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This study investigated maturation and stability levels during composting of Quercus robur (QR) woodchips mixed with different nitrogen sources (horse manure, HM and lake mud, LM) for potential agronomic utilisation. The woodchips were mixed with HM and LM, respectively, at mixing ratios of 1QR:2HM or 1QR:2LM. The experiment was conducted in a greenhouse over 100 days. During composting, organic carbon and its fractioning and humification constituents were quantified. In the final compost product, pH, organic matter (OM), cation exchange capacity (CEC) and selected available nutrients were measured. Thermostability of compost, compared with that of soil and compost mixed with charcoal were also quantified. Results showed OM evolution during the composting process with total organic and extractable carbons and humification indices decreasing, while the degree of humification increased. Compost produced from the 1QR:2HM mix resulted in the highest available nutrients, CEC and OM content values in the final product. Thermogravimetric profiles indicated that compost OM thermostability was higher than that in soil and higher for the 1QR:2HM than the 1QR:2LM mix. Application of charcoal revealed no additional stabilising effect of OM in wood compost.

MSc (Microbiology), North-West University, Potchefstroom Campus Root-knot nematodes continue to be a global problem in agriculture causing major economic losses. Various Bacillus spp. have the potential to inhibit the Meloidogyne root-knot nematode populations. Although many studies conclude that the secondary metabolites of relevant Bacillus spp. are responsible for nematicidal activity, the specific metabolites are not characterised. Subsequently, the efficacy and reproducibility of biocontrol products are questionable. The aim of this study was to compare the metabolic profiles of Bacillus spp. with known nematicidal activity to bacteria without nematicidal activity. For this purpose, four Bacillus spp. known to exhibit inhibitory effects towards second-stage juveniles (J2) of Meloidogyne incognita, namely B. cereus, B. firmus, B. subtilis and B. pumilus were compared to two bacteria without known nematicidal activity – Escherichia coli and B. soli. Bacterial strains were cultivated in two types of media, namely Luria-Bertani (LB) broth as a complex medium and minimal broth (MB) as a simpler medium. This was done to evaluate the effect of the medium composition on bacterial metabolism and also on metabolomics analyses performed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). The first step was to do nematicidal bioassays to confirm the nematicidal activity of cell-free filtrates obtained from the bacteria. During the bioassays the motile and paralysed J2 nematodes were quantified after exposure to different concentrations of cell-free filtrates presumably containing secondary metabolites of the bacteria. From the results obtained it was evident that all of the selected Bacillus spp., as well as E. coli, had nematicidal effects on the M. incognita J2. When cultivated in LB broth with optimised incubation times, B. firmus and B. pumilus showed the highest nematode paralysis in the bioassays. However, when cultivated in MB, B. firmus and B. cereus showed the highest nematode paralysis. Due to the nematicidal activity observed for E. coli and B. soli, the bioassays were repeated using E. coli OP50 as control. This strain caused the lowest levels of paralysis in all assays and was therefore a more appropriate control that should be used in further studies. Untargeted metabolomics distinguished between metabolite profiles from the different Bacillus spp. Moreover, there was a clear difference between profiles when bacteria were cultivated in the different media. Bacterial cultures produce extracellular metabolites in response to their surrounding environment and it is critical that specific bacterial species are matched with the optimal cultivation media to ensure reproducible production of compounds of interest before identification of metabolites is attempted. National Research Foundation (NRF) Masters

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2017 Palabora Mining Company (PMC) in the Limpopo Province has exploited unique ultramafic rock formations in the Phalaborwa Igneous Complex. Subsequently, mining activities has created ‗mountains‘ of processed materials (tailings and dumps). Efficient reclamation approaches have stabilized the degraded landscape and improved the ecosystem functionality of these facilities. However, successful rehabilitation of copper mine tailings requires an in depth understanding of the biotic and abiotic factors most limiting to vegetation establishment and growth. Knowledge of the factors that promote effective rehabilitation is important, as it allows mine management to make informative decisions to address rehabilitation shortcomings by means of appropriate mitigation measures. Therefore, to achieve sustained rehabilitation success, knowledge of aboveground and belowground factors form a crucial link in assessing rehabilitation progress on post-mining sites. The objectives of this study were therefore to determine the effect of different post rehabilitation ages, aspects and topographic positions on; i) soil microbial biomass and community structure, ii) species composition, diversity, biomass and cover of