The purpose of this research was: (1) to determine the extent of bacterial tolerance to grow in the presence of different concentrations of toxic selenium and tellurium oxyanions; (2) to identify organo-selenium compounds in the headspace of metalloid-resistant bacteria; and (3) to develop a simple, quick, and reliable method for selenium and tellurium oxyanion analysis in live bacterial cultures.
Using optical density to follow bacterial population and growth rate, a Bacillus bacterial species was examined in the presence of different concentrations of selenium and tellurium oxyanions. At regular time intervals, the optical density of these samples was measured at 525 nm using a spectrophotometer. Specific growth rates, which reflect relative metalloidal oxyanion toxicity, were determined using the slope of the growth curve. Zone of inhibition experiments were also used to measure the relative toxicity of selenium and tellurium oxyanions to the test organism. Bacteria were grown in Petri dishes with a paper disk placed in its center which was saturated with a metalloid solution of various concentrations. A zone of growth inhibition observed around this disk was used as a measure of relative toxicity of these oxyanions. The second set of experiments carried out included the identification of organo-selenium compounds in the headspace of metalloid-resistant bacteria and the determination of bacterial ability to reduce and methylate toxic metalloidal salts. Solid phase micro extraction was used to collect headspace gases above live bacterial cultures. Analysis of the headspace samples was performed by a fluorine-induced sulfur chemiluminescence detector coupled to a gas chromatograph. Gas chromatography/mass spectrometry was used to confirm the identity of the compounds collected above the cultures of the bacteria. The final set of experiments performed included analysis of selenium and tellurium oxyanions using UV/visible spectrophotometry. Sodium borohydride (NaBH4) was used as a reducing agent to convert soluble selenium and tellurium oxyanions to their elemental forms whose optical density was measured at 500 nm.
Specific growth rate and zone of inhibition experiments showed that tellurite was the most toxic among the selenium and tellurium oxyanions examined. Headspace experiments resulted in the detection of two new organo-metalloidal components previously unreported in the literature: dimethyl diselenenyl sulfide and dimethyl triselenide. In addition a wide range of organo-sulfur and -selenium compounds were detected. Spectrophotometric determination of selenium and tellurium oxyanions in bacterial growth media using NaBH4 as the reducing agent provided a good linear range for selenate, selenite, and tellurite. The linear range for tellurate analysis using the borohydride method was quite small but this may have been caused by the poor solubility of this oxyanion in growth media. The detection limits of this method for metalloidal oxyanions are modest in relation to other modern instrumental methods; however, borohydride reduction was shown to be a quicker and more straightforward method compared to most other analytical techniques, with simple instrumental requirements and the need for few reagents.
Keywords: zone of inhibition, specific growth rate, selenocyanate, borohydride