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Inorganic Selenium and Tellurium Speciation in Aqueous Medium of Biological Samples

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1 INORGANIC SELENIUM AND TELLURIUM SPECIATION IN AQUEOUS MEDIUM OF BIOLOGICAL SAMPLES ________________________ 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 Rukma S. T. Basnayake December, 2001 2 INORGANIC SELENIUM AND TELLURIUM SPECIATION IN AQUEOUS MEDIUM OF BIOLOGICAL SAMPLES by Rukma S.T. Basnayake _______________________________ APPROVED: ________________________________ Thomas G. Chasteen, Thesis Director ________________________________ Paul A. Loeffler ________________________________ Benny E. Arney Jr. APPROVED: _____________________________ Dr. Brian Chapman, Dean College of Arts and Sciences 3 ABSTRACT Basnayake, Rukma ST, Inorganic Selenium and Tellurium Speciation in Aqueous Medium of Biological Samples, Master of Science (Chemistry), December 2001, Sam Houston State University, Huntsville, Texas, 60 pp. Purpose The purpose of this research was to develop methods to study the ability of bacteria, Pseudomonas fluorescens K27 to detoxify tellurium and selenium salts by biotransformation processes under anaerobic conditions. Another purpose was to make an effort to separate biologically produced Se0 from cells. Methods Pseudomonas fluorescens K27 was grown in TSN3 medium (tryptic soy broth with 0.3% nitrate) under anaerobic conditions and the production of elemental tellurium and elemental selenium was observed when amended with inorganic tellurium salts and selenium salts, respectively. The amount of soluble tellurium species in the culture medium also was determined. Samples from a 2.75 L bioreactor were taken after cultures had reached the stationary growth phase and were centrifuged in order to separate insoluble species (elemental tellurium, elemental selenium) from soluble species (oxyanions of tellurium, oxyanions of selenium). In tellurium samples, supernatant consisting of soluble tellurium and the sediment consisting of insoluble tellurium were analyzed separately by oxidation and reduction procedures followed by using hydride generation atomic absorption spectroscopy (HGAAS). Sediment from selenium bioreactor samples was used to find free elemental selenium and elemental selenium inside and/or on K27 cells. A sucrose density gradient with overlaid precipitate was prepared, centrifuged, fractionated and analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) to find the concentration of Se0 and also analyzed by UV/Vis spectrometry to find the distribution of cells in the sucrose gradient. 4 Findings K27 grew well in TSN3 medium under anaerobic conditions. Bacterial cells survived and continued their growth when poisoned with 1 mM tellurate or 10 mM selenite and produced elemental tellurium or elemental selenium. The calibration range for Te analysis using the HGAAS instrumental method was narrow (~0Ð20 ppb) with a detection limit of 3 ppb. The calibration range for Se analysis using ICP-AES instrumental method was wide (~0Ð1000 ppb) with a detection limit of 500 ppb. Approximately 66% of added tellurium was recovered in the liquid medium and 34% was recovered in the solid phase. According to the sucrose density gradient experiment, it was not clear whether selenium was distributed outside the K27 cells as well as inside the K27 cells; either Se0 has the same density as cells or Se0 and cells are bound together. Approved: ______________________________ Thomas G. Chasteen Thesis Director 5 ACKNOWLEDGMENTS I would like to express my appreciation to Dr. Thomas Chasteen, for the great amount of time, effort, support and guidance. He was not only my thesis advisor but also my graduate advisor through out my studies at Sam. I am deeply grateful for his understanding and patience during the research, the thesis writing and the analytical chemistry lecture course. Specially, without his support and help I could not even have come to the United States. I extend my