Theses and Dissertations (Biosciences)

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Characterisation of Amaranthus Tricolor mutant plants with increased drought-tolerance
(Vaal University of Technology, 2010-02) Kgang, Itumeleng Eugenia; Van Emmenis, Lynelle, Dr.; Laloo, Neelan
Amaranthus tricolor (A. tricolor) is a nutritious vegetable crop that is used as a subsistence and cash crop in the rural areas in Africa. Its yield and production is severely limited by abiotic stresses such as drought. Mutation technology, using gamma irradiation, was previously employed as a tool to create genetic variation in order to select for lines with improved drought-tolerance. During irradiation, 160 Gy (Gray) was selected as the optimal dosimetry that allowed subsequent seed germination. The resulting mutant lines were screened over several generations under field and greenhouse conditions and seven promising drought-tolerant lines were selected. Here we report on physiological and morphological studies of two of these Amaranthus mutant lines (#2 and #5) to confirm the enganced drought-tolerance. Plants were grown in the greenhouse in plastic pots containing germination mix with fertiliser. They were exposed to 21 days of well-watered condition, 19 days of drought-stress conditions and 7 days of re-watering. shoot height, leaf area, protein content and relative water content (RWC) of the fresh and dry material were determined colorimetrically under well-watered and drought-stress conditions, while anthocyanin was only measured during well-watered conditions. Shoot height, leaf area, number of leaves per plant and the protein content were significantly reduced under water-stress conditions. Under well-watered condition mutant #5 grew faster with the shoot length significantly higher than mutant #2 and the wild type. Even though drought adversely affected shoot lenght, mutant#5 still performed better than mutant #2 and the wild type under drought-stress conditions. While under both well-watered and drought-stress conditions, the wild type plants had bigger leaf area compared to the two mutant lines. After 16 days of drought-stress conditions, all the leaves of the wild type plants were dried out, as a result no wild type plants recovered after 8 days re-watering. Meanwhile, both mutant #2 and #5 plants recovered significantly after 8 days of re-watering. The wild type was tolerant compared to the two mutant lines. Protein content for mutant #2 plants was higher under both well-watered and drought-stress conditions but was not significantly different from mutant #5 plants compared to the wild type plants after 19 days of drought-stress conditions. Furthermore, genetic diversity was examined in all the Amaranthus lines using random amplified polymorphic DNA (RAPD) analysis. Nineteen arbitrary RAPD markers were used of which two detected polymorphisms (OPA) 07 and OPA 16).
The effects of biofouling on a reverse osmosis membrane purification system at Sasol, Sasolburg
(Vaal University of Technology, 2011-06) Takaidza, Samkeliso; Van Wyk, C. S.; Stegmann, P., Dr.
Reverse osmosis (RO) membranes are widely used in water purification. The presence of biofilms in water and industrial water purification systems is prevalent. As a result, biofouling which is a biofilm problem causes adverse effects on reverse osmosis process, which include flux decline, shorter membrane lifetime and an increase in energy consumption The effect of biofouling on RO membranes was investigated at a water treatment facility at Sasol, Sasolburg by investigating the quality of water purified by the RO system and the extent of fouling that is attributed to biofouling. Chemical and microbiological data was averaged based on the results obtained from water analysis and samples from a fouled membrane. Bacteriological plate counts ranged between log 1.5 to 4 cfu/ml in water samples and log 3.9 to 4.5 cfu/cm2 on biofilm from the membrane surface. Water analysis indicated a high conductivity of 121 µS/cm in the feed and 81 ppm of the TDS, whereas in the permeate conductivity was found to be around 6 µS/cm and 3.8 ppm of the TDS. This indicated that components present in the feed were retained by the membrane. This was supported by membrane autopsy which showed that the bacteria and elements found in the feedwater were also present on the membrane surface, hence contributing to fouling. An average of 33% of cellular ATP was measured on the biofilm from membrane sample, showing that the fouling bacteria are metabolically active in situ. The results clearly indicated that an important biological activity occurred at the membrane surface.
