The formation of an objectionable "swampy" odour in drinking water distribution systems in Perth, Western Australia, was first described by Wajon and co-authors in the mid-1980s (Wajon et al., 1985; Wajon et al., 1986; Wajon et al, 1988). These authors established that the odour, variously described as "swampy", "sewage" or "cooked vegetable" was caused by dimethyltrisulfide (DMTS) which has an odour threshold concentration of 10 nanograms per litre (ng/L). Investigations described in the present Thesis extend the work of Wajon and co-workers in attempting to establish the origin and cause of DMTS formation in Perth drinking water distribution systems.The DMTS problem appeared to be confined to water originating from a particular type of groundwater, specifically groundwater sourced from shallow, unconfined aquifers, which contain relatively high concentrations of sulfide, dissolved natural organic matter (NOM) and dissolved iron. DMTS was not present in the groundwater, but only formed in the distribution system, after treatment of groundwater via alum coagulation-filtration and oxidation processes. One objective of the present work was to determine the reasons for the observed association between DMTS formation and this specific groundwater type. A primary focus was to investigate the chemistry and biochemistry of sulfur species and NOM which might act as precursors to DMTS. The work was driven by the view that increased understanding of the problem might lead to more effective and acceptable treatment solutions than those presently in use.The observation that DMTS forms in distributed water that originates from groundwater, but not in water from surface sources has led to the hypothesis that groundwater NOM may contain precursor(s) to DMTS For example, it was proposed that methyl esters and ethers within humic substances might be a source of methyl groups that ++ / could participate in DMTS formation in distributed water (Wajon and Heitz, 1995; Wajon and Wilmot, 1992). Further, comparison of levels of reduced sulfur with levels of dissolved organic carbon (DOC) in groundwaters feeding Wanneroo GWTP revealed that a positive correlation between these two parameters existed. This observation provided further impetus to examine the nature of NOM in these groundwater systems. In the present study (discussed in Chapter 3), NOM from two Perth drinking water sources was isolated and characterised, with the aim of identifying major differences in structure and/or functional groups that might influence DMTS formation. NOM was isolated from water samples using ultrafiltration, and characterised using pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) and offline- thermochemolysis/methylation (TCM). Pyrolysis of groundwater NOM yielded a high proportion of organosulfur compounds, primarily methyl thiophenes and sulfur gases, but did not yield detectable amounts of methoxy-aromatic compounds. Analysis by TCM yielded sulfur compounds tentatively identified as the methyl esters of methylthiopropanoate and methylthiobutanoate, compounds that may arise as degradation products of dimethylsulfoniopropionate (DMSP), an algal odmoregulator Compounds such as DMPS could potentially undergo reactions to form DMTS in distributed water.The task of investigating the formation of nanogram-per-litre concentrations of DMTS demanded the development of new analytical procedures that could be used to determine similarly low concentrations of DMTS precursors. Evidence existed to suggest that inorganic polysulfides could be plausible precursor compounds, and since no technique existed to analyse and quantify individual polysulfide homologues a new technique needed to be developed and verified. The technique, first used in a semiquantitative manner by ++ / Wajon and Heitz (1995), utilizes methyl iodide to derivatise polysulfides in-situ. The technique was developed further and shown to be quantitative and specific for inorganic polysulfides. Further, a new procedure for the determination of d i methyl polysulfides (DMPSs; CH3SnCH3, where n = 2-5), based on purge and trap was developed. In this new procedure analytes were trapped on a "Grob" activated charcoal tube, which was integrated into a commercially available, automated purge and trap instrument. Perdeuterated analogues of the DMPS analytes were synthesized and used as internal standards. These modifications resulted in a more rapid and robust procedure than the previously used procedures, vii which were based on closed loop stripping analysis (CLSA). Validation of the precision, accuracy, linearity and robustness of the new procedures for both inorganic polysulfides and dimethylpolysulfides is described in Chapter 4.Previous authors (Wajon and Heitz, 1995; Wajon and Wilmot, 1992; Wilmot and Wajon, 1997) hypothesized that DMTS could arise in the distribution system from residual polysulfides or other reduced sulfur compounds originating from groundwater. The latter authors showed that a small proportion of sulfide in the