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Fox, Bala Krishna Pathem, and Thomas G. Chasteen Sam Houston State University Russell Gerads and Hakan Grleyk Applied Speciation and Consulting, LLC, Tukwilla, WAb?(Abstract  The genetically modified bacteria Escherichia coli 1VH carry a plasmid containing genes conferring resistance to the antibiotic ampicillin as well as resistance to common, yet highly toxic oxyanions of selenium and tellurium. For instance, selenate and tellurite are two common biospheric forms of these metalloids. In addition to this resistance, volatile sulfur, selenium and tellurium compounds are observed in the headspace of cultures amended with the corresponding metal salts. The growth rates of the bacteria were examined with amendments of various concentrations of sodium selenite, sodium selenate, and potassium selenocyanate using the optical density of each culture as a measure of culture population. In addition to this the headspace of the cultures were examined using gas chromatography coupled with a fluorine-induced chemiluminescence detector. Analysis of Se-containing oxyanions such as SeCN- were carried out using ion chromatography with inductively coupled plasma/mass spectrometry. Experiments involving bacterial amendments with or bacterial production of SeCN- are important because of the interest in this anion's determination in industrial waste streams contaminated with selenium.b "_  }P O~ Bacterial and Sample Preparation!!( E. coli 1VH  Escherichia coli JM109 cells were modified to express the genes encoded in a 3.8-kb chromosomal DNA fragment from a metalloid-resistant thermophile, Geobacillus stearothermophilus V.This metalloid resistant organism, 1VH, was cloned in Dr. Claudio Vsquez s laboratory at University of Santiago, Chile. LB Medium  The nutrient broth for the bacteria was made by combining tryptone, NaCl and yeast extract in water. The pH of the media was adjusted to 7 and the media sterilized. Before inoculation, ampicillin was added to the medium. Pre-cultures of 1VH were prepared by adding a single colony from a plated culture to 100-200 mL of LB medium. These were incubated at 37 C for approximately 24 hours to allow the bacteria to reach stationary phase. To prepare the metalloid solutions, 100 mM solutions were made for sodium selenite, sodium selenate and potassium selenocyanate. A 1.0 mM solution of sodium tellurite was also made. Each solution was sterile-filtered. Pi" "  "  G  R-Sample Preparation cont. (for growth curves)".(RTo prepare samples for growth curve analyses, LB medium was distributed into test tubes (9 mL for the control samples and 8 mL for the samples to be tested with metalloid amendments). The batch was sterilized and, once cooled, 0.1 mL of ampicillin solution was added to each test tube. To the control was added 1.0 mL of stationary 1VH from the pre-culture broth. For the amended solutions, 1.0 mL of the sterile metalloid solution and 1.0 mL of stationary 1VH were added. All samples were sealed with screw-caps and incubated at 37 C between readings in a constant-temperature water bath."S"1Sample Preparation cont. (for headspace analysis)"2(&Headspace samples were prepared by distributing LB medium in 9.0 mL aliquots into test tubes. After sterilization, 0.1 mL of ampicillin solution was added to each test tube. To the control was added 1.0 mL of stationary 1VH. To each of the amended solution was added 1.0 mL of stationary 1VH and 0.1 mL of a 100 mM metalloid solution. Each test tube was capped with an open-ended screw cap equipped with a Teflon-lined septum. These samples were incubated at 37 C for approximately 72 hours in order to allow headspace gasses to accumulate. 2'*"; Growth Curve AnalysisfThe growth curve analysis was performed using liquid culture absorbance/scatter at 526 nm. Absorbance readings were taken at regular intervals, using sterile solutions of each of the different media for blanks. The readings continued until cultures had reached the stationary growth phase. Log phases of growth were estimated as the linear portion of the log absorbance versus time plot. A short lag phase was assumed. The slope of the linear least squares fit, the specific growth rate, gave a clear idea about the relative toxicity of each of the amendments. Lower specific growth rates suggest higher toxicity. gZg Headspace AnalysisHeadspace sampling was accomplished using a solid-phase microextraction (SPME) fiber (carboxen-polydimethysiloxane with 75 micrometer thickness) exposed to the headspace of the bacteria for 30 minutes. The fiber was then inserted into the injection port (275 C) of the GC using a temperature program that held 30 for 2 minutes, ramped 15 /min and held 275 C for five minutes. A Sievers 300 fluorine-induced sulfur chemiluminescence detector was coupled with the GC in order to detect organo-sulfur, -selenium and  tellurium compounds in the sample. Compounds were identified based on retention times of commercial standards or via GC/MS."4 " ""  $*$((X,,00b8d /The Fluorine-Induced Chemiluminescence Detector00(Growth Curve ResultsGrowth Curve Results cont. Growth Curve Results cont. Growth Curve Results cont. Growth Curve Results cont. %Headspace Results for Control Culture&&(,Headspace Results for 10 mM Selenite Culture--(,Headspace Results for 10 mM Selenate Culture--(*Headspace Results for 1.0 mM KSeCN Culture++( /lIon Chromatography- Inductively Coupled Plasma/Mass Spectrometry Analysis of Se-Amended Bacterial Cultures*"ml(M7 Conclusions  NEach of the selenium-amended solutions had a pronounced effect on the specific growth rate of 1VH. Of those oxyanions, selenite had the most toxic effect. The relative toxicity of the selenocyanate anion was similar to that of the selenate anion, only slightly more toxic. The tellurite-amended solution had the most toxic effect of all the amended solutions, reducing the growth rate by almost 86% compared to the control culture. Headspace analysis showed a diverse production of sulfur- and selenium-containing volatiles. Of these, the culture amended with KSeCN produced the widest array of compounds in the largest quantities. Selenite-amended cultures of P. fluorescens produced detectable amounts of selenocyanate, SeCN1-. 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Fox1, Bala Krishna Pathem1, Russell Gerads2, Hakan Grleyk2 and Thomas G. Chasteen1, (1)Sam Houston State University, Huntsville, TX, (2)Applied Speciation and Consulting, LLC, Tukwilla, WA  James D. Foxn oLevelD.Dr. Thomas Chasteen51 Microsoft Office PowerPoint@{pB@Z@#y@c*Gb/PICT/Z HH HH  r, &fff&fff&fff&fff&fff&fff&fff&fff&fff&fff&fff&fff""BBBBBBBBBBBBBB<BBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB  BBBBBBBBBBBBBBBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBB  BBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB BBBBBB    BBBBB BBBBBBB BBBBBBBBBBBBBBBBB BBBBBBBBBBBBB    (B+BB""sBBBBBBBB*BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB{ BBBBBBBBBBBBBBBBBB BBBBBB!BBBBBBBBBBBBBBBBBBBBBBBBB  !  !{BBBBBBBBBBBBBBBBBBB BBBBBB%BBBBBBBBBBBBBBBBBBBBBBBBB % %fBBBBBBBBBBBBBBBBBBBBBBBB BBBBBBBBBBBBBB  7BBB:BBBB"""BBBBBBBBBBBBBBH BBBBBBBB BBBBBBBBBBBBBBBBB BBBBBBBBBBBBBBBB    rBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBoBBBBBBBBBBBBBBBBBBBBBBBBBBBB"BBBBBBBBBBBBBBBBBBBB""HBBBBBBBBBBBBBBBB BBBBBBBBBBBBBBB BBBBBB    CBBBBFBBBBB"""vBBBBBBBBBBBBBBBBBBBBBBBBBBBBB BBBBBBBBB BBBBBBBBBBBBBBBBBBBB    BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB4BB:BBBB"""""""""""":""""܁3   4""   ("""""""(""ց   ^"""""""""""""""""" lurium compounds in the headspace of genetically-modified organisms using GC/SCD Abstract!Bacterial and Sample Preparation.Sample Preparation cont. (for growth curves)2Sample Preparation cont. (for headspace analysis)Growth Curve AnalysisHeadspace Analysis0The Fluorine-Induced Chemiluminescence DetectorGrowth Curve ResultsGrowth Curve Results cont.Growth Curve Results cont.Growth Curve Results cont.Growth Curve Results cont.&Headspace Results for Control Culture-Headspace Results for 10 mM Selenite Culture-Headspace Results for 10 mM Selenate Culture+Headspace Results for 1.0 mM KSeCN CulturemIon Chromatography- Inductively Coupled Plasma/Mass Spectrometry Analysis of Se-Amended Bacterial Cultures* ConclusionsAcknowledgements  Fonts UsedDesign TemplateEmbedded OLE Servers Slide Titles+_Dr. Thomas Chasteen