the herbaceous layer, and iii) physical and chemical soil properties of tailings facilities. The herbaceous layer of two of the PMC tailings facilities, namely the Rock Dump (RD) and Tailings Dam (TD), were sampled by means of the fixed quadrat method. Quadrats were placed in a stratified manner to sample the different age levels, aspects and topographic positions of both the facilities. A total of 174 quadrats were sampled, and the herbaceous plant richness, abundance, cover and biomass was documented for each quadrat. Soil sampling was conducted simultaneously to vegetation sampling. A total of 91 soil, and soil microbial, samples were collected and analysed. PRIMER 6, PAST and STATISTICA 11 were used for data analyses, which included Non-metric Multidimensional Scaling (NMDS) ordinations based on the Bray-Curtis index, One-way Analysis of Similarities (ANOSIM), Similarity Percentage Analysis (SIMPER), One-way Analysis of Variance (ANOVA) and Tukey‘s post-hoc HSD for unequal N. Results indicated high levels of vegetation cover on the oldest and intermediate post rehabilitation ages on the RD and TD respectively, while the intermediate age on the RD and youngest age on the TD had the highest biomass. Regarding aspect and topography, the RD and TD reacted similarly, with cover highest on the slope positions on the eastern aspects, and biomass highest on the terrace positions on the western aspects. Terrace positions revealed the highest species richness and diversity, especially on the eastern aspect of the RD and southern aspect of the TD. Highest species richness and diversity was recorded for the oldest age on the RD and youngest age on the TD. Benchmark vegetation results indicated that all tested vegetation indices of natural Mopaneveld were significantly higher than both the RD and TD. Slope positions of eastern aspects on the oldest post rehabilitation ages maintained the highest microbial biomass; however, precarious patterns of microbial community structure were identified. Highly variable physical and chemical soil properties were noticeable on both the RD and TD. Best performing soils were found on the oldest soils on slopes with an eastern aspect on the RD and the oldest soils on terraces of the western aspect on the TD. This study revealed that different post rehabilitation ages, aspects and topographic positions do not affect floristic composition to such an extent that a significant dissimilarity could be identified across the RD and TD. Most of the tested vegetation variables, although with erratic occurrence of significant differences, were recorded between terrace and slope topographic positions, eastern and western aspects, and oldest and youngest post rehabilitation ages of the RD and TD. Overall, enhanced species richness, diversity, physical and chemical soil and soil microbial properties were revealed under combined conditions of slope positions on the RD and terrace positions on the TD on the eastern aspects of the oldest post rehabilitation ages. This study provides valuable information regarding patterns of herbaceous species diversity, microbial community structures and physical and chemical soil characteristics on copper mine tailings and serves as benchmark for long-term monitoring of biotic and abiotic environmental factors Masters

PhD (Environmental Sciences), North-West University, Potchefstroom Campus, 2016 Although the importance of phospholipid fatty acid (PLFA) analysis to characterise microbial communities has been recognised over the years, the vast number of modifications to this traditional method as well as discrepancies regarding its data interpretation have given rise to scepticism about the accuracy of PLFA analysis in environmental studies. Therefore, comprehensive literature-based studies were necessary to investigate these uncertainties. Additionally, new and modernised high-throughput approaches, such as metabolomics, holds promise for investigating microbial communities and there was a need to evaluate such an approach against traditional methods for characterisation of fatty acid methyl esters (FAMEs). The literature reviews conducted for this study evaluated i) different methods and their modifications in terms of the main steps of PLFA analysis, namely extraction, fractionation, derivatisation and quantification; and ii) recent applications of PLFA analysis in environmental studies with specific reference to data interpretation. Both reviews made significant contributions to summarise and clarify a vast body of literature on the topics and were published in the Journal of Applied Microbiology. During the experimental work, targeted analysis of selected FAMEs to characterise both homogenised soil and pure bacterial cultures were evaluated by comparing three methods, namely fractionated PLFA, total lipid extract (TLE) and metabolomics analysis. As an additional outcome, untargeted analysis was also investigated. The results showed that the extraction method and different derivatisation techniques had an effect on FAME concentrations and on repeatability between sample replicates. Furthermore, extraction method had a greater influence when analysing pure culture samples, while derivatisation technique was more important when analysing soil samples. The most appropriate derivatisation technique for fractionated PLFA analysis proved to be mild alkaline methanolysis, while methanolysis with oximation