The aim of this study was to determine the effects of the heavy metal pollution on microbial diversity along the gradient from a platinum mine tailings dam using culture-dependent (plating methods) and molecular methods. Tailings and soil samples were collected from seven sites (6 samples per site) at increasing distances from the tailings dam. Samples were collected over a two year period and included two rainy and two dry periods. Concentrations of various heavy metals were determined using an inductively coupled plasma mass spectrometer (ICP-MS). The results demonstrated that seasonal variations in metal concentrations occurred and also that concentrations were significantly different'(P < 0.05) between the experimental sites for each metal. The relative relationship between metals was in the following order: Al > Ni > Cu > Cr. Since soil metal concentration benchmarks for South Africa are lacking, the concentrations were compared to the Canadian microbial benchmarks (MB) and Netherlands maximum permissible concentrations (MPC). Concentrations of most of the heavy metals exceeded the MB and MPC. Levels and diversity of culturable fungi and bacteria at each site were determined using plate count methods. Results indicated that levels of bacteria and fungi were not suppressed by high concentrations of heavy metals. Significantly higher levels (P < 0.05) of fungi were found at the sites on the tailings dam (higher concentrations of heavy metals), compared to sites more than 300 m away. A commonly used soil health index (Shannon-Weaver diversity index) was used to compare microbial community diversity at each site and to evaluate whether or not the heavy metal contamination impacted negatively on these soil bacterial and fungal communities. Shannon-Weaver diversity indices were higher at sites on and close to the tailings dam than sites more than 300 m away. However, ratio of fungal to bacterial levels as determined by plate counts was inconsistent. Representatives of bacterial species that were grouped using colony morphology and whole cell protein profiles were identified by 16S rDNA sequences as Bacillus barbaricus (B. barbaricus) and -Paenibacillus lautus {P. Lautus). Restriction enzyme digest, SDS-PAGE and random amplified polymorphic DNA (RAPD) analyses provided supporting evidence that representatives were correctly grouped. Cluster analysis results demonstrated that the RAPD profiles of the metal tolerant P. lautus representatives were sufficiently dissimilar to discriminate between individuals from the spatially separated sites. The spatially separated sites also represented sites with high and low heavy metal concentrations. Observed genetic variability was thus also associated with varying levels of heavy metals. In conclusion, this study demonstrated the potential of using RAPD analysis as biomarkers for genotoxic effects of heavy metals on bacterial genomes. / Masters / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2009.

The aim of this study was to determine the effects of the heavy metal pollution on microbial diversity along the gradient from a platinum mine tailings dam using culture-dependent (plating methods) and molecular methods. Tailings and soil samples were collected from seven sites (6 samples per site) at increasing distances from the tailings dam. Samples were collected over a two year period and included two rainy and two dry periods. Concentrations of various heavy metals were determined using an inductively coupled plasma mass spectrometer (ICP-MS). The results demonstrated that seasonal variations in metal concentrations occurred and also that concentrations were significantly different'(P < 0.05) between the experimental sites for each metal. The relative relationship between metals was in the following order: Al > Ni > Cu > Cr. Since soil metal concentration benchmarks for South Africa are lacking, the concentrations were compared to the Canadian microbial benchmarks (MB) and Netherlands maximum permissible concentrations (MPC). Concentrations of most of the heavy metals exceeded the MB and MPC. Levels and diversity of culturable fungi and bacteria at each site were determined using plate count methods. Results indicated that levels of bacteria and fungi were not suppressed by high concentrations of heavy metals. Significantly higher levels (P < 0.05) of fungi were found at the sites on the tailings dam (higher concentrations of heavy metals), compared to sites more than 300 m away. A commonly used soil health index (Shannon-Weaver diversity index) was used to compare microbial community diversity at each site and to evaluate whether or not the heavy metal contamination impacted negatively on these soil bacterial and fungal communities. Shannon-Weaver diversity indices were higher at sites on and close to the tailings dam than sites more than 300 m away. However, ratio of fungal to bacterial levels as determined by plate counts was inconsistent. Representatives of bacterial species that were grouped using colony morphology and whole cell protein profiles were identified by 16S rDNA sequences as Bacillus barbaricus (B. barbaricus) and -Paenibacillus lautus {P. Lautus). Restriction enzyme digest, SDS-PAGE and random amplified polymorphic DNA (RAPD) analyses provided supporting evidence that representatives were correctly grouped. Cluster analysis results demonstrated that the RAPD profiles of the metal tolerant P. lautus representatives were sufficiently dissimilar to discriminate between individuals from the spatially separated sites. The spatially separated sites also represented sites with high and low heavy metal concentrations. Observed genetic variability was thus also associated with varying levels of heavy metals. In conclusion, this study demonstrated the potential of using RAPD analysis as biomarkers for genotoxic effects of heavy metals on bacterial genomes. / Masters / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2009.

Multicellular organisms are not single individuals but carry a complex natural microflora with them. This complex's diversity and function can be considered a distinct ecosystem. Traditional methods of isolation and identification miss >90% of the actual diversity. This study uses the gut microflora of the Madagascar hissing roach, Gromphadorhina portentosa, as a model to examine this ecosystem. Isolated cultured bacteria were used to establish methods for identifying members of the microflora based on ribosomal RNA sequences. Universal primers for Eubacterial, Archaeal, and Eukaryotic 16s/18s rRNA were then used for PCR amplification of total DNA isolated from gut contents. Sequences from isolates were compared using BLAST, ClustalW, and other programs to recognize the isolates' identities and place them using a phylogenetic tree analysis. Eubacterial, Archaeal, and Eukaryotic organisms were found present in the hissing roach gut which can serve as a model ecosystem since it houses Eubacterial, Archaeal, and Eukaryotic organisms.