Wastewater has been shown to contain the necessary nutritive requirements for the growth of microorganisms. The term, oleaginous, has been given to a classification of microorganisms know to produce up to twenty percent of the weight as oil. This study is designed to examine the potential accumulation of lipids within an oleaginous consortium grown on synthetic wastewater. Potential of the fluorescent stain, Nile red, as a lipid detector is also emphasized. Percentages of extractables greater than thirtyive percent were achieved within the oleaginous consortium using a nitrogen-limited medium. Low pH was found to increase the percentages of extractables. Xylose was shown to be a more optimal carbon source for accumulation than glucose. Nile red was shown to bind to intracellular inclusions and may be useful in monitoring lipid accumulation in industrial settings.

Nile red and Nile blue are intensely colored fluorescent dyes from the benzophenoxazine family. Modification of the donor amine with varying alkyl substituents was achieved with the principle aim being to investigate differences, both optical and physical, between dye scaffolds outfitted with secondary amines versus tertiary amines. These changes to the dyes’ architectural framework gave holistic effects to the bulk molecule by modulating hydrophobicity, optical properties, and protein binding constants to human serum albumin.

A set of three benzophenoxazine dyes, two completely soluble and one partially soluble in aqueous media, has been prepared and their spectroscopic properties examined. These dyes can be used as either donor or acceptor in synthesis of through-bond energy transfer cassettes. Structural modifications prevented aggregation in water and improved their fluorescence properties in water. Their absorption and emission were studied in both organic and aqueous media. Two of the three dyes have superior quantum yields in aqueous media as compared to other reported dyes. Improved quantum yield makes these dyes attractive candidates for biological studies in aqueous media. We have also prepared alkynes and iodo derivatives of benzophenoxazines, which can be used for synthesis of water-soluble, through-bond, energy transfer cassettes. Alkynes were prepared via Sonogashira coupling.

This project investigates the potential of the 27 Everglades green algal strains for producing biodiesel. The five potential strains chosen by measuring the neutral lipid content using the Nile red method were Coelastrum 46-4, Coccoid green 64-12, Dactylococcus 64-10, Stigeoclonium 64-8 and Coelastrum 108-5. Coelastrum 108-5 and Stigeoclonium 64-8 yielded the same amount of lipids in both Gravimetric and Nile red method. A linear relationship between algal biomass and lipid accumulation was seen in Coelastrum 46-4, Coccoid green 64-12, Stigeoclonium 64-8 and Coelastrum 108-5 indicating that increase in algal biomass increased the lipid accumulation. Nitrogen and phosphorous stress conditions were also studied where higher lipid accumulation was observed significantly (p < 0.05) in 64-8 Stigeoclonium and 64-12 Coccoid green. Collectively, it could be summarized that Coelastrum 108-5, Coccoid green 64-12 and Stigeoclonium 64-8 were promising in some aspects and could be used for further studies.

An assay based on Nile red fluorescence was developed for quantitative analysis of triglycerides, a common cellular component with important biological functions and is routinely analyzed for diagnosis of metabolic disorders and as an important feedstock of food industry and biodiesel production. Based on studies on the Nile red fluorescence of pure, binary, and ternary systems of triglycerides, ethanol, and water, 20% ethanol aqueous solution was determined to be the most suitable solvent for lipid fluorescence measurement. Excellent linearity was established for lipid samples in the range of 0.1- 0.5 mg/ml with several different lipid standards and vegetable oils. Results also suggest that the fluorescence of triglycerides was not sensitive to the fatty acid composition of lipids. This finding is important since it implies that the assay could potentially be used for the measurement of triglyceride content of different oil crops without causing significant variations. The results of this method were then verified by comparing with the results of the conventional gravimetric methods. The results of the fluorescence assay were consistently lower than that of the gravimetric method by approximately 10%. This phenomenon was tentatively attributed to the fact that the gravimetric method measures the total amount of lipophilic materials in samples while the fluorescence assay is selective to glycerides. Attempts were also made to apply this assay to estimate the lipid content of green alga Neochloris oleoabundans. However, the results were less than ideal due to the existence of interfering components in the extract of microalga samples that could significantly repress the fluorescence of lipids.

