Thesis (MSc Food Sc (Food Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Over the last decade, studies on the quality of the water in many of South Africa.s rivers revealed an increase in pollution levels (DWAF, 1996; DWAF, 2000). In agricultural areas of the Western Cape many farmers draw water for irrigation from nearby rivers without knowing what the microbial pollution level is. In order to investigate the current quality of river water in some of the Western Cape.s major rivers and to explore the possibility of pathogen carryover from water onto produce, this research project was initiated. In an exploratory study over a five month period, the microbiological and water chemistry of three selected sites from the upper Berg and two from the Plankenbrug Rivers were assessed. Studied organisms included Salmonella, Staphylococcus, Listeria, endosporeformers, coliforms, Escherichia coli and intestinal Enterococci, while the chemical analysis consisted of pH, alkalinity, conductivity and chemical oxygen demand (COD). Faecal coliform counts ranging from 540 to 1 700 000 cfu.100ml-1 and 490 to 160 000 cfu.100ml-1 were found for the Berg and Plankenbrug Rivers, respectively. The water temperature ranged from 12º to 21ºC with COD values always below 100 mg.L-1. Potential human pathogens such as Salmonella, Staphylococcus, Listeria, endosporeformers, E. coli and intestinal Enterococci were frequently isolated from all five sites that were sampled. These results are of great concern to farmers, fresh produce retailers and consumers alike as the river water is regularly drawn for irrigation of produce that is later consumed raw or after a minimal processing step. From the exploratory study it was concluded that the water from all the sites were not suitable for use in irrigation practices as they regularly exceeded the guidelines for faecal coliforms and E.coli as set out by South African authorities. Irrigation with faecally polluted river water is one way that fruit and vegetables can become contaminated with foodborne pathogens. The risk of disease transmission from potential pathogens present in the irrigation water is influenced by the microbial load present and the numbers carried over to the produce. In this study the carry-over and survival of Escherichia coli on green beans, sugar-snap peas and cocktail tomatoes was assessed under controlled laboratory conditions. The produce was exposed to E.coli under different combinations of exposure times (5, 15 or 30 min), drying times (30 or 120 min) and different inoculum concentration ranges (107, 105, 104, 10³ and 10ª). In all cases a reduction of at least one log value in original inoculum number was found with the 10ª inoculums showing no survivors. Follow-up studies with an exposure time of 60 min and increased drying times of 6 or 12 h. Neither of these parameter changes affected the variation in numbers for the same inoculum or the E.coli survivors. Similar E.coli loads to those detected on the green beans were detected on the sugar-snap peas. The number of survivors on the cocktail tomatoes was much lower than found for the beans and peas. The number of survivors on the cocktail tomatoes was much lower than found for the beans and peas. This was attributed the 'smooth'surface of the tomato skin probably making attachment of the E.coli bacteria difficult. With the exception of the 10ª inoculum range, all E.coli survivors detected on the three types of produce studied exceeded the guideline numbers set for fresh produce. If similar survival patterns are to be found in the environment then results from this study should serve as a warning that the Plankenburg river water is unsafe for use in the irrigation of fresh produce. Some farmers are already treating river water with chlorine prior to irrigation to eliminate the chance of pathogen transfer onto produce. However, this is not a feasible solution for most farmers due to the high cost of implementing a system such as this. / AFRIKAANSE OPSOMMING: Oor die afgelope tien jaar het studies oor die gehalte van die water in baie van Suid-Afrika se riviere 'n toename in besoedelingsvlakke getoon. Baie boere in landbou gebiede van die Wes-Kaap wat watertrek vir