Microalgal oil is a potential energy source because it can be easily converted to fatty acid methyl ester or hydrocarbon type of diesel, and it is produced with relatively higher productivity compared with oil from plants and animals. Heterotrophic growth of microalgae is superior due to its high final product concentration; however, the cost of the raw materials is unacceptable if sugar is utilized as the carbon source. The aim of this study is to optimize the lipid accumulation of Chlorella protothecoides by using carbon sources other than glucose in heterotrophic and mixotrophic cultures. Different factors such as different carbon sources, carbon to nitrogen ratio, initial pH level, salinity, and rotational speed are studied in affecting the cell growth and the oil accumulation. Our experiments revealed that the heterotrophic and mixotrophic cultures of C. protothecoides grew better than autotrophic cultures. C. protothecoides can grow on glycerol or acetate, as well as on glucose. Several stress factors were confirmed or discovered to significantly increase the lipid content of microalgae cells. The replacement of glycerol and acetate as carbon sources for microalgae cultivations provides potential for waste utilization: glycerol from biodiesel industry and acetate from biohydrogen production.

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Cultivation of microalgae to utilize CO 2 and nutrients in the wastewater to generate biofuel products is a promising research objective. However, the process faces tremendous technical difficulties, especially the harvest of microalgae cells, an economically challenging step. Several researchers recently reported co-culturing of filamentous fungi with microalgae so that microalgae cells can be co-pelletized in order to facilitate the cell harvest. This algae pelletization via the filamentous fungi represents an innovative approach to address both the cost and sustainability issues in algae biofuel production and also has potential with direct commercial applications. This paper reviews the current research status in this area and some possible drawbacks of this method in order to provide some possible directions for the future research.

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