Optimization and verification of changes made to US-EPA 1623 Method to analyse for the presence of Cryptosporidium and Giardia in water
(Vaal University of Technology, 2011-08-03) Khoza, M. N. L. (Mtetwa); Stegmann, P.
Methods for detecting the presence of Cryptosporidium oocysts and Giardia cysts have been developed and continuous improvement is being done to improve the recovery rate of the target protozoa. Rand Water has adopted their method for isolation and detection of Cryptosporidium oocysts and Giardia cysts in water from United State Environmental Protection Agency (US-EPA) Method 1623, 1999. In 2005 changes were made by US-EPA to the Method 1623. A study was done to improve the performance of the Rand Water Method 06 (2007) used for isolation and detection of Cryptosporidium oocysts and Giardia cysts. Three methods namely: Rand Water Method 06 (2007), US-EPA Method 1623 (2005) and Drinking Water Inspectorate standard operating procedures (2003) were compared and key different steps in the methods were identified (wrist action speed, centrifuge speed, immunomagnetic separation procedures and addition of pre-treatment steps). Different experiments were conducted to verify and evaluate the difference between two wrist action shaker speeds, three different centrifuge speeds, two slightly different immunomagnetic separation procedures and when a pre-treatment step was included in the method. Three different types of water matrices (reagent grade water, drinking water and raw water) were used for the experiments and secondary validation. Data obtained from the experiments and secondary validation was statistically analyzed to determine whether there was a significant difference in the recovery of Cryptosporidium oocysts and Giardia cysts. Secondary validation of the Rand Water Method 06 (2007) was performed by implementing the study experiments‟ findings into the method. The results indicated an increase in the recovery rate of Cryptosporidium oocysts and Giardia cysts when data was compared with the previous secondary validation report. The mean recovery of Cryptosporidium oocysts in reagent grade water samples increased from 31% to 55%, drinking water samples increased from 28% to 44% and raw water decreased from 42% to 29%. The mean recovery of Giardia cysts in reagent grade water samples increased from 31% to 41%, drinking water samples increased from 28% to 46% and raw water decreased from 42% to 32%. Furthermore, even though the recovery rate of raw water decreased the use of pre-treatment buffer reduced the number of IMS performed per sample by reducing the pellet size. Enumeration of microscope slides was also easier as there was less background interference. The optimization of the Rand Water Method 06 (2007) was successful as the recovery rate of Cryptosporidium oocysts and Giardia cysts from water increased. All the changes that were verified and that increased the recovery rate were incorporated into the improved Rand Water Method 06.
Is the shelf life of bottled water a cause for concern?
(Vaal University of Technology, 2011-10-27) Liee, Yvone Lieketseng; Ncube, Esper Jacob, Dr.; Van Wyk, Christa
Bottled water like any drinking water used for human consumption should be safe and wholesome to ensure adequate public health protection. This is due to potential health effects of concern such as endocrine disruption, toxicity teratogenicity, mutagenicity and carcinogenicity. Despite the number of regulatory bodies, publications on bottled water and speculations on its public health significance, many questions remain to be answered. One of the questions is whether the shelf life of bottled water is a cause for concern. The aim of the study was to determine the shelf-life of various commercial bottled waters by monitoring the variation in microbiological, chemical and aesthetic qualities of bottled water. A total of five commercial bottled water brands (A, B, C, D, E) each containing bottles from the same batch consisting of spring water, mineral water and bottled tap water were purchased directly after being bottled from different distributors around Gauteng in South Africa. All samples were stored at room temperature with artificial lighting and controlled temperature for a year thus mimicking typical conditions in retail outlets, supermarkets and in homes. Analyses were conducted over a period of 12 months, at monthly intervals. Within days of being purchased, high Heterotrophic plate counts (HPC) bacteria exceeding drinking water alert level >5 000 cfu/ml was common in four bottled water brands. Growth succession occurred during the period of study as various algal species were growing and accumulating on all bottled water tested. Total coliforms (TC), faecal coliforms (FC) and E.coli were not detected in all the bottled water tested. Yeasts and moulds were also not detected in all the bottled water. There were insignificant variations during the period of study for turbidity, pH, TDS, conductivity, and colour. These did not indicate any potential impact on aesthetic quality of bottled water. Two bottled water brands had hardness measures as low as 11mg/ℓ as CaCO3 making the water too soft which has an effect on taste. Radioactive substances, trihalomethanes, heavy metals, pesticides and other chemical contaminants were not found at levels that can be detrimental to human health.
