Many microalgae are reported to accumulate TAG under high concentrations of bicarbonate and nitrogen deficiency, but their physiological responses remain unknown and the associated applied technology is rarely explored. We found that single high concentration of bicarbonate did not enhance lipid content, except when combined with nitrogen limitation in Desmodesmus sp. NMX451, and the lipid contents obtained were independent from nitrogen limitation and alkalinity stress. Further analysis showed that adding bicarbonate not only retarded the cell cycle, repressed starch accumulation but also promoted polar lipid content. Nitrogen limitation meantime caused protein and chlorophyll degradation with a corresponding increase in total lipid content. Following the aforementioned insights, combined cultivation was performed at an outdoor 5 L scale, and higher biomass and lipid productivities were obtained. These results suggest that bicarbonate can be used as a "trigger" for rapid lipid accumulation in microalgae during nitrogen depletion, and this is an effective new strategy for the cultivation of oleaginous microalgae.