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 of all MIC and MBC data for the bacterium TM1b obtained for all five
metalloidal anions amendments...... 19
4 ICP analysis results of pellets collected from the two E. coli strains, gshA and gshB.
DL indicates that the reading was below the detection limit of the instrument...... 24
5 Cd and Te content in the as-prepared NPs as determined by ICP ...... 24
viii
LIST OF FIGURES
Figure Page
1 Rows A and B (horizontal) contain a bacterial control consisting of only LHVE and
the resazurin dye...... 15
2 Rows C and D contain resazurin dye, LHVE, and concentrations of selenocyanate
starting with 1 M and decreasing two-fold across the width of the plate...... 15
3 Rows E and F contain resazurin dye, LHVE, and concentrations of selenocyanate
starting with 750 mM and decreasing two-fold across the width of the plate...... 16
4 Rows G and H contain resazurin dye, LHVE, and concentrations of selenocyanate
starting with 400 mM and decreasing two-fold across the width of the plate...... 16
5 LB plus agar plates containing LHVE amended with (A) 400 mM selenocyanate
after incubation overnight, (B) 500 mM selenocyanate after incubation overnight,
(C) 425 mM selenocyanate after incubation overnight, and (D) 400 mM
selenocyanate after incubation for 96 h showing formation of red, elemental
selenium...... 17
6 Graph showing selenite consumption by TM1B (diamond), BW (square) and LHVE
(triangle)...... 20
7 Graph showing tellurite consumption by TM1B (diamond), BW (square) and LHVE
(triangle)...... 21
8 gshA fluorescence. From left to right: Control, Cd alone, Te alone, and Cd+Te
amended samples...... 22
ix
9 gshB fluorescence. From left to right: Control, Cd alone, Te alone, and Cd+Te
amended samples...... 23
10 Green, yellow, and red QDs...... 25
x
1
CHAPTER I
INTRODUCTION
Introduction to Selenium
Selenium was first recognized in 1817 by J.J. Berzelius and is the 66th most abundant element in the earth’s crust at only 50 ppb (Earnshaw & Greenwood, 1997). It
2- is most commonly found in soluble forms as oxyanions selenite (SeO3 ) and selenate
2- (SeO4 ); however it also exists in Se-containing amino acids selenocysteine and selenomethoionine, as well as in an anion known as selenocyanate (SeCN-). The main source of commercial Se is the anode slime deposited during the electrolytic refining of copper. Selenium is also recovered from the sludge accumulating in sulfuric acid plants and from electrostatic precipitator dust collected during the processing of copper and lead
(Earnshaw & Greenwood, 1997). One well-known area with high concentrations of selenium is the San Joaquin Valley of California with approximately 230-640 ppb Se in subsurface drainage water (Cantafio et al., 1996). The source of this selenium is irrigation of agricultural lands in the San Joaquin Valley and has resulted in the leaching of naturally occurring selenium—from the weathering of nearby marine shales—into these subsurface draining waters. Selenium has been found to have many uses in industry in photoelectric cells, light meters, solar cells and photocopiers (Emsley, 2002). Selenium can be an essential micronutrient to biological systems at lower concentrations; however, it becomes toxic at more elevated levels (Kuo and Jiang, 2008). Selenium plays an essential dietary role in animals and also in humans due to its ability to form the enzyme glutathioine peroxidase which is involved in fat metabolism (Schwarz and Foltz, 1957;
Muth et al., 1958). In trace amounts selenium is beneficial to the human body; the
2 average human dietary intake of Se in the USA is said to be approximately 150 µg daily
(Earnshaw & Greenwood, 1997). When optimum levels are exceeded Se has negative effects on both humans and animals, and, at concentrations above 5 ppm, causes severe disorders such as selenosis as was the case with an endemic of selenium intoxication that occurred in China. (Wrobel et al., 2005; WHO, 2011; Earnshaw & Greenwood; Yang et al., 1983). According to the World Health Organization (WHO), the concentration of selenium in drinking water should not exceed 10 µg/L (WHO, 2011).
Selenium and Selenocyanate Toxicity
The concentration range in which selenium is considered toxic or essential is very constricted. Of all the elements, selenium has one of the narrowest ranges between dietary deficiency (< 40 µg per day) and toxic levels (>400µg per day), this makes it necessary to carefully control the intake of selenium by humans and other animals
(Fordyce, 2005). Selenium poisoning, also known as selenosis, may be acute or chronic.
Acute exposure can be caused by occupational exposure from workers of the industrial sector (Pedrero and Madrid, 2009). Chronic exposure is due to the presence of high levels of selenium in food and water and results in discoloration of the skin, hair loss, deformation of nails, excessive tooth decay and discoloration, a garlic odor to the breath, weakness and a lack of mental alertness. An episode of long-term exposure to selenium intoxication was detected in several areas of China from the consumption of products that contained selenium (Pedrero and Madrid, 2009). Selenium is not only toxic to mammals but to other organisms in the environment as well. Selenium is almost always present in water and soil at some concentration and the presence of selenium represents a combination of naturally occurring forms as well as the forms of selenium that were put
3 back into the environment by human activity (Palmer, 1998). Selenium is present in varying quantities in the environment all around us as a result of natural and man-made processes. Animals and humans are exposed to environmental selenium via dermal contact, the inhalation of air and via the ingestion of water, plants, and animals who have a diet that contains food produced on soil containing selenium (Fordyce, 2005)
Selenocyanate is not as well-known as the oxyanions of selenium. However, its detection in the environment has prompted research concerning its toxicity.
