphodiacylglycerides, or a combination thereof and at least 5% by weight of fatty acids are DHA, EPA, or a combination thereof.
2. The method as claimed in claim 1, wherein the microalgae are cultivated in the range of about 20°C to about 40°C.
3. The method as claimed in claim 1, wherein CO2 is bubbled into the open ponds during cultivation.
4. The method of claim 1, wherein the microalgae are diatoms.
5. The method as claimed in claim 4, wherein said diatoms are Thalassiosira sp. or Chaetoceros sp.
6. The method as claimed in claim 4, wherein the EPA yield is at least 5 mg/liter culture.
7. The method as claimed in claim 4, wherein at least 10% by weight of the fatty acids are EPA.
8. The method as claimed in claim 4, wherein the diatoms are cultivated photoautotrophically outdoors in open ponds in batch system under filtered sunlight.
9. The method as claimed in claim 1, wherein the microalgae are Chlorophyta.
10. The method as claimed in claim 9, wherein said Chlorophyta are Tetraselmis sp.
11. The method as claimed in claim 9, wherein the EPA yield is at least 5 mg/liter culture.
12. The method as claimed in claim 9, wherein the Chlorophyta are cultivated photoautotrophically outdoors in open ponds in batch system under filtered sunlight.
13. The method as claimed in claim 1, wherein the microalgae are Prymnesiophyta.
14. The method as claimed in claim 13, wherein said Prymnesiophyta are Isochrysis >sp. or Pavlova sp.
15. The method as claimed in claim 13, wherein the DHA yield is at least 3 mg/liter culture.

16. The method as claimed in claim 13, wherein the DHA is at least 5% by weight of total fatty acids
17. The method as claimed in claim 13, wherein the Prymnesiophyta are cultivated photoautotrophically outdoors in open ponds in batch system under filtered sunlight. ,
18. A concentrated microalgae composition prepared by the process as claimed in claim 1.
19. The concentrated microalgae composition as claimed in claim 18, wherein said microalgae is selected from a group consisting of diatoms, Chlorophyta, Prymnesiopyta and a combination thereof.
20. The concentrated microalgae composition as claimed in claim 19, wherein the diatoms are Thalassiosira sp. or Chaetoceros sp, the Chlorophyta are Tetraselmis sp and the Prymnesiophyta are Isochrysis sp. or Pavlova sp.
21. The concentrated microalgae composition as claimed in claim 18, wherein atleast 5% of fatty acids in the lipid composition are EPA.
22. The concentrated microalgae composition as claimed in claim 18, wherein atleast 3% of fatty acids in the lipid composition are DHA.
23. A food grade dietary supplement for human consumption comprising the concentrated microalgae composition of claim 18.
24. An aquaculture or animal feed comprising the concentrated microalgae composition of claim 18.
25. A lipid composition prepared by a process comprising: extracting lipids from the concentrated microalgae composition as claimed in 18, wherein the lipids comprise at least 5% by weight EPA or DHA or a combination thereof.
26. The lipid composition of claim 25, wherein at least 5% of fatty acids in the lipid composition are EPA.
27. The lipid composition of claim 25, wherein at least 5% of fatty acids in the lipid composition are DHA.
28. The lipid composition of claim 25, wherein at least 40% of the EPA or DHA or both are in glycodiacylglycerides or phosphodiacylglycerides or a combination thereof.

29. A lipid composition comprising at least 5% by weight ot bFA or UM or a comumauun thereof, wherein said lipid composition is prepared from the concentrated microalgae composition as claimed in 18.

To
The Controller of Patents
Patent Office at Chennai