and silylation provided the best results for TLE analysis. The metabolomics-based approach benefited from the inclusion of an oximation step for derivatisation of a complete metabolite profile. An assessment of the applicability of the different methods to distinguish between soil microbial communities exposed to various soil fumigant treatments in a greenhouse study, showed that even though TLE analysis gave higher FAME yields than PLFA analysis, its discrimination potential between treatments were much lower. Therefore, PLFA analysis was recommended for FAME characterisation in microbial communities. The untargeted metabolomics analysis has potential in differentiating between different treatments, despite representing the larger soil community and not microbial communities per se. Based on all the obtained results, a standard operating procedure for the targeted analysis of FAMEs and the untargeted analysis of all metabolites from soil is proposed Doctoral

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015 The evolution of pest resistance to Cry proteins threatens the long-term use of Bt crops. Busseola fusca developed resistance to Bt maize in South Africa but the mechanism of resistance is not well understood. According to the gut microbiota theory, extensive cell lysis caused by Cry proteins provide gut microbes access to the more favourable environment of the hemocoel where they germinate and reproduce, causing septicemia and death of the host. This theory brought on questions about the role of gut microbes in the efficacy of Bt maize against target lepidopteran pests. The aim of this study was to determine whether microbes present in the mid-gut of B. fusca influence the efficacy of Cry 1Ab proteins. Larvae were collected from 30 different geographical locations, dissected to excise the mid-gut and mid-gut content which was separated according to morphological types. The morphological types were used to test the antibiotic susceptibility of the bacteria and proved that ciprofloxacin, ampicillin and doxycycline were the most effective bacteriostatic and bactericidal antibiotics. These three antibiotics were exposed to the morphological types at different concentrations to visualise the possible deleterious effects of the antibiotics on the bacteria. This visualisation was performed by observing the growth curve of the bacteria in the presence of the combination of antibiotics. The antibiotics concentration of 500 μg/ml showed the highest efficacy compared to the other concentrations tested. An antibiotic concentration of 500 μg/ml of ciprofloxacin, ampicillin and doxycycline was incorporated into an artificial diet for the larvae to feed on for 7 days. This method was used to rid the larvae of gut microbes before allowing them to feed on Bt maize (MON810) plant material expressing Cry proteins. The results suggests that by placing antibiotic reared larvae on a Bt plant, the absence of the mid-gut microbes contributed to larvae survival on Bt maize. This observation will contribute to understanding the role of gut microbes on the efficacy of Cry proteins. Masters

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015 Biofumigation is an important crop protection practice that uses a plant’s natural defence mechanisms to control agricultural crop pathogens and diseases. Glucosinolates are volatile compounds found in most Brassica species and when hydrolysed, it forms a range of natural toxins including isothiocyanates that act as biofumigants. Research suggests that biofumigation is a good alternative to chemical fumigants as it is effective in controlling plant pests but with lower health and environmental risks. Several studies have confirmed the effectiveness of the breakdown products, especially isothiocyanates, as fungicidal, bactericidal and nematicidal products against a series of plant pests. However, very little information is available on the effects of glucosinolates and its breakdown products on non-target and beneficial soil organisms. Negative effects on beneficial soil organisms can have serious negative impacts on soil quality especially when essential ecosystem functions such as nutrient cycling and soil bioturbation are affected. Three biofumigants, broccoli, mustard and oilseed radish, and two chemical fumigants, metham sodium and cadusafos, were investigated for possible effects on non-target and essential soil organisms such as earthworms and the soil microbial community. Sublethal endpoints, including growth and reproductive success of the earthworms, were monitored. The genotoxicity of the biologically active compounds found in the fumigants, towards earthworms, was evaluated by means of the comet assay. The DNA damage was quantified by tail intensity parameters. Furthermore, the changes in the soil microbial community function and structure were evaluated by means of community level physiological profiling (CLPP) and phospholipid fatty acid (PLFA) analyses respectively. All exposures were done in artificial soil prepared according to the OECD standard guidelines. In the biofumigant treated soils, results varied and different effects were observed on the non-target soil organisms. Broccoli reduced cocoon production and the number of hatchlings while mustard induced more DNA strand breaks in earthworm cells compared to the control. All the biofumigants stimulated microbial growth but broccoli and oilseed