veral mechanisms that contribute to the high lipid contents of microalgal cells under N deprivation.
Figure 6. Schematic diagram indicating effects of N deprivation on processes involved in TAG accumulation based on transcriptional changes. Square and elliptical boxes indicate up-regulation and down-regulation, respectively. “ѳ” indicates inhibition of the corresponding process.
The results also provide indications of strategies that could be used to enhance biodiesel production from microalgal cultures. The two-step culture method applied here dramatically increased neutral lipid accumulation, up to 2.4-fold, more than previous N limitation or continuous deficiency strategies. Furthermore, the inhibition of light harvesting under N deprivation indicates that high light intensity is not required for lipid accumulation, and the increase in carbon fixation suggests that increasing CO2 supplies during this stage should be advantageous.
In summary, results of this study show that diatom cultures with high levels of neutral lipids and increased cell proliferation rates can be obtained by nitrogen deprivation during the late exponential phase. They also provide indications of strategies that could be applied to manipulate the biosynthetic pathways of microalgae to generate cultures with high levels of lipids that may be suitable for biodiesel production.
Materials and methods
Marine diatom Phaeodactylum tricornutum, with a fusiform morphotype, was obtained from the Freshwater Algal Culture Collection of the Institute of Hydrobiology, China (No. FACHB-863), and sub-cultured weekly, using a quarter of the preceding cultures as inocula, in Erlenmeyer flasks containing medium sterilized by passage through 0.22-μm filters (Millipore). The growth medium (f/2-Si) contained 75 mg NaNO3, 5.65 mg NaH2PO4 · 2H2O, 4.16 mg Na2 EDTA, 3.15 mg FeCl3 · 6H2O, 0.01 mg CuSO4 · 5H2O, 0.022 mg ZnSO4 · 7H2O, 0.01 mg CoCl2 · 6H2O, 0.18 mg MnCl2 · 4H2O, 0.006 mg Na2MoO4 · 2H2O, 0.0005 mg vitamin B12, 0.1 mg vitamin B1 and 0.0005 mg Biotin per 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