Influence of matrix effect of selected organochlorine pesticide residues in water from the Jukskei River catchment
(Vaal University of Technology, 2011-11) Rimayi, Chengetayi Cornelius; Mtunzi, F., Dr.; Odusanya, A. O. D., Dr.; Van Wyk, C. S.
One of the major problems encountered in qualitative and quantitative determination of residual pesticides by gas chromatography is the matrix effects. Matrix components have a considerable effect on the way analysis is conducted and the quality of results obtained, introducing problems such as inaccurate quantification, low analyte delectability and reporting of false positive or even false negative results. It was aimed to develop and validate a suitable method for counteracting the matrix effects so as to improve the detection and quantification of selected organochlorine pesticide residues from real water samples. The real water samples used were sampled from three points along the Jukskei River catchment area in Gauteng, South Africa for a period of 7 months from January to July 201 0 so as to create a representative sample. An automated solid phase extraction (SPE) method coupled to Gas ChromatographyMass Spectrometry (GC-MS) method for the analysis of 20 selected organochlorine pesticides was developed and validated for the purposes of studying the matrix effects. The analytical method showed a significant degree of validity when tested against parameters such as linearity, repeatability and sensitivity. Endosulphan beta, 4,4' Dichlorodiphenyldichloroethane, and Heptachlor-epoxide had the broadest linear calibration ranges of 1 ppm- 0.0156 ppm. Benzene hexachloride (BHC) delta and Lindane had the lowest statistical limits of detection of 0.018 ppm. Statistical hypothesis testing indicated that there was significant linearity in all selected organochlorine calibration curves. Four different reversed sorbent phases, including LC18, SC18- E and Strata-X (styrene divinyl benzene) were tested for organochlorine retention efficiency. The LC-18 200 mg cartridge proved to be the most robust and effective sorbent phase as it produced better recoveries varying from 90-130% for most analytes. A breakthrough volume of 100 ml for the LC-18 200 mg cartridge was determined using an optimum matrix load curve. It was then concluded that the method developed was suitable for further research towards the influence of the matrix on selective determination of the selected organochlorine pesticides. Four different calibration methods, namely matrix-free external standard, matrixmatched external standard, matrix-free internal standard and matrix-matched internal standard were applied to test the efficiency of computing recoveries. All calibration curves for the 20 organochlorine pesticides showed significant linearity > 0.99 when plotted on both Chemstation and Excel. The calibration methods were tested on three different matrices composed of a high sample matrix (synthetic matrix), a low sample matrix (real sample matrix) and a no sample matrix (ultrapure water). Statistical hypothesis testing led to the decision that there are significant differences between the mean recoveries of the three water sample matrices and also that the differences in the mean recoveries of the three sample matrices are independent of the both the two calibration techniques (internal standard and external standard) and calibration types (matrix-matched and matrix-free) applied. This led to the overall conclusion that the matrix effects have an overwhelming influence on the selective determination of the selected organochlorine pesticides.
The environmental impact of NATREF refinery storm water on the Taaibosch spruit
(Vaal University of Technology, 2012-01) Ramotsehoa, Motsehoa Cynthia; Van Wyk, C. S.; Stegmann, P., Dr.
