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
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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.iiiACKNOWLEDGEMENTÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.vTABLE OF CONTENTSÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.vi
LIST OF TABLESÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉ.viiLIST 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..14III. 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 fourduplicate bioreactor experiments.Each run involved four replicatesÉÉÉÉÉÉÉÉÉÉÉÉÉ..25
Table V Se calibration data in 0.05 M sucrose mediumfrom ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...26
Table VI Se calibration data in 0.67 M sucrose mediumfrom ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...27
Table VII Se calibration data in 0.75 M sucrose mediumfrom ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...28
Table VIII Se calibration data in 0.83 M sucrose mediumfrom ICP-AES experimentsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ29
Table IX ICP-AES analysis of sucrose gradients. Observed concentrationsof selenium in each fraction-Bioreactor experiment 1ÉÉÉÉÉ...30
Table X UV/Vis analysis of sucrose gradients. Optical density ofeach fraction at 526 nm-Bioreactor experiment 1ÉÉÉÉÉÉÉ..31
Table XI ICP-AES analysis of sucrose gradients. Observed concentrationsof selenium in each fraction-Bioreactor experiment 2ÉÉÉÉÉ...33
Table XII UV/Vis analysis of sucrose gradients. Optical density ofeach fraction at 526 nm-Bioreactor experiment 2ÉÉÉÉÉÉÉ..34
Table XIII ICP-AES analysis of sucrose gradients. Observed concentrationsof selenium in each fraction-Bioreactor experiment 3ÉÉÉÉÉ...36
Table XIV UV/Vis analysis of sucrose gradients. Optical density ofeach fraction at 526 nm-Bioreactor experiment 3ÉÉÉÉÉÉÉ..37
8
Table XV UV/Vis analysis of unamended cultures. Optical density ofeach fraction at 526 nmÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉ42
Table XVI Variation of optical density at 526 nmwith the concentration of sucrose solutionsÉÉÉÉÉÉÉÉÉ...44
Table XVII Observed concentrations for 13.38 ppbtellurium samples from HGAAS methodÉÉÉÉÉÉÉÉÉÉ..45
Table XVIII Observed concentrations for 600 ppb seleniumsamples from ICP-AES methodÉÉÉÉÉÉÉÉÉÉÉÉÉÉ45
Table XIX Observed data for deionized water samplesfrom 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, beforecentrifugation. Culture suspension is on the top most layerÉÉÉ..17
Figure 3 Sucrose gradients after centrifugation. Culture suspensionhas 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 mediumusing ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.26
Figure 6 Calibration curve for Se analysis in 0.67 M sucrose mediumusing ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.27
Figure 7 Calibration curve for Se analysis in 0.75 M sucrose mediumusing ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.28
Figure 8 Calibration curve for Se analysis in 0.83 M sucrose mediumusing ICP-AESÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..29
Figure 9 Tube profile showing the average distribution of selenium and cells in each sucrose layer for three tubes from thesame 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 thesame bioreactor -Bioreactor experiment 2ÉÉÉÉ.ÉÉÉÉÉÉ35
Figure 11 Tube profile showing the average distribution of seleniumand cells in each sucrose layer for three tubes from thesame bioreactor -Bioreactor experiment 3ÉÉÉÉÉÉÉÉÉÉ38
Figure 12 Tube profile for replicate bioreactor experimentsshowing the distribution of selenium in each sucrose layerÉÉÉ..39
Figure 13 Tube profile for replicate bioreactor experimentsshowing the distribution of cells in each sucrose layerbased on optical densityÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ40
Figure 14 Tube profile showing the distribution of cellsUnamended 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 of its
resemblance to tellurium. Tellurium is an element, which had been discovered earlier and
was named from the Latis word, tellus, meaning Òthe earthÓ. The chemistry of Se and Te
are similar.
Selenium and tellurium show are of allotropic. There are three allotropic forms of
selenium (Rosenfeld and Beath, 1964): (1) ÒmetallicÓ hexagonal, crystalline-stable form,
lustrous gray to black in color; (2) red selenium, monoclinic crystals; (3) amorphous
selenium as black, amorphous red or colloidal selenium.
Compared to selenium, tellurium is more metallic and its stable crystalline form is
hexagonal rhombohedral. Tellurium prepared by reduction of a solution of TeO2 with
sulfurous acid may be amorphous. Tellurium molecules in the vapor phase (at 6000C) are
partially diatomic. Vapor density measurements indicated Te2 molecules (Brasted, 1966).
Selenium occurs naturally in soils, igneous and sedimentary rocks, and waters and is
rarely present in any materials in concentrations exceeding 500 ppm (Muth et al., 1967).
