oding PAP-fibrillin-1 and PAP-fibrillin-2) decreased 4- and 5-fold, respectively. This could at least partially explain the decrease of plastoglobuli in N-deprived cells.
The overall goal of this study was to use genomic information and various analytical techniques to obtain an overview of the molecular mechanisms responsible for neutral lipid accumulation in the model diatom P. tricornutum induced by N deprivation. N-deprived cultures of the diatom showed up to 2.4-fold increases in neutral lipid contents per cell, slightly higher cell density than controls and exhibited ultrastructural changes. In addition, the lipid composition of the N-deprived cells was more suitable for biodiesel production as they had higher proportions of saturated fatty acids.
Although the level of transcription is not directly proportional to the enzyme and the catalytic activity, recent study on another diatom T. pseudonana has shown a good correlation of proteomic and transcriptomic data . In combination with biochemical investigations, our results could provide a global view of biosynthetic metabolic fluxes and unraveled significantly altered key metabolic pathways in the diatom following N deprivation. As summarized in Figure 6, it induced genes involved in carbon fixation, the TCA cycle and glycerolipid metabolism, but inhibited genes involved in light harvesting and photosynthesis. Energy resources necessary for carbon fixation and energy demand associated with the neutral lipid synthesis could be replenished from other sources such as elevated TCA cycle. A possible catabolism of carbohydrate reserves including chrysolaminarin may largely explain the neo-synthesis of TAG, in accordance with the up-regulation of glycolysis, coupled with the suppression of critical enzymes, mostly involved in lipid catabolism. The gene expression analyses, in combination with the ultrastructural and biochemical investigations, indicate that additional fatty acids could be produced by membrane remodeling. Thus, the findings provide indications of several 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 