NATREF is the only inland refinery in South Africa and as such has unique water disposal challenges since it does not have the advantage of marine outlet like many other refineries. Most of its process streams are treated by the Sasol water treatment facility, leaving the concern of water that collects and drains off during rain fall events from the refinery site. Two sampling points were used during this study. Temperature and pH were measured in situ while bacterial counts and algal bioassays were performed in VUT laboratory. The area experienced a total of 485 mm of rain during the study period with 75 % thereof during spring and summer, there rest in autumn and winter receiving no rain. The average seasonal pH of the samples remained between 8 and 9.3 and this was found to fall within TWQR as well as being within normal range for natural waters. The temperature changes followed a typical summer/winter pattern, with Taaibosch Spruit showing greater variations from 10.95 ˚C in winter to 21.4 ˚C in summer due to its shallow nature. TDS, Nitrates & phosphates were all above the TWQR. Higher HPC & FC counts were observed during spring when rain storms began with Taaibosch Spruit had the higher of the two. As the rainfall continued into summer, the most of the bacterial counts decreased up to the lowest in winter. Higher than expected coliform counts (between 0 and 8 x 104) were observed, indicating a possible source of pollution which has to be studied. Chlorophyll a values ranged from 2.85 μg/L during spring to100 μg/L in winter indicating the potential for stimulation of the algae and possible algal blooms. The algal bioassays showed inhibition potential of the water during spring, summer and autumn with recovery by the winter. This meant that the storm water from NATREF does have a potential to cause chemical & biological pollution of the Taaibosch Spruit although the actual source of storm water pollution has to be properly studied.
The construction and evaluation of a novel tubular photobioreactor at a small pilot plant scale
(Vaal University of Technology, 2012-07) Kutama, Makonde; Van Wyk, C.; Stegmann, P., Dr.
The mass production of algae for commercial purposes has predominately been carried out in open ponds systems. However, open ponds systems have a number of disadvantages such as poor light utilization, requirement for large areas of land and high risks of contamination. On the other hand, photobioreactors have attracted much interest because they allow a better control of the cultivation conditions than open systems. With photobioreactors, higher biomass productivities are obtained and contamination can be easily prevented. Photobioreactors can also be engineered to manipulate the light and dark photosynthetic reactions thus enhancing biomass productivity. The main objective of this study was to construct a novel tubular photobioreactor which had the ability to expose the cultured alga to light and dark phases with the aim of optimizing the algal biomass production. A novel tubular photobioreactor with the ability to manipulate the cultured alga’s light and dark photosynthetic reactions was constructed in this study. The alga Spirulina platensis was chosen as the test organism in this novel tubular photobioreactor due to a number of reasons such as its globally socioeconomic importance, its tolerance of higher pH and temperature values which makes it almost impossible to contaminate. The cultivation process of Spirulina in the photobioreactor was investigated through alternating light and dark cycles in an attempt to increase the photosynthetic efficiency of the culture. The effect of different light intensities on the growth of Spirulina in the novel tubular photobioreactor was investigated and it was found that the best light condition that favored higher biomass formation was at 600 μ mol m-2 s-1. Five different light/ dark ratios were evaluated at a light intensity of 600 μ mol m-2 s-1 during a batch mode of operation of the novel tubular photobioreactor. The light/ dark ratio of 1:0.25 was found to be the best ratio because it gave the highest biomass in the shortest period of time when compared to the other ratios used. These results seem to suggest that longer light cycle relative to dark cycle results in higher biomass production. The ratio of 1:0.25 was then used to operate the novel tubular photobioreactor in a continuous mode. A maximum biomass productivity of 25 g/m2/day was achieved which corresponded to a net photosynthetic efficiency of 5.7 %. This result was found to be higher than what most photobioreactors could achieve but it was 2.8 g/m2/day lower than the highest ever reported productivity in a photobioreactor when Spirulina is cultivated. The 2.8 g/m2/day lower was attributed to the different materials used in the construction of these two photobioreactors. The photobioreactor which achieved 27.8 g/m2/day was made up of a clear glass whereas the novel tubular photobioreactor was made up of a PVC tubing. PVC tubes tend to change from clear to a milky colour after a certain period when it is used at higher temperature and pH values hence blocks a certain amount of light. Therefore the main recommendation in this study is to use a PVC tubing with a longer life span when used at a higher temperature and pH values.
The efficiency of three shRNAs in silencing the galactose-1-phosphate uridyl transferase gene
(Vaal University of Technology, 2013) Nokoane, Mmateisi Patricia; Van Wyk, Christa; Lebea, Phiyane J., Dr.; Pillay, M., Prof.