groundwater was not completely oxidised to sulfate during the water treatment process and proposed that this residual reduced sulfur fraction, which they referred to as non-sulfide reduced sulfur (NSRS) could contain precursors to DMTS. In a review of the chemistry of sulfide oxidation (Chapter 2) it was shown that the most likely forms of sulfur comprising the NSRS that enters the Wanneroo distribution system are organosulfur compounds and elemental sulfur, probably associated with organic matter in the form of a sulfur sol.Analysis of inorganic polysulfides in treated water, using the newly described method in Chapter 4, revealed that small ++ / amounts of these compounds (20-80 ng/L) were occasionally present in some samples. However, it was concluded that, since inorganic polysulfides could not survive water treatment processes, these compounds probably arose from traces of biofilm or pipe sediment that may have entered the water during sampling. It was proposed that the presence of biofilm particulates in water samples probably also accounted for observations that DMTS appeared to form in some water samples during storage of the sample. These studies are discussed in Chapter 5.The primary method of control of DMTS formation in the distribution system has been to maintain free chlorine residuals. However, the mechanisms by which this occurs have not been studied; the effectiveness of DMTS oxidation by chlorine, or how chlorine affects microbial processes that might form DMTS is not known. These issues are addressed in the final section of Chapter 5. Experiments to determine the effectiveness of oxidation of dimethyldisulfide (DMDS) and DIVITS (5 mu g/L) by free chlorine (0.2 to 0.6 mg/L) in distributed water showed that these substances are rapidly and completely oxidised in water containing a chlorine residual of more than 0.4 mg/L. However, slow regeneration of traces of DMDS and DIVITS after dissipation of free chlorine to non-detectable levels showed that these compounds were incompletely oxidised at the lower chlorine concentrations~ This provides some rationale for field observations that DIVITS occurs even where low, but measurable, chlorine residuals appear to exist (<0.2 mg/L).As was established in a review of the chemistry of reduced sulfur compounds Chapter 2), reducing conditions not present in the oxic bulk water are required for DMTS to form and to persist. It was therefore proposed that microbial reduction processes could generate anoxic microniches in the distribution system, within which ++ / DMTS production could occur. This hypothesis was investigated in Chapter 6; the new methods for analysis of organic and inorganic polysulfides were applied to the study of biofilms and deposits of colloidal material found in distribution pipes and storage reservoirs. The study demonstrated that these materials contained concentrations of methylated and inorganic polysulfides four to six orders of magnitude higher than those ever found in the bulk water phase. The results indicated that reducing conditions most probably exist within the biofilms and pipewall deposits, where these polysulfides were formed. The iron-rich pipe slimes appeared to protect the sulfur compounds against the oxidative effects of chlorine and dissolved oxygen. It was concluded that the organic and inorganic polysulfides most probably arise through microbial sulfate reduction processes that occur in anoxic microenvironments within the slimes and deposits.Microbial processes that lead to the formation of polysulfides and dimethylpolysuifides under conditions approximately representative of those in distribution systems were investigated in work described in Chapter 7. The aim of this work was to investigate the role of biofilms in the formation of DMTS and to determine the nature of chemical precursors which might stimulate these processes. Biofilms, artificially generated on synthetic supports within chambers filled with water from Wanneroo GWTP, were exposed to compounds thought to be potential DMTS precursors. The response of the systems in terms of production of methylated sulfur compounds was monitored. Conclusions of the study were that, under the test conditions, production of DMDS and DMTS could occur via several mechanisms and that these dimethyloligosulfides could be formed even without the addition of compounds containing sulfur or methyl moieties. DMTS did not form in the absence of ++ / biofilms and it was therefore concluded that minimisation of biofilm activity was a key in preventing DMTS formation. Outcomes of the work imply that environments within distribution systems are complex and dynamic, as perhaps manifested by the intermittent nature of the DMTS problem.Finally, in Chapter 8 the conclusions to the present studies are summarised. It is shown how they underpin the rationale for proposed new treatment solutions aimed at preventing DMTS problems in the Wanneroo zone, primarily by minimising microbial activity and biofilm formation within distribution systems.