Elevated selenium concentrations in water are found mainly in drain water, groundwater,
ponds and wetlands. There were some wildlife problems, at Kesterson Wildlife Refuge in
California, attributed to irrigation water contaminated with selenium that leached from
cultivated soil (Lange and Berg, 2000). Although selenium is a toxic element, it is an
essential trace nutrient required for the prevention of a number of serious deficiency
11
diseases in various species of livestock and poultry. Selenium deficiency in animals
affects fertility and produces muscular dystrophy, leucocyte inefficiency and liver
necrosis. Inorganic selenium compounds such as sodium selenite or sodium selenate are
be involved together with vitamin E in the prevention of a many nutritional deficiency
diseases (Gunther, 1973). However in excess it causes cancers, deformation of hair and
nails, giddiness, depression and nervousness. Health problems can arise from both
excesses and deficiencies of selenium and there is only a narrow range between
essentiality and toxicity (Table I, Lange and Berg, 2000). While in some regions of the
world part of the daily food intake is artificially enriched with Se for health reasons, other
regions (e.g. some parts of San Joaquin Valley in central California) are polluted with
selenium (Fergusson, 1982).
Table I
Intake of selenium in relation to health (Fergusson, 1982).
Effect of Se Human beings
(mg/day)
Rats (mg/day) Plants (mg/L) of
nutrient solution
Deficient < 0.006 < 0.0003 < 0.02
Normal 0.006 - 0.2 0.0003 Ð 0.004 < 1
Toxic > 5 > 0.004 > 1- 2
Lethal > 1 Ð 2
12
In seleniferous regions the element occurs in foods of animal origin such as milk,
eggs and meat, as well as in vegetables and cereal grains (Table II). A concentration of
5 ppm in common foods or one-tenth this concentration in milk or water is potentially
dangerous. Selenium concentration in drinking water is federally regulated not to exceed
0.01 mg/L, 10 ppb (Rosenfeld and Beath, 1964).
Table II
The selenium content of water, milk, eggs meat and bread (Smith et al., 1936; Smith and
Westfall, 1937).
Material Total no. of Number of samples showing Selenium (ppm)
Samples No Se Traces
(£ 2 ppm)
Positive
(> 2 ppm)
Minimum Maximum
Water 44 20 14 10 0.05 0.33
Milk 50 0 6 44 0.16 1.27
Eggs 32 0 0 32 0.25 9.14
Meat 6 0 0 6 1.17 8.00
Bread 11 0 5 6 0.25 1.00
It is shown that out of 44 samples of drinking water from different wells only
about 23 percent showed the presence of selenium in the relatively small amounts of 5 to
33 mg per 100 g or cc. Milk, of which 50 samples were obtained, showed some selenium
in every instance, the amounts varying usually from 16 to 127 mg per 100 g. Of the
samples of eggs, 22 percent contained less than 100 mg per 100 g and 78 percent
contained in excess of 100 mg per 100 g. Of the 6 samples of meat, selenium was detected
in each sample varying from 117 to 800 mg per 100 g (Smith and Westfall, 1937).
13
Human activities, such as coal mining and fuel refining, as well as industrial uses
of selenium (e.g. in photocopy machines, electronics, glass manufacturing, chemicals,
pigments, flame-proofing agents for textiles, sensitizer in photographic emulsions,
addition to steel to increase machineability) effect the biological availability of selenium.
Intensive explorations in the United States have not been successful in exposing large
deposits of selenium. Normally both selenium and tellurium are chiefly obtained
commercially as a by-product of electrolytic refining of Cu (Rosenfeld and Beath, 1964).
The natural abundance of tellurium in the earthÕs crust is small (2 ppb). Therefore
the usage of tellurium is not as extensive as selenium but there are some applications.
Tellurium and its compounds are used in the semiconductor industry and electronics, the
production of thermoelements, photoelements, and other devices in automation
equipment. The increasing demand for new and different semiconductors necessitates
research work on the application of various tellurium compounds as semiconductor
components (Craig, 1986).
The fate of selenium (Se) in natural environments is affected by many physical,
chemical and biological factors, which are associated with changes in its oxidation state.
Selenium can exist in four different oxidation states and as a component of organic
compounds in natural environments. These include the following: for Se(IV): SeO32-,
HSeO3-, H2SeO3, CaSeO3, and MgSeO3; for Se(VI): SeO4
2-, HSeO4-, H2SeO4, CaSeO4,
and MgSeO4 and for Se(II): Se2-, HSe-, H2Se, CaSe, MgSe, (CH3)2Se and (CH3)2Se2. The
different chemical forms of Se can effect Se solubility and availability to organisms. The
Se species of major environmental concern are selenite [SeO32-, Se(IV)] and selenate
14
[SeO42-, Se(VI)]. Selenate is the most oxidized form of selenium and is highly soluble in
water. Selenite occurs in oxic to suboxic environments and is less available to organisms
because of its affinity to sorption sites of sediment and soil constituents. Under anoxic
conditions, elemental Se [Se0 ] and selenide [Se(II)] are the thermodynamically stable
forms. Elemental selenium is relatively insoluble and selenide precipitates as metal
selenides of very low solubility (Reddy et al., 1995; Zhang et al., 1993). The toxicity of
selenium oxyanions and the volatile products varies depending on the organism.