Selenocyanate is produced by the petrochemical refining of crude oils from crustal strata that contain selenium (Appleton & Cain, 1995; Meng & Beng, 2002) and understanding how to eliminate it from the environment is an important task. In a recent study by Petrov et al. (2012), 13 different Se species were found in flue gas desulfurization (FGD) waters from coal-fired power plants. Of those 13, two of them were conclusively identified, for the first time, as being selenosulfate and selenocyanate. Selenocyanate has also been detected in petroleum refinery waste water and in gold mine waste water in a study by
Wallschläger & Bloom (2001). Ion chromatography-hydride generation-atomic fluorescence spectrometry (IC-HG-AFS) was used to determine Se speciation and concentrations of 1.48 mg/L (petroleum refinery waste water) and 20.8 µg/L (gold mine waste water) SeCN- were reported.
Introduction to Bioremediation and Biodegradation
Much attention has been given to methods for cleaning Se-containing contaminates from toxic sites. Conventional wastewater treatment processes (coagulation with ferric salts) are not effective for the removal of SeCN- due to its low affinity for iron hydroxide at neutral pH (Tonietto et al., 2010). Methods such as phytoremediation,
4 bioremediation and biodegradation can be useful when chemical methods are not.
Bioremediation is the use of microorganisms, such as bacteria, to remove pollutants, like
Se-containing species, and biodegradation is the use of bacteria to breakdown materials.
Sources suggest that biological treatment has emerged as a leading technology for selenium treatment in the environment (Cain, 1994; Golder, 2009). In order to help protect the environment, treatment processes are necessary to remove selenium from wastewaters and drinking water (Manceau et al., 1997; Boegel et al., 1986). Heavy metals and metalloids such as selenium are major contaminants in 40% of hazardous waste sites.
Since there is expected to be a strong correlation between heavy metal contaminated sites and toxic levels of selenium, remediation of selenium is an important ongoing challenge
(Sandrin et al., 2000; Hasan et al., 2010). In order to help improve bioremediation, the use of metalloid-resistant bacteria has been used before (Zhang and Frankenberger, 2006;
Zhang et al., 2004; Cantafio et al., 1996; Frankenberger & Arshad, 2001; Adams &
Picket, 1998). It has been suggested that biological processes for treating toxic effluents are more cost effective than chemical and physical methods due to their efficiency (Paul et al., 2005; de Souza et al., 2002).
Evaluation of Toxicity Studies
The relative toxicity of oxyanions of selenium and tellurium and that of selenocyanate has previously been studied albeit in a general way. Through a series of zone of inhibition (ZOI) and growth curve experiments Burra et al. (2009) found that tellurite was more toxic to metalloid-resistant bacteria LHVE than tellurate and both the
Te-containing compounds were much more toxic than selenate, selenite and selenocyanate. However, no quantitative data for selenocyanate has ever been presented.
5
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are quantitative measures that are commonly used to evaluate the toxicity of a chemical species (Islam et al., 2008; Primm and Franzblau, 2007; Di
Gregorio et al., 2006; Harrison et al., 2005). The MIC is the concentration at which growth of the bacteria is halted or inhibited, whereas the MBC is the concentration at which the bacteria are killed and no growth can be seen. Determining the toxicity of anions such as selenocyanate to specific bacteria can help to improve the implementation of biodegradation and bioremediation. In order to successfully carry out bioremediation, a monoculture of the necessary bacteria will need to be grown in the areas where bioremediation is needed. Since the areas are non-sterile, in order to insure growth of only the desired bacteria (the bacteria that is intentionally put in to bioremediate), the bacteria will need to be fixed with a resistance marker of some sort to help ensure that only the desired bacteria is present. The bacteria are capable of reducing SeCN- to the insoluble elemental Se and previous studies have shown that selenate and selenite operate in this same manner (Frankenberger and Arshad, 2001; Losi and Frankenberger, 1997a;
Losi and Frankenberger, 1997b; Dungan and Frankenberger, 1998; Mishra et al., 2011)
Introduction to Nanoparticle Biosynthesis
Although nanoparticles (NPs) are considered a modern discovery, they have a history that dates back to 1857 when Michael Faraday provided the first scientific description of the optical properties of nanometer-scale metals. It was not until 1903 and again in 1908 when the topic was revisited by Beilby and Turner respectively, where they investigated gold and silver NPs. Nanoparticles have recently begun to gain much attention as researchers are finding that they can be useful in many ways. Nanoparticles
6 are defined as materials with at least two dimensions between 1 and 100 nm.
Nanomaterials possess unique mechanical, catalytic, optical, and electric conductivity properties because of their nano size (Domingos et. al., 2008, Klaine et. al., 2008). Due to these characteristics, there has been exponential growth over the past decade in the development of new manufactured or engineered nanomaterials and they can be classified according to their chemical composition. For example, classifications can include: organics, metal oxides, semiconductors (including quantum dots), zero-valent metals such as iron, silver, and gold, and nanopolymers such as dendrimers. A second method of their classification is according to morphology: nanoparticles, nanofibers, nanowires and nanosheets (Klaine et. al., 2008).
Semiconductor nanoparticles (NPs) or quantum dots (QDs) are bimetallic structures of elements like Cd, S, Se or Te that have many applications due to their physiochemical and optoelectric properties including broad-range excitation, size-tunable narrow emission spectra and high photostability. Tellurium, like selenium, is a metalloid and it can be utilized along with cadmium to synthesize and biosynthesize cadmium telluride (CdTe) NPs. Cadmium telluride NPs have several applications. Specifically,
CdTe QDs are used in electronic and optoelectronics, as an important tool for new solar cell technology (photovoltaic panels), and in biomedicine (Azzazy et al., 2007; Bang &
Kamat, 2009; Liu and Yu, 2010). Typically NPs have several potential medical applications including nanodiagnostics, imaging, targeted drug delivery, and photodynamic delivery, so understanding methods to synthesize them can be extremely useful (Azzazy et al., 2007).
