Thesis (DTech (Biomedical Technology))--Cape Peninsula University of Technology, 2008. / Bibliography: leaves 208-236. Also available online.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Chemical Engineering in the Faculty of Engineering at the Cape Peninsula University of Technology 2014 / Environmental legislation focusing on wastewater disposal in industries that utilise cyanide and/or cyanide-related compounds has become increasingly stringent worldwide, with many companies that utilise cyanide products required to abide by the Cyanide International Code associated with the approval of process certifications and management of industries which utilise cyanide. This code enforces the treatment or recycling of cyanide-contaminated wastewater. Industries such as those involved in mineral processing, photo finishing, metal plating, coal processing, synthetic fibre production, and extraction of precious metals, that is, gold and silver, contribute significantly to cyanide contamination in the environment through wastewater. As fresh water reserves throughout the world are low, cyanide contamination in water reserves threatens not only the economy, but also endangers the lives of living organisms that feed from these sources, including humans. In the mining industry, dilute cyanide solutions are utilised for the recovery of base (e.g. Cu, Zn, Ni, etc.) and precious metals (e.g. Au, Ag, etc.). However, for technical reasons, the water utilised for these processes cannot be recycled upstream of the mineral bioleaching circuit as the microorganisms employed in mineral bioleaching are sensitive to cyanide and its complexes, and thus the presence of such compounds would inhibit microbial activity, resulting in poor mineral oxidation. The inability to recycle the water has negative implications for water conservation and re-use, especially in arid regions. A number of treatment methods have been developed to remediate cyanide containing wastewaters. However, these chemical and physical methods are capital intensive and produce excess sludge which requires additional treatment. Furthermore, the by-products that are produced through these methods are hazardous. Therefore, there is a need for the development of alternative methods that are robust and economically viable for the bioremediation of cyanide-contaminated wastewater. Biological treatment of free cyanide in industrial wastewaters has been proved a viable and robust method for treatment of wastewaters containing cyanide. Several bacterial species, including Bacillus sp., can degrade cyanide to less toxic products, as these microorganisms are able to use the cyanide as a nitrogen source, producing ammonia and carbon dioxide. These bacterial species secrete enzymes that catalyse the degradation of cyanide into several end-products. The end-products of biodegradation can then be utilised by the microorganisms as nutrient sources. This study focused on the isolation and identification of bacterial species in wastewater containing elevated concentrations of cyanide, and the assessment of the cyanide biodegradation ability of the isolates. Thirteen bacterial isolates were isolated from electroplating wastewater by suppressing the growth of fungal organisms and these species were identified as species belonging to the Bacillus genus using the 16S rDNA gene. A mixed culture of the isolates was cultured in nutrient broth for 48 hours at 37°C, to which FCN as KCN was added to evaluate the species‟ ability to tolerate and biodegrade cyanide in batch bioreactors. Subsequently, cultures were supplemented solely with agro-waste extracts, that is, Ananas comosus extract (1% v/v), Beta vulgaris extract (1% v/v), Ipomea batatas extract (1% v/v), spent brewer‟s yeast (1% v/v) and whey (0.5% w/v), as the primary carbon sources. Owing to the formation of high ammonium concentration from the cyanide biodegradation process, the nitrification and aerobic denitrification ability of the isolates, classified as cyanide-degrading bacteria (CDB) was evaluated in a batch and pneumatic bioreactor in comparison with ammonia-oxidising bacteria (AOB). Furthermore, the effects of F-CN on the nitrification and aerobic denitrification was evaluated assess the impact of F-CN presence on nitrification. Additionally, optimisation of culture conditions with reference to temperature, pH and substrate concentration was evaluated using response surface methodology. Using the optimised data, a continuous