M. Tech. / Background: Waterborne disease contributes significantly to the total global disease burden. Populations in rural areas of South Africa depend on untreated waters for consumption and sanitation. Contamination of public water supplies by harmful bacteria such as pathogenic E. coli poses a major risk for public health. Ingestion of these pathogenic microorganisms present in the contaminated and untreated waters could cause infection, leading to systemic inflammatory responses manifested by the production of various proinflammatory cytokines. To date, there is no human system test available to detect whether water, following ingestion, would cause disease (i.e. whether the water is infectious). The current water testing methods only test for the presence of indicator organisms, such as faecal coliforms, total coliforms, and Escherichia coli. A reliable in-vitro bioassay that could assess whether the water would cause an inflammatory response was investigated in this study. Objectives: Pro-inflammatory cytokines and whole-blood have been used in similar studies to detect the inflammatory responses following exposure to specific stimulants such as dust, lipopolysaccharide (LPS), E. coli and various others. It has been reported that larger numbers of these contaminants induced higher levels of pro-inflammatory cytokine expression. This implies that the pro-inflammatory cytokine expression could be used as a marker of infection since, inflammation occurs in response to infection. Successful infection is thus necessary for inflammation to occur, and high levels of pro-inflammatory cytokine expression confirm that infection has occurred. Thus if pro-inflammatory cytokines could serve as indicators for infection, these cytokines could be used as indicators for bacterial pathogenicity of water.

M.Sc. / The prevalence of natural organic matter (NOM) in water remains a huge challenge for water treatment companies and municipalities. NOM, however, is not a stand-alone problem as it affects water quality in many ways. NOM is largely responsible for the formation of disinfection by-products (DBPs) via its interaction with disinfectants during water disinfection. It is implicated for the undesirable colour, taste and odour of water and NOM even inhibits precipitation precursors which form the backbone of drinking water treatment. There is therefore no question that NOM, which is either a precursor to or direct cause of the problems highlighted above, should be considered as one of the critical design parameters to be considered for drinking water treatment. In our laboratories, research that involves the use of cyclodextrin (CD) polyurethanes for the removal of organic pollutants from water has been extensively investigated, and the CD polyurethanes have demonstrated the ability to effectively remove the organic species from water at low (ppb) concentrations. CDs, which are cyclic oligomers consisting of glucopyranosyl units linked together through α-1,4 glycosidic linkages, behave like molecular hosts. They are capable of forming inclusion complexes with guest molecules and hence can be utilised for the removal of organic contaminants from water. Their solubility in water, however, limits their application in the removal of organic contaminants from water. This limitation is easily resolved by polymerising the CDs into water-insoluble polymers with bifunctional linkers such as hexamethylene diisocyanate (HMDI). In this study, CD polyurethanes and ion exchange resins were used for the removal of NOM and possibly its DBPs from water. This study first investigated the local NOM source to establish its type and character, hence the study involved the characterization of the bulk water samples and fractionating the NOM using ion exchange resins for further characterization. The water samples were found to consist mainly of humic substances in the form of hydrophobic NOM, with the hydrophilic basic (HpiB) fraction being the most abundant NOM fraction in all samples. Trihalomethanes (THMs) were used as a representative of DBPs in this vii study. THMs formation studies of the individual NOM fractions indicated that all six NOM fractions were found to form THMs but in varying proportions. The HpiB fraction was found to be the most reactive precursor fraction for THMs formation. The ozonation process was also independently evaluated for its ability to remove the NOM fractions from water. Ozonation was found not to be very effective at NOM removal since it only resulted in a 12% overall decrease of the NOM in the water samples. The CD polyurethanes, when used alone, were also not very effective at removing the NOM fractions from water (maximum of 33% NOM removal). On the other hand, the combination method (i.e. the use of CD polyurethanes and ozonation for NOM removal) resulted in a good capability of the CD polyurethanes at decreasing some NOM fractions in water as evidenced by a 73% and 88% decrease of the HpiB and hydrophilic acid (HpiA) fractions, respectively. The 73% reduction of the HpiB fraction demonstrates a great success of the combination approach employed herein, as this study reports this fraction as the most reactive precursor fraction for THM formation.

