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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.

10.1007/s11270-008-9719-7 http://www.springerlink.com/content/6171x71459w22456/?p=c5990faa540b4cfaa9aee12e99b5c534&pi=25

http://dx.doi.org/10.1007/s00254-008-1250-z http://www.springerlink.com/content/923m4315n10w2407/fulltext.pdf

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

Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2004. Mining activities cause severe disturbance to the soil environment in terms of soil quality and productivity and are of serious concern worldwide. Under South African legislation, developers are required to ecologically rehabilitate damaged environments. The application of agronomic approaches for the rehabilitation of coal discard sites has failed dismally in the arid areas of southern Africa. It is obvious that compliance with mitigation and rehabilitation requirements cannot be enforced without a thorough understanding of the ecological principles that ensure ecological stability and subsequent sustainability of soil ecosystems. Soil micro organisms are crucial role-players in the processes that make energy and nutrients available for recycling in the soil ecosystem. Poor management practices and other negative impacts on soil ecosystems affect both the physical and chemical properties of soil, as well as the functional and structural properties of soil microbial communities. Disturbances of soil ecosystems that impact on the normal functioning of microbial communities are potentially detrimental to soil formation, energy transfers, nutrient cycling, plant reestablishment and long-term stability. In this regard, an extensive overview of soil properties and processes indicated that the use of microbiological and biochemical soil properties, such as microbial biomass, enzymatic activity and the analysis of microbial community structure by the quantification of specific signature lipid biomarkers are useful as indicators of soil ecological stress or restoration properties because they are more responsive to small changes than physical and chemical characteristics. In this study, the relationship between the physical and chemical characteristics and different biological indicators of soil quality in the topsoil covers of seven coal discard sites under rehabilitation in South Africa, as well as three reference sites was investigated. Through the assimilation of basic quantitative data and the assessment of certain physical, chemical and biological properties of the topsoil covers obtained from the various coal discard sites as well as the reference sites, the relative success or progress of rehabilitation and the possible correlation between the biological indicators of soil quality and the establishment of self sustaining vegetation covers was determined. Results from soil physical and chemical analyses and percentage vegetation cover were correlated with the results obtained for the functional and structural diversity of microbial communities at the various sites. All results were investigated through statistical and multivariate analysis and the most prominent physical and chemical parameters that influence the biological and biochemical properties of the soil and possibly the establishment of self-sustainable vegetation cover on these mine-tailing sites were identified. Results obtained from this study indicated no significant difference (p>0.05) between the various discard sites based on conventional microbiological enumeration techniques. However, significant differences (p

Mining activities cause severe disturbance to the soil environment in terms of soil quality and productivity and are of serious concern worldwide. Under South African legislation, developers are required to ecologically rehabilitate damaged environments. The application of agronomic approaches for the rehabilitation of coal discard sites has failed dismally in the arid areas of southern Africa. It is obvious that compliance with mitigation and rehabilitation requirements cannot be enforced without a thorough understanding of the ecological principles that ensure ecological stability and subsequent sustainability of soil ecosystems. Soil micro organisms are crucial role-players in the processes that make energy and nutrients available for recycling in the soil ecosystem. Poor management practices and other negative impacts on soil ecosystems affect both the physical and chemical properties of soil, as well as the functional and structural properties of soil microbial communities. Disturbances of soil ecosystems that impact on the normal functioning of microbial communities are potentially detrimental to soil formation, energy transfers, nutrient cycling, plant reestablishment and long-term stability. In this regard, an extensive overview of soil properties and processes indicated that the use of microbiological and biochemical soil properties, such as microbial biomass, enzymatic activity and the analysis of microbial community structure by the quantification of specific signature lipid biomarkers are useful as indicators of soil ecological stress or restoration properties because they are more responsive to small changes than physical and chemical characteristics. In this study, the relationship between the physical and chemical characteristics and different biological indicators of soil quality in the topsoil covers of seven coal discard sites under rehabilitation in South Africa, as well as three reference sites was investigated. Through the assimilation of basic quantitative data and the assessment of certain physical, chemical and biological properties of the topsoil covers obtained from the various coal discard sites as well as the reference sites, the relative success or progress of rehabilitation and the possible correlation between the biological indicators of soil quality and the establishment of self sustaining vegetation covers was determined. Results from soil physical and chemical analyses and percentage vegetation cover were correlated with the results obtained for the functional and structural diversity of microbial communities at the various sites. All results were investigated through statistical and multivariate analysis and the most prominent physical and chemical parameters that influence the biological and biochemical properties of the soil and possibly the establishment of self-sustainable vegetation cover on these mine-tailing sites were identified. Results obtained from this study indicated no significant difference (p>0.05) between the various discard sites based on conventional microbiological enumeration techniques. However, significant differences (p

Aflatoxin contamination remains one of the most important threats to food safety and human health. Aflatoxins are mainly found in soil, decaying plant material and food storage systems and are particularly abundant during drought stress. Regulations suggest the disposal of aflatoxin-contaminated crops by incorporation into the soil for natural degradation. However, the fate and consequences of aflatoxin in soil and on soil organisms providing essential ecological services remain unclear and could potentially pose a risk to soil health and productivity. The protection of soil biodiversity and ecosystem services are essential for the success of the declared United Nations Decade on Ecosystem Restoration. The focus of this study was to investigate the toxicological consequences of aflatoxins to earthworms’ survival, growth, reproduction and genotoxicity under different temperature and moisture conditions. Results indicated an insignificant effect of aflatoxin concentrations between 10 and 100 µg/kg on the survival, growth and reproduction but indicated a concentration-dependent increase in DNA damage at standard testing conditions. However, the interaction of the toxin with different environmental conditions, particularly low moisture, resulted in significantly reduced reproduction rates and increased DNA damage in earthworms.

