AU2007237174B2 - A process for producing lipids - Google Patents

Description

Regulation 3.2 Revised 2/98 AUSTRALIA Patents Act, 1990 ORIGINAL COMPLETE SPECIFICATION TO BE COMPLETED BY THE APPLICANT NAME OF APPLICANT: Martek Biosciences Corporation ACTUAL INVENTOR: William R Barclay ADDRESS FOR SERVICE: Peter Maxwell and Associates Level 6 60 Pitt Street SYDNEY NSW 2000 INVENTION TITLE: A PROCESS FOR PRODUCING LIPIDS DETAILS OF ASSOCIATED APPLICATION NO(S): Divisional of Australian Patent Application No. 2004 229 116 filed on 15 November 2004 which is a divisional of Australian Patent No. 776,655 filed on 19 March 2002; which is a divisional of Australian Patent No. 742,163 filed on 20 January 2000; which is a divisional of Australian Patent No. 711,828 filed on 25 November 1997; which is a divisional of Australian Patent No. 687,016 filed on 12 October 1993 The following statement is a full description of this invention including the best method of performing it known to us: m:\docs\931280\131399.doc 2 This invention relates to a process for producing lipids during growth of microorganisms in a saline fermentation medium. Omega-3 highly unsaturated fatty acids (HUFAs) are of significant commercial interest in that they have been recently recognised as important 5 dietary compounds for preventing arteriosclerosis and coronary heart disease, for alleviating inflammatory conditions and for retarding the growth of tumour cells. These beneficial effects are a result both of omega-3 HUFAs causing competitive inhibition of compounds produced from omega-6 fatty acids, and from beneficial compounds produced directly from the omega-3 HUFAs 10 themselves (Simopoulos et al 1986). Omega-6 fatty acids are the predominant HUFAs found in plants and animals. Currently, a commercially available dietary source of omega-3 HUFAs is from certain fish oils which can contain up to 20 30% of these fatty acids. The beneficial effects of these fatty acids can be obtained by eating fish several times a week or by daily intake of concentrated 15 fish oil. Consequently large quantities of fish oil are processed and encapsulated each year for sale as a dietary supplement. However, there are several significant problems with these fish oil supplements, including bioaccumulation of fat- soluble vitamins and high levels of saturated and omega-6 fatty acids, both of which can have deleterious health effects. 20 Another source of omega-3 HUFAs is the microflora Thraustochytrium and Schizochytrium which are discussed in detail in related U.S. Patent No. 5,130,242. These microflora have the advantages of being heterotrophic and capable of high levels of omega-3 HUFA production. There still exists a need however for improved methods for fermentation of these microflora and 25 identification of improved uses of the microflora product.

3 The present invention is directed to a new process for culturing a euryhaline microorganism in a low chloride medium. According to one aspect of the invention, there is provided a process for culturing microorganism of the order Thraustochytriales in a low chloride 5 medium, the process comprising: culturing microorganism of the order Thraustochytriales in a culture medium having a non-chloride source of sodium, having a chloride concentration of less than 3 grams of chloride per liter of said culture medium, and having a sodium concentration between 1 g/L and 50 g/L, wherein less 10 than 50% of the sodium in the fermentation medium is supplied as sodium chloride. In a preferred form of the invention the lipids comprise at least one highly unsaturated fatty acid which may be selected from the group consisting of DHA, EPA, DPA(n-6) and mixtures thereof. 15 Preferably the non-chloride source of sodium is in an amount less than 25g/L. Less than 25% of the sodium in the fermentation medium may be supplied as sodium chloride. In a preferred form of the invention, the microorganism produces about 12% dwt or less of omega-3 fatty acids. It is further preferred that the micro 20 organism replicates by successive bipartition. In a further preferred form of the invention, the microorganism replicates by forming sporangia and releasing zoospores. In a further preferred form of the invention the micro-organism is Thraustochytrium, Schizochytrium or mixtures thereof. 25 The microorganism may be cultured at a temperature from about 5 0 C to about 48'C and a pH from about pH 5.0 to about pH 11.0.

4 Preferably, the chloride concentration is less than 1gram of chloride per litre of the culture medium and more preferably the chloride concentration is less than 500mg of chloride per litre of the culture medium. In a further preferred form of the invention, the culture medium 5 comprises between about 60mg and about 120mg of chloride per litre of culture medium. The non-chloride source of sodium may be sodium sulphate. The concentration of sodium expressed as grams of sodium per litre of the culture medium is preferably between 2 g/L and 25 g/L. 10 It is further preferred that the process includes the step of recovering fatty acids from the microorganism. The present process is particularly useful in commercial production because the chloride content in the medium can be significantly reduced, thereby avoiding the corrosive effects of chloride on fermentation equipment. 15 In addition the present invention is particularly useful for production of food products for use in aquaculture as euryhaline microorganisms, including Thraustochytrium , Schizochytrium and mixtures thereof, cultured in such media form much smaller clumps than those cultured in high chloride media and are thus more available as a food source for larval shrimp. In particular, 20 euryhaline microorganisms grown as a result of this process have cell aggregates of an average size of less than about 150 microns in diameter. The small cell aggregate size allows the microorganisms to be eaten by the larval shrimp, brine shrimp, rotifers and molluscs. The present invention is also useful in aquaculture as the euryhaline 25 microorganisms produced have a sterol content of at least about 0.1 % ash free dry weight and a cholesterol content of at least about 15% of the total sterol 4a content. Both sterol and cholesterol are necessary for larval shrimp development. The euryhaline microorganisms may be grown at a temperature of between about 5 0 C to about 48 0 C and a pH of about 5 to about 11. 5 Fig. 1 is a graphical representation of HUFA production in newly isolated strains, represented by -, and previously isolated strains represented by +. Each point represents a strain, the position of each point is determined by the percent by weight of total fatty acids which were omega-3 HUFAs (abscissa) and the percent by weight of total fatty acids which were omega-6 10 fatty acids (ordinate). Only those strains were plotted wherein less than 10.6% (w/w) of total fatty acids were omega-6 and more than 67% of total fatty acids were omega-3. Fig. 2 is a graphical representation of HUFA production in newly isolated strains, represented by -, and previously isolated strains, represented 15 by +. Each point represents a strain, the position of each point is determined by the percent by weight of total fatty acids which were omega-3 HUFAs (abscissa) and percent of weight of total fatty acids which were eicosapentaenoic acid (EPA C20:5n-3) (ordinate). Only those strains were plotted wherein more than 67% (w/w) of total fatty acids were omega-3 and 20 more than 7.8% (w/w) of total fatty acids were C20:5n-3.

5 Fig. 3 is a graphical representation of omega-3 HUFA composition in newly isolated, represented by 0, and previously isolated strains, represented by +. Each point represents a separate strain. Values on the abscissa are weight fraction of total omega-3 HUFAs which were C20:5n-3 and on the 5 ordinate are weight fraction of total omega-3 fatty highly unsaturated acids which were C22:6n-3. Only strains were plotted having either a weight fraction of C20:5n-3 28% or greater, or a weight fraction of C22:6n-3 greater than 93.6%. Fig. 4 is a graph showing growth of various newly isolated strains and 10 previously isolated strains, at 250C and at 300C. Growth rates are normalised to the growth rate of strain U-30 at 250C. Previously isolated strains are designated by their ATCC accession numbers. Fig. 5 is a graph of total yields of cellular production after induction by nitrogen limitation. Each of ash-free dry weight, total fatty acids and omega-3 15 HUFAs, as indicated, was plotted, normalised to the corresponding value for strain 28211. All strains are identified by ATCC accession numbers. Fig. 6 is a graph of fatty acid yields after growth in culture media having the salinity indicated on the abscissa. Strains shown are newly isolated strains S31 (ATCC 20888) (0) and U42-2 (ATCC 20891) (+) and previously isolated 20 strains, ATCC 28211 (Q) and ATCC 28209 (A) . Fatty acid yields are plotted as relative yields normalised to an arbitrary value of 1.00 based on the average growth rate exhibited by S31 (ATCC 20888) (0) over the tested salinity range. Fig. 7 is a graph of increases in the omega-3 HUFA content of the total lipids in the brine shrimp, Artemia salina, fed Thraustochytrid strain (ATCC 25 20890) isolated by the method in Example 1. EPA = C20:5n-3; DHA = C22:5n 3.

6 Fig. 8 is a graph of increases in the omega-3 HUFA content of the total lipids in the brine shrimp, Artemia saina, fed Thraustochytrid strain (ATCC 20888) isolated by the method in Example 1. EPA = C20:5n-3; DHA = C22:5n 3. 5 For purposes of definition throughout the application, it is understood herein that a fatty acid is an aliphatic.

monocarboxylic acid. Lipids are understood to be fats or oils including the glyceride esters of fatty acids along with associated phosphatides, sterols, alcohols, hydrocarbons, ketones, and related compounds. 5 A commonly employed shorthand system is used in this specification to denote the structure of the fatty acids (e.g., Weete, 1980). This system uses the letter "C" accompanied by a number denoting the number of carbons in the hydrocarbon chain, followed by a colon and a number 10 indicating the number of double bonds, i.e., C20:5, eicosapentaenoic acid. Fatty acids are numbered starting at the carboxy carbon. Position of the double bonds is indicated by adding the Greek letter delta (A) followed by the carbon number of the double bond; i.e., C20:5omega 15 3,5,8,11,14,1 7 . The "omega" notation is a shorthand system for unsaturated fatty acids whereby numbering from the carboxy terminal carbon is used. For convenience, n-3 will be used to symbolize "omega-3, " especially when using the numerical shorthand nomenclature described herein. Omega-3 highly 20 unsaturated fatty acids are understood to be polyethylenic fatty acids in which the ultimate ethylenic bond is 3 carbons from and including the terminal methyl group of the fatty acid. Thus, the complete nomenclature for eicosapentaenoic acid, an omega-3 highly unsaturated fatty acid, would be 25 C20:5n-3AS 8

