Diarrhoea caused by strains of Escherichia coli is recognised as a significant cause of morbidity and mortality world-wide. Efficient isolation of these bacteria from water is necessary for effective management of available water sources and for the control and prevention of waterborne disease. Methods that are currently available do not allow for the efficient isolation of the diarrhoeagenic E. coli. Therefore, the aims of the current study were: to determine the efficacy of Chromocult® Coliformen Agar for identification of diarrhoeagenic E. coli; correlate virulence gene carriage with biochemical profiles of diarrhoeagenic E. coli; and develop an enrichment method for the selective isolation of diarrhoeagenic E. coli. Chromocult® Coliformen Agar proved unreliable for identifying diarrhoeagenic E. coli – 30% of strains would have been incorrectly identified using this medium. The carriage of pathotype-specific virulence genes by enteroaggregative E. coli strains was associated with the production of ornithine decarboxylase and the fermentation of sucrose. The enrichment procedure detailed in this study was an improvement upon an existing method. In addition, the improved method proved effective for the isolation of five diarrhoeagenic E. coli pathotypes. In contrast, the original procedure was only shown to be effective when isolating enterohaemorrhagic E. coli. The improved method was applied on environmental water and two samples yielded colonies positive for genes carried by enteropathogenic E. coli and enterotoxigenic E. coli, respectively. As confirmation of these results PCR products were sequenced and corresponded to published sequences of the virulence genes. / Ms. P.S. Kay Dr. T. G. Barnard Prof. P. Jagals

D.Phil. (Chemistry) / Please refer to full text to view abstract

D.Phil. (Chemistry) / The application of dendritic-based materials has attracted great interest. For the first time this research has investigated the feasibility of poly (propyleneimine) (PPI) dendrimers and hyperbranched polyethyleneimine (HPEI) in combination with beta-cyclodextrin (β-CD) embedded in polysulfone (PSf) membrane for water treatment. The advantage of embedding these conjugates (β-CD-PPI and β-CD-HPEI) in PSf membranes is the presence of numerous nanocavities which can act as water channels allowing easy water passage through the membrane improving water permeability. Secondly, the presence of functional groups such as –OH and –NH greatly improves hydrophilicity of membranes. Commercial polysulfone (PSf) ultrafiltration membranes were crosslinked with β-cyclodextrin-poly (propyleneimine) (β-CD-PPI) and β-cyclodextrin-hyperbranched polyethyleneimine (β-CD-HPEI) using trimesoyl chloride (TMC) by interfacial polymerisation. These membranes were used in the rejection of Aldrich humic acid (molecular weight: 4.1 kDa) from synthetic water samples prepared in the laboratory. Moreover, β-cyclodextrin-poly (propyleneimine) (β-CD-PPI) was used as a host for the preparation of Fe/Ni nanoparticles. The new membranes were synthesised by crosslinking β-CD-PPI with trimesoyl chloride and subsequently loading Fe/Ni nanoparticles and this was supported on a commercial polysulphone (PSf) layer for the degradation of 2,4,6-trichlorophenol (2,4,6-TCP). The membrane surfaces were characterised using Fourier transform infrared/attenuated total reflectance (FT-IR/ATR) spectroscopy , scanning electron microscopy (SEM), atomic force microscopy (AFM), high resolution transmission electron microscopy (HR-TEM), water-contact angle, and water-intake capacity...