No description available.

A simulation-optimization methodology was used to assess monitoring strategies for a drinking water distribution network. Multiple simulation trials of contamination events were used to create input data for an integer optimization problem. A network model, based on the Blacksburg, VA water distribution system, was used as the basis for a case study of contaminant transport under conditions of uncertainty. The model was not calibrated due to the lack of reliable field data. Optimization of monitoring plans was performed within the context event based simulation trials. This precluded the design of monitoring plans that were directly compatible with requirements of water quality regulations. However, the results of the optimization did provide information that may be of use to the broader problem of compliance monitoring. Optimal plans were assessed in comparison with several alternative plans using a separate set of simulation trials. Optimization of monitoring plans derived from simulated source node contamination events was generally effective at choosing points that provided better detection of source node contamination than alternative plans based on random sampling or judgement sampling. Optimal monitoring plans derived from simulated random node contamination events were ineffective at detecting random node contamination. The results of optimization and the separate analysis of monitoring plan performance indicated that the number of simulation trials may have been inadequate to completely describe the stochastic behavior of the system. Additionally, comparison of these results with those obtained from a previous simulation study indicate that the results of any simulation of distribution system contamination may be very sensitive to the level of contaminant loading and the size and layout of the system. / Master of Science

The main aim of the study was to analyse the impact of physico-chemical parameters on drinking water quality, biofilm formation and antibiotic resistant bacteria in the drinking water distribution system in Mafikeng, North West Province, South Africa. Another objective was to isolate and characterise Pseudomonas and Aeromonas species from drinking water distribution system and detect the virulence gene determinants in the isolates by PCR analysis. The physico-chemical data obtained were subjected to statistical analysis using Excel 2007 (Microsoft) and SPSS (version 14.0) programmes. Pearson’s correlation product of the moment was used to determine the correlation between EC, TDS, pH and temperature. The two tailed test of significance (p<0.05) was used in order to determine the significance of the result. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. Cluster analysis based on the antibiotic inhibition zone diameter data of different organisms isolated from different sites was determined and was expressed as dendograms using Wards algorithm and Euclidean distance of Statistica version 7. Specific PCR was used to determine the identities of presumptive Pseudomonas and Aeromonas species through amplification of the gyrB, toxA and the ecfX gene fragments. Virulence gene determinants for the confirmed Pseudomonas and Aeromonas species were detected by amplifying the exoA, exoS and exoT genes and the aerA and hylH gene fragments, respectively. A Gene Genius Bio imaging system (Syngene, Synoptics; UK) was used to capture the image using GeneSnap (version 3.07.01) software (Syngene, Synoptics; UK) to determine the relative size of amplicons. Physico-chemical parameters were monitored from three drinking water sources three times a week and bacteriological quality was monitored weekly for four months from raw and treated drinking water. Water samples were analysed for pH, temperature, total dissolved solids (TDS) and electric conductivity (EC). Bacterial consortia from drinking water samples were isolated using selective media and enumerated. The results revealed a good chemical quality of water. However, the microbial quality of the water is not acceptable for human consumption due to the presence of Pseudomonas, Aeromonas, faecal coliforms (FC), total coliforms (TC) and Heterotrophic bacteria. The results showed that the drinking water is slightly alkaline with pH value ranging between7.7 to 8.32. What is of concern was the microbial quality of the water. Pseudomonas sp., faecal coliforms (FC), total coliforms (TC) and heterotrophic bacteria were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas sp.