7
Currently, CdTe QD synthesis involves chemical procedures that require high temperature, anaerobic conditions, toxic substrates and are energy intensive (Azzazy et al., 2007). Therefore, QDs produced by these methods are not highly biocompatible and are in turn not useful if their biocompatibility is low. Currently, the development of new, simpler, less toxic and environmentally friendly synthesis procedures is a subject of growing interest (Kumar et al., 2007). Using bacteria or microorganisms to synthesize
QDs is cost effective, non-toxic, reproducible and can be done at ambient temperatures and pressure. Kumar et al. (2007) synthesized highly luminescent CdSe QDs at room temperature by the fungus Fusarium oxysporum by incubating the microbe with a mixture of CdCl2 and SeCl4. Bai et al. (2006) successfully synthesized ZnS by use of bacteria and Kowshik et al. (2002) were able to synthesize both PbS and CdS by using a
Schizosaccharomyces pombe strain.
Introduction to Nanoparticle Synthesis
NPs that are produced by chemical methods can be synthesized in a way that mimics biological conditions. This can be done by capping the NPs that are chemically synthesized with glutathione (GSH) and thus, does not involve the direct use of the bacteria itself. These glutathione-capped NPs can then be used as molecular probes, for instance, for the detection of heavy metal ions. As a result of specific interactions, the fluorescence intensity of GSH-capped NPs is selectively reduced in the presence of heavy metal ions such as Pb2+, As3+, and Hg2+ (Ali et al., 2007; Wang et al., 2011; Yang et al.,
2011).
When synthesizing NPs, the quantification of tellurium and cadmium present in
CdTe NPs is important because it determines the extent to which the NPs can be used.
8
Knowing the Cd and Te ratios present in the NPs is necessary because different synthesis procedures produce NPs with different Cd content (Pérez-Donoso, 2011). Determining the amounts of Cd and Te present in fluorescent CdTe NPs will help with future applications in heavy metal bioremediation. A higher Cd/Te ratio is important because when biosynthesizing CdTe NPs, those with a higher Cd/Te ratio will be more fluorescent and most likely more beneficial when it comes to their utilization in different applications. When synthesizing or biosynthesizing CdTe NPs, even though specific concentrations of cadmium and tellurium are added, one cannot be sure as to the exact amount of these two elements that are present in the actual NPs produced. Therefore, inductively coupled plasma atomic emission spectrometry (ICP-AES) is a valuable tool for determining the concentrations of Cd and Te that are present inside the biosynthesized and synthesized NPs.
9
CHAPTER II
MATERIAL AND METHODS
Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial
Cells
Reagents
Reagents used in these experiments include: BactoTM tryptone (Becton Dickson,
Sparks, MD, USA), yeast extract (EMD Chemicals, Gibbstown, NJ, USA), sodium chloride (BDH Chemicals, West Chester, PA, USA), potassium selenocyanate, sodium selenate, sodium selenite, potassium tellurate, potassium tellurite (Sigma-Aldrich, St.
Louis, MO, USA), agar (Amresco, Solon, OH, USA), resazurin sodium salt (Alfa Aesar,
Ward Hill, MA, USA), and pre-sterilized 15-mL centrifuge tubes (VWR International,
LLC, Radnor, PA, USA). A RiOs 3 water purification system from Millipore (Billerica,
MA, USA) was used for water deionization.
Determination of MIC and MBC
A preculture of LHVE strain, isolated from Huerquehue National Park in southern
Chile, was grown overnight at 37°C under anaerobic conditions in Lysogeny Broth (LB) medium (Sambrook et al, 1989). TM1B is another bacterial strain isolated from Chile that produces volatile organo-metalloids when grown in capped cultures amended with selenium-containing salts (results not shown), as has been also described for LHVE strain
(Burra et al, 2010). The OD at 525 nm was measured for the preculture after 24 h of incubation. When the OD was measured to be between 0.4 and 0.5, the preculture was diluted to 0.005 OD525 in media with a total adjusted volume of 20 mL. One hundred and
10
fifty µL of the 0.005 OD525 diluted culture was loaded into each well in a sterilized 96- well plate (Becton Dickson, Sparks, MD, USA). A desired amount of metalloid was loaded into the first row on the 96-well plate. The final volume of each well in the first row was 300 µL. If the volume of the toxicant was less than 150 µL then LB was added to make up the difference in volume. Each metalloid toxicant concentration was run in duplicate.
A 2-fold serial dilution was performed across the width of the plate. A multi-well pipette was used to transfer 150 µL to the next well and then mixed vigorously by pipetting that well’s volume up and down 10-12 times. After mixing, 150 µL from a well was transferred to the next row and this step was repeated for the width of the entire plate. After the last row, 150 µL was left remaining in the pipette and this was discarded.
The completed 96-well plate was stored in a zip-lock bag for 24 h at 37°C. After 24 h, 10
µL of resazurin sodium salt (1 mg/10 mL) was added to each well.
After another 24 h incubation period, the plates were checked for color change. A pink color demonstrates bacterial metabolic activity and the retention of a blue color (the unreduced resazurin color) means that the bacterial growth was severely inhibited due to the presence of the added metalloid. The MIC is recorded from these resulting data; it is determined to be the very lowest concentration of compound that inhibited growth after
24 h.