D.Phil. (Chemistry) / Please refer to full text to view abstract

Background: The cholera epidemic experienced in the province of KwaZulu-¬Natal between August 2000 and July 2001 resulted in 105, 000 reported cases of cholera and 220 human deaths. Objectives: The primary objective of this study was to make comparison between districts with cholera and districts with no cholera in rural KwaZulu-Natal on the basis of known risk factors for cholera and diarrhoeal diseases. Comparison was made with regards to key factors such as the prevalence of diarrhoeal diseases, the provision of safe water supply, ownership and effective utilization of sanitary facilities, knowledge and practice on water purification and prevention of cholera. Methods and materials: This was a descriptive, cross- sectional, ecological and comparative study among households of KwaZulu-Natal. The communities were stratified into two groups. One group had cholera (Group 1) and the other group (Group 2) had no cholera. Thirty communities were selected by systematic random selection from each of the two groups. From each community, eligible households were selected using simple random sampling technique. Trained field workers used a pre-tested questionnaire to collect data during the months of November and December 2001. Statistical procedures such as two-sample tests on means and proportions, Pearson's chi-square tests of association, odds ratios, binary logistic regression analysis, sensitivity tests, specificity tests and ROC (receiver of characteristics) analysis were used for data analysis. Results: A total of 1420 households from both groups were included in the study. The response rate for Group 1 and Group 2 was 84%,92%. Female respondents (70%) predominated male respondents (30%). Tap water supply was less common in Group 1 (54%) than in Group 2 (72%), (p< 0.05). A higher percentage of households in Group 1 (27%) used dam or river water compared to Group 2 (20%) (p<0.05). Household knowledge on water purification by boiling was (71%) in Group 1 and (87%) (p<0.05) in Group 2.38% of households in Group 1 practiced water purification by using disinfectant JIK. The corresponding figure for Group 2 was 50% (p<0.05). Groups 1 and 2 were similar with respect to ownership of toilets (84% and 85% respectively). Groups 1 and 2 showed a marked difference with respect to utilization of toilet facilities by all family members (70% and 89% respectively). The prevalence of diarrhoeal diseases for Group 1 was higher (14.3%) (p<0.05) than the Group 2 was (11.1%). Factors found to be helpful for protection against diarrhoeal diseases were the boiling of water (OR=0.41, 95% CI, 0.19-0.90) and the use of disinfectant JIK (OR=0.45, 95% CI, 0.19-0.94). The study also showed that the use of dam or river water was significantly associated with diarrhoeal diseases (OR=2.92, 95% CI, 1.06-7.80). Conclusion: The results showed that there was significant difference between the two groups of households in regards to basic provision of safe water, knowledge and practice of good hygiene, ownership and effective utilization of sanitary facilities. Findings from this study could be useful as baseline information for future planning, monitoring and evaluation of ongoing programmes. / Dissertation (MSc(Epidemiology))--University of Pretoria, 2005. / School of Health Systems and Public Health (SHSPH) / unrestricted

Inadequate sanitation is one of the leading causes of water pollution and consequently illness in many underdeveloped countries, including South Africa and, specifically, the Eastern Cape Province, where cholera has become endemic. As modern wastewater treatment processes are often energy intensive and expensive, they are not suitable for use in these areas. There is thus a need to develop more sustainable wastewater treatment technologies for application in smaller communities. The integrated algal ponding system (IAPS) was identified as a possible solution to this wastewater management problem and was investigated for adaptation to local conditions, at the Rhodes University Environmental Experimental Field Station in Grahamstown, South Africa. The system was monitored over a period of nine years, with various configuration adjustments of the high rate algal pond (HRAP) unit operation investigated. Under standard operating conditions, the system was able to achieve levels of nutrient and organic removal comparable with conventional wastewater treatment works. The mean nitrate level achieved in the effluent was below the 15mg.l-1 South African discharge standard, however, nitrate removal in the IAPS was found to be inconsistent. Although the system was unable to sustain chemical oxygen demand (COD) removal to below the 75mg.l-1 South African discharge standard, a removal rate of 87% was recorded, with the residual COD remaining in the form of algal biomass. Previous studies in the Eastern Cape Province have shown that few small wastewater treatment works produce effluent that meets the microbial count specification. Therefore, in addition to the collation of IAPS data from the entire nine year monitoring period, this study also investigated the use of the HRAP as an independent unit operation for disinfection of effluent from small sewage plants. It was demonstrated that the independent high rate algal pond (IHRAP) as a free standing unit operation could consistently produce water with Escherichia coli counts of 0cfu.100ml-1. The observed effect was related to a number of possible conditions prevailing in the system, including elevated pH, sunlight and dissolved oxygen. It was also found that the IHRAP greatly enhanced the nutrient removal capabilities of the conventional IAPS, making it possible to reliably and consistently maintain phosphate and ammonium levels in the final effluent to below 5mg.l-1 and 2mg.l-1 respectively (South African discharge standards are 10mg.l-1 and 3mg.l-1 in each case). The quality of the final effluent produced by the optimisation of the IAPS would allow it to be used for irrigation, thereby providing an alternative water source in water stressed areas. The system also proved to be exceptionally robust and data collected during periods of intensive and low management regimes were broadly comparable. Results of the 9 year study have demonstrated reliable performance of the IAPS and its use an appropriate, sustainable wastewater treatment option for small communities.