biodegradation process was carried out in a dual-stage packed- bed reactor combined with a pneumatic bioreactor for the biodegradation of F-CN and subsequent nitrification and aerobic denitrification of the formed ammonium and nitrates. The isolated bacterial species were found to be gram positive and were able to produce endospores that were centrally located; using the 16S rDNA gene, the species were found to belong to the Bacillus genus. The species were able to degrade high cyanide concentration in nutrient broth with degradation efficiencies of 87.6%, 65.4%, 57.0% and 43.6% from 100 mg F-CN/L, 200 mg F-CN/L, 300 mg F-CN/L, 400 mg F-CN/L and 500 mg F-CN/L respectively over a period of 8 days. Additionally, the isolates were able to degrade cyanide in an agro-waste supported medium, especially in a medium that was supplemented with whey which achieved a degradation efficiency of 90% and 60% from 200 mg F-CN/L and 400 mg F-CN/L, respectively over a period of 5 days. The nitrification ability of the isolates was evaluated and the removal of NH4 +/NO3 - by the CDB and AOB in both shake flasks and pneumatic bioreactor was determined to be pH dependent. The maximum NH4 +/NO3 - removal evaluated over a period of 8 days for CDB and 15 days for AOB, observed at pH 7.7 in shake flasks, was 75% and 88%, respectively, in the absence of F-CN. Similarly, the removal of NH4 +/NO3 - in a pneumatic bioreactor was found to be 97.31% for CDB and 92% for AOB, thus demonstrating the importance of aeration in the designed process. The nitrification by CDB was not inhibited by cyanide loading up to a concentration of 8 mg FCN/ L, while the AOB were inhibited at cyanide loading concentration of 1 mg F-CN/L. The CDB removed the NH4 +/NO3 - in PBSs operated in a fed-batch mode, obtaining efficiencies >99% (NH4 +) and 76 to 98% (NO3 -) in repeated cycles (n = 3) under F-CN (≤8 mg F-CN/L). The input variables, that is, pH, temperature and whey-waste concentration, were optimised using a numerical optimisation technique where the optimum conditions were found to be: pH 9.88, temperature 33.60 °C and whey-waste concentration 14.27 g/L, under which 206.53 mg CN-/L in 96 h can be biodegraded by the microbial species from an initial cyanide concentration of 500 mg F-CN/L. Furthermore, using the optimised data, cyanide biodegradation in a continuous mode was evaluated in a dual-stage packed-bed bioreactor connected in series to a pneumatic bioreactor system used for simultaneous nitrification including aerobic denitrification. The whey-supported Bacillus sp. culture was not inhibited by the free cyanide concentration of up to 500 mg F-CN/L, with an overall degradation efficiency of ≥99% with subsequent nitrification and aerobic denitrification of the formed ammoniu and nitrates over a period of 80 days.

Eutrophication is a nutrient enrichment of dams and lakes. Increased eutrophication in dams results in blooms of cyanobacteria. Cyanobacteria are troublesome as they form massive surface scums, impart taste and odour to the water. Some strains of cyanobacteria such as Microcystis aeruginosa are dangerous to humans and animals. They produce toxins that can kill animals drinking the contaminated water and have also been implicated in human illnesses. The study investigated the effectiveness of techniques deployed in controlling cyanobacterial growth in Rietvlei, Roodeplaat and Hartbeespoort Dams. This was done by interpreting data from April 2010 to March 2012. The conditions in the three dams show that Microcystis produced toxins in the summer season and all the variables analysed were favourable for the production of toxins. The methods deployed to rehabilitate the dams do not completely solve the problems of toxins experienced by the dams. / Environmental Sciences / M. Sc. (Environmental Management)

Eutrophication is a nutrient enrichment of dams and lakes. Increased eutrophication in dams results in blooms of cyanobacteria. Cyanobacteria are troublesome as they form massive surface scums, impart taste and odour to the water. Some strains of cyanobacteria such as Microcystis aeruginosa are dangerous to humans and animals. They produce toxins that can kill animals drinking the contaminated water and have also been implicated in human illnesses. The study investigated the effectiveness of techniques deployed in controlling cyanobacterial growth in Rietvlei, Roodeplaat and Hartbeespoort Dams. This was done by interpreting data from April 2010 to March 2012. The conditions in the three dams show that Microcystis produced toxins in the summer season and all the variables analysed were favourable for the production of toxins. The methods deployed to rehabilitate the dams do not completely solve the problems of toxins experienced by the dams. / Environmental Sciences / M. Sc. (Environmental Management)

