Schizochytrium SP and uses thereof

Abstract

The present disclosure describes an isolated Schizochytrium sp. strain and uses thereof. Also disclosed are fermentation methods, methods of producing compositions of lipids comprising docosahexaenoic acid (DHA), as well as methods of producing compositions of lipids comprising odd chain fatty acids thereof using the isolated Schizochytrium sp. strain.

Description

SCHIZOCHYTRIUM SP AND USES THEREOFFIELD OF THE INVENTION

[0001] The present invention relates generally to the field of biotechnology. In particular, the present invention relates to isolated strains of Schizochytrium sp. and uses thereof in the production of fatty acids.DEPOSITION OF MICROORGANISM ACCORDING TO BUDAPEST TREATY

[0002] The Schizochytrium sp. strain PLU-D, as disclosed herein, has been deposited at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, under the accession number DSM34665 on 16 June 2023.BACKGROUND

[0003] Components of dietary supplements are traditionally either harvested or chemically synthesised. In the current climate, alternative sources of such dietary supplements are being sought after to replace traditional chemical syntheses. This is evident in the application of such alternative methods in synthesizing compounds such as medium-chain triglycerides, dicarboxylic acids, and structured lipids. There is a growing demand for omega-3 long-chain fatty acids, particularly docosahexaenoic acid (DHA, C22:6n) and eicosapentaenoic acid (EPA, C20:5n). These are increasingly being consumed as dietary supplements due to their recognized health benefits. In 2019, the global omega-3 fatty acid market was assessed at USD 2.49 billion, and the compound’s annual growth rate is currently projected to be 7% from year 2020 to 2027.

[0004] Odd chain fatty acids, including pentadecanoic acid and heptadacanoic acid, have recently gained attention as new, emerging bioactive lipids. These fatty acids have shown potential in mitigating several metabolic disorders, having been shown to have a positive impact on diabetes, enhancing insulin sensitivity and glucose regulation. Furthermore, these odd chain fatty acids have been shown to exhibit a protective role against fatty liver, contributing to its prevention and management. They have been implicated in having cardiovascular benefits, as they have been shown to reduce the risk of cardiovascular diseases.

[0005] Traditionally, the major source of these fatty acids, particularly DHA, is from marine fisheries, specifically fish oil from these marine fisheries, which are rich in EPA and DHA. However, there are concerns that the stable supply of fish oil may be disrupted from unsustainable fish farming, varied lipid composition and oil quality, and potential contamination from hazardous environmental pollutants.

[0006] Thus, there is an unmet need in providing oleaginous microbial strains and developing methods for the production of lipid compositions.SUMMARY

[0007] In one aspect, the present disclosure refers to a fermentation method of producing a composition of lipids using a Schizochytrium sp. strain deposited under accession no. DSM34665, wherein the method comprises culturing the Schizochytrium sp. strain to produce the composition of lipids.

[0008] In another aspect, the present disclosure refers to a composition of lipids obtained from the fermentation method as disclosed herein.

[0009] In another aspect, the present disclosure refers to an isolated strain of Schizochytrium sp., deposited under the accession no. of DSM34665.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0011] Fig. 1 shows the information pertaining to the mangrove samples disclosed herein. Fig. 1 A is a geographical map, showing the location of Pulau Ubin (arrow); Fig. IB shows pictures of the mangrove and seawater samples collected.

[0012] Fig. 2 is a plot showing exemplary growth of Schizochytrium sp. PLU-A, Schizochytrium sp. PLU-B, Schizochytrium sp. PLU-C, and Schizochytrium sp. PLU-D strains isolated from the Singapore mangrove habitat when cultured in glucose and yeast extract (GY) medium in shake flask.

[0013] Fig. 3 is an unrooted phylogenetic tree of the isolated Schizochytrium sp. PLU strains and other publicly available microalgae strains based on their 18S rRNA sequences. An unrooted phylogenetic tree is one that only shows the relatedness between organisms, as opposed to a rooted phylogenetic tree which also shown organism ancestry. The tree was constructed by neighbour -joining method in MEGA 11 software. A Bootstrap value of 500 was used for the analysis.

[0014] Fig. 4 comprises photomicrographs showing the morphology of Schizochytrium sp. PLU-D strain when cultured in glucose yeast extract (GY) medium for 3 days. Fig. 4A shows the Schizochytrium sp. PLU-D as vegetative cells; Fig. 4B shows Schizochytrium sp. PLU-D as vegetative cells and an amoeboid cell (dark arrow); Fig. 4C shows the release of zoospores from zoosporangium; Fig. 4D shows clusters of cells released from binary cell division of an octad (white arrow) and a decad (black arrow); Fig. 4E shows an amoeboid cell (black arrow) and a diad from binary cell division (white arrow). The scale bars in Fig. 4A, Fig. 4B, and Fig. 4C are 20 pm, and the scale bars in Fig. 4D and Fig. 4E are 10 pm.

[0015] Fig. 5 is a plot showing the change in biomass of Schizochytrium sp. PLU-D and the amount of lipid yield over a period of about 93 hours when the Schizochytrium sp. PLU-D strain was cultured in a 2-litre fermenter. The pH of the culture medium was controlled at pH 7.0 with 2 M NaOH and 0.5 M citric acid and the temperature was maintained at 28°C. In this example, the glucose batch phase of the claimed method can be considered to take place from 0 hours to about 45 hours of the timelineshown in this figure. For this example, the glucose fed-batch phase was initiated at about 45 hours, as indicated by the “initiate feeding” arrow.DETAILED DESCRIPTION

[0016] Docosahexaenoic acid (DHA, C22:6n) and odd chain fatty acids (OCFAs), such as but not limited to pentadecanoic acid and heptadecanoic acid, have garnered attention due to their potential in mitigating a spectrum of metabolic disorders.

[0017] DHA is an essential omega-3 fatty acid found in the human brain and eyes. It is known to play a role in the fetal development of the brain and retina during pregnancy and up to two years of infancy. Apart from infant development, the preventive effects of DHA on cardiovascular disease, a reduced risk of Alzheimer's disease and improved cognitive performance of Alzheimer’s patients with DHA consumption, as well as its ability to reduce cancer cell viability and suppress cell proliferation have been previously reported.

[0018] Fatty acids, particularly docosahexaenoic acid (DHA, C22:6n) are usually obtained from fish oil. However, fish are not able to synthesize fatty acids de novo, but obtain fatty acids from their food intake, which can include microorganism such as, but not limited to, marine microalgae. Examples of such oleaginous microbial strains can be, but are not limited to, Yarrowia lipolytica, Rhodotorula, Crypthecodinium conhii, and Schizochytrium sp.

[0019] Marine microalgae belonging to the Schizochytrium genus have the ability to produce high docosahexaenoic acid (DHA) content, amounting to about 26-55% of total fatty acids produced. There are two pathways used to synthesize DHA in marine microalgae strains, namely the aerobic elongase- desaturase pathway and the anaerobic polyketide-like synthase (PKS) pathway.

[0020] Saturated fatty acids, for example, either C16:0 or C18:0, are first synthesized in a stepwise manner by the canonical fatty acid synthetase (FAS) pathway, which is initiated by the condensation of a molecule of acetyl-CoA and a molecule of malonyl-CoA. In the aerobic elongase-desaturase pathway, DHA is then synthesized by modifying C16:0 or C18:0 fatty acids through a series of alternate elongation and desaturation reactions. These reactions are catalysed by different elongases and desaturases, whereby desaturases require molecular oxygen in order to introduce a double bond into the fatty acids.

