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A research report submitted to the Faculty of Science, in partial fulfilment of the requirements for the degree of Master of Science, University of the Witwatersrand, Johannesburg, March 2016. / The study aims to assess the impacts of Acid Mine Drainage (AMD) polluted soils on Acacia karroo seed germination and viability, seed dry mass and predation, in comparison with trees from the same provenance growing on non-polluted soils. The study was undertaken within the Vaal River Operations mining rights area. This area is bisected by the Vaal River which separates the polluted area from the non-polluted area. Contamination of soils on the northern section of the Vaal River is a result of mining operations, historical tailings spillage as well as an existing pollution plume which has resulted in AMD polluted soils. The rehabilitation of disturbed land is often hindered due to low seedling establishment. The success of germination is one of the most important first steps for seedling establishment and growth and hence towards establishing a self-sustaining vegetation cover over disturbed areas. Dry seed mass was slightly higher from trees in non-polluted (0.051±0.009g) compared to the polluted areas (0.046±0.009g), however no significant difference was found. Seeds collected from the non-polluted area had highest proportion of seeds in the seed mass class 0.0455-0.0904g, compared to the seeds from the polluted areas which were highest in the smaller seed mass class 0.0155-0.454g. At the tree level, the Coefficient of Variation (CV) for dry seed mass was higher for seeds collected from the polluted area (20.5%) compared to the non-polluted area (17.9%), however, no significant difference was found. However, percentage seed predation was significantly lower in the polluted (35±15.76%) relative to the non-polluted areas (48±14.69%). Percentage seed germination was significantly higher in the non-polluted (92±9.35%) compared to the polluted areas (81±20.42%), with a significantly more rapid germination rate of 4.2±0.19 days compared to 4.7±0.45 days, respectively. In conclusion, despite their lower dry seed mass, seeds collected from AMD polluted soils still had high percentage germination, while exhibiting a lower percentage of seed predation compared to those growing on unpolluted soils. Due to A. karroo’s apparent tolerance to the poor conditions on the AMD polluted soils and its regeneration capabilities, it is likely to be a good species for rehabilitation of AMD polluted sites. Further studies should aim to determine seedling performance from those seeds collected from polluted areas in terms of seedling establishment, rates of growth and survival over time when established in AMD polluted soils as well as non-polluted soils, to determine their likely success.

