WO2012033855A1

WO2012033855A1 - Commercial production docosahexaenoic acid using phototrophic mecroalgae

Info

Publication number: WO2012033855A1
Application number: PCT/US2011/050724
Authority: WO
Inventors: Qiang Hu; Milton Sommerfeld
Original assignee: Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University
Priority date: 2010-09-07
Filing date: 2011-09-07
Publication date: 2012-03-15

Abstract

Processes to produce the omega-3 polyunsaturated fatty acid docosahexaeoic acid (DHA) or DHA plus eicosapentaenoic acid (EPA) by photosynthetic microalgae grown in an open pond or a closed photobioreactor under indoor or outdoor conditions.

Description

COMMERCIAL PRODUCTION DOCOSAHEXAENOIC ACID USING PHOTOTROPHIC MICROALGAE

RELATED APPLICATIONS

[0001] [Not Applicable]

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] [Not Applicable]

FIELD OF THE INVENTION

[0003] The present invention relates to the commercial production of docosahexaenoic acid by phototrophic microalgae.

BACKGROUND OF THE INVENTION

[0004] Docosahexaenoic acid (DHA) (22:6) is an omega-3-fatty acid, so called because it has a double-bond 3 carbon atoms away from the methyl end of the molecule. All the fatty acids which are essential in the human diet are either omega-3 or omega-6. Although DHA can be synthesized in the body from alpha-linolenic acid (a simpler omega-3 found in the linseed oil and perilla oil), the capacity for the synthesis declines with age. The omega-3 and omega- 6 family of fatty acids are essential because they cannot be readily synthesized in the body, but must be obtained in the diet. Fatty acids are contained in the membranes of every cell in the body, but essential fatty acids are particularly concentrated in the membranes of the brain cells, heart cells and the immune system cells.

[0005] Interest in the Omega-3 polyunsaturated fatty acids (n-3 PUFAs), specifically DHA and eicosapentaenoic acid (EPA), has increased substantially in recent years. DHA and EPA have been credited with improving human cardiovascular health and disease prevention, cancer prevention and treatment, physical and cognitive development of infants, anti-aging, the treatment of mental health disorders, lessening the impact of rheumatoid arthritis, and prevention of liver disease. DHA and EPA have also been credited with improving survival rate and development and reproduction of animals, particularly cultured marine animals (e.g., fish, shrimp, and bivalves). Realized and potential human health and animal nutrition benefits are becoming more publicized almost daily as additional research studies are completed.

[0006] Cardiovascular Health and Disease Cardiovascular health and disease studies comprise the greatest number of DHA and EPA benefit studies. The correlation between recommended DHA/EPA consumption and better cardiovascular health and cardiovascular disease prevention has been documented for decades. Numerous studies have shown that diets high in DHA/EPA lead to higher survival rates in individuals with chronicled cardiovascular disease (Lee et al. 2008, Marchioli et al. 2002 & Burr et al. 1989). Origasa et al. (2010) created a five-year predictive model illustrating that patients with hypercholesterolemia had a 51 % reduction of risk for future cardiac death or myocardial infarction when they exhibited adherence to recommended EPA doses. Regular doses of DHA and EPA in elderly subjects decreased triglycerides, a known cardiovascular disease marker, without impacting insulin resistance (Olza et al., 2010). Mattar and Obeid (2009) also observed a reduction in plasma triglycerides when subjects took more than 2 g/day of DHA plus EPA. A lower incidence of atrial fibrillation was observed in elderly adults consuming increased DHA plus EPA (Circ. 1 10:368-373(2004). Stirban et al. (2010) completed a study that showed Omega-3 fatty acids improved both microvascular and macrovascular function in human subjects with type 2 diabetes mellitus.

[0007] The mechanisms for health benefits of DHA and EPA are not yet fully understood. However, it is believed that these Omega-3 PUFAs are incorporated into membrane phospholipids (Clandinin et al., 1994), resulting in increased production of series 3-eicosonoids. The 3-eicosanoids prostaglandin I3, thromboxane A3, and series 5 leukotriene B5 that are known to reduce inflammation, decrease blood platelet aggregation and increase vasodilation (Gallai et al., 1995). Omega-3 PUFAs appear to shift lipid production away from triglycerides towards phospholipids (Harris & Bulchandani, 2006).

[0008] Infant Development A significant body of research exists supporting the benefits of DHA and EPA for both physical and cognitive development in infants. Some have shown that supplementing diets of expectant mothers with DHA contributes to increased birth weight, length and head circumference in newborns (Smuts et al. 2003 & Olson et al. 2002). Dunstan et al. (2008) reported consuming DHA (2.2 g/day) and EPA (1 .1 g/day) during pregnancy led to increased hand-eye coordination in children at age 2.5 years. DHA and EPA supplements for mothers that breast feed have a positive effect on the visual acuity of their newborns (Innis et al. 2001 & Lauritzen et al. 2004). Birch et al. (2000) reported that full-term infants receiving formula supplemented with DHA exhibiting increased cognitive development based on the Mental Development Index (MDI).

