THE BACTERIAL TOXICITY OF SELENOCYANATE AND THE INCORPORATION OF TELLURIUM AND SELENIUM IN BACTERIAL CELLS, AND THE SYNTHESIS AND BIOSYNTHESIS OF CADMIUM TELLURIDE NANOPARTICLES AND THEIR ELEMENTAL QUANTIFICATION VIA ICP-AES __________ A Thesis Presented to The Faculty of the Department of Chemistry Sam Houston State University __________ In Partial Fulfillment of the Requirements for the Degree of Master of Science __________ by Rebecca Ann Montes May, 2012 THE BACTERIAL TOXICITY OF SELENOCYANATE AND THE INCORPORATION OF TELLURIUM AND SELENIUM IN BACTERIAL CELLS, AND THE SYNTHESIS AND BIOSYNTHESIS OF CADMIUM TELLURIDE NANOPARTICLES AND THEIR ELEMENTAL QUANTIFICATION VIA ICP-AES by Rebecca Ann Montes _______________________________________ APPROVED: _____________________________ Dr. Thomas G. Chasteen Thesis Director _____________________________ Dr. Donovan C. Haines _____________________________ Dr. David E. Thompson Approved: ___________________________________ Dr. Jerry Cook, Dean College of Science ABSTRACT Montes, Rebecca Ann, The bacterial toxicity of selenocyanate and the incorporation of tellurium and selenium in bacterial cells, and the synthesis and biosynthesis of cadmium telluride nanoparticles and their elemental quantification via ICP-AES. Master of Science (Chemistry), May, 2012, Sam Houston State University, Huntsville, Texas. Purpose The purpose of this research was: (1) to determine the bacterial toxicity of the selenocyanate anion by determining minimal inhibitory and bactericidal concentrations and to determine how much selenium and tellurium three different types of bacteria could incorporate into their cells when exposed to sodium selenite and potassium tellurite; and (2) to determine the ratios of cadmium and tellurium present in cadmium telluride nanoparticles by using ICP-AES. Methods Minimal inhibitory and minimal bactericidal concentrations (MIC and MBC respectively) were determined for three different bacteria by using a 96-microwell plate. A blue color indicated that those bacteria contained no metabolic activity; a pink color indicated that bacteria were functioning metabolically. The smallest concentration to retain its blue color was designated the MIC. Wells surrounding the MIC were plated onto LB agar plates and left to incubate overnight. The MBC was determined to be the plated concentration that contained no growth of bacteria. For bacterial consumption experiments, each strain of bacteria was left to incubate at certain time intervals with a specific concentration of metalloidal salt. Samples were centrifuged and pellets were taken to dryness and resuspended in dilute nitric acid. The samples were then analyzed via ICP-AES. iii Cadmium telluride NPs were biosynthesized and synthesized according to methods developed by collaborators at the University of Chile and University of Santiago. Upon being biosynthesized or synthesized, nanoparticle fluorescence was observed by using a UV trans illuminator. The samples were analyzed via ICP-AES. Findings The toxicity experiments showed that selenocyanate had 1) an MIC of 400 mM for LHVE, 250 mM for the wild-type E. coli species, and 125 mM for TM1b, and 2) an MBC of 450 mM for LHVE, 300 mM for the wild-type E. coli species, and 200 mM for TM1b. Through these experiments, to our knowledge, the first evidence for the biological production of elemental Se by a metalloid-resistant bacterium amended with selenocyanate was discovered. Consumption experiments showed the incorporation of selenium and tellurium oxyanions by all three types of bacteria. Analysis by ICP-AES demonstrated that LHVE more successfully incorporates Te and Se into cells as compared to E. coli or TM1B. The nanoparticle synthesis and biosynthesis experiments showed that fluorescent cadmium telluride nanoparticles were successfully produced both biologically and chemically. The ratios of cadmium and tellurium in these nanoparticles were determined. Keywords: metalloid-resistant bacteria, minimum inhibitory, minimum bactericidal concentrations, glutathione, fluorescence iv ACKNOWLEDGEMENTS First of all I would like to thank Dr. Thomas G. Chasteen. Without him, this would not be possible. He has taught me more about chemistry and life in general than I could have ever thought possible. Without him, I wouldn’t know about fittings, ferrules, regulators or about how to change pump oil or helium tanks. He has truly been an amazing mentor throughout all of this. I would also like to acknowledge Dr. Donovan C. Haines and Dr. David E. Thompson for their help with this thesis as well. I would also like to thank Radhika Burra, Andira Hight, Desire’ Lopez, Iris Porter, Heather Jennings and Gayan Adikari. Without their help and assistance in the laboratory I would not have accomplished so many tasks. In addition, I would like to thank the research group of Dr. Claudio C. Vásquez of the Laboratorio de Microbiologia Molecular of the Facultad de Química y Biología, Universidad de Santiago de Chile as well as Dr. José Pérez-Donoso at the Universidad de Chile for their much needed knowledge in the field of microbiology and biochemistry. Lastly, I would like to thank my friends and especially my family. Without their support, love and encouragement I would not be where I am today nor would I be the person I am today. This thesis is specifically dedicated to my grandfather David Montes Jr. who passed away a year before I started this journey. Even though he is no longer with me I know I always had and will always have his endless love and encouragement. v TABLE OF CONTENTS Page ABSTRACT.......................................................................................................................iii ACKNOWLEDGEMENTS................................................................................................ v TABLE OF CONTENTS................................................................................................... vi LIST OF TABLES...........................................................................................................viii LIST OF FIGURES ........................................................................................................... ix CHAPTER I INTRODUCTION............................................................................................................ 1 Introduction to Selenium................................................................................................. 1 Selenium and Selenocyanate Toxicity ............................................................................ 2 Introduction to Bioremediation and Biodegradation....................................................... 3 Evaluation of Toxicity Studies........................................................................................ 4 Introduction to Nanoparticle Biosynthesis...................................................................... 5 Introduction to Nanoparticle Synthesis........................................................................... 7 II MATERIAL AND METHODS ...................................................................................... 9 Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial Cells....... 9 Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP- AES ............................................................................................................................... 11 III RESULTS .................................................................................................................... 15 vi Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial Cells .... 15 Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP- AES ............................................................................................................................... 21 IV DISCUSSION.............................................................................................................. 26 Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial Cells .... 26 Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP- AES ............................................................................................................................... 30 V CONCLUSIONS........................................................................................................... 33 Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial Cells .... 33 Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP- AES ................................................................................................................................... BIBLIOGRAPHY............................................................................................................. 35 APPENDIX....................................................................................................................... 44 VITA................................................................................................................................. 45 vii LIST OF TABLES Table Page 1 Summary of all MIC and MBC data for the bacterium LHVE obtained for all five metalloidal anions amendments. ............................................................................... 18 2 Summary of all MIC and MBC data for the bacterium E. coli BW25113 obtained for all five metalloidal anions amendments. ................................................................... 18 3 Summary