(>100/100ml), but also that when one considers the TDS it demonstrates that water from the Modimola Dam has an impact on the quality of the mixed water. The prevalence and antibiotic resistance profiles of planktonic and biofilm bacteria isolated from drinking water were determined. The susceptibility of these isolates was tested against 11 antibiotics of clinical interest and the multiple antibiotic resistance (MAR) patterns were compiled. The most prevalent antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All isolates from all samples were susceptible to ciprofloxacin. However, all faecal coliforms and Pseudomonas spp. were susceptible to neomycin and streptomycin. On the contrary all organisms tested were resistant to erythromycin (100%) trimethoprim and amoxycillin. Cluster analysis based on inhibition zone diameter data could not differentiate the various isolated into sample types. The highest prevalence of antibiotic resistant isolates was observed in Modimola Dam and Molopo eye. Biofilms were investigated in both raw water and treated drinking water sources for the presence of faecal coliforms, total coliforms, Pseudomonas spp., Aeromonas spp. and heterotrophic bacteria based on conventional microbiology and molecular methods. Drinking water biofilms were grown twice and the biofilm developing device containing copper and galvanized steel coupons were utilized. The Mini Tap filter, a home water treatment device which can be used at a single faucet, under constant flow was used during the second collection of treated water samples from cold water taps. Scanning electron micrograph revealed the existence of biofilms in all the sites investigated and the highest density was obtained on galvanized steel coupons. Isolates were tested against the antibiotics ampicillin (10μg), cephalothin (5μg), streptomycin (10μg), erythromycin (15μg), chloramphenicol (30μg), neomycin (30 μg), amoxycillin (10 μg), ciprofloxacin (5 μg), trimethoprim (25μg), kanamycin (30μg), and oxytetracycline (30μg). The multiple antibiotic resistance profiles and the presence of virulence related genes were determined. Various types of drug resistance and presence of virulence genes were observed. The most prevalent resistance phenotype observed was KF-AP-C-E-OT-TM-A. In conclusion, the results indicated the occurrence of faecal indicator bacteria in the drinking water destined for human consumption. Faecal indicator bacteria are the major contributors of poor drinking water quality and may harbour opportunistic pathogens. This highlighted survival of organisms to treatment procedures and the possible regrowth as biofilms in plumbing materials. The detection of large proportion of MAR Aeromonas and Pseudomonas species which possessed virulent genes was a cause of concern as these could pose health risks to humans. The data obtained herein may be useful in assessing the health risks associated with the consumption of contaminated water. / PhD (Microbiology), North-West University, Potchefstroom Campus, 2014

The main aim of the study was to analyse the impact of physico-chemical parameters on drinking water quality, biofilm formation and antibiotic resistant bacteria in the drinking water distribution system in Mafikeng, North West Province, South Africa. Another objective was to isolate and characterise Pseudomonas and Aeromonas species from drinking water distribution system and detect the virulence gene determinants in the isolates by PCR analysis. The physico-chemical data obtained were subjected to statistical analysis using Excel 2007 (Microsoft) and SPSS (version 14.0) programmes. Pearson’s correlation product of the moment was used to determine the correlation between EC, TDS, pH and temperature. The two tailed test of significance (p<0.05) was used in order to determine the significance of the result. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. Cluster analysis based on the antibiotic inhibition zone diameter data of different organisms isolated from different sites was determined and was expressed as dendograms using Wards algorithm and Euclidean distance of Statistica version 7. Specific PCR was used to determine the identities of presumptive Pseudomonas and Aeromonas species through amplification of the gyrB, toxA and the ecfX gene fragments. Virulence gene determinants for the confirmed Pseudomonas and Aeromonas species were detected by amplifying the exoA, exoS and exoT genes and the aerA and hylH gene fragments, respectively. A Gene Genius Bio imaging system (Syngene, Synoptics; UK) was used to capture the image using GeneSnap (version 3.07.01) software (Syngene, Synoptics; UK) to determine the relative size of amplicons. Physico-chemical parameters were monitored from three drinking water sources three times a week and bacteriological quality was monitored weekly for four months from raw and treated drinking water. Water samples were analysed for pH, temperature, total dissolved solids (TDS) and electric conductivity (EC). Bacterial consortia from drinking water samples were isolated using selective media and enumerated. The results revealed a good chemical quality of water. However, the microbial quality of the water is not acceptable for human consumption due to the presence of Pseudomonas, Aeromonas, faecal coliforms (FC), total coliforms (TC) and Heterotrophic bacteria. The results showed that the drinking water is slightly alkaline with pH value ranging between7.7 to 8.32. What is of concern was the microbial quality of the water. Pseudomonas sp., faecal coliforms (FC), total coliforms (TC) and heterotrophic bacteria were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas sp.