This study seeks to design and test specific short hairpin RNA (pshRNA) for their efficiency in knocking down the GALT gene RNA products thereby limiting the resultant enzyme activity. The following objectives were followed in designing the current study: 1. Designing a shorthairpin RNA (pshRNA) to target different regions of the coding sequence of the target GALT gene. 2. Propagating the pshRNAs in Escherichia coli (E.coli) and subsequently isolation of the respective plasmids for transfection. 3. Transfection of HeLa cells to test the efficiency of relevant pshRNAs in knocking down the GALT gene expression. 4. Transfection was followed by extraction of total mRNA, purification and quantification of total mRNA. 5. The GALT gene expression was qualitatively quantified against a house-keeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) to evaluate efficiency of knockdown using real time PCR. The three newly designed pshRNA (pshRNA2, pshRNA3 and pshRNA4) targeting the GALT gene expression showed a knockdown efficiency of 171 %, 48 % and 200 %, respectively. The results of this study will be useful for future evaluation of the possible long term glycosylation patterns under proper UDP glucose/UDP galactose levels compared with variable defective GALT gene levels.
Assessing the genetic diversity of South African sweetpotato germplasm using DNA and protein markers
(Vaal University of Technology, 2013-06) Selaocoe, Maleshoane Ellen; Adebola, Patrick, Dr.; Pillay, Michael, Prof.
Sweetpotato is one of the most important food crops in developing countries including South Africa. Currently two major types of cultivars are grown in South Africa: one is the orange-fleshed sweetpotato (OFSP) which has high β-carotene content, a precursor of vitamin A. The second type is the cream-fleshed sweetpotato (CFSP) which has low β-carotene content but is high in dry matter. Most South Africans prefer the CFSP although the OFSP offers more advantages. This presents a challenge to plant breeders to develop new varieties that will combine the desirable qualities of both the cultivars. To achieve this goal, plant breeders need knowledge about the genetic variation of the crop to develop an efficient breeding programme. This study assessed the genetic relationships of 28 orange- and cream-fleshed sweetpotato accessions by (i) examining the variation in leaf proteins, (ii) using random amplified polymorphic DNA (RAPD) and, (iii) using variation of the ITS region. The analysis of proteins, RAPD and variation of the ITS region polymorphism levels were 55.6%, 98% and 16.5%, respectively. Dendrograms generated from all the analyses generally clustered the accession according to their flesh colour and country of origin. Analysis of molecular variance (AMOVA) found a significant difference between OFSP and CFSP and a significant difference between the South African and non-South African germplasm. The high genetic diversity in the South African sweetpotato germplasm is a positive indicator for a breeding programme that has a number of targets such as breeding for nutritional improvement, disease resistance and drought tolerance
Genetic diversity analysis and determination of Calcium Oxalate Crystals in South African Taro (Colocasia Esculenta) accessions
(Vaal University of Technology, 2014) Nguluta, Mwamba; Adebola, P., Dr.; Pillay, M., Prof.
Taro [Colocasia esculenta (L) Schott] belongs to the family Araceae. It is an important staple food crop grown mainly by small scale farmers in many parts of the world. Taro is also grown in South Africa from the costal parts of the northern Eastern Cape to the KwaZulu-Natal north coast. Although it is an important staple crop in South Africa, very little information exists on the genetic diversity of the crop. Knowledge of the genetic diversity of a crop is important for breeding programmes. The aim of this study is to assess the genetic diversity of taro using morphological and molecular techniques and to determine the calcium oxalate content of 25 South African taro accessions. This study showed that the aerial portions of taro are variable for most quantitative characters. Most of the morphological variation was due to lamina length, petiole length, lamina width and plant girth that explained 54% of the variance in principal component analysis. The number of raphides was able to divide the accessions into two groups, one with relatively low counts and the other with high counts. Ntumeni had the lowest raphide count of only 27 ±12 raphides and Modderfontein had the highest count with 1150 ±104 raphides. Twelve accessions having low raphide counts ranging from 27 ±12 to 147 ±28 raphides per cell have been identified. RAPD data separated the accessions into three main groups that were further divided into five subgroups. The accessions did not group according to geographical locations. The ITS2 sequence generated clustering patterns that were similar to that obtained from RAPDs. The variation in the ITS2 secondary structure of taro included one common motif that was present in all 25 accessions. Some motifs were only present in some accessions. The discovery of these motifs strengthens the potential of the ITS2 region as a taxonomic marker and a powerful barcode for taro. The ITS2 motifs provide the means of identifying each of the 25 accessions of taro. The high genetic diversity, morphological variation and accessions with low calcium oxalate content found in this study provide taro breeders a selection of parent crops for the improvement of taro.
