- Oleaginous microalgae are considered as a promising resource for the production of biofuels. Especially diatoms arouse interest as biofuel producers since they are most productive in carbon fixation and very flexible to environmental changes in the nature. Naturally, triacylglycerol (TAG) accumulation in algae only occurs under stress conditions like nitrogen-limitation. We focused on Phaeodactylum strain Pt4 (UTEX 646), because of its ability to grow in medium with low salinity and therefore being suited when saline water is less available or for wastewater cultivation strategies. Our data show an increase in neutral lipids during nitrogen-depletion and predominantly 16:0 and 16:1(n-7) accumulated in the TAG fraction. The molecular species composition of TAG suggests a remodeling primarily from the betaine lipid diacylglyceroltrimethylhomoserine (DGTS), but a contribution of the chloroplast galactolipid monogalactosyldiacylglycerol (MGDG) cannot be excluded. Interestingly, the acyl-CoA pool is rich in 20:5(n-3) and 22:6(n-3) in all analyzed conditions, but these fatty acids are almost excluded from TAG. Other metabolites most obviously depleted under nitrogen-starvation were amino acids, lyso-phospholipids and tricarboxylic acid (TCA) cycle intermediates, whereas sulfur-containing metabolites as dimethylsulfoniopropionate, dimethylsulfoniobutyrate and methylsulfate as well as short acyl chain carnitines, propanoyl-carnitine and butanoyl-carnitine increased upon nitrogen-starvation. Moreover, the Calvin cycle may be de-regulated since sedoheptulose accumulated after nitrogen-depletion. Together the data provide now the basis for new strategies to improve lipid production and storage in Phaeodactylum strain Pt4.