Thesis submitted in fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology 2008 / The quality of South Africa’s water sources is fast deteriorating due to an influx of pollutants from industrial and agricultural areas. In addition, urbanisation has led to the establishment of informal settlements along river systems. This study focuses on the importance of maintaining water quality and the management of water resources in order to ensure its sustainability in South Africa. The primary aim of this study was to determine the extent of bacterial contamination in three rivers namely the Berg-, Plankenburg- and Diep Rivers in the Western Cape, South Africa and to investigate the application of a bioremediation system as a possible treatment technology. Several aspects contributing to the contamination were addressed and different approaches were studied and reviewed. In all three rivers, four sampling sites were identified, which were sampled over a period of 9 to 12 months. Contamination levels for the three rivers were evaluated by applying various enumeration techniques, which could provide an accurate indication of the planktonic bacterial pollution load in the river systems. The Most Probable Number (MPN) technique was used to determine the level of faecal coliforms and E. coli. The highest MPN, faecal coliform and E. coli counts of 3.5 x 107 micro-organisms/100 m , 3.5 x 107 micro-organisms/100 m and 1.7 x 107 micro-organisms/100 m , respectively, were recorded at Site B2 in week 37 in the Berg River. Results showed that in all the river water sampled and evaluated, the total MPN count mostly exceeded the maximum limit of 2000 micro-organisms/100 m (SABS, 1984) stipulated for river water throughout the study period. The heterotrophic plate count (HPC) method was used to determine the number of culturable micro-organisms in planktonic samples, while the flow cytometry (FCM) and epifluorescence microscopy (EM) with different fluorochromes (Acridine orange and BacLight™ Live/Dead stain) were employed to evaluate total bacterial counts in planktonic (water) samples. The highest HPC at the various sites sampled was 1.04 x 106 micro-organisms/m (Berg River, Site B2), 7.9 x 104 micro-organisms/m (Plankenbrug River, Site A) and 1.7 x 105 micro-organisms/m (Diep River, Site B). Total cell counts as high as 3.7 x 107 micro-organism/m (Berg River, Site B2), 5.5 x 108 micro-organism/m (Plankenburg River, Site D) and 2.5 x 109 micro-organisms/m (Diep River, Site B) were obtained by the FCM technique, which were significantly (p < 0.05) higher than the total counts obtained by epifluorescence microscopy. The results thus show that the FCM technique was the most reliable method for determining the total cell count in river water samples. This technique makes use of computer software whereas epifluorescence microscopy involves manual counting which may lead to human error. In addition, the impact of residential, agricultural and industrial areas situated along these rivers was also investigated. Even though exact point sources of pollution could not be determined, it was found that all the sources, such as the storm water drainage pipes, the industrial as well as the agricultural areas, could contribute to increased MPN, heterotrophic and total bacterial counts. This study also aimed at investigating and comparing the microbial contamination levels at various sites in the Plankenburg and Diep Rivers in the Western Cape, South Africa. Sampling of sites along the Plankenburg River started in June 2004 and continued for a period of one year until June 2005. Sampling of the Diep River sites started in March 2005 and continued for a period of nine months until November 2005. Faecal coliform (FC) and E. coli (EC) counts were determined by means of the Most Probable Number technique, the number of culturable cells were determined using the heterotrophic plate count (HPC) technique and total microbial counts were evaluated by Flow cytometric analysis (FCM). The highest microbial counts for the Plankenburg River were observed at site B where the highest MPN, FC, E. coli and total