sincere thanks to Dr. Harry Kurtz, professor in Biology for letting me to use the centrifuge in Biology department and for his guidance and help for this research. I also thank to the faculty who taught me in last two years; and to Ms. Pat Johnson for her daily help. I thank Ms. Janet Bius and Mr. Suminda Hapuarachchi for all their help, kindness and friendship during these two years at Sam. Special thanks go to my parents for their love and bringing me up here, to this level. Finally, I thank my husband, Ananda Bandulasiri who is a graduate student at department of statistics at Sam, for providing me constant support whenever I need it. I dedicate this thesis to my parents, my husband and my son Sithija. This is the least I can do to show you how much I love you all. 6 TABLE OF CONTENTS PAGE ABSTRACTÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.iii ACKNOWLEDGEMENTÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.v TABLE OF CONTENTSÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.vi LIST OF TABLESÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉ.vii LIST OF FIGURESÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..Éix CHAPTERS I. INTRODUCTIONÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.1 II. EXPERIMENTAL...ÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...8 Part 1 ReagentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.8 Part 2. InstrumentationÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ12 Part 3. Bioreactor ExperimentsÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ..13 Part 4. ProceduresÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ..14 III. DATA AND RESULTSÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..22 IV. DISCUSSION AND CONCLUSIONSÉÉÉÉÉÉÉÉÉÉÉÉ...49 BIBLIOGRAPHYÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ55 APPENDIXÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..59 Chemical Abstract Service Registry NumbersÉÉÉÉÉÉÉÉÉÉÉÉÉ..59 VITAÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ60 7 LIST OF TABLES Table I Intake of selenium in relation to healthÉÉÉÉÉÉÉÉÉÉÉ...2 Table II The selenium content of water, milk, eggs meat and breadÉÉÉÉ3 Table III Te calibration data from HGAASÉÉÉÉÉÉÉÉÉÉÉÉÉ..23 Table IV Distribution of Te among supernatant and solid phase in four duplicate bioreactor experiments. Each run involved four replicatesÉÉÉÉÉÉÉÉÉÉÉÉÉ..25 Table V Se calibration data in 0.05 M sucrose medium from ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...26 Table VI Se calibration data in 0.67 M sucrose medium from ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...27 Table VII Se calibration data in 0.75 M sucrose medium from ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...28 Table VIII Se calibration data in 0.83 M sucrose medium from ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ29 Table IX ICP-AES analysis of sucrose gradients. Observed concentrations of selenium in each fraction-Bioreactor experiment 1ÉÉÉÉÉ...30 Table X UV/Vis analysis of sucrose gradients. Optical density of each fraction at 526 nm-Bioreactor experiment 1ÉÉÉÉÉÉÉ..31 Table XI ICP-AES analysis of sucrose gradients. Observed concentrations of selenium in each fraction-Bioreactor experiment 2ÉÉÉÉÉ...33 Table XII UV/Vis analysis of sucrose gradients. Optical density of each fraction at 526 nm-Bioreactor experiment 2ÉÉÉÉÉÉÉ..34 Table XIII ICP-AES analysis of sucrose gradients. Observed concentrations of selenium in each fraction-Bioreactor experiment 3ÉÉÉÉÉ...36 Table XIV UV/Vis analysis of sucrose gradients. Optical density of each fraction at 526 nm-Bioreactor experiment 3ÉÉÉÉÉÉÉ..37 8 Table XV UV/Vis analysis of unamended cultures. Optical density of each fraction at 526 nmÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉ42 Table XVI Variation of optical density at 526 nm with the concentration of sucrose solutionsÉÉÉÉÉÉÉÉÉ...44 Table XVII Observed concentrations for 13.38 ppb tellurium samples from HGAAS methodÉÉÉÉÉÉÉÉÉÉ..45 Table XVIII Observed concentrations for 600 ppb selenium samples from ICP-AES methodÉÉÉÉÉÉÉÉÉÉÉÉÉÉ45 Table XIX Observed data for deionized water samples from HGAAS methodÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉ..46 9 LIST OF FIGURES Figure 1 Marked polycarbonate tube in 450 angleÉÉÉÉÉÉÉÉÉÉ...16 Figure 2 Sucrose gradients overlain with culture suspension, before centrifugation. Culture suspension is on the top most layerÉÉÉ..17 Figure 3 Sucrose gradients after centrifugation. Culture suspension has distributed among the sucrose layersÉÉÉÉÉÉÉÉÉÉ.18 Figure 4 Calibration curve for Te analysis using HGAASÉÉ.