[0009] Cancer Prevention and Treatment A variety of studies have suggested Omega-3 fatty acids play a positive role in the prevention and treatment of many types of cancers. Several research groups have concluded that diets rich in EPA and DHA reduce the risk of colon tumorigenesis (Anti et al. 1992, Bartram et al. 1993, Chang et al. 1997, Fernandez et al. 1999, Rao et al. 2001 , Phillips et al. 2006, & Courtney et al. 2007). Increased EPA intake has suppressed pancreatic cancer cell growth in vitro (Funahashi et al., 2008). Saw et al. (2010) reported a synergistic anti-inflammatory and anti-oxidative stress effect when very lose doses of curcumin where taken with DHA and EPA. Schley et al. (2007) reported that Omega-3 fatty acids possess anti-breast cancer properties. Siddiqui et al. (2008) discussed the potential properties of oxidation products from DHA in fighting cancer. They emphasize the difficulty of assessing the 1000s of oxidation products from DHA, the "longest, most unsaturated and hence most oxidizable PUFA found in nature".

[0010] Mental Health Research suggests that DHA EPA consumption results in the production of eicosanoids that improve brain health and function (Uauy & Dangour, 2006). DHA and EPA may also contribute to an increase in neurotransmitter production, thus increasing brain function (Fenton et al., 2000). Omega-3 PUFA supplements may increase brain function in the elderly and lessen the impact of Alzheimer's disease (Cunnane et al., 2009). Katani et al. (2006) observed immediate increases in memory scores of patients with Alzheimer's disease when their diets were supplemented with DHA. [0011] Benefits from DHA and EPA dietary supplements have been noted with respect to depression, dementia, Alzheimer's disease, attention deficit hyperactivity disorder and aggressive and psychopathic behavior. Several studies have reported encouraging results from DHA/EPA consumption regarding childhood and adult depression. Nemets et al., 2006, observed a pronounced reduction in childhood depression when study participants consumed 600 mg/day of DHA plus EPA for a 16-week period (AM. J. of Psychiatry, 163:1098-1 102). A decrease in depression, suicidality and perception of daily stresses was reported when subjects who exhibited repeated self-harm took 1 .2 g/day EPA plus 0.9 g/day DHA for 12 weeks (Hallahan et al., 2007). An intake of 9.6 g/day of DHA and EPA in a 2 to 1 proportion, respectively, showed a significant effect on bipolar depression patients (Stoll, 1999).

[0012] Studies of DHA and EPA supplementation in regard to attention deficit hyperactivity disorder (ADHD) yield a variety of results. However, one study indicated that when children with ADHD took 80 mg/day of EPA and 480 mg/day of DHA a significant decrease was observed when parents rated conduct problems and their teachers observed increased attention symptoms (Stevens et al., 2003). Similarly, children diagnosed with learning disabilities showed decreased hyperactivity and increased attention after taking EPA at 186 mg/day and DHA at 480 mg/day for 12 weeks (Richardson & Puri, 2002). In a study of Dutch prisoners that took EPA and DHA supplements, prison officials noted a decrease in aggressive behavior and rule-breaking in the inmate population (Zaalberg et al., 2010).

[0013] Other Health Benefits Dietary supplementation with DHA and EPA has shown significant effects on the prevention and treatment of several additional diseases and disorders not described above. One study suggests that increasing daily DHA intake reduces the rate of Retinitis pigmentosa (Retina, 25:552-554 (2005)). Research suggests that low DHA concentrations in spermatozoa may be responsible for infertility in males (Safarinejad et al. 2010 & Aksoy et al. 2006). Mii et al. (2007) studied the effects of perioperative doses of EPA in vein graft patients. They concluded that the subjects taking EPA had significantly higher primary patency at 1 , 3 and 5 years when compared to the subjects that did not take perioperative EPA. Increasing Omega-3 intake may provide some protection against chemotherapy-related hair loss, a condition called alopecia. It has been suggested that increased DHA EPA doses may help patients suffering from psoriasis. A longitudinal study by Iwasaki et al. (2010, in press) provided evidence that increased doses of DHA in the elderly led to decreased progression of periodontal disease. Supplementations DHA and EPA have contributed to improvement in rheumatoid arthritis patients due to anti-inflammatory properties (Goldberg & Katz, 2007). Dietary supplements with DHA and EPA may exhibit anti-aging properties by increasing gene expression of collagen and elastic fibers in the skin of both young and older humans (Kim et al., 2006). The build-up of advanced glycation end-products (AGES) in human tissue leads to diabetic complications. Sun et al. (2010) have demonstrated that the PUFAs from microalgal extracts, including DHA, have strong antiglycative capabilities. EPA has been reported to prevent steatosis (non-alcoholic liver disease) and hepatic fibrosis in rats (Kajikawa et al., 2010). Masterton et al. (2010) reported similar results in rats and humans, but state that serious inadequacies must be addressed in the human trials.