FCM counts of 9.2 x 106 (week 14), 3.5 x 106 (week 39) and 3.5 x 106 micro-organisms/100 m (week 39) and 2.1 x 108 micro-organisms/m (weeks 1 and 39) respectively, were recorded. The highest HPC recorded for the Plankenburg River was 7.9 x 106 micro-organisms/100 m (week 44, site A). Site B is situated close to an informal settlement where waste effluents from storm water drainage pipes enter the river system. In addition, other possible contamination sources included agricultural (site A) and industrial (site C) areas bordering the Plankenburg River. The highest total MPN, FC and E. coli counts in the Diep River were 5.4 x 106 (week 23) and 1.6 x 106 micro-organisms/100 m [FC and E. coli, respectively (both in week 23)], recorded at site B. The highest HPC and total FCM counts of 1.7 x 107 micro-organisms/100 m (week 14) and 2.5 x 109 microorganisms/ m (week 23), respectively, were also recorded at site B. This site was identified as the most contaminated site along the Diep River and served as an accumulation point for waste effluents from the residential and industrial areas, which included paint and machine manufacturers. Other sources situated along the Diep River included storage and maintenance facilities for steel containers, a waste water treatment plant and an oil-refinery. Most of the bacterial counts obtained for the Plankenburg and Diep Rivers exceeded the accepted maximum limit for river water for most of the sampling period. Bacterial species from the Berg- and Plankenburg Rivers were isolated and identified. The presence of various Enterobacteriaceae species isolated at all the sites in both rivers confirmed faecal contamination of these water sources over the entire sampling period. Opportunistic pathogens such as Klebsiella sp., Serratia sp., Enterobacter sp., Shewanella sp., Aeromonas sp., Pseudomonas sp., Acinetobacter sp. and Citrobacter freundii as well as pathogens such as Bacillus cereus and B. anthracis were also identified in both river systems. All the respective articles are presented in the required format of the journal in which the article has been published or submitted to.

Thesis submitted in fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology 2008 / River systems can become contaminated with micro-organisms and metals and the routine monitoring of these rivers is essential to control the occurrence of these contaminants in water bodies. This study was aimed at investigating the metal contamination levels in the Berg-, Plankenburg- and Diep Rivers in the Western Cape, South Africa, followed by the remediation of these rivers, using bioreactor systems. Sampling sites were identified and samples [water, sediment and biofilm (leaves, rocks and glass, etc.)] were collected along the Berg- and Plankenburg Rivers from May 2004 to May 2005 and for the Diep River, from February 2005 to November 2005. The concentrations of aluminium (Al), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb) and zinc (Zn) were determined using the nitric acid digestion method and analysed by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). For the Berg River, the highest concentrations in water samples were recorded for Al, Mn and Fe at the agricultural area (Site A – chapter 2). In the sediment and biofilm samples, the highest metal concentrations were once again recorded for Al and Fe. The concentrations of Al and Fe were significantly higher (p < 0.05) than than Cu, Zn, Pb, Ni and Mn in water, sediment and biofilm samples, and were mostly higher than the quality guidelines recommended by the Department of Water Affairs and Forestry (DWAF, 1996) and the Canadian Council for the Ministers of the Environment (CCME, 2001). Possible sources of contamination in the Berg River could be due to the leaching or improper discarding of household waste from the informal- and established residential areas, as well as the improper discarding of pesticides at the agricultural area. For both the Plankenburg and Diep Rivers the Al and Fe concentrations were higher than all the other metals analysed for in sediment and water samples. The highest concentrations recorded in the Plankenburg River was 13.6 mg.l-1 (water - Week 18, Site B) and 15 018 mg.kg-1 (sediment - Week 1, Site C) for Al and 48 mg.l-1 (water - Week 43, Site A) and 14 363.8 mg.kg-1 (sediment - Week 1, Site A) for Fe. The highest concentrations recorded in the Diep River was 4 mg.l-1 (water - Week 1, Site A) and 19 179 mg.kg-1 (sediment - Week 