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The Mechanism of Thiosulfate Oxidation by Thiobacill Us Thioox7dms 8085

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The Mechanism of Thiosulfate Oxidation by Thiobacill Us Thioox7dms 8085 THE MECHANISM OF THIOSULFATE OXIDATION BY THIOBACILL US THIOOX7DMS 8085 BY ROSEMARIE JEFFERY Y. MASAU A Thesis submitted to the Faculty of Graduate Studies in Partial FulFient of the Requirements for the degree of MASTER OF SCIENCE Department of Microbiology University of Manitoba Winnipeg, Manitoba National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington OîtawaON K1AON4 OttawaON K1A ON4 Canada Canada Your file Votre réUrence Our file Noire refdmce The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of ths thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author7s ou autrement reproduits sans son permission. autorisation. Canada TEE mITYOF MANITOBA FACULTY OF GRADUATE STUDIES ***** COPYRIGHT PERMISSION PAGE The Mechanism of Thiosulfate Ondation by ThiobaciIIzs thwoxiihns 8085 BY Rosemarie Jeffery Y. Masau A Thesislfracticum snbmitted to the Faculty of Graduate Studies of The University of Manitoba in partial fuifiüment of the requirements of the degree of Master of Science Rosemarie Jeffery Y. MasalaOl999 Permission bas been granted to the Library of The University of Manitoba to tend or seii copies of this thesidpracticum, to the National Library of Canada to microfilm thh thesis and to lend or seU copies of the film, and to Dissertations Abstracts International to publish an abstract of this thesidpracticum. The author reserves other publication rights, and neither this thesis/practicum nor extensive extracts from it may be printcd or otherwise reproduced without the anthor's -en permission. Abstract Growth of Thiobacillus thiooxidans 8085 on 9K medium containing thiosulfate instead of sulfur, as the sole energy source, was successfully attained The optimum growth pH was maintained at 5.0 during cultivation. The cultivation period for T. thiooxidans on thiosulfate was 4 - 5 days at 28OC. The final ce11 yield with 1 % thiosulfate in a 4 Mer reactor was 425 mg wet celWL. The culture was continuously maintained with fiesh medium. The harvested cells were washed and stored in 0.1M sodium citrate pH 6.0 buffer at 4OC where full activiîy of the cells remained afier 3 weeks. The mechanism of thiosulfate oxidation in T, thiooxidans 8085 was studied at pH 5.0 as well as pH 2.3 and pH 7.0.The oxidation of other reduced inorganic sulfur substrates tetrathionate, sdulfur dissolved in dimethyl sulfoxide (DMSO), sulfur suspended in Tween 80 and sulfite were also studied. The oxygen consumption rate and the total oxygen consumed were determined for al1 substrates in the presence of various metal chelators, inhibitors and uncouplers. Results showed that metal chelations had an effect on the substrate oxidation depending on specific substrate. Most substrate oxidation showed stimulation with the addition of metal chelators except sulfite oxidation which was inhibited Stoichiometnc studies in the presence of inhibitors 2-n-heptyl-4- hydroqquinoline N-oxide (HQNO) and N-ethylmaleimide (NEM) in the oxidation of thiosulfate showed that in 0.1M sodium citrate at pH 5.0, thiosulfate was initially cleaved to sulfur and sulfite by rhodanese before Meroxidation. In 9K medium and 0.1M sodium sulfate at pH 2.3, thiosulfate was oxidized to tetrathionate by thiosulfate- oxidizing e-e. Sulfite was established as an interinediate in sulfiu oxidation. The cytochrome system of T. ihiooxidans 8085 in both whole cells and cell-free crude extracts was also studied with various substrates, inhibitors and uncouplers. Full reduction of the whole-ce11 cytochromes with thiosulfate, tetrathionate, DMSO sulfiu and sulfite was achieved in 0.1M sodium citrate at pH 5.0. However, full cytochrome reduction of cell-fiee cmde extracts codd only be achieved with sulfite in 0.1M Tris-HC1 pH 7.5, while thiosulfate, tetrathionate and DMSO sulfbr could reduce only cytochrome c and only in 0.1M sodium citrate at pH 5.0. The cytochrome system of T. thiooxzdam 8085 was tentatively identified by respective absorption peaks : cy-420,6,433, aXd,)-442, cg-525, 6,435, c,-555. 