It is also noticeable that the overall rates of oil production might be lower in the case of nutrient deficiency due to the overall low- er biomass achieved. Therefore factors other than nitrogen should be considered altogether. Initial carbon to nitrogen (C/N) ratio in the medium has signif- icant impact on the biosynthesis of lipids in microalgae. With the addition of glucose as organic carbon source to the medium and the tremendous decrease of nitrogen source in the medium, a crude lipid content up to 55.2% was achieved in heterotrophic C. protothecoides, which was about 3.4-fold that in photoautotrophic C. protothecoides [41]. Chen and Johns [34] also investigated the ef- fect of C/N ratio and aeration on the fatty acid composition of het- erotrophic Chlorella sorokiniana. When C/N ratio of approximately 20, cell lipid content was at a minimum and increased at both higher and lower C/N values.