Analysis of seaweed as a vector for microplastics is an integral part of understanding the formation and deposition of micro-sized plastic waste in seawater. The project itself originated due to the influx of seaweed (and mismanaged plastic waste) residing on the shores of St. Kitts and Nevis and the constant deposition of plastic pollution intertwined within the seaweed. The natural occurrence of the two together lead to the consideration of fragmented plastics remaining on the seaweed. The objective of this research is to stain, identify, and quantify the concentration of microplastics sourced from both store-bought and environmental seaweed samples. A Nile red solution dissolved in either acetone or methanol was used to stain the microplastics, as per a proven method. The fluorescence of the stained microplastics was measured (excitation: 523-543 nm and emission: 580-640 nm) to identify potential dissolution. The seaweed was washed of microplastics and the solid particles collected were evaluated using infrared (IR) spectroscopy. The fluorescence and infrared spectrum results were compared to spectra within the Spectral data base system (SDBS) for the most common plastics: polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polypropylene (PP), polycarbonate (PC). The use of a fluorescence microscope allowed for direct quantification of microplastics over a specific area of the sample and therefore allowed for further identification of microplastic presence.

Gas detection is vital in different fields including environmental applications, clinical analysis, and homeland security. To perform these tasks the sensors need to be stable, sensitive, selective, operating at room temperature, rapidly responding, and easy to regenerate. On the other hand, most chemical sensors often suffer from a lack of selectivity, i.e., reacting more or less similarly to a collection of substances. As a result, these sensors may lead to false alerts. Even worse, the molecules to be detected could be masked by some interfering compounds which may result in failure to detect the targets. The goal of this research is to develop a portable gas-sensing device that integrates a zeolite/dye unit with an optoelectronic detector. At nano-scale the sensor is expected to be more accurate, more sensitive, and can better differentiate and detect one chemical component in a mixture of different gases. This could be achieved by incorporating fluorescent dyes into the zeolites' cavities, measuring gas absorption, desorption and photo-chromic interaction of dye and gases, interfacing the zeolite/dye sensor arrays with light source and electronic detectors and fully integrating the sensor arrays into a portable unit. This research addresses many of the above-sated threads. The highly fluorescent organic dye, nile red, was successfully included in the supercages of different zeolites Y (ammonium Y, hydrogen Y, and sodium Y) via chemical reaction. The research also developed an effective method to clean the synthesized inclusions, which is a combination of ultrasound and centrifuge. The cleaned inclusions were baked to remove any gases and/or moisture trapped inside the zeolites' structure. The spectra of the baked inclusions were used as references. The cleaned inclusions were optically characterized in terms of light absorption and fluorescence emission. When exposed to acetone, ethanol, methanol, and de-ionized water, the fluorescence emission spectra of zeolite-sodium-Y/nile-red inclusion showed a similar spectral shift compared to the reference spectrum. On the other hand, the fluorescence emission spectra of zeolite-hydrogen-Y/nile-red inclusion and zeolite-ammonium-Y/nile-red inclusion showed different spectral shifts compared to the reference spectra. This shows the successful proof of encapsulating the nile red dye in zeolites Y's cages, cleaning the zeolite/nile-red combinations, and measuring the desorption and fluorescence emission of the combinations. The optical characteristics of the nile red adsorbing to the external surface of the zeolites Y were studied as well. The research also included the design of the optical system to excite the sensing elements (zeolite/nile-red inclusions), and to collect the fluorescence response, the design and simulation of electronic circuits to condition and process electrical signal, and overall design of an integrated gas detector onto a pressed ceramic optical bench. / Electrical and Computer Engineering

