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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.
This dissertation describes the creation and development of a chemical genetic assay to screen small molecules in microalgae, with the goal of increasing growth and lipid yields. Chapter one describes the motivation of research to investigate green energy sources such as microalgae by giving background information regarding the current fuel situation and by discussing how microalgae chemical genetics could potentially solve thisproblem. From there it describes the use of mass spectrometry to profile the changes induced in microalgae lipid profiles, since this analytical tool is essential for the verification of lipid modification. Chapter two describes the development of the chemical genetic assay. Beginning with the initial screening experiments, the development and verification of the efficacy of screening methods are discussed. Test screens are run with positive control compoundsbased on previous publications to assist in method development. Then 54 compounds are chosen for the initial screen, and the results between four different algae species are discussed and compared. Lead compounds for additional dose response screening areselected and screened as well to determine which compounds will be selected to progress to batch cultures. Chapter three details the development of a 500 mL batch culture screening. Lead compounds selected and optimized in lead compound dose response screening are then screened in larger culture volumes to see if results remain consistent over volume changes. This additional screening in 500 mL batch cultures of the lead compounds found earlier allows for gravimetric analysis of nonpolar extracts which allow for direct
This book provides the reader with an updated comprehensive view of the rapidly developing and fascinating field of fluorescence spectroscopy and microscopy. In recent years, fluorescence spectroscopy and microscopy have experienced rapid technological development, which has enabled the detection and monitoring of single molecules with high spatial and temporal resolution. Thanks to these developments, fluorescence has become an even more popular method in physical, biological and related fields. This book guides the reader through both basic and advanced fluorescence spectroscopy and microscopy approaches with a focus on their applications in membrane and protein biophysics. Each of the four parts: A - Fluorescence Spectroscopy, B - Fluorescence Microscopy, C - Applications of Fluorescence Spectroscopy and Microscopy to biological membranes and D - Applications of Fluorescence Spectroscopy to protein studies are written by experts within the field. The book isintended for both complete beginners who want to quickly orient themselves in the large number of existing fluorescent methods, as well as for advanced readers who are interested in particular methods and their proper use. Chapter “Dynamics and Hydration of Proteins Viewed by Fluorescence Methods: Investigations for Protein Engineering and Synthetic Biology” is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
Lipophilic dye probes are widely used for labelling of cells, organelles, liposomes, viruses and lipoproteins. The lipophilic dye diffuses in the membrane and stains the cell and cells even tolerate the lipophilic dye in high concentration. The fluorescence of styryl dyes increases after insertion into the hydrophobic environment of the lipid membrane compared their fluorescence in the aqueous phase solution. The alkyl chains of the fluorescent styryl dye probe insert into membranes and are used to understand their biophysical properties and their behavior in lipid bilayers. The mechanism of incorporation of the dyes into cell membranes, or vesicle model systems, is not resolved. In this study we used a modified dialkylaminostyryl fluorescent lipid, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA), replacing the I- counterion with the Cl- anion to make DiA-Cl increase hydration of the polar head and to enable self-assembling in water and formation of vesicles. Vesicles composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine)/DiA, DPPC (1,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine) /DiA, DSPC (1,2-distearoyl-sn-glycero-3- phosphatidylcholine) /DiA, DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine)/DiA, DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine)/DiA and DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)/DiA have been prepared in mole ratios between 100/0 to 0/100, in order to investigate the effects of chain length and headgroup type on chain packing and phase separation in these mixed amphiphilic systems, using nanocalorimetry, dynamic light scattering and fluorescence data, as well as confocal laser scanning microscopy (CLSM) and cryo-transmission electron microscopy (Cryo-TEM). In addition, we report the self-assembly of DiA-Cl, to form H-aggregates of lipid bilayers in aqueous solution, beyond a critical vesicle concentration. Lipid bilayers can be fused onto silica nanoparticles (NPs) to form supported lipid bilayer (SLB)-NPs. (SLB)-NPs have a varous interdisciplinary applications from medicine to environmental fields and agriculture sciences. Here, the lipids on the nanoparticles were used for two applications. One was to adsorb polycyclic aromatic hydrocarbons (PAHs) from the environment and the other was as vehicles for foliar delivery of nutrients to plants. Silica SLB nanoparticles can increase the solubility of Benzo[a]Pyrene (BaP) in order to extract the BaP from soil for in situ biodegradation. Initial studies were begun on the effect of foliar application of silica SLBs nanoparticles on plants. The SLBs to be used were prepared using both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and DiA, in order to determine whether the lipid increased the entry of the silica into the plant leaves and whether the lipids also entered.