6,- 566, a,-602 and da-635. In the presence of inhibitors, HQNO, cyanide and azide, results suggested that a branched chain electron transport system similar to that of Azotobacter vineIandii existed. The cytochrome d is the main terminal oxidase of the branch chain system. The second branch of the system iç believed to cary a much lower flux of electrons to cytochrome a terminal oxidase. Acknowledgment 1wouid like to extend my deepest gratitude and sincerest thanks to my advisor, Dr. Isamu Suniki, for his guidance, teaching and support in both inspirational and hancial throughout the research and the preparation of this thesis. Also many thanks to the members of the department of Microbiology and in particular Dr. P. Y. Maeba, Dr. R M. Lyric and Dr. L. Van Caeseele for serving as my committee members. For the utmost, 1 would like to thank Goci, my mother, my father and my two brothers for their never ending love and support for the past 7 years of my life here in Canada. 1 would like to dedicate my thesis to rny mother and father, Miand Mrs. Dora and Jeffery Masau, to whom I am forever grateful. Tabie on contents Page Abstract 1 Acknowiedgment Table of contents List of figures List of tables List of abbreviation Introduction History Microbial leaching Oxidation of inorganic sulfùr compounds pathway 8 General chemistry of thiosulfate IO Theory - Thiosulfate oxidation 13 Theory - Tetrathionate oxidation 16 Materials and methods Materials Chernical Culture medium Met hods Adaptation of sulfûr grown T. thiooxzdum 8085 to thiosulfate medium 20 Transfer of flask culture to a reactor 21 Cultivation Ce11 harvesting Preparation of cell-fkee extract Oxidation of thiosuifate and other substrates by whole cells and cell-fkee cmde extract Meta1 chelation effects Inhibition studies Determination of thiosulfate Deterrnination of tetrathionate Modified tetrathionate determination Cold cyanolysis Determination of sulfite Modified sulfite determination Determination of elernental sulfur Cytochrome studies using Shimadni Multipurpose recording spectophotometer Results Cultivation of T. thiooxidans 8085 on thiosulfate Oxidation studies Effect of meta1 chelators on substrate oxidation Effect of azide and cyanide on substrate oxidation Effect of inhibitors and uncouplers on thiosulfate and tetrathionate oxidation Stoichiometric study : Thiosulfate oxidation - HQNO Tetrathionate oxidation - HQNO Sulfur oxidation - HQNO Thiosulfate oxidation - NEM Tetrathionate oxidation - NEM Thiosulfate oxidation - HQNO + NEM Cytochrome study : Reduction of cytochromes with dithionite Reduction of cytochromes with substrates Effect of EDTA on the reduction/oxidation of cytochromes Effect of inhibitor HQNO on the reduction of cytochromes Effect of inhibitor NEM on the reduction of cytochromes Effect of inhibiton azide and cyanide on the reduction of cytochromes Effect of CCCP and 2,4DNP on the reduction of cytochromes Reduction of cell-fiee crude extract cytochromes with substrates Effect of various inhibitors on the reduction of cell-free cmde extract with sulfite Discussion Cultivation Substrate oxidation Metal chelation effect Stoichiornetric studies Cytochrome studies Reduction of cytochromes by substrates Effect of EDTA on the reduction of cytochrome Effect of HQNO on the reduction of cytochrome Effect of azide and cyanide on the reduction of cytochrome CelCfiee cmde extract cytochrome reduction by substrates Effect of inhibitors and uncouplers on the reduction of cytochrome of cell-free crude exctract References List of figures Page History Figure I Pathway of oxidation of inorganic sulfîir compounds Materials and methods Figure 2 Culture fermentor (reactor) Olridation st udies Figure 3 Growth medium expeximent Figure 4 Optimum pH for growth and oxidation of thiosulfate Figure 5 0.1 pmole of thiosulfate oxdation Figure 6 1.O prnole of thiosulfate oxidation Figure 7 10.0 pmole of thiosulfate oxidation Figure 8 Tetrathionate oxidation Figure 9 DMSO sdfiir oxidation Figure 10 Tween 80 sulfur oxidation Figure 1 1 Sulfite oxidation Figure 12 The effect of EDTA on thiosulfate oxidation Figure 13 The effect of metal chelators on thiosulfate oxidation in citrate buf5er Figure 14 The effect of metal chelators on thiosulfate oxidation in 9K medium Figure 15 The effect of metal chelators on tetrathionate oxidation in citrate buffer Figure 16 The effect of metal chelators on tetrathionate oxidation in 9K medium Figure 17 The eKect of metal chelators on DMSO sulfur oxidation in citrate b&er Figure 18 The effect of metal chelaton on DMSO sulfur oxidation in 9K medium Figure 19 The effect of metai chelators on sulfite oxidation in citrate buffer Figure 20 The effect of metal chelators on sulfite oxidation in 9K medium Figure 2 1 The effect of azide and cyanide on thiosulfate oxidation
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