The MBC was determined by transferring 75 µL of solutions in wells on either side of the determined MIC concentration to an LB-agar sterile plate that was free of metalloid salts. Duplicate agar plates were also made for each concentration that was plated. The solution was spread using a sterile, glass L-rod and each plate was inverted
11 and incubated for 24 h at 37°C. In order to determine the MBC, the colonies were counted and the concentration plates that contained no colonies at this point were designated the MBC.
Te and Se Consumption by Bacteria
Pre-sterilized 15-mL centrifuge tubes were weighed and used to grow bacterial pre-cultures for approximately five hours (the amount of time needed to reach stationary phase). Upon reaching stationary phase, the bacterial cultures were inoculated with 5 mM sodium selenite or 50 µM potassium tellurite to a total volume of 10 mL. After inverting the tubes several times at time zero, a tube inoculated with potassium tellurite or a tube inoculated with sodium selenite were centrifuged (3100xg for 15 min.), the supernatant was removed and the tube weighed again to obtain the weight of the pellet that remained.
The pellet was then digested with concentrated nitric acid, taken to dryness and then re- suspended in 10 mL of 10% nitric acid. This procedure was then repeated for replicate tubes at 0.5, 1, 1.5, 2, 2.5, 4, 6 and 24 h. The tubes of re-suspended pellet were then analyzed via inductively coupled plasma atomic emission spectrometry (ICP-AES) using a Spectro CIROS Vision ICP-OES instrument. The nitric acid content of all samples was
10% and the analytical lines used were 196.090 nm for Se and 214.281 nm for Te.
Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP-AES
Reagents
Reagents used in these experiments include: BactoTMtryptone (Becton Dickson,
Sparks, MD, USA), yeast extract (EMD Chemicals, Gibbstown, NJ, USA), sodium chloride (BDH Chemicals, West Chester, PA, USA), potassium tellurite (Sigma-Aldrich,
12
St. Louis, MO, USA), cadmium nitrate (Sigma-Aldrich, St. Louis, MO, USA), chloramphenicol (Alfa Aesar, Ward Hill, MA, USA), sodium citrate (Sigma-Aldrich, St.
Louis, MO, USA), sodium borate (Sigma-Aldrich, St. Louis, MO, USA), L-glutathione reduced (GSH) (Sigma-Aldrich, St. Louis, MO, USA), and pre-sterilized 15-mL centrifuge tubes (VWR International, LLC, Radnor, PA, USA). A RiOs 3 water purification system from Millipore (Billerica, MA, USA) was used for water deionization.
Biosynthesis of Cadmium Telluride NPs
The E. coli strains gshA and gshB were from the ASKA (A complete Set of E. coli
K-12 ORF Archive) collection (Kitigawa et al., 2005). Cells were grown at 37 °C in
Luria-Bertani (LB) medium with shaking. Chloramphenicol (25 µg/mL) was added to the medium to prevent any other bacterial strains from growing; only cells that had incorporated the resistance gene for chloramphenicol were grown. Growth in liquid media was started with 1/100 dilutions of overnight cultures prepared in pre-sterilized 15- mL centrifuge tubes. The induction of gshA and gshB genes was carried out in the presence of 0.5 mM isopropyl-ß-D-1-thiogalactopyranoside (IPTG). IPTG was not added until the cultures reached stationary phase (~5 h). After the cells were left to grow for 24 h they were pelleted (spun down) via centrifugation for 10 minutes at 3500xg. The pellets were then washed twice with deionized water. After washing, cells were resuspended in water and cadmium nitrate (Cd(NO3)2) and/or potassium tellurite (K2TeO3) were added at final concentrations of 4 µg/mL and 0.5 µg/mL respectively. Four different types of samples were created: cells with no amendment, cells with only Cd(NO3)2 amendment, cells with only K2TeO3 amendment, and cells with both Cd(NO3)2 and K2TeO3
13
amendment. The cell suspensions along with the Cd(NO3)2 and K2TeO3 were left to incubate for two days at 37 °C to allow for NP synthesis. After two days, the cells were centrifuged and washed twice in order to remove any unincorporated Cd and Te, and the pellets were checked for fluorescence by using a UV trans illuminator at both 302 nm and
365 nm.
Quantification via ICP-AES
The samples were dissolved in concentrated nitric acid and taken to dryness twice. The remaining CdTe NPs were then suspended in a 10% HNO3 solution and analyzed via inductively coupled plasma atomic emission spectrometry (ICP-AES) using a Spectro CIROS Vision ICP-OES instrument. The nitric acid content of all samples was
10%.
Synthesis of Cadmium Telluride NPs
Stock solutions of citrate-borax buffer (30 mM in deionized water), 100 mM
Cd(NO3)2 (in citrate-borax buffer) and 100 mM K2TeO3 (in citrate-borax buffer) were prepared. In a test tube, 0.046 g of GSH was dissolved in 9.5 mL of 30 mM citrate-borax buffer at a pH of ~9.0. The tube was vortexed until all of the GSH was dissolved. After the GSH was dissolved, 400 µL of Cd(NO3)2 was added and the test tube was inverted to dissolve. Finally, 100 µL of K2TeO3 was added and a slight green appearance to the solution was observed. The solution was then incubated at 90 °C for ~2-4 h to synthesize the green NPs, ~4-6 h to synthesize the yellow NPs, and ~6-8 h to synthesize the red NPs.