Biochemical and molecular characterization of heavy metal resistant bacteria isolated from the Klip River, South Africa
(Vaal University of Technology, 2014) Chihomvu, Patience; Stegmann, P., Dr.; Pillay, M., Prof.
The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.
Co-production of inulinase by Kluyveromyces marxianus and Saccharomyces cerevisiae in solid state fermentation
(Vaal University of Technology, 2014-02) Molefe, Nnana Mantsopa; Reddy, P.; Padayachee, T., Prof.
Solid-state fermentation (SFF) has emerged as a good method for the production of microbial enzymes such as inulinases. The use of low-cost agricultural plants and agro-industrial residues as substrates in SSF processes provides a value adding alternative to these otherwise under/or un-utilised vegetation. Production of inulinases, using various inulin-containing plant materials as carbon sources was studied using pure and mixed cultures of yeast strains. All substrates resulted in different levels of enzyme activity. A mixed culture of Kluyveromyces marxianus and Saccharomyces cerevisiae produced an extracellular exoinulinase when grown on different types of inulin-containing plant materials. Initial inulinase production was achieved as follows: 10 IU/gds (garlic cloves), 15 IU/gds (parsnips), 10 IU/gds (wheat bran) and 7 IU/gds (amadumbe) by K. marxianus and S. cerevisiae in a mixed culture. The production of inulinases by a mixed culture of K. marxianus and S. cerevisiae under SSF was further optimized by investigating initial moisture content, temperature, carbon source, nitrogen source, inoculum volume and inoculum ratio. The highest inulinase activity attained was in garlic cloves (85 IU/gds), followed by parsnips (65 IU/gds), wheat bran (37 IU/gds) and amadumbe (25 U/gds). The activities yielded 5.6 fold higher inulinase than in preliminary studies. The optimum pH and temperature of the crude enzyme were 5.0 and 50 oC, respectively. The pH and temperature stability of the enzyme was steady for 1 hour retaining about 64% activity. The average inulinase/invertase activity (I/S) ratio of 1.0 by crude inulinases was also observed after 48 hours. The crude extracellular enzyme extracts from the garlic cloves, parsnips, amadumbe and wheat bran were partially purified by ammonium sulphate precipitation and showed a specific activity of 9.03 U/mg, 0.08 U/mg, 4.12 U/mg and 0.133 U/mg respectively. The Km and Vmax values of the inulinase were 21.95 mM and 2.09 μM/min; 19.79 mM and 1.38 μM/min; 31.59 mM and 0.51 μM/min; and 25.74 mM and 0.23 μM/min, respectively. All extracts demonstrated potential for large-.scale production of inulinase and fructose syrup.
Development of an "A" genome-specific sequence characterised amplified region (SCAR) marker in Musa L. (bananas and plantains)
(Vaal University of Technology, 2014-09) Mabonga, Lloyd; Pillay, M., Prof.
