avelengths, respectively, with 10-30 min staining time . The fluorescence intensity per cell was significantly (10%) higher than that of the controls after one day of N deprivation, and 2.4-fold higher after two days (Figure 2), which was basically in line with the lipid contents of 24.13% (control) and 53.33% (−N) obtained after –N for 2 days by the gravimetric determination (Table 1). These results confirmed that N-deprivation induces substantial neutral lipid accumulation and that P. tricornutum is a promising biodiesel resource. However, the growth rate of the N-deprived cultures was slightly lower than control rates following the medium changes, although higher than that of the cultures kept in the same medium throughout the experiment. The findings indicate that a two-step culture strategy with N deprivation could deliver much higher neutral lipid yields than strategies based simply on continuous N deficiency .
Figure 2. Relative fluorescence intensity of diatom cells under N deprivation. Triplicate samples of diatom cells were stained with Nile Red after 1 and 2 days of N deprivation, their fluorescence was measured and their relative fluorescence intensity was calculated by subtracting the autofluorescence of non-stained microalgae and Nile red. The left and right columns in each day reprensent the control and –N, respectively.
Table 1. Fatty acid composition of P. tricornutum following N deprivation
Changes in fatty acid composition under N deprivation
As shown in Table 1, substantial differences in fatty acid composition between N-deprived and control cells were detected by GC-MS analysis: proportions of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) were 45% and 61% higher in the former, respectively, while the proportion of polyunsaturated fatty acids (PUFA) was 22% lower. The proportions of other fatty acids also markedly declined in the N-deprived cultures. The total unsaturated lipid content decreased by 16% under N deprivation, and proportions of the long-chain fatty acids C20:5 and C22:6 were 43% and 40% lower, respectively. The changes in fatty acid composition were largely due to dramatic increases in palmitic and palmitoleic acids, with accompanying losses of eicosapentaenoic acid, which is generally one of the most abundant fatty acids in P. tricornutum.
Observation of oil bodies by confocal microscopy
The size and number of oil bodies in sampled cells were observed every day during the culture cycle using a laser scanning confocal microscope. Substantially more oil bodies were observed in the N-deprived cells than in controls, but their sizes were similar (Figure 3), with diameters ranging from 0.2 to 2.0 μm. Thus, the total volume of oil bodies was clearly higher in the N-deprived cultures than in the controls, in accordance with the neutral lipid increase, as shown in Figure 2.
Figure 3. Representative confocal microscope images of the diatoms, showing oil bodies. Cells sampled during a 7-day culture cycle were stained with Nile Red and photographed under the confocal microscope daily. –N, cells subjected to the N deprivation treatment five days after subculture; Control, cells grown in f/2-Si medium and switched to fresh medium five days after subculture. Bar = 2 μm.
High-throughput analysis of differential gene expression
To characterize the effects of N deprivation further, we examined differences in gene expression before and after N-deprivation, using RNA-Seq. The complete dataset is listed in Additional file 1, and also available at http://guanlab.ccmb.med.umich.edu/data/Yang_N_deprivation/ webcite, but briefly 1213 genes were found to be up-regulated, with an FDR cutoff of 0.1%, and 4527 genes were down-regulated, indicating that the deprivation induced wide re-programming of regulation. Distribution of the number of reads across different mRNA sequence size indicates a significant correlation (Additional file 2: Figure S1). To validate the findings we applied quantitative RT-PCR (qPCR) to a randomly selected set of genes, including β-actin as a housekeeping marker, to t