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Thesis Lopez.FINAL STUDY OF THE TOXICITY OF SYNTHETICALLY- AND BIOLOGICALLY-PRODUCED CADMIUM TELLURIDE NANOPARTICLES AND AN EXAMINATION OF THE EFFECTS OF S- ADENOSYL METHIONINE AMENDMENT ON bnf05 BACTERIAL HEADSPACE 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 Desiré A. Lopez May, 2014 STUDY OF THE TOXICITY OF SYNTHETICALLY- AND BIOLOGICALLY-PRODUCED CADMIUM TELLURIDE NANOPARTICLES AND AN EXAMINATION OF THE EFFECTS OF S- ADENOSYL METHIONINE AMENDMENT ON bnf05 BACTERIAL HEADSPACE by Desiré A. Lopez APPROVED: Dr. Thomas G. Chasteen Thesis Director Dr. Donovan C. Haines Dr. David E. Thompson Approved: Dr. John B. Pascarella, Dean College of Sciences ABSTRACT Lopez, Desiré A., Study of the toxicity of synthetically- and biologically-produced cadmium telluride nanoparticles and an examination of the effects of S-adenosyl methionine amendment on bnf05 bacterial headspace. Master of Science (Chemistry), May, 2014, Sam Houston State University, Huntsville, Texas. Purpose The purpose of this research was: (1) to determine the toxicity of both synthetically- and biologically-produced nanoparticles (NPs); and (2) to determine if K2TeO3 could be reduced and methylated by a metalloid-resistant bacterium isolated from Antarctica, bnf05. Methods Cadmium telluride NPs were made both synthetically and biologically. Biologically-made NPs were grown under three separate growing conditions, 1) aerobic, 2) microaerobic, and 3) aerobic for the initial growth and microaerobic after the introduction of a lacZ gene inducer, to determine which condition produced the most nanoparticle fluorescence. MIC (Minimal Inhibitory Concentration) experiments were conducted on both synthetically- and biologically-made NPs for two different bacteria, BW and LHVE, using a colony counting technique. The headspace of live cultures including the Antarctic bacterium bnf05, S- adenosyl methionine, and K2TeO3, incubated for 24 or 48 hours were analyzed via gas chromatography using solid phase microextraction. Findings Exciting the fluorescent biologically-made NP solutions with UV light showed that when grown in completely aerobic conditions the most fluorescent NPs were produced. The toxicity experiments for synthetically-made NPs showed that LHVE could iii grow in the presence of much higher NP concentrations compared to a wild type E. coli strain: an MIC of 475 mg/mL for LHVE and 35 mg/mL for BW. Through these experiments the first evidence that LHVE—known to be metalloid resistant—is also cadmium resistant was discovered. Steps were taken to determine the MICs for biologically-made NPs, but the chemicals used for lysing cells and dialyzing the NP solution back to a more neutral pH proved to interfere with MIC results. Upon using the precipitation and drying technique adopted for synthetically-produced NPs, the biologically-produced NPs were successfully isolated from their lysis and dialysis solution and this process could be used for future nanoparticle MIC determinations. The culture headspaces of SAM (S-adenosyl methionine) and Te amended samples of bnf05 bacteria headspace showed that SAM added at two different culture concentrations could not be used to methylate the elemental tellurium produced by the bacterium to form DMTe over the 24 and 48 hr incubation periods examined. KEY WORDS: Quantum dots, toxicology, metalloids, Antarctic bacteria. iv ACKNOWLEDGMENTS Firstly I would like to thank Dr. Red Chasteen. He helped me learn, most of all, to be independent. This was a very hard project to take on and he helped me to find the answers myself. That is the most important skill I will take from him; not to complain, but to evolve and move on. And from insisting for me to turn the music up in the lab, to reminding me to back up my work in two places, I will not forget what he has taught me, because the music is never too loud and there are only two kinds of people in the world: those who have lost their data and those who will. I also want to acknowledge Dr. Donovan C. Haines and Dr. David E. Thompson for their tips and support. A special thanks goes to Rebecca Montes, Gayan Adikari, Poorna Wansapura, and Juan Pablo Monrás, Universidad de Chile. I would also like to thank Vicente Durán- Toro, Universidad de Chile for helping me embrace the fact that I am part microbiologist. Without all of their help I would not have been able to accomplish so much in the lab. I would also like to thank the research group of Dr. Claudio C. Vásquez of the Laboratorio de Microbiologia Molecular of the Facultad de Química y Biolgía, Universidad de Santiago de Chile for teaching me more microbiology and biochemistry than I ever thought I needed to know, but am glad to have learned. Most of all I would like to thank my parents for their continued support throughout this process; from helping me take care of my dinosaur children, to being there when I needed someone to vent to, I could not beat their support. They taught me to be strong, a trait of which I could not do without in earning this degree. v TABLE OF CONTENTS Page ABSTRACT ....................................................................................................................... iii ACKNOWLEDGMENTS .................................................................................................. v TABLE OF CONTENTS ................................................................................................... vi LIST OF FIGURES ......................................................................................................... viii CHAPTER I INTRODUCTION .................................................................................................. 1 Environmental Metalloids ....................................................................................... 1 Tellurium ................................................................................................................. 1 Biodegradation and Bioremediation ....................................................................... 4 S-Adenosyl Methionine Amendment Experiments ................................................ 5 Nanoparticles: Introduction and Applications ........................................................ 6 Nanoparticles: Synthesis and Biosynthesis ............................................................. 8 Nanoparticle Toxicity ............................................................................................. 9 Bacterial Strains .................................................................................................... 10 II MATERIALS AND METHODS .......................................................................... 12 Synthetically- and Biologically-Produced Cadmium Telluride Nanoparticles Synthesis and MIC Determination ........................................................................ 12 Effects of S-Adenosyl Methionine Amendment on bnf05 Bacterial Headspace .. 19 III RESULTS ............................................................................................................. 21 Synthetically- and Biologically-Produced Cadmium Telluride Nanoparticle Synthesis and MIC Determination ........................................................................ 21 vi MIC Determination ............................................................................................... 24 Additional Drying Step ......................................................................................... 32 Effects of S-Adenosyl Methionine Amendment on bnf05 Bacterial Headspace .. 33 IV DISCUSSION ....................................................................................................... 38 Synthetically- and Biologically-Produced Cadmium Telluride Nanoparticles Synthesis and MIC Determination ........................................................................ 38 Effects of S-Adenosyl Methionine Amendment on bnf05 Bacterial Headspace .. 40 V CONCLUSION ..................................................................................................... 42 Synthetically- and Biologically-Produced Cadmium Telluride Nanoparticles Synthesis and MIC Determination ........................................................................ 42 Effects of S-Adenosyl Methionine Amendment on bnf05 Bacterial Headspace .. 43 BIBLIOGRAPHY ............................................................................................................. 44 APPENDIX A ................................................................................................................... 51 VITA ................................................................................................................................. 52 vii LIST OF FIGURES FIGURE Page 1 The experimental steps for production of synthetically- and biologically-made NPs. ........................................................................................................................13 2 The fluorescent supernatant solutions after cell lysis of the three growing methods are shown above. Red capped 1A, 2A, and 3A are the controls with no metals or metalloids added. Method 1) foil over the flask, 2) in a closed flask, and 3) foil over the flask for initial growth and closing the flask after addition of IPTG. 1B, 2B, and 3B contain the NP solutions with cadmium and tellurite added. .............21 3 The background spectrum collected with a clean, empty cuvette. ........................22 4 The fluorescence spectrum of NP
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