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Long-term monitoring of post-mining rehabilitation is inherently difficult and cannot be replicated, making the application of traditional experimental and statistical approaches for quantitative assessment of rehabilitation progress unattainable. An alternative approach to monitoring through time is space-for-time substitution; a technique used to infer a temporal trend from a study of different aged sites. In this investigation, space-for-time substitution was applied to two chronosequences of coal discard sites to determine the applicability of this approach to show the long-term effect of rehabilitation age on microbial communities. Sites at different stages of rehabilitation at separate locations (space) were identified to obtain a chronosequence of ages (time). Two chronosequences of rehabilitation ages from 1-11 and 6-17 years, respectively, were included, each with its own management regime. The long-term effect of the management regimes on soil microbial communities was investigated in terms of community function (enzymatic assays) and structure (phospholipid fatty acid profiles). Results showed no trends consistent with the rehabilitation ages of the respective sites for any of the investigated parameters. However, multivariate statistical analysis indicated a clear distinction between the chronosequences based on management regimes. This study shows the value of alternative statistical approaches in monitoring to elucidate long-term effects of management that might otherwise not be apparent.

The rehabilitation of degraded soils, such as those associated with post-mining sites, requires knowledge of the soil ecosystem and its physical, chemical, and biological composition in order for rehabilitation efforts to fulfil the long-term goal of reconstructing a stable ecosystem for rehabilitated mine soil. This study addresses the need for appropriate assessment criteria to determine the progress of rehabilitation and subsequently the success of management practices. Significant contributions made by this investigation included the establishment of minimum and maximum values for microbial community measurements from two case studies of rehabilitated coal discard sites. Furthermore, it was shown that there was no relationship between changes in microbial community function and structure and the rehabilitation age of the sites. Following this, the considerable impact of management practices on microbial communities was illustrated. The first part of the study investigated the temporal changes in microbial community function and structure in a chronosequence of rehabilitated coal discard sites aged 1 to 11 years. The most important observation made during the investigation of the microbial communities in the different aged soil covers of the rehabilitated coal discard sites, was that there was no relationship between rehabilitation age and microbial activity or abundance of certain microbial groups. What was responsible for a clear differentiation between sites and a shift in microbial community attributes was the management practices applied. A comparison of two chronosequences of rehabilitated coal discard sites was achieved by an application of the 'space-for-time' hypothesis. Sites of different ages and at separate locations ('space') were identified to obtain a chronosequence of ages ('time'). The two chronosequences included sites aged 1 to 11 years (chronosequence A) and 6 to 17 years (chronosequence B), respectively. Sites in the same chronosequence were managed identically, while there was a distinct difference in management practices applied to each chronosequence. The long-term effect of the different management regimes on the soil microbial community function and structure was investigated. Again, there was no relationship between rehabilitation age and microbial community measurements. Fluctuations of selected microbial properties occurred in both chronosequences and similar temporal trends existed over the rehabilitation periods. However, the less intensively managed chronosequence (8) seemed more stable (less fluctuation occurred) over the rehabilitation period than the more intensively managed chronosequence (A). It was therefore concluded that the microbial communities in the less managed sites maintained their functional and structural integrity within bounds in the absence of management inputs or disturbance. While there was similarity in the trends over time for individual microbial community measurements, the seemingly more stable conditions in chronosequence 6 are important in terms of the goal of rehabilitation. Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007

Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007 The rehabilitation of degraded soils, such as those associated with post-mining sites, requires knowledge of the soil ecosystem and its physical, chemical, and biological composition in order for rehabilitation efforts to fulfil the long-term goal of reconstructing a stable ecosystem for rehabilitated mine soil. This study addresses the need for appropriate assessment criteria to determine the progress of rehabilitation and subsequently the success of management practices. Significant contributions made by this investigation included the establishment of minimum and maximum values for microbial community measurements from two case studies of rehabilitated coal discard sites. Furthermore, it was shown that there was no relationship between changes in microbial community function and structure and the rehabilitation age of the sites. Following this, the considerable impact of management practices on microbial communities was illustrated. The first part of the study investigated the temporal changes in microbial community function and structure in a chronosequence of rehabilitated coal discard sites aged 1 to 11 years. The most important observation made during the investigation of the microbial communities in the different aged soil covers of the rehabilitated coal discard sites, was that there was no relationship between rehabilitation age and microbial activity or abundance of certain microbial groups. What was responsible for a clear differentiation between sites and a shift in microbial community attributes was the management practices applied. A comparison of two chronosequences of rehabilitated coal discard sites was achieved by an application of the 'space-for-time' hypothesis. Sites of different ages and at separate locations ('space') were identified to obtain a chronosequence of ages ('time'). The two chronosequences included sites aged 1 to 11 years (chronosequence A) and 6 to 17 years (chronosequence B), respectively. Sites in the same chronosequence were managed identically, while there was a distinct difference in management practices applied to each chronosequence. The long-term effect of the different management regimes on the soil microbial community function and structure was investigated. Again, there was no relationship between rehabilitation age and microbial community measurements. Fluctuations of selected microbial properties occurred in both chronosequences and similar temporal trends existed over the rehabilitation periods. However, the less intensively managed chronosequence (8) seemed more stable (less fluctuation occurred) over the rehabilitation period than the more intensively managed chronosequence (A). It was therefore concluded that the microbial communities in the less managed sites maintained their functional and structural integrity within bounds in the absence of management inputs or disturbance. While there was similarity in the trends over time for individual microbial community measurements, the seemingly more stable conditions in chronosequence 6 are important in terms of the goal of rehabilitation. Doctoral

