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The present study sought to characterize the synthesis pathways producing the essential polyunsaturated fatty acid (PUFA) 20:5n-3 (EPA). For this, the incorporation of 13C was experimentally monitored into 10 fatty acids (FA) during the growth of the diatom Chaetoceros muelleri for 24 h. Chaetoceros muelleri preferentially and quickly incorporated 13C into C18 PUFAs such as 18:2n-6 and 18:3n-6 as well as 16:0 and 16:1n-7, which were thus highly 13C-enriched. During the experiment, 20:5n-3 and 16:3n-4 were among the least-enriched fatty acids. The calculation of the enrichment percentage ratio of a fatty acid B over its suspected precursor A allowed us to suggest that the diatom produced 20:5n-3 (EPA) by a combination between the n-3 (via 18:4n-3) and n-6 (via 18:3n-6 and 20:4n-6) synthesis pathways as well as the alternative &omega, 3 desaturase pathway (via 20:4n-6). In addition, as FA from polar lipids were generally more enriched in 13C than FA from neutral lipids, particularly for 18:1n-9, 18:2n-6 and 18:3n-6, the existence of acyl-editing mechanisms and connectivity between polar and neutral lipid fatty acid pools were also hypothesized. Because 16:3n-4 and 20:5n-3 presented the same concentration and enrichment dynamics, a structural and metabolic link was proposed between these two PUFAs in C. muelleri.

International audience; Carbon isotopic fractionation was studied during the development of the diatom Chaetoceros muelleri grown in batch culture with 13C-depleted CO2 addition. Cellular and growth parameters and isotopic composition of dissolved inorganic carbon and particulate organic carbon were monitored every two days, while the content and isotopic composition of individual fatty acid in polar lipid and neutral lipid were measured on the 5th day (end of exponential phase), 10th and 14th days (stationary phase). Continuous addition of petrochemical CO2 to the algae led to a rapid and strong modification of dissolved inorganic carbon isotopic composition with cascading effects on particulate organic carbon and fatty acid isotopic compositions. Carbon isotope fractionation in Chaetoceros muelleri ranged from 17‰ to 25‰ and changed according to culture ages. Isotopic fractionation into fatty acids, overall, was similar between polar and neutral lipids, and was systematically higher than in particulate organic carbon. At the end of the exponential growth phase, the isotopic composition of individual fatty acids varied from −51.3‰ to −58.4‰. At this culture age, large differences in the isotopic compositions between fatty acids were observed. Polyunsaturated fatty acids such as 16:3n-4, 18:4n-3, and 20:5n-3 were more strongly 13C-depleted than other fatty acids such as 14:0, 16:0, 16:1n-7 or 18:1n-9. These results showed how isotopic effects occur during the desaturation and elongation phases. Such isotopic effects were also supported by the lower δ13C of averaged δ13C of saturated fatty acids and monounsaturated fatty acids as compared to those of polyunsaturated fatty acids. However, during the stationary phase, fatty acid isotopic compositions were less variable and closer to particulate organic carbon, while saturated and monounsaturated fatty acids were more depleted than polyunsaturated fatty acids. Our study underlined the importance of consideration of phytoplankton physiological status when conducting ecological and biogeochemical studies as they appeared to strongly control phytoplankton carbon isotopic composition.

The synthetic pathways responsible for the production of the polyunsaturated fatty acids 22:6n-3 and 20:5n-3 were studied in the Dinophyte Alexandrium minutum. The purpose of this work was to follow the progressive incorporation of an isotopic label (13CO2) into 11 fatty acids to better understand the fatty acid synthesis pathways in A. minutum. The Dinophyte growth was monitored for 54 hours using high-frequency sampling. A. minutum presented a growth in two phases. A lag phase was observed during the first 30 hours of development and had been associated with the probable temporary encystment of Dinophyte cells. An exponential growth phase was then observed after t30. A. minutum rapidly incorporated 13C into 22:6n-3, which ended up being the most 13C-enriched polyunsaturated fatty acid (PUFA) in this experiment, with a higher 13C atomic enrichment than 18:4n-3, 18:5n-3, 20:5n-3, and 22:5n-3. Overall, the 13C atomic enrichment (AE) was inversely proportional to number of carbons in n-3 PUFA. C18 PUFAs, 18:4n-3, and 18:5n-3, were indeed among the least 13C-enriched FAs during this experiment. They were assumed to be produced by the n-3 PUFA pathway. However, they could not be further elongated or desaturated to produce n-3 C20-C22 PUFA, because the AEs of the n-3 C18 PUFAs were lower than those of the n-3 C20-C22 PUFAs. Thus, the especially high atomic enrichment of 22:6n-3 (55.8% and 54.9% in neutral lipids (NLs) and polar lipids (PLs), respectively) led us to hypothesize that this major PUFA was synthesized by an O2-independent Polyketide Synthase (PKS) pathway. Another parallel PKS, independent of the one leading to 22:6n-3, was also supposed to produce 20:5n-3. The inverse order of the 13C atomic enrichment for n-3 PUFAs was also suspected to be related to the possible &beta, oxidation of long-chain n-3 PUFAs occurring during A. minutum encystment.