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.

An investigation into the quality of surface water and ground water was conducted during 1988 in the 38,8 km² Hout Bay River catchment near Cape Town. The main objective of the study was to determine those areas and activities which constitute a pollution source and to ascertain the relative proportion which each contributes to the pollution problem and health risk of the surf zone of the beach at Hout Ba . The objective was achieved by monitoring the chemical and microbiological attributes of the Hout Bay River, its tributaries and stormdrains in wet and dry conditions on a routine basis and during storm events. Hout Bay is a rapidly developing residential area in which sewage disposal occurs by means of septic tank soakaway systems. Ground water quality was monitored to investigate the contribution to contamination by septic tank effluent. Results showed that stormdrain effluent in dry and wet conditions and surface runoff during rainfall were the main vectors of pollution. Although the pollution concentration was high during dry conditions the greatest pollution discharge to the surf zone of Hout Bay occurred during storm events. In view of the proposed residential development it is imperative that pollution control measures be undertaken so as to secure the future recreational and aesthetic value of Hout Bay

Climate is an important factor of agricultural productivity and many rural dwellers in developing countries depend on agriculture and are highly affected by climate change and variability. The world is currently experiencing climatic changes and variability conditions which results in high temperatures, low rainfall patterns, shortage of water and drought persistence. Climate change and variability is affecting weather patterns and shifting seasons which results in serious repercussions on smallholder farmers. Smallholder farmers are extremely vulnerable to climate change and variability because their farming and production systems are climate sensitive and are not rebound to climate stresses. These adverse effects in developing countries arise from different climate change and variability-related causes, notable extreme weather events, food security, increased health risks in agriculture from vector home diseases, and temperature-related morbidity in environments. The study was carried in King Sabata Dalindyebo Municipality in the Eastern Cape Province in South Africa. This study examines farmers’ awareness of climate change and variability and its effects on agricultural productivity in King Sabata Dalindyebo municipality using a Descriptive Statistics, Binary and Ricardian Model fitted to data from a cross-sectional survey of 200 farmers in King Sabata Dalindyebo Municipality. Both primary and secondary data was used. This research study estimates the effects of climate change and variability on King Sabata Dalindyebo agricultural productivity using a continental dimension of Ricardian analysis. Results revealed that local farmers were aware of climate change and variability and perceived changes in average temperatures and rainfall. The changes in average temperatures and rainfall had adverse effects on crop and livestock production. However, farmers’ awareness of climate change is not to an extent that they presume adaptation to climate change as a necessity and crucial. The results show that climate change and variability affects farm income and there is a non-linear relationship existing between climatic variables (temperature and precipitation) and farm income which depicts U-shaped. The study results indicated that climate change and variability affect agricultural productivity and have an effect on agricultural productivity in King Sabata Dalindyebo Municipality. In view of the research findings, several policy proposals are suggested. The study findings suggest that climate change and variability must be taken seriously and monitored. Policy makers and government officials must support farmers with information distribution,education, market access, well trained extension agents, credit and information about mitigation strategies to climate change and variability which includes institutional and technological methods, particularly smallholder farmers.

MRDV / Institute for Rural Development / See the attached abstract below

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

MENVSC / Department of Geography and Geo-Information Sciences / South Africa was badly affected by the recent 2015/16 severe drought. Water levels in dams declined drastically resulting in decimation of livestock herds and widespread crop failure. Mopani District Municipality is comprised of many agricultural activities that contribute to the economy and social development of the country. The study evaluated the nature of the drought hazard - its impacts, vulnerability and adaptation strategies employed by rural communities of Mopani District. The study used a mixed method approach with both quantitative and qualitative datasets. The district was divided into two distinct climatic areas, the eastern lowveld which includes the Greater-Giyani, Ba-Phalaborwa and Maruleng Local Municipalities and the western highveld which includes Greater- Tzaneen and Greater- Letaba Local Municipalities. Questionnaires were administered among community members whilst key informant interviews were conducted among relevant government and municipal officials. Anomalies in long term climate data were analysed to determine the frequency and intensity of drought in the district. Drought characterisation was done using a Standardised Precipitation and Evapotranspiration Index whilst vegetation anomaly maps, maize yields and dam level data were used to analyse the impacts of drought across the district. Levels of vulnerability to drought were determined using the Household Vulnerability Index. Spatially distinct patterns of drought conditions across the district were remarkable with wet conditions on the western highveld along the escarpment and harsh dry conditions towards the eastern lowveld. It was found that nearly half the time there is some form of drought or another in the district which may be linked to the remote El Nino phenomenon. Community vulnerabilities have a direct impact on human welfare and different strategies are employed to adapt to drought hazards both at community and district levels. The study showed a link between drought hazard extent and vulnerability. Community members are adapting using conservation agriculture, selling fire-wood, accessing boreholes and rearing chickens, amongst other means to survive in these harsh climatic conditions. Local government intervention strategies include supply of seeds and fertilisers, selling fodder at a cheaper price and supplying water using trucks. The findings of this study contribute to disaster risk reduction efforts in Mopani District Municipality / NRF

