The primary objective of this study was to investigate the use of water table management (WTM) as a microbial delivery system for in-situ bioaugmentation of contaminated soils. In addition, the use of Rhizobium ( R.) for PCB degradation in soils was evaluated. / First, the presence and isolation of a variety of strains of Rhizobium meliloti was demonstrated using plant nodulation tests on alfalfa plants in soils that were contaminated for over 15 years with PCBs, PAHs and heavy metals. Next, R. meliloti, strain A-025, was selected based on its membrane (hydrophobicity, adhesion) characteristics and its potential to transform PCBs. This strain was delivered and implanted in sod columns, 200 mm in diameter x 1000 mm in length, packed with a sandy loam soil, using surface and subirrigation. The results of this study showed that subirrigation led to a higher number and a more uniform distribution of the bacterial cells in the soil at 60, 300, 500, and 700 mm depths, than surface irrigation. / In a different setup, similar column were packed with a PCB contaminated soil. These soil columns were bioaugmented with three bacterial cultures, i.e., R. meliloti (strain A-025), Comomonas testosteroni (strain B-356) and an indigenous bacterial consortium using subirrigation. The results indicated that bioaugmentation of the PCB contaminated soil was possible by using subirrigation. Bioaugmentation with the indigenous culture was observed to be more effective in the biodegradation of PCBs than with A-025 and B-356 cultures at 140 and 340 mm depths. However, at 590 mm depth, bioaugmentation with strain A-025 was observed to be better than the other treatments. Sequential aerobic and anaerobic cycles appear to be of significance for effective dechlorination of PCB congeners to lower chlorinated congeners. / In a separate exploratory study, the rhizospheric effects of alfalfa plants on R. meliloti for PCB depletion were investigated. The results suggest that the growth of alfalfa plants and bioaugmentation of soil with R. meliloti, strain A-025, increased the depletion of PCB congeners in the soil as compared to bioaugmentation alone. In other preliminary studies, the results showed that the presence of PCBs in a sandy loam soil increases the filtration of bacterial cells. Also, soil type and the presence of PCBs affected water infiltration, moisture, and hardness of the soil. Furthermore, water table management system along with bioaugmentation of soil columns with R. meliloti, strain A-025, decreased the concentration of atrazine by 31% during anaerobic and aerobic cycles and reduced the concentration of nitrate by 87% and 78% in the absence and presence of atrazine, respectively, in the drainage water. / The overall results of this work indicate that water table management (subirrigation) can be used for bioaugmentation of contaminated soils. Also, use of R. meliloti may prove to be an interesting option for soils contaminated with PCBs, atrazine and nitrate.

Thesis (M. Tech.: Biotechnology)-Dept. of Biotechnology and Food Technology, Durban University of Technology, 2008. xiv, 103 leaves : ill. / Phytoremediation is an emerging technology where specially selected and engineered metal-accumulating plants are used for bioremediation. Amaranthus dubius (marog or wild spinach) is a popular nutritious leafy vegetable crop which is widespread especially in the continents of Africa, Asia and South America. Their rapid growth and great biomass makes them some of the highest yielding leafy crops which may be beneficial for phytoremediation. This study was undertaken to evaluate the potential of A. dubius for the phytoremediation of Chromium (Cr), Mercury (Hg), Arsenic (As), Lead (Pb), Copper (Cu) and Nickel (Ni). Locally gathered soil and plants of A. dubius were investigated for the metals from a regularly cultivated area, a landfill site and a sewage site. Metals were extracted from the samples using microwave-digestion and analyzed using Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS). Further experiments were conducted with plants from locally collected seeds of A. dubius, in a tunnel house under controlled conditions. The mode of phytoremediation, the effect of the metals on the plants, the ability of the plant to extract metals from soil (Bioconcentration Factor - BCF), and the ability of the plants to move the metals to the aerial parts of the plants (Translocation Factor - TF) were evaluated for the different metals. Finally, A. dubius was micro-propagated in a tissue culture system with and without exposure to the metal, and the effect was studied by electron microscopy.

