Docosahexanoic Acid (DHA) Production in the Aurantiochytrium Species T66 and S61
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Enhanced growth of aquaculture is completely dependent of a sufficient supply of the essential omega-3 fatty acid, docosahexanoic acid (DHA), which has beneficial effects on human health. Increasing DHA demand requires new and alternative sources that are sustainable. The marine heterotrophic protists, thraustochytrids, have been found to produce commercial amounts of triacylglycerol with a high content of DHA and other polyunsaturated fatty acids (PUFA). The strains T66 and S61 of the thraustochytrid genus, Aurantiochytrium, were used to study the DHA production in shake flask cultivation and fermentation. The aim of this thesis was to achieve a better understanding of the DHA production during different nitrogen limitation strategies throughout the fermentation of T66 and S61. In the batch cultures, 15 g/l Na-glutamate was added from the start. It was attempted to sustain the same total Na-glutamate concentration in the fed-batch cultures by continuous feeding at minimum concentrations. Additionally, the strains were examined for individual differences in the DHA-production. Also, the activity of malic enzyme in cell-free extracts of T66 and S61 was investigated. This was done to reveal synthesis of NADPH, which a reducing power used in the fatty acid synthesis. First of all, shake flask cultivations of T66 were executed for the determination of the carbon source and yeast extract (YE) concentrations in the fermentation media, with regards to an optimized DHA production. Four different YE concentrations (0.2, 0.5, 1.0 and 2.0 g/l) were compared to investigate the influence on the fatty acid (FA) production. The YE concentration had a considerable effect on the final cell density and the production of palmitic acid (C16:0), which decreased the relative DHA fraction (%, w/w) of the total fatty acid (TFA). Increased YE concentration gave somewhat greater DHA concentration and a relatively constant DHA content of TFA. A shake flask cultivation of T66 was performed to investigate the DHA productivity as a result of a mixed carbon source (MCS) strategy. This was compared to glucose or glycerol as a main carbon source. The results indicated that glycerol gave a higher DHA concentration which compensated for a slightly longer lag-phase. In the fermentation experiment, the fed-batch fermentation increased the DHA concentration and fraction of TFA compared to the batch cultures of both strains. For the S61 strain, fed-batch fermentation increased the DHA concentration with 46 % compared to the batch cultures, while there was a 31 % increase in T66. The maximum DHA concentrations obtained were 22.0 and 11.3 g/l in the fed-batch cultures of S61 and T66, respectively. Similarly, the highest DHA fraction of TFA obtained was respectively 34 and 27 % (w/w). Between the strains, S61 achieved the highest DHA content and concentration. The concentration was 49 % higher than in T66. In addition, other FAs such as myristic acid (C14:0), palmitoleic acid (C16:1) and oleic acid (C18:1) were present in greater amounts in T66 (20-25 %) than in S61 (<10 %). Malic enzyme activity in cell-free extracts of T66 and S61 was determined through continuous spectrophotometric rate determination. Further work on establishing the method on how to make cell-free extracts has to be done due to the low soluble protein content of ≥1 % (w/w) of the lipid-free dry weight, and the low enzymatic activity of ≥8 mU/mg protein in both strains. In this study it was found that feeding of Na-glutamate at limiting concentrations was more effective on the DHA concentration and content of TFA than in the batch fermentation. Findings indicate that the S61 strain is a promising candidate for DHA production, in combination with a fed-batch fermentation strategy. This knowledge may contribute towards future industrial fermentation processes of omega-3 PUFA production in microorganisms.