radish changed the microbial functional diversity. Mustard had no lasting effect on the functional diversity but altered the microbial community structure. The chemical fumigants had a marked negative impact on the survival, growth, reproduction and the genotoxicity of the earthworms with metham sodium causing greater harm than cadusafos. The effects on the microbial community varied. Both chemicals had an inhibitory effect on the microbial growth in terms of the viable biomass determined by PLFA and the average well colour development in the Biolog™ Ecoplates. No lasting effects were observed in the community structure. Overall, cadusafos had a more pronounced effect on the microbial community functional diversity than metham sodium. Results indicated that each bioindicator species illustrates effects at their own level of organisation. Masters

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015 Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca.However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect's adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. Masters

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014 Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. Masters

Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2012. One of the major constrains in beekeeping is damage caused by the greater wax moth (Galleria mellonella). Due to problems and concerns regarding the use of chemical insecticides, biopesticides have been introduced to manage problematic insects. For the biological control of greater wax moth, Bacillus thuringiensis subsp. aizawai (B.t.a) has been widely researched and used. Large scale production of B.t.a as a biopesticide is expensive because of the high cost of commercial media formulations. In this study, three raw materials, horse manure, soy meal and maize chops, were selected for evaluation as raw materials for media production based on their economical feasibility and availability and compared to two commercial media (Tryptone soy broth and bactopeptone). Sixty three raw material formulations and two commercial media were evaluated in terms of spore yield and toxicity towards G. mellonella. For each formulation the spore and vegetative cell yield was determined using direct microscopy. From the results obtained from the 63 formulations, the 12 highest spore yielding formulations were selected and re–evaluated for confirmation of the results. Spores obtained from the 12 highest spore yielding formulations were used in a bioassay to determine their toxicity against G. mellonella larvae and the protein concentration were also determined. Spore morphology was investigated for the 12 highest spore yielding formulations. Formulations containing 3.75 g horse manure, 5 g maize chops and with soy meal ranging between 1.25 g and 3.75 g yielded the highest spore yields. In particular the combination of 3.75 g horse manure, 5 g maize chops and 1.25 g soy meal had the highest spore yields but also resulted in 100 % mortality against G. mellonella larvae within 3 days. The yields obtained were also significantly higher than those for the commercial media against which they were evaluated. As such it was concluded that the selected formulation represents a feasible alternative to commercial media for the production of B.t.a. Masters

Thesis (M.Sc. (Environmental Science))--North-West University, Potchefstroom Campus, 2010. The asbestos mining industry has left a legacy of pollution that continues to poison former mining areas and surrounding land – posing a significant health risk to local communities. The rehabilitation of sites disturbed by mining activities, aims to negate the adverse effects associated with these post-mining landscapes and to achieve the return of a disturbed site to a degree of its former state or to a sustainable usable condition. In order to assist the effective rehabilitation of derelict and ownerless asbestos mines it was critical to develop a scientific database to indicate the status of rehabilitation at specific sites. The Rehabilitation Prioritisation Index (RPI) was developed in 2007 to indicate the sequence for rehabilitation of asbestos pollution by quantifying the risk associated with a specific pollution site. The use of the RPI has been implemented by the South African Department of Minerals and Energy as part of an integrated approach towards the rehabilitation of the asbestos legacies of the past. In this study, a multidisciplinary approach was applied to sites in three provinces as identified in the RPI, to facilitate the development of the Rehabilitation Monitoring Index (RMI). It is envisioned that this index, as part of a larger monitoring database, would assist in the successful monitoring and long-term rehabilitation of asbestos mines. During the monitoring process, the most prominent aspects governing the rehabilitation process were identified from comprehensive assessments of quantitative and qualitative data. Quantitative parameters included cover depth, physical and chemical soil properties, soil microbial activity, vegetation properties and small mammal surveys. Qualitative data included the footprint area, land use, erosion or flood damage, secondary pollution and water control structure damage. From the quantitative data, those parameters which had the greatest influence on the rehabilitation process were identified. In order of most to least important