Abstract


McCarty, Steven L., Design and application of a dynamic headspace sampling system for the study of bioremediation of toxic metalloids by bacteria. Master of Science (Chemistry ), December, 1994, Sam Houston State University Huntsville, Texas. 42 pp. (pdf version of this thesis)

Purpose
The purpose of the experiments describe here was to follow the output of reduced chemical species produced by metalloid-resistant bacteria.
Method
In order
1) to remove the volatile biological wastes produced as these organisms biodegrade selenium oxyanions and

2) to maintain an anaerobic environment,

the bacterial cultures studied had to be continuously purged with an inert gas. This therefore required the trapping and concentration of the gas phase components before analyses. Variable trapping times allowed for the changes in headspace component concentrations associated with the continuous fluctuation in the bacterial efficiency of bioremediation.

The trapping apparatus consisted of a double focussing cryogenic system with an initial trap external to the gas chromatograph. The second cryogenic trapping was performed by the oven of the gas chromatograph. A bioreactor was designed that interfaced with the purge and trap system. It allowed for anaerobic sampling and continuous purging and mixing of the bacterial culture with sterile nitrogen. The methylated selenium and sulfur compounds released by these bacteria were separated by capillary gas chromatography and detected by fluorine-induced chemiluminescence.

Findings
A dynamic headspace sampling apparatus was designed and constructed. Headspace gases above anaerobic bacterial cultures were sampled over long times course experiments. Large volumes of headspace gases could be collected, cryogenically concentrated, and then cryofocussed in an external subambient cryotrap and again in the GC oven. Organosulfide and organoselenide compounds could be determined from that headspace. Repeated sampling of the same culture over time permitted a time based examination of the bioremediation of the oxyanions of selenium by anaerobic bacteria.

The high boiling organo-chalcogens were examined routinely over experiments up to 100 hours long using the same culture. Plotted over time, these analyses showed that the production of dimethyl disulfide and dimethyl diselenide tracked the growth curve of the facultative anaerobe examined, Pseudomonas fluorescens K27.


Thomas G. Chasteen
Thesis Director


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