The heterotrophic nitrogen (N) deprivation (HND) was the first employed to culture Chlorella protothecoides for microalgal lipids, which are regarded as one of the most promising feedstocks for biodiesel production. First, C. protothecoides was cultivated heterotrophically to achieve high biomass, and the broth was then transferred to N deprivation environment for lipid accumulation. This study aims to investigate proteomic changes in C. protothecoides cells and identify the molecular pathways responsible for lipid storage with HND. Approximately 72% of biomass (13.89 g/L) accumulated as lipids after 240 h, which equates to a lipid productivity of 10.0 g/L. This result represents an increase of 79.5% compared with the lipid yield from the simple heterotrophic mode. Furthermore, 33 altered proteins in HND-cultured algal cells were successfully identified, including 13 down-regulated proteins involved in photosynthesis, protein synthesis and folding, gene regulation and β-oxidation of fatty acids; 15 up-regulated proteins related to carbohydrate metabolism, stress response and defense, amino acid biosynthesis and secondary metabolite biosynthesis; and 5 hypothetical proteins. Analysis using the Kyoto encyclopedia of genes and genomes showed that the carbohydrate metabolism and inhibition of fatty acid catabolism are major routes for lipid accumulation in algal cells. Our results proved that the combination of heterotrophism and N deprivation can increase lipid productivity for algal-based biodiesel. In future studies, further functional analysis of these altered proteins would help elucidate the complicated relationship between cell growth and lipid accumulation in microalgae.

Microalgae can grow heterotrophically with glucose as a carbon source to accumulate high amounts of lipids, which are one of the most promising feedstock for biodiesel production. However, glucose is one of the main contributors in the high cost of microalgae culture. This study was to evaluate the feasibility of using potato starch hydrolysate (PSH) instead of glucose as a carbon source for microalgal lipid production. When PSH was used as a carbon source for the heterotrophic microalga Chlorella protothecoides, the highest biomass and total lipid yields were 20.23 and 10.43 g/L, respectively, representing 1.3- and 1.4-fold higher than those obtained when glucose is used as a carbon source. A noticeable increase in the number of oil droplets was observed in the PSH-feeding algal cells. In addition, the calorific value was significantly increased to 29.08 kJ/g compared to the calorific value of 22.65 kJ/g from the cells grown in glucose medium. These findings confirmed that the lipid content greatly improved in PSH-feeding cells. Moreover, PSH-feeding cells produced significantly elevated levels of 17:0, 17:2Δ9,12, 19:1Δ9, and 19:2Δ9,12 fatty acids (over 84%), which are feasible ingredients for biodiesel production. These results demonstrated that green alga fatty acids from heterotrophic C. protothecoides obtained by PSH feeding possess properties that are beneficial for biodiesel production.