Exploring lipid 2H/1H fractionation mechanisms in response to salinity with continuous cultures of the diatom Thalassiosira pseudonana

The hydrogen isotopic (2H/1H) composition of lipids in microalgae is significantly depleted relative to extracellular water. While a variety of growth conditions influence the magnitude of 2H-depletion, the effect of salinity is of particular interest due to the paleohydrological applications of lipid 2H/1H. In previous studies, lipid-water 2H/1H fractionation was shown to decrease as salinity increased, a response largely independent of lipid type, species, or setting. The mechanism responsible for this response remains uncertain, primarily because salinity is rarely isolated as the sole variable in laboratory cultivation experiments investigating hydrogen isotope systematics in microalgae. Here we report the lipid-water 2H/1H fractionation response to salinity in nutrient-replete continuous cultures of the centric diatom Thalassiosira pseudonana. In six cultures with the same growth rate at salinities between 14-40 ppt, lipid-water 2H/1H fractionation decreased linearly as salinity increased by 1.3‰/ppt in fatty acids (C14:0, C16:0, C16:1) and by 1.0‰/ppt in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol. A constant growth rate between cultures reveals that the fractionation response to salinity is independent of growth rate. Sensitivity tests using a simple hydrogen flux model indicated that at high salinity a greater proportion of metabolic NAD(P)H in lipids at the expense of photosynthetic NADPH can cause 2H-enrichment. Additionally, increased exudate release or decreased hydrogen transport can enrich both lipids and cell-water in 2H. The 1.0-1.3‰/ppt increase in lipid-water 2H/1H fractionation observed in T. pseudonana is within the 0.8-2‰/ppt range observed in field studies and culture studies, supporting the application of algal lipid 2H/1H as a paleosalinity proxy.