To track gene transcription in the oleaginous microalga N. oleoabundans, cells were first grown under+N and — N conditions as a method to pro­duce differential cellular enrichments of TAGs. Cells were harvested after 11 days. This sampling time corresponded to below detection level con­centrations for NO3- and a reduction in growth rate in the -N reactors (Fig­ure 1A, B). The maximum growth rate for the — N cultures was 113 ± 4 (std. err.) mgl-1 day-1 and decreased to 34 ± 0.7 mgl-1 day-1 once nitrogen became limited in the reactor. Total lipids extracted under the+N and — N scenarios revealed a statistically significant increase (p < 0.05) from 22% DCW in+N to 36% in the — N condition (Figure 1C). Extracted lipids were transesterified and fatty acid methyl esters (FAMEs) (FAMEs assumed to

be equivalent to TAGs content [22]), were quantified. Compared to the+N condition, the FAME or TAG content per cell mass increased by five times in the — N case (p <0.05), demonstrating that the additional lipids produced during N limitations were mostly TAGs (Figure 1C). Estimates of total cell mass based on direct microscopic counts and DCW determinations revealed that the average mass of a cell in — N was 81% of that in+N, confirming that the change in TAG was independent of changes in DCW. FAME profiles are presented in Figure 1D, and show a 50% decrease in the proportion of unsaturated fatty acids (i. e. C16:2, C16:3, C16:4, C18:2, and C18:3) under nitrogen limitation. The most significant change was in the amount of oleic acid (C18:1), which increased over 5 times, while the quantity of a-linoleic acid (C18:3) decreased by 4.8-fold under — N conditions. This trend toward a greater proportion of C18:1 is consistent with prior investigations of the oleaginous microalgae N. oleoabundans and Chlorella vulgaris FAME contents under nitrogen limitations [13,22].

To aid in interpreting how photosynthetically fixed carbon was directed into major metabolic pathways, the chlorophyll, protein, and starch con­tent of N. oleoabundans were also measured under the — N and+N scenarios (Table 1). Nitrogen deprivation lead to a reduction in nitrogen-containing chlorophyll content. This loss of chlorophyll is consistent with the light green color of chlorosis observed in the cultures under nitrogen limitation (Figure 1A inset). Also under nitrogen limitation, a decrease in cellular protein content and an increase in cellular starch content were observed. The observed changes in metabolite and biomolecule contents suggest the redirection of metabolism in N. oleoabundans during nitrogen limitation to reduce nitrogen-containing compounds (protein and chlorophyll) and fa­vor the accumulation of nitrogen free storage molecules TAGs and starch.