M.Tech. (Biotechnology) / Please refer to full text to view abstract

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[pt] O impacto ambiental causado pela queima de combustíveis por veículos automotivos, atualmente, atua como restrição na especificação de parâmetros de projetos de motores em vários Países. O uso de etanol hidratado no Brasil criou a necessidade de determinar o comportamento das emissões de poluentes sob condições típicas de projeto, auxiliando o estabelecimento de uma política de controle de poluição. Nesta pesquisa, as emissões de um motor monocilíndrico foram medidas, basicamente óxido nítrico, monóxido de carbono e aldeídos. Usou-se gasolina comercial e etanol hidratado como combustíveis. A influencia da taxa de compressão, da relação combustível-ar e da quantidade de água no etanol, na performance do motor e no comportamento das emissões de acetaldeído foi desenvolvido para cálculos em computador digital, concordando bem com os resultados experimentais. Usando este modelo, valores de emissões de poluentes por motores de combustão interna, usando etanol hidratado foram calculados, mostrando uma tendência bem definida que pode ser útil para políticas de controle de poluição. / [en] The impact on the environment caused by the burning of fuels from automotive vehicles, nowadays, acts as a restriction on the specification of the engine designe parameters in several countries. The use of ethyl acohol-ewater blends in Brazil has created the necessity of determining the pollutant emission pattern under typical design operating conditions aiming the stablishment of a pollution control policy. In this research, emissions from a monocylinder engine were measured, bassicaly ntric oxide, carbon monoxide and aldehydes. The engine was fueled with commercial gasoline and ethyl alcohol-water blends. The influence of the compression ratio, air-fuel ratio and water content of blens, on the perfomance of the engine and pollutant pattern was studied. A Kinetic model, taking into account CO and NO formations mechanisms, as well as acetaldehyde emissions was developed for digital computer calculations, matching the experimental results to the calculated ones. The, using this model, values of the pollutant emissions from internal combustion engines, fueled with ethyl alcohol-water blends were calculated showing a very well defined trend, which can be useful for pollution control policies.

[pt] O presente trabalho investigou a dispersão de poluentes na forma de gases e material particulando provinientes de uma chaminé sujeita a ventos transversais. Os campos de concentração, velocidade e temperatura foram determinados através numérica das equação de conservação de massa, quantidade de movimento linear, energia e espécie química nas suas formas tridimensionais para regime permanente. Os efeitos da força de empuxo devido aos gradientes de temperatura foram considerados através da solução simultânea das equações de quantidade de movimento linear e energia.O escoamento foi considerado parabólico na direção principal e foi utilizado o modelo k--e de turbulência. O modelo desenvolvido neste trabalho, incorporou os efeitos produzidos pelo campo gravitacional para o caso de material particulado. Os campos de concentração obtidos, apresentam boa concordância com os resultados do Modelo de Pluma Gaussiana. Um estudo paramétrico revelou a influência das razões de velocidade e temperatura entre gases da emissão e o vento sobre os campos de concentração, velocidade e temperatura. A velocidade de deposição das partículas também mostrou influência sobre o campo de concentração. / [en] The present work numerically investigated the dispersion of pollutants in the form of gases or particulate matter emitted from a single stack subjected to horizontal winds. The concentration, velocity and temperature fields were determinate by the solution of the equations governing the conservation of mass, linear momentum, energy and chemical species for steady-state, three-dimensional flow conditions. Buoyancy effects due to temperature gradients were considered by the simultaneous solution of the linear momentum and energy equations. The flow was considered to be the parabolic in the principal direction and the K-E Turbulence Model was employed. The model developed accounted for gravitational setting of particulate matter. The predictions for the concentration field the influence of the wind-to-discharge velocity and temperature ratios on the concentration, velocity and temperature fields. The particle settling velocity was also shown to influence the concentration field.