[0021] In contrast, the synthesis of polyunsaturated fatty acids via the anaerobic polyketide-like synthase (PKS) pathway is a more efficient pathway, as it is independent of molecular oxygen and requires fewer Nicotinamide adenine dinucleotide (NAD(P)H; a reducing agent that functions as a coenzyme in metabolic pathways). In the PKS pathway, DHA synthesis starts with acetyl-CoA as a precursor, and then undergoes repeated cycles of condensation, ketoreduction, dehydration, and enoyl - reduction reactions, with the regulation of chain length factor protein to control the chain length.

[0022] Schizochytrium sp. strains typically synthesize DHA anaerobically, via the PKS pathway. This is reflected by their long-chain polyunsaturated fatty acid composition, the main components of whichare, but are not limited to, DHA and docosapentaenoic acid (DPA). This shows their suitability as microorganisms for high DHA production.

[0023] Singapore is an island country with several coastal mangrove habitats. However, there are limited reports on the isolation of novel marine microalgae strains from this location. In the present disclosure, the isolation and characterization of isolated strains of microalgae from mangrove samples collected from Pulau Ubin, Singapore are described. As described herein, four isolated strains of Schizochytrium sp. with high lipid productivity and DHA content were obtained.

[0024] In order to characterise the strains obtained as outlined in the present disclosure, cell morphological phenotypes were observed under a light microscope. The isolated microalgae strains were grown in glucose yeast extract (GY) medium at 25°C, 200 rpm. Morphologies of the microalgae isolates were characterized with respect to vegetative cells, sporangium, ectoplasmic net, zoospores, amoeboid cells, and binary divisions (see Fig. 4).

[0025] Phylogenentic analysis using 18S rRNA showed that the isolated Schizochytrium sp. strains appeared to be closely related to other Schizochytrium sp. strains, such as Schizochytrium sp. LY-2012 (JX847369.1), Schizochytrium sp. SH104 (KX379459.1), and Schizochytrium sp. ABC101 (MK265698). Thus, the four strains identified herein were identified to be previously unknown strains of Schizochytrium sp. The 18S rRNA sequence of the Schizochytrium sp. strains disclosed herein were designated as Schizochytrium sp. PLU-A (OP808218), Schizochytrium sp. PLU-B (OP808219), Schizochytrium sp. PLU-C (OP808220), and Schizochytrium sp. PLU-D (OP763747), respectively.

[0026] In the present disclosure, use of an isolated Schizochytrium sp. strain in the production of a composition of lipid is described. In one example, the isolated Schizochytrium sp. strain is, but not limited to, PLU-A, PLU-B, PLU-C and PLU-D. In one example, the Schizchytrium sp. is PLU-D. In another example, the isolated strain is a Schizochytrium sp., as deposited under the accession no. of DSM34665 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (DSMZ) on 16 June 2023.

[0027] As shown in the experimental section, after 84 hours of culturing the isolated PLU-D strains using the fermentation method as described herein, resulted in a biomass of about 175 g / 1 of dry cell weight, which corresponds to a lipid yield of about 93.6 g / 1 of lipids. Thus, in one example, the fermentation method of producing a composition of lipids using the Schizochytrium sp. strain as described herein, wherein the biomass of the isolated strain is increased from the start of culturing the Schizochytrium sp. strain up to about 84 hours.

[0028] Comparing to other fermentation studies with Schizochytrium sp. known in the art, high cell density fermentation of the Schizochytrium sp. G13 / 2S strain was reported to reach a biomass of up to about 65 g / 1 dry cell weight and a lipid yield of about 16 g / 1, while the culturing of the Schizochytrium sp. ABC101 strain isolated from Korea produced about 86 g / 1 of dry cell weight and a lipid yield of about 37.2 g / 1 under its optimized condition for about 84 hours . In contrast, the biomass and lipid yieldobtained within about 84 hours from fermentation method using Schizochytrium sp. PLU-D was higher than other strains disclosed herein.

[0029] In other words, comparing to other microalgae strains know in the art, culmring the four isolated Schizochytrium PLU strains disclosed herein resulted in a higher biomass under similar culture conditions. For example, the dry cell weight obtained from culturing the Schizochytrium sp. F26-b strain known in the art was only about 3.5 g / 1, which was lower than the biomass obtained from culturing the PLU strains under similar conditions. Further, when comparing to other microalgae strains isolated in the mangrove of Southeast Asia, the biomass obtained from culturing the cells of the PLU strains was also higher. For example, as known in the art, the biomass of Thraustochytrids sp. strains isolated from Thailand ranged from about 0.6 to about 3.1 g / 1 of dry cell weight when cultivated under shake-flask conditions. Similarly, the biomass of Thraustochytrids sp. strains isolated from Malaysian mangrove habitats were found to be in the range of about 1 to about 5.8 g / 1 of dry cell weight, while culmring the isolated Aurantiochytrium sp. AW1 strain from Malaysian coastal waters in a separate study obtained a biomass of about 13.17 g / 1 of dry cell weight. Thus, the biomass productivity of PLU-A, PLU-B, PLU-C and PLU-D strains was found to be higher than the isolated microalgae strains from Southeast Asia known in the art when cultured under similar shake-flask conditions.

[0030] As shown in the present disclosure, the four isolated strains of Schizochytrium sp. disclosed herein showed similar growth characteristics when cultured in glucose yeast extract (GY) medium (see Fig. 2). The dry cell weight of all four strains was between about 13 to about 14 g / 1 in GY medium when cultured for about 96 hours (see Table 1).

[0031] In one example, the biomass obtained using the methods disclosed herein ranges between less than 1 g / 1 to about 200 g / 1 dry cell weight. In another example, the biomass is between about 9 to about 15 g / 1, between about 10 to about 14 g / 1, between about 12 to about 15 g / 1, between about 13 to about 15 g / 1 dry cell weight, between about 10 to about 20 g / 1 dry cell weight, between about 15 to about 30 g / 1 dry cell weight, between about 25 to about 50 g / 1 dry cell weight, between about 45 to about 80 g / 1 dry cell weight, between about 75 to about 100 g / 1 dry cell weight, between about 80 to about 150 g / 1 dry cell weight, between about 120 to about 200 g / 1 dry cell weight, or between about 150 to about 180 g / 1 dry cell weight. In one example, the biomass obtained from culturing the PLU strains disclosed herein in glucose yeast extract (GY) is between about 13 to about 14 (g / 1) dry cell weight. In on example, the biomass obtained using the methods disclosed herein is about 15 g / 1, about 20 g / 1, about 25 g / 1, about 30 g / 1, about 35 g / 1, about 40 g / 1, about 45 g / 1, about 50 g / 1, about 55 g / 1, about 60 g / 1, about 65 g / 1, about 70 g / 1, about 75 g / 1, about 80 g / 1, about 85 g / 1, about 90 g / 1, about 95 g / 1, about 100 g / 1, about105 g / 1, about 110 g / 1, about 115 g / 1, about 120 g / 1, about 125 g / 1, about 130 g / 1, about 135 g / 1, about140 g / 1, about 145 g / 1, about 150 g / 1, about 155 g / 1, about 160 g / 1, about 165 g / 1, about 170 g / 1, about175 g / 1, about 180 g / 1, about 185 g / 1, about 190 g / 1, or about 195 g / 1. In another example, the biomass is 175 g / 1 dry cell weight.

[0032] As used herein, the term “biomass” refers to the total quantity, mass, or weight of organisms in a given volume of liquid. The liquid can be, for example, but not limited to, a cell culture media or a buffer solution. In the present disclosure, biomass is expressed in terms of “grams per litre (g / 1) of dry cell weight. As a person skilled in the art will appreciate, the amount of biomass generated is dependent on the amount of time spent in culture, as well as the concentration of glucose given.

