Analyses of lipidic bodies from green microalgae.
This study presents the analyses of oil body components in microalgae which may be involved in oil droplet assembly including certain triacylglycerol precursors which can be processed to biodiesel, an alternative fuel source. Stress induction of microalgae, Chlorella vulgaris CCAP 211/11B and Dunaliella primolecta CCAP 11/34 was achieved by exclusion of nitrates in growth media. Contrary to other forms of nitrogen depletion, this condition did not greatly enhance lipid biosynthesis in the microalgae. Confocal microscopy and fluorescent dyes nile red and bodipy were employed for the visualization of lipidic body components. The fluorescence hues emitted by neutral lipids and phospholipids were differentiated from those due to autofluorescence and chlorophyll using ZEN software to analyse images from a Zeiss LSM 710 confocal microscope. Oil from both algae, which were subjected to transesterification and gas chromatography, revealed a predominant fatty acid, namely palmitic acid (C16:0). D. primolecta produced linolelaidic acid (C18:2n6t) under growth conditions involving both nitrate supplementation and exclusion; whilst the longest fatty acid, docosanoic acid (C22:0 chain) was produced by the alga C. vulgaris only under conditions of nitrate supplementation. Nitrate limitation had minimal effect on the oil hydrocarbon yield which increased only 0.02% and 0.01% for C. vulgaris and D. primolecta, respectively. The highest biodiesel yield of 26.11 % was recorded from D. primolecta when grown under conditions of nitrate exclusion. The protein concentrations extracted from oil of the former alga ranged from 1.87 - 1.95 Gg/ml when grown under nitrate supplemented conditions and 1.74 - 1.90 Gg/ml when nitrate was excluded from the media. The protein concentrations extracted from oil of D. primolecta ranged from 1.91 - 2.23 Gg/ml and 1.88 - 1.98 Gg/ml, respectively, when the algae were grown in the presence and exclusion of nitrates. In the adaptation of protocols for protein extraction from oil, sunflower and salmon oils were initially used. Sunflower oil extracts produced by 10% (w/v) SDS treatment, yielded protein bands of 198, 96, 70 and 58 KDa on 10% (w/v) polyacrylamide gels while 6M urea treatment yielded a band of 200 KDa. Salmon oil treated with 10% (w/v) SDS and 6 M urea yielded bands of 195 and 27 KDa, and 198 KDa, respectively, as well as common bands of 68 and 64 KDa. In comparison, the extraction of discrete proteins from algal oil proved to be difficult as the extractants SDS and urea could have denatured protein components into subunit structure.