Bioethanol and biodiesel are currently produced from food crops such as sugar beet, sugar cane, soybean or rapeseed, thus competing with land use for food production and urging to find new feedstocks . Due to their high surface productivity, microalgae are considered as a promising way of producing biofuels [2–4]. Under standard growth conditions, microalgal biomass is mainly composed of proteins, cell wall carbohydrates and membrane lipids. Accumulation of energy-rich reserve compounds such as starch and storage lipids (oil) occurs in many microalgae under conditions of nutrient shortage such as nitrogen (N) deficiency [3, 5].
Oil is largely composed of long-chain triacylglycerols (TAG) and represents a form of energy storage 2.25 times greater than starch on a weight basis . TAGs can be converted to biodiesel by chemical transesterification of its fatty acids and is thus a highly desirable storage compound. Some microalgae species accumulate up to 50% TAG on a dry weight basis in response to N deficiency [2, 7]. Despite high biomass productivity and ability to accumulate high oil amounts, microalgal biodiesel is currently not competitive for several reasons. Firstly, contamination of microalgal cutltures by bacteria, viruses and other microalgae is a common issue. Secondly, the cost of microalgae cultivation, biomass harvest and oil extraction contribute significantly to the overall costs. Thirdly, intracellular oil accumulation requires a phase of nutrient starvation, which severely decreases the overall productivity of the system [2, 3]. It is therefore clear that competitiveness of microalgal biodiesel will depend on improvements in both cultivation and harvesting technologies  as well as in strain performances .
Improving microalgal strain performances requires a better understanding in model microalgae of the mechanisms and regulations of carbon fixation, carbon allocation between biosynthetic pathways and induction by stresses. The green unicellular alga Chlamydomonas reinhardtii is a widely recognized model organism to investigate numerous biological functions, including photosynthesis , starch metabolism [5, 9] or flagella . The recent sequencing of its whole genome , the availability of numerous molecular tools including transformation of the three (nuclear, plastid and mitochondrial) genomes, and the existence of a sexual cycle allowing genetic studies make C. reinhardtii an attractive model for molecular investigations . In addition to its well known starch reserves, C. reinhardtii has also been observed to accumulate intracellular oil droplets under N limiting conditions [13, 14]. Pathways of TAG biosynthesis are still poorly documented in microalgae including in C. reinhardtii and most putative reactions are based on similarity of microalgal sequences to characterized proteins from bacteria, yeasts and higher plants . Starch biosynthesis on the other hand has been particularly well-studied in this organism due to the isolation of several starchless mutants .
Whether starch synthesis competes with oil synthesis for carbon precursors is an important question. If such a competition exists, shutting down starch biosynthesis could be a simple way to increase the amount of oil stored in microalgal cells. Increased amount of oil on a dry weight basis has been reported for starchless mutants in Chorella pyrenoidosa  and more recently in Chlamydomonas reinhardtii  but no direct quantitative estimates of the oil content per cell were provided. Another study reports a 1.5 to 2.0 fold increase in oil per cell in a Chlamydomonas starchless mutant , which suggests a competition between oil and starch syntheses. However, recent data showing that complemented strains of C. reinhardtii starchless mutants have both high oil and high starch content  seems inconsistent with a competition hypothesis. The first studies of oil mutants in Chlamydomonas thus highlight the gaps existing in our understanding of oil deposition in this species and on the factors that might be critical when assessing the effect of mutations on oil content. Gaining insights into these issues is important because Chlamydomonas will increasingly be used as a model to study oil synthesis and isolate oil mutants.
Here, we characterize the oil accumulation process in C. reinhardtii by investigating the kinetics of oil deposition and mobilization comparatively to starch, as well as the changes occurring in major plastidial membrane lipids during TAG accumulation. We also show that common laboratory strains of Chlamydomonas widely used as references in mutant comparisons have up to 5-fold variation in their capacity to accumulate oil. Comparison of starchless mutants using appropriate reference strain on a per cell basis shows that blocking starch synthesis has no significant effect on oil accumulation in the cw15 background. Finally, it is shown that in C. reinhardtii CC124 (137C) wild-type strain, oil accumulation can be induced by salt stress, which could advantageously replace nitrogen depletion in mutant screens.