(>100/100ml), but also that when one considers the TDS it demonstrates that water from the Modimola Dam has an impact on the quality of the mixed water. The prevalence and antibiotic resistance profiles of planktonic and biofilm bacteria isolated from drinking water were determined. The susceptibility of these isolates was tested against 11 antibiotics of clinical interest and the multiple antibiotic resistance (MAR) patterns were compiled. The most prevalent antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All isolates from all samples were susceptible to ciprofloxacin. However, all faecal coliforms and Pseudomonas spp. were susceptible to neomycin and streptomycin. On the contrary all organisms tested were resistant to erythromycin (100%) trimethoprim and amoxycillin. Cluster analysis based on inhibition zone diameter data could not differentiate the various isolated into sample types. The highest prevalence of antibiotic resistant isolates was observed in Modimola Dam and Molopo eye. Biofilms were investigated in both raw water and treated drinking water sources for the presence of faecal coliforms, total coliforms, Pseudomonas spp., Aeromonas spp. and heterotrophic bacteria based on conventional microbiology and molecular methods. Drinking water biofilms were grown twice and the biofilm developing device containing copper and galvanized steel coupons were utilized. The Mini Tap filter, a home water treatment device which can be used at a single faucet, under constant flow was used during the second collection of treated water samples from cold water taps. Scanning electron micrograph revealed the existence of biofilms in all the sites investigated and the highest density was obtained on galvanized steel coupons. Isolates were tested against the antibiotics ampicillin (10μg), cephalothin (5μg), streptomycin (10μg), erythromycin (15μg), chloramphenicol (30μg), neomycin (30 μg), amoxycillin (10 μg), ciprofloxacin (5 μg), trimethoprim (25μg), kanamycin (30μg), and oxytetracycline (30μg). The multiple antibiotic resistance profiles and the presence of virulence related genes were determined. Various types of drug resistance and presence of virulence genes were observed. The most prevalent resistance phenotype observed was KF-AP-C-E-OT-TM-A. In conclusion, the results indicated the occurrence of faecal indicator bacteria in the drinking water destined for human consumption. Faecal indicator bacteria are the major contributors of poor drinking water quality and may harbour opportunistic pathogens. This highlighted survival of organisms to treatment procedures and the possible regrowth as biofilms in plumbing materials. The detection of large proportion of MAR Aeromonas and Pseudomonas species which possessed virulent genes was a cause of concern as these could pose health risks to humans. The data obtained herein may be useful in assessing the health risks associated with the consumption of contaminated water. / PhD (Microbiology), North-West University, Potchefstroom Campus, 2014

An ideal drinking water distribution system (DWDS) must supply safe drinking water with free chlorine residual (FCR) in the form of HOCI and OCIֿ at a required concentration level. Meanwhile the FCR is consumed in the bulk liquid phase and at the DWDS pipes wall as the result of chemical reactions. Because of these, an optimized chlorine dosing for the DWDS using model-based predictive control (MBPC) is developed through the steps of modelling the FCR transport along the main pipes of the DWDS, designing chlorine dosing and implementing a multiple-input multiple-output system control scheme in Matlab 7.0.1 software. Discrete time-space models (DTSM) that can be used to predict free chlorine residual (FCR) concentration along the pipes of the DWDS over time is developed using explicit finite difference method (EFDM). Simulations of the DTSM using step and rectangular pulse input show that the effect of water flow rate velocity is much stronger than the effect of chlorine effective diffusivity coefficient on the FCR distribution and decay process in the DWDS main pipes. Therefore, the FCR axial diffusion in single pipes of the DWDS can be neglected. Investigating the effect of injection time, initial chlorine distribution, and overall chlorine decay rate constant involved in the process have provided a thorough understanding of chlorination and the effectiveness of all the parameters. This study proposed a