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4dosed with them (Strotmann et al., 1994; Yu et al., 1997), as well as selenium’s effect onpersister cells (Harrison et al., 2005).Analytical Techniques for Detecting Selenium CompoundsA number of instrumental techniques have been applied in the search for variousselenium species. X-ray absorption near-edge structure (XANES) spectroscopy has beensuccessfully implemented in the detection of various forms of selenium, includingelemental, aqueous and volatile forms (Pickering et al., 1995; Sarret et al., 2005). Ionchromatography–inductively coupled plasma–mass spectrometry (IC-ICP-MS) has alsobeen used in the detection of trace amounts of water soluble selenium species. Thismethod has the added benefit of low detection limits (0.04-0.09 ng) in the presence ofcomplex samples (Miekeley et al., 2004). Hydride generation-atomic absorptionspectroscopy (HG-AAS) was also used with positive results. In a study by Losi et al.(1997), the microbiological conversion of selenate to selenite and then to elementalselenium was observed.For this research, several different methods of selenium speciation were applied:Gas Chromatography with Fluorine-Induced Sulfur Chemiluminescence DetectionThere are a number of benefits in using gas chromatography with fluorineinduced sulfur chemiluminescence detection (GC/SCD) to look for volatile organo-sulfur,-selenium, and -tellurium compounds. Given their non-polar nature and relatively largedifferences in boiling points, volatile methylated selenides have been found to separateeasily on several different gas chromatographic columns (Basnayake et al., 2001; Meija

5et al., 2002; Hunter and Kuykendall, 2004; Swearingen, Jr. et al., 2006). In the SCD,gaseous, elemental fluorine is produced in situ by flowing sulfur hexafluoride across an1100 V volt discharge at approximately 26 kHz of alternating current. A substantialportion of the resulting gas phase fragments reform as F2 and continue into the reactionvessel. It is there that they react with methylated chalcogens to produce excited-statehydrogen fluoride, which, upon de-excitation, emits a photon that is detected via aphotomultiplier tube. The pressure in the reaction vessel is maintained at less that onetorr to ensure low collisional deactivation.Because of the intense reaction of methylated chalcogens with F2 compared toother molecules, the background signal for this instrument remains low enough and theselectivity high enough to obtain detection limits in the picogram range for this family ofcompounds (Chasteen, 1993).Capillary Electrophoresis (CE)Much work has been done in the task of separating and detecting various watersoluble selenium species using capillary electrophoresis. Some methods include couplingCE with inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) (Deng etal., 2007), conductivity detection (Kubá et al., 2004) or simple ultraviolet/visibleabsorption spectroscopy (Walker et al., 1996). CE has provided an efficient means ofseparating and detecting these selenium species in solution. It is because of this that CEseems ideal for use with broth cultures of selenium-resistant bacteria. Recently, work byPathem et al. (2007) showed the time-dependant conversion of selenate to selenite andelemental selenium by a strain of Escherichia coli.

6The principles on which electrophoresis operates are simple, yet effective. Themethod’s capillary, similar to a capillary column in GC, is made of fused silica with apolyimide coating for thermal and electrical insulation. The inner diameters usuallyrange from 25-75 µm and the length is between 50-75 cm. Because of this, the samplevolume needed is typically 1-50 nL. This allows smaller overall sample sizes. ForUV/Vis detection, a small portion of the polyimide coating is removed so that light canpass through with minimal absorbance by the capillary itself. This is the capillarywindow.Before separation can occur, the capillary is filled with a buffer solution that actsto ionize the silanol groups along its interior. The more basic the buffer solution, themore ionization occurs thus raising the overall negative charge along the capillary walls.This buffer solution will also act as an electrolyte to carry the current during separation.Positively charged ions will coat the interior of the capillary in two layers: one that isaffixed to the wall and one that will move in the electric field. This second layer causes amass movement of molecules within the capillary, thus establishing the electro-osmoticflow (EOF). Typically the cathode is on the same end as the detector, causing positivelycharged molecules to move toward this electrode the fastest, followed by neutral and thennegatively charged molecules dragged along by the EOF.In these experiments, a photodiode array was used for detection, allowing for thesimultaneous monitoring of absorbance values between 190 and 300 nm. As themolecules pass the capillary window, their absorbance was measured as a function oftime and wavelength. This was useful for distinguishing between CE peaks whoseretention times are similar but differ in their absorbance profiles.

7In the case of the water-soluble selenium species, negatively charges ions are ofinterest. Therefore a modification to the EOF was necessary. To accomplish this, asurfactant (tetradecyltrimethylammonium bromide) was used to coat the interior of thecapillary, reversing its charge. Once this was done, the polarity of the electric field wasalso reversed. This caused anions to elute first, followed by neutral molecules and thencations.With broad linear ranges and low detection limits, CE is ideal for the monitoringof trace amounts of selenium species produced by selenium-resistant microorganisms.Solid-Phase Extraction (SPE)While CE provides low detection limits, it may be necessary to pre-concentratethe samples in order to better see them in the electropherogram at even lowerconcentrations or in the presence of interfering species. In order to accomplish this, SPEwas utilized to collect selenocyanate from broth cultures of selenium-resistant organisms.To this day, SPE is typically used as a cleanup method for complex biologicalsamples (Shimelis et al., 2007). However, this method was shown to be effective atretaining and recovering analytes when, among others, Monohan et al. (1995) used stronganion exchange (SAX) SPE in order to collect the herbicide Dacthal for analysis via GCwith electron capture detection.This research made use of aminopropyl-coated SPE cartridges. The pKa of theamine group on the cartridges was approximately 9.8. Solvation of the cartridgeprotonated the amine groups to generate a positive charge and ionic binding betweenthem and negatively charged components of the solvation buffer. Once solvated, a

8sample was passed through the cartridge, at which time negatively charged seleniumcontaining species attached to the amino groups and were retained on the cartridge’s solidphase. Then, a buffer (pH 11.8) was passed through, causing the deprotonation of theamino groups and the elution of the analytes. This allowed an amount of analyte to becollected and deposited into a smaller volume of liquid, effectively raising theconcentration.Microorganisms UsedFor this research, data from three different metalloid-resistant, anaerobicmicroorganisms were examined: 130404 (a species of the genus Alcaligenes), Bacillusspp. and Escherichia coli 1VH. The organism 130404 was provided by collaboratingresearchers at the University of Santiago, Chile. E. coli 1VH was prepared by Araya etal. (2004). The Bacillus spp. was isolated in the Chasteen research group at Sam HoustonState University and its genus determined in Chile. All organisms display some degreeof selenium resistance as measured by their ability to grow in millimolar solutions ofselenium oxyanions.Relevance of This ResearchMuch instrumentation has already been successfully applied to examine theconversion of water-soluble selenium into other forms. However, many of these methodshave looked solely at the conversion to elemental or volatile selenium. Interesting workexamining the intermediate biological steps as selenium-containing oxyanions arebioprocessed to less oxidized forms has been reported (Walker et al., 1996; Pathem et al.,

92007). This may be important in understanding the final destination of selenium as it isprocessed by these resistant organisms.IC-ICP-MS experiments by Gürleyük et al. (2006) indicated that one possiblebiological product that had been overlooked was selenocyanate. Until this point, theoccurrence of selenocyanate in contaminated areas was attributed to one of two things:the reduction of other selenium species via environmental chemistry or the presence ofoil waste. Locating a microbiological source of selenocyanate may prove useful to thoseinterested in the removal of selenium from areas of high concentration.For the work in this thesis, a method was developed to examine thebacteriological production of selenocyanate using solid-phase extraction and capillaryelectrophoresis. In addition to this, the relative toxicities of the various water-solubleselenium species and the production of volatile headspace gases was also determined.

10CHAPTER IIEXPERIMENTALBacterial GrowthReagentsThe chemical reagents used in these experiments were obtained from thefollowing sources: pancreatic digest of casein and yeast extract from Difco Laboratories(Detroit, MI, USA); tryptic soy broth from Mediatech, Inc. (Herndon, VA, USA); sodiumselenite, sodium selenate and sodium hydroxide from Sigma-Aldrich (Milwaukee, WI,USA); potassium selenocyanate from Acros Organics (New Jersey, USA); potassiumnitrate from EM Science (Gibbstown, NJ, USA); sodium chloride from BDH (WestChester, PA, USA); agar from Amresco (Solon, OH, USA). All water used in theexperiments was purified via a RiOs 3 water purification system from Millipore(Billerica, MA, USA).Bacterial Cultures and Growth MediaBacterial cultures of Escherichia coli 1VH and the Gram negative bacterium130404 were grown anaerobically in Lauria-Bertani (LB) medium at 37º C in a waterbath. The LB medium was prepared by adding 10.0 g of pancreatic digest of casein, 5.0g of yeast extract and 5.0 g of sodium chloride to 1.0 L of water and mixing thoroughly.If needed, the pH of the solution was adjusted to 7.0 with 1.0 M sodium hydroxide. Thismedia was then distributed into flasks and sterilized at 121º C and 15 psi for 20 min.Once cooled, the media was inoculated with 1VH from a stab culture using a sterile loopand allowed to incubate for 24 hours. Plate cultures were prepared by adding 20 g of

11agar to 1.0 L of LB medium and sterilized as above. Before cooling, the agar wasdistributed to Petri dishes and amended with 1.0 mM sodium selenite. These plates werestreaked using a sterile loop dipped in samples of the prepared liquid cultures andallowed to incubate for 24-36 hours (until single, red bacterial colonies could be seen).Precultures were prepared by inoculating LB medium with a single colony and allowed itto incubate for 24 hours. Experimental cultures were made by distributing sterile LBmedium into test tubes and inoculating with a 1:10 v/v inoculum from the preculture.Experimental cultures were amended with sodium selenate, sodium selenite or potassiumselenocyanate to yield a final concentration of 1.0 or 10.0 mM.The Bacillus spp. was grown anaerobically in tryptic soy medium with a 0.3%nitrate content (TSN3) at 30º C in a water bath. TNS3 was prepared by adding 10.0 g oftryptic soy broth (TSB) and 3.0 g of potassium nitrate to 1.0 L of water and mixedthoroughly. Plate cultures, precultures and experimental cultures were made with TSN3using the same steps as above.Headspace SamplingApparatusFor the headspace analysis, a Hewlett-Packard 5890 Series II gas chromatograph(GC) with a Sievers 300 fluorine-induced sulfur chemiluminescence detector (SCD)(Ionics Instruments, Boulder, CO, USA) was used. The GC was equipped with a DB-1column (100% polydimethylsiloxane, 5.0 µm stationary phase) that measured 30 m x0.32 mm i.d. (J&W Scientific, Folsom, CA, USA). Data acquisition was accomplishedusing a Hewlett-Packard 3396 Series III integrator. The headspace samples were

12collected using 75 µm Carboxen -polydimethylsiloxane (PDMS) solid-phasemicroextraction (SPME) fibers from Supelco (Bellfonte, PA, USA).GC ParametersHelium was used as a carrier gas at a flow rate of 1.0 mL per minute. All sampleswere injected in splitless mode into a 275º C injection port. The split valve opened at2 min. The initial oven temperature was held at 30º C for 2 min and then raised15º C/min until a final temperature of 275º C was reached. This temperature was held for5 min.SPME SamplingCultures (10 mL liquid) used for headspace samples were grown in 16 mL testtubes capped with open-top screw caps with Teflon /silicone liners purchased formAlltech (Deerfield, IL, USA). The SPME syringe was used to pierce the septa and thefiber was then exposed to the volume of gases above the liquid culture for approximately30 min in order to adsorb the headspace gases. The fiber was then retracted and thenintroduced into the injection port of the GC and exposed for the duration of thetemperature program (initiated immediately after exposure of the fiber).CE SamplesReagentsThe reagents used in the CE analysis were obtained from the following sources:potassium dihydrogen phosphate (KH2PO4) from Sigma-Aldrich;tetradecyltrimethylammonium bromide from Acros Organics; concentrated hydrochloric

13acid (Mallinckrodt, Paris, Kentucky, USA); and methanol from Burdick & Jackson(Muskegon, MI, USA).ApparatusA P/ACE MDQ CE system (Beckman Coulter, Fellerton, CA, USA) was used inall CE experiments. This system was equipped with an auto sampler and temperaturecontrol for the sample trays and capillary. Fused silica capillaries (also purchased fromBeckman Coulter) with dimensions of 57 cm total length, 50 cm effective length, 75 µmi.d. and 375 µm o.d. were used.CE AnalysisAll CE samples were run according to the methods developed by Pathem et al.(2007).The run

the analysis of selenocyanate from the broth cultures of selenium-resistant bacteria using solid-phase extraction and capillary electrophoresis