,

1 1

,

14

,

17 . For the sake of brevity, the double bond locations (A 5 ,a',1, 4

''

7 ) will be omitted. Eicosapentaenoic acid is then designated C20:5n-3, Docosapentaenoic acid (C22:5n 3A7,10,13,16,1 9 ) is C22:5n-3, and Docosahexaenoic acid (C22:6n- 3A 4 ,7,10, 13

,

16 ,19) is C22:6n-3. The nomenclature "highly unsaturated fatty acid" means a fatty acid with 4 or more double bonds. "Saturated fatty acid" means a fatty acid with 1 to 3 double bonds. 5 A collection and screening process has been developed to readily isolate many strains of microorganisms with the following combination of economically desirable character istics for the production of o-mega-3 HUFAs: 1) capable of heterot-rophic growth; 2) high content of omega-3 HUFAs; 3) SuT T f low content of saturated and omega-6 HUFAs;. 5) preferably nonpigmented, white or essentially colorless cells; 6) preferably thermotolerant (ability to grow at temperatures above 30*C); and 7). preferably euryhaline (able to grow over a wide range of salinities, but especially at low salinities) - This process is described in detail in related U.S. Patent No. 5,130,242. Using the collection and screening process, strains of unicellular microflora can be isolated which have fatty acid contents up to about 45% total cellular dry weight percent (%dwt), and which exhibit growth over a temperature range from 15-48'C and grow in a very low salinity culture medium. Many of the very high omega-3 strains are very slow growers. Strains which have been isolated by the method outlined above, and which exhibit rapid growth, good production and high omega-3 HUFA content, have omega-3 unsaturated fatty acid contents up to approximately 12% dwt. This specification describes the growth of Thraustochytrium, Schizochytrium, and mixtures thereof with high omega-3 HJFA content, in fermentation medium containing non-chloride containing sodium salts and preferably sodium sulfate. More particularly, a significant portion of the sodium requirements of the fermentation are supplied as non 5 chloride containing sodium salts. For example, less than about 75% of the sodium in the fermentation medium is supplied as sodium chloride, more preferably less than about 50% and more preferably less than about 25%. A particular advantage of the present invention is that the medium provides the source of sodium needed by the microflora to grow in the absence of a significant amount of chloride which can corrode the vessel in which the microflora are being grown and other fermentation or downstream processing equipment. It has been surprisingly found that the micro flora described herein can be grown at chloride concen trations of less than about 3 gl/l, more preferably less than about 500 mg/l, more preferably less than about 250 mg/l and more preferably between about 60 mg/i and about 120 mg/i while still attaining high yields of biomass per sugar of about 50% or greater- The specification also describes the production of microflora that are high in cmega-3 HUFA - content but have a small enough cell aggregate size to be consumed by larval shrimp, brine shrimp, rotifers and mollusks. Non-chloride containing sodium salts can include soda ash (a mixture of sodium carbonate and sodium oxide) , sodium carbonate, sodium bicarbonate, sodium sulfate and mixtures thereof, and preferably include sodium sulfate. Soda ash, 10 sodium carbonate and sodium bicarbonate tend to increase the pH of the fermentation medium, thus requiring control steps to maintain the proper pH of the medium. The concentration of sodium sulfate is effective to meet the salinity requirements of the microflora, preferably the sodium concentration is 5 (expressed as g/I of Na) greater than about 1.0 g/l, more preferably between about 1.0 g/I and about 50 g/l, and more preferably between about 2.0 g/I and about 25 g/l. It has been surprisingly found that fermentation of the strains in the presence of a non-chloride containing sodium salt, and particularly sodium 10 sulfate, limits the cell aggregate size of the strains to less than about 150 microns, preferably less than about 100 microns, and more preferably less than about 50 microns. As used herein, the term cell aggregate size refers to the approximate average diameter of clumps or aggregates of cells in a fermentation medium of a microfloral culture. Typically, greater than about 25 15 percent of the cell aggregates in a microfloral culture have cell aggregate size below the average size, more preferably greater than about 50 percent and more preferably greater than about 75 percent. Microfloral cells produced in accordance with the processes described herein meet cell aggregate size parameters described above while in fermentation medium as well as after 20 freezing and/or drying of the biomass if resuspended in liquid or physically agitated, such as by a blender or vortexer. These processes are particularly important for microflora which replicate by successive bipartition (wherein a single cell replicates by dividing into two cells which each divide into two more, etc.) because as cells repeatedly and rapidly undergo this process, the cells tend to clump forming multi-cell aggregates which are often outside the cell aggregate size parameters identified 5 above. Schizochytrium replicate by successive bipartition and by forming sporangia which release zoospores. Thraustochytrium, however, replicate only by forming sporangia and releasing zoospores. For Thraustochytrium which replicate by sporangia/zoospore formation, clumping 2 can be a problem as well, particularly because even though the number of -cells in an aggregate may not be as great as aggregates formed by successive bipartition, the individual cell sizes of Thraustochytrium tend to be larger, and thus, clumps of a small 'number of cells are larger. However, one deposited -strain of Thraustochytrium, ATCC 26185, has been identified which does not exhibit significant aggregation. The specification also describes the finding that by restricting the oxygen content of the fermentation medium during the growth of Thraustochytrium, Schizochytrium, and mixtures thereof, the lipid content of the strains can be increased. The optimum oxygen concentration for lipid production can be determined for any particular microflora by variation of the oxygen content of the medium. In particular, the oxygen content of the 5 fermentation medium is maintained at an oxygen content of less than about 40% of saturation and preferably between about 5% of saturation and about 40% of saturation.

-12 Growth of the strains by these processes can be effected at any temperature conducive to satisfactory growth of the strains; for example, between about 5'C and about 48 * C, preferably between about 15"C and about- 40" C, and more preferably between about 25'C and about 35'C. The culture medium typically becomes more alkaline. - during the fermentation if pH is not controlled by acid addition or buffers. The strains will grow over a pH range from 5.0-11.0 with a preferable range of about 6.0-8.5. Various fermentation parameters for inoculating, growing and recovering microflora are discussed in detail in U.S. Patent No. 5,130,242- The biomass harvested from a fermentation run can be dried (e.g., spray drying, -tunnel drying, vacuum drying, or a similar process) and used as a feed or food supplement for any animal whose meat or products are consumed by humans. Similarly, extracted omega-3 HUFAs can be used as a feed or food supplement. Alternatively, the harvested and washed biomass can be used directly (without drying) as a feed supplement. To extend its shelf life, the wet biomass can be acidified (approximate pH = 3.5-4.5) and/or pasteurized or flash heated to inactivate enzymes and then canned, bottled or packaged under a vacuum or non oxidizing atmosphere (e.g., N 2 or CO 2 ). The term "animal means any organism belonging to the kingdom Animalia and includes, without limitation, any animal from which poultry' meat, seafood, beef, pork or lamb is derived. Seafood is derived from, without limitation, fish, shrimp and shellfish. The term "products" includes any product other than meat derived from such animals, including, without ~limitation, eggs or other products. When fed to such animals, omega-3 HUFAs in the harvested biomass or extracted omega-3 HUFAs are incorporated into the flesh, eggs or other products of such animals to increase- the omega-3 HUFA content thereof. This specification still further describes the use of the harvested biomass as a food product for larval shrimp, brine shrimp, rotifers and mollusks and in particular, larval shrimp. During the larval stage of development, shrimp larvae are unable to use some food sources because the food source is too large. In particular, at certain stages of development, shrimp larvae are unable to use a food source having a diameter greater than about 150 microns. Thus, microflora grown in fermentation medium containing a non-chloride sodium salt, and particularly sodium sulfate, as broadly discussed above, are suitable for use as a shrimp food product. As discussed above, microflora grown under such conditions typically have a cell aggregate size less than about 150 microns, preferably less than about 100 microns, and more preferably less than about 50 microns. A further advantage of the use of 'the microflora as described herein as a food source for shrimp -is that such microflora have a significant sterol content including cholesterol, which is a primary feed requirement for shrimp. These microflora -- typically have a. sterol content of preferably at least- about 0.i%1 ash-free dry weight (afdw) , more preferably at least about 0.5% afdw, and even more preferably at least about 1.0% afdw. In 14 addition, these typically have a cholesterol content of preferably at least about 15% of the total sterol content, more preferably at least about 25% of the total sterol content, and even more preferably at least about 40% of the total sterol content. Further, the microfloral biomass as described herein also provide 5 shrimp with additional nutritional requirements such as omega-6 fatty acids, protein, carbohydrates, pigments and vitamins. The microbial product described in this specification is of value as a source of omega-3 HUFAs for fish, shrimp and other products produced by aquaculture. The product can be used as a food product as described above 10 for shrimp; or added directly as a supplement to the feed for shrimp and fish, generally; or it can be fed to brine shrimp or other live feed organisms intended for consumption by an aquacultured organism. The use of such microflora in this manner enables the shrimp farmer to obtain significantly higher growth rates and/or survival rates for larval shrimp and to produce post-larval shrimp 15 which are more hardy and robust. For most feed applications, the fatty acid content of the harvested cells will be approximately 15-50% dwt with the remaining material being largely protein and carbohydrate. The protein can contribute significantly to the nutritional value of the cells as several of the strains that have been evaluated 20 have all of the essential amino acids and would be considered a nutritionally balanced protein.