Quantification via ICP-AES
After being synthesized, the NPs were centrifuged for 10 minutes at 3500xg and washed twice, and the pellets were checked for fluorescence by using a UV trans
14 illuminator. The samples were dissolved in concentrated nitric acid and taken to dryness twice. The remaining CdTe NPs were then suspended in a 10% HNO3 solution and analyzed via inductively coupled plasma atomic emission spectrometry (ICP-AES) using a Spectro CIROS Vision ICP-OES instrument. The nitric acid content of all samples was
10%.
15
CHAPTER III
RESULTS
Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial
Cells
The results for the MIC were easily determined based on color change using the method based on resazurin dye (Primm & Franzblau, 2007). By looking at Figures 1-4, a microwell plate for selenocyanate additions, it can be seen that the MIC falls somewhere around 400 mM (lowest concentration to retain its blue color), as seen specifically in
Figure 4.
Figure 1. Rows A and B (horizontal) contain a bacterial control consisting of only LHVE and the resazurin dye
Figure 2. Rows C and D contain resazurin dye, LHVE, and concentrations of selenocyanate starting with 1 M and decreasing two-fold across the width of the plate.
16
Figure 3. Rows E and F contain resazurin dye, LHVE, and concentrations of selenocyanate starting with 750 mM and decreasing two-fold across the width of the plate.
Figure 4. Rows G and H contain resazurin dye, LHVE, and concentrations of selenocyanate starting with 400 mM and decreasing two-fold across the width of the plate.
From this microwell plate, the concentrations of 500 mM and 400 mM were subsequently plated onto LB-agar plates (Figure 5, A and B respectively). The plates that contained bacteria amended with 500 mM selenocyanate were devoid of bacterial colonies after 24 h. The plates that contained bacteria amended with 400 mM selenocyanate were covered by bacteria demonstrating that the MBC was higher than 400 mM but lower than 500 mM.
17
Figure 5. LB plus agar plates containing LHVE amended with (A) 400 mM selenocyanate after incubation overnight, (B) 500 mM selenocyanate after incubation overnight, (C) 425 mM selenocyanate after incubation overnight, and (D) 400 mM selenocyanate after incubation for 96 h showing formation of red, elemental selenium.
Based on the results seen in Figure 5, the range in which the MBC fell was narrowed down by plating 425, 450 and 475 mM selenocyanate concentrations on LB- agar plates, and 24 h after replating 450 mM selenocyanate, those cultures had no visible colonies but the 425 mM plate did. Therefore the MBC was determined to be 450 mM.
The 425 mM-amended LB plus agar plates can be seen in Figure 5C in which the LB plus agar plates contained very few bacterial colonies due to growth inhibition from the
18 metalloid to which the bacteria were exposed from the MIC portion of the experiment (no additional toxicant was ever added to the LB-agar plates). Data for all five metalloid- containing anions are included in Table 1 for LHVE, Table 2 for BW (wild-type E. coli) and Table 3 for TM1b. An unexpected result was obtained with TM1b strain, in which high levels of tellurite-resistance but low levels of SeCN- tolerance were determined.
This result could indicate that both compounds interact with TM1b cells in different ways, probably in terms of cellular incorporation. That is, the highly toxic compound tellurite is less incorporated in TM1B cells as compared to other bacterial strains (E. coli or LHVE).
Table 1. Summary of all MIC and MBC data for the bacterium LHVE obtained for all five metalloidal anions amendments.
Metalloid-containing anion MBC MIC Selenate 600 mM 400 mM Selenite 200 mM 150 mM Tellurate 400μM 350 μM Tellurite 12 μM 10 μM Selenocyanate 450 mM 400 mM
Table 2. Summary of all MIC and MBC data for the bacterium E. coli BW25113 obtained for all five metalloidal anions amendments. “ >” indicates limits of solubility
Metalloid-containing anion MBC MIC Selenate > 1.5 M 1.5 M Selenite 300 mM 250 mM Tellurate > 1 mM > 1 mM Tellurite 63 µM 31 µM Selenocyanate 300 mM 250 mM
19
Table 3. Summary of all MIC and MBC data for the bacterium TM1b obtained for all five metalloidal anions amendments. “>” indicates limits of solubility.
Metalloid-containing anion MBC MIC Selenate 1.5 M 1 M Selenite 750 mM 500 mM Tellurate > 1 mM >1 mM Tellurite 750 µM 500 μM Selenocyanate 200 mM 125 mM
In Figures 6 and 7, consumption data can be seen for both selenite and tellurite, respectively. These experiments were carried out to see how relative bacterial toxicity compared to the relative incorporation of metalloids from these toxicants into cells. Each figure shows all three bacterial species consumption data for the two elements over a period of 24 h. Consumption in this context means the incorporation in or on the spun- down cells or precipitation of the toxicant added to those growing cultures.
20
2- SeO3 consump on by TM1B, LHVE and BW 2.5
2
1.5
TM1b 1 BW LHVE
0.5 [Se]/mg cells (ppm/mg)
0 0 5 10 15 20 25 30 Time (h)
Figure 6. Graph showing selenite consumption by TM1B (diamond), BW (square) and
LHVE (triangle).
21
2- TeO3 consump on by TM1b, LHVE and BW 0.08
0.07
0.06
0.05
0.04 BW
0.03 TM1b LHVE 0.02 [Te]/mg cells (ppm/mg) 0.01
0 0 5 10 15 20 25 30 Time (h)
Figure 7. Graph showing tellurite consumption by TM1B (diamond), BW (square) and
LHVE (triangle).
Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP-AES
Figure 8 shows fluorescence from spun-down bacterial cells of gshA of tubes that contain no metal amendments (control), cadmium nitrate alone, potassium tellurite alone, and cadmium nitrate plus potassium tellurite. Fluorescence can be seen in the tubes that contain Cd alone and in the tube that contains Cd plus Te. No fluorescence is observed in the control or Te tubes.
22
Figure 8. gshA fluorescence. From left to right: Control, Cd alone, Te alone, and Cd+Te amended samples.
Figure 9 shows fluorescence from gshB tubes that contain no metals (control), cadmium nitrate alone, potassium tellurite alone, or cadmium nitrate plus potassium tellurite. Some fluorescence can be seen in the tube that contains Cd alone, however, no fluorescence is seen in any of the other tubes (control, Te, Cd plus Te).
23
Figure 9. gshB fluorescence. From left to right: Control, Cd alone, Te alone, and Cd+Te amended samples.
Table 4 shows ICP data regarding the amounts of Cd and Te present in each tube. Based on the results seen in Table 4, GshA incorporates more Cd and Te into its cells more so than gshB does.
24
Table 4. ICP analysis results of pellets collected from the two E. coli strains, gshA and gshB. DL indicates that the reading was below the detection limit of the instrument.
*Indicates a corrected value based on dilutions
gshA gshB Cd Te Cd Te concentration concentration concentration concentration (ppm) (ppm) (ppm) (ppm) Control DL 0.109 0.105 DL Cd 6.680* 0.082 4.881 DL Te DL 0.272 DL DL Cd+Te 5.990 0.159 4.88 0.249
Table 5, shows ICP data regarding the amounts of Cd and Te present in each set of synthesized NPs, the Cd/Te ratio is represented here as a molar ratio. Based on the results seen in Table 5, the larger the NPs get (the longer they are incubated at 90 °C), the higher the Cd/Te molar ratio.
Table 5. Cd and Te content in the as-prepared NPs as determined by ICP
Cd/Te molar NPs µg Te/mg µg Cd/mg ratio Green 4.3 9.8 2.6 Yellow 3.0 9.1 3.4 Red 2.1 11.9 6.4
Figure 10 shows the green, yellow, and red synthesized QDs. These QDs were synthesized by only using four reagents. A UV trans illuminator is used to show fluorescence.
25
Figure 10. Green, yellow, and red QDs
26
CHAPTER IV
DISCUSSION
Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial
Cells
The results reported here suggest that the toxicity of selenocyanate is comparable to that of the selenate oxyanion but less toxic than selenite using the metalloid-resistant bacterium LHVE as the test organism to determine MIC and MBC values. Also, by using these measures of toxicity, tellurite was found to be more toxic to LHVE than tellurate; however, both the Te-containing compounds were much more toxic than selenate, selenite and selenocyanate (Table 1). The relative order of these toxicity data agrees with the ZOI and growth curve data previously obtained by Burra et al. (2009); however, we can also provide quantitative data as MIC and MBC values on important environmental species that had heretofore only been referred to as “extremely toxic” (Ye et al., 2003). In a more general way, although LHVE is obviously very resistant to the anions tested, it is reasonable to assume that non metalloid-resistant bacteria will probably exhibit this same relative order of toxicity for the anions studied though with much lower MIC/MBC values (Araya et al, 2004). However, according to the results obtained in Table 2 for the wild-type E. coli—which is considered non metalloid-resistant—the MIC/MBC values for all tested metalloids are higher than those for LHVE with the exception of selenocyanate. In addition, in Table 3, MIC/MBC values for TM1B are higher than those for LHVE and either higher or about the same as the values obtained for the E. coli wild type, again with the exception of selenocyanate. In this context, wild-type E. coli, may
27 not be useful as a remediation organism because it does not convert selenate or selenite to insoluble elemental selenium (data not shown) like LHVE has demonstrated (Figure 5D).
Knowing the MIC and MBC values of selenocyanate helps to establish at which concentration the growth of LHVE will be inhibited by these toxicant (MIC) and at which concentration growth will be completely halted and result in death of the bacteria (MBC).
Others have found utilitarian value in metalloid-resistant microbes with MIC values much lower. For instance, when Frankenberger and coworkers were isolating Se-resistant bacteria for a pilot bioremediation project involving the Kesterson Reservoir Se- contamination problems, their process involved selecting for microbes which would grow on 50 µM selenate-amended plates (Thompson-Eagle et al, 1989). While an overview by
Lenz et al. of selenium sampling at mine sites, shale acid seeps, and flue gas desulfurization suggested an Se range of 0.28 to 5.1 µM concentrations (Lenz et al.,
2008a), all these sites almost certainly involve multiple Se-containing chemical species, that is, multiple anionic Se-containing forms to which bacteria used in a bioremediation would be exposed. And while our MICs values are for single toxicant exposures, we and others have shown that these anions can have a synergistically negative biological effect
(Basnayake et al., 2001; Lenz et al., 2008b) and so metalloid-resistant microbes put into service in bioremediation will require high metalloid resistance to be viable in mixed- toxicant waste streams. Flue gas desulfurization waste water, for instance, has been found
2- 2- - to contain a mixture of SeO4 , SeO3 , and SeCN (Applied Speciation, 2011).
Finally, methods of bioremediation in aqueous systems and soils have previously involved using Se-resistant microbes to reduce Se chemical species to the (red) insoluble elemental form and/or to reduce and methylate metalloid contamination to volatile forms
28 like dimethyl selenide or dimethyl diselenide (Flury et al., 1997; Park et al., 2011).
