Theses and dissertations (Applied Sciences)
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Item Isolation and characterization of Bacillus spp. for use in the remediation of petroleum waste residues(2020) Masika, Wendy Snoyolo; Kudanga, Tukayi; Santosh, Ramchuran; Moonsamy, GhaneshreePetroleum hydrocarbons are toxic to all forms of life; therefore, environmental pollution caused by petroleum is of great concern. Environmentally friendly strategies are required for the remediation of the contaminated sites. Microbial populations comprising of several different genera have been detected in soil and water environments that have been contaminated with petroleum. This suggests that these organisms are able to use hydrocarbon compounds as a substrate for survival and could be harnessed in bioremediation of contaminated sites. The first stage of this research was focussed on the isolation, purification, screening and selection of putative Bacillus spp from environmental samples. Samples were collected from different sites around the Gauteng province in South Africa. Samples from both soil and water were obtained from selected sites including environments that were contaminated by oil. Isolate selection was based on the growth rate of the isolates, the degree of sporulation and the rate of oil degradation. The identities of the potential isolates as well as their safety status were clarified in order to reduce possible risk to end users or the environment. Once suitable isolates were identified, those that possessed inherently strong biodegradation ability were assessed for their efficacy as well as compatibility to perform in a consortium. Various organism combinations were assessed and compared to the efficacy of individual isolates, in order to formulate a bioremediation consortium. Of the 115 isolates, the top performing isolates, identified as GPA 11.2, GPA 7.1, GPA 3.5, GPA 8.3 and GPB 4.4, were obtained from a car workshop in Midrand and a taxi rank in Silverton. GPA 8.3 and GPA 4.4 were, however, eliminated due to their low sporulation efficiency. The selected Bacillus isolates were identified using 16s rDNA sequencing and GPA 7.1 and GPA 11.2 were identified as B. subtilis, while GPA 3.5 was identified as B. methylotrophicus. These isolates were further assessed for enterotoxin production and the presence of anthrax virulent plasmids pX01 and pX02. After conducting the biosafety assays, the isolates were rendered safe for use. The bioremediation potential of the consortium was evaluated using industrial effluents that contained hydrocarbons. Degradation of hydrocarbons using all three consortiums (Gen 3.1, Gen 3.2, Gen 3.3) in the respective industrial effluents were determined by measuring the rate of degradation for each hydrocarbon compound using Gas Chromatography (GC). Results indicated that the bulk of the contaminants were removed during the first 48 hours; and removal (%) did not increase significantly after 72 hours. The total petroleum hydrocarbons (TPH) (C8-C28) removal rates from synthetic effluent after 48 hours of treatment using the Gen 2, Gen 3.1, Gen 3.2 and Gen 3.3 consortia were 0.8, 0.26, 0.07 and 0.58 mg.L-1.h-1, respectively. The TPH (C8- C28) removal rate from true effluents after 48 hours of treatment using the Gen 2 (benchmark), Gen 3.1, Gen 3.2 and Gen 3.3 consortia were 0.23, 0.25, 0.12 and 0.17 mg.L-1.h-1, respectively. The results showed that the best performing consortium was Gen 3.1.This study has demonstrated the potential application of Bacillus as bioremediation agents for the treatment of hydrocarbon-contaminated sites. This technology could potentially also be utilised for addressing the challenges of a wider range of different hydrocarbon effluents.Item Bioaugmentation with Bacillus spp. for bioremediation of synthetic wastewater using a fluidized-bed reactor(2020-03-25) Roets, Yrielle; Bux, Faizal; Lalloo, Rajesh; Kumar, SheenaSouth Africa’s freshwater resources, including rivers, man-made lakes and groundwater are under severe threat due to an ever-expanding population and economy, which is depleting these resources. The increase in population has a direct correlation with the increase in wastewater generated. The remaining fresh water resources need to be preserved therefore recycling of wastewater, to replenish our water supplies and preserve the environment, is a solution to the problem. For a developing country, it is important to use treatment methods that are cost effective and do not exert a negative impact on the environment, such as biological wastewater treatment options. One of the systems commonly used in biological wastewater treatment is the fluidized-bed bioreactor (FBBR) due to its advantages such as higher biomass concentration and a higher mass transfer thus resulting in a higher rate of