-15 The specification also describes the production of a food product using Thraustochytrium, Schizochytrium, and mixtures thereof, -combined with- an additional component selected 5 from the group consisting of rapeseed, flaxseed, soybean and avocado meal. A-particular advantage of this. -embodiment is that the food product contains both short chain omega-3 HUFAs from the additional component and long chain omega-3 HUFAs from the microflora. Food products having flaxseed, rapeseed, soybeans and avocado meal are known to be useful for supplying a source of short chain omega-3 HUFAs and for additionally supplying a source of short chain omega-3 *HUFAs, which can be elongated by the humans and animals that ingest them. Such food products, however, have the disadvantages of having high choline contents from the additional component, which can form primary amines and result in an unpleasant fish smell; and toxic compounds from the additional component, which at high levels can, for example, inhibit the laying of eggs by hens or cause animals to go off of their feed. As such, the food product as described herein has the advantage of a lowered flaxseed, rapeseed, soy bean or avocado meal content because the organism ingesting the food product does not need high levels of short chain omega-3 HUFAs for the purpose of converting them to p long chain HUFAs. Thus, the lowered content of the flaxseed and rapeseed of the food product results in lowered amounts of choline and/or inhibitory toxic compounds present in the food product.

The amount of Thraustochytrium, Schizochytrium, and mixtures thereof, used in the food product can range from between about 5% to about 95% by weight. The additional component can be present- in the food product at a range of 5 between about 5% to about 95% by weight. Additionally, the food product can include other components as well, including grains, supplements, vitamins, binders and preservatives. The above food product is preferably produced using an extrusion process. The extrusion process ) involves.mixing the microflora with the additional component, thereby reducing the moisture in the microfloral biomass by the amount of the additional component mixed. The food product is extruded under heat, thus removing further moisture from the food product. The resulting product which has a low moisture content can be air dried or dried by relatively short baking times thereby reducing the overall energy requirements of drying and the potential degradation of omega-3 HUFAs due to long time periods at high temper atures. In addition, heat from the extrusion process can degrade some of the unwanted toxic compounds commonly found in the additional component which can, for example, inhibit egg laying by hens or cause animals to go off of their feed. Species meeting the selection criteria described above have not been described in the prior art. By employing these selection criteria, over 25 potentially promising strains have been isolated from approximately 1000 samples screened. Out of the approximate 20,500 strains in the American Type Culture Collection (ATCC) ; 10 strains were later identified as belonging to the same taxonomic group as the strains isolated. Those strains still viable in the Collection were procured and used to 5 compare with strains isolated and cultured by the disclosed procedures. The results of this comparison are presented in- Examples 4 and 5 below. The most recent taxonomic theorists' place Thraustochydrids with the algae or algae-like protists. All of the strains of unicellular microorganisms disclosed herein -are members of the order Thraustochytriales (Order: Thraustochytriales; Family: Thraustochytriaceae; Genus: Thraustochytrium or Schizochytrium)- For general purposes of discussion herein, these microorganisms will be called microflora to better denote their uncertain exact taxonomic position. The novel strains identified below were deposited under the Budapest Treaty on the International Recognition of the Deposit of . Microorganisms for the Purpose of Patent Procedure.

18 The microorganisms described in this specification have all of the identifying characteristics of the deposited strains and, in particular, the identifying characteristics of being able to produce omega-3 HUFAs as described herein and having cell aggregate size characteristics when cultured 5 under conditions as described herein. In particular, the microorganisms as described herein refer to the following deposited microorganisms and mutants thereof. Strain ATCC NO. Deposit Date Schizochytrium S31 20888 5/8/88 Schizochytrium S8 20889 5/8/88 Thraustochytrium 12B 20890 5/8/88 Thraustochytrium U42-2 20891 5/8/88 Thraustochytrium 23B 20892 5/8/88 10 The following examples and test results are provided for the purposes of illustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1. Collection and Screening A 150ml water sample was collected from a shallow, 5 inland saline pond and stored in a sterile polyethylene bottle. Special effort was made to include some of the living plant material and naturally occurring detritus (decaying plant and animal matter) along with the water sample. The sample was placed on ice until return to the 10 laboratory. In the lab, the water sample was shaken for 15 30 seconds, and 1-10ml of the sample was pipetted or poured into a filter unit containing 2 types of filters: 1) on top, a sterile 47mm diameter Whatman #4 filter having a pore size about 25pm; and 2) underneath the Whatman filter, a 47mm 15 diameter polycarbonate filter with about 1.Opm pore size. Given slight variations of nominal pore sizes for the filters, the cells collected on the polycarbonate filter range in size from about 1.0pm to about 25pm. The Whatman filter was removed and discarded. The 20 polycarbonate filter was placed on solid F-i media in a petri plate, said media consisting of (per liter): 600ml seawater (artificial seawater can be used), 400ml distilled water, log agar, lg glucose, ig protein hydrolysate, 0.2g yeast extract, 2ml 0.1 M KH 2

PO

4 , lml of a vitamin solution (A-vits) 25 (Containing 100mg/l thiamine, 0.5mg/l biotin, and 0.5mg/l cyanocobalamin) , 5ml of a trace metal mixture (PII metals, containing per liter: 6.Og Na 2 EDTA, 0.29g FeCl 3 6H 2 0, 6.84g

H

3 B0 3 , 0.86 MnCl 2 4H 2 0, 0.06g ZnCl 2 , 0.026g CoCl 2 6H 2 0, (0.052g NiSO 4

H

2 0, 0.002g CuSo 4 5H 2 0, and 0.005g Na 2 Moo 4 2H 2 0, and 500mg each of streptomycin sulfate and penicillin-G. The agar plate was incubated in the dark at 30*C. After 2-4 days numerous colonies appeared on the filter. Colonies of unicellular 5 microflora (except yeast) were picked from the plate and restreaked on a new plate of similar media- composition. Special attention was made to pick all colonies consisting of colorless white cells. The new plate was incubated at 30*C and single colonies picked after a 2-4 day incubation 10 period. Single colonies were then picked and placed in 50ml of liquid medium containing the same organic enrichments as in the agar plates. These cultures were incubated for 2-4 days at 30*C on a rotary shaker table (100-200 rpm). When the cultures appeared to reach maximal density, 20-40ml of 15 the culture was harvested, centrifuged and lyophilized. The sample was then analyzed by standard, well-known gas chromatographic techniques (e.g., Lepage and Roy, 1984) to identify the fatty acid content of the strain. Those strains with omega-3 HUFAs were thereby identified, and cultures of 20 these strains were maintained for further screening. Using the collection and screening process outlined above, over 150 strains of unicellular microflora have been isolated which have high omega-3 HUFA contents as a percent of total fatty acids and which exhibit growth over a 25 temperature range from 15-48*C. Strains can also be isolated which have less than 1% (as % of total fatty acids) of the undesirable C20:4n-6 and C22:5n-6 HUFAs for some applications. Strains with high omega-6 content can also be isolated. Strains of these microflora can be repeatedly isolated from the same location using the procedure outlined above. A few of the newly isolated strains have very similar fatty acid profiles. The possibility that some are duplicate 5 isolates of the same strain cannot be ruled out at present. Further screening for other desirable traits such as salinity tolerance or ability to use a variety of carbon and nitrogen sources can then. be carried out using a similar process. LO Example 2. Maintaining Unrestricted Growth: P0, and Yeast Extract Cells of Schizochytrium aggregatum (ATCC 28209) were picked from solid F-1 medium and inoculated into 50ml of FFM medium. (Fuller et al., 1964). This medium contains: .5 seawater, 1000ml; glucose, 1.Og; gelatin hydrolysate, 1.0g; liver extract, 0.01g; yeast extract, 0.lg; PII metals, 5ml; Iml B-vitamins solution (Goldstein et al., 1969); and 1ml of an antibiotic solution (25g/l streptomycin sulfate and penicillin-G) . 1.0ml of the vitamin mix (pH 7.2) contains: !0 thiamine HCl, 200pg; biotin, 0.5yg; cyanocobalamin, 0.O5yg; nicotinic acid, 100pg; calcium pantothenate, lOOg; riboflavin, 5.0pg; pyridoxine HCl, 40.Opg; pyridoxamine 2HCl, 20.Opg; p-aminobenzoic acid, 10pg; chlorine HCl, 500pg; inositol, 1.0mg; thymine, 0.8mg; orotic acid, 0.26mg; folinic 25 acid, 0.2pg; and folic acid, 2.5pg. The culture was placed on a rotary shaker (200 rpm) at 27*C. After 3-4 days, iml of this culture was transferred to 50ml of each of the following treatments: 1) FFM medium (as control) ; and 2) FFM medium with the addition of 250mg/l KH 2