[0014] During a 1999 National Institute of Health (NIH) workshop sponsored by the International Society for the Study of Fatty Acids and Lipids (ISSFAL) in Bethesda, Maryland, experts established DHA and EPA intake guidelines for the average American, as well as some specific segments of the population. Workshop participants proposed an average daily intake of combined DHA plus EPA of at least 650 mg/day. Exact proportions of the two fatty acids were not critical provided that a minimum of 220 mg/day of EPA and 220 mg/day of DHA was consumed. The American Heart Association (AHA) recommends even higher average daily intake of DHA plus EPA. The AHA suggests that individuals should consume a minimum of 900 mg/day of DHA plus EPA to prevent cardiovascular disease and 1000 mg/day for patients with a history of coronary heart disease. [0015] Unfortunately, the average daily intake of DHA and EPA falls woefully shy of the recommended daily dietary targets established by both NIH workshop participants and the American Heart Association. According to the EPA^»DHA Omega-3 Institute (2010), the average American intake of DHA plus EPA is 120-150 mg/day which is approximately 15% of the amount recommended by the AHA. It is estimated that 1/3 of Americans consume less than 100 mg/day of DHA plus EPA. ISSFAL also recommended an average daily intake of 300 mg of DHA during pregnancy. The typical North American diet consists of an average of 50 mg/day of EPA and 80 mg/day of DHA. The United States Food and Drug (USFDA) Administration has determined EPA plus DHA is safe to consume in concentrations up to 3,000 mg/day.

[0016] Since DHA and EPA are not produced endogenously in humans, fresh fish and some shellfish have been the primary sources of dietary EPA and DHA. For example, a 100 g serving of salmon contains 1 ,200 mg of DHA plus EPA. Human adults are able to convert a very small amount of a-linolenic acid (ALA) consumed in their diets into DHA and EPA, 5-10% and 2-5%, respectively (Wijendran & Hayes, 2004). ISSFAL suggests a less efficient conversion of ALA into DHA of only 1 % in healthy infants and even less in healthy adults (Brenna et al., 2009).

[0017] Although dietary supplements containing DHA and EPA from fish oil provide a viable source of these n-3 PUFAs, the fears related to potential contaminants in fish oils produce the greatest concerns over using raw fish as the source of EPA and DHA. The United States Environmental protection Agency (USEPA) has released warnings that shark, swordfish, king mackerel and tile fish may contain high concentrations of methyl mercury and PCBs in their tissue (No. EPA-823-R-04-005). Others studies recommend a decrease in DHA and EPA consumption due to concerns over elevated concentrations of dioxins in fish tissue (Bays 2007). Concerns also exist from a medical condition standpoint. It has been reported that human DHA and EPA intake in excess of 3 grams per day may lead to excessive bleeding in consumers (Harris 2007). Consumption of fish oils has also been associated with fishy burp and nausea side-effects. [0018] Concerns also exist regarding the global supply of fish tissue for DHA and EPA production and even C0₂ emissions associated with shipping fresh fish to processing plants far away from coastal regions. Sustainability of current fishing practices to provide enough fish oil for the production of EPA and DHA to adequately supply the needs of the increasing Omega-3 fatty acid supplement market is another major concern regarding the fish source of DHA and EPA.

[0019] Thus, there exists a need for the sustainable production of DHA and EPA that does not rely on fish. The present application provides methods and compositions for the commercial production of DHA by phototrophic microalgae that fulfills the need for such sustainable production of DHA.

BRIEF SUMMARY OF THE INVENTION

[0020] The present application relates to a process for the production of the omega-3 polyunsaturated fatty acid docosahexaeoic acid (DHA) or DHA plus eicosapentaenoic acid (EPA) by photosynthetic microalgae grown in a closed photobioreactor under indoor or outdoor conditions. Five major technical components may include: 1 ) selected naturally occurring and genetically modified DHA-producing strains Isochrysis sp. and Pavlova sp.; 2) culture media for mass cultivation of the selected strains; 3) photobioreactor design; 4) optimized operation/maintenance protocols for sustainable high-yield cultivation of the selected strains; and 5) downstream processes to harvest and dewater algal cells from the culture suspension. In addition, an extraction method developed by the inventors can be applied to extraction of DHA from algal biomass. This process is designed for commercial production of DHA from microalgae for human health and animal nutrition.