ÉÉÉÉÉ.23 Figure 5 Calibration curve for Se analysis in 0.05 M sucrose medium using ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.26 Figure 6 Calibration curve for Se analysis in 0.67 M sucrose medium using ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.27 Figure 7 Calibration curve for Se analysis in 0.75 M sucrose medium using ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.28 Figure 8 Calibration curve for Se analysis in 0.83 M sucrose medium using ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..29 Figure 9 Tube profile showing the average distribution of selenium and cells in each sucrose layer for three tubes from the same bioreactor-Bioreactor experiment 1ÉÉÉÉÉÉÉÉÉÉ.32 Figure 10 Tube profile showing the average distribution of selenium and cells in each sucrose layer for three tubes from the same bioreactor -Bioreactor experiment 2ÉÉÉÉ.ÉÉÉÉÉÉ35 Figure 11 Tube profile showing the average distribution of selenium and cells in each sucrose layer for three tubes from the same bioreactor -Bioreactor experiment 3ÉÉÉÉÉÉÉÉÉÉ38 Figure 12 Tube profile for replicate bioreactor experiments showing the distribution of selenium in each sucrose layerÉÉÉ..39 Figure 13 Tube profile for replicate bioreactor experiments showing the distribution of cells in each sucrose layer based on optical densityÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ40 Figure 14 Tube profile showing the distribution of cells Unamended bioreactor experimentÉÉÉÉÉÉÉÉÉÉÉÉÉ43 10 CHAPTER I INTRODUCTION Selenium was identified as an element in 1817 by the Swedish chemist, Berzelius. It was named from the Greek word, selene, meaning Òthe moonÓ, because
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  • The Control of the Grain Size of Zinc

    The Control of the Grain Size of Zinc

    Montana Tech Library Digital Commons @ Montana Tech Bachelors Theses and Reports, 1928 - 1970 Student Scholarship 5-8-1936 The onC trol of the Grain Size of Zinc Oswald J. Wick Follow this and additional works at: http://digitalcommons.mtech.edu/bach_theses Part of the Ceramic Materials Commons, Environmental Engineering Commons, Geology Commons, Geophysics and Seismology Commons, Metallurgy Commons, Other Engineering Commons, and the Other Materials Science and Engineering Commons Recommended Citation Wick, Oswald J., "The onC trol of the Grain Size of Zinc" (1936). Bachelors Theses and Reports, 1928 - 1970. 67. http://digitalcommons.mtech.edu/bach_theses/67 This Bachelors Thesis is brought to you for free and open access by the Student Scholarship at Digital Commons @ Montana Tech. It has been accepted for inclusion in Bachelors Theses and Reports, 1928 - 1970 by an authorized administrator of Digital Commons @ Montana Tech. For more information, please contact [email protected]. I Wick,O.J. THE CONTROL OF THE GRAIN SIZE OF ZINC by OSWALD J. WICK A ThesIs SubmItted to the Department of Metallurgy in partIal FulfIllment of the Requirement for the Degree of BaChelor of Science in Metallurgical Engineering MONTANA SCHOOL OF MINES BUTTE, MONTANA MAY8, 1936 ~NTANA sCHga~Of MJNf! UBIWlY. .. - .;..... THE CONTROL OF THE GRAIN SIZE OF ZINC by OSWALD J. WICK A T~esis Subm1 tted to the Dep,artment of Metallurgy in partial Fu1fillment of the Requirement for the Degree ot Bachelor of Scienc~ in Metallurgioal Engineering I /17A7 ,I MONTANA SCHOOL OF MINE~ LlBBAB~o . - -. ...., MONTANA SCijOOL OF MINES BUTTE. MONT ANA MAY 5, 1936 I ...