these groups were analysed by multivariate statistical ordination and classified into four groups: success parameters > essentials to be addressed > reasons for failure > non-distinguishable entities. The qualitative data indicated that the Limpopo Province was in the highest state of degradation after rehabilitation and that site history plays an important role in rehabilitation planning. Quantitative and qualitative parameters were assessed for all sites and applied in the RMI as weighted factors from which the rehabilitation status of a specific site can be calculated. Qualitative data was given a weight of 25% and quantitative data a weight of 75%. RMI values were calculated for each parameter and sites were distributed across a range which classifies the sites according to their rehabilitation status. Once again the Limpopo Province was identified as the province with the least successful rehabilitation. The results from this investigation show that a multidisciplinary approach is a step in the right direction for the successful monitoring of rehabilitated post-mining sites such as asbestos mines. It is however necessary that the RMI must be validated and the weights allocated to qualitative parameters must be reconsidered for the future development of this tool. While the RPI and RMI cannot be compared directly, it might be of great revelation to reassess the RPI values of all the sites after rehabilitation and compare this data to the RMI values. Masters

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2011 The diversity of microorganisms and the influence of their enzymatic activities in soil are critical to the maintenance of good soil health. Changes in these parameters may be the earliest predictors of soil quality changes, potentially indicating anthropogenic influences. The goal of this study was to investigate the soil microbial diversity and function of grasslands along an urbanization gradient. Soil samples were collected in the Potchefstroom municipal area, South Africa, at specific sites. Sampling sites were described as urban, suburban and rural - according to the V-I-S (Vegetation-Impervious surface-Soil) model of Ridd (1995). Soil samples were collected over a warmer, wet season (May) and a colder, dry season (August) over two years (2007 and 2008). Collected soil samples were characterised using certain physical and chemical parameters. Plant species composition and abundance were determined at each site, along with basic site data (soil compaction, percentage ground cover, percentage bare ground, percentage organic material present). The Shannon-Weaver diversity index was used to calculate biodiversity values for all the investigated sites regarding collected plant species composition. The microbial component of the soil was quantified and characterized using culture-dependent and culture-independent techniques. Culture-dependent techniques included the investigation of the aerobic heterotrophic bacteria and fungi. Organisms were plated out on different media, and the bacterial component was broadly grouped using morphology. Dominant organisms were identified by sequencing of PCR amplified 16S ribosomal DNA fragments. Shannon-Weaver index for bacterial diversity was determined for each of the sites. Denaturing gradient gel electrophoresis (DGGE) profiling of selected bacterial communities were also conducted. Microbial community function was determined using enzyme assays of five major groups of enzymes, namely (i) dehydrogenase; (ii) β-glucosidase; (iii) acid phosphatase, (iv) alkaline phosphatase and (v) urease. Plant species results were then brought into context with microbiological diversity and functionality results using multivariate statistics. Physical and chemical parameters of the collected soil samples revealed patterns present along the urbanization gradient. The pH values were mostly higher in the sub-urban and urban sites than in the rural sites. Electrical conductivity values were generally highest in the sub-urban sites. Plant species composition revealed trends along the urbanization gradient. Ordinations clearly grouped the plant species into rural, sub-urban and urban groups regarding plant species composition. Rural sites had the highest number of plant species. Shannon-Weaver values regarding the plant diversity supported the plant species composition data indicating higher plant diversity in the rural areas, followed by the sub-urban and the urban areas. Plant structural data indicated that forbs were most numerous in the rural sites, and less so in the urban sites. Higher average aerobic heterotrophic bacterial levels were present in the urban soil samples. The bacterial levels were lower in the sub-urban and rural soil samples. Subsequent identification of the dominant bacteria in the soil samples revealed organisms of the genus Bacillus dominated the aerobic heterotrophic bacterial communities in the soil samples. Bacillus species dominated the soil samples along the urbanization gradient. Shannon-Weaver indices based on culture-dependent methods indicated that urban sites had the highest biodiversity. These results could have been exaggerated, because of an overestimation of the number of bacterial morphotypes present in samples. Fungal levels were higher in the soil from samples collected at the rural samples sites. The culture-independent method (DGGE) was not optimized and inconclusive