The research reported here investigated the use of a Baffle Reactor in order to study aspects of the biological sulfur cycle, where a floating sulfur biofilm formation occurs and where complex organic compounds provide electron donor sources. The development of a laboratory-scale Baffle Reactor model system satisfied the requirements for sulfate reducing bacterial biomass growth and sulfur biofilm formation. Since relatively little is known about the microbial ecology of floating sulfur biofilm systems, this study was undertaken to describe the sulfate reducing sludge population of the system together with its performance. A combination of culture- and molecular-based techniques were applied in this study in order to investigate the microbial ecology of the sulfate-reducing bacteria component of the system. These techniques enabled the identification and the analysis of the distribution of different sulfate reducing bacterial strains found within the sludge bioreactors. Strains isolated from the sludge were characterised based on culture appearance, gram staining and scanning electron microscopy morphology. Molecular methods based on the PCR-amplified 16S rRNA including denaturing gradient gel electrophoresis were employed in order to characterise sulfate-reducing bacteria within the reactors. Three novel Gram negative sulfate-reducing bacteria strains were isolated from the sludge population. Strains isolated were tentatively named Desulfomonas rhodensis, Desulfomonas makanaiensis, and Clostridium sulforhodensis. Results obtained from the Baffle Reactor showed that three dominant species were isolated from the DNA extracted from the whole bacterial population by peR. Three of these were similar to those mentioned above. The presence of these three novel unidentified species suggest that there are a range of other novel organisms involved in sulfate reduction processes.

Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.

The removal of lead from aqueous solution and lead-acid battery manufacturing waste-water by the non-viable biomass of the water fern Azolla filiculoides was investigated in both batch and column reactors. The maximum lead uptake by the Azolla biomass at a pH value of approximately 5, was found to be 100 mg lead/g biomass from aqueous solution. Lead removal varied from 30% of the initial lead concentration at pH 1.5 to approximately 95% at pH values of 3.5 and 5.6. Lead removal from aqueous solution decreased to 30% of the initial lead concentration if the lead concentration was initially over 400 mg/l. At initial lead concentrations of less than 400 mg/l, percentage lead removal was found to be over 90% of the initial lead concentration. Lead removal remained at approximately 90% between 10°C and 50°C. Biomass concentration (4-8 mg/l) had little effect on lead removal. The presence of iron (Fe) and lead, copper (Cu) and lead or all three metal ions in solution at varying ratios to each other did not appear to have any significant effect on lead removal. Percentage lead, copper and iron removal from aqueous solution was 80-95, 45-50 and 65-75% respectively for the different multiple-metal solutions studied. No break-through points were observed for lead removal from aqueous solutions in column reactors, with initial lead concentrations of less than 100 mg/l at varying flow rates of 2, 5 and 10 ml/min. This suggested that flow rate, and therefore retention time, had little effect on percentage lead removal from aqueous solution, which was more that 95%, at low initial lead concentrations (less than 100 mg/l). At initial lead concentrations of 200 mg/l or more, an increase in flow rate, which equates to a decrease in column retention time, resulted in break-through points occurring earlier in the column run. Percentage lead removal values, from lead-acid battery efiluent in column systems, of over 95% were achieved. Desorption of approximately 30% and 40% of bound lead was achieved, with 0.5 M HNO₃ in a volume of 50 ml, from two lead-acid battery. Repeated adsorption and desorption of lead by the Azalia biomass over 10 cycles did not result in any decrease in the percentage lead removal from effluent, which strongly suggested that the Azalla biomass could be re-used a number of times without deterioration in its physical integrity, or lead removal capacity. No evidence of deterioration in the Azolla biomass's physical integrity after 10 successive adsorption and desorption procedures was observed using scanning electron microscopy. The Azolla filiculoides biomass was, therefore, found to be able to effectively remove lead from aqueous solution and lead-acid battery effluent repeatedly, with no observed reduction in it's uptake capacity or physical integrity.

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