According to McConnell and Portman (1952) the median lethal dose (LD50) of dimethyl selenide was determined to be 1.8 g dimethyl selenide for the mouse and 2.2 g dimethyl selenide for the rat, each value was for every kg of body weight. Our work with LHVE has shown that along with high MIC values, this microorganism also converts Se oxyanions and selenocyanate to volatile and less toxic methylated compounds such as dimethyl selenide, dimethyl selenenyl sulfide, and dimethyl diselenide (Burra et al.,
2009), as well as producing red elemental Se in plate experiments as seen in Figure 5D.
As far as we know this is the first evidence for the biological production of elemental Se by a metalloid-resistant bacterium amended with selenocyanate. The production of elemental selenium was only observed with selenocyanate, no elemental production was observed with any other compounds at the concentrations that were tested. The production of Se0 by LHVE cells exposed to SeCN- could be a cellular process involving new reducing enzymes or maybe requires the enzymatic machinery that produces Se0 in
2- 2- cells amended with SeO3 or SeO4 . Experiments to study this phenomenon will be conducted in our laboratory.
In Figures 6 and 7, it can be seen that LHVE is clearly better for cellular Te and
Se incorporation or precipitation as compared to BW, particularly at longer time exposure. Also, for LHVE, Se consumption at first, appears to be saturated between 4 and
6 h; however after 24 h it is still able to bioprocess more Se. This is not observed for BW which is saturated at shorter times (4h) and incorporates/precipitates smaller amounts of
Se. Consumption of Se and Te by TM1B is more similar to that of BW than it is to
LHVE. This suggests that TM1B and BW do not have the ability to consume as much Se
29 or Te as LHVE does indicating that LHVE could be better for bioremediation due to its capacity to incorporate much higher amounts of Se and slightly higher amounts of Te.
Probably, this improved capacity of Se and Te incorporation observed in LHVE could be related with the presence of Se/Te-reductases that favor the intracellular detoxification of the toxicants by forming Se0, Te0 or other less toxic forms. Determining the cellular mechanism involved in resistance and Te/Se incorporation of LHVE could help to find or develop new bacterial strains with improved capacities for the bioremediation of this toxic compounds. The shape of the consumption curves (Figures 6 and 7) indicate that there might be two different processes that occur based on the rate that the bacteria consume either Se or Te. For example, when looking at Figure 6, for LHVE at 6 h, the consumption of Se appears to happen rather quickly, whereas once it starts to approach
24 h, the consumption of Se appears to happen much more slowly. This could be due to the amount (biological) reagents available initially in bacteria cells that cause a faster consumption rate. Prospective cellular biological reducing agents would be methanethiol and reasonable methylation reagents would S-adenosyl methionine. When this initial cellular pool of bioreagents is used up the metalloidal conversion rate slows down to a rate at which the bacteria can produce new chemicals in order to assist in the consumption of Se and Te. Also, from Figures 6 and 7, an inverse relationship between resistance and toxicant incorporation is observed. TM1B cells that are resistant to tellurite avoid tellurite incorporation which probably corresponds to a mechanism of toxic defense. This observation is also very interesting since the way in which tellurite is incorporated into bacterial cells has not been determined to date, and the existence of a bacterium with decreased cellular uptake of tellurite represents an ideal candidate to
30 study this process and to understand the proteins or cellular structures involved in this process. Also, the knowledge of the mechanism present in TM1B avoiding oxyanions uptake could be useful to construct new strains with improved properties for bioremediation by mutating the involved genes.
Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP-AES
Most NP-synthesizing procedures described to date are expensive, require dangerous compounds and produce NPs with elevated toxicity (Peng and Peng, 2001;
Hardman, 2006). The use of non-toxic stabilizing agents that reduce the oxidant properties of NPs and increase their solubility in aqueous solutions is an alternative to NP biocompability (Hoshino et al., 2007). Based on the experimental conditions of a biomimetic protocol developed by Perez-Donoso et al. (2012), and the known interactions of GSH with CdCl2 and K2TeO3, an E. coli strain with increased levels of
GSH was used to biosynthesize CdTe NPs or QDs in the presence of Cd(NO3)2 and
K2TeO3. The E. coli gshA strain contains higher glutathione content than the gshB strain since the gshA strain catalyzes the final and limiting step in GSH synthesis (Murata and
Kimura, 1982). Therefore, this strain was an excellent candidate to test the role of GSH as a reducing and stabilizing agent in the biological synthesis of CdTe QDs. Cells that overexpressed the gshA gene displayed fluorescence under UV excitation (Figure 5). In contrast, no fluorescence was observed with the strain that overexpressed the gshB gene, showing that GSH favors the synthesis of fluorescent structures. Fluorescent cells displayed higher Cd and Te amounts (Table 4); this correlates fluorescence with the incorporation of metal substrates for QD biosynthesis. This observation could be the
31 basis for future applications in heavy metal bioremediation associated with the production of QDs of biotechnological interest. It is important to study CdTe QDs because they could potentially be used as molecular probes for the detection of heavy metal ions (Ali et al., 2007; Wang et al., 2011; Yang et al., 2011). As a result of specific interactions, the fluorescence intensity of GSH-capped NPs is selectively reduced in the presence of heavy metal ions such as Pb2+, As3+, and Hg2+ (Ali et al., 2007; Wang et al.,
2011; Yang et al., 2011). This work will be explored more in our laboratory.
