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Background  Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass, and biofuel production because of their allowance of high cell density levels, as well as simple production technology.  Tetradesmus bernardii , a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of  T. bernardii  in heterotrophic batch culture under two nitrogen sources (NaNO 3  and CO(NH 2 ) 2 ). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of  T. bernardii  in heterotrophic culture.    Results   T. bernardii  can accumulate high biomass concentrations in heterotrophic batch culture where the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass exceeded 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was beneficial to lipid accumulation at a C/N ratio between 110 and 130. NaNO 3  used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of  T. bernardii . As the nitrogen was consumed in the medium, nitrogen metabolism-related genes were significantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely come from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time.    Conclusion   T. bernardii  exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid.

biotechnology for biofuels

Biomass, lipid accumulation kinetics, and the transcriptome of heterotrophic oleaginous microalga Tetradesmus bernardii under different carbon and nitrogen sources