The efficacies ofsoil extraction methods, namely, Soxhlet, sonication, agitation, alkaline digestion and the ethyl acetate micro-method, for monitoring soil bioremediation were evaluated using three soil types, Swartland, Rensburg and Hutton, encompassing the mineralogical range prevalent in Kwa Zulu Natal. Phenol, atrazine and the BTEX component of petrol were the molecules used in this study and were extracted under different spiking concentrations, after prolonged ageing times up to 21 days and after changing the composition of the spiking solution. It was concluded that extraction methods must be validated for the specific conditions under which they would be used, taking into consideration, soil type, spiking solutions, moisture content, weathering times and the analyte(s) in question. A preliminary appraisal of atrazine degradation in a Hutton soil was then made under the conditions of sterilized, fertilized/non-fertilized and non-sterilized, fertilized/nonfertilized soils. The predominant pathway of atrazine degradation was deemed to be chemically/abiotically mediated due to the soil pH and the presence of iron and aluminium oxides as well as the high levels of manganese in the soil. The results obtained prompted further study into atrazinecatabolism using soil-slurry reactors, under the conditions of carbon-limitation, nitrogen limitation, carbon/nitrogen non-limitation and carbon/nitrogen limitation. A comparison was made between inoculated and non-inoculated bioreactors. The ability of the indigenous microbial population to return the Hutton soil to its original pristine state was confirmed. The expense of inoculation and culture maintenance could be avoided since carbon and nitrogen supplementation would be as equally effective as inoculation. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Most natural products and xenobiotic molecules, irrespective of their molecular or structural complexity, are degradable by some microbial species/associations within particular environments. Atrazine- and selected petroleum hydrocarbon (benzene, toluen~ and 0-, m- and p-xylene (BTX))-degrading associations were enriched and isolated"trom atrazine- and petroleum hydrocarbon (PHC)-contaminated KwaZuluNatal loamy and sandy soils, respectively. In total, eight pesticide- and forty BTXcatabolizing associations were isolated. Electron microscopy revealed that, numerically, rods constituted the majority of the populations responsible for both atrazine and PHC catabolism. Cocci and, possibly, spores or fungal reproductive bodies were observed also. For the BTX-catabolizing associations, the population profiles appeared to be dependent on the enrichment pH and the molecule concentration. After combining selected associations, to ensure that all the isolated species were present, batch cultures were made to determine the optimum pH and temperature for growth; With an atrazine concentration of 30 mgr1, the highest specific growth rates, as determined by biomass (OD) changes, were recorded at 30DC and pH 4 although the rate§ at 25DC and pH 5 were comparable. For the BTX (50 mgr1)-catabolizing associations, the highest growth rates were recorded at pH 4 for the four temperatures (15, 20, 25 and 30DC) examined. The sole exception was p-xylene with the highest specific growth rate recorded at pH 5 and 30De. Batch and continuous (retentostat) cultivations in the presence/absence of methanol and under C- and N-limited conditions were used to investigate the impacts of the solvent and the catabolic potentials of a combined atrazine-catabolizing culture (KRA30). In general, different degradation rates were recorded for the culture in response to element limitation. Addition of citrate as the primary carbon source / effected atrazine (100 mg!"l) degradation rates comparable to that of Pseudomonas sp. strain ADP while succinate addition effected herbicide co-metabolism. Carbon supplementation may, therefore, be considered for site amelioration practices. To complement conventional culture-based microbiological procedures, molecular techniques were employed to explore the diversities and analyze the structures of the microbial communities. In parallel, anaerobic microbial associations which targeted atrazine were also characterized. The soil DNA isolation/characterization protocol adopted consisted of a clean-up step followed by the polymerase chain reaction (peR) and 16S rDNA fingerprinting by denaturing-gradient gel electrophoresis (DGGE). The preliminary results suggested that despite different, but chemically similar, petroleum hydrocarbon molecules, the common selection pressures of the primary enrichments effected the isolation of similar and complex aerobic microbial associations. Some similar numerically-dominant bands characterized the aerobic and anaerobic atrazine-catabolizing associations although distinct differences were also recorded on the basis of the enrichment/isolation pH value and the concentration of the herbicide. Cloning and sequencing were then used to identify some of the numerically-dominant and non-dominant association members. Community-level physiological profiling (CLPP) for physiological fingerprinting was made with Biolog EcoPlates and highlighted the differences in the isolated aerobic atrazine-catabolizing associations depending on the enrichment pH and molecule concentration. Logarithmic-phase cultures of the combined atrazine- and BTX-catabolizing associations were used to explore the association profiles following pH and temperaiure optImIzation. Although some common numerically-dominant components were maintained, differences in numerical and, possibly, activity dominance were observed in the 16S rDNA profiles in response to changes in pH and temperature. This indicated that environmental parameter optimization and characterization of catabolic association structure must precede bioaugmentation so that control of key variables will facilitate maintenance of the dominant site-specific species. Following KRA30 cultivation in the presence/absence of methanol and under carbon and nitrogen-limited conditions, the population fingerprints showed that the presence of methanol effected shifts in species numerical dominance and, possibly, changes in atrazine catabolic capacity. Also, Coulter counter results, optical density readings and 16S rDNA characterization by DGGE indicated that degradation rate changes were accompanied by shifts in species numerical/activity dominance within the association. Although N-limitation effected the highest rates of herbicide catabolism, a potential versatility of the combined association for bioaugmented and/or biosupplemented remediation with acceptable rates regardless of any elemental limitation was recorded. To determine if the contaminated and pristine source soils contained comparable catabolic populations and, thus, offered potential for intrinsic bioremediation, PCRDGGE was used to characterize the populations in comparison with the enriched/isolated associations. Some similar dominant bands characterized the contaminated soils and the enriched/isolated associations. The significance of this, in relation to a possible correlation between numerical and activity dominance in the component species, is discussed with respect to the use of PCR-DGGE to identify natural attenuation potential and monitor sustained intrinsic and enhanced (bioaugmented and biosupplemented) bioremediation. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 156-170).

The present study was carried out to determine the levels of heavy metals, Arsenic (As), Chromium (Cr), Cadmium (Cd) and Lead (Pb) in samples of water, sediments, and specimens from sheep known to graze and drink from Wonderfontein stream around the Khutsong area in the North West Province of South Africa. Determination of heavy metal levels was carried out using Atomic Absorption Spectrophotometer. Abundance of metals in water samples followed the trend As>Pb>Cd>Cr, while that in sediments followed the trend: Pb>As>Cr>Cd. Faecal levels were highest for Cr, followed by Cd, As then Pb, while serum levels were highest for As, Cd Cr and then Pb. The metal concentration in liver, kidney and muscle showed the following trends respectively: As>Cd>Cr>Pb ; As>Pb>Cd>Pb and As>Cd >Cr> Pb. The liver, kidney and muscle samples had higher concentration of As compared to other heavy metals. Generally, most samples showed a higher concentration in As. The mean concentrations of heavy metals in ppm were compared with European Commission Regulation, World Health Organisation, Korean Soil Environmental Conservation Act and the Australian New Zealand Food Standards maximum acceptable levels. The metal levels generally tended to be higher than the permissible levels and thus, public health risks. A survey conducted also revealed that the community in Khutsong does not have the knowledge on environmental contamination due to mining effluents and the effects thereof. The varying levels of water and sheep specimen contamination with As, Cd, Cr and Pb revealed in this study imply public health risks. Further biomonitoring, public and animal health studies are therefore indicated in this area. / Thesis (Msc in Agric Animal Health) North-West University, Mafikeng Campus, 2012

published_or_final_version / Real Estate and Construction / Master / Master of Science in Real Estate and Construction