 contents of P. tricornutum was first tested, by staining samples of the cultures with Nile Red (Sigma) following Chen et al., with modifications . Nile red (30 μL of a 0.1 mg mL-1 acetone solution) was added to 3 mL portions of cell cultures in triplicate, the resulting suspensions were mixed by rapid inversion, and incubated in darkness for 20 min at room temperature. The stained cell cultures were then transferred to cuvettes to determine their fluorescence intensity using a fluorescence spectrophotometer with 480 nm excitation wavelength. The optimal emission wavelength (592 nm) and staining time (20 min) were determined empirically, using non-stained cultures as auto-fluorescence controls. The relative fluorescence intensity values reported here reflect the differences in neutral lipid contents between stained and non-stained cells.
Fatty acid composition analysis
For fatty acid profiling analysis, total lipids were extracted from three independent biological replicates according to the method of Lepage and Roy , with modifications. Portions (250 mL) of the cultures were harvested after two days of N deprivation by centrifugation at 4400 rpm at 4°C for 10 min. Pellets were transferred to 10 mL tubes. Then 5 mL KOH-CH3OH solution was added to each tube, the cells were lysed by ultrasonication in an ice bath, the tubes were infused with nitrogen for 1 min, tightly sealed, agitated and incubated at 75°C for 10 min. After cooling to room temperature the upper phase was transferred to a 50 mL tube. The lower phase was subjected to two further washes with 5 mL KOH-CH3OH, lysis and incubation, as described above, the resulting upper phases from each sample were pooled in a single tube, then 15 mL of HCl-CH3OH solution was added, the solution was thoroughly mixed and incubated again at 75°C for 10 min. N-hexane (4 mL) was added, the mixture was vortex-mixed, and allowed to settle. The upper phase was then transferred to a new 10 mL tube, while the lower phase was re-extracted with hexane. Then, the upper phases were pooled in a 10 mL tube, dried under a nitrogen stream in a Nitrogen Evaporator (Organomation, USA) and the residues were weighed and stored at −80°C until analysis. Fatty acids were determined by gas chromatography–mass spectrometry (GC-MS) at the Institute of Microbiology, Guangdong, China. The chromatographic column used was a 30 m × 0.25 mm × 0.25 μm DB-5 quartz capillary column. The column temperature was held at 60°C for 1 min, raised by 10°C min-1 to 160°C, and to a final temperature of 250°C at 2.5°C min-1. The injector temperature was 280°C, and 1 μL samples were injected, splitless. The mass spectrum transmission line temperature was 200°C, and fatty acids were identified using the equipped NBS spectrum library, and quantitatively analyzed by determining integrated peak areas. Relative (percentage) contents of detected fatty acids were calculated by using the normalization method.
For lipid profiling analysis, total algal lipids were extracted following a protocol for Arabidopsis leaf tissue  with modifications. 200 ml algal culture was harvested and the resulting pellets were quickly immersed in 3 ml preheated (75°C) isopropanol with 0.01% butylated hydroxytoluene (BHT) for 15 min. The algal cells were then subjected to 80 rounds (5 s) of ultrasonication in an Ultrasonic Crasher Noise Isolating Chamber, with 200 W output energy and 7 s intervals, during which the samples were placed in an ice bath to prevent lipid degradation. Then, 1.5 ml chloroform and 0.6 ml water were added to the cell detritus, and the suspensions were vortex-mixed and agitated in a shaking incubator at low temperature at 4°C for 40 min. The mixtures were centrifuged at 4400 rpm for 8 min to separate phases, and the lower phase was transferred to a new 10 ml centrifuge tube. The remaining debris was extracted twice with 3 ml chloroform/methanol (2:1) with 0.01% BHT, after which it was white. All extracts from samples representing cultures subjected to the same treatment were pooled, 1 ml of 1 M KCl was added, the resulting mixture