In 1990, an oil recycling plant situated in Hammarsdale, South Africa, was decommissioned and a decision was taken by management to rehabilitate the site in preparation for resale. The heavily impacted area covered over two hecatares and oil contamination penetrated soil to depths in excess of three metres, making excavation and removal of the soil very expensive. The options for remediation of the site were limited. No facility for incineration of contaminated soil exists in South Africa, and landfilling was not permitted. The emphasis in developing a remediation strategy, therefore, focussed upon the possibility of in situ remediation with minimal excavation of soil. This study, the first of its kind in South Africa, was subsequently initiated to assess the feasibility of this approach, the results of which would underpin a full-scale cleanup programme. The development of such a strategy involved four key stages of work : (1) a comprehensive site investigation to evaluate and fully understand the particular problems at the site; (2) treatability studies to determine the potential for biological treatment of the contaminated soil and the optimisation of such treatments, particularly in terms of time and cost; (3) the testing of some of the more effective treatments on a pilot-scale; and (4) recommendations for full-scale bioremediation of the contaminated site. various conditions unique to South Africa had to be considered at each stage viz. the lack of funds and remediation experience, which created numerous problems and emphasised the requirement for a simple, "low-tech" approach. Site investigations revealed that in situ remediation may be possible due to the high permeability of the sandy soils and low concentrations of heavy metals. Laboratory experiments also showed that a mixed association of indigenous microorganisms was present which, once stimulated by nutrient supplementation at C:N:P, ratios of between 10:1:1 and 20:1:1, was capable of degrading total petroleum hydrocarbons at an average rate of 11% week -1. Further experimentation, aimed at reducing the cost of remediation and improving the soil quality, focussed on the efficacy of oil solubilisers, a soil ameliorant (composted pine-bark), indigenous fungi and higher plants in the remedial process. Three commercial surfactants (Arkopal N-050, N-060 and E2491) and one natural solubiliser (soybean lecithin) were tested for their biotoxicity, solubilisation and biodegradability at various concentrations (0.01 - 1.0%). Formulation E2491 was able to support a microbial population and was selected as the preferred commercial surfactant if soil washing was to be recommended; however, lecithin was considered to be more useful in situ because of its localised solubilising effect, biological origin and nutritional contribution. The use of fungi was of particular interest in addressing the persistent organic compounds, such as the heavy fractions of oil, for which bacterial remediation methods have been slow or ineffective. While it was not possible, however, to demonstrate in the laboratory that the indigenous fungi contributed significantly towards the degradation of the contaminating oil, the basic trends revealed that the fungal component of the indigenous microbial population was readily stimulated by the addition of nutrient supplements. The bulking-up process was also a success and additional exploratory work was proposed in the form of a larger scale composting design. Finally, the potential for using higher plants and 20% (v / v) composted pinebark (in addition to nutrients) to increase the microbial degradation of the contamination was investigated in both greenhouse and field plot studies. Greenhouse investigations employed soybeans which were postulated to have soil quality and cost benefits. However, although the soybeans were found to significantly enhance the remedial process, the complex soil-contaminant- plant interactions gave rise to strange nutritional effects and, in some cases, severe stunting. In contrast, the field studies employed grasses that had previously established on the site and which ultimately demonstrated a better tolerance for the contaminated conditions. Scanning electron microscopy revealed that there were considerable differences between the root tips of soybean plants which had been grown in contaminated soil and those which had been grown in uncontaminated soil. It was concluded that toxicity symptoms, which are readily observed in the root, could be used as an early indicator for determining the suitability of vegetation for remediation purposes. In both instances, despite the differences, the addition of composted pine-bark and nutrients (nitrogen and phosphorus) resulted in total petroleum hydrocarbon reductions of >85%, illustrating the benefits of plant establishment and oxygen availability. The need to link results from laboratory or pilot-scale experiments to achieve reliable predictions of field-scale behaviour was an essential component of this research. The results of the field study provided evidence, similar to that found in the pot trial, of the accelerated disappearance of organic compounds in the rhizosphere. All experiments incorporated parallel measurements of hydrocarbon residues, microbial activity and pH changes in the contaminated soil, the results of which strongly supported the argument that biodegradation was the dominant component of the remediation process. Thus, after consideration of the significant interactions which dominated the study (time-contaminant-nutrient; time-contaminant-pine-bark; and time-contaminant- pine-bark-plant), it was clear that, aside from these limiting factors, little should preclude the in situ bioremediation of the impacted soil. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