1, Site C) for Al and 513 mg.l-1 (water - Week 27, Site A) and 106 379.5 mg.kg-1 (sediment - Week 9, Site C) for Fe. For most of the metals analysed the concentrations were higher than the recommended water quality guidelines as stipulated by the Department of Water Affairs and Forestry (DWAF, 1996b), the Canadian Council for the Ministers of the Environment (CCME, 2001) and the ‘World average’ (Martin and Windom, 1991). Point sources of pollution could not conclusively be identified, but the industrial and residential areas could have influenced the increased concentrations. Metal concentrations should be routinely monitored and the guidelines should be updated and revised based on the current state of the rivers and pollution influences. Micro-organisms isolated from flow cells after exposure to varying metal concentrations were investigated for possible metal-tolerance. A site where high metal concentrations were recorded along the Plankenburg River was investigated. The micro-organisms isolated from the flow cells were cultured and identified using the Polymerase Chain Reaction (PCR) technique, in conjunction with universal 16SrRNA primers. The phylogeny of the representative organisms in GenBank, were analysed using the Neighbour-joining algorithm in Clustal X. After exposure, the channels were stained with the LIVE/DEAD BacLightTM viability probe and visualised using Epifluorescence Microscopy. The results revealed that when exposed to the highest concentrations of Al (900 mg.l-1), Fe (1000 mg.l-1), Cu (10 mg.l-1) and Mn (80 mg.l-1), the percentage of dead cells increased, and when exposed to the lowest concentrations of Al (10 mg.l-1), Cu (0.5 mg.l-1), Mn (1.5 mg.l-1) and Zn (0.5 mg.l-1), no significant differences could be distinguished between live an dead cells. When exposed to the highest concentrations of Zn (40 mg.l-1) and Ni (20 mg.l-1), no significant differences between the live and dead cell percentages, were observed. The phylogenetic tree showed that a diverse group of organisms were isolated from the flow cells and that some of the isolates exhibited multiple metal resistance (Stenotrophomonas maltophilia strain 776, Bacillus sp. ZH6, Staphylococcus sp. MOLA:313, Pseudomonas sp. and Delftia tsuruhatensis strain A90 exhibited tolerance to Zn, Ni, Cu, Al, Fe), while other isolates were resistant to specific metals (Comamonas testosteroni WDL7, Microbacterium sp. PAO-12 and Sphingomonas sp. 8b-1 exhibited tolerance to Cu, Ni and Zn, respectively, while Kocuria kristinae strain 6J-5b and Micrococcus sp. TPR14 exhibited tolerance to Mn). The efficiency of two laboratory-scale and one on-site bioreactor system was evaluated to determine their ability to reduce metal concentrations in river water samples. The laboratory-scale bioreactors were run for a two-week and a three-week period and the on-site bioreactor for a period of ten weeks. Water (all three bioreactors) and bioballs (bioreactor two and on-site bioreactor) were collected, digested with 55% nitric acid and analysed using ICP-AES. The final concentrations for Al, Ni and Zn (bioreactor one) and Mn (bioreactor two), decreased to below their recommended concentrations in water samples. In the on-site, six-tank bioreactor system, the concentrations for Fe, Cu, Mn and Ni decreased, but still exceeded the recommended concentrations. The concentrations recorded in the biofilm suspensions removed from the bioballs collected from bioreactor two and the on-site bioreactor, revealed concentrations higher than those recorded in the corresponding water samples for all the metals analysed, except Fe. The bioballs were shown to be efficient for biofilm attachment and subsequent metal accumulation. The species diversity of the organisms isolated from the bioreactor (bioreactor two) experiment after three days (initial) differed from the organisms isolated after 15 days (final). Hydrogenophaga sp., Ochrobactrum sp, Corynebacterium sp., Chelatobater sp. and Brevundimonas sp. were present only at the start of the bioreactor experiment. The surviving populations present both in the beginning and at the end of the bioreactor experiment belonged predominantly to the genera, Pseudomonas and Bacillus. Metal-tolerant organisms, such as Bacillus, Pseudomonas, Micrococcus and Stenotrophomonas, amongst others, could possibly be utilised to increase the efficiency of the bioreactors. The bioreactor system should however, be optimised further to improve its efficacy.

