Strains, cell culture and harvest

The cc5373-sta6 strain of the C. reinhardtii mt⁻ progeny of the original BAFJ5 sta6 starchless mutant crossed into a 21gr background was obtained from the Chlamydomonas Resource Center (www.chlamy.org). Cells were maintained in Tris-acetate-phosphate (TAP) media agar plates at 25°C under continuous low intensity white light (60–80 µE m^− 2 s^− 1). Cells were grown in 50 mL TAP medium, which included acetate (17.4 mM) as a carbon source and Tris-base (20 mM) as a buffer, under the same conditions as cell maintenance on a rotary shaker at 120 rpm and used as the algal cell stock for N, S and NS deprivation treatments. A calibration curve was constructed by measuring the optical density (OD) at 750 nm (Bausch and Lomb, Model Spectronic 20) over the course of six days, and OD750 = 0.8 was used for log phase [30, 32].

Once the cells reached the log phase, the cells were harvested via centrifugation at 2,900 g for 5 min at 10^oC (FIBERLite TM F15-8 × 50c). The supernatant was removed, and pelleted cells were washed thrice with their respective media: TAP-N media to remove nitrogen, TAP-S media to remove sulfur and TAP-NS media to remove nitrogen and sulfur [22]. The pelleted cells were resuspended in 50% of their original volume using the respective media and centrifuged at 2,900 g for 5 min (FIBERLite TM F15-8 × 50c) to harvest the cells. After all the washing steps, the pellet was resuspended in 100 mL of its respective media, and 25 mL of cells was transferred into 250 mL sterilized flasks and placed on a rotary shaker. Cells were collected from these flasks for analysis of growth, chlorophyll, protein, lipid bodies and gene expression. All analyses were conducted in duplicate for each of three blocks.