Microalgae isolation, genetic improvement and transcriptional profiling for high-efficiency lipid production

Abstract

Interest in a sustainable alternative to fossil fuels has recently intensified as the effects of rising oil costs and dangers of increasing CO2 levels are becoming more apparent. Microalgae-derived biodiesel provides a promising alternative, as theoretical calculations of areal microalgal oil production are at least 10 to 20-fold greater than any other biofuel crop. Importantly, microalgae cultivation can achieved without competing with precious arable land or rainforests and freshwater resources. At present, large-scale microalgal oil production is not economically viable, and many technical and biological barriers still need to be overcome in order to improve lipid productivity and reduce cost of production. The main objective of this thesis was to improve microalgal lipid productivity and gain a deeper understanding of the molecular mechanisms behind lipid biosynthesis. In the first part of the present work, numerous microalgal strains were collected from coastal water in South East Queensland, Australia. After isolation of pure strains, the fastest growing algae were compared to each other using a specially developed standardised lipid induction assay. This assay combined rapid exponential growth with a nutrient starvation phase to induce lipid biosynthesis, a survival mechanism of microalgae under adverse conditions. Based on their lipid productivity and fatty acid profile, several strains, including Nannochloropsis sp. BR2 and several Tetraselmis sp,, were identified as potential feedstock cultures for biodiesel production. As the identified cultures can be considered as undomesticated, one method of further increasing algal lipid productivity is via mutation and selection of high-lipid yielding algal strains. Instead of using a transgenic approach, this research used adaptive evolution methods, incorporating mutagenesis and high-throughput selection to select for high-lipid yielding algal strains. UV-C and different laser beams were used as mutagens, followed by a selection method encompassing flow cytometry and microplate readers to effectively select individual cells with high lipid contents, but also uncompromised growth. After several generations of mutation and selection, higher lipid accumulation potential was observed in several strains. This research also focused on understanding the underlying mechanisms of nitrogen- starved lipid induction in Tetraselmis sp. M8 through various growth phases.  Transcriptional profiling using RNA-Seq and quantitative real-time PCR analysis of this previously unsequenced genus, combined with physiological measurements after nutrient starvation, revealed that early lipid accumulation was predominately due to a reduced fatty acid degradation rate, while the rate of lipid biosynthesis remained unchanged. At 48 h onwards however, the expression of lipid biosynthesis genes was significantly upregulated, indicating lipid accumulation was now an effect of active triacylglyceride (TAG) synthesis. This first report on the molecular mechanisms of lipid accumulation in Tetraselmis sp. identified potential bottlenecks and target genes for metabolic engineering to maximise lipid accumulation in microalgae. Apart from strain improvement, culturing and lipid induction techniques offer further scope to optimise lipid productivity. Current efforts in the development of cost-effective harvesting and algal oil extraction procedures may further position microalgae as a significant feedstock for economical biodiesel production.