liter in natural seawater, acquired from Daya Bay, Huizhou, China. The synchronized cultures were routinely cultivated in an artificial climate incubator at constant irradiance (200 μmol photons · m–2 · s–1) and temperature (21 ± 0.5°C) with 12 h/12 h (1ight/dark) photoperiods.
For the experiments reported here, 1.5 L of algal culture in late-logarithmic growth phase was harvested 5 days after subculturing at 14:00 by centrifugation (4400 rpm for 10 min at 4°C). The supernatant was discarded, the cell pellet was washed twice with NaNO3-free f/2-Si medium to completely remove the N. Then the cells were collected by centrifugation (4400 rpm for 15 min at 4°C), and the resulting pellet was re-inoculated into 1.5 L N-free f/2-Si medium. The resuspended culture was divided into two aliquots. One aliquot was split into six equal portions, which were transferred into six flasks, each containing 125 mL N-free medium, and cultured under the above conditions (designated N-deprived cultures). The other aliquot was also divided, transferred to six flasks and similarly treated, except that the medium was supplemented with N, to provide controls. To maintain sampling consistency with respect to the diel cycle, cells were sampled at 14:00 every day.
Cell density determination
Cell numbers were counted using an Olympus microscope and Brightline Hemocytometer at the same time every day, in triplicates. Cell density (cell mL-1) was calculated as follows:
where CD is cell density and N is the cell number per 80 grid cells.
To facilitate counting, each sample was diluted 2-fold if the cell density exceeded 4 × 106 cells mL-1.
Neutral lipid content analysis
Nile red is a soluble phenoxazone lipid dye that partitions to cytoplasmic oil bodies in cells, and becomes fluorescent , the intensity of the fluorescence providing robust and convenient indications of the amount of neutral lipids present in various species, including the green microalga Chlorella[14,41,42]. In this study, the validity of the method for determining cellular lipid 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