In order to develop a simple protocol for the aqueous synthesis of CdTe QDs,
2+ 2- Cd(NO3)2 and K2TeO3 were used as the Cd and Te sources since the effects that these two compounds have on microorganisms are relatively well known (Pérez et al., 2006;
Pérez et al., 2007; Chasteen et al., 2009). Taking into consideration the redox properties of GSH, and its abundance in cells, this biological thiol was tested as a reducing and capping agent for the aqueous synthesis of CdTe QDs. As a result, a simple protocol requiring only Cd(NO3)2, K2TeO3 and GSH was generated. This protocol was developed based on the protocol seen in Pérez-Donoso et al. (2012). A reaction carried out at 90 °C in citrate-borax buffer and pH 9.0, allowed the generation of highly fluorescent green, yellow and red NPs after approximately 2-12 h depending on the length of time the reaction mixture was held at 90 °C. In order to analyze the Cd/Te ratios in each color of
NPs, ICP experiments were carried out. Results confirmed the presence of Cd and Te at the expected ratio in the synthesized NPs (Table 5). In addition to ratios, size is an important factor that affects potential biological applications of the NPs. Dynamic light scattering (DLS) measurements of aqueous CdTe-GSH QDs were carried out by Pérez-
Donoso et al. (2012). Results of DLS measurements showed that CdTe-GSH QDs ranged
32 from 3-6 nm in size with green and red NPs exhibiting average sizes of 3.3 and 5 nm respectively and yellow NPs exhibited intermediate sizes. Since this method uses lower temperatures than other protocols, GSH decomposition and the concomitant production of CdS are decreased, thus favoring NP biocompatibility (Pérez-Donoso et al., 2012).
33
CHAPTER V
CONCLUSIONS
Bacterial Toxicity of SeCN- and the Incorporation of Se and Te in Bacterial
Cells
The relative toxicities of selenocyanate along with selenate, selenite, tellurate and tellurite were evaluated using a series of experiments to determine an MIC and MBC for each compound by using three different bacteria: LHVE, TM1b and a wild-type E. coli
2- 2- 2- species designated as BW. The relative toxicities for LHVE are: TeO3 >TeO4 > SeO3
2- - 2- 2- 2- >SeO4 ≈SeCN . The relative toxicities for TM1b are: TeO3 >TeO4 > SeO3
2- 2- 2- 2- ≈SeCN>>SeO4 . The relative toxicities for BW are: TeO3 >TeO4 >SeCN > SeO3
2- >SeO4 . Upon determining the relative toxicities of many selenium and tellurium containing anions we also discovered, what we believe to be, the first evidence for the biological production of elemental Se by a metalloid-resistant bacterium amended with selenocyanate. LHVE, amended with 400 mM selenite, produced red, elemental Se after
96 hours, when grown on an agar plate.
All three bacterial species were exposed to selenite and tellurite salts to see how much Se and Te they could incorporate into their cells. It was concluded that LHVE is clearly better for cellular Te and Se incorporation or precipitation as compared to BW.
Consumption of Se and Te by TM1B is more similar to metalloid consumption of BW than it is to LHVE. This suggests that LHVE could be better for bioremediation due to its ability to incorporate or precipitate much higher amounts of Se and slightly higher
34 amounts of Te. In addition, it can also be concluded that an inverse relationship between resistance and toxicant incorporation was observed for the bacteria studied.
Synthesis and Biosynthesis of CdTe QDs and Their Elemental Quantification via ICP-AES
An E. coli strain with increased levels of GSH was used to successfully biosynthesize CdTe NPs in the presence of Cd(NO3)2 and K2TeO3. Cells that overexpressed the gshA gene displayed fluorescence after being excited by UV light.
Those cells that expressed the gshB gene did not exhibit any fluorescence. It was determined by ICP-AES that those cells that did fluoresce showed higher Cd and Te amounts than those that did not fluoresce.
A novel protocol was followed and green, yellow and red NPs were successfully produced by using only GSH, Cd(NO3)2, K2TeO3, and a citrate-borax buffer at a pH of
9.0. These conditions mimic those that are used to biologically synthesize CdTe NPs.
Upon ICP-AES analysis it was determined that the larger NPs (produced by longer times in a hot water bath) contain higher ratios of Cd and Te.
35
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APPENDIX
Chemical Abstract Service Registry Numbers
Compound CAS Number sodium selenate 13410-01-0 sodium selenite 101202-18-8 potassium selenocyanate 3425-46-5 potassium tellurate 26006-71-3 potassium tellurite 123333-66-4 agar 9002-18-06 sodium chloride 7647-14-5 tryptone 73049-73-7 yeast extract 8013-01-2 cadmium nitrate 10022-68-1
L-glutathione reduced 70-18-8
Resazurin sodium salt 62758-13-8 isopropyl-ß-D-1-thiogalactopyranoside 367-93-1
45
VITA
Rebecca Ann Montes was born on September 24, 1986 in Houston, TX to David and Maria Montes. She is the fourth of five children and the second in her family to pursue a degree beyond that of a Bachelor’s degree. In August of 2005 she began working on her undergraduate studies at Sam Houston State University. She went from wanting to be a medical doctor to wanting to be a chemist. She graduated with her B.S. in
Chemistry in May of 2010 and began working on her Master’s degree in Chemistry.
Throughout her graduate career she continued doing research with Dr. Thomas G.
Chasteen who she had previously enjoyed working with at the end of her undergraduate career. During her graduate studies she presented at the 2011 University Wide Graduate
Research Exchange and has had the privilege of working with individuals from both Iran and Chile. She has been co-author on articles that have been published in Analytical
Biochemistry and Applied Environmental Microbiology.