[0033] Lipid content is another parameter used when evaluating microalgae strains for their lipid productivity in scale-up production. As described herein, lipid productivity of an isolated strain can be evaluated by determining the lipid content in cells. As used herein, the term “lipid content” refers to the percentage of lipid extracted from cells, whereby the cells are measured in dry cell weight. In the context of the four isolated PLU strains disclosed herein, their lipid content ranges between about 8.9% and about 32.3%, with Schizochytrium sp. PLU-D demonstrating the highest lipid content of about 32.3% (see Table 1).

[0034] Thus, in one example, the lipid content of the isolated PLU strains described herein can be between about 5% to about 40% of dry cell weight. In another example, the lipid content is between about 10% to about 20% of dry cell weight, between about 20% to about 30% of dry cell weight, or between about 30% to about 40% dry cell weight. In yet another example, the lipid content is between about 9% to about 35% of dry cell weight, between about 8% to about 33% of dry cell weight, or between about 8.9% and about 32.3% of dry cell weight. In another example, the lipid content is about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, or about 40%. In one example, the lipid content in the isolated strains is about 8.9% of dry cell weight. In another example, the lipid content in the isolated strain is about 12.7% of dry cell weight. In another example, the lipid content of the isolated strain is about 19.2%. In yet another example, the lipid content of the isolated strains is about 32.3% of dry cell weight. In one example, the Schizochytrium sp. PLU-D strain comprises a lipid content of about 32.3% of dry cell weight.

[0035] Comparing to other isolated microalgae strains from Southeast Asia known in the art, the lipid contents of the isolated PLU strains were also higher than both the Malaysian isolated thraustochytrids sp. strains (about 3.29 to about 15.76% of dry cell weight) and the Thailand isolated thraustochytrids sp. strains (about 3.5 to about 19.4% of dry cell weight).

[0036] As described herein, lipid productivity can be expressed in terms of lipid yield. As used herein, the term “lipid yield” refers to the total amount of the lipid harvested from the culture cells of the microalgae. The microalgae are usually defined in volumetric units. This yield is commonly expressed in grams of lipid per litre of culture (g / 1), but can also be expressed in other equitable units such as, but not limited to, mg / ml or mg / 1. The lipid yield disclosed herein can range between about 0.5 g / 1 to about 10 g / 1, between about 1 g / 1 to about 5 g / 1, between about 1.5 g / 1 to about 5 g / 1, between about 2 g / 1 to about 4 g / 1, between about 3 g / 1 to about 5 g / 1, between about 4 g / 1 to about 5 g / 1 or between about 4.5 g / 1 to about 5 g / 1. In another example, the lipid yield is about 1 g / 1, about 1.5 g / 1, about 2 g / 1, about 2.5 g / 1, about 3 g / 1, about 3.5 g / 1, about 4 g / 1, about 4.5 g / 1, about 5 g / 1, about 6 g / 1, about 6.5 g / 1, about 7g / 1, about 7.5 g / 1, about 8 g / 1, about 8.5 g / 1, about 9 g / 1, about 9.5 g / 1, or about 10 g / 1. In one example, culturing the isolated Schizochytrium strains as described herein results in a lipid yield of between about 1.29 to about 4.43 g / 1. In another example, the lipid yield is about 1.29 g / 1. In another example, the lipid yield is about 1.70 g / 1. In another example, the lipid yield is about 2.62 g / 1. In yet another example, the lipid yield is about 4.43 g / 1.

[0037] The isolated strains described herein can be used to produce or obtain compositions of lipids as disclosed herein. In one example, there is described a method of producing a composition of lipids using a Schizochytrium sp. strain. In another example, the method disclosed herein is a fermentation method. As used herein, the Schizochytrium sp. strain is cultured under conditions known in the art to produce a composition of lipids. As described herein, the Schizochytrium sp. strain can be a strain deposited under accession no. DSM34665. Thus, the present disclosure also describes a fermentation method of producing a composition of lipids using a Schizochytrium sp. strain deposited under accession no. DSM34665, wherein the method comprises culturing the Schizochytrium sp. strain to produce the composition of lipids.

[0038] As used herein, the term “fermentation” refers to an enzyme -catalysed, anaerobic breakdown of an energy-rich compound, such as, for example, but not limited to, a carbohydrate, into carbon dioxide and an alcohol or to an organic acid, using microorganisms. Such microorganisms can be, for example, bacteria, yeast or microalgae.

[0039] In one example, the fermentation method comprises a glucose batch phase for the accumulation of biomass, and a glucose fed-batch phase. In an exemplary method, the first phase of the method is batch fermentation, which takes place until both the glucose and nitrogen sources have been depleted. Once these sources have been depleted, the cells would then be batch-fed with additional glucose and nitrogen sources. Thus, in one example, glucose is present in both phases of the disclosed method. In one example, the glucose batch phase is considered to be complete when the glucose in the medium is fully consumed. This can be indicated, for example, by a rise or spike in the dissolved oxygen level, or any other method available to a person skilled in the art. It is thought that changing the process from glucose batch phase to glucose fed-batch phase at this point promotes lipid accumulation and further increases in biomass. In one example, the accumulation of biomass is obtained using a glucose batch phase, wherein the cells of the Schizochytrium strain are cultured in a medium for about 36 to about 48 hours. In another example, the cells of the Schizochytrium strains are cultured in a medium for about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours or about 48 hours. In one example, the cells are cultured for about 36 to about 46 hours, about 36 to about 44 hours, about 36 to about 42 hours, about 36 to about 40 hours, about 38 to about 48 hours, about 38 to about 46 hours, about 38 to about 44 hours, about 38 to about 42 hours, about 38 to about 40 hours, about 40 to about 48 hours, about 40 to about 46 hours, about 40 to about 44 hours, about 40 to about 42 hours, about 42 to about 48 hours, about 42 to about 46 hours, about 42 to about 44 hours, about 44 to about 48 hours,about 44 to about 46 hours or about 46 to about 48 hours. In another example, the feed solution is provided for about 45 hours. In another example, the cells of the Schizochytrium strain are cultured for about 45 hours. In another example, glucose batch phase is performed at about 28°C and at a pH of 7.0. In another example, the pH is maintained at pH 7.0 with 2 M NaOH and 0.5 M citric acid.

[0040] As shown in the experimental section below, an initial glucose concentration supplied to the fermenter was about 200 g / 1 and was almost fully consumed at around 45 hours, as indicated by a dissolved oxygen spike. This glucose concentration resulted in a biomass of about 128 g / 1 of dry cell weight (DCW) with a lipid yield of about 67.4 g / 1 of lipid.