MESMEG / Department of Mining and Environmental Geology / Many recent studies have shown that some of the greatest water needs occur in areas underlain by crystalline rocks with complex hydrogeology. Crystalline basement rocks underlie over 60% of the South African surface, and the Limpopo Province of South Africa is no exception. Previous attempts to develop the lithologies of Limpopo for groundwater abstraction without the use of sound scientific methodologies resulted in low yielding boreholes and a higher rate of borehole failure. The complexity of the lithologies in the region necessitates the use of sound scientific methodologies for the delineation of promising groundwater potential zones. Therefore, the principal objective of the present study was to delineate groundwater potential zones through an integrated approach of remote sensing, geophysics, as well as the use of ancillary datasets. The area of focus is located in the northeastern section of Limpopo province, covering an area of about 16 800km2. Geologically, it is underlain by three Lithostratigraphic domains comprised of Archean-aged basement rocks, Soutpansberg volcano-sedimentary succession and subsidiary basins of the main Karoo young sedimentary cover. In general, the groundwater potential of a region is a function of factors such as lithology, lineaments, slope, climate and land use/ land cover. Thus, the present study used parameters such as lineaments, lithologies, slope, and land use/ land cover to produce a groundwater potential zone map. The thematic layers were prepared from raw datasets, which include; LANDSAT 8 OLI, ASTER-DEM, aeromagnetic data, geological maps, and land use/land cover data, which were overlaid in a GIS environment. The resultant groundwater map revealed the presence of five distinct classes of groundwater potential zones, which were categorised into excellent, good, moderate, low and very low. Interpretation of the results shows that the study area is dominated by areas that may be regarded as moderate water potential zones, covering about 52% of the total area. On the other hand, low and good groundwater potential zones occur in almost equal proportions of 19.52 % and 24 % respectively. The results obtained were validated using GRIP borehole dataset, and a number of follow-up geophysical surveys. iii Overlaying of the boreholes dataset on the map showed positive correlation between borehole yields groundwater potential zones. On the other hand, follow-up Vertical Electrical Sounding surveys revealed the presence of conductive layers in some selected target areas. The groundwater potential zone map and validation results provided a meaningful regional assessment of groundwater distribution in the study area. Thus, the results of this study can be used as a guideline for future groundwater exploration projects. / NRF

Thesis (M. Sc. (Geography)) -- University of Limpopo, 2021 / Acid Mine Drainage (AMD) is the acidic water emanating from the mine tailing dams into the surrounding environment. AMD is regarded as a major environmental threat associated with mining. The lower Olifants River in the Kruger National Park (KNP) is considered an environmentally sensitive area, which exhibits high levels of aquatic ecosystems and supports a variety of terrestrial ecosystems within and around the KNP. The Phalaborwa mining industries have been discharging the acid mine drainage contaminated-water into the Ga-Selati River, a tributary to the Olifants River. Although the impacts in the upper Olifants River catchment have been well documented, it was the amount of AMD witnessed at KNP and the dying of fish within the lower Olifants River that raised issues of concerns. Hence, the study investigated the impact of acid mine drainage on water quality of the lower Olifants River, modelled the distribution of the dissolved heavy metals in the stream, and evaluated the applied mine wastewater management strategies at Phalaborwa mining industries. In this study, water samples were collected seasonally (winter, spring, and summer) from 2019 to 2020, and the analytical methods and procedures were optimized for the determination of selected elements in the water samples. During the study, ion chromatography (IC) was used to detect chloride (Cl), sulphate (SO4 - ), nitrate (NO3), and fluoride (F), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) was used to detect pH, turbidity, electrical conductivity (EC), total dissolved solids (TDS), magnesium (Mg), manganese (Mn), sodium (Na), potassium (K), aluminium (Al) and calcium (Ca). Modelling of the distribution of dissolved heavy metals was performed using the inverse distance weighted (IDW) interpolation technique available in ArcGIS 10.8 software. The range of pH across four sampling sites was between 7.77 and 9.11, indicating an alkaline pH. The concentration of measured parameters elevated downstream points with some exceeding the target water quality range (TWQR) for aquatic ecosystems. The elevated concentration of SO4 - at sites 3 and 4 (downstream points) showed that the acid mine drainage is still a matter of concern at the lower Olifants River catchment. However, the GIS models showed a decreasing trend of the concentration of heavy metal towards the KNP.

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.