[0021] In specific embodiments, the methods relate to producing an algal lipid fraction enriched for DHA and/or EPA, said method comprising

(i) culturing a first algal culture consisting of a strain from the algal genus Isochrysis or a mutant thereof wherein said first algal strain produces at least a first fatty acid subset wherein at least 10% of the fatty acids in said subset are of a chain length of Docosahexaenoic acid (DHA) (22:6), wherein the culturing is conducted under conditions suitable to promote production of the Docosahexaenoic acid (DHA) (22:6);

(ii) culturing a second algal culture consisting of a strain from the algal genus Pavlova or a mutant thereof wherein said second algal strain produces at least a second fatty acid subset wherein at least 10 % of the fatty acids in said subset are Docosahexaenoic acid (DHA) (22:6), wherein the culturing is conducted under conditions suitable to promote production of the of chain length of Docosahexaenoic acid (DHA) (22:6); and

(iii) extracting oil from the first algal strain and the second algal strains to produce a fatty acid composition enriched for C20 or C22 fatty acids;

[0022] More particularly, the culturing in step (i) is performed photoautotophically in an open pond of various designs (such as raceway pond, circular pond) or a closed photobioreactor, wherein said cultures are supplied with C0₂ as a carbon source and light as an energy source. The culturing in step (ii) is performed photoautotophically in a closed photobioreactor, wherein said cultures are supplied with C0₂ as a carbon source and light as an energy source.

[0023] The first algal strain and the second algal strains may be cultured as separate cultures or may be cultured as a co-culture. More particularly, the Isochrysis strain may be selected from the group consisting of Isochrysis sp. UTEX LB 2307 and Isochrysis galbana UTEX LB 987. These strains can be obtained from UTEX The Culture Collection of Algae at the University of Texas at Austin (http://web.biosci.utexas.edu/utex) and the Pavlova strain may be selected from the group consisting of Pavlova gyrans UTEX LB 992 and Pavlova lutheri UTEX LB 1293.

[0024] The methods described herein further comprise isolating algal biomass. [0025] Also contemplated is a composition comprising a high DHA- producing Isochrysis strain or a mutant thereof and an a high DHA-producing Pavlova strain or mutant thereof wherein the DHA content of the Isochrysis and Pavlova strains makes up at least 15% of total fatty acids in the algae.

[0026] Also contemplated is a substantially pure culture comprising:

(a) growth medium; and

(b) the composition comprising a high DHA-producing Isochrysis strain or a mutant thereof and an a high DHA-producing Pavlova strain or mutant thereof wherein the DHA content of the Isochrysis and Pavlova strains makes up at least 15% of total fatty acids in the algae.

[0027] In addition, there is contemplated a composition comprising lipids extracted from the first and second algal strains according to the method of the invention.

[0028] A dietary supplement comprising a lipid fraction enriched for DHA and/or EPA prepared according to the methods described herein also is a useful embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Microalgae are the primary and original producers of DHA and EPA in nature. Table 1 list a number of DHA- and DHA/EPA-producing algal strains reported in peer-reviewed scientific journals. Potential advantages of using microalgae as a reliable and predictable alternative to fish tissue are numerous. Microalgae are cultured under controlled environmental and physiological conditions, thus eliminating the potential of contamination by such chemicals as methyl mercury, PCBs and dioxins often found in fish tissue. The side-effect of fishy burps from fish oil consumption would also be eliminated. Microalgae possess much simpler fatty acid composition and fewer fatty acids than that of fish oils, making the extraction, purification and concentration of DHA and EPA easier and more cost-effective. Because of lacking of the odors and tastes of fish oils, microalgae-based DHA/EPA products would be more acceptable by consumers. TABLE 1 . Docosahexaeoic Acid (DHA) and Eicosapentaenoic Acid (EPA) Concentrations (% Total Fatty Acids) in selected algal strains.

[0030] The use of microalgae in lieu of fish tissue as the alternate source of DHA and EPA will also lead to a reduction in greenhouse gas emissions. Microalgae remove C0₂ from the atmosphere to perform photosynthesis. In addition, culturing and processing microalgae on the same site will eliminate the C0₂ emissions associated with transporting fish tissue from sea ports to DHA and EPA processing facilities.