model-based chlorine dosing design (MBCDD) based on a conventional-optimum design process (CODP) (Aurora, 2004), which is created for uncertain water demand based on the DTSM simulation. / In the MBCDD, the constraints must be met by designing distances between chlorine boosters and optimal value of the initial chlorine distribution in order to maintain the controlled variable (CV), i.e. FCR concentration with a certain degree of robustness to the variations of water flow rate. The MBCDD can cope with the simulated DWDS (SDWDS) with the conditions; the main pipe is 12 inch diameter size with the pipe length of 8.5 km, the first consumers taking the water from the point of 0.83 km, the assumed pipe wall chlorine decay rate constant of 0.45 m/day, and the value of chlorine overall decay rate constants follow Rosman's model (1994), by proposing a set of rules for selecting the locations for additional chlorine dosing boosters, and setting the optimal chlorine dosing concentrations for each booster in order to maintain a relatively even FCR distribution along the DWDS, which is robust against volumetric water supply velocity (VWS) variations. An example shows that by implementing this strategy, MBCDD can control the FCR along the 8.5 km main pipe of 12 inch diameter size with the VWS velocity from 0.2457 to 2.457 km/hr and with the assumed wall and bulk decay constants of 0.45 and 0.55 m/day, respectively. An adaptive chlorine dosing design (ACDD) as another CODP of chlorine dosing which has the same concept with the MBCDD without the rule of critical velocity is also proposed in this study. The ACDD objective is to obtain the optimum value of initial chlorine distribution for every single change in the VWS. Simulation of the ACDD on the SDWDS shows that the ACDD can maintain the FCR concentration within the required limit of 0.2-0.6 mg/1. / To enable water quality modelling for studying the effectiveness of chlorine dosing and injection in the form of mass flow rate of pure gaseous chlorine as manipulated variable (MV), a multiple-input multiple-output (MIMO) system is developed in Simulink for Matlab 7.0.1 software by considering the disturbances of temperature and circuiting flow. The MIMO system can be used to design booster locations and distribution along a main pipe of the DWDS, to monitor the FCR concentration at the point just before injection (mixing) and between two boosters, and to implement feedback and open-loop control. This study also proposed a decentralized model-based control (DMBC) based on the MBCDD-ACDD and centralized model predictive control (CMPC) in order to optimize MV to control the CV along the main pipe of the DWDS in the MIMO system from the FCR concentration at just after the chlorine injection (CVin) to the FCR concentration (CVo) before the next chlorine injection with the constraints of 0.2-0.6 ppm for both the CVin and CVo. A comparison of the performances of decentralized PI (DPI) control, DMBC and CMPC, shows that the performances of the DMBC and CMPC in controlling the MIMO system are almost the same, and they both are significantly better than the DPI control performance. In brief, model-based predictive control (MBPC), in this case a decentralized model-based control (DMBC) and a centralized predictive control (CMPC), enable optimization of chlorine dosing for the DWDS.

The purposes of this study include: (1) investigating the reasons why drinking water quality degrades during transportation in the distribution system and developing an easy and effective tool to evaluate the status of distribution system; (2) investigating residents¡¦ satisfaction with advanced treated drinking water. It is found that the main reason of drinking water degradation is that most people don¡¦t flush the drinking water storage facilities routinely. It is also found that although most respondents are satisfied with advanced treated drinking water, nearly 40% of local residents still buy bottle water instead of drinking tap water. Therefore, Taiwan Water Supply Corp. (TWSC) should let people know the importance of flushing water storage facilities routinely and what TWSC has done to improve drinking water quality. The LSI (Langelier Saturation Index) of most water samples is negative, which means that the drinking water is corrosive when too much hardness is removed to comply with the regulations. A simple, efficient and cost-effective method is developed to provide TWSC sufficient information to solve the problems regarding water quality degradations in distribution systems. By using contour maps of different water quality parameters, TWSC can easily identifies locations with potential problems and easily assesses the necessity and appropriate locations of building re-chlorination stations, even though the lack of information regarding pipeline material, hydraulic conditions, thickness of biofilm¡Ketc.

