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Investigation into the bacterial pollution in three Western Cape rivers, South Africa and the application of bioremediation strategies as clean-up technologyPaulse, Arnelia Natalie January 2008 (has links)
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.
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Investigation into the metal contamination of three rivers in the Western Cape and the subsequent application of a bioreactor system as remediation technologyJackson, Vanessa Angela January 2008 (has links)
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.
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Comparison of diagnostic tools and molecular based techniques for the rapid identification of Escherichia coli and coliforms in contaminated river waterNdlovu, Thando January 2013 (has links)
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.
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