Diana Meiying Wong
Published: 2013
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The high demands for transportation fuel and depleting supply of petroleum have pushed the search for more sustainable liquid fuels. Biofuels derived from microalgae have the ability to displace petroleum-derived diesel fuel compared to other oil crops because microalgae are rich in oil, grow extremely rapid, and do not compromise the production of food. Intracellular lipids (oils) in microalgae consist of triacylglycerides (TAGs) that can be converted to biodiesel in the form of fatty acid methyl esters (FAMEs). This dissertation describes research to develop analytical tools for lipid visualization and chemical genetic screening to identify chemical triggers that increase microalgal lipid productivity for biofuel applications. Chapter one provides a background of the benefits of microalgae as a feedstock for biofuel production and how lipids can be converted to biodiesel. I introduce the concept of using chemical genetic methods of phenotypic screening to increase lipid production in microorganisms. This chapter also describes how analytical tools are utilized to measure intracellular lipids in microalgae and to understand the formation of lipid bodies. This chapter introduces two lipophilic dyes, Nile red (9-(diethylamino)-5H-benzo-alpha-phenoxazin-5-one) and BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) to analyze lipids using our analytical techniques. I also describe the use of laser scanning confocal microscopy and image quantification software to understand the formation of lipid bodies (LBs) in different microalgal strains. Chapter two describes the process of optimizing a high-throughput and rapid method for phenotypic screening of lipids in microalgae and how I translated the method to various applications. In this chapter, I demonstrate how I optimized a Nile red (NR) protocol to monitor intracellular lipids in microplates for phenotypic assays. Subsequently, I describe the use of this optimized method in the following investigations: 1) to discover chemical triggers that enhance lipid levels in microalgae, 2) to identify the optimal day to harvest for maximum lipids using aliquots from a culture, and 3) to measure intracellular lipids for a variety of yeast species. Chapter three discusses the fact that treatment with ethylenediaminetetraacetic acid (EDTA) significantly enhances the fluorescence intensity for intracellular lipid staining in T. suecica using Nile Red dye. In this chapter, I compare the effect of EDTA to common additives DMSO and glycerol, and found that EDTA was specific to T. suecica. Using optimized conditions with either DMSO or EDTA, I investigated six different strains of oleaginous microalgae in microplates and compared chemical treatments for phenotypic screening of intracellular lipids by directly using suspensions of microalgae in growth media. I demonstrate that EDTA is recommended as a treatment to enhance lipid staining for T. suecica because it maintains microalgal viability, is non-toxic, and is cost-effective. Chapter four discusses the first phenotypic screening with microalgae to study lipid metabolism pathways and discover organic small molecules as chemical triggers that increase growth and lipid production. I developed a microplate assay for analysis of intracellular lipids using Nile Red fluorescence in order to screen a collection of diverse bioactive organic molecules (e.g. kinase inhibitors) with four strains of oleaginous microalgae. I performed statistical analysis on all assay data and showed how lead compounds were identified in microplate screening for evaluation in larger cultures to compare lipid production and composition using gravimetric analysis and secondary lipid screening methods. I discuss, in detail, the compounds that I investigated in dose response screening, which consisted of lipoxygenase inhibitors, protein tyrosine phosphatase inhibitors, and protein tyrosine kinase inhibitors. This work demonstrates that small molecules can increase lipid productivity without decreasing overall growth rate and biomass production, and are effective within the context of larger batch culture experiments. Chapter five describes a method to study the accumulation patterns of lipid bodies (LBs) in different microalgae strains and culture conditions utilizing laser scanning confocal microscopy (LSCM) with BODIPY 505/515 staining, in parallel with NR fluorescence analysis of intracellular lipids in microplates. Microalgae contain LBs composed of triacylglycerols, which can be converted to biodiesel. Phaeodactylum tricornutum and Tetraselmis suecica were selected as model organisms and monitored throughout the growth phases in standard and nitrogen- deficient growth conditions. Utilizing image quantification techniques, the number and morphology of LBs suggest that P. tricornutum accumulates lipids by merging with existing LBs, while T. suecica synthesizes new LBs. I observed that T. suecica accumulates a higher number of LBs and total volume of lipids per cell, while P. tricornutum accumulates only 1-2 LBs with a larger volume per LB. LSCM analysis complements NR methods because LSCM provides three- dimensional images of lipid accumulation at a cellular level, while NR analysis can quickly monitor the total levels of intracellular lipids for phenotypic screening. Using NR analysis, I observed that the optimal harvest date for P. tricornutum and T. suecica in standard cultivation conditions is 24 and 42 days, respectively. Comparison with nitrogen-deficient growth conditions is utilized as a model to confirm that LSCM and NR analysis can be used to study lipid storage and productivity for diverse growth conditions and various strains of microalgae.