>Magister Scientiae - MSc / The global lack of clean water for human sanitation and other purposes has become an emerging dilemma for human beings. The presence of organic pollutants in wastewater produced by textile industries, leather manufacturing and chemical industries is an alarming matter for a safe environment and human health. For the last decades, conventional methods have been applied for the purification of water but due to industrialization these methods fall short. Advanced oxidation processes and their reliable application in degradation of many contaminants have been reported as a potential method to reduce and/or alleviate this problem. Lately, it has been assumed that incorporation of some metal nanoparticles such as magnetite nanoparticles as photocatalyst for Fenton reaction could improve the degradation efficiency of contaminants. Core/shell nanoparticles, are extensively studied because of their wide applications in the biomedical, drug delivery, electronics fields and water treatment. The current study is centred on the synthesis of silver-doped Fe₃O₄/SiO₂/TiO₂ photocatalyst. Magnetically separable Fe₃O₄/SiO₂/TiO₂ composite with core–shell structure were synthesized by the deposition of uniform anatase TiO₂ NPs on Fe₃O₄/SiO₂ by using titanium butoxide (TBOT) as titanium source. Then, the silver is doped on TiO₂ layer by hydrothermal method. Integration of magnetic nanoparticles was suggested to avoid the post separation difficulties associated with the powder form of the TiO₂ catalyst, increase of the surface area and adsorption properties. Lastly and most importantly magnetic nanoparticles upsurge the production of hydroxyl groups or reduced charge recombination. The a synthesized catalysts were characterized using Transmission Electron Microscopy, X-ray Diffraction; Infra-red Spectroscopy, Scanning Electron Microscope and Energy Dispersive Spectroscopy. Other characterization techniques includeVibrating Sample Magnetometry, Brunauer Emmett Teller analysis and Thermogravimetric analysis. The average size of the particles size is 72 nm. Furthermore the photocatalytic performances of the magnetic catalysts were assessed in comparison with that commercial titanium dioxide for the degradation of methylene blue using photochemical reactor under ultra violet light. The results showed that the photocatalytic activity was enhanced using Fe₃O₄/SiO₂/TiO₂ and Ag-Fe₃O₄/SiO₂/TiO₂ compared with that for Fe₃O₄, commercial titanium dioxide powder.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants which are of concern due to their potential human toxicity. They are formed during numerous combustion processes, including biomass burning and diesel vehicular emissions, which are of relevance in developing countries. A novel analytical screening method for atmospheric polycyclic aromatic hydrocarbons (PAHs) was developed in this study based on laser induced fluorescence (LIF) of samples on quartz multi-channel polydimethylsiloxane (PDMS) traps. A tunable dye laser with a frequency doubling crystal provided the excitation radiation, and a double monochromator with a photomultiplier tube detected emitted fluorescence. The method allowed for the rapid (<5 min), cost effective analysis of samples. Those yielding interesting results could be further analysed by direct thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS, with limits of detection of ~0.3 ng.m-3), as photodegradation was minimal (<10% over 5 min irradiation). Without any signal optimization, a LIF detection limit of ~1 ìg.m-3 was established for naphthalene using a diffusion tube (diffusion rate of 2 ng.s-1) and 292 nm excitation. Gas standards which facilitated the uniform distribution of analyte across each of the 22 PDMS tubes were provided by easily constructed diffusion tubes for naphthalene and by a gas chromatographic fraction collection method for the less volatile target PAHs. The methods developed were successfully tested in a number of applications which are of relevance to southern Africa, as emissions from sugar cane burning, household fires, diesel vehicles and industries were monitored. The LIF method allowed for the differentiation between impacted and non-impacted industrial sites, and the importance of naphthalene as an indicator for atmospheric PAHs was verified in that this PAH was the most abundant in the various applications which were investigated. The multi-channel silicone rubber traps were also evaluated theoretically and practically in the denuder configuration, in order to monitor PAHs in both the gas and particle phases, which is important in terms of human health effects. The novel LIF method developed in this study has the potential to serve as a screening tool to avoid the comprehensive and costly analysis of samples which do not contain appreciable levels of PAHs. The experimental procedure is simple and rapid, with acceptably low limits of detection, even with the initial, unoptimized optical arrangement and without extensive time-averaging. LIF also provides selectivity without the need for sample clean-up and separation processes. The LIF method could be further optimized by improving the laser energy stability, as well as by the investigation of possible time resolution techniques. As equipment cost considerations were important, it is possible that the LIF screening method could find application in a centralized environmental laboratory for the southern African region. This would facilitate the widespread monitoring of atmospheric PAHs in a cost effective manner. / Thesis (PhD)--University of Pretoria, 2010. / Chemistry / unrestricted