There is currently growing concern about the presence of heterotrophic plate count (HPC) bacteria in drinking water. These HPC may have potential pathogenic features, enabling them to cause disease. It is especially alarming amongst individuals with a weakened immune system. South Africa, the country with the highest incidents of HIV positive individuals in the world, mainly uses these counts to assess the quality of drinking water in terms of the number of micro-organisms present in the water. These micro-organisms may be present in the bulk water or as biofilms adhered to the surfaces of a drinking water distribution system. The current study investigated the pathogenic potential of HPC bacteria occurring as biofilms within a drinking water distribution system and determined the possible presence of these micro-organims within the bulk water. Biofilm samples were taken from five sites within a drinking water distribution system. Fifty six bacterial colonies were selected based on morphotypes and isolated for the screening of potential pathogenic features. Haemolysin production was tested for using sheep-blood agar plates. Of the 56, 31 isolates were ?-haemolytic. Among the 31 ?-haemolytic positive isolates 87.1% were positive for lecithinase, 41.9% for proteinase, 19.4% for chondroitinase, 9.7% for DNase and 6.5% for hyaluronidase. All of the ?-haemolytic isolates were resistant to oxytetracycline 30 ?g, trimethoprim 2.5 ?g and penicillin G10 units, 96.8% were resistant to vancomycin 30 ?g and ampicillin 10 ?g, 93.5% to kanamycin 30 ?g, 74.2% to chloramphenicol 30 ?g, 54.8% to ciprofloxacin 5 ?g, 22.6% to streptomycin 300 ?g and 16.1% to erythromycin 15 ?g. Nineteen isolates producing two or more enzymes were subjected to Gram staining. The nineteen isolates were all Gram-positive. These isolates were then identified using the BD BBL CRYSTALTM Gram-positive (GP) identification (ID) system. Isolates were identified as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus and Kocuria rosea. 16S rRNA gene sequencing was performed to confirm these results and to obtain identifications for the bacteria not identified with the BD BBL CRYSTALTM GP ID system. Additionally identified bacteria included Bacillus thuringiensis, Arthrobacter oxydans and Exiguobacterium acetylicum. Morphological properties of the different species were studied with transmission electron microscopy (TEM) to confirm sequencing results. All the isolates displayed rod shaped cells with the exception of Arthrobacter oxydans being spherical in the stationary phase of their life cycle. Bulk water samples were taken at two sites in close proximity with the biofilm sampling sites. The DNA was extracted directly from the water samples and the 16S rRNA gene region was amplified. Denaturing gradient gel electrophoresis (DGGE) was performed to confirm the presence of the isolates from the biofilm samples in the bulk water samples. The presence of Bacillus pumilus and Arthrobacter oxydans could be confirmed with DGGE. This study demonstrated the presence of potentially pathogenic HPC bacteria within biofilms in a drinking water distribution system. It also confirmed the probable presence of two of these biofilm based bacteria in the bulk water. / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2010.

The presence of heterotrophic plate count (HPC) bacteria in drinking water distribution systems is usually not considered harmful to the general consumer. However, precautions must be taken regarding the immunocompromised. All water supply authorities in South Africa are lawfully required to provide consumers with high-quality drinking water that complies with South African-and international standards. This study mainly focused on the isolation, identification and characterization of HPC and other bacteria from biofilm-and bulk water samples from two sampling points located within the Potchefstroom drinking water distribution system. Based on five main objectives set out in this study, results indicated that the bulk water at the J.S. van der Merwe building was of ideal quality fit for lifetime consumption. Application of enrichment-and selective media allowed for the isolation of 12 different bacterial morphotypes. These were identified by way of biochemical-and molecular methods as Bacillus cereus, Bacillus subtilis, Brevundimonas spp., Clostridiaceae, Corynebacterium renale, Flavobacteriaceae, Kytococcus sedentarius, Leuconostoc lactic, Lysinibacillus sphaericus, Pseudomonas spp., Staphylococcus aureus and Staphylococcus capitis. The greatest diversity of bacteria was detected early autumn 2008, while the lowest diversity occurred during mid-winter 2007. Bacillus cereus, Kytococcus sedentarius and Staphylococcus capitis displayed potential pathogenic properties on blood agar. Kytococcus sedentarius could be classified as potentially the most pathogenic among the isolates. All isolates displayed multiple-resistant patterns towards tested antibiotics. Corynebacterium renale and Staphylococcus aureus were least resistant bacterial species and Lysinibacillus sphaericus the most resistant. All isolates were susceptible to ciprofloxacin (CIP) and streptomycin (S), but most were resistant to erythromycin (E). Transmission electron microscopy (TEM) allowed for detailed examination of Brevundimonas spp., Pseudomonas spp. and Staphylococcus spp. The capability of Brevundimonas spp. to produce slime and store nutrients within inclusion bodies, suggests the ability of this bacterium to form biofilm and persist in the drinking water for prolonged periods. Despite the inhibitory or toxic effect of copper against bacterial growth, scanning electron microscopy (SEM) revealed the presence of biofilms as well as diatoms on red-copper coupons. Biofilm activity was also observed on reverse-osmosis (RO) filters. Since corrosion was evident on red-copper coupons, it is recommended that prospective studies also look into the significance of microbial induced corrosion (MIC) within the Potchefstroom drinking water distribution system. Other prospects include determining minimum inhibitory concentrations of isolates against antibiotics and the application of culture independent methods such as SSCP and DGGE to investigate biofilm development. The use of diatoms as an index of the drinking water quality is also suggested. / Thesis (M.Sc. (Environmental Science))--North-West University, Potchefstroom Campus, 2010.

