compounds, such as polyunsaturated fatty acids (PUFAs), extracellular polymeric substances and cell coatings . Hence, they have attracted both biological and medical attention. Recently, the marine diatom Phaeodactylum tricornutum has emerged as a potential microalgal energy source. It grows rapidly, has a short life cycle, and accumulates TAGs in late exponential phase; storage lipids constitute about 20-30% of its dry cell weight under standard culture conditions . Furthermore, it is a model diatom species and its genome has been fully sequenced and available at the JGI (http://genome.jgi-psf.org/Phatr2/Phatr2.home.html webcite) .
The accumulation of neutral lipids and changes in lipid profiles under nutrient starvation have been monitored in P. tricornutum and another model diatom, Thalassiosira pseudonana[5-7]. Nitrogen is a major constituent of proteins and nucleic acids. Accordingly, pigment and protein losses have been observed in Antarctic sea ice diatoms under nitrogen limitation , and while N-starved P. tricornutum cells can accumulate high levels of lipids, their division reportedly ceases and cell density increases only marginally [1,9,10]. Furthermore, changes in fatty acid profiles of the green alga Chlamydomonas reinhardtii under N deprivation have been reported recently , and associated shifts in fluxes through metabolic pathways have been inferred from changes in transcript abundance in C. reinhardtii. However, the biochemical and molecular mechanisms involved in diatom responses to losses of nitrogen availability are still unclear. Therefore, in the study reported here P. tricornutum was cultured under standard laboratory conditions then subjected to nitrogen deprivation in the late exponential phase to achieve both high neutral lipid contents and high cell biomass. Then, to assess the holistic effects of N deprivation and identify the mechanism involved in the diatoms, we examined changes in their levels of neutral lipids, oil bodies, fatty acid profiles, chloroplast structure, photosynthetic parameters, 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 w