Le bassin versant de l’Escaut (20 000 km²) est caractérisé par forte densité de population (plus de 500 habitants par km2) et une activité agro-pastorale intensive. Les rivières de ce bassin sont sévèrement affectées par les rejets d’eaux usées domestiques, les effluents d’élevage et les eaux de ruissellement des sols agricoles. Le but de cette étude est :(i) d’évaluer la qualité microbiologique de ces rivières ;(ii) d'identifier et de quantifier les différentes sources de contamination fécale à l’échelle du bassin de l’Escaut; (iii) d’étudier les processus qui contrôlent le devenir des bactéries fécales en rivière; (iv) de développer des modèles numériques sur la base des travaux expérimentaux permettant de prédire la concentration des bactéries indicatrices dans les rivières du bassin de l’Escaut.<p>L’évaluation de la qualité microbiologique des principales rivières du bassin est basée sur le dénombrement de deux indicateurs de contamination fécale (Escherichia coli et entérocoques intestinaux). Les abondances des deux indicateurs dans les principales rivières du bassin indiquent très clairement que les eaux et les sédiments de ces rivières sont fortement contaminés par des micro-organismes entériques. Les sources prédominantes de la pollution fécale de ces rivières sont les rejets des effluents des stations d’épuration. Les niveaux de contamination les plus élevés sont observés dans la Senne en aval de Bruxelles et s’expliquent par le faible débit de la Senne comparé aux débits des effluents des deux stations d’épuration de Bruxelles. Les niveaux de contamination atteignent leur maxima à l’aval de Bruxelles par temps de pluie en raison des surverses de réseaux unitaires.<p>Les connaissances acquises sur les apports des bactéries indicatrices par les sources ponctuelles et les sources diffuses et sur le devenir des bactéries indicatrices ont permis de développer un module décrivant la dynamique des E. coli dans les rivières. Ce module est original par le fait de considérer trois compartiments de bactéries fécales (libres, attachées aux particules dans la colonne d’eau et présentes dans les sédiments) qui sont affectés différemment par les processus de transport et de disparition. Ce module a été couplé à deux modèles décrivant l’hydrodynamique respectivement de l’ensemble des rivières du bassin (SENEQUE-EC) et de la partie fluviale de l’Escaut sous l’influence de la marée et son estuaire (SLIM-EC2). Ces deux modèles permettent de décrire la distribution temporelle et spatiale des E. coli dans les eaux de surfaces et de prévoir les modifications de la qualité microbiologique des eaux suite à des changements de gestion des eaux usées. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Pathogens, such as Escherichia coli and fecal coliforms, are causing the majority of water quality impairments in U.S., making up ~87% of this grouping's violations. Predicting and characterizing source, transport processes, and microbial survival rates is extremely challenging, due to the dynamic nature of each of these components. This research built upon current analytical methods that are used as exploratory tools to predict pathogen indicator counts across regional scales. Using a series of non-parametric methodologies, with spatially explicit predictors, 6657 samples from non-estuarine lotic streams were analyzed to make generalized predictions of regional water quality. 532 frequently sampled sites in the Oregon Coast Range Ecoregion, were parsed down to 93 pathogen sampling sites in effect to control for spatial and temporal biases. This generalized model was able to provide credible results in assessing regional water quality, using spatial techniques, and applying them to infrequently or unmonitored catchments. This model's 56.5% explanation of variation, was comparable to other researchers' regional assessments. This research confirmed linkages to land uses related to anthropogenic activities such as animal operations and agriculture, and general riparian conditions.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Environmental Health in the Faculty of Applied Sciences at the Cape Peninsula University of Technology, 2013 / Water is an important daily requirement and in a clean, pure form, it promotes health and well-being. In addition to South Africa being one of the driest countries in the world, water availability is also being compromised by massive pollution of remaining water sources. The Berg- and Plankenburg Rivers are two of the surface water sources in the Western Cape, South Africa, which are highly polluted by sewage, industrial and agricultural run-off. The current investigation was aimed at comparing diagnostic tools, which are employed by municipalities and food industries, and molecular based techniques to routinely monitor water for indicator organisms in time- and cost-effective manner. These rivers were sampled twice a month (July 2010 to January 2011) at the sites closest to the informal settlements of Kayamandi in Stellenbosch (Plankenburg River) and