[0031] It is expected that marine and freshwater microalgae may eventually become the primary source of DHA and EPA in dietary supplements due to their potential Omega 3 production capabilities (Table 1 :). Currently, there are only a handful of microalgae-based edible products rich in Omega 3 PUFAs on the market. Accordingly, the present application discloses methods for producing DHA from selected photosynthetic microalgae, comprising: high DHA- and DHA EPA-containing photosynthetic microalgal strains from the genera of Isochrysis and Pavlova that were selected from a pool of candidate strains obtained from the public microalgal culture collections. [0032] It has been discovered that co-culturing a first algal culture consisting of a strain from the algal genus Isochrysis, wherein said first algal strain produces at least a first fatty acid subset wherein at least 10% of the fatty acids in said subset are of a chain length of Docosahexaenoic acid (DHA) (22:6), with a second algal culture consisting of a strain from the algal genus Pavlova, wherein said second algal strain produces at least a first fatty acid subset wherein at least 10% of the fatty acids in said subset are of a chain length of Docosahexaenoic acid (DHA) (22:6), can reliably produce large quantities of DHA or DHA plus EPA. It is very difficult to grow two or more strains in a culture system/process because the culture conditions that determine or maintain co-proliferation of sub-populations is difficult to establish and the fate of species succession over time is not well understood. Thus, an inventive step is made over known processes for algal production of DHA or DHA/EPA.

[0033] The content of DHA or DHA EPA in the selected strains is thought to be further increased by means of chemical mutagenesis. The mutagenesis of selected Isochrysis and Pavlova strains may be conducted using the mutagenic agent ethyl methane sulphonate (EMS) at a final concentration of 50 μΙ_ΛηΙ_ for 30 minutes.

[0034] The selected strains of Isochrysis and Pavlova were grown in optimized synthetic culture media that can sustain high cell density culture and promotion of DHA or DHA/EPA formation of the selected strains. The culture media are the modified versions of F/2 culture medium by Guillard and Ryther (1962). The F/2 culture medium may be made as follows:

For I L Total

1 . To approximately 950 mL of non-pasteurized seawater ( 30-35ppt), add each of the components in the order specified (except vitamins) while stirring continuously.

2. Bring total volume to 1 L with non-pasturized seawater.

*For 1 .5% agar medium add 15 g of agar into the flask; do not mix.

3. Cover and autoclave medium.

4. When cooled add sterile vitamins.

*For agar medium add vitamins, mix, and dispense before agar solidifies.

5. Store at refrigerator temperature. Stock Solution Final

# Component

Concentration Concentration

, NaN0₃ (Fisher BP360-

7.5 g/100 mL dH20 880 μΜ 500)

2 NaH₂P0₄- H₂0(MCIB 742) 1 mL 0.5 g/100 mL dH20 36 μΜ

„ Na₂Si0₃-9H₂0 (Sigma , ,

3 g/100 mL dH20 106 μΜ 307815)

4 Trace Metals Solution 1 mL/L

5 Vitamin Bi? 1 mL/L

6 Biotin Vitamin Solution 1 mL/L

7 Thiamine Vitamin Solution 1 mL/L

Modifications to the F/2 medium are made mainly on the concentration of nitrogen, phosphate, salt, and trace elements. Thus, for example, 20-1 00 mg/L N-nitrate is added in the culturing methods disclosed herein.

[0035] The algal strains described herein are grown in suitable open raceway ponds or modular photobioreactors for large-scale cultivation of DHA- or DHA/EPA producing strains under indoor or outdoor conditions. The operation and maintenance protocols that facilitate high performance of the selected strains to achieve simultaneous high-yield production of both algal biomass and DHA or DHA plus EPA are optimized, which include, but are not limited to, the light path of photobioreactors, light intensity, temperature, rate of culture aeration/mixing, pH, nutrient concentration, and C0₂ concentration. Thus, for example, suitable culture conditions for induction of high DHA/EPA synthesis are:

1 . Light intensity: 100 to 500 mmol m^"2 s^"1 ;

2. Light period: either continuous illumination or a light-dark cycle (e.g., Iight:dark=14h:1 0h)

3. Temperature range: 15-35 °C;

4. C0₂ concentration: 0.5-1 .5% of air bubbles; 5. Nitrogen level in growth medium: 20-100 mg/L N-nitrate;

6. The light path (culture depth) of ponds/photobioreactors: 5-25 cm;

7. pH: 7-9

[0036] Two examples of especially favorable culture conditions are:

1 . Continuous illumination/light intensity: 100 mmol m^"2 s^"1/ temperature: 25 °C/20 mg/L nitrogen (N in a form of nitrate )/1 % CCVbioreactor light path: 5 cm.

2. Light:dark = 12h:12h/light intensity: 500 mmol m^"2 s^"1/ temperature: 30 °C/40 mg/L nitrogen (N in a form of nitrate )/2% CCVbioreactor light path: 10 cm.

