Nitrogen limitation can induce neutral lipid accumulation in microalgae, as well as inhibiting their growth. Therefore, to obtain cultures with both high biomass and high lipid contents, and explore the lipid accumulation mechanisms, we implemented nitrogen deprivation in a model diatom Phaeodactylum tricornutum at late exponential phase.
Neutral lipid contents per cell subsequently increased 2.4-fold, both the number and total volume of oil bodies increased markedly, and cell density rose slightly. Transcriptional profile analyzed by RNA-Seq showed that expression levels of 1213 genes (including key carbon fixation, TCA cycle, glycerolipid metabolism and nitrogen assimilation genes) increased, with a false discovery rate cut-off of 0.001, under N deprivation. However, most light harvesting complex genes were down-regulated, extensive degradation of chloroplast membranes was observed under an electron microscope, and photosynthetic efficiency declined. Further identification of lipid classes showed that levels of MGDG and DGDG, the main lipid components of chloroplast membranes, dramatically decreased and triacylglycerol (TAG) levels significantly rose, indicating that intracellular membrane remodeling substantially contributed to the neutral lipid accumulation.
Our findings shed light on the molecular mechanisms of neutral lipid accumulation and the key genes involved in lipid metabolism in diatoms. They also provide indications of possible strategies for improving microalgal biodiesel production.
Keywords:Microalga; Nitrogen deprivation; Lipid; Membrane remodeling; Transcriptomics
Concerns about global climate change and rises in prices of fossil fuels have prompted intense interest in the ability of microalgae to produce lipids that can be easily converted to biodiesel. Diatoms account for up to 40% of primary productivity in marine ecosystems  and some species are known to accumulate neutral lipids. Moreover, diatoms are metabolically versatile since they can synthesize and accumulate wide ranges of valuable 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 p