results were obtained. Enzyme assays revealed that potential dehydrogenase, β-glucosidase and urease activity followed a trend along the urbanization gradient, with urban samples registering the highest values and rural sites the lowest. Enzymes involved in carbohydrate catabolism (β-glucosidase and dehydrogenase) registered significantly higher potential activity in urban sites than the sub-urban and rural sites. The results could indicate that urban sites have the potential to lose carbon at higher rates than the rural sites. This aspect may need further investigation. Higher potential urease activity could indicate higher N-cycling in the urban soil environment. Ordination results for soil-, plant- and microbial diversity as well as microbial functionality indicated certain trends along the urbanization gradient. Plant species composition and structure data indicated that urbanization has a definite effect on the plant communities in the urban ecosystem. Results regarding aerobic heterotrophic bacteria populations and potential enzyme activity of the dehydrogenase, β-glucosidase (both active in the carbon cycle) and urease (active in the nitrogen cycle) illustrated clear trends along the urbanization gradient. In conclusion, results indicated that urbanization has an effect on plant species composition, and the population and function of aerobic heterotrophic bacteria and the fungal population. Furthermore, this study demonstrated the potential of using microbial diversity and activity as tools to investigate carbon utilization and storage along an urban-rural gradient. Masters

Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009. South Africa is one of the most important mining countries in the world, hosting the world's largest reserves of platinum group metals (PGMs). Even though mining is clearly an important activity in South Africa, contributing approximately US$ 7.4 billion annually to the countries' gross domestic product (GDP), the costs to the environment are not insignificant. One of the most severe environmental aspects associated with mining is the storage of mineral waste on tailings storage facilities due to their impacts on air quality, ground water quality, aesthetics and land use. It is also unknown whether the environmental effects of tailings storage facilities increase or decrease over time. The aim of this study was to determine the ecotoxicity of platinum tailings storage facilities of different ages by means of soil physical and chemical analysis, earthworm ecotoxicological studies, dehydrogenase activity and soil mesofauna studies. Samples were obtained from three platinum tailings storage facilities of different ages of which two were already rehabilitated while the third was still operational at the time this study was performed. The latter was used as a negative control for the purpose of the study. Soil samples were physically and chemically analysed. Earthworm ecotoxicological studies were conducted to determine changes in biomass, reproduction, mortality, neutral red retention times and tissue metal concentrations. Dehydrogenase activity was determined before the introduction of earthworms and manure, after introductions of manure and after introductions of earthworms and manure. Soil mesofauna were extracted and identified in order to determine species richness, diversity, abundance and functional grouping. Soil chemical analysis indicated that concentrations of certain heavy metals, especially chrome (Cr), present in platinum tailings materials could have a potential effect on microorganisms, microbial processes and earthworms. Earthworm ecotoxicological results indicated that earthworms that bioaccumulated higher levels of heavy metals showed poor hatchability of cocoons. Dehydrogenase activity indicated that earthworms play a significant role in increasing the number and biomass of soil microbes because significant increases in dehydrogenase activity were noticed after the addition of earthworms to platinum tailings materials. Results from the earthworm ecotoxicological studies, dehydrogenase activity, and soil mesofauna composition indicated that environmental impacts of tailings storage facilities did not increase with age, but is more likely to be an indication of the rehabilitation measures administered to the different tailings storage facilities. Masters

Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006. The disposal of solid waste material produced by mining activities can have vastly negative environmental impacts with regards to soil ecosystems. Global and South African legislation requires the mining company to rehabilitate the disturbed area in such a manner that sustainable ecosystem stability is achieved. Rehabilitation of co-disposed diamond tailings at Finsch mine (De Beers) entails the application of organic matter in the form of vermi-compost, organic and inorganic fertilizers, as well as different grass seed mixtures. Optimal amounts of chemical ameliorants were determined in a previous experimental trial prior to the current field study at the mine. The aim of this study was to evaluate the rehabilitation progress by analysis of soil microbial communities as biological indicators of sustainability. Soil quality can be determined by the analysis of physical, chemical and biological soil properties. Chemical analysis of the tailings material was done by 1 :2 water extract, as well as ammonium acetate extraction techniques. Enzymatic analysis (dehydrogenase, acid and alkaline phosphatase, f3-glucosidase and urease) as well as substrate induced respiration (SIR) was used to determine the soil microbial community function. Soil microbial community structure was evaluated by the analysis of specific microbial lipid biomarkers. The vegetation response towards the rehabilitation effort was evaluated by determination of subjective ratings for plant species frequency and density, and plant biomass. Plant vitality was determined by analysis of photosynthetic activity, which is known to be extremely sensitive towards environmental disturbance. In terms of overall enzymatic activity (dehydrogenase), the application of 90-tonlha vermi-compost in the experimental plots showed the best results. No statistically significant differences in SIR results were noted in the experimental plots. However, the plots treated with 60-tonslha vermi-compost showed the best results in terms of plant vitality and plant biomass. The determination of soil microbial community structure showed that fungal to bacterial ratios were increasing over time although actual ratio values are much less than one, and that overall bacterial population dominance is evident in the experimental plots. Masters

Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006. Platinum mining activities contribute substantially to South Africa's economy since it exceeded gold as economical contributor in 2001. Mining activities contribute to large amounts of waste production in the form of tailings and rock waste, deposited in the surrounding environment of the mine premises. Mining companies are held responsible for damages caused to the surrounding environment. These companies are required to introduce the cost of ecological rehabilitation in their operation costs as well as compile an environmental management plan. Numerous attempts to rehabilitate mine waste have proven unsuccessful. New and improved rehabilitation techniques are required to facilitate in the rehabilitation of these mine spoils. Woodchip-vermicompost produced from platinum mining wastes (woodchips and sewage sludge) was used as an alternative amendment to inorganic fertilisers during the rehabilitation of platinum mine tailings. The effectiveness of the woodchip-vermicompost as an alternative amendment during the platinum mine tailings rehabilitation were monitored using different ecosystem components. A natural veldt in the vicinity of the mine area was randomly selected to serve as a reference site. These ecosystem components selected have previously been shown to be effective as indicators of ecosystem quality. The components selected for this study includes the use of microbial enzymatic activity, microbial community structure, nematode trophic structures, and other mesofaunal groups such as micro-arthropods. The physical and chemical properties of the platinum mine tailings and reference area as well as the vegetation cover of the platinum mine tailings were determined. Statistical and multivariate analyses were use to determine the correlation between the dependent microbial components and dominate independent chemical properties. Nematode trophic structure, Maturity Index, and Plant-Parasitic nematode Index were used to compare the two rehabilitation techniques in terms of nematodes as indicators. Microarthropods family structures were used to compare the two amendments in terms of diversity and abundance. Enzymatic activity was positively affected by the addition of woodchip-vermicompost, than in the sites treated with inorganic fertilisers. The microbial community structure showed no statistically significant (p < 0.05) differences between the two amendments. A higher abundance of nematodes especially plant-parasitic nematodes and bacterivorous nematodes were observed in the woodchip-vermicompost sites than in the inorganic fertilised sites. According to the Maturity Index, both amendments became more enriched during the study period, while the Plant-Parasitic nematode Index showed that the carrying capacity for plantparasitic nematodes on the woodchip-vermicompost sites increased while it decreased in the inorganic fertilised sites, which can be related to the decrease in vegetation cover on the inorganic fertilised sites. Both coloniser (Prostigmata) and persister (Cryptostigmata and Mesostigmata) groups of the micro-arthropods, as well as a higher diversity of micro-arthropods, were present on the woodchip-vermicompost sites whereas the inorganic fertilised sites showed only the presence of colonisers, with a decrease in diversity and abundance of micro-arthropods over the study. The colonisation of micro-arthropods may have been affected by the addition of woodchip-vermicompost and vegetation cover, which contribute to the establishment of suitable microhabitats for these soil biota. By intercorrelating the results, it may be concluded that the addition of woodchip-vermicompost may be an essential part of the rehabilitation process, by contributing to soil organic material to the ecosystem system, which may improve the recolonisation of soil biota and ecosystem processes. However further studies need to be conducted in order to determine the long-term sustainability of the woodchip-vermicompost in providing organic material and sustaining the ecosystem processes. The study also showed the necessity to integrate various ecosystem components when evaluating ecosystem development due to the unique role each component plays and the impact it may have on other components. Masters

Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2005 Increased amounts of pesticide production and application of pesticides for agriculture, plant protection and animal health has resulted in soil, water and air pollution, consequently relating a serious risk to the environment and also to human health. Pesticides include several groups of compounds, herbicides, insecticides, rodenticides and fumigants consisting of several hundred individual chemicals. Herbicides are an integral pan of modem agriculture and for industries requiring total vegetation control. Most herbicides are soil applied and more and more concern is raised that herbicides not only affect target organisms but also the microbial community present in soil. The ESKOM sub-station Zeus, in Mpumalanga (South Africa) used to apply an industrial weed control program for the eradication of vegetation, which led to the contamination of soil by several herbicides. These herbicides consisted of Bromacil, Tebuthiuron and Ethidimuron which are all photosynthesis inhibitors, more specifically, they disrupt the plastoquinone protein during electron transport at photosystem ll (PSII). In this study the effect of biostimulation and bio-augmentation of a specific bioremediation agent (B350) as prescribed by ESKOM, on residual herbicides, Bromacil, Tebuthiuron and Ethidimuron was evaluated by monitoring the soil physical and chemical properties, microbial attributes, including potential microbial activity and community structure, as well as the physiological effect experienced by plants (Cynodoh dactylon and Zea mays). Results from soil physical and chemical analyses were correlated with results obtained for the functional and structural diversity of microbial communities. All results were investigated through statistical and multivariate analysis and the most prominent soil physical and chemical parameters that influence the biological and biochemical properties of the soil were identified. Results obtained from this study indicated that there were no significant difference (p < 0.05) between the treatments, with bioremediation agent, irradiated agent and without the agent based on results obtained from soil microbial properties and plant physiology. Before the trial started the uncontaminated soil showed an active microbial function, characterised by dehydrogenase, urease and arylsulphatase activity, but community structure was not very diverse. The contaminated soil, irradiated contaminated soil and silica sand showed less enzymatic function and was characterised by phospholipid fatty acid groups, mid-branched saturated fatty acids, terminally branched saturated fatty acids, normal saturated fatty acids and monosaturated fatty acids which are indicative of microorganisms that survive better in harsh environments. Three weeks after the addition of the specific bioremediation took place, the uncontaminated soil showed an increase in P-glucosidase activity and percentage organic carbon (%C), which could be a result of the presence of available plant material. Furthermore, an increase in major PLFA groups were seen, suggesting that an increase in diversity within the soil community occurred. The contaminated soil, irradiated contaminated soil and silica sand once again was characterised by a low microbial function and diversity, showing no improvement. Fluorescence data clearly show a decline in PS ll function that result in the decline of the rate of photosynthesis, which was seen from COz gas exchange rates. Furthermore, the decrease in photosynthetic activity after three weeks was too severe to supply additional information about the mechanism within photosynthesis or the photoprotective mechanisms. A detailed study was conducted in which a 3: 1 dilution of contaminated soil with silica sand, was also monitored for changes within plant physiology. Results revealed that inhibition of PS ll function already takes place within a few days time and the decline in photosynthesis is as a result of electron transport that does not supply adenosine triphosphate (ATP) and P-nicotinamide adenine dinucleotide (NADPH) to the Calvin cycle (or Reductive Pentose Phosphate pathway). It does not appear that rubulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) is affected within the Calvin cycle. As a result of PS II function failure, reaction centres are damaged by the production of harmful singlet oxygen and photoprotective mechanisms (xanthophyll cycle) can not be activated. Thus, except for dealing with ineffective electron transport, additional damage is caused to physiological functions. After six weeks a decrease in the estimated viable biomass for all growth mediums was found. Results of the of trans- to cis- monoenoic fatty acids and cyclopropyl fatty acids to their monoenoic precursors ratios indicated that the soil microbial community for the contaminated growth mediums, all experienced nutritional stress throughout this trail. The specific bioremediation agent (B350) used, seemed to have no effect on the microbial function and community structure within soil and as agent had no effect on the residual herbicides or the plant physiology which experienced an extreme decline in major metabolic functions. Masters