PO

4 and 250mg/l yeast extract. These cultures were placed on a rotary shaker (200 rpm) at 27"C for 48 hr. The cells were harvested and the yield of cells quantified. In treatment 1, the final concentration of cells on an ash-free dry weight basis was 5 616mg/l. In treatment 2, the final concentration of cells was 1675mg/1, demonstrating the enhanced effect-of increasing PO4 and yeast extract concentrations in the culture medium. Example 3. Maintaining Unrestricted Growth: Substitution 10 of Corn Steep Liquor for Yeast Extract Cells of Schizochytrium sp. S31 (ATCC No. 20888) were picked from solid F-1 medium and placed into 50ml of M-5 medium. This medium consists of (on a per liter basis): yeast extract, lg; NaCl, 25g; MgSO 4 - 7H 2 0, 5g; KCl, lg; CaCl 2 , 15 200mg; glucose, 5g; glutamate, 5g; KH2 P0 4 , 1g; PII metals, 5ml; A-vitamins solution, lml; and antibiotic solution, lml. The pH of the solution was adjusted to 7.0 and the solution was filter sterilized. Sterile solutions of corn steep liquor (4g/40ml; pH 7.0) and yeast extract (lg/40ml; pH 7.0) 20 were prepared. To one set of M-5 medium flasks, the following amount of yeast extract solution was added: 1) 2ml; 2) 1.5ml; 3) lml; 4) 0.5ml; and 5) 0.25ml. To another set of M-5 medium flasks the yeast extract and corn steep liquor solutions were added at the following levels: 1) 2ml yeast 25 extract; 2) 1.5ml yeast extract and 0.5ml corn steep liquor; 3) 1.0mil yeast extract and 1.0ml corn steep liquor; 4) 0.5ml yeast extract and 1.5ml corn steep liquor; and 5) 2ml corn steep liquor. One ml of the culture in F-1 medium was used to inoculate each flask. They were placed on a rotary shaker at 27*C for 48 hr. The cells were harvested by centrifugation and the yield of cells (as ash-free dry weight) was determined. The results are shown in Table 1. The results indicate the addition of yeast extract up to 0.8g/l of medium 5 can increase the yield of -cells. However, addition of corn steep liquor is even more effective and results in twice the yield of .treatments with added yeast extract. This is very advantageous for the economic production of cells as corn steep liquor is much less expensive than yeast extract. 10 Table 1. Treatment (Amount Nutrient Ash-Free Dry Weight 15 Supplement Added) (mg/l) 2.Oml yeast ext. 4000 20 1.5ml yeast ext. 4420 1.0ml yeast ext. 4300 0.5ml yeast ext. 2780 25 0.25ml yeast ext. 2700 2.Oml yeast ext. 4420 30 1.5ml yeast ext. + 0.5ml CSL* 6560 1.Oml yeast ext. + 1.0ml CSL 6640 35 0.5ml yeast ext. + 1.5ml CSL 7200 2.Oml CSL 7590 40 *CSL = corn steep liquor Example 4. Enhanced HUFA Content of Strains Isolated by Method in Example 1 Compared to ATCC Strains (Previously Known Strains) A battery of 151 newly isolated strains, selected 5 according to the method described in Example 1, were sampled in late exponential phase growth and quantitatively analyzed for HUFA content by gas-liquid chromatography.- All strains were grown either in M1 medium or liquid FFM medium, which ever gave highest yield of cells. Ml medium has the same 10 composition as M5 medium, except that the concentrations of glucose and glutamate are 1 g/l. Additionally, five previously isolated Thraustochytrium or Schizochytrium species - were obtained from the American Type Culture Collection, representing all the strains which could be 15 obtained in viable form from the collection. These strains were: T. aureum (ATCC No. 28211) , T. aureum (ATCC No. 34304) , T. roseum (ATCC No. 28210), T. straitum (ATCC No. 34473) and S. aggregatum (ATCC No. 28209). The strains all exhibited abbreviated growth in conventional media, and generally 20 showed improved growth in media of the present invention, including M5 medium and FFM medium. The fatty acid production of each of the known strains was measured as described, based upon the improved growth of the strains in media of the invention. 25 Fatty acid peaks were identified by the use of pure compounds of known structure. Quantitation, in terms of percent by weight of total fatty acids, was carried out by integrating the chromatographic peaks. Compounds identified were: palmitic acid (C16:0), C20:4n-6 and C22:1 (which were not resolved separately by the system employed), C20:5n-3, C22:5n-6, C22:5n-3, and C22:6n-3. The remainder, usually lower molecular weight fatty acids, were included in the combined category of "other fatty acids." Total omega-3 5 fatty acids were calculated as the sum of 20:5n-3, 22:5n-3 and 22:6n-3. Total omega-6 fatty acids were.calculated as the sum of the 20:4/22:1 peak and the 22:5n-6 peak. The results are shown in Tables 2-3 and illustrated in Figs. 1-3. From Table 2 it can be seen that large numbers LO of strains can be isolated by the method of the invention, and that large numbers of strains outperform the previously known strains by several important criteria. For example, 102 strains produced at least 7.8% by weight of total fatty acids C20:5w3, a higher percentage of that fatty acid than .5 any previously known strain. Strains 23B (ATCC No. 20892) and 12B (ATCC No. 20890) are examples of such strains. Thirty (30) strains of the invention produced at least 68% by weight of total fatty acids as omega-3 fatty acids, more than any previously known strain. Strain 23B (ATCC No. 0 20892) is an example of such strains. Seventy-six (76) strains of the invention yielded not more than 10% by weight of total fatty acids as omega-6 fatty acids, considered undesirable components of the human diet, lower than any previously known strain. Strains 23B (ATCC No. 20892) and ?5 12B (ATCC No. 20890) are examples of such strains. In addition, there are 35 strains of the invention that produce more than 25% by weight of total fatty acids as omega-6 fatty acids, more than any previously known strain. While such strains may have a more narrow range of uses for dietary purposes, they are useful as feedstock for chemical synthesis of eicosanoids starting from omega-6 fatty acids. In addition, the data reveal many strains of the 5 invention which produce a high proportion of total omega-3 fatty acids as C22:6n-3. In Table 3, 48 of the.strains shown in Table 2 were compared to the previously known strains, showing each of C20:5n-3, C22:5n-3 and C22:6n-3 as percent by weight of total omega-3 content. Fifteen strains had at 10 least 94% by weight of total omega-3 fatty acids as C22:6n-3, more than any previously known strain. Strain S8 (ATCC No. 20889) was an example of such strains. Eighteen strains had at least 28% by weight of total omega-3 fatty acids as C20:5n-3, more than any previously known strain. Strain 12B L5 (ATCC No. 20890) was an example of such strains.

