The nitrate concentration of culture media was determined daily by pas­sage through a 0.2 pm pore-size filter and analysis on an ion chromato­graph equipped with conductivity detection [50]. Microalgae growth was monitored daily by measuring the optical density of the cultures at 730 nm (OD730) using a spectrophotometer (HP 8453, Hewlett Packard, Palo Alto, CA, USA). Biomass samples for analysis of cellular constituents (starch, proteins, chlorophyll and lipids), and extraction of total RNA were harvested on day-11 by centrifugation at 10,000 g for 5 min at 4°C. Cell pellets were snap-frozen in liquid nitrogen and immediately transferred to -80°C until further analysis. The dry cell weight (DCW) of cultures was determined by filtering an aliquot of cultures on pre-weighed 0.45 pm pore size filters and drying the filters at 90°C until constant weight was reached. For analysis of starch content, 109 cells ml-1 were suspended in deionized water in 2 ml screw-cap tubes containing 0.3 g of 0.5 mm glass beads, and disrupted by two cycles of bead-beating at 4800 oscillations per minute for 2 min, followed by three freeze/thaw cycles. The suspension was then incubated in a boiling water bath for 3 min and autoclaved for 1 hour at 121°C to convert starch granules into a colloidal solution. After samples were cooled to 60°C, cell debris was removed by centrifugation at 4,000 g for 5 min. The concentration of starch in the supernatant was measured enzymatically using the Sigma Starch Assay Kit (amylase/amy — loglucosidase method, Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s instruction. Chlorophyll a and b were measured by the N, N’-dimethylformamide method and calculated from spectrophoto — metric adsorption measurement at 603, 647, and 664 nm, as previously reported [51,52]. The total protein content of cells was determined with minor modifications to the original Bradford method [53] as described in [54]. Starch, chlorophyll, and protein measurements were performed in at least triplicates, and averages and standard deviations are reported as a percent of DCW.

The total lipid content of the cells was determined using a modified Bligh and Dyer method utilizing 2:1 chloroform:methanol [55]. To deter­mine the profile of fatty acids, lipid samples were transesterified [56] and the resulting fatty acid methyl esters (FAME) were analyzed using a liq­uid chromatography-mass spectrometer (Varian 500-MS, 212-LC pumps, Agilent Technologies, Santa Clara, CA, USA) equipped with a Waters nor­mal phase, Atlantis® HILIC silica column (2.1 x 150 mm, 3 pm pore size) (Waters, Milford, MA, USA), and atmospheric pressure chemical ioniza­tion [56]. Identification was based upon the retention time and the mass to charge ratio of standard FAME mixtures. The sum of FAME was used as a proxy for TAG content [22].