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Optimization and Characterization of the Growth Of OPTIMIZATION AND CHARACTERIZATION OF THE GROWTH OF THE PHOTOSYNTHETIC BACTERIUM BLASTOCHLORIS VIRIDIS AND A BRIEF SURVEY OF ITS POTENTIAL AS A REMEDIATIVE TOOL A Dissertation Submitted to the Graduate School of the University of Notre Dame in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Darcy Danielle LaClair, B.S. ___________________________________ Agnes E. Ostafin, Director Graduate Program in Chemical and Biomolecular Engineering Notre Dame, Indiana April 2006 OPTIMIZATION AND CHARACTERIZATION OF THE GROWTH OF THE PHOTOSYNTHETIC BACTERIUM BLASTOCHLORIS VIRIDIS AND A BRIEF SURVEY OF THEIR POTENTIAL AS A REMEDIATIVE TOOL Abstract by Darcy Danielle LaClair The growth of B. viridis was characterized in an undefined rich medium and a well-defined medium, which was later selected for further experimentation to insure repeatability. This medium presented a significant problem in obtaining either multigenerational or vigorous growth because of metabolic limitations; therefore optimization of the medium was undertaken. A primary requirement to obtain good growth was a shift in the pH of the medium from 6.9 to 5.9. Once this shift was made, it was possible to obtain growth in subsequent generations, and the media formulation was optimized. A response curve suggested optimum concentrations of 75 mM carbon, supplemented as sodium malate, 12.5 mM nitrogen, supplemented as ammonium sulfate, Darcy Danielle LaClair and 12.7 mM phosphate buffer. In addition, the vitamins p-Aminobenzoic acid, Thiamine, Biotin, B12, and Pantothenate were important to achieving good growth and good pigment formation. Exogenous carbon dioxide, added as 2.5 g sodium bicarbonate per liter media also enhanced growth and reduced the lag time. The optimized medium enhanced the total growth of cells slightly, and increased the culture optical density and pigmentation by a factor of 2 and 3, respectively, bringing the culture performance in the defined medium above the performance in the rich, undefined medium. A total of 31 chemicals from 17 chemical families were screened at either 10 mM or 2 mM concentration to determine their effect on the growth and pigment formation of B. viridis. Most alcohols surveyed caused significant enhancement to growth rate, and the primary carbon in a secondary alcohol was proposed as the site of attack allowing usage of these compounds. Aromatic compounds were not as successfully cultured, and potential mechanisms for cell damage and compound degradation were discussed. B. viridis demonstrated the ability to grow unimpaired in the presence of MEK, methyl chloride, mimethyl sulfoxide, dimethyl formamide, chloroethane, acetonitrile, and dioxane. Several methods for working with bacteria were presented. The Two-Point method, a rapid and reliable method to compare cell growth and health, was demonstrated. A mechanized device for spin plating was also introduced, both significantly reducing operator fatigue and injury, and increasing the consistency of plate count data. © Copyright by Darcy Danielle LaClair 2006 All rights reserved. To my parents, Lance and Christine, who instilled in me a love of learning, made it a priority from day one, and moved heaven and earth to let me pursue it. To my sister, Caitlin, end of discussion. To my husband, Eric, without whom I would have laughed much less and lost my perspective much faster and more often. ii CONTENTS FIGURES ........................................................................................................................viii TABLES .........................................................................................................................xvi ACKNOWLEDGMENTS ............................................................................................…xx CHAPTER 1: INTRODUCTION, MOTIVATION, AND BACKGROUND ................…1 1.1 Introduction ...................................................................................................…1 1.2 The Toxin Problem .......................................................................................…3 1.2.1 Hazardous substances .....................................................................…3 1.2.2 Sources of hazardous substances ...................................................…4 1.2.3 Release rates of hazardous substances ...........................................…4 1.2.4 Determining acceptable levels of hazardous substances ...............…5 1.3 Current Methods for Toxin Recovery ...........................................................…8 1.3.1 Overview ........................................................................................…8 1.3.2 Non-biological ...............................................................................…9 1.3.3 Biological..........................................................................................12 1.3.4 Why are new methods needed? ........................................................18 1.4 Types of Bacteria Utilized in Processing Waste .............................................20 1.5 Purple Non-Sulfur Bacteria (PNSB) ...............................................................24 1.5.1 Classification ....................................................................................24 1.5.2 Known toxins degraded by PNSB ...................................................27 1.5.3 Natural environment of PNSB..........................................................30 1.5.4 Relation of B. viridis in PNSB group ...............................................31 1.5.5 Why B. viridis for toxins? ................................................................32 1.6 Brief Overview of This Work .........................................................................33 1.6.1 Measured parameters .......................................................................33 1.6.2 Behavior in RMPABA and Minimal medias ...................................34 1.6.3 Optimizing growth conditions .........................................................35 1.6.4 Examining the effect of target compounds ......................................35 1.6.5 Choice of compounds ......................................................................36 1.7 References .......................................................................................................39 iii CHAPTER 2: QUANTIFYING CELL GROWTH ..........................................................47 2.1 Overview .........................................................................................................47 2.2 Background on Methods Used to Quantify Bacterial Cell Growth ................48 2.2.1 UV-Vis Spectrophotometry .............................................................48 2.2.2 Colony Forming Unit (CFU) count ..................................................51 2.3 Methods Not Adopted for Use in this Project .................................................53 2.3.1 Particle counters ...............................................................................53 2.3.2 Cell mass measurements ..................................................................54 2.3.3 Microscope methods ........................................................................54 2.3.4 TOC ..................................................................................................57 2.4 Materials and Methods ....................................................................................59 2.4.1 Media composition ...........................................................................59 2.4.2 Culture preparation ..........................................................................60 2.4.3 UV-Vis spectrophotometry ..............................................................61 2.4.4 Colony Forming Unit (CFU) counting ............................................62 2.5 Results and Discussion ...................................................................................65 2.5.1 Determining the culture density range for most accurate UV-Vis measurement ....................................................................................65 2.5.2 A Correlation for the relationship between A660 and CFUs ...........69 2.6 Conclusions .....................................................................................................78 2.7 References........................................................................................................80 CHAPTER 3: STANDARD GROWTH BEHAVIOR OF B. VIRIDIS ............................84 3.1 Introduction .....................................................................................................84 3.1.1 Previous work and the next step ......................................................84 3.1.2 Goals & Justification ........................................................................85 3.1.3 Background on plotting culture growth ...........................................87 3.2 Materials & Methods ......................................................................................90 3.2.1 Culture and sample preparation .......................................................91 3.2.2 Characterization of cell growth and health for all performed experiments ......................................................................................92 3.2.3 Developing and testing the Two Point Method ...............................93 3.3 Results & Discussion ......................................................................................95 3.3.1 Growth curve for RMPABA, Minimal, and a 50/50 RMPABA
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