Determination of the Hydrocarbon Synthesis of the Algae, Emiliania Huxleyi

Discovering the reaction pathway that Emiliania huxleyi utilizes to synthesize hydrocarbons will allow for the engineering of advanced biofuels.  Previous work on the algae, Botryococcus braunii, indicates that hydrocarbons are synthesized by elongation of the carboxylic acid followed by decarboxylation/decarbonylation of the carboxyl carbon. The objectives for this experiment consisted of determining whether E. huxleyi has a similar reaction pathway to B. brauniiI and if oleic acid is a precursor to its hydrocarbon classes. Two classes of hydrocarbons are synthesized by E. huxleyi naturally. These are C-31, C-33 dienes with double bonds in the 1, 2, 3 and ω9 position in the cis configuration and C37 and C38 trienes with double bonds in the 1, ω15, ω22 and ω29 positions in the trans configuration. To analyze how these products were synthesized, nonadecenoic acid, acetate (isotopically labeled and unlabeled) and propionate were each added to separate flasks containing E. huxleyi cultures that had been grown for seven days on F2 media. Sodium bicarbonate was also added at this time to stimulate fatty acid and hydrocarbon production, and the cells were then harvested after seven days. After harvesting, the cells were extracted using a mixture of hexane and chloroform and hydrocarbons isolated from the extraction were analyzed using gas chromatography and nuclear magnetic resonance (NMR) spectroscopy. When comparing the NMR spectroscopy results using isotopically labeled acetate from the E. huxleyi strain 1516 to B. braunii, it was found that both have peaks representing terminal vinylic carbons (114.1 ppm & 139.2 ppm) and have no splitting at the terminal carbon (114.1 ppm) indicating that the hydrocarbons were created by elongation followed by decarboxylation/decarbonylation. Incorporation of odd-chain fatty acids in the pathway from nonadecenoic acid and propionate resulted in the production of even chain hydrocarbons, further indicating that E. huxleyi undergoes elongation followed by decarbonxylation/decarbonylation during hydrocarbon synthesis. The E. huxleyi fed nonadecenoic acid and propionate followed a similar pathway for oleic acid, indicating that unsaturated fatty acids are precursors for the synthesis of hydrocarbons.