The introduction of genetically modified glyphosate-resistant (GR) crops led to increased use of glyphosate, with potential long-term effects on biological soil communities. Abundance and diversity of plant-parasitic and non-parasitic nematodes, as well as microbial community structure in the rhizosphere of GR and conventional soybean (CS) plants as well as native plants (mainly grasses) that grew in natural veld (NV), were investigated during one growing season. No significant differences (p ≥ 0.05) existed between the non-parasitic nematode trophic groups (dominated by bacterivores) and different plant-parasitic nematodes (dominated by the family Hoplolaimidae) that were present in all soils where GR and CS crops were grown or in NV areas. Faunal analysis of non-parasitic nematodes indicated that 83% of the sites contained enriched, but unstructured soil food-webs while 17% were identified as enriched and structured. No significant differences (p ≥ 0.05) existed for microbial community structures among the GR, CS and NV ecosystems with soils from all three primarily dominated by bacteria. Positive relationships were found between nematode trophic groups and their corresponding microbial prey. This emphasises the value of these organisms as indicators of soil quality and also the impact that agricultural practices can have on soil food webs

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 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

Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2012. Sunflower cultivation in South Africa is threatened to a large extent by the fungal parasite, Sclerotinia sclerotiorum, which causes extensive head rot and crop losses of up to 100%. This is a major problem for commercial farmers since it leads to a lower farm income, as the use of fungicides minimises crop damage, but increases production costs and can lead to several environmental problems. Therefore, an alternative is needed which can still control crop diseases, without harbouring health and environmental risks. Due to their biofumigation potential, Brassica plant species incorporated into the soil as green manures can be applied as alternatives for chemical pesticides. These plants produce glucosinolates that are hydrolysed upon tissue disruption by the enzyme, myrosinase, into active products for example isothiocyanates. Since isothiocyanates are highly toxic, it can be used instead of conventional pesticides for the inhibition of soil–borne pathogens. However, little is known about the effect of such biofumigants on the natural soil microbial communities required to maintain soil functions. A greenhouse experiment was conducted to assess the influence of canola (Brassica napus) green manure on soil microbial community function and structure. The study consisted of 32 pots containing four treatments of eight replicates each. The treatments included 1) only sunflowers in soil (control), 2) sunflowers in soil incorporated with canola green manure; 3) sunflowers in soil incorporated with canola and inoculated with S. sclerotiorum and 4) sunflowers in soil inoculated with S. sclerotiorum. The experiment was conducted for 120 days. From the soil physico–chemical properties conducted before the treatments were applied and after experiment completion, it was evident that the initial stimulating effect of canola manure on the soil carbon, total nitrogen and organic carbon content was not long-lasting. The overall microbial activity assessed with dehydrogenase assays and Biolog® Ecoplates, varied in relation to plant growth cycles, as root secretions differed. Multivariate analysis of the substrate utilisation patterns, distinguished among the treatments. Utilisation profiles illustrated that although different members of microorganisms were active in the various treatments, similar trends could still be observed. All four treatments showed similar diversity profiles after 120 days. Phospholipid fatty acid results indicated a significant increase in microbial biomass for all four treatments over time. The microbial community structure differed to a lesser extent between treatments, but changed over time within each treatment. Community function varied according to the changing structure. Fatty acid stress ratios for all treatments showed significantly lower stress levels just after manure amendments, as the added organic matter might have stimulated microbial growth. Chlorophyll a fluorescence measurements showed shifts occurring in the photosynthetic efficiency of the sunflowers among the treatments. S. sclerotiorum had a suppressive effect on photosystem II functionality leading to lower electron transport and ATP production. Canola green manure amendments had a slight negative effect on sunflower vitality. Overall the results obtained from this study suggest that incorporation of canola green manure into the soil has an effect on soil microbial community function and structure. Nonetheless, this biofumigation effect is short–lived and microbial communities returned to their initial compositions after the disturbance. The methods applied during this investigation indicated a possible suppressive effect of the canola manure on S. sclerotiorum. Masters