M.Sc. / The main aim of this study is to determine the water quality of water sampling points situated in Region Five of The City of Johannesburg. The water quality is studied over a three-year period from July 2000 until June 2003; this includes a dry, normal and rainy year. Region Five falls within the Upper Klipriver sub-catchment, which forms part of the Klip river catchment. The physical, chemical and microbiological sampling results are obtained from Rand Water. These results are compared with the water quality guidelines as set by the Department of Water Affairs and Forestry namely for domestic use, recreational use and aquatic ecosystems, as well as the guidelines set by Rand Water. These results of the variables that were selected are depicted visually in the form of graphs. A scientific approach is followed with respect to the water quality results. The significance of the data is statistically evaluated by using the Student’s t-test. The seasons are divided into two groups namely the more dry and cool season (autumn/winter) and the more rainy and hot season (spring/summer). This is done to determine if the seasons have a significant effect on the water quality results in comparison to each other. The water quality results are then discussed with respect to the different sampling sites. Sampling point K9, the stream at Durban Roodepoort Deep mine delivered the most problematic results of the different sampling points studied, and indicated that acid mine drainage was taking place during the sampling period. The other sampling points are less problematic. Point and non-point source pollution are elaborated on, and recommendations are made to improve the water quality at the sampling points selected.

Ph.D. / The primary objective of the study was to determine the ecological integrity of the Klip River. The protocols applied during the study gave a reliable and good reflection of the overall ecological integrity, as well as the state of different components determining the overall integrity. The ecological integrity of the most recent assessment (February 1999) is summarized in Figure 8.2. It was decided to keep the different components determining ecological integrity (physico-chemical, physical, biological) separate and not to combine everything into one score. When expressed separate, such as in the case of Figure 8.2, it is possible to observed deterioration in overall ecological integrity at a site, and it is also evident which of the components are responsible for the degradation. As mentioned previously, biological communities, and thus biotic integrity, are the best indicators of overall ecological integrity, due to the fact that they integrate both water and habitat related stresses over time. Habitat and water quality assessments are indications of the conditions prevailing at the time of sampling, while biota give an indication of the conditions prevailing over the long term. Invertebrates have shorter life cycles, and in many cases have a terrestrial phase, therefore they recolonise quicker than fish may be able to do after a pollution incident. Invertebrates can therefore be seen as indicators of short-term biological integrity, while fish indicate long-term biological integrity of a river. / Prof. G.J. Steyn

M. Tech. (Department of Biotechnology, Faculty of Engineering and Technology) Vaal University of Technology / The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.