TABLE2: LIST OF STRAINS AND COMPOSITIONS UNDER STANDARD SCREENING CONDITIONS PER CENT OF TOTAL FATTY ACIDS Total Total Strain 16:0 C20:4w6 C20:5w3 C22:5w6 C22:5w3 C22:6w3 Other FA Omega3 Omega6 0.4% 2.8% 6.6%- 3.2% 0-2% 8.3% 48-5% 15.1% 6.0% 21 2.9% 0.4% 2.3% 15.5% 0.5% 47.0% 11.5% 49.7% 15.9% ATCC20889 4.9% 6.51 12.0% 11.8% 0.4% 49.7% 4.7% 62.1% 18.3% U40-2 0.3% 1.7% 3.8% 8.6% 0.0% 8.2% 37-4% 12.0% 10.2% 21B 3.7% 0-4% 7.8% 0.0% 0.0% 1.1% 70.1% 8.--9% 0.4% BG1 5.0% 5.7% 1.51 9.7% 0.7% 9.7% 46.7% 11.9% 15.41 56A 5.4% 1.4% 10.0% 1.9% 2.2% .46.4% 21.8% 58.6% 3.3% 11A-1 3.7% 3.3% 10.5% 1.9% 1.8% 29.9% 28.9% 42.2 5.2% 4A-1 3.7% 6.9% 9.2% 11-91 3.2% 25.2% 24.9% 37.5% 18.8% 178 -4% 4.2% 7.3% 9-5% 0.9% 51.2% 11.6%- 59.3% 13.7% ATCC208 9 1 .3% 3.9% 7.6% 23.5% 0.5% 22.1% 20.2% 30.2% 27.4% S44 -4% 2.3% 15.0% 18.4% 0.7% 43.8% 5.5% 59.4% 20.7% U30 1% 7.8% 3-1% 12.7% 1.0% 14.9% 38.3% 19.0% 20.5% 59A 11% 2.3% 6.9% 9.1% 0.8% 52.2% 10.6% 59.9% 11-4% U37-2 .8^ 3.9% 8.8% 11.6% 1.2% 53.3% 5.5% 63.3% 15.51 S50W -7% 3.8% 6.3% 6.9% 0.6% 43.0% 15-6% 50.0% 10.7% ATCC20891 .0% 0.0% 0-0% 0.0% 0.0% 0.0% 90.0% 0.0% 0.0% Ux .6% 6.3% 11.9% 13.3% 1.7% 43.0% 7.31 56.6% 19.5% LW9 .3% 2.3% 8.4% 11.4% 0.7% 53.6% 6.5% 62.6% 13.6% C32-2 .8% 1.2% 6.41 2.5% 1.9% 34.4% 29-8% 42.6% 3-7% 5A-1 .1% 5.21 11.1% 7.6% 2.2% 27.2% 29.6% 40.4% 12.9% BG1 .4% 2.2% 9.6% 7.0% 1.1% 46.0% 8.8% 56.7% 9.1% U3 .9% 12.0% 6.6% 16.2% 0.4% 25.1% 22.8% 32.1% 28.2% 558 .3% 2.6% 8.6% 2.0% 2.5% 32.4% 25.5% 43.5% 4.6% 18A .4% 0.3% 9.8% 0.0% 0.3i 38.4% 31.7% 48.6% 0.3% 328 .0% 16.7% 8.6% 18.4% 0.0% 22.5% 17.7% 31.1% 35.1% 56B .6% 7.7% 11.4% 3.6% 4.3% 31.7% 22.7% 47.4% 11.2% SX2 .8% 4.4% 16.2% 6.4% 3.7% 33.6% 17.8% 53.5% 10.9% 538 .8% 2.7% 13.81 20.5% 1-41 39.3% 5.5% 54.4% 23.3% 549 .8% 8.0% 8.9% 6.4% 1.7% 33.9% 20.3% 44.5% 14.4% S3 .8% 0.3% 3.7% 3.9% 0.0% 69.9% 7.4w 73.6% 4.2% 3A-1 -11 5.2% 12.7% 3.27 0.9% 20.9% 29.0% 34.51 8.4% 15A -9% 0.7% 8.51 1.0% 0.01 35.8% 33.0% 44.3% 1.7X 9A-1 .71 10-21 8.8% 13.4% 1.5% 23.9% 26.3% 34.3% 23.7% 51B -21 11.2% 7.8% 16.4% 1.51 20.4% 26.5 29.7I 27.6% 8A-1 -5% 5.51 8.6% 4.8% 2.7% 28.7% 29.2% 40.01 10.3% 13A-1 .11 13.61 11.1% 16.0% 0.0% 28.4% 14.8% 39.4% 29.6% 24B-2 -91 7.31 16-4% 6.1% 0.01 40.8% 12.41 57.2% 13.4% 248-1 -2% 0.0% 10-91 1.0% 0.01 56.5% 15.5I 67.4% 1.0% 3B -01 0.0% 5.0% 2.3% 0.0% 73.4% 2.3I 78.3% 2.3% SBG5 -81 4.5I 5.8% 3.8% 1.0% 22.7% 41.3; 29.5 8-4% 16B .0% 14.0% 8.3% 18.9% 0.7% 23.9% 15-2% 32.91 32.9% 6A-1 .0% 0.31 10.1% 0.0% 0.0% 48.9% 22.7% 59.01 0.3% 33B PER CENT OF TOTAL FATTY ACIDS Total Total Strain C16:0 C2O:4w6 C20:5w3 C22:5w6 C22:5w3 C22:6w3 Other FA Omega3 Omega6 16.7% 5.51 14.8% 8,51 1.7% 31-81 21.0% 48.3% 13.9% 840 15.0% 1.01 11-7% 2.1% 0.9% 62.3% 6.9% 74.9% 3.1% 28A 17.8% 18.5% 8.11 20.5% 0.01 22.1% 12.9% 30.2% 39.0% 43B 16.9% 0.0% 3-4% 2-71 0.01 61.2% 15.8% :64.6% 2.7% 1A-I 15.6% 2.7% 11.4% 10-91 0.8% 53.71 4.9% 65.9% 13.6% U41-2 16.5% 0-7% 3.9% 3.9% 0.-0. 68.4% 6.7% 72.2% 4.6% 568 14-4% 0.9% 10.9% 2-5% 1.0% 66.41 3.8% 78.3% 3.41 46A 17.6% 0.0% 2.4% 3.3% 0.0% 66.31 10.4% 68.7% 3.3I 15A-1 25.0% 0.0% 3.3% 0.0% 1.4% 53.2; 17.1% 57.9% 0.0% 13A 16.1% 13.4% 9.3% 13-6% 0.0% 32.3% 15.3% 41.6% 27.0% 378 16.5% 9.1% 13.2% 6.7% 0.0% 38.9% 15.6% 52.1% 15.9% 438 16.1% 12.4% 12.0% 15.7% 0.8% 30-5% 12.5% 43.3% 28.1% 178 13.8% 0.8% 11.-51 2-8% 0.0% 67.0% 4.11 78.6% 3.6% 27A 17.51 18.6% 9.0% 19.5% 0.0% 21.7% 13.7% 30.7% 38.1% 468 21.4% 1.4% 18.9% 0.0% 5-0% 43.5% 9-91 67.3% 1.4% ATCC20890 17-7% 0.0% 0.61 4.41 0.0% 68.2% 9.1% 68.8% 4-4% 5A 17-.6% 16.0% 9.6% 18.8% 0.0% 25.6% 12.4% 35.2% 34.8% 288-2 14.0% 0-91 13-2% 1.6% 0.0% 64.7% 5.5% 77.9x 2.6% 278 19.5% 2.9% 16.6% 1I.I 1.6% 30.2% 28.1% 48.5% 4.0% 498 17.2% 0.7% 6.8% 2.7% 0.0% 63.0% 9.6% 69.8% 3.4% 188 14.4% 3.5% 13.5% 26.0% 1.0% 37-2% 4-41 51.6% 29.51 549-2 16.1% 2.2% 15.7% 21.6% 0.0% 36.7% 7.8% 52.4% 23.7% 208 17.3% 4.7% 14.3% 7.21 2.9% 30.2% 23.5% 47.3% 11.9% 88 11.5% 3.3% 11.3% 6.51 1.1% 59.9% 6.5% 72.2% 9.8% 138 16.6% 0.7% 10.7% 1.61 0.01 59.7% 10.8% 70.4% 2.2% 26A 16.1% 3.31 13.5% 23-8% 0.0% 38.7% 4.7% 52.2% 27.1% 542 15.6% 0.6% 12.11 0.01 0.0% 60.2% 11.5% 72.3% 0.6% 358 19.5% 0.0% 1.4% 3.4% 0.1 66.6% 9.1% 68.0% 3.4% 42A 18.9% 3.5% 12.7% 25.0% 0.0% 35.0% 5.0% 47.6% 28.5% 40A 25.2% 3.31 9.3% 21.8% 0.01 30.3% 10.1% 39.6% 25.1% SSOC 17.6% 11.1% 13.2% 14.1% 1.3% 28.7% 14.01 43.2% 25.2% 59A 19-9% 0.0% 5.5% 1.9% 0.0% 66.8% 6.0% 72.3% 1.9% SBG9 15.4% 3.1% 13.2% 26.1% 0-01 35.8% 6.5% 49.1% 29.11 218 18.9% 0.7% 11.6% 0.0% 0.0% 59.1% 9.7% 70.7% 0.7% 28 14.1% 1-1% 12.4% 2.0% 0.0% 65.2% 5.2% 77.6% 3.11 18 22.21 16.2% 6.31 17-7% 0.0% 18.1% 19.5% 24.4% 33.8% 558 16.01 1.01 4.51 0.0% 0.01 69.5% 9.01 74.0% 1.0% 3A 17.0% 4.31 12.4% 29.81 0.0% 34.0% 2.5% 46.41 34.1% 98 15-4% 4.31 8.71 13.2% 0.0% 53.2% 5-11 62.0% 17.51 U24 14.2 3-1% 12.0% 20.0% 1.1% 35.2% 14.31 48.3% 23-21 U28 16.8% 14.6% 10.11 16.01 0.6% 27.7% 14.0% 38.5% 30.7% 288-1 23.2% 1-9% 8.3% 1.11 2.3% 22.7% 40-4% 33.3% 3.01 44B 24.6% 15.8% 8.7% 16.0% 0.01 15.3% 19.6% 24.0% 31.8% 548 15-5% 0.01 1.3% 2.91 0.0% 72.7% 7.6% 74.0% 2.9% 55A 18-4% 1-01 5.01 3.01 0.01 66.2% 6.41 71.3% 3.91 49A 18-61 15.3% 9.4% 18.0% 0.0% 27.3 11.41 36.7% 33.31 51A 23.5% 13.11 7.3% 17.9% 0.0% 26.7% 11.4% 34-0% 31.0% 14A-1 13.31 1.1% 14.5% 0-91 0.0% 64.61 5.61 79-1% 2.01 25B 22.9% 2.4% 10-31 21.51 0.01 26.5% +6.4I 36.9% 23.9 41A 16.8% 1.01 9.7% 2.71 0.0% 58.31 11.5% 68.0% 3.7% 24A 0.41 8.5% 14.1% 10.2% 2.11 27.6% 37.0% 43.8% 18.8% 61A PER CENT OF TOTAL FATTY ACIDS Total Total Strain .6:0 C20:4w6 C20:5w3 C22:5w6 C22:5w3 C22:6w3 Other FA Omega3 Omega6 1.5% 0.0% 7.11 0-01 0.01 0.6% 61.8% 7.7% 0.0% BRBG .2% 14.9% 8.3% 18.7% 0.0% 24-4% 15.5% 32.7% 33.6% 17A .4% 2.0% 9.3% 2-8% 0.0% 55.7% 12.7% 65.0% 4.9% 60A .1% 0-8% 13.0% 1.2% 0.0% 67.8% 3.1% 80.8% - 2.01 268 .8% 5.0% 6.9% 15.0% 1.5% 47.4% 6.4% 55.8% 20.0% ATCC208 88 .0% 0.0% 1.8% 2.0% 0.0% 70.8% 9.4% 72.6% 2.0% 2A .6% 0.0% 4.0% 0.0% 0.0% 49.4% 22.0% 53.4% 0.0% 44A .4% 1.8% 0.0% 2.9% 0.0% 55.3% 23.3% 55.3% 4.6% 14A .3% 1.3% 4.6% 0.0% 0.0% 12.6% 58.11 17.3% 1.31 418 .3% 0.0% 1.1% 3.8% 0.0% 66.6% 9.1% 67.8% 3.8% 66A .6% 15.6% 8-3% 17.1% 1.1% 24.6% 14.8% 33.9% 32.7% 11A .6% 5-1% 10-1% 27.2% 0.0% 27.5% 10.6% 37.51 32.3% 2X .7% 2.4% 14.0% 25.7% 0.0I 36.7% 5-4% 50.8% 28.1% 33A .6% 1.5% 13-51 0-0% 0.0% 66.0% 4-3% 79.5% 1.5% ATCC2089 2 PRIOR STRAINS PER CENT OF TOTAL FATTY ACIDS Total Total Strain :0 C20:4w6 C20:5w3 C22:5w6 C22:5w3 C22:6w3 Other FA Omega3 Omega6 7% 3.9% 3.7% 8.1% 0.0% , 55.1% 13.5% 58.8% 12.0% ATCC3434 21 1.6% 6.9% 11.4% 0.0% 17.8% 34.1% 24.7% 12.9% ATCC24473 Z% 2.9% 7.7% 9.8% 0.6% 54.6% 9.2% 62.9% 12.7% ATCC28211 ? 10.71 4.3% 12.6% 1.5% 20.6% 27.% 26.4% 23.4% ATCC28210 6-3% 6-9% 4-3% 0.0% 60-1%. 9..1% 67.0% 10.6% ATCC28210 Krj en n Ln Ln -- en e 1 n UN J C'J en T 0 O - 1 en M~ rCDe Lfl4 -4Cen I-- C)r M~- CM C7 fn-C C)jf men 2: 2- u) r4 -; 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L < D: " Ab.4 b - A 0 : CI-LCiI C)C C ) oC < - c)0 - r c; C o % 0) C) - mm Mc o M'. C)C1MCD D m -0 LA LL H H C)~~~~ z r.0 c-. - ' n%.0 LU ~ ~ ~ ~ ~ ~ ~ L r H H Hi C- Kr 0 m) CD WN 0: C)C M.0o T r14 - C uLi U' (NJ .- M C0 L '.0 M m M %0 %.0 ko C H HHD "0 C 22 222 ll .:r 41 1 U' ra-- c-i (J r c'i C-i~L r) C.j Ln c-i r4 - TABLE 3: COMPOSITION OF OMEGA 3 FATTY ACID FRACTION EPA OPA DHA Strain CZO:5w3 C22:5w3 C22:6w3 44.0% 1.1% 54.9% 21 4.6% 0.9% 94.5% ATCC20889 19.31 0.7% 80.0% U40-2 31.9% 0.0% 68.1% 218 87.9% 0.0%~-. 12.1% BRBG1 12.5% 6.1% 81.5% 56A 17.0% 3.7% 79.3% 11A-1 24.9% 4.3% 70.8% 4A-1 24.4% 8.4% 67.2% 17B 12.2% 1.5% 86-3% ATCC20891 25.1% 1.7% 73.2% S44 25.2% 1.1% 73.7% U30 16.21 5.4% 78.4% 59A 11-5% 1.4% 87.1% U37-2 14.0% 1.9% 84.2% 55OW 12.7% 1.3% 86.01 ATCC20891 --- --- --- Ux 21.0% 2.9% 76-1% LWN9 13.4% 1.0% 85.6% C32-2 15.0% 4.3% 80.7% SA-1 27.4% 5.4% *67.2% BRBGl 17.0% 1.9% 81.1% U3 20.5% 1.3% 78.2% 55B 19.8% 5.8% 74.4% 18A 20.1% 0.7% 79.2% 32B 27.8% 0.0% 72.2% 56B 24.1% 9.1% 66.9% SX2 30.3% 6.9% 62.8% 538 25.3% 2.5% 72.2% S49 19.9% 3.8% 76.31 53 5.0% 0.0% 95.0% 3A-1 36.9% 2.6% 60-5% ISA 19.3% 0.0% 80.71 9A-1 25.8% 4.41 69.8% 518 26.3% 5.01 68-71 8A-1 21.6% 6.7% 71-71 13A-1 28.0% 0.0% 72.0% 248-2 28.7% 0.0% 71.31 248-1 16.2% 0.0% 83-8% 3B 6.3% 0.0% 93.7% SBG5 19.7% 3.3% 77.0% 16B 25.Z% 2.1% 72.61 6A-1 17.1% 0.0% 82.91 33B 30.5% 3.6% 65.91 840 15.6% 1.2% 83.1% 28A EPA OPA DHA Strain C20:5w3 C22:5w3 C22:6w3 26.8% 0.0% 73.2% 438 5.2% 0.0% 94.8% 1A-1 17.4% 1.2% 81.5% U41-2 5.4% 0.0% 94.6% 568 13.9% 1.3% 84.81 46A 3.5% 0.0% 96.5% 15A-1 5.8% 2.4% 91.8% 13A 22.3% 0.0% 77.7% 378 25.4% 0.0% 74&6% 43B 27.7% 1.9% 70.3% 178 14.7% 0.0% 85.3% '27A 29.2% 0.0% 70.8% 468 28.0% 7.5% 64.5% ATCC20890 0.9% 0.0% 99.1% 5A 27.3% 0.0% 72-7% 288-2 16.9% 0.0% 83.1% 27B 34.3% 3.4% 62.3% 498 9.7% 0.0% 90.3% 188 26.1% 1.9% 71.9% S49-2 29.9% 0.0% 70.1% 208 30.1% 6.2% 63.7% 8B 15.6% 1.5% 82.9% 13B 15.2% 0.0% 84.8% 26A 25.9% 0.0% -74.1% S42 16.7% 0.0% 83.3% 35B 2.1% 0.0% 97.9% 42A 26.6% 00% 73.4% 40A 23.4% 0.0% 76.6% S5OC 30.6% 2-9% 66.4% 59A 7.6% 0.0% 92.4% SBG9 27.0% 0.0% 73.0% 218 16.4% 0.0% 83.6% 28 15.9% 0.0% 84.1% 18 25.9% 0.0% 74.1% 558 6.0% 0.0% 94.0% 3A 26.7% 0.0% 73.3% 98 14.1% 0.0% 85.9% U24 24.9% 2-2% 72.9% U28 26.4% 1-5% 72.1% 28B-1 24.8% 6.9% 68-3% 448 36.4% 0.0% 63.6% 548 1.8% 0.0% 98.2% 55A 7.1% 0.0% 92.9% 49A 25.6% 0.0% 74.41 51A 21.5% 0.0% 78.5% 14A-1 18.4% 0.0% 81.6% 258 28.1% 0.0% 71.9% 41A 1-4--3% 0.01 85.7% 24A 32.3% 4.8% 63.0% 61A 91.6% 0.0% 8.4% BRBG EPA DPA DHA Strain C20:5w3 C22:5w3 C22:6w3 25.5% 0.0% 74.5% 17A 14.4% 0.0% 85.6% 60A 16.1% 0.0% 83.9% 268 12-4% . 2.7% 84.9% -ATCC20888 2.5% 0.0% 97.5% 2A 7.5% 0.0% 92.5% 44A 0.0% 0.0% 100.0% 14A 26.7% 0.0% 73.3% 418 1.7% 0.0% 98.3% 66A 24.5% 3.1% 72.4% 11A 26.8% 0.0% 73.2% 2X 27.6% 0.0% 72.4% 33A 17.0% 0.0% 83.0% ATCC20892 PRIOR STRAINS EPA OPA DHA Strain C20:5w3 C22:5w3 C22:6w3 6.4% 0.0% 93.6% ATCC34304 27.9% 0.0% 72.1% ATCC24473 12.2% 1.0% 86.8% ATCC28211 16.4% 5.6% 78.1% ATCC28209 10.3% 0.0% 89.7% ATCC28210 Fig. 1 illustrates the set of strains, isolated by the method in Example 1, that have more than 67% omega-3 fatty acids (as % of total fatty acids) and less than 10.6% omega-6 fatty acids (as % of total fatty acids). All of the 5 previously known strains had less than 67% omega-3 fatty acids (as % of total fatty acids) and greater than 10.6% omega-6 (as % of total fatty acids). Fig. 2 illustrates the set of strains, isolated by the method in Example 1, that have more than 67% omega-3 fatty 10 acids (as % of total fatty acids) and greater than 7.5% C20:5n-3 (as % of total fatty acids). All of the previously known strains had less than 67% omega-3 fatty acids (as % of total fatty acids) and less than 7.8% C20:5n-3 (as % of total fatty acids). 15 Example 5. Enhanced Growth Rates of Strains Isolated by Method in Example 1 Compared to ATCC Strains (Previously Known Strains) Cells of Schizochytrium sp. S31 (ATCC No. 20888), 20 Schizochytrium sp. S8 (ATCC No. 20889), Thraustochytrium sp. 542, Thraustochytrium sp. U42-2, Thraustochytrium sp. S42 and U30, (all isolated by the method of Example 1) and Thraustochytrium aureum (ATCC #28211) and Schizochytrium aggregatum (ATCC 428209) (previously known strains) were 25 picked from' solid F-1 medium and placed into 50ml of M-5 medium. The pH of the solution was adjusted to 7.0 and the solution was filter sterilized. After three days of growth on an orbital. shaker (200 rpm, 27'C), 1-2ml of each culture was transferred to another flask of M-5 medium and placed on the shaker for 2 days. The cultures (1-2ml) were then transferred to another flask of M-5 medium and placed on the shaker for 1 day. This process ensured that all cultures were in the exponential phase- of growth. These~later cultures 5 were then used to inoculate two 250ml flasks of M-5 medium for each strain. These flasks were than placed on shakers at 25'C and 30'C, and changes in their optical density were monitored on a Beckman DB-G spectrophotometer (660nm, 1cm path length). Optical density readings were taken at the 10 following times: 0, 6, 10, 14, 17.25, 20.25 and 22.75 hours. Exponential growth rates (doublings/day) were then calculated from the optical density data by the method of Sorokin (1973). The results are presented in Table 4 and illustrated (normalized to the growth of strain U30 at 25'C) in Fig. 4. 15 The data indicate that the strains isolated by the method in Example 1 have much higher growth rates than the previously known ATCC strains at both 25'C and 30'C, even under the optimized phosphate levels essential for continuous growth. Strains of Thraustochytriales isolated from cold 20 Antarctic waters have not been shown to grow at 30'C.