[0041] As described herein, the purpose of the glucose fed-batch phase is to promote lipid accumulation in a cell and to further increase in biomass. In one example, the glucose fed-batch phase is initiated after the glucose batch phase is complete. A person skilled in the art would be able to ascertain when the glucose batch phase is complete using methods known in the art. In one example, the glucose fed-batch phase can be initiated 24 hours after the glucose batch phase had been initiated. In another example, the glucose fed-batch phase is initiated about 24 hours to about 45 hours after the glucose batch phase had been initiated. In yet another example, glucose fed-batch phase is initiated about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours or about 45 hours after glucose batch phase has started. In another example, glucose fed-batch phase is initiated about 25 to about 44 hours, about 25 to about 42 hours, about 25 to about 40 hours, about 25 to about 38 hours, about 25 to about 36 hours, about 25 to about 34 hours, about 25 to about 32 hours, about 25 to about 30 hours, about 25 to about 28 hours, about 28 to about 40 hours, about 28 to about 36 hours, about 28 to about 32 hours, about 28 to about 30 hours, about 30 to about 42 hours, about 30 to about 38 hours, about 30 to about 34 hours, about 32 to about 42 hours, about 32 to about 36 hours, about 32 to about 34 hours after glucose batch phase has started. In one example, the glucose fed-batch phase is initiated about 45 hours after glucose batch phase has started. In another example, the glucose fed-batch phase is initiated by introducing a feed solution. In one example, the feed solution is provided at a single timepoint. In another example, the feed solution is provided continuously, that is, over time. In one example, the feed solution comprises glucose and yeast extract. In another example, the feed solution is provided to the cells of the Schizochytrium strain for about 36 to about 48 hours. In one example, the feed solution is provided for about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours or about 48 hours. In one example, the feed solution is provided for about 36 to about 46 hours, about 36 to about 44 hours, about 36 to about 42 hours, about 36 to about 40 hours, about 38 to about 48 hours, about 38 to about 46 hours, about 38 to about 44 hours, about 38 to about 42 hours, about 38 to about 40 hours, about 40 to about 48 hours, about 40 to about 46 hours, about 40 to about 44 hours, about 40 to about 42 hours, about 42to about 48 hours, about 42 to about 46 hours, about 42 to about 44 hours, about 44 to about 48 hours, about 44 to about 46 hours or about 46 to about 48 hours. In another example, the feed solution is provided for about 45 hours.

[0042] In another example, the fermentation method further comprises culturing the isolated strain in the presence of sodium propionate. In another example, culturing the isolated strain in the presence of sodium propionate results in a higher concentration of odd chain fatty acids (OCFA) in the composition of lipids disclosed herein. A person skilled in the art would be able to ascertain the specific concentration, yield, or composition of the compounds and lipid compositions disclosed herein using methods known in the art such as, for example and not limited to, high pressure liquid chromatography (HPLC), gas chromatography (GC), gas chromatography -mass spectrometer (GCMS), gas chromatography-mass spectrometer coupled with flame ionization detector (GCMS / FID) or any other suitable quantitative or qualitative, analytic method.

[0043] In another example, sodium propionate is used to increase the yield of odd-chained fatty acids in the lipid compositions disclosed herein. In one example, the concentration of sodium propionate is 1 to 20 g / 1. In another example, the concentration of sodium propionate is about 1 g / 1, about 2 g / 1, about 3 g / 1, about 4 g / 1, about 5 g / 1, about 6 g / 1, about 7 g / 1, about 8 g / 1, about 9g / l, about 10 g / 1, about 11 g / 1, about 12 g / 1, about 13 g / 1, about 14 g / 1, about 15 g / 1, about 16 g / 1, about 17 g / 1, about 18 g / 1, about 19 g / 1 or about 20 g / 1. In another example, the concentration of sodium propionate is about 5 to about 20 g / 1, about 5 to about 15 g / 1, about 5 to about 10 g / 1, about 2.5 to about 7.5 g / 1, about 2.5 to about 5 g / 1 , about 1 to about 5 g / 1 or about 1 to about 2.5 g / 1. In another example, the concentration of sodium propionate is about 5 g / 1. In one example, the fermentation method as described herein comprises the steps of culturing the cells of the Schizochytrium strain in the presence of sodium propionate for about 5 days at about 25°C.

[0044] Taken together, the fermentation method as disclosed herein results in an increase in concentration of the composition of lipid compared to concentration if the composition of lipids obtained using a control strain. In one example, culturing the Schizochytrium sp. strain results in an increase in the concentration of (docosahexaenoic acid) DHA as compared to a control strain. In one example, culturing the Schizochytrium sp. strain results in an increase in the concentration of odd chain fatty acids (OCFAs) as compared to a control strain. In one example, the control strain is Schizochytrium sp. ABC101 or Schizochytrium sp. G13 / 2S.

[0045] Comparing the fatty acid profile of the lipid composition obtained from the fermentation method using Schizochytrium sp. PLU strains to the fatty acid profiles of two commercially available DHA algae oils in the market, namely Nordic Naturals and GNC (see Table 2), the fatty acid profile of the composition of lipid obtained from the fermentation method as disclosed herein differs from the fatty acid profile of both the commercial algae oils.

[0046] Also described herein, the fermentation method as described herein produces a composition of lipids comprising polyunsaturated fatty acids and polysaturated fatty acids. Thus, it is also described herein a composition of lipids obtain from the fermentation method as described herein.

[0047] As described herein, the composition of lipids obtained from culturing the cells of the four isolated Schizochytrium PLU strains were also analysed for their fatty acid profile. The term “composition of lipids” and lipid composition are used interchangeably in the present disclosure.

[0048] In one example, the composition of lipids comprises docosahexaenoic acid (DHA). In another example, the composition of lipids comprises odd chain fatty acids (OCFAs). In one example, there is described a composition of lipids obtained from culturing the isolated PLU strains disclosed herein. In another example, the composition of lipids is obtained from the fermentation method disclosed herein.

[0049] In another example, the composition of lipids comprises about 45% to about 50% of docosahexaenoic acid (DHA, C22:6) of the total lipid yield. In another example, the lipid composition comprises about 32% of palmitic acid (C16:0). In another example, the composition of lipids comprises about 1% to about 3.35% of eicosapentaenoic acid (EPA, C20:5). In another example, the composition of lipids comprises about about 4.5% of docosapentaenoic acid (DPA, C22:5).

[0050] Isolated Thraustochytrium sp. strains from Japan and Fiji have been previously reported to contain five different types of polyunsaturated fatty acid profile in their lipid composition, namely DHA / DPA, DHA / DPA / EPA, DHA / EPA, DHA / DPA / EPA / arachidonic acid (AA), and DHA / DPA / EPA / AA / docosatetranoic acid. On the other hand, the polyunsaturated fatty acid profiles of the four isolated PLU strains comprise DHA / DPA / EPA. As described in the experimental section, the DHA content of the isolated PLU strains is in the range of about 44.51 to about 48.72% of total lipid yield, which is higher than other strains know in the art such as Schizochytrium sp. ATCC20888, comprising about 15.68 to about 38.30% of DHA of total lipid yield, Schizochytrium sp. ATCC20889 comprising about 15.56 to about 26.25% of DHA of total lipid yield, and Schizochytrium mangrovei FBI comprising about 37.8 to about 39.14% of DHA of the total lipid yield.

[0051] As described herein, culturing the isolated PLU strains as described herein also produced odd chain fatty acids in their lipid compositions. In one example, culturing the cells of the Schizochytrium strain in the presence of sodium propionate produces a shift in ratio of composition of lipids compared to culturing the cells of the Schizochytrium strain in the absence of sodium propionate. That is to say, culturing the cells or performing the methods disclosed herein in the absence sodium propionate would still yield the desired lipid composition. In other words, the presence of sodium propionate has been shown to increase the amount of odd-chained fatty acids in the resulting lipid composition. In one example, the lipid composition comprises about 3% to about 25% of C15:0 of its total lipid yield when the cells of the Schizochytrium strain are cultured in the absence of sodium propionate. In another example, the percentage of C15:0 in the lipid composition from culturing the cells of the Schizochytrium strain in the absence of sodium propionate is about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 25%, about 10% to about 20%, about 10%to about 15%, about 15% to about 25%, about 15% to about 20% or about 20% to about 25%. In another example, the percentage of C15:0 in the lipid composition from culturing the cells of the Schizochytrium strain in the absence of sodium propionate is about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18% or about 20%.