[0037] Once the algae are grown in culture and are producing the DHA and DHA/EPA, a downstream process is used to harvest and dewater the algal cells from the culture. The method further involves a process to treat and recycle used culture media for cost-reduction and maximum resource utilization with no or minimum discharge of treated wastewater into the environment.

[0038] Ultimately, the harvested algal strains are used to extract oil from the algae, wherein the oil is enriched in DHA or DHA plus EPA.

[0039] It should be noted that the methods described herein may ideally use multiple algal strains that possess the ability to produce DHA or DHA plus EPA.; in this embodiment, one strain may produce DHA only or produce both DHA and EPA, which is strain-specific. In a further embodiment, the method comprises blending two or more oils enriched in DHA or DHA plus EPA.

[0040] In another aspect, compositions comprising a blended plurality of algal cell fractions enriched in DHA or DHA plus EPA are provided. In a further embodiment, the compositions comprise DHA or DHA plus EPA.

[0041] In a first aspect, methods for producing algal oil enriched in DHA or DHA plus EPA are provided, comprising:

[0042] (a) culturing one or more algal strains from the genera of Isochrysis and Pavlova that can produce and accumulate large quantities of DHA or DHA plus EPA under conditions suitable to promote accumulation of DHA or DHA plus EPA; and [0043] (b) extracting oil from the algal strains, wherein the oil is enriched in DHA or DHA plus EPA.

[0044] As used herein, the phrase "DHA and EPA" refers to the omega-3 polyunsaturated fatty acids docosahexaeoic acid and eicosapentaenoic acid, repsectively. The one or more algal strains used can produce large quantities of DHA and EPA. "Large quantities" means that 15% or more of total fatty acids produced by the algal strain are DHA and DHA plus EPA. In a further embodiment, the one or more algal strains produce and accumulate at least 20% of the fatty acids produced as DHA or DHA plus EPA; more preferably, at least 25%. Those of skill in the art will understand that while the algal strains employed produce DHA and EPA, they may also produce other chain-length fatty acids. Table 2 shows the fatty acid composition of Isochrysis sp. UTEX2307 grown under our selected and favorable culture conditions.

[0045] Table 2. Fatty acid composition of Isochrysis sp. UTEX 2307.

[0046] The cell samples were harvested from early exponential growth phase (day 2), cultured under continuous illumination of 35 μΕ m^"2s^"1 and aeration of 0.5% C0₂ (Nitrogen was provide as nitrate at 50 mg/l).

[0047] Previous efforts to produce algal oil enriched in DHA by heterotrophic fermentation of specific microalgae using organic compounds such as starch and proteins as carbon and energy source. In this disclosure, selected DHA- and DHA EPA-producing microalgae are grown photoautotrophically, i.e., using C0₂ as a carbon source and light (artificial light or sunlight) as the energy source. The methods disclosed herein can produce DHA or DHA plus EPA more cost-effectively and energy-efficiently than the fermentation methods.

[0048] Previous efforts were also made to produce algal oil enriched in DHA by photoautotrophic culture or heterotrophic culture mode. However, the methods disclosed herein can produce DHA or DHA plus EPA more cost- effectively, because of the superior strains from the genera of Isochrysis and Pavlova and photobioreactor and cultivation process used.

[0049] As used herein, the term "microalgae" includes naturally occurring and genetically modified Isochrysis and Pavlova strains.

[0050] Growth of the algae can be in any type of system or photobioreactor. As used herein, a "photobioreactor" is an industrial-scale culture vessel made of transparent clear materials (e.g., glass, acrylic, polycarbonate, PVC, etc.) in which algae grow and proliferate. For use in this aspect of the invention, any type of system or photobioreactor can be used, including but not limited to open raceways (i.e., shallow ponds with water levels ca. 15 to 30 cm high) each covering an area of 1000 to 5000 m² constructed as a loop in which the culture is circulated by a paddle-wheel (Richmond, 1986)), closed systems, i.e., photobioreactors made of transparent tubes or containers in which the culture is mixed by either a pump or air bubbling (Lee 1986; Chaumont 1993; Richmond 1997; Tredici 2004), tubular photobioreactors (For example, see Tamiya et al. (1953), Pirt et al. (1983), Gudin and Chaumont 1983, Chaumont et al. 1988; Richmond et al. 1993)) and flat plate-type photobioreactors, such as those described in Samson and Leduy (1985), Ramos de Ortega and Roux (1986), Tredici et al. (1991 , 1997) and Hu et al. (1996, 1998a,b,c).

[0051] As used herein, "promote accumulation" means that the conditions employed result in algal production of DHA or DHA plus EPA equal to at least 1 % total dry cell weight, and preferably 2%, 3%, 4%, 5%, or more.

