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LIGHT INTENSITY INFLUENCES on ALGAL PIGMENTS, PROTEINS and CARBOHYDRATES: IMPLICATIONS for PIGMENT-BASED CHEMOTAXONOMY by Cidya Grant

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LIGHT INTENSITY INFLUENCES on ALGAL PIGMENTS, PROTEINS and CARBOHYDRATES: IMPLICATIONS for PIGMENT-BASED CHEMOTAXONOMY by Cidya Grant LIGHT INTENSITY INFLUENCES ON ALGAL PIGMENTS, PROTEINS AND CARBOHYDRATES: IMPLICATIONS FOR PIGMENT-BASED CHEMOTAXONOMY by Cidya Grant A Dissertation Submitted to the Faculty of The Charles E. Schmidt College of Science in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Florida Atlantic University Boca Raton, FL December 2011 ACKNOWLEDGEMENTS Special thanks to my research advisor Dr. J. W. Louda, for his guidance and support during this dissertation research. To the members of my dissertation committee: Drs. J. E. Haky, C. Parkanyi and S. Hagerthey, for answering pertinent questions and steering me on the right path to fulfilling the objectives and goals of this research. To the FAU-Harbor Branch Oceanographic Institute for NMR sample analyses: special thanks to Dr. Amy Wright for granting permission for instrument use and to her post- doctoral associate Dr. P. Winder for her assistance with experiment set-up. To the West natural products research group at FAU, particularly Dr. L. West, his post-doctoral associate Dr. P. Gupta and graduate student T. Vansach: thank you for the technical assistance with LC-MS analyses and NMR interpretation. To my teaching supervisors and mentors at FAU: Drs. D. Chamely-Wiik and E. Rezler, thank you for always challenging me to reach the highest academic standards, in research and teaching. The encouragement and assistance were all greatly appreciated. Funding for this material is based in part upon work supported by the National Science Foundation under Grant no. DGE: 0638662. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author and do not reflect the views of the National Science Foundation. iii ABSTRACT Author: Cidya Grant Title: Light Intensity Influences on Algal Pigments, Proteins and Carbohydrates: Implications for Pigment-Based Chemotaxonomy Institution: Florida Atlantic University Dissertation Advisor: Dr. J. W. Louda Degree: Doctor of Philosophy Year: 2011 Phytoplankton Chlorophyll a (CHLa), total protein, colloidal carbohydrates, storage carbohydrates and taxonomic pigment relationships were studied in two cyanophytes (Microcystis aeruginosa and Synnechococcus elongatus), two chlorophytes (Dunaliella tertiolecta and Scenedesmus quadricauda), one cryptophyte (Rhodomonas salina), two diatoms (Cyclotella meneghiniana and Thalassiosira weissflogii) and one dinophyte (Amphidinium carterae) to assess if algal biomass could be expressed in other indices than just chlorophyll a alone. Protein and carbohydrates are more useful currencies for expressing algal biomass, with respect to energy flow amongst trophic levels. These phytoplankton were grown at low light (LL = 37 µmol photons m-2 s-1), medium light (ML = 70-75 µmol photons m-2 s-1), and high light (HL= 200 µmol photons m-2 s-1). Even though pigment per cell increased with increasing light intensity, iv statistically light had very little effect on the CHL a: taxonomic marker pigment ratios, as they covaried in the same way. Protein, colloidal carbohydrates and storage carbohydrates per cell all increased with increasing light intensity, but they did not co- vary with CHLa. Statistical data showed that light intensity had a more noticeable effect on protein: CHL a, colloidal carbohydrate: CHLa, storage CHO: CHLa, therefore a general mathematical expression for these relationships cannot be generated. This study showed that light intensity does have an influence on these biomass indices, therefore, seasonal and latitudinal formulas may be required for meaningful algal biomass estimation. However, more studies are needed if that goal is to be realized. While studying the effects of light intensity on algal pigment content and concentration, a new pigment was isolated from a cyanophyte (Scytonema hofmanii) growing between 300-1800 µmol photons·m-2·s-1 and from samples collected in areas of the Florida Everglades. This pigment was characterized and structurally determined to possess indolic and phenolic subunits that are characteristic of scytonemin and its derivatives. In addition, the pigment has a ketamine functionality which gives it its unique polarity and spectral properties. Based on the ultra violet/visible absorbance data, this pigment was postulated to be protecting the chlorophyll a and cytochrome Soret bands as well as α and β bands of the cytochromes (e.g. cyt-c562) in the photosynthetic unit. v LIGHT INTENSITY INFLUENCES ON ALGAL PIGMENTS, PROTEINS AND CARBOHYDRATES: IMPLICATIONS ON PIGMENT-BASED CHEMOTAXONOMY LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ X I. INTRODUCTION ........................................................................................................... 1 The working hypothesis .................................................................................................. 4 BACKGROUND ............................................................................................................ 4 Methods for estimating algal biomass ........................................................................ 4 Converting CHLa to biomass.................................................................................... 10 Select algal metabolites which may serve as biomass indices .................................. 17 Photosynthesis overview ........................................................................................... 23 Novel sunscreen pigment .............................................................................................. 30 Overall goals of this study ............................................................................................ 33 II. MATERIALS AND METHODS ................................................................................. 34 Experimental organisms................................................................................................ 34 Algal culturing .............................................................................................................. 36 Culture conditions ..................................................................................................... 37 Cell counting. ................................................................................................................ 38 Chemical Analyses........................................................................................................ 39 Algal protein extraction ................................................................................................ 39 Algal protein measurement ....................................................................................... 39 Algal colloidal and storage carbohydrate extraction .................................................... 40 Algal colloidal and storage carbohydrate measurement ........................................... 40 Algal total organic carbon (TOC) extraction ................................................................ 41 Colorimetric determination of extracted TOC samples ............................................ 42 vi Nutrient analyses ........................................................................................................... 42 Pigment Analyses.......................................................................................................... 43 Ultra Violet - Visible (UV/Vis) Analyses of Extracts .............................................. 45 High Performance Liquid Chromatography (HPLC) ................................................... 46 HPLC Data Calculations ........................................................................................... 47 Statistical analyses ........................................................................................................ 49 Isolation and characterization of a new pigment. ............................................................. 50 IR analysis ................................................................................................................. 52 Mass Spectrometry .................................................................................................... 52 NMR analyses ........................................................................................................... 54 Acetylation reactions ................................................................................................ 54 Deuterium exchange reactions .................................................................................. 55 III. RESULTS - STATISTICAL ANALYSES ................................................................. 56 Significance of the algal species used in this study .................................................. 56 Analyses overview .................................................................................................... 59 Synechococcus elongatus .............................................................................................. 60 Microcystis aeruginosa ................................................................................................. 70 Dunaliella tertiolecta .................................................................................................... 78 Scenedesmus quadricauda ............................................................................................ 87 Rhodomonas salina ......................................................................................................
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