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Inorganic sulfur-oxidising Bacteria are present throughout the Proteobacteria and inhabit all environments of Earth. Despite these facts they are still poorly understood in terms of taxonomy, physiology, biochemistry and genetics. Using phylogenetic and chemotaxonomic analysis two species that were erroneously classified as Thiobacillus trautweinii spp. in 1921 and 1934 are in fact novel chemolithoheterotrophic species for which the names Pseudomonas trautweiniana sp. nov. and Achromobacter starkeyanus sp. nov. are proposed, respectively. These species were found to oxidise thiosulfate in a “fortuitous” manor when grown in continuous culture and increases in maximum theoretical growth yield (YMAX) and maximum specific growth rate (μMAX) were observed. Cytochrome c linked thiosulfate-dependent ATP production was confirmed in both species, confirming “true” chemolithoheterotrophy. Evidence is presented that the ATP concentration governs the benefits of chemolithoheterotrophy. There were significant changes in enzyme activities, including enzymes of the TCA cycle that might be affecting amino acid synthesis. This is strong evidence that chemolithoheterotrophy gives a strong physiological boost and evolutionary advantage over strictly heterotrophic species. An autotrophic species that was historically placed in Thiobacillus was also shown to be a novel species for which the name Thermithiobacillus parkerianus sp. nov. is proposed. The enzyme profiles of Thermithiobacillus parkerianus differed significantly between different inorganic sulfur growth substrates and was the first time all TCA cycle enzymes were assayed in a member of the Acidithiobacillia. The properties of thiosulfate dehydrogenase varied significantly between Pseudomonas sp. Strain T, Achromobacter sp. Strain B and Thermithiobacillus sp. ParkerM both in terms of optimal parameters and the effect of inhibitors. This evidence adds to the increasing body of work indicating there to be at least two thiosulfate dehydrogenases present in the Bacteria.

Due to their superior thermal and chemical stability, polyimides are often used as adhesives in hazardous environments. This study examines the effect of thioether sulfur in the polyimide backbone on bond strength. X-ray photoelecton spectroscopy (XPS) and reflectance infrared spectroscopy indicated that certain metals catalyze the oxidation of the thioether sulfur. It was believed that this oxidation could lead to direct metal-oxygen-sulfur bonds across the polymer/metal interface which would serve to enhance interfacial strength. Bonds were made using substrates that were believed to catalyze the oxidation strongly (steel) and minimally (aluminum). In addition, non-sulfur containing polyimides with similar Tg were also studied for comparison. The polymer/metal interface was studied using both the T-peel and wedge tests. In some cases, oxidized sulfur was detected on the failed surfaces via XPS. No apparent effect was observed in the T-peel test, where the T-peel strengths of non-sulfur and sulfur containing polyimides were similar. In the wedge test, however, the sulfur containing BDSDA/ODA bonded to steel had an initial crack length of 34 mm. Even after eleven days the crack length was only 47 rnm, which was the initial crack length for the next best polyimide. Thus, metal-catalyzed oxidation of sulfur did take place, but not to an extent to have a noticeable effect on peel strength. / Ph. D.