The wood-degrading basidiomycete, Phanerochaete chrysosporium, has been studied as a model organism in elucidating the mechanisms and pathways enabling this white-rot fungus to degrade recalcitrant lignin. These same mechanisms are implicated in the mineralisation of environmentally persistent, toxic phenolic chemicals. For this reason, P. chrysosporium has been exploited in a number of environmentally sound technologies, including the degradation of the indigestible lignin component in agricultural waste for the generation of digestible animal feedstocks or high sugar content raw materials for ethanol production; brightening processes in the pulp and paper industry; the detoxification and decolourisation of industrial effluents; and the bioremediation of hazardous waste sites. The improvement of these technologies is dependant on ongoing research involving strain selection, strain development using genetic engineering approaches and process development. Strain improvement using non-recombinant methods is beneficial in that it does not limit the inherent robustness observed amongst natural variants. In this research, through a breeding programme, ten P.chrysosporium sibling strains were screened for variable ligninase activities and pollutant degradation capabilities in order to further describe previously identified differences between these organisms. A conventional stationary liquid culture technique was effectively miniaturised from 10 ml flask cultures to a 96-well microtitre plate format, for the assessment of multigenic traits amongst sibling strains. Using the 96-well microtitre plate method, the relationships between P. chrysosporium growth kinetics, peroxidase production, pollutant sensitivity and pollutant degradation was explored. Significant correlations were primarily associated with P. chrysosporium growth [P < 0.05]. Percentage p-cresol removal and tannic acid tolerance were both correlated with a shorter lag phase in growth [tannic acid: r = 0.7698, P < 0.05; p-cresol: r = 0.7584, P < 0.05] and lower stationary phase biomass levels [tannic acid: r = 0.8177, P < 0.05; p-cresol: r = 0.7803, P < 0.05]. A significant correlation (linear relationship) was also detected between percentage Poly-R478 decolourisation and time of onset of MnP [r = 0.9689, P < 0.001]. No correlation was observed between dye decolourisation, p-cresol degradation, lignin degradation and lignin peroxidase (LiP) or manganese peroxidase (MnP) activities [P > 0.05]. These results imply that differences in the biosynthetic pathways for biomass accumulation in sibling strains play a significant role in the intraspecific variation observed in pollutant sensitivity, pollutant degradation, and enzyme production. Categorical analysis of intraspecific differences was assessed according to four criterions. These included growth, extracellular peroxidase activities, tolerance to toxic pollutants and the biodegradation of model pollutants. Sibling strains showing the most variable responses in three or more of the selective criterion were recommended for further studies. These strains include P. chrysosporium ME446, BS 2.52, BS 13, BS 17, BS 18, and BS 24. Interestingly, BS 2.52 (a dikaryotic strain generating from the crossing of two haploid progeny) showed significantly lower degradation capabilities than the wildtype parent strain ME446. The inherited variability observed between sibling strains is to be further explored through proteome and transcriptome analysis and genetic linkage studies aimed at describing the mechanisms or pathways conferring tolerance to or degradation of environmental pollutants. In examining fewer organisms at this next level, the number of replicates examined can be increased and thus the power of detection of experimental procedures improved, enabling the detection of multigenic traits amongst genetically related organisms. Growth was shown to play a significant role in the intraspecific differences detected in pollutant sensitivity and degradation between sibling strains. Little is known about the mechanism of growth and differentiation, or the role of differentiation in regulating the lignolytic activity in this organism. The membrane gradostat bioreactor and a unique plug-flow membrane bioreactor were evaluated as novel tools with which to further explore the relationship between secondary metabolism, pollutant degradation and biofilm development in sibling strains. High yield MnP production at levels as high as 1478.8 U.l-1 was achieved using a laboratory scale membrane gradostat bioreactor. Furthermore, extensive mycelial differentiation and tissue formation are reported for P. chrysosporium in both the membrane gradostat bioreactor and plug-flow membrane bioreactor. Intraspecific differences in the extent of this differentiation were observed in strains ME446, BS 13, BS 17 and BS 26 cultured using the membrane gradostat bioreactor, highlighting the potential of these techniques as a platform for future strain improvement strategies.

Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.