biodegradation. This study focused on evaluating the efficacy of augmenting with Bacillus spp. to enhance the bioremediation of wastewater using a FBBR. Bacillus spp. used in this study were isolated from a municipal wastewater treatment plant (10 isolates) and the remaining three isolates were selected from the CSIR Bacillus database. The isolates (13 in total) were screened for 1) their ability grow in wastewater, 2) ability to reduce high concentrations of COD, ammonium, nitrates and phosphates in flask studies containing synthetic wastewater (SWW) and 3) ability to produce common enzymes such as amylase, cellulase, lipase and protease. Isolates showed varying bioremediation potential for different compounds analysed. Isolate B006 showed the highest phosphate removal rate (3.290 mg.L-1.h-1) where as D005 showed the highest growth rate (0.955 h-1), COD reduction rate (55 mg.L-1.h-1) and cellulase activity (5.485 mm) among all the isolates. Isolate D014 presented the highest ammonium removal rate (12.43 mg.L-1.h-1), amylase (5.00 mm) and protease (10.00 mm) activity whilst B001 displayed the highest nitrate removal rate (9.4 mg.L-1.h-1). The results for the individual assays were assessed and weighted in a matrix and the isolates that scored above 50% were selected for consortium studies. Four Bacillus spp. that scored above 50% in the scoring matrix were then evaluated for their ability to co-exist as a consortium. The consortium studies were then compared with results obtained for individual isolates. The selected Bacillus isolates were identified and assessed for their safety to the environment and to the end user. Identification was conducted using 16s rDNA sequencing and results showed that B006 identified as B. cereus, D005 as B. cereus and D014 as B. subtilis. Isolates, B006 and D005 were further assessed for enterotoxin production and the presence of anthrax virulent plasmids pX01 and pX02. After conducting the biosafety assays, the isolates were rendered safe for use. The isolates were then cryopreserved as spores in 25% glycerol and stored at -80 °C. The impact of the cryopreservation method and the storage conditions on the viability of the isolates was assessed after six months of storage and it was established that the isolates were still viable and that the method was adequate. The bioremediation potential of the consortium was further evaluated using a 17 L Pilot scale fluidised-bed bioreactor. The reactors were fed at three different flow rates of 1.5 L.h-1, 2 L.h-1 and 3 L.h-1 over steady state conditions (~3months). The results showed that the FBBR augmented with the selected Bacillus isolates, resulted in improved nutrient (COD, ammonium and phosphates) removal efficiencies compared to the non-bioaugmented control. The highest ammonium removal (62.8%) was observed at a flow rate of 1.5 L.h-1 (11.30 h retention time), whereby there was an overall 29.8% improvement in ammonia removal in comparison to the non-augmented control. Similarly, an overall improvement in phosphate (14.73%) was observed at a flow rate of 2 L.h-1 (8.48 h retention time) with 50% removal efficiency. The highest COD removal was observed at a flow rate of 1.5 L.h-1 (11.30 h retention time) whereby 74.5% COD was reduced with a 32.6% improvement when compared to the non-bioaugmented control. Our work has demonstrated the potential application of Bacillus as bioaugmentation agents to enhance wastewater treatment efficiency as a potential solution to water challenges in developing countries. This technology could also be utilized for addressing the challenges of a wider range of different effluents.Item Phytoremediation of heavy metals using Amaranthus dubius(2008) Mellem, John JasonPhytoremediation 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.Item Microbial degradation of polychlorinated biphenyls(2007) Mustapha, ShubnumThe aromatic compounds Polychlorinated Biphenyls (PCBs) are one of the largest groups of environmental pollutants. The greatest concern is the release of PCBs in the water systems by industrial effluent, accidental spillages or leaks. PCBs are able to bioaccumulate in the fatty tissues of animals, fish and humans. The impact on human health due to PCBs has prompted interest in their degradation. The application of microbial degradation of PCBs can transform many PCB metabolites. There are a wide variety of microorganisms that can degrade PCBs or utilise them as sole carbon sources. This study focused on isolating microrganisms from industrial wastewater capable of aerobic degradation of PCBs. The degradation potential of the selected isolates were investigated by using different analytical techniques viz. ultra violet or visible spectrophotometer (UV/Vis), thin layer chromatography (TLC) and gas chromatography electron capture detector (GC-ECD).