[0052] The methods disclosed herein comprise extracting oil enriched with DHA or DHA plus EPA from selected algal strains from the genera of Isochrysis and Pavlova. Initially, algae are harvested from liquid culture in the photobioreactor using a suitable harvesting method (such as centrifugation, dissolved air floatation, membrane filtration, etc, singularly or in combination). The resulting wet algal cell paste can be subjected to oil extraction. The harvested algae can also be dried using any suitable technique (such as sun- drying, drum-drying, freeze drying, or spray-drying). The resulting dried algae can be in any useful form, including but not limited to a form of algal flour.

[0053] Any suitable process for extracting oil from the algae can be used, including but not limited to solvent extraction and supercritical fluid extraction.

[0054] As will be apparent to those of skill in the art, oil extraction from algae can be accompanied by extraction of other algal biomass that is separated from the oil during the extraction process. Thus, in another embodiment, the methods of the invention further comprise isolating algal biomass residue from the oil extraction process. Such biomass residue can include, but is not limited to, bulk products (useful, for example, for animal feed and biofertilizer); ethanol and methane (requires subsequent fermentation; useful, for example, in energy production); and specialty products, including but not limited to pigments (chlorophyll), polymers, carotenoids (e.g., beta- carotene, zeaxanthin, lutein, and astaxanthin), and polyunsaturated fatty acids.

[0055] In specific embodiments, the DHA and/or EPA compositions isolated from the algal strains as described herein may be used to prepare nutritional supplements. The compositions may be prepared as separate supplements or may be prepared in the form of a supplement comprising one or more additional nutritional supplements. For example, the composition of the present invention may be used to prepare a soft gel nutritional supplement which comprises Calcium Carbonate 150 mg; DHA isolated from the algal biomass as described herein:150 mg, carbonyl iron 27 mg, Linolenic acid 30 mg, Linoleic acid 30 mg, Sunflower oil 30 mg, Vitamin C 25 mg, Vitamin B6 25 mg, Folic acid 1 mg, Vitamin D3 170 IU, Vitamin E 30 IU.

[0056] A soft gelatin supplement is prepared by first combining mineral oil and soybean oil in a first vessel and blending it to form a uniform oil mixture, heating the oil mixture to 45°C, and then adding propylene glycol. In a second vessel preheated to 70 °C, yellow beeswax and soybean oil are added and blended until a uniform wax mixture is formed. The wax mixture was cooled to 35 °C and then added to the oil mixture. To this combined oil and wax mixture the active ingredients listed above are then added and blended together to form a uniform biologically active mixture. The mixture was then cooled to 30 °C to form a viscous biologically active core composition, after which time the composition was ready for encapsulation in a soft gelatin shell.

[0057] A soft gelatin shell is prepared by heating purified water in a suitable vessel and then adding 175 bloom gelatin. This water gelatin mixture is mixed until the gelatin is fully dissolved, and then glycerin, preservative, one or more flavors, and one or more colorants are added. This gelatin mixture is blended well and cooled. The shells are then filled with the core composition and formed in accordance with soft gelatin techniques commonly used and well known to persons of skill in the art. The resulting soft gelatins were recovered and stored for future use.

EXAMPLES:

[0058] The present disclosure describes a process to produce the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) by photosynthetic microalgae grown in a closed photobioreactor under indoor or outdoor conditions. More specifically, the process consists of five major technical components: 1 ) selected naturally occurring and DHA-overproduction strains Isochrysis sp. and Pavlova sp. derived from chemical mutagenesis; 2) culture media for mass cultivation of the selected strains; 3) photobioreactor design; 4) optimized operation/maintenance protocols for sustainable high-yield cultivation of the selected strains; and 5) downstream processes to harvest and dewater algal cells from the culture suspension. In addition, a proprietary extraction method developed by the inventors can be applied to extraction of DHA from algal biomass. This process is designed for commercial production of DHA from microalgae for human health and animal nutrition. No recombinant strains will be developed or deployed. Potential DHA- overproduction mutants are generated using a standard, conventional chemical mutagenesis. Note that mutants obtained from chemical mutagenesis do not belong to the GMO category. [0059] Strains of Isochrysis sp. and Pavlova. It was found that the following strains of Isochrysis and Pavlova were particularly useful at production of DHA: Isochrysis sp. UTEX LB 2307; Isochrysis galbana UTEX LB 987; Pavlova gyrans UTEX LB 992; Pavlova lutheri UTEX LB 1293.

[0060] Culture media. The strains are grown in modified F/2 culture media.

[0061] Photobioreactor design. DHA-producing strains of Isochrysis and Pavlova can be cultivated using closed photobioreactors of various designs and configurations, such as tubular, column, and flat panel photobioreactors.