Table 4. Exponential Growth Rate (doublings/day) Strain 25'C 30*C 5 S31* (ATCC No. 20888) 8.5 9.4 U40-2* 5.8 6.0 10 S8* (ATCC No. 20889) 7.1 8.8 S42* 6.6 8.3 U30* 5.5 7.3 15 28209** 4.6 5.0 28210** 3.5 4.5 20 28211** 4.2 5.7 34304** 2.7 3.7 25 24473** 4.6 5.3 strain isolated by method in Example 1 30 ** previously known ATCC strain Example 6. Enhanced Production Characteristics (Growth and Lipid Induction) of Strains Isolated by Method in Example 35 1 Compared to ATCC Strains (Prior Art Strains) Cells of Schizochytrium sp. S31 (ATCC No. 20888), Schizochytrium sp. S8 (ATCC No. 20889) (both isolated by the method of Example 1) and Thraustochytrium aureum (ATCC #28211) and Schizochytrium aggregatum (ATCC #28209) (prior 40 art strains) were picked from solid F-1 medium and placed into 50ml of M-5 medium (see Example 3). The pH of the solution was adjusted to 7.0 and the solution was filter sterilized. After three days of growth on an orbital shaker (200 rpm, 27'C), 1-2ml of each culture was transferred to 45 another flask of M-5 medium and placed on the shaker for 2 days. The ash-free dry weights for each of these cultures were then quickly determined and then 3.29mg of each culture was pipetted into two 250ml erlenmeyer flasks containing 50ml of M-5 medium. These flasks were placed on-a rotary shaker 5 (200 rpm, 27*C). After 24 hours 20ml portions of each culture were then centrifuged, the supernatants discarded, and the cells transferred to 250ml erlenmeyer flasks containing 50 ml of M-5 medium without any glutamate (N source). The flasks were placed back on the shaker, and 10 after another 12 hours they were sampled to determine ash free dry weights and quantify fatty acid contents by the method of Lepage and Roy (1984) . The results are illustrated (normalized to the yields of ATCC No. 28211, previously known strain) in Fig. 5. The results indicate that the strains 15 isolated by the method of Example 1 produced 2-3 times as much ash-free dry weight in the same period of time, under a combination of exponential growth and nitrogen limitation (for lipid induction) as the prior art ATCC strains. In addition, higher yields of total fatty acids and omega-3 20 fatty acids were obtained from strains of the present invention with strains S31 (ATCC No. 20888) producing 3-4 times as much omega-3 fatty acids as the prior art ATCC strains. 25 Example 7. Enhanced Lower Salinity Tolerance and Fatty Acid Production by Strains Isolated by Method in Example 1 Strains of 4 species of Thraustochytrids, Schizochytrium sp. S31 (ATCC No. 20888) and Thraustochytrium sp. U42-2 (ATCC No. 20891) (both isolated and screened by the method of Example 1) , and S. aggregatum (ATCC 28209) and T. aureum (ATCC 28210) ' (obtained from the American Type Culture Collection) were picked from solid F-1 medium and incubated for 3-4 days at 27"C on a rotary shaker (200 rpm). A range 5 of differing salinity medium was prepared by making the following dilutions of M medium salts (NaCl, 25g/l; MgSO 4 -7H 2 0, 5g/l; KCl, lg/l; CaCl 2 , 200mg/l: 1) 100% (w/v m medium salts; 2) 80% (v/v) M medium, 20% (v/v) distilled water; 3) 60% (v/v) M medium, 40% (v/v) distilled water; 4) 10 40% (v/v) M medium, 60% (v/v) distilled water; 5) 20% (v/v) M medium, 80% distilled water; 6) 15% (v/v) M medium, 85% (v/v) distilled water; 7) 10% (v/v) M medium, 90% (v/v) distilled water; 8) 7% (v/v) M medium, 93% (v/v) distilled water; 9) 3% (v/v) M medium, 97% (v/v) distilled water; 10) 15 1.5% (v/v) M medium, 98.5% (v/v) distilled water. The following nutrients were added to the treatments (per liter): glucose, 5g; glutamate, 5g; yeast ext., lg; (NH 4 )2SO 4 , 200 mg; NaHCO , 200 mg; PII metals, 5ml; A-vitamins solution, lml; and antibiotics solution, 2ml. Fifty ml of each of 20 these treatments were inoculated with lml of the cells growing in the F-1 medium. These cultures were placed on an orbital shaker (200 rpm) and maintained at 27'C for 48 hr. The cells were harvested by centrifugation and total fatty acids determined by gas chromatography. The results 25 are illustrated in Fig. 6. Thraustochytrium sp. U42-2 (ATCC No. 20891) isolated by the method of Example 1 can yield almost twice the amount of fatty acids produced by T. aureum (ATCC 28211) and over 8 times the amount of fatty acids produced by S. aggregatum (ATCC 28209). Additionally, U42-2 appears to have a wider salinity tolerance at the-upper end of the salinity range evaluated. Schizochytrium sp. S31 (ATCC No. 20888), also isolated by the method in Example 1, 5 exhibited both a high fatty acid yield (2.5 to 10 times that of the previously known ATCC strains) and a much wider range of salinity tolerance than the ATCC strains. Additionally, Schizochytrium sp. S31 (ATCC No. 20888) grows best at very low salinities. This property provides a strong economic LO advantage when considering commercial production, both because of the corrosive effects of saline waters on metal reactors, and because of problems associated with the disposal of saline waters. .5 Example 8. Cultivation/Low Salinity Fifty ml of M/10-5 culture media in a 250ml erlenmeyer flask was inoculated with a colony of Schizochytrium sp. S31 (ATCC No. 20888) picked from an agar slant. The M/10-5 media contains: 1000ml deionized water, 2.5g NaCl, 0.5g MgSO 4 7H 2 0, !0 0.1g KC1, 0.02g CaCl 2 , 1.0g KH 2