There is currently growing concern about the presence of heterotrophic plate count (HPC) bacteria in drinking water. These HPC may have potential pathogenic features, enabling them to cause disease. It is especially alarming amongst individuals with a weakened immune system. South Africa, the country with the highest incidents of HIV positive individuals in the world, mainly uses these counts to assess the quality of drinking water in terms of the number of micro-organisms present in the water. These micro-organisms may be present in the bulk water or as biofilms adhered to the surfaces of a drinking water distribution system. The current study investigated the pathogenic potential of HPC bacteria occurring as biofilms within a drinking water distribution system and determined the possible presence of these micro-organims within the bulk water. Biofilm samples were taken from five sites within a drinking water distribution system. Fifty six bacterial colonies were selected based on morphotypes and isolated for the screening of potential pathogenic features. Haemolysin production was tested for using sheep-blood agar plates. Of the 56, 31 isolates were ?-haemolytic. Among the 31 ?-haemolytic positive isolates 87.1% were positive for lecithinase, 41.9% for proteinase, 19.4% for chondroitinase, 9.7% for DNase and 6.5% for hyaluronidase. All of the ?-haemolytic isolates were resistant to oxytetracycline 30 ?g, trimethoprim 2.5 ?g and penicillin G10 units, 96.8% were resistant to vancomycin 30 ?g and ampicillin 10 ?g, 93.5% to kanamycin 30 ?g, 74.2% to chloramphenicol 30 ?g, 54.8% to ciprofloxacin 5 ?g, 22.6% to streptomycin 300 ?g and 16.1% to erythromycin 15 ?g. Nineteen isolates producing two or more enzymes were subjected to Gram staining. The nineteen isolates were all Gram-positive. These isolates were then identified using the BD BBL CRYSTALTM Gram-positive (GP) identification (ID) system. Isolates were identified as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus and Kocuria rosea. 16S rRNA gene sequencing was performed to confirm these results and to obtain identifications for the bacteria not identified with the BD BBL CRYSTALTM GP ID system. Additionally identified bacteria included Bacillus thuringiensis, Arthrobacter oxydans and Exiguobacterium acetylicum. Morphological properties of the different species were studied with transmission electron microscopy (TEM) to confirm sequencing results. All the isolates displayed rod shaped cells with the exception of Arthrobacter oxydans being spherical in the stationary phase of their life cycle. Bulk water samples were taken at two sites in close proximity with the biofilm sampling sites. The DNA was extracted directly from the water samples and the 16S rRNA gene region was amplified. Denaturing gradient gel electrophoresis (DGGE) was performed to confirm the presence of the isolates from the biofilm samples in the bulk water samples. The presence of Bacillus pumilus and Arthrobacter oxydans could be confirmed with DGGE. This study demonstrated the presence of potentially pathogenic HPC bacteria within biofilms in a drinking water distribution system. It also confirmed the probable presence of two of these biofilm based bacteria in the bulk water. / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2010.