Microorganisms have the potential to remove heavy metals from polluted waters and effluents and may be used in clean-up processes. Microbial associations were enriched for and adapted to grow in nutrient solutions containing various concentrations of different metals. As immobilised cells are known to be more stable and more efficient in metal uptake than are corresponding planktonic or free-living cells the attachment of the microbial associations was investigated using a model stream and it was found that biofilm development was better on rough surfaces such as ground glass and polystyrene than on smooth surfaces such as unetched glass plates and glass beads. When comparing metal uptake by planktonic and attached microorganisms, attached populations were found to have a greater metal-uptake capacity. The uptake of individual metals from various metal combinations was tested with various proportions of pregrown metal-adapted microbial populations as inoculum and it was found that a particular metal was taken up more readily by microbial associations which had previously been exposed to that metal. Lead (Pb2+) appeared to be taken up more readily than copper (Cu2+) or cadmium (Cd2+) while Cd2+ was more actively removed than Cu2+ from solution. pH also affected metal uptake and the optimum range for Cu2+ uptake by the Cu2+ -adapted microbial association was found to be between 5.8 and 7.0. Dead microbial biomass was investigated and found to have efficient metal uptake capacity. Living mycelium from an isolated Aspergillus species showed poor uptake of Cu2+ initially, but when this fungus was pregrown and subsequently killed by moist heat treatment the non-living mycelium was efficient in removal of Pb2+ and Cu2+ ions. The optimum mycelial biomass concentration for metal uptake was also determined. The mechanism of metal uptake by this Aspergillus species was determined, using electron microscopy and EDX techniques, to be metabolism-independent biosorption onto the hyphal surface. Thus the microbial associations and fungal cultures used in this study were shown to have the potential for use in the removal of heavy metals from polluted waters. / Thesis (Ph.D.)-University of Natal, Pietermartizburg, 1995.

Hyperthermophilic proteins are of great interest in both the academic and industrial world in understanding how these proteins are capable of retaining their biological activity under such harsh environmental conditions. This thesis studies a tetrabrachion stalk domain from Staphylothermus marinus, know as Right Handed Coiled Coil (RHCC). This protein is of interest due to its extreme thermostability and its affinity for heavy metals. We aim to better understand the reason for the extreme thermal stability of the protein and to take advantage of the proteins affinity for heavy metals with a view to developing a novel approach to bioremediate Hg2+, a major environmental pollutant. Our results clearly indicated that the protein is more thermostable in alkaline conditions in comparison to acidic conditions. This observation can be explained by careful inspection of the high resolution structure. Our data also clearly show that RHCC is able to bind ionic mercury compounds such as mercury nitrate and dipotassium mercury iodide.

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Salt marshes are one of the most difficult environments to remediate due to their sensitive and important ecosystems. Traditional cleanup methods can do more harm to the marsh than the oil itself. Bioremediation is the preferred cleanup approach for these delicate environments. Typically bioremediation has been carried out by the addition of nutrients but the results have been inconsistent. Previous laboratory studies conducted in oiled seawater demonstrated that clay minerals enhanced microbial growth and hence oil degradation. However, this had not been tested at field sites or on oiled marine sediments where oil is known to persist. The main objectives of this research were to test clay minerals as a bioremediation alternative for enhancing degradation of oiled salt marsh sediments and evaluate the geochemistry of sediment profiles for heavy metal enrichment. Laboratory experiments were carried out to evaluate the application of montmorillonite or kaolinite clay minerals on salt marsh sediments impacted by diesel oil. Oil biodegradation in control experiments was significantly more successful than in clay amended experiments. Clay minerals may have caused an increase in pH which inhibited enzyme-catalyzed processes required for metabolism. Field experiments were carried out in control and clay treatment plots in Louisiana salt marshes impacted by the Deepwater Horizon oil spill. In this research it was demonstrated for the first time that biodegradation of n-alkanes and PAHs was significantly enhanced by the addition of montmorillonite compared to controls. Vegetated treatment plots were slightly more effective than non-vegetative treatment areas. It is suggested that bivalent cations adsorbed to montmroillonite’s surface suppressed the diffuse double layer. This allowed the oil along the clay’s surface to be accessible to the bacteria where oil could be readily consumed. Clay minerals may serve as a new and unique bioremediation strategy for oiled salt marsh sediments. Geochemical sediment profiles from Louisiana salt marshes showed elevated concentrations of selected heavy metals (Zn > Cu > Pb > V > Cr > Fe > Ni). The Deepwater Horizon oil spill is a likely source of metal enrichment as many of these same metals are also constituents of the Deepwater Horizon oil.