PO

4 , I.Og yeast extract, 5.0g glucose, 5.0g glutamic acids, 0.2g NaHCO 3 , 5ml PII trace metals, 2ml vitamin mix, and 2ml antibiotic mix. The culture was incubated at 30'C on a rotary shaker (200 rpm). After 2 days the culture was at a moderate density and actively 25 growing. 20ml of this actively growing culture was used to inoculate a 2 liter fermenter containing 1700ml of the same culture media except the concentration of the glucose and glutamate had been increased to 40g/l (M/10-40 media). The fermenter was maintained at 30*C, with aeration at 1 vol/vol/min, and mixing at 300 rpm. After 48 hr, the concentration of cells in the fermenter was 21.7g/l. The cells were harvested by centrifugation, lyophilized, and 5 stored under

N

2 . The total fatty acid content and omega.-3 fatty acid content was determined by gas chromatography. The total fatty acid content of the final product was 39.0% ash-free dry weight. The omega-3 HUFA content (C20:5n-3, C22:5n-3 10 and C22:6n-3) of the microbial product was 25.6% of the total fatty acid content. The ash content of the sample was 7.0%. Example 9. Diversity of Fatty Acid Content Growth and gas chromatographic analysis of fatty acid 15 production by various strains as described in Example 4 revealed differences in fatty acid diversity. Strains of the present invention synthesized fewer different fatty acids than previously available strains. Lower diversity of fatty acids is advantageous in fatty acid purification since there 20 are fewer impurities to be separated. For food supplement purposes, fewer different fatty acids is advantageous because the likelihood of ingesting unwanted fatty acids is reduced. Table 5 shows the number of different HUFAs present, at concentrations greater than 1% by weight of total fatty acids 25 for previously known strains, designated by ATCC number and various strains of the present invention.

Table 5. No. of Different Fatty Acids at 1% or Greater 5 Strain % of Total Fatty Acids 34304** 8 10 28211** 8 24473** 10 28209** 13 15 28210** 8 S31* 5 20 S8* 6 79B* 6 25 strain isolated by the method in Example 1 ** previously known ATCC strain 30 Example 10. Recovery Fifty ml of M5 culture media in a 250 ml erlenmeyer flask was inoculated with a colony of Schizochytrium sp. S31 (ATCC No. 20888) picked from an agar slant. The culture was incubated at 30*C on a rotary shaker (200 rpm). After 2 days 35 the culture was at a moderate density and actively growing. 20ml of this actively growing culture was used to inoculate a 1 liter fermenter containing 1000ml of the same culture media except the concentration of the glucose and glutamate had been increased to 40g/l (M20 media) . The fermenter was 40 maintained at 30'C and pH 7.4, with-aeration at 1 vol/min, and mixing at 400 rpm. After 48 hr, the concentration of the cells in the fermenter was 18.5g/1. Aeration and mixing in the fermenter was turned off. Within 2-4 minutes, the cells flocculated and settled in the bottom 250 ml of the fermenter. This concentrated zone of cells had a cell concentration of 72g/l. This zone of cells can be siphoned from the fermenter, and: (1) transferred to another reactor 5 for a period of nitrogen limitation (e.g., combining the highly concentrated production of several fermenters) ; or (2) harvested directly by centrifugation or filtration. By preconcentrating the cells in this manner, 60-80% less water has to be processed to recover the cells. 10 Example 11. Utilization of a Variety of Carbon and Nitrogen Sources Fifty ml of M5 culture media in a 250ml erlenmeyer flask was inoculated with a colony of Schizochytrium sp. S31 (ATCC 15 No. 20888) or Thraustochytrium sp. U42-2 (ATCC No. 20891) picked from an agar slant. The M5 media was described in Example 3 except for the addition of 2ml vitamin mix, and 2ml antibiotic mix. The culture was incubated at 30'C on a rotary shaker (200 rpm). After 2' days the culture was at 20 a moderate density and actively growing. This culture was used to inoculate flasks of M5 media with one of the following substituted for the glucose (at 5g/l): dextrin, sorbitol, fructose, lactose, maltose, sucrose, corn starch, wheat starch, potato starch, ground corn; or one of the 25 following substituted for the glutamate (at 5g/l): gelysate, peptone, tryptone, casein, corn steep liquor, urea, nitrate, ammonium, whey, or corn gluten meal. The cultures were incubated for 48 hours on a rotary shaker (200 rpm, 27'C).

The relative culture densities, representing growth on the different organic substrates, are illustrated in Tables 6-7. Table 6. Utilization of-Nitrogen Sources 5 N-Source Strains Thraustochytrium Schizochytrium sp. U42-2 sp. S31 10 ATCC No. 20891 ATCC No. 20888 glutamate +++ +++ 15 gelysate +++ +++ peptone ++ ++ tryptone ++ ++ 20 casein ++ ++ corn steep liquor +++ +++ 25 urea + ++ nitrate ++ 30 ammonium + whey +++ +++ corn gluten 35 meal +++ +++ = high growth ++ = medium growth 40 + = low growth 0 = no growth Table 7. Utilization of Organic Carbon Sources C-Source Strains 5 Thraustochytrium Schizochytrium sp. U42-2 sp. S31 ATCC No. 20891 ATCC No. 20888 10 glucose +++ +++ dextrin +++ +++ sorbitol + + 15 fructose + +++ lactose + + 20 maltose +++ + sucrose + + corn starch +++ + 25 wheat starch +++ + potato starch +++ + 30 ground corn +++ 0 +++ = high growth ++ = medium growth 35 + = low growth 0 = no growth Example 12. Feeding of Thraustochytrid-Based Feed Supplement 40 to Brine Shrimp to Increase Their Omega-3 HUFA Content Cellular biomass of Thraustochytrium sp. 12B (ATCC 20890) was produced in shake flasks in M-5 medium (see Example 3) at 25'C. Cellular biomass of Thraustochytrium sp. S31 (ATCC 20888) was produced in shake flasks in M/10-5 45 medium (see Example 8) at 27'C. The cells of each strain were harvested by centrifugation. The pellet was washed once with distilled water and recentrifuged to produce a 50% solids paste. The resulting paste was resuspended in sea water and then added to an adult brine shrimp culture as a feed supplement. The brine shrimp had previously been reared on agricultural waste products and as a result their omega-3 HUFA content was very low, only 1.3 - 2.3% of total fatty 5 acids (wild-caught brine shrimp have an average omega-3 HUFA content of 6 - 8% total fatty acids). The brine shrimp (2 3/mL) were held in a 1 liter beaker filled with sea water and an airstone was utilized to aerate and mix the culture. After addition of the feed supplement, samples of the brine LO shrimp were periodically harvested, washed, and their fatty acid content determined by gas chromatography. The results are illustrated in Figs. 7 and 8. When fed the thraustochytrid-based feed supplement as a finishing feed, the omega-3 content of the brine shrimp can be raised to that L5 of wild-type brine shrimp within 5 hours if fed strain 12B or within 11 hours when fed strain S31. The omega-3 HUFA content of the brine shrimp can be greatly enhanced over that of the wild type if fed these feed supplements for up to 24 hours. Additionally, these feed supplements greatly increase .0 the DHA content of the brine shrimp, which is generally only reported in trace levels in wild-caught brine shrimp. Example 13. Use of Sodium Sulfate in Culture Medium This example illustrates that omega-3 production and 25 total fatty acid content is not harmed and can be the same or better when using sodium sulfate instead of sodium chloride as the sodium salt in a fermentation medium.