[0052] In another example, the percentage of C15:0 in the lipid composition from culturing the cells of the Schizochytrium strain in the presence of sodium propionate is between about 20% to about 55%. In another example, the percentage of C15:0 in the lipid composition from culturing the cells of the Schizochytrium strain in the presence of sodium propionate is between about 20% to about 30%, about 25% to about 35%, about 34% to about 45%, or about 42% to about 54%. In another example, the percentage of Cl 5:0 in the lipid composition from culturing the cells of the Schizochytrium strain in the presence of sodium propionate is at least about 30% to at least about 50%, at least about 30% to at least about 45%, at least about 30% to at least about 40%, at least about 30% to at least about 35%, at least about 35% to at least about 50%, at least about 35% to at least about 50%, at least about 35% to at least about 45%, at least about 35% to at least about 40%, at least about 40% to at least about 55% at least about 40% to at least about 50%, at least about 40% to at least about 45%, at least about 45% to at least about 55%, at least about 45% to at least about 50%, or at least about 50% to at least about 55% of the total lipid yield. In a further example, the percentage of C15:0 in the lipid composition from culturing the cells of the Schizochytrium strain in the presence of sodium propionate is at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49% or at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54% or at least about 55%. In another example, percentage of C15:0 is about 47%. Given the presence of these OCFAs in the isolated PLU-strains, a lipid composition obtained from the isolated PLU strains therefore differs from other producers of high - DHA microalgae oil.

[0053] To date, Schizochytrium sp. strains have been approved for DHA algae oil production in countries and regions including but not limited to United States, European Union, Australia, and China. For example, the multi-national corporation giant, DSM, has recently commercialized their Schizochytrium sp. -derived DHA products such as Life’s DHA® and Life’s™ OMEGA. Further, new strains and processes associated with Schizochytrium vp-derived DHA algae oil production have also been developed. For example, the Schizochytrium sp. ABC101 strain, which was isolated from seawater samples near Jeju Island, South Korea, obtained about 86 grams per litre (g / 1) of dry cell weight, about 37.2 g / 1 of lipid, and about 16.7 g / 1 of DHA under an optimized fermentation process. In another study, the Schizochytrium sp. HX-308 strain isolated from seawater samples in China produced about 19 g / 1 of lipids which contained 60% DHA through the addition of about 4 g / 1 of malic acid at the lipid accumulation stage. Continuous optimization of the the Schizochytrium sp. HX-308 strain, throughadaptive laboratory evolution further resulted in a DHA yield of about 38.1 g / 1 under fermentation conditions. Further, the Schizochytrium sp. PKU#Mn4 strain, isolated from the mangrove of coastal waters in China, through atmospheric and room temperature plasma (ARTP) mutagenesis, increased its DHA content of its lipids from about 42.6% to about 60.7%, resulting in a yield of about 0.98 g / 1 of DHA in a shake-flask. In this regard, the discovery and isolation of new Schizochytrium sp. strains with high biomass and lipid and DHA productivity is therefore desirable.

[0054] In one example, the fermentation method as described herein produces a composition of lipids comprising docosahexaenoic acid (DHA, C22:6). In one example, the percentage of DHA in the composition of lipids obtained from the fermentation method as disclosed herein is at least about 30% to at least about 55% of the total lipid yield Thus, the percentage of DHA content in the composition of lipids obtained from the fermentation method using the Schizochutrium sp. strain is at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49% or at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54% or at least about 55% of the total lipid yield. In one example, the percentage of DHA at least about 35% to at least about 50%, at least about 35% to at least about 45%, at least about 35% to at least about 40%, at least about 40% to about 55%, at least about 40% to at least about 50%, at least about 45% to at least about 50% or at least about 45% to at least about 50% of the total lipid yield. In one example, there is described a composition of lipid described herein comprising docosahexaenoic acid (DHA). In one example, the composition is as described herein, wherein the percentage of docosahexaenoic acid (DHA) ranges from about 45% to about 50%. In another example, the composition is as described herein, wherein the percentage of docosahexaenoic acid (DHA) is about 44.5%. In another example, the lipid composition as described herein comprises docosahexaenoic acid (DHA) at about 44.95%. In another example, the percentage of docosahexaenoic acid (DHA) is about 45.43% in the lipid composition described herein. In yet another example, the composition of lipids described herein comprises about 48.72% of docosahexaenoic acid (DHA).

[0055] Utilization of the Schizochytrium sp. PLU-D strain in a fermentation method as disclosed herein produced a DHA amount of about 25.6 g / 1. In contrast, the amount of DHA produced using Schizochytrium sp. G13 / 2S and Schizochytrium sp. ABC101 are about 6 g / 1 and about 16.7 g / 1, respectively. Thus, the fermentation method described herein results in an increase concentration of docosahexaenoic acid (DHA) as compared to a control strain.

[0056] In another example, the fermentation method as disclosed herein has been shown to produce a composition of lipids comprising, among others, odd chain fatty acids. The percentage of C15:0 odd chain fatty acid in the composition of lipid obtained is at least about 25% to at least about 55% of the total lipid yield. Thus, the percentage of C15:0 fatty acid level in the composition of lipid from culturingthe Schizochutrium sp. strain is at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55% of the total lipid yield. In one example, the percentage of C15:0 fatty acid is at least about 25% to at least about 55%, at least about 25% to at least about 50%, at least about 25% to at least about 45%, at least about 25% to at least about 40%, at least about 25% to at least about 35%, at least about 25% to at least about 30%, at least about 30% to at least about 55%, at least about 30% to at least about 50%, at least about 30% to at least about 45%, at least about 30% to at least about 40%, at least about 30% to at least about 35%, at least about 35% to at least about 50%, at least about 35% to at least about 50%, at least about 35% to at least about 45%, at least about 35% to at least about 40%, at least about 40% to at least about 55% at least about 40% to at least about 50%, at about least 40% to at least about 45%, at least about 45% to at least about 55%, at least about 45% to at least about 50%, or at least about 50% to at least about 55% of the total lipid yield. In one example, there is described a composition of lipid described herein comprising odd chain fatty acids (OCFAs). In one example, the composition as described herein, wherein the percentage of odd chain fatty acids (OFCAs) ranges from about 30% to about 55%. In another example, the percentage of OCFA in the lipid composition is about 47%. In another example, the percentage of OCFA is about 51%.

[0057] In another example, the composition of lipids obtained from the fermentation method disclosed herein further comprises, but is not limited to, the following fatty acids: Cll:0. C12:0, C13:0. C14:0. C15:0, C16:0. C17:0. C18:0 fatty acids, eicosapentaenoic acid (EPA, C20:5), docosapentaenoic acid (DPA, C22:5), docosahexaenoic acid (DHA, C22:6), and combinations thereof.

[0058] Also as described herein is a composition of lipids comprising Cl 1:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:5, C22:5 or C22:6 fatty acids. In one example, the percentage of Cl 1:0 fatty acids is at least about 0.01% to at least about 0.03% of the total lipid yield the percentage of C12:0 fatty acids is at least about 0.23% to at least about 0.33% of the total lipid yield; the percentage of C13:0 fatty acid is at least about 0.08% to at least about 0.12% of the total lipid yield; the percentage of C14:0 fatty acid is at least about 3.25% to at least about 4.34% of the total lipid yield; the percentage of C15:0 fatty acid is at least about 4.43% to at least about 4.97% of the total lipid yield; the percentage of C16:0 fatty acid is at least about 32.24% to at least about 33.7% of the total lipid yield; the percentage of C17:0 fatty acid is at least about 0.82% to at least about about 1.03% of the total lipid yield; the percentage of C18:0 fatty acid is at least about 0.71% to at least about about 1.09% of the total lipid yield; the percentage of eicosapentaenoic acid (EPA, C20:5) is at least about 1.07% to at least about 3.35% of the total lipid yield; the percentage of docosapentaenoic acid (DPA, C22:5) is at least about4.43% to at least about 4.9% of the total lipid yield; and the percentage of docosahexaenoic acid (DHA, C22:6) is at least about 44.95% to about at least about about 48.72% of the total lipid yield

[0059] In another example, the percentage of Cl 1 :0 fatty acid is at least about 0.18% to at least about 0.40%; C12:0 fatty acids is at least about 0.09% to at least about 0.22%; the percentage of C13:0 fatty acid is at least about 1.4% to at least about 3.0%; the percentage of C14:0 fatty acid is at least about 0.63% to at least about 0.96%; the percentage of C15:0 fatty acid is at least about 27% to at least about 32%; the percentage of C16:0 fatty acid is at least about 9.6% to at least about 13%; the percentage of C17:0 fatty acid is at least about 1.9% to at least about 2.4%; the percentage of C18:0 fatty acid is at least about 2.5% to at least about about 5.8%; the percentage of eicosapentaenoic acid (EPA, C20:5) is at least about 2.1% to at least about about 3.2%; the percentage of docosapentaenoic acid (DPA, C22:5) is at least about 2.8% to at least about 4.2%; and the percentage of docosahexaenoic acid (DHA, C22:6) is at least about 35% to about at least about 43%.