[0062] Optimization of protocols for selected strains. Optimization of the production and maintenance protocols for sustainable high-yield production of DHA in selected Isochrysis and Pavlova strains can be realized through monitoring and maintaining a suitable amount of nitrogen in the growth medium, a suitable level of light intensity; and a suitable cell population throughout the entire cultivation period. Such conditions include:

Light intensity: 100 to 500 mmol m^"2 s^"1;

Light period: either continuous illumination or a light-dark cycle (e.g., Iight:dark=14h:10h)

Temperature range: 15-35 °C;

C0₂ concentration: 0.5-1 .5% of air bubbles;

Nitrogen level in growth medium: 20-100 mg/L N-nitrate;

The light path (culture depth) of ponds/photobioreactors: 5-25 cm;

pH: 7-9

[0063] Downstream processing and harvesting of alga: DHA-rich cells from the mass culture of Isochrysis and pavlova cultures can be harvested and dewatered using a various physical and chemical approaches, such as membrane ultrafiltration, dissolved air flotation, centrifugation, and flocculation. The wet algae pastes or slurries can be dried using a drum dryer, a spray dryer, or freeze dryer.

Claims

1 . A method for producing an algal lipid fraction enriched for DHA and/or EPA, said method comprising

(i) culturing a first algal culture consisting of a strain from the algal genus Isochrysis, wherein said first algal strain produces at least a first fatty acid subset wherein at least 10% of the fatty acids in said subset are of a chain length of Docosahexaenoic acid (DHA) (22:6); and

(ii) culturing a second algal culture consisting of a strain from the algal genus Pavlova, wherein said second algal strain produces at least a second fatty acid subset wherein at least 10 % of the fatty acids in said subset are Docosahexaenoic acid (DHA) (22:6).

2. The method of claim 1 , further including extracting oil from the first algal strain and the second algal strains to produce a fatty acid composition enriched for C20 or C22 fatty acids.

3. The method of claim 1 , wherein said culturing steps are performed photoautotophically in an open pond or a closed photobioreactor, wherein said cultures are supplied with C0₂ as a carbon source and light as an energy source.

4. The method of claim 1 , wherein the first algal strain and the second algal strains are cultured together as a co-culture.

5. The method of claim 1 , wherein said Isochrysis strain is selected from the group consisting of Isochrysis sp. UTEX LB 2307 and Isochrysis galbana UTEX LB 987.

6. The method of claim 1 , wherein said Pavlova strain is selected from the group consisting of Pavlova gyrans UTEX LB 992 and Pavlova lutheri

UTEX LB 1293.

7. The method of claim 1 , further comprising isolating algal biomass.

8. A composition comprising lipids extracted from the first and second algal strains cultured according to the method of claim 1 .

9. A dietary supplement comprising a lipid fraction enriched for DHA and/or EPA obtained according to the culturing method of claim 1 .

10. The method of claim 1 , wherein said culturing steps are performed under the following conditions:

light intensity or 100 to 500 mmol m^"2 s^"1;

light period of continuous illumination;

temperature range of about 15-35 °C;

C0₂ concentration of about 0.5-1 .5% of air bubbles;

nitrogen level in growth medium of about 20-100 mg/L N-nitrate;

a light path (culture depth) of ponds or photobioreactors of about 5-25 cm; and a pH of about 7-9.

1 1 . The method of claim 4, wherein said culturing steps are performed under the following conditions:

light intensity or 100 to 500 mmol m^"2 s^"1;

light period of continuous illumination;

temperature range of about 15-35 °C;

C0₂ concentration of about 0.5-1 .5% of air bubbles;

nitrogen level in growth medium of about 20-100 mg/L N-nitrate;

12. A composition comprising a co-culture of a high DHA-producing Isochrysis strain and an a high DHA-producing Pavlova strain, wherein the DHA content of the Isochrysis and Pavlova strains makes up at least 15% of total fatty acids in the algae.

13. A method for producing an algal lipid fraction enriched for DHA and/or EPA, said method comprising: culturing a strain from an algal genus Isochrysis or genus Pavlova in an open pond or a closed photobioreactor, wherein the culturing is conducted under conditions suitable to promote production of at least 15% of the fatty acids of the Docosahexaenoic acid (DHA) (22:6).

14. The method of claim 13, wherein said culturing is performed under the following conditions:

light intensity or 100 to 500 mmol m^"2 s^"1;

light period of continuous illumination;

temperature range of about 15-35 °C;

C0₂ concentration of about 0.5-1 .5% of air bubbles;

nitrogen level in growth medium of about 20-100 mg/L N-nitrate;