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

With an increase in the global population, a protein-rich crop like soybean can help manage food insecurity in sub-Saharan Africa (SSA). The expansion of soybean production in recent years lead to increased land requirements for growing the crop and the increased risk of exposing this valuable crop to various pests and diseases. Of these pests, plant-parasitic nematodes (PPN), especially Meloidogyne and Pratylenchus spp., are of great concern. The increase in the population densities of these nematodes can cause significant damage to soybean. Furthermore, the use of crop rotation and cultivars (cvs.) with genetic resistance traits might not be effective for Meloidogyne and Pratylenchus control. This review builds on a previous study and focuses on the current nematode threat facing local soybean production, while probing into possible biological control options that still need to be studied in more detail. As soybean is produced on a global scale, the information generated by local and international researchers is needed. This will address the problem of the current global food demand, which is a matter of pressing importance for developing countries, such as those in sub-Saharan Africa.

Aflatoxins are secondary metabolites produced by specific strains of fungi, especially Aspergillus spp. These natural toxins are mainly found in soil, decaying vegetation and food storage systems and are particularly abundant during drought stress. Aflatoxin contamination is one of the most important threats to food safety and human health due to its toxic, mutagenic and carcinogenic properties. Therefore, most research focuses on post-harvest contamination of aflatoxins in feed and food commodities but very limited information is available about aflatoxin contamination and its toxicological consequences in the soil ecosystem. Current regulations provide minimal options for the disposal of aflatoxin-contaminated crops, amongst which is the incorporation of residues into the soil for natural degradation. This form of mycotoxin loading into the soil could potentially change its physicochemical characteristics and biotic parameters. Recent studies suggest that as climate conditions change, the occurrence and geographical distribution of aflatoxins might increase, posing significant health risks to the soil ecosystem, food crop production and human health. This review will focus on studies that look at the environmental and toxicological consequences of aflatoxin contamination with the aim of clarifying the risk that aflatoxin contamination poses to soil ecosystems. Many aspects of aflatoxin occurrence, degradation and the effects of its transformation products in the soil environment are still unknown and remain an important area of research for soil health and productivity. A climatic approach, in terms of changes in soil moisture and air temperature, is important for future risk assessments of aflatoxin contamination.

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

Root-knot nematodes are destructive phytopathogens that damage agricultural crops globally, and there is growing interest in the use of biocontrol based on rhizobacteria such as Bacillus to combat Meloidogyne species. It is hypothesized that nematicidal activity of Bacillus can be attributed to the production of secondary metabolites and hydrolytic enzymes. Yet, few studies have characterized these metabolites and their identities remain unknown. Others are speculative or fail to elaborate on how secondary metabolites were detected or distinguished from primary metabolites. Metabolites can be classified based on their origin as either intracellular or extracellular and based on their function, as either primary or secondary. Although this classification is in general use, the boundaries are not always well defined. An understanding of the secondary metabolite and hydrolytic enzyme classification of Bacillus species will facilitate investigations aimed at bionematicide development. This review summarizes the significance of Bacillus hydrolytic enzymes and secondary metabolites in bionematicide research and provides an overview of known classifications. The importance of appropriate cultivation conditions for optimum metabolite and enzyme production is also discussed. Finally, the use of metabolomics for the detection and identification of nematicidal compounds is considered.

Agricultural crops are severely damaged by root-knot nematodes causing extensive financial losses globally. Historically, agrochemicals have been the preferred method to combat these pests; however, threats to humans and the environment posed by these agrochemicals led to the need for developing new biocontrol agents. Importantly, the latter should adhere to biosafety regulations while being highly effective. Root-knot nematodes live in soil and thus the use of rhizobacteria such as Bacillus for biocontrol development have shown potential. Although various Bacillus species have been tested in this capacity, little is known about their secondary metabolites and the mechanisms of action responsible for their nematicidal activity. If these secondary metabolites can be qualitatively and quantitatively characterised, metabolic features could be synthetically engineered and used to combat root-knot nematodes. Although there is great potential for bionematicides, the commercialisation and development of such products can be difficult. This review summarises the importance of Bacillus species as natural antagonists of root-knot nematodes through the production of secondary metabolites. It provides an overview of the significance of root-knot nematodes in agriculture and the advances of chemical nematicides in recent years. The potential of Bacillus species as biocontrol agents, the known mechanisms of action responsible for the nematicidal activity demonstrated by Bacillus species, non-target effects of biocontrol agents and the commercialisation of Bacillus-based bionematicides are discussed

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. 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

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. 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