[0060] As disclosed herein, four microalgae strains were isolated from decayed mangrove samples collected from Pulau Ubin, Singapore and were identified as Schizochytrium sp. through phylogenetic analysis. Culturing these PLU isolated strains in a shake -flask experiment resulted in a higher biomass, a higher lipid content and a higher docosahexaenoic acid (DHA) content as compared to other wildtype microalgae strains isolated from Southeast Asia. Further, culturing the PLU isolated strains in the presence of sodium propionate produces OCFA. When the Schizochytrium sp. PLU-D strain was cultured under conditions for fed-batch fermentation, it was able to produce a significantly higher amount of biomass and a higher amount of lipids, which have comparable or higher concentrations of DHA when compared to two commercially available algae oils. This makes Schizochytrium sp. PLU- D strain a suitable host for the commercial production of DHA.

[0061] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0062] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.

[0063] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0064] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0065] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub -generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0066] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0067] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.EXPERIMENTAL SECTIONSample collection and strain isolation

[0068] Decayed mangrove samples including fallen leaves, stems, flowers, and fruits were collected from Pulau Ubin (1.4126°N, 103.9577°E), an island located off Singapore’s north-eastern coast (see Fig. 1). The samples were first washed with sterilized seawater containing 0.5 grams per litre (g / 1) of penicillin G, and 0.5 g / 1 of streptomycin sulfate. The suspension was then filtered through a 40 pm filter to remove the debris before being streaked onto glucose, peptone, yeast extract (GPY) plates for isolation. The GPY plates contained 1 g / 1 peptone, 2 g / 1 of yeast extract, 4 g / 1 of glucose 0.5 g / 1 of penicillin G, 0.5 g / 1 of streptomycin sulfate, 0.1 g / 1 of fluconazole and 10 g / 1 of agar in half-strength seawater. The plates were then sealed and incubated at 25°C in the dark for 2 to 5 days. Microalgae-likecolonies were aseptically picked and streaked onto new GPY plates. Single colonies were isolated repeatedly, until axenic cultures were obtained. Cell morphologies were then examined under a light microscope equipped with a colour camera to verify the authenticity of microalgae -like morphology. The pure isolates were stored in 20% glycerol at -80°C. Four axenic strains of microalgae-like microorganisms, designated PLU-A, PLU-B, PLU-C and PLU-D were obtained after a few rounds of cultivation and purification. The strains formed colonies with opaquely white and flat creamy appearance on agar plates after incubation for 4 days at 25 °C.Stain cultivation and measurement of dry cell weight (DCW)

[0069] The isolated strains of microalgae were cultured in a 250 ml baffled flask with 50 ml of glucose yeast extract (GY) medium containing 30 g / 1 of glucose, 10 g / 1 of yeast extract, and half-strength seawater, at pH 6.0. The cultures were incubated in a rotary shaker at 200 rpm and 25°C, and the cell growth was monitored by measuring the wet cell mass. At the end of the cultivation, the cells were harvested for lipid extraction to determine the lipid yield and fatty acid compositions.

[0070] To determine the dry cell weight, 20 ml of culture broth was collected, and the cells were pelleted by centrifugation at 3500 g for 10 minutes at room temperature with a centrifuge. The cell pellet was then washed twice with 20 ml distilled water and recentrifuged as above, before being dried at 60°C until the weight remained constant.

[0071] The dry cell mass, lipid content and lipid yield of the isolated Schizochytrium sp. strains, PLU- A, PLU-B, PLU-C and PLU-D are shown in Table 1.Table 1PLU-A PLU-B PLU-C PLU-DDCM* (g / 1) 13.4 14.5 13.6 13.7Lipid content* (%) 12.7 8.9 19.2 32.3Lipid yield (g / 1) 1.70 1.29 2.62 4.43*DCW: dry cell weight; *Lipid content: lipid weight over DCWStrain identification by 18S rRNA sequences and morphological analysis

[0072] Genomic DNA was extracted from the isolated Schizochytrium sp. strains. The 18S rRNA sequences were amplified with primers PF: 5’-CTGGTTGATCCTGCCAGTAGTC-3’ and PR: 5’- GTTAAGACTACGATGGTATCTAA-3’ from the genomic DNA. Each PCR reaction mixture contained 100 ng template genomic DNA, lx PCR buffer, 1.0 mM MgSO4, 0.2 mM dNTPs, 0.3 pM of each primer and 1.0 U KOD DNA polymerase in a 50 pl reaction volume. PCR amplification was carried out using the following program: Initial Denaturation at 94°C for 2 minutes, 30 cycles of [Denaturation at 94°C for 15 seconds, Annealing at 52°C for 30 seconds, Extension at 68°C for 1 minute] and a Final Extension at 68°C for 5 minutes. The PCR products were then analysed by agarose gelelectrophoresis. The desired bands were excised from the gel and purified. The purified products were then sequenced.

[0073] BLAST analysis was performed on the resulting 18S rRNA sequences against available 18S rRNA sequences in GenBank database using nucleotide BLAST. Several 18S rRNA sequences were selected for multiple sequence alignment analysis, which was performed using the MUSCLE algorithm using MEGA 11 software. An unrooted phylogenetic tree, which shows the relationships between species, but do not depict their common ancestor, was then generated using the neighbour joining algorithm, results of which are shown in Fig. 3.

[0074] The 18S rRNA sequences of PLU-A, PLU-B, PLU-C and PLU-D isolated strains were deposited to the NCBI database and the corresponding GenBank Accession numbers were obtained. Phylogram analysis illustrated three clusters, with the four PLU strains found in a tight cluster with Schizochytrium sp. TK6 (OK244290.1), as shown in Fig. 3. The cell morphological phenotypes were observed under a light microscope. The selected microalgae isolated strains were grown in glucose yeast extract (GY) medium at 25°C, 200 rpm. Morphologies of the microalgae isolates were characterized with respect to vegetative cells, sporangium, ectoplasmic net, zoospores, amoeboid cells, and binary divisions (see Fig. 4).