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes single-molecule tracking (SMT) studies for the quantitative characterization of one-dimensional (1D) solvent-filled surfactant-templated mesoporous silica (STMS) materials and other nanostructured materials, such as double-stranded DNA. SMT permits the simultaneous and quantitative assessment of the nanoscale and microscale morphologies and mass-transport properties of the materials with nanometer-scale spatial resolution. The efficiency and selectivity of catalytic reactions and chemical separations occurring in liquid-filled mesoporous materials are governed by the translational and orientational mobilities and surface interactions of the incorporated reagents and analytes. Polarization dependent SMT results demonstrate that the dye molecules used as probes of materials nanostructure are tightly confined within the one-dimensional (1D) pores of surfactant-templated mesoporous silica films. Spectroscopic single molecule tracking (sSMT) data reveal that the hydrophobic probe dyes are confined within nonpolar regions of the nanomaterials For this dissertation, surfactant templated mesoporous silica films were prepared by the spin coating of acid catalyzed tetramethoxysilane (TMOS)-based silica sols on glass substrates in the presence of Cetyltrimethylammonium bromide (CTAB). Cylindrical CTAB micelles formed during evaporation of the solvent acted as a structure directing template, forming nanometer-sized one-dimensional pores within the silica films. SMT experiments were performed using a wide-field fluorescence microscope that was sufficiently sensitive to allow detection of the fluorescence from individual dye molecules. A series of perylene diimide (PDI) dyes was employed for basic structural characterization of the silica materials. Single molecule fluorescence was recorded in the form of fluorescence videos. These videos revealed the presence of immobile dye molecules, along with those diffusing in one and two dimensions (1D and 2D). The 1D diffusing molecules provided basic evidence for the confinement mass transport of the dye molecules within the silica mesopores. Spectroscopic single molecule tracking (sSMT) studies served as an extension of basic SMT experiments and were employed to determine the location of the molecules. The polarity sensitive dye Nile Red (NR) was employed in these studies. It exhibits 1D diffusion, consistent with its confinement to the cylindrical pores, as was also the case for the PDI dyes. The sSMT data revealed that the majority of NR molecules were found in nonpolar environments having polarities similar to that of n-hexane. Single molecule emission polarization (SMEP) measurements were employed to explore the orientational confinement of the dyes. The results of these experiments demonstrated that the PDI and NR molecules diffuse with their long axes aligned parallel to the long axis of the pores. All of the dyes employed were found to be orientationally confined to ∼1 nm diameter pathways within the pores. The diffusion coefficient for the dyes was also shown to be ∼10^3 -fold smaller than in bulk solution. The results of the NR studies demonstrate that the dye molecules were confined to the hydrophobic cores of the micelles, and provide support for the conclusion that the PDI dyes are similarly confined. These studies afford an enhanced understanding of how nanostructuring of the pore-filling medium in solvent- and surfactant-filled mesoporous materials governs the mass transport and surface interactions of incorporated reagents and analytes. The dependence of molecular confinement on dye charge and structure was also explored in this dissertation. The confined translational and orientational motions of a series of four different PDI dyes diffusing along one dimension (1D) within individual cylindrical silica mesopores were investigated in these studies. Specifically, the motions of cationic and anionic PDI dyes were compared to those of two uncharged PDIs having different alkane tail lengths. All four dyes exhibited populations that were immobile, along with separate populations that diffused in either 1D or 2D. The anionic and cationic PDI dyes exhibited the largest and smallest populations, respectively, of immobile molecules, suggesting that electrostatic interactions between the charged dyes and the cationic surfactant head groups play a significant role in limiting molecular motion. The cationic and anionic PDI dyes also exhibit the largest populations of 2D diffusing molecules, suggesting they may more readily pass between the cylindrical micelles and through the silica pore walls. All four dyes also emit strongly polarized fluorescence as they move in 1D, indicating they are orientationally confined within the nanochannels. Nile Red dye was used to determine the dielectric constant, ε, of nonpolar microenvironments in double-stranded DNA (ds-DNA) single molecules both in aqueous buffer solution and when adsorbed on amine-modified chemical gradient surfaces. The value of ε within the DNA decreased with increasing buffer concentration. Values of ε ∼ 6.75 and ∼3.00 were obtained in 0.1 mM phosphate buffered saline (PBS) and in 10 mM PBS, respectively. Similar effects were observed upon adsorption to chemically graded amine-modified silica surfaces. Under 1 mM buffer, ε was measured to be ∼2.84 and ∼1.90 at the low amine (high silica), and high amine (low silica) ends of the gradient, respectively. An increase in the buffer concentration again led to a decrease in ε, but only at the low amine end. It is concluded that high buffer concentrations and binding to an amine surface cause condensation of the ds-DNA, forming less polar microenvironments within its structure. These results provide important knowledge of the factors governing the polarity of DNA microenvironments to which intercalators bind.