besproeiing uit die nabygelee riviere is onbewus van die mikrobiese besoedelingsvlak van die rivier. Hierdie navorsingsprojek is geinisieer ten einde die huidige gehalte van die water in sommige van die Wes-Kaap se riviere te monitor, asook die moontlikheid van patogeen oordrag van die water na vars produkte te ondersoek. Met behulp van 'n verkennende studie oor . vyf maande tydperk, is die mikrobiologiese en water chemie kenmerke van drie geselekteerde moniteringspunte uit die boonste Bergrivier, en twee uit die Plankenburgrivier bemonster. Die bestudeerde organismes het ingesluit: Salmonella, Staphylococcus, Listeria, endosporeformers, fekale kolivorme, Escherichia coli en intestinale Enterococci. Die chemiese parameters het bestaan uit pH, alkaliniteit, geleiding, temperatuur en chemiese suurstof behoeftes (CSB). Fekale kolivorme tellings het gewissel van 540 - 1 700 000 cfu.100ml-1 en 490 tot 160 000 cfu.100ml-1 is gevind vir die Berg- en Plankenburgriviere, onderskeidelik. Die water temperatuur het gewissel van 12 º tot 21ºC met die CSB-waarde altyd onder 100 mg.L-1. Potensiele menslike patogene soos Salmonella, Staphylococcus, Listeria, endosporeformers, E. coli en intestinale Enterococci is dikwels geisoleer uit al vyf die moniteringspunte. Hierdie resultate is van groot belang vir boere, kleinhandelaars en varsprodukte verbruikers omdat die riviere se water gereeld onttrek word vir besproeiing van produkte wat later gebruik word in rou of in minimale geprosesseerde vorm. Vanuit die verkennende en uit die langer termyn studie (12 maande) is die gevolgtrekking gemaak dat die water vanuit die riviere nie geskik is vir gebruik in besproeiingspraktyke waar die riglyne vir fekale kolivorme en E. coli, soos uiteengesit deur die Suid-Afrikaanse owerhede, gereeld oorskry word nie. Besproeiing met fekale besoedelde rivierwater is een manier waarop groente en vrugte besmet kan word met voedsel patogene. Die risiko vir die oordrag van moontlike patogene teenwoordig in die besproeiingswater word beinvloed deur die mikrobiese lading teenwoordig en die getalle wat oorgedra word na die produkte. In hierdie studie is die oordrag en oorlewing van E. coli op groenboontjies, ertjies en tamaties onder beheerde laboratoriumtoestande beoordeel. Die produkte is blootgestel aan E. coli onder verskillende kombinasies van blootstellingstydperke (5, 15 of 30 min), drogingstye (30 of 120 min) en verskillende inokulum konsentrasies (107, 105, 104, 10³ en 10ª). In elke geval is . afname van ten minste een log waarde van die oorspronklike inokulum konsentrasie gevind. Die 10ª inokulum reeks het geen oorlewende selle getoon nie. Opvolg studies is gedoen met 'n blootstellingstyd van 60 min en verlengde drogingstye van 6 of 12 uur. Daar is gevind dat hierdie parameter veranderings die variasie in getalle vir dieselfde inokulum of die oorlewende E. coli nie beinvloed het nie. Soortgelyke E. coli ladings wat op die groenbone aangetref is, is ook op die ertjies aangetref. Die aantal oorlewendes op die tamaties was heelwat laer as die resultate vir die boontjies en ertjies. Dit word toegeskryf aan die "gladde" oppervlak van die tamatie-vel wat waarskynlik die aanhegting van die E. coli selle bemoeilik. Met die uitsondering van die 10ª inokulum reeks, het al die E. coli oorlewende selle gevind op die drie tipes produkte, die riglyn getalle oorskry wat vir vars produkte gestel is. As soortgelyke oorlewingspatrone gevind word in die omgewing, behoort die resultate van hierdie studie as waarskuwing te dien dat die water van die Plankenburgrivier ongeskik is vir die besproeiing van vars produkte. Sommige boere is reeds besig met chloor behandeling van die rivierwater voor besproeiing, om die kans van patogeenoordrag na die produkte uit te skakel. Dit is egter nie 'n haalbare oplossing vir die meeste boere nie as gevolg van die hoe kostes verbonde aan die implementering van' n stelsel soos hierdie.

Thesis (DTech (Biomedical Technology))--Cape Peninsula University of Technology, 2008. / Includes bibliographies. Also available online.

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 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.

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.