[0075] The Schizochytrium sp. PLU-D strain was further selected for morphological study. When examined under the microscope, the vegetative cells showed a typical spherical shape of Schizochytrium sp. strains. These vegetative cells were dispersed as single cells in the culture, with their sizes falling in the range of 9 to 20 pm in diameter (Fig. 4A & 4B). The vegetative cells underwent a series of binary divisions to form diads, octads, and decads etc., which formed small clusters of cells (Fig. 4D & Fig. 4E). Zoosporangia developed from vegetative cells were larger in size with a diameter of around 10 to 25 pm. Zoospores were released from sporangia upon rupture of the zoosporangium wall (Fig. 4C). Amoeboid cells in elongated shaped with diameters around 15-20 pm were also observed (Fig. 4B & Fig. 4E). Amoeboid cells would become spherical and mature into zoosporangium that would release zoospores.Measurement of total lipids and fatty acid composition

[0076] The microalgae cells of the Schizochytrium sp. from 40 ml of crude cell culture of each the four PLU isolate strains were first pelleted by centrifugation at 3500 G for 10 minutes. The cell pellet was then resuspended with 20 ml of 0.1 M TrisHCl at pH 8.0, followed by addition of 100 pL Alcalase and 100 pL cellulase (yolume / volume) to the cell suspension to initiate enzymatic hydrolysis. To promote complete enzymatic hydrolysis, the suspension was incubated at 50°C for 6 hours with constant agitation. Following which, the lipids were extracted with an equal volume of hexane and the extraction was repeated two more times. The hexane organic layer was then combined, and the organic solvent was removed by evaporation with reduced pressure using a concentrator. The extracted lipid compositions were weighed and stored in -20°C until further analysis.

[0077] The extracted lipids were derivatized to fatty acid methyl esters (FAMEs) prior to fatty acid composition analysis. 50 mg of lipid was added to 1 ml of 3M hydrogen chloride solution in methanol, incubated at 60°C in a water bath for 20 minutes, and then cooled to room temperature. Next, 400 pl hexane and 200 pl water were added to the reaction mixture and vortexed for 1 minute, followed by centrifugation of the mixture at 3500 g for 5 minutes. The upper layer was then aliquoted to a fresh vial for analysis of FAMEs.

[0078] The FAMEs were analysed by gas chromatography-mass spectrometer coupled with flame ionization detector (GCMS / FID). The gas chromatography is equipped with a 100 m x 0.25 mm ID x 0.25 pm film thickness HP-88 column and a mass spectrometer. Samples were injected in a split ratio of 1:10. Helium was used as the carrier gas, and the column flow rate was set at 1.3 ml per minute (ml / min). The initial oven temperature was 100°C and the inlet temperature was 250°C. After 5 minutes, the oven temperature was increased by 4°C per minute to reach 200°C and held at 200 C for 10 minutes, followed by a further increase to 220°C at a rate of 1°C per minute. The temperature for flame ionization detector (FID) was set at 250°C with a makeup flow of 30 ml / min, a hydrogen flow of 40 ml / min and an air flow of 400 ml / min. The injection volume was set at 1 pl. Peaks were then identified based on retention times and mass spectrometry of FAME standards. The FAME standard is a commercially available, certified reference material, and comprises the following analytes: cis-13,16-Docosadienoic acid methyl ester 2 weight (wt.) %; cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester 2 wt. %; cis-11,14-Eicosadienoic acid methyl ester 2 wt. %; cis-5, 8,11,14, 17-Eicosapentaenoic acid methyl ester 2 wt. %; cis-8,l l,14-Eicosatrienoic acid methyl ester 2 wt. %; cis-11, 14, 17-Eicosatrienoic acid methyl ester 2 wt. %; Methyl arachidate 4 wt. %; Methyl arachidonate 2 wt. %; Methyl behenate 4 wt. %; Methyl butyrate 4 wt. %; Methyl decanoate 4 wt. %; Methyl cis-13-docosenoate 2 wt. %; Methyl dodecanoate 4 wt. %; Methyl cis-ll-eicosenoate 2 wt. %; Methyl elaidate 2 wt. %; Methyl heneicosanoate 2 wt. %; Methyl heptadecanoate 2 wt. %; Methyl cis-10-heptadecenoate 2 wt. %; Methyl hexanoate 4 wt. %; Methyl linoleate 2 wt. %; Methyl linolelaidate 2 wt. %; Methyl linolenate 2 wt. %; Methyl y-linolenate 2 wt. %; Methyl myristate 4 wt. %; Methyl myristoleate 2 wt. %; Methyl octanoate 4 wt. %; Methyl oleate 4 wt. %; Methyl palmitate 6 wt. %; Methyl palmitoleate 2 wt. %; Methyl pentadecanoate 2 wt. %; Methyl cis-10-pentadecenoate 2 wt. %; Methyl stearate 4 wt. %; Methyl tetracosanoate 4 wt. %; Methyl cis-15-tetracosenoate 2 wt. %; Methyl tricosanoate 2 wt. %; Methyl tridecanoate 2 wt. %; Methyl undecanoate 2 wt. %. One example of the FAME standard is commercially available from Sigma-Aldrich (F.A.M.E. Mix, C4-C24, 18919-1AMP). The composition of fatty acids in the extracted lipid compositions of the isolated Schizochytrium sp. strains are shown in Table 2.

[0079] The two most dominant fatty acids in the extracted lipid compositions were docosahexaenoic acid (DHA, C22:6) and palmitic acid (Cl 6:0) as shown in Table 2, which accounted for about 45% and 32% of total fatty acids present in the extracted lipid composition, respectively. Besides DHA, the PLU- strains also synthesized other types of polyunsaturated fatty acids including eicosapentaenoic acid (EPA,C20:5) and docosapentaenoic acid (DPA, C22:5), which accounted for 1.07 to 3.35% and about 4.5%, respectively.Table 2Fatty Acids PLU-A PLU-B PLU-C PLU-D CO1* CO2#Cl 1:0 0.02 0.03 0.02 0 0 0C12:0 0.28 0.33 0.24 0.23 0.09 0.24C13:0 0.12 0.09 0.11 0.08 0.02 0.01C14:0 3.98 4.34 3.99 3.25 1.48 7.63C15:0 4.88 4.97 4.53 4.43 0.37 0.32C16:0 32.24 32.68 33.7 32.84 20.35 19.2C17:0 0.82 0.89 0.73 1.03 0.08 0.06C18:0 1.09 1.03 0.71 0.74 1.69 0.94C18:l 0 0 0 0 11.98 12.64EPA (C20:5) 3.35 2.79 2.8 1.07 19.84 1.26DPA (C22:5) 4.61 4.43 4.9 4.45 1.81 15.77DHA (C22:6) 44.51 44.95 45.43 48.72 39.26 38.43Others 4.09 3.49 2.85 3.17 3.03 3.51Total 100 100 100 100 100 100*CO-1 refers to Algae Omega from Nordic Naturals; *CO-2 refers to GoldMinds DHA 200 from GNC; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid. All values provided in percentage (“%”).Fed-batch fermentation for producing DHA

[0080] The isolated Schizochytrium sp. PLU-D strain was subjected to fed-batch fermentation in a 2- litre Biostat B bioreactor to determine its lipid and DHA productivity. Seed culture was first prepared in a 500 ml shake-flask containing 100 ml culture medium containing 90 grams per litre (g / 1) of glucose, 22.5 g / 1 of yeast extract, and 20 g / 1 of sea salt, and was incubated at 28°C and shaken at 150 rpm for 3 days. 70 ml of the seed culture was then inoculated into 700 ml of fermentation medium containing 50 g / 1 of yeast extract, 2.5 g / 1 of NaCl, 4 g / 1 of KH2PO4, 2.5 g / 1 of MgSO-r7H2O, 0.5 g / 1 of KC1, 9.7 g / 1 of Na2SC>4, 0.1 g / 1 of CaCk, 200 g / 1 of glucose, 10 ml / 1 of trace element solution and 2 ml / 1 of vitamin solution. The trace element solution contains 6 g / 1 of EDTA-Na2, 0.174 g / 1 of FeCL, 6.84 g / 1 of H3BO3 , 0.55 g / 1 of MnCL. 60 milligrams per litre (mg / 1) of Z.11CI2. 26 mg / 1 of COQ2 6H2O, 52 mg / 1 of NiSO4-6H2O, 2 mg / 1 of CUSO4 5H2O and 4.26 mg / 1 of Na2MoO4, and was filter-sterilized with a 0.22 pm membrane, while the vitamin solution contains 100 mg / 1 of thiamine and 0.5 mg / 1 of biotin, and was filter-sterilized with a 0.22 pm membrane. Feed solution containing 100 g / 1 of yeast extract and400 g / 1 of glucose was fed when a spike or rise in the dissolved oxygen (DO) level was observed. The feed solution flowrate was first set at providing 0.14 ml per minute (ml / min) of feed solution and was increased every 8 h to ensure that the overall feed solution flowrate was maintained at 0.185 ml per litre fermentation volume per minute. A total of 500 ml of feed solution was fed into the fermenter after 90 hours of fermentation. During fermentation, the air flowrate was set at 2.5 litres per minute (1 / min) and was reduced to 1.5 1 / min upon initiation of feeding, while the O2 flowrate was kept at 0.5 1 / min throughout the fermentation. Throughout the entire fermentation process, the pH was maintained at 7.0 with 2 M of NaOH and 0.5 M of citric acid and the temperature was kept at 28 °C, while the agitation was set at 700 rpm.

