arameters, gene expression patterns and inferred shifts in fluxes through key metabolic pathways.
Results and discussion
Effects of N deprivation in late exponential growth phase on growth and neutral lipid accumulation in P. tricornutum
To monitor their growth, the P. tricornutum cultures were sampled at the same time (14:00) daily and cells in the samples were counted using a hemacytometer. As shown in Figure 1, the cultures grown solely under standard conditions showed typical growth curves. Switching to fresh medium (controls) in late exponential phase stimulated growth, and switching to N-free medium (N deprivation, -N) induced an intermediate response; slightly lower growth than in the controls, but higher than that of the standard continuous cultures. These findings indicate that N deprivation in late exponential phase can stimulate further increases in cell biomass, and intracellular N reserves can be used by diatom cells to fuel growth even if the extracellular N concentration is low. It should be noted that the cells showed typical growth characteristics in the absence of silicon, in accordance with findings by De Martino et al. . Thus, silicon was omitted from the culture medium used in further experiments, to prevent the formation of frustules and thus facilitate cell breakage.
Figure 1. Growth of P. tricornutum under standard and N-free conditions. Normal, cells grown under standard conditions, i.e. continuous culture in f/2-Si medium; Control, cells cultured in f/2-Si medium, with a transfer to fresh medium 5 days after subculture; -N, cells cultured in f/2-Si medium, with a transfer to fresh N-free medium 5 days after subculture.
The lipid contents of the cultures were assessed, rapidly and conveniently, using Nile Red and a fluorimetric spectrophotometer with a 480 nm excitation wavelength. The optimal emission and staining time were empirically found to be 592 nm and 20 min, respectively, similar to previously optima for neutral lipid determination: 480 nm and 580–610 nm emission and excitation wavelengths, 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: