Agal-fermentation-based microbio-diesel production was realized through high-cell-density fermentation of Chlorella protothecoides and efficient transesterification process. Cell density achieved was 16.8 g l(-1) in 184 h and 51.2 g l(-1) in 167 h in a 5-l bioreactor by performing preliminary and improved fed-batch culture strategy, respectively. The lipid content was 57.8, 55.2, and 50.3% of cell dry weight from batch, primary, and improved fed-batch culture in 5-l bioreactor. Transesterification was catalyzed by immobilized lipase, and the conversion rate reached up to 98%. The properties of biodiesel from Chlorella were comparable to conventional diesel fuel and comply with US standard for Biodiesel. In a word, the approach including high-density fermentation of Chlorella and enzymatic transesterification process were set up and proved to be a promising alternative for biodiesel production.

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The green alga Chlorella protothecoides has received considerable attention because it accumulates neutral triacylglycerols, commonly regarded as an ideal feedstock for biodiesel production. In order to gain a better understanding of its metabolism, tracer experiments with [U-13 C]/[1-13 C]glucose were performed with heterotrophic growth of C. protothecoides for identifying the metabolic network topology and estimating intracellular fluxes. Gas chromatography-mass spectrometry analysis tracked the labeling patterns of protein-bound amino acids, revealing a metabolic network consisting of the glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle with inactive glyoxylate shunt. Evidence of phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme activity was also obtained. It was demonstrated that the relative activity of the pentose phosphate pathway to glycolysis under nitrogen-limited environment increased, reflecting excess NADPH requirements for lipid biosynthesis. Although the growth rate and cellular oil content were significantly altered in response to nitrogen limitation, global flux distribution of C. protothecoides remained stable, exhibiting the rigidity of central carbon metabolism. In conclusion, quantitative knowledge on the metabolic flux distribution of oleaginous alga obtained in this study may be of value in designing strategies for metabolic engineering of desirable bioproducts.