It is well-known that photosynthetic cells of various microalgae species display distinct fluorescent properties. The efficiency of self-fluorescence excitation and emission at different wavelengths depends on the structure of photosynthetic system and particularly on the structure of antenna complex of specific strains. The peculiar structure of blue-green algae light-harvesting complex allows to discriminate and classify known and new cells up to species/strain level by means of microscopic spectroscopy. In this chapter, a novel fluorescent spectroscopic technique for microalgae species discrimination will be presented. This method is based on a special data processing of a set of fluorescent spectra, obtained from a single photosynthetic cell of microalgae, particularly from cyanobacterial cells. According to the presented technique, single-cell self-fluorescence spectra are recorded by means of confocal laser scanning microscopy (CLSM), and data processing is conducted via linear discriminant analysis (LDA) and artificial neural networks (ANN).
Lipids are crucial biomolecules for the well being of humans. Altered lipid metabolism may give rise to a variety of diseases that affect organs from the cardiovascular to the central nervous system. A deeper understanding of lipid metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of lipid diseases. Lipid visualization remains a complex task because of the perturbative effect exerted by traditional biochemical assays and most fluorescence markers. Coherent Raman scattering (CRS) microscopy enables interrogation of biological samples with minimum disturbance, and is particularly well suited for label-free visualization of lipids, providing chemical specificity without compromising on spatial resolution. Hyperspectral imaging yields large datasets that benefit from tailored multivariate analysis. In this thesis, CRS microscopy was combined with Raman spectroscopy and other label-free nonlinear optical techniques to analyze lipid metabolism in multiple biological systems. We used nonlinear Raman techniques to characterize Meibum secretions in the progression of dry eye disease, where the lipid and protein contributions change in ratio and phase segregation. We employed similar tools to examine lipid droplets in mice livers aboard a spaceflight mission, which lose their retinol content contributing to the onset of nonalcoholic fatty-liver disease. We also focused on atherosclerosis, a disease that revolves around lipid-rich plaques in arterial walls. We examined the lipid content of macrophages, whose variable phenotype gives rise to contrasting healing and inflammatory activities. We also proposed new label-free markers, based on lifetime imaging, for macrophage phenotype, and to detect products of lipid oxidation. Cholesterol was also detected in hepatitis C virus infected cells, and in specific strains of age-related macular degeneration diseased cells by spontaneous Raman spectroscopy. We used synthesized highly-deuterated cholesterol to track its compartmentalization in adrenal cells, revealing heterogeneous lipid droplet content. These examples illustrate the potential of label-free nonlinear optical microscopy for unveiling complex physiological processes by direct visualization of lipids. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine.
Producing biofuels and bioproducts from microalgae is a promising path for low-carbon energy and products. Microalgal biomass is an attractive feedstock for the generation of carbon neutral biofuels and high-value bioproducts because of the high growth rate and lipid content of many microalgae species. Understanding the downstream processing of converting microalgal biomass to valuable products is a critical step in the biofuel industry. In this thesis, a novel microfluidic platform capable of precise control of processing parameters and providing optical access to reactions at high temperature and pressure was developed and applied to observe and quantify the biomass-to-bioproducts conversions in three distinct studies. First, for bioenergy application, hydrothermal liquefaction of microalgae was performed on this microfluidic platform monitored using fluorescence microscopy. A strong shift in the fluorescence signature from the algal slurry at 675 nm (chlorophyll peak) to a post-HTL stream at 510 nm is observed for reaction temperatures at 260°C, 280°C, 300°C and 320°C (P = 12 MPa), and occurs over a timescale on the order of 10 min. Biocrude formation and separation from the aqueous phase into immiscible droplets is directly observed and occurs over the same timescale. Second, many algal bioproduct efforts currently focus on high-value products such as astaxanthin due to the much-improved economics over producing fuels. Hydrothermal disruption of the cell wall for astaxanthin extraction from wet biomass using high temperature and pressure was demonstrated and studied using this microfluidic platform. Hydrothermal disruption at a temperature of 200 °C was shown to be highly effective, resulting in near-complete astaxanthin extraction from wet biomass - a significant improvement over traditional methods. Third, supercritical CO2 has relatively low critical temperature and pressure (31.1 °C and 7.4 MPa) is considered a greener solvent for bioactive compounds extraction. Supercritical CO2 extractions of astaxanthin with and without co-solvents (ethanol and olive oil) were performed on the microfluidic platform to study the extraction mechanism in each case. Astaxanthin extraction using ScCO2 achieved 92% recovery at 55 °C and 8 MPa applied over 15 hours. With the addition of co-solvents, ethanol and olive oil, the timescales of extraction process are reduced significantly from 15 hours to a few minutes, representing the fastest complete astaxanthin extraction at such low pressures. The direct observation of these complex reaction processes was made possible for the first time here, allowing visual characterization, fluorescence spectroscopy, and quantitative imaging of the conversion at the single-cell scale during all stages. This level of insight has simply not been possible with previous conventional reactors. Although batch reactors have advantages in, for instance, quantifying yields requiring large volumes of products, microfluidic reactors have advantages with respect to process control and visualization at cellular level - providing high resolution, real-time data on complex reactions. The innovative platform and results presented in this thesis provide new insight in the challenging area of biomass-to-bioproduct conversion, and provide insight that can inform larger scale operations.