[0081] The Schizochytrium sp. PLU-D strain was selected for fermentative production of DHA microalgae oil. The fermentation process started with a glucose batch phase for the accumulation of biomass, followed by a glucose fed-batch phase when the glucose is fully consumed, as indicated by a spike in the dissolved oxygen level, to promote lipid accumulation and further increase in biomass. As shown in Fig. 5, the biomass increased slightly from 2.0 to 7.3 grams per litre (g / 1) of dry cell weight in the first 10 hours, followed by a sharp increase from 7.3 to 109.9 g / 1 of dry cell weight from 10 to 38 hours. The initial glucose concentration supplied to the fermenter was 200 g / 1 and was almost fully consumed at around 45 hours, as indicated by a dissolve oxygen spike, and resulted in a biomass of 128 g / 1 of dry cell weight with lipid accumulation of 67.4 g / 1 of lipid. After 45 hours, the feed solution was progressively introduced into the fermenter for another 45 hours, leading to a slower and gradual increase in biomass from 45 to 93 hours. At around 84 hours, fermentation of the PLU- strain obtained a high biomass of 175 g / 1 of dry cell weight, which corresponded to a lipid yield of 93.6 g / 1 of lipid.Odd chain fatty acid production in shake flask

[0082] The cells of Schizochytrium sp. PLU-D were cultured in 50ml of medium containing 30 g / 1 glucose, 10 g / 1 yeast extract and 5 g / 1 sodium propionate in 250ml shake flask. After 5 days cultivation at 25°C, the cells were harvested and the lipids in the cells were extracted. The fatty acid composition of the extracted lipids was measured by GC-MS. The concentration of each fatty acid in the lipid extract is provided in Table 3. All values indicated in percentage of total of fatty acid composition. Odd chain fatty acids reached 31.5% for C15:0 fatty acid and 2.3% for C17:0 fatty acid.Table 3Fatty Acids PLU-A PLU-B PLU-C PLU-DCll:0 0.39 0.32 0.40 0.18C12:0 0.20 0.22 0.18 0.09C13:0 2.82 2.44 3.01 1.44C14:0 0.98 0.80 3.96 0.63C15:0 30.78 27.68 30.85 31.56C16:0 12.71 13.93 13.96 9.64C17:0 2.24 1.91 2.24 2.33C18:0 5.11 5.77 5.57 2.56EPA (C20:5) 2.22 3.13 2.9 2.10DPA (C22:5) 3.62 4.16 2.82 3.80DHA (C22:6) 37.58 38.19 35.52 42.62EPA: eicosapentaenoic acid; DPA: docosapentaenoic id; DHA: docosahexaenoic acid; all values indicated in percentage (“%”) of total of fatty acid composition.Fed-batch Fermentation for odd chain fatty acid production

[0083] Schizochytrium was cultivated in 2-litre fermenter with fermenting media containing 150 g / 1 to 200 g / 1 glucose, 30 to 50 g / 1 yeast extract, 5 g / 1 sodium propionate, and trace element solution. The trace element solution used herein contains 6 g / 1 of EDTA-Na2, 0.174 g / 1 of FeCh, 6.84 g / 1 of H3BO3, 0.55 g / 1 of MnCh, 60 milligrams per litre (mg / 1) of ZnCE. 26 mg / 1 of COCI2 6H2O, 52 mg / 1 of NiSCU^EkO, 2 mg / 1 of CuSOvSPEO and 4.26 mg / 1 of Na2MoC>4. Glucose was almost fully consumed at around 40 hours to 45 hours, as indicated by a dissolved oxygen spike. Subsequently, the feed solution comprising 400 g / 1 to 500 g / 1 glucose, 70 to 100 g / 1 yeast extract, and 5 g / 1 to 15 g / 1 sodium propionate was progressively introduced into the fermenter for another 40 hours to 60 hours. The final dry cell weight obtained was 65 g / 1, lipid yield 40 g / 1, the percentage of odd chain fatty acid in the lipid composition is approximately 51%, comprising 47% of C15:0 fatty acids and 4% of C17:0 fatty acid, while the percentage of DHA in the lipid composition is around 24%.SEQUENCE LISTING

Claims

CLAIMS1. A fermentation method of producing a composition of lipids using a Schizochytrium sp. strain deposited under accession no. DSM34665, wherein the method comprises culturing the Schizochytrium sp. strain to produce the composition of lipids.

2. The fermentation method of claim 1, wherein the composition of lipids comprises docosahexaenoic acid (DHA).

3. The fermentation method of claim 1, wherein the composition of lipids comprises odd chain fatty acids (OCFAs).

4. The fermentation method of claim 3, the method further comprising culturing the isolated strain in the presence of sodium propionate.

5. The fermentation method of claim 4, wherein culturing the isolated strain in the presence of sodium propionate results in a higher concentration of odd chain fatty acids (OCFA) in the composition of lipids.

6. The fermentation method of any one of claims 1 to 3, wherein biomass of the isolated strain is increased from the start of culturing the Schizochytrium sp. strain up to 84 hours.

7. The fermentation method of any one of claims 1 to 6, wherein culturing the Schizochytrium sp. strain results in an increase in concentration of the composition of lipids compared to the concentration of the composition of lipids obtained using a control strain.

8. The fermentation method of any one of claims 1 to 7, wherein culturing the Schizochytrium sp. strain results in an increase in the concentration of docosahexaenoic acid (DHA) as compared to a control strain.

9. The fermentation method of any one of claims 1 to 7, wherein culturing the Schizochytrium sp. strain results in an increase in the concentration of odd chain fatty acids (OCFAs) as compared to a control strain.

10. The fermentation method of any one of claims 7 to 9, wherein the control strain is Schizochytrium sp ABC101 or Schizochytrium sp G13 / 2S.

11. A composition of lipids obtained from the fermentation method of claim any one of claims 1 to 9.

12. The composition of claim 10 comprising Cl 1:0, C12:0, C13:0, C14:0, Cl 5:0, C16:0, C17:0, C18:0, C20:5, C22:5 or C22:6 fatty acids.

13. The composition of claim 10 comprising docosahexaenoic acid (DHA).

14. The composition of claim 10 comprising odd chain fatty acids (OCFAs).

15. The composition of claim 12, wherein the concentration of docosahexaenoic acid (DHA) ranges from 35% to 55%.

16. The composition of claim 13, wherein the concentration of odd chain fatty acids (OCFAs) ranges from 30% to 55%17. An isolated strain of Schizochytrium sp., deposited under the accession no. of DSM34665.

Images