Generally, selenate is less toxic than selenite and dimethyl selenide, is less toxic than
both of these oxyanions. Toxicity experiments using the bacterium K27, which is also
used in this research, have confirmed that selenite (LD50=2370 ppm Se) is more toxic
than selenate (LD50=15,800 ppm Se) as can generally be expected from a review of the
literature (Yu et al., 1997).
Biological transformations for both Te and Se follow the same pathways, which
include methylation and reduction by metalloid resistant microbes. Oxyanions of
selenium can be biotransformed into the volatile forms of dimethylselenide (DMSe),
dimethylseleneylsulfide (DMSeS) and dimethyldiselenide (DMDSe). These are
prominent processes for Se movement in the environment. These Se volatilization
processes have been characterized to some extent in some animals, terrestrial plants, and
soil microbes. The production of DMSe in animals is viewed as a detoxification
mechanism (Fan et al., 1997).
Toxic Se oxyanions can be made less toxic by their conversion to elemental Se
(Se0), which is insoluble and therefore, for the most part, biologically unavailable.
Chemical detoxification of metal and metalloid polluted sites has proven to be expensive
15
and often results in secondary effects in the environment. There are microorganisms
capable of reducing SeO32- to Se0. Although the majority of microorganisms have been
isolated and described metabolically, it is unclear what reductive processes are involved.
These may involve multiple detoxification processes during reduction of selenite to
elemental selenium by microorganisms since Se0 deposits inside the cytoplasm in the
periplasmic space and outside the cell. The Se0 found outside the cells are released by cell
lysis (Levine, 1925; Dungan and Frankenburger, 1998; Kessi et al., 1999).
The reduction of sodium selenite or selenate by microorganisms. P. chrysogenun
and P. notatum could reduce selenite or selenate to dimethylselenide in two hours in
bread cultures to which selenite or selenate solutions (whose concentrations were 265
mM or 319 mM respectively) had been added (Bird and Challenger, 1939). In 1977, a
strain of Corynebacterium sp. was isolated from soil. This bacterium reduced selenate,
selenite, and elemental selenium to dimethylselenide when the concentration of selenium
in each solution was about 0.3 mM at 290C (Doran and Alexander, 1977). In 1987,
approximately, 200 selenite resistant isolates were mainly obtained from Kesterson
Reservoir in California, a Se-polluted site. Only three isolates which were selenate-
reducing strains produced red colonies when exposed to 10 mM selenate. These three
strains belonged to the genus Citrobacter, Flavobacterim, and
Pseudomonas (Burton et al., 1987). Similar results with several isolates from samples
from the Kesterson Reservoir area in California were obtained. Although all isolates had
the ability to reduce selenite, only one microorganism was able to reduce selenate in pure
culture. That isolate could reduce selenate in concentrations of up to 100 mg/L to less
oxidize forms (Maiers et al., 1988). Pseudomonas stutzeri rapidly reduced both selenite
16
and selenate to elemental selenium at initial concentrations of both anions of up to 48.1
mM at temperatures of 25-350C (Lortie et al., 1992). Reduction of selenate to elemental
selenium was observed in cultures of sediment samples (Steinberg and Oremland, 1990).
Tomei et al. (1992) reported that Wolinella succinoens was inhibited by selenate
or selenite. However, through adaptation, a culture resistant to 1 mM selenite or 10 mM
selenate was obtained. It was able to reduce selenate to elemental selenium and deposit it
as a discrete granule in the cytoplasm. Bacteria capable of energy conservation via
selenate or selenite respiration were identified (Tomei et al., 1992).
Since the biological uptake and toxicity of selenium are controlled by its chemical
form, an evaluation of this chemical speciation in particulate matter is needed. The
removal of selenite from Se-contaminated water through its bioreduction to Se0 may
prove to be a feasible and cost effective remediation technology. A strain of
Pseudomonas fluorescens K27 which reduces both selenite and selenate to elemental
selenium and volatile selenium (Burton et al., 1987) and tellurite and tellurate to
elemental tellurium and volatile tellurium was studied in our lab. This study was
undertaken to find the detoxification ability of K27 under anaerobic conditions by a
biotransformation process and to find the soluble and insoluble Se and Te species as well
as the Se0 inside and outside the cells of K27. To determine the Se0 inside and outside
bacterial cells, it was necessary to separate free Se0 from cells; hence a sucrose density
gradient was performed. Metalloid determination was carried out using hydride
generation atomic absorption spectrometry (HGAAS) or inductively coupled plasma
atomic emission spectrometry (ICP-AES).
17
CHAPTER II
EXPERIMENTAL
Part 1. Reagents
The reagents, except sucrose, used throughout this research were analytical grade
chemicals and were used without further purification. Tryptic soy broth (TSB) was
obtained from DIFCO Laboratories (Detroit, MI USA). Sodium tellurate, sodium
Weres O, Cutter GA, Yee A, Neal R, Moesher H, Tsao L. Standard methods for theexamination of water and wastewater, 17th Ed. American Public Health Association:Washington, D.C.1989; Chapter 3500-Se.
Weres O, Jaouni A, Tsao L. Appl. Geochem. 1989; 4:543-563.