Becer, Eser, Isolation and investigation of antimony-resistant bacterial cultures. Master of Science (Chemistry), May, 1997, Sam Houston State University, Huntsville, Texas. 89 pp. (pdf version of this thesis)

The purpose of this study was to isolate and investigate the antimony-resistant bacterial cultures which had played a role in the biological methylation of antimony compounds in the soils and cultures examined.
Related experiments were carried out in order to investigate antimony-resistant bacterial cultures and also to try to enrich and isolate the ones which were responsible from the methylation of inorganic antimony compounds. After mixed bacteria cultures were amended with different antimony compounds (potassium antimonyl tartrate and potassium hexahydroxy antimonate) in various growth media (DMN, trypticase soy broth with nitrate), the ones which were resistant to antimony grew in anaerobic environment and produced trimethylstibine. Individual colonies were separated from the rest of the mixed colony by striking the solution that contained the bacterial colonies on agar plates that amended with potassium hexahydroxy antimonate; and then after they grew, they were picked off the plates and re-inoculated in sterile bacterial medium.

After the amendment of these monocultures with the specified antimony compounds, the headspace of the bacterial solutions were analyzed by gas chromatography coupled with fluorine-induced chemiluminescence detection. Bacterial growth was monitored by optical density.

In addition, theoretical treatment of retention time correlation experiments were designed and utilized for the determination of retention times of different organo-sulfur, and -selenium compounds of whose standard retention times could not be determined because of their commercial unavailability. Also this correlation method was employed to evaluate the standard retention times of organo-sulfur, and -antimony compounds when a different chromatographic temperature program was required in our analyses.

Trimethylstibine production (via biological methylation) from mixed bacterial cultures were identified for different soil samples (Swiss soil and Body Shop soil). Re-enrichment of these cultures was achieved; moreover, the separated colonies (from soil) also produced trimethylstibine when they were amended with antimony compounds. The lack of trimethylstibine production in the control groups (ones which were not amended with antimony) supported the conclusion that organo-antimony production by these organisms was a result of biological activity (biomethylation).

Mixed bacterial polycultures were isolated successfully; the individual isolated colonies were also found to be antimony-resistant. However, a solid conclusion could not be made on the antimony reducing capabilities of these isolated monocultures even though they were found to produce trimethylstibine since organo-antimony concentration which was produced by these isolated colonies was relatively low, and the trimethylstibine peak in the headspace analyses of these isolated cultures hardly rose above the level of 3 times signal to noise ration which is a benchmark in our laboratory for detection. Retention time correlation technique was successfully employed to two major projects; in the Garlic experiment, 5 mixed organo-sulfur and -selenium compounds were identified. The practical use of this method allowed us to determine the retention times of volatile and relatively reactive organo-sulfur and -selenium species whose standard were not available commercially.

Determination of retention times of organo-sulfur and -antimony compounds were also calculated with the same method (retention time correlation technique) when a different chromatographic temperature ramp was used in our analyses.

Thomas G. Chasteen
Thesis Director

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