This research study's the use of biological sulfate reduction technologies for the treatment of Sasol Secunda's coal-mine drainage (CMD) using Fischer-Tropsch Reaction Water (FTRW) as a cost-efficient carbon source. The research aims to develop a prototype dynamic model that describes this co-treatment of FTRW and CMD in both a continuously stirred tank reactor (CSTR) biological sulfate reduction (BSR) system and a BSR gas-lift (BSR-GL) integrated system. The BSR-GL system recovers elemental sulfur (S0 ) from the H2S produced and stripped from the BSR unit. Furthermore, this study aims to use the prototype model for a quantitative comparison of the CSTR-BSR and BSR-GL systems. Two bench-scale 5-litre CSTR-BSR and a 20-litre BSR-GL system were operated, under varying feed COD concentrations and hydraulic retention times (HRTs), to generate datasets for use in verification and a rudimentary validation of the prototype model. The BSR-GL integrated system includes 1) a 1-litre H2S gas reactive absorption (ABS) unit utilising an aqueous ferric solution for the recovery of elemental sulfur (S0 ) from sulfide and 2) ferrous biological oxidation reactor to regenerate ferric from the ferrous for re-supply to the ABS unit. The datasets generated in the experimental study allowed for the identification, mathematical modelling and reaction verification of 32 components that interact as reactants and products in 23 reactions observed in the two BSR systems. The prototype model is presented in a mass and charge balanced Gujer matrix that includes, i) 5 SRB mediated processes, ii) 2 liquid-gas mass transfer processes, iii) 3 processes describing the ABS and Fe2+ bio-oxidation units, iv) 4 processes describing sulfide and elemental sulfur oxidation and v) the S0 and poly-sulfide aqueous equilibrium and vi) 9 processes describing death regeneration and BPO hydrolysis. This prototype model was implemented in the DHI WEST® software for initial stage simulation trials. The experimental datasets allowed for the first-stage estimation of the best-fit reaction rate equations and the calibration of the kinetic parameters related to the 23 reactions, using MATLAB® curve fitting toolbox. A pre-processor that describe the pH and equilibrium chemistry of the components of the artificially prepared FTRW+CMD feed mixture batches under varying total concentrations have also been developed in this research. This was done to generated influent file to the DHI WEST® simulations that incorporated the dynamics related to the FTRW+CMD feed mixtures. The sulfate utilisation rate (gSO4 -2 .l-1 .d-1 ) of the GL-BSR and CSTR-BSR systems were compared to determine which system had the best sulfate removal. The results were found to be as follows; a. On comparison it was found that the sulfate substrate utilisation rate for the CSTR_BSR system is 39.28% of that of the BSR-GL_N2 system, where both systems were fed at feed mixture of COD of 2500mgCOD/l, where the COD:SO4 2- was 0.7, b. For the same systems fed a feed mixture of COD at 5000mgCOD/l (COD:SO4 2- = 0.7), the sulfate substrate utilisation rate for the CSTR_BSR system was found to be 17.86% less than that of the BSR_GLN2 system. c. Finally, it was also found that the substrate utilisation rate for the CSTR_BSR system was 30.06% less than that of the BSR_GLN2 system at Se of 4gCOD/l, for both systems fed substrate at 5000mgCOD/l. Thus, it can be concluded that the sulfate substrate utilisation rate for the BSR-GL system is higher than that of the CSTR_BSR system, for systems fed COD feed mixtures at 2.5 or 5gCOD/l where both systems have the same effluent substrate concentrations. However, the difference in the comparative substrate utilisation rate is less at higher feed substrate concentrations. This is the influence of substrate inhibition on the active SRB biomass, which increases with higher effluent substrate concentrations. Finally, this research found that the use of gas-lift reactor technologies is superior to CSTR technologies in the treatment of coal-mine drainage utilising biological sulfate reduction (BSR). The CSTR-BSR system, fed sulfate between 1.6 to 14gSO4 2- /l, produced effluent with high dissolved H2S concentrations, on average 285mgS/l and maximum at >600mgS/l. Releasing this effluent to the environment would be hazardous to aquatic and human health and corrosive to infrastructure. As such, the effluent from the CSTR-BSR system requires further treatment to stabilise the water for any use. The BSR-GL technology allows for the conversion of the H2S produced during BSR reactions to form elemental sulfur, which is a resource recovered from this process, thus complying to the circular economy aim of this study.

Many bacteria use inorganic nitrogen and sulfur compounds for energy metabolism. These compounds are often toxic and so bacteria must adapt to survive their deleterious effects. Bacteria use specific proteins in order to metabolise, sense and detoxify these compounds. In this thesis protein interactions with inorganic nitrogen and sulfur compounds are examined at the mechanistic level. Intermediates in the Sox sulfur oxidation pathway are covalently attached to a cysteine on the swinging arm of the substrate carrier protein SoxYZ. An interaction between the Sox pathway enzyme SoxB and the carrier protein SoxYZ is demonstrated. A crystal structure of a trapped SoxB-SoxYZ complex at 3.3 Å resolution identifies two sites of interaction, one between the SoxYZ carrier arm and the SoxB active site channel and the other at a patch distal to the active site. The presence of a distal interaction site suggests a mechanism for promiscuous specificity in the protein-protein interactions of the Sox pathway. Using biophysical methods it is shown that SoxB distinguishes between the substrate and product forms of the carrier protein through differences in interaction kinetics and that the carrier arm-bound substrate group is able to out-compete the adjacent C-terminal carboxylate for binding to the SoxB active site. The thiosulfate dehydrogenase TsdA has an unusual His/Cys coordinated heme. TsdA catalyses oxidative conjugation of two thiosulfate molecules to form tetrathionate. Mass spectrometry and UV/visible spectroscopy are used to identify an S-thiosulfonate reaction intermediate which is covalently attached to the cysteine heme ligand. A catalytic mechanism for TsdA is proposed using a crystal structure of TsdA at 1.3 Å resolution alongside site-directed mutagenesis of active site residues. Nitric oxide is produced by the mammalian immune response to kill bacterial pathogens. Part of the killing mechanism occurs through the reaction of nitric oxide with protein-bound iron-sulfur clusters. However, the same type of reaction is also exploited by nitric oxide-sensing bacterial proteins. An infrared spectroscopy approach is developed to detect the products of iron-sulfur protein nitrosylation. Using this methodology it is shown that the presence of trace O2 strongly impacts which products are formed in these nitrosylation reactions. These observations are of physiological relevance because bacteria are often exposed to NO under aerobic conditions during an immune response.