Diacylglycerol acyltransferase (DGAT) is a key for the synthesis of triacylglycerol (TAG) from diacylglycerol in the unicellular green alga Chlamydomonas reinhardtii.Acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS) are responsible for the synthesis of fatty acids. We found the TAG and fatty acid synthesis related genes in C. reinhardtii, including five DGAT (DGAT1 (JGI 184281), DGAT2 (JGI 400751), DGAT3 (JGI 285889), DGAT4 (JGI 141301), and DGAT5 (JGI 190539)), three £] ketoacyl-ACP reductase isoforms ( (JGI 153976), (JGI 153976), and (JGI 194728)) and two £] ketoacyl-ACP synthase isofroms ( (JGI 139619) and (JGI 205887)) for FAS, and ACC £\ (NCBI XP_001696945.1), ACC £] (NCBI XP_001703187.1) and ACC biotin carboxylase ( NCBI XP_001702319.1)) for ACCase in C. reinhardtii. This investigation designed the primers of the above genes to determine whether acetate influences their mRNA expression levels in cell-wall less strain CC400 in the nitrogen starvation condition. The results showed that the absence of nitrogen in the medium triggered the lipid accumulation for the strains of CC400 in the condition of 50 £gE light. DGAT3 mRNA levels were increased by nitrogen starvation. For the FAS genes, in the strain of CC400 showed no increased mRNA levels upon exposure to nitrogen starvation. The mRNA levels of ACC£\, ACC £] and ACC biotin carboxylase were more or less decreased by nitrogen starvation in CC400 strains. Thus, the responses of DGAT gene expression to acetate supplement were checked. The absence of acetate from the medium partly inhibited the nitrogen starvation induced increases in lipid and DGAT3 mRNA levels, and the mRNA levels of DGAT1 and DGAT2 in the nitrogen starvation condition. However, DGAT4 mRNA levels were significantly induced by the absence of acetate from the medium. In conclusion, the present study demonstrate that acetate is required for the nitrogen starvation induced DGAT3 gene expression (mRNA levels) and lipid accumulation in C. reinhardtii.

This study was to determine the effects of several selected environmental factors (temperature, salinity, light intensity, and acetate) on the nitrogen starvation induced lipid accumulation in Chlamydomonas reinhardtii CC 400 by the Nile Red staining of lipid in the cells. Nitrogen starvation induced lipid accumulation, the extent of lipid accumulation increased as nitrogen concentrations in the medium decreased. For the 9.4 mM NH4Cl of HS medium as 100% N, the absence of NH4Cl from the medium will show the maximum induction in the lipid accumulation. This was also observed in the treatment of algal cells in mid-log phase by the absence of NH4Cl in the medium. A decrease in temperature down to 15oC depressed the nitrogen starvation induction in lipid accumulation for the algal cells from the mid-log phase, while the elevation in the light intensity up to 300 £gmol photons • m-2 • s-1 also showed an inhibitory effect. However, the transfer to darkness for the nitrogen starvation also inhibited the lipid increase. The addition of 100 mM NaCl enhanced the nitrogen starvation induced lipid accumulation but the NaCl level up to 200 mM inhibited the increment. The nitrogen starvation induction of lipid increase was partly inhibited due to the absence of acetate, whereas the increase in acetate concentrations in the medium did not have effect on lipid accumulation as compared to normal acetate addition in the medium. Overall, the results of the present study show that light and acetate are essential factors for the maximum lipid accumulation in C. reinhardtii by nitrogen starvation.