| Agricultural crops including corn, sugar cane, and oil palm have been investigated as potential sources for biofuel; however, they produce only a fraction of the oil percent biomass as compared to that of microalgae. Growth and lipid production by microalgae is regulated by a variety of environmental factors, including light intensity, availability of nutrients, temperature regime and salinity. We assessed 14 strains of the saltwater algae Dunaliella spp. (Teodoresco) in unialgal cultures within four species to determine a best strain or strain(s) as potential feedstock for biofuels. The taxonomy of these 14 strains was elucidated by comparing both physiological characteristics and the ITS2 and 18S regions. After careful analysis, the data suggest that the 14 strains grouped within four species: D. tertiolecta, D. pseudosalina, D. salina, and D. viridis. In addition, the isolation and accurate quantification of neutral lipids in Dunaliella was developed from existing techniques. Nile Red was optimized as a qualitative stain to rapidly screen and visualize neutral lipids. Direct transesterification was determined to be the best quantitative method because it yielded high amounts of neutral lipids with precise and reproducible results when compared to conventional extraction methods. Seven strains were selected for further efforts to enhance lipid production using salinity stress, nutrient limitation, pH stress, continuous light, and bubbling with carbon dioxide (CO2). High salinity yielded the maximum total fatty acid (FA) content (up to 65% by dry weight) in comparison to controls (∼10-25% total FAs). High pH x low salinity, low pH, and continuous light x CO2 yielded near maximum FA content (56%, 43%, and 42%, respectively). Nitrogen and/or phosphorus limitation and 12:12 (light:dark photoperiod) x CO 2 did not significantly enhance FA production (23% and 31%, respectively). Results were strain-specific with high intraspecific variation observed within each environmental stressor. Glycerol production, a known mechanism of osmoregulation in Dunaliella, was measured in a short-term salinity stress experiment and found to significantly increase 30 min to 24 hr after exposure. In addition, the glycerol biosynthesis gene, glycerol-3-phosphate dehydrogenase or GPDH, was significantly expressed 30 min to 2 hr in response to hyperosmotic stress. The data suggest that Dunaliella strains may incorporate a proportion of glycerol as triacylglycerol (TAG) under short-term, high-salinity stress. High lipid-producing strains were grown in mass culture, but at this time the commercialization of harvesting has not been proven economically feasible. Autoflocculation, electro-flocculation, and hollow-fiber filtration were compared as potential harvesting mechanisms for the mass culture of Dunaliella spp. Hollow-fiber filtration (>99% biomass recovery) as harvesting mechanism offers many attractive advantages (i.e. reuse of filtrate as culture medium) when compared to auto-flocculation and indirect electroflocculation (>95% biomass recovery). This research provides evidence that Dunaliella can be used as a source of biofuel because these strains can be mass-cultured; their lipids enhanced through a simple high-salinity adjustment; and commercially harvested. |
