 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 was shaken and centrifuged, and the upper phase was discarded. Then 2 ml water was added to wash the extract, the mixture was centrifuged (12000 rpm for 10 min) and the upper phase was removed. Finally, the organic solvents were evaporated by nitrogen and the residues were stored in a −80°C refrigerator until analysis. Total lipid extracts were separated into lipid classes according to Demandre et al. with modifications . The dried algal lipid extracts were re-dissolved in 1 mL buffer A (isopropanol-hexane, 4:3), and the solutions were centrifuged at 14000 rpm for 10 min to remove solid impurities, then transferred to a glass vial. Lipid classes were subsequently separated by HPLC using a silica column and a mobile phase consisting of a 20-min linear gradient from 100% solution A to 100% B (isopropanol-hexane-water, 8:6:1.5), followed by isocratic solution B for a further 25 min, with the column and sample collection system set at 20°C. The total running time, including a 5 min re-equilibration step, was about 50 min. The flow rate was 1.0 mL min-1 and 10 μL samples were injected. Eluting fractions of lipids were detected spectrophotometrically at 205 nm, manually collected in separate glass tubes, then stored at −20°C. Compounds in the lipid classes fractionated were diluted in isopropanol and identified using a Micromass Q-TOF micro system (Waters Co., UK) equipped with a syringe pump, LockSpray and ESI interface, operated in positive ionization mode. The mass spectrometer conditions were as follows: electrospray capillary voltage, 3.2 kV; ion source temperature, 110°C; desolvation temperature, 350°C; cone voltage, 15V; cone and collision gases, nitrogen and argon, at 60 L h-1 and 600 L h-1 flow rates, respectively. Sodium formate solution was used to calibrate the TOF mass spectrometer in positive electrospray ionization (ESI+) mode, and leucine enkephalin (L-EK) was used as an external mass calibration standard. Masses were scanned from 100 to 1100 amu at approximately 0.4 scan s-1, and data were collected f