Schizochytrium ATCC No. 20888 was grown in medium, pH 7.0, containing 2.36 grams of sodium per liter of medium, 1.5-3.0 grams of a nitrogen source per liter of medium, and 3.0 grams of glucose per- liter of medium. 'The cells were 5 incubated at 28'C, at 200 rotations per minute, for 48 hours. The results are shown in Table 8. Table 8. Effect of Sodium Sulfate Compared With Sodium Chloride on Fatty Acid Content 1.0 A) Na salt = sodium chloride; N source = sodium glutamate total biomass N source omega-3 fatty acid yield (g/L) (% dwt) (% dwt) (g/L) 3.0 6.0 11.2 1.74 2.5 5.8 10.8 1.71 0 2.0 5.8 11.0 1.65 1.5 7.5 20.3 1.39 B) Na salt sodium chloride; N source = peptone 5 total biomass N source omega-3 fatty acid yield (g/L) (% dwt) (% dwt) (g/L) 3.0 7.9 21.9 1.34 2.5 9.4 27.4 1.21 2.0 6.7 28.9 1.18 1.5 11.1 42.1 1.16 5 C) Na salt sodium sulfate; N source = sodium glutamate total biomass 3 N source omega-3 fatty acid yield (g/L) (% dwt) (% dwt) (g/L) 3.0 9.3 31.9 1.34 5 2.5 10.1 38.6 1.35 2.0 10.1 41.4 1.30 1.5 9.5 43.6 1.26 As seen in Table 8, omega-3 and total fatty acid production when using sodium sulfate is comparable to or better than when using sodium chloride as a sodium salt. 5 Example 14. Production of Schizochytrium in Low Sal inity Culture Medium This Example illustrates the fermentation of Schizochytrium in a low salinity culture medium while maintaining high biomass yields and high omega-3 and fatty LO acid production. Schizochytrium ATCC No. 20888 was grown in medium, containing 3 .33g/l of peptone as a nitrogen source, 5.0g/l of glucose as a carbon source, with varying sodium concentrations. The cells were fermented at 30'C with an 5 inoculum of about 40mg/L dwt for a period of 48 hours. The sodium was supplied as sodium chloride. The results of this run are shown in Table 9. Table 9. Production of Schizochytrium in Low Salinity Culture Medium Bnomass Fatty. final a conc. C conc. YieLd acids omega-3 glucose 5 9//L 9/1 % dwt X dwt g/L 4.88 7.12 1.76±0.60 35.4±1.0 10.2±0.6 0.00 3.90 5.70 1.72±0.67 37.0±0.7 11.1±0.3 0.15 2.93 4.27 1.70±0.42 43-0±0.2 12.1±0.1 0.22 0 1.95 2.85 1.66±0.57 29.8±0.7 9.3±0.1 1.55 0.98 1.42 0.40±0.61 10.6±2.4 4.0±1.0 4.31 As can be seen from the results in Table 9, high biomass 5 yields and production of omega-3 fatty acids and total fatty acids can be achieved at sodium concentrations of greater than about 1.0 g/l.

Example 15. Cultivation of SchizochYtrium in Medium with Low Chloride Cntent This Example illustrates the fermentation of microflora of the present invention at minimal chloride concentrations 5 while achieving high biomass yields based on starting sugar concentration. Schizochytrium ATCC No. 20888 was cultured in shake flasks at 200 rpm and 28'C in 50ml aliquots of the following medium. 1000ml deionized water; 1.2g Mg S04.7H20; 0.

0 67g 0 CaCO 3 ; 3.0q glucose; 3.0g monosodium glutamate; 0.2g

KH

2

PO

4 ; 0.4g yeast extract; 5.Oml PII metals, 1.0 vitamin mix; and O.lg each of penicillin-G and streptomycin sulfate. The chloride concentration was varied by adding differing amounts of KCl to each treatment. The potassium concentration in all of the treatments was held constant by additions of potassium citrate. Sodium concentration was either 2 .37g/l or 4.0 g/l through addition of sodium sulfate. The results of these fermentations are shown below in Table 10. Table 10. Fermentation of Schizochytrium at Minimal Chloride Concentrations Nah 2.37 g/L Na 4.0 g/L Chloride conc. Biomass Yield Bioass Yield (mg/L) (mg/L) (mg/L) 0.1 198 ± 21 158 t 48 7.1 545 ± 120 394 + 151 15.1 975± 21 758 163 30.1 1140 ± 99 930 ± 64 59.1 1713 ± 18 1650 ± 14 119.1 1863 ± 53 1663 ± 46 238.1 1913 ± 11 1643 ± 39 As can be seen from the results shown in Table 10, high yields of biomass per sugar can be achieved at low chloride concentrations. For example, at a chloride concentration of greater than 59.lmg/L, yields of greater than 50% are 5 achieved. Example 16. Variation of Sodium Sulfate Concentration at Low Chloride Concentrations This Example illustrates the effect of varying sodium 10 sulfate concentration in a fermentation at low chloride concentration. Schizochytrium ATC 20888 was cultured in shake flasks at 200 rpm and 28*C in 50 ml aliquots of the following medium: 1000ml deionized water; 1.2g MgSO 4 .7H 2 0; 0.125g KCl; 15 0.067g CaCO 3 ; 3.Og glucose; 3.Og monosodium glutamate; 0.2g KH2PO 4 ; 0.4g yeast extract; 5.Oml PII metals; 1.0ml vitamin mix; and 0.lg each of penicillin-G and streptomycin sulfate. The sodium sulfate concentration was varied in the treatments from 3.0g/1 to 30.2g/l. The results of the fermentation runs 20 are shown below in Table 11. Table 11. Variation of Sodium Sulfate Concentration at Low Chloride Content 25 Sodium Sulfate Biomass yield (g/1) (g/1) 3.0 0.78 30 6.0 1.13 9.1 1.72 12.1 1.88 15.1 1.89 22.7 1.91 35 30.2 1.63 The results shown in Table 11, illustrate that at a low chloride concentration of about 59g/l, high biomass yields from glucose of greater than 50% can be obtained by selection of an appropriate sodium sulfate concentration.

Claims (18)

1. A process for culturing microorganism of the order Thraustochytriales in a low chloride medium, the process comprising: culturing microorganism of the order Thraustochytriales in a culture medium having a non-chloride source of sodium, having a chloride concentration of less than 3 grams of chloride per litre of said culture medium, and having a sodium concentration between 1 g/L and 50 g/L, wherein less than 50% of the sodium in the fermentation medium is supplied as sodium chloride.

2. The process of claim 1, wherein the lipids comprise at least one highly unsaturated fatty acid (HUFA).

3. The process of claim 2, wherein the at least one HUFA is selected from the group consisting of DHA, EPA, DPA(n-6), and mixtures thereof.

4. The process of claim 3, wherein the at least one HUFA comprises DHA.

5. The process of claim 1, wherein the non-chloride source of sodium is in an amount less than 25 g/L.

6. The process of any one of claims 1 to 5, wherein less than 25% of the sodium in the fermentation medium is supplied as sodium chloride.

7. The process of any one of claims 1 to 6, wherein the microorganism produces about 12% dwt. or less of omega-3 fatty acids. 19/11/07 DI

8. The process of any one of claims 1 to 7, wherein the microorganism replicates by successive bipartition.

9. The process of any one of claims 1 to 7, wherein the microorganism replicates by forming sporangia and releasing zoospores.

10. The process of any one of claims 1 to 9, wherein the microorganism is Thraustochytrium, Schizochytrium or mixtures thereof

11. The process of any one of claims 1 to 10, comprising culturing the microorganism at a temperature from about 50C to about 480C and a pH from about pH 5.0 to about pH 11.0.

12. The process of any one of claims 1 to 11, wherein the chloride concentration is less than 1 gram of chloride per litre of said culture medium.

13. The process of any one of claims 1 to 11, wherein the chloride concentration is less than 500 mg of chloride per litre of said culture medium.

14. The process of any one of claims 1 to 11, wherein the chloride concentration is less than 250 mg of chloride per litre of said culture medium.

15. The process of any one of claims 1 to 11, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per litre of culture medium.

16. The process of any one of claims 1 to 15, wherein the non chloride source of sodium is sodium sulphate. 19/11/07

17. The process of any one of claims 1 to 16, wherein the concentration of sodium, expressed as grams of sodium per litre of said culture medium is between 2 g/L and 25 g/L.

18. The process of any one of claims 1 to 17, further comprising the step of recovering fatty acids from the microorganism. Dated this 19 day of November 2007 Martek Biosciences Corporation Patent Attorneys for the Applicant PETER MAXWELL AND ASSOCIATES 19/11/07