Arginine-fed cultures generates triacylglycerol by triggering nitrogen starvation responses during robust growth in Chlamydomonas

Under nitrogen (N) starvation, certain microalgae increase carbon storage in the form of lipid droplets while also downregulating photosynthesis and eventually terminating growth. To improve lipid yield, we asked whether lipid droplets and N starvation responses can be induced without limiting growth or photosynthesis. In the chlorophyte Chlamydomonas reinhardtii, N starvation induces gametogenesis alongside lipid droplet accumulation, and gametogenesis has been observed in arginine-fed cultures wherein the arginine was provided as the sole N source. We therefore assessed whether arginine-fed cultures displayed N starvation responses other than gametogenesis in mixo- and phototrophic conditions, representing two primary modes of nutrition that may affect N starvation responses. We showed that arginine-fed cultures using two lab strains of C. reinhardtii supported normal mixotrophic growth, constitutively turned on N starvation-induced genes at the equivalent level to N-starved cells, and increased the triacylglycerol content of total fatty acids by 125–400% relative to ammonium-fed cultures. The lipid profile of triacylglycerol in these arginine-fed mixotrophic cultures exhibited 3 to 5.5-fold enrichment of saturated and monounsaturated fatty acids, a preferred characteristic of biodiesel precursors. Arginine-fed phototrophic cultures likewise turned on N starvation-induced genes, accumulated lipid droplets, and led to a 50% reduced growth rate per day while reaching a 3 to 6.5-fold more cell density at the stationary phase relative to ammonium-fed cultures. To test the applicability of our result to algae outside the green lineage, we conducted similar experiments with the diatom Phaeodactylum tricornutum, which also accumulated 2 to 7-fold more neutral lipids in arginine-fed phototrophic cultures without growth impairment relative to nitrate-fed cultures. We document a system wherein N starvation responses are induced without compromising photosynthesis or growth, thereby suited to the production of valuable chemicals and biofuel precursors without requiring stressors in microalgae.