Mbekweni in Paarl (Berg River). The contamination levels of the two river systems were evaluated by the enumeration of Escherichia coli and coliforms using the Colilert 18® system, Membrane Filtration (MF) and Multiple Tube Fermentation (MTF) techniques. The highest faecal coliform count of 9.2 × 106 microorganisms/100 ml was obtained in weeks 21 and 28 from the Plankenburg River system by the MTF technique, while the lowest count of 1.1 × 103 microorganisms/100 ml was obtained in week one for both river systems by the MTF technique. The highest E. coli count of 1.7 × 106 microorganisms/100 ml was obtained from the Berg River system (week 9) using the MTF technique, while the lowest count of 3.6 × 102 microorganisms/100 ml was obtained by the MF technique from the Plankenburg River system. The coliform and E. coli counts obtained by the enumeration techniques thus significantly (p > 0.05) exceeded the guidelines of 2000 microorganisms/100 ml stipulated by the Department of Water Affairs and Forestry (DWAF, 1996) for water used in recreational purposes. Overall the results obtained in this study showed that the water in the Berg- and Plankenburg River systems is highly polluted, especially where these water sources are used for irrigational and recreational purposes. For the coliform and E. coli counts obtained using the three enumeration techniques, it was noted that the MTF technique was more sensitive and obtained higher counts for most of the sampling weeks. However, the media (Membrane lactose glucuronide agar) used in the MF technique also effectively recovered environmentally stressed microbial cells and it was also better for the routine selection and growth of coliforms and E. coli. While E. coli and total coliforms were detected utilising the Colilert 18® system, accurate enumeration values for these two indicator groups was not obtained for the entire sampling period for both river systems. It has previously been shown that dilutions (up to 10-3) of highly polluted waters increase the accuracy of the Colilert 18® system to enumerate colifoms and E. coli in marine waters. As the results obtained utilising the Colilert 18® system were also not comparable to the MF and MTF techniques it is recommended that highly polluted water samples be diluted to increase the accuracy of this system as a routine enumeration technique. Water samples were directly inoculated onto MacConkey, Vile Red Bile (VRB) agar and the Chromocult Coliform agar (CCA) and single colonies were inoculated onto nutrient agar. Chromocult coliform agar proved to be more sensitive than MacConkey and VRB agar for the culturing of E. coli and coliforms. Preliminary identification of these colonies was done using the RapID ONE and API 20 E systems. The most isolated Enterobacteriaceae species by both systems, included Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli and Enterobacter cloacae in both river systems. The API 20 E system was more sensitive in the preliminary identification of the various isolates, as greater species diversity was obtained in comparison to the RapID ONE system. The Polymerase Chain Reaction (PCR) was firstly optimised using positive Enterobacteriaceae species. The optimised method was then applied to the analysis of river water samples, which were centrifuged to harvest the bacterial cells, with DNA extracted using the boiling method. The extracted DNA was amplified using conventional PCR with the aid of species specific primers. The Enterobacteriaceae species that were detected throughout the study period in both river systems include Serratia marcescens, Escherichia coli, Klebsiella pneumoniae and Bacillus cereus. Conventional PCR was the most reliable and sensitive technique to detect Enterobacteriaceae to species level in a short period of time when compared to RapID ONE and the API 20 E systems. Multiplex PCR was optimised using the positive pathogenic E. coli strains namely, Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAEC). It was then employed in river water sample analysis and enabled the detection of EAEC, EHEC, and EIEC strains in Berg River system, with only the EAEC detected in the Plankenburg River system. Real-time PCR was used to optimise the multiplex PCR in the amplification of E. coli strains and successfully reduced the time to obtain final results when using control organisms. Real-time PCR was found to be more sensitive and time-effective in the identification of E. coli strains, and also more pronounced DNA bands were observed in real-time PCR products compared to conventional-multiplex PCR amplicons. To sustain the services provided by the Berg- and Plankenburg Rivers in the Western Cape (South Africa), these water sources should frequently be monitored, results assessed and reported according to the practices acknowledged by responsible bodies. It is therefore recommended that the enumeration techniques be used in conjunction with the very sensitive PCR technique for the accurate detection of coliforms and E. coli in river water samples.