Most cultivated bananas and plantains (Musa spp. sect. Eumusa), originated from two wild diploid species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), which contributed the A and B genomes, respectively. The two genomes confer different traits to a banana plant. Intra- and interspecific hybridization between the wild diploid species, somatic mutations and selection over many thousands of years has given rise to considerable genetic variability in cultivated bananas. Bananas are classified according to its genome composition and a number of morphological traits are used to identify the genomes of a plant. Morphological classification can be misleading since the morphology of plants can be affected by environmental factors. Molecular techniques to identify the genomes of banana have many advantages. The objective of this study was to develop a SCAR (sequence characterized amplified region) marker from a previously reported A genome-specific RAPD fragment that distinguish the A genome of banana from the B genome. This fragment designated OPA17600 was cloned, sequenced and used to design longer 20-mer SCAR primers. Verification of the SCAR primers for its fidelity to the A genome was carried out on a sample of 22 homo-and heterogenomic accessions representing landraces and hybrids of different ploidy and genome combinations. Out of six primers sets that were tested one set (SC3) produced a unique 600 bp in all the A genome containing banana accessions. However, these primers also amplified an 800 bp fragment in all the BB genotypes and some accessions containing the A and B genomes. While previous reports suggested that there was considerable differentiation between the A and B genomes, recent evidence points to the contrary. The presence of the A genome fragment in the B genome genotypes and accessions may be due to recombination between the two genomes, translocations and substitutions. The study concluded that the 600-bp SCAR sequence is conserved across the A genome in Musa and can be used to identify the A genome in banana classification and Musa breeding programmes.
Antimycobacterial activity of synthetic compounds isolated from South African medicinal plants against mycobacterium tuberculosis
(Vaal University of Technology, 2014-11) Ledwaba, Elizabeth Ramadimetsa; Bapela, N. B., Dr.; Van Wyk, Christa
Tuberculosis (TB) remains one of the most difficult infectious diseases to control in the world today. The disease spreads easily in overcrowded, badly ventilated places and among people who are undernourished. Trends in the incidence of TB together with the development of multi-drug (MDR-TB) and extensively drug resistant (XDR-TB) strains of TB raises the need to intensify the search for more efficient drugs to combat this disease. Herbal remedies used in traditional medicine provide an interesting and largely unexplored source for the discovery of potentially new drugs for infections such as TB. The aim of the study was to evaluate the in vitro antimycobacterial activity of synthesized compounds from medicinal plants against Mycobacterium tuberculosis (M. tuberculosis). About 40 synthesized compounds isolated from South African medicinal plants were screened against H37RV using microplate alamar blue assay (MABA). Identified active compounds were screened against resistant strains of M. tuberculosis (MDR, XDR and pre-XDR) and sensitive clinical isolates of TB. Cytotoxicity and synergistic drug combination studies were done on active compounds to validate their toxicity and synergy levels. Cytotoxicity was done by sulforhodamine assay (SRB) against the C2C12 cell line. Only six compounds showed activity against M. tuberculosis with minimum inhibitory concentration (MIC) below 10μg/ml. The results obtained indicated that the cytotoxicity effects of the three compounds on C2C12 cells demonstrated marginal toxicity except for MVB 282/61215 which showed a high toxicity at the lowest concentration of 0.156μg/ml with over 100% viable cells at the highest concentration (5μg/ml). MVB 282/61271 had the highest percentage cell viability (65%) at the lowest concentration. Only two compounds had a higher potency evoking a bigger response at low concentrations with treated cells still viable after 3 days of incubation with the compound which was comparable with the treatment of isoniazid (INH). Synergistic activity of the six compounds was less in INH combination as compared to the rifampicin’s (RIF) combination. The results demonstrated that the synergistic interaction between the compounds and RIF could the antituberculosis acitivity. In conclusion the synergistic effects with RIF translate to lower dosing requirements of the compounds and the potential to combat multidrug resistant TB. In deed there is no doubt that natural products, with their range of interesting chemical structures and powerful antimycobacterial effects are certain to remain important participants in the development of new generations of antimycobacterial drugs.
Assessing the genetic diversity of Alternaria Bataticola in South Africa using molecular markers
(Vaal University of Technology, 2015) Chalwe, Joseph Musonda; Adebola, Patrick, Dr.; Pillay, Michael, Prof.
Sweetpotato (Ipomoea batatas) is an important food crop that is grown in many countries. A number of viral and fungal sweetpotato diseases have been reported worldwide. One of the major and most economic diseases of the sweetpotato is Alternaria blight which is caused by the fungal pathogen Alternaria bataticola. This disease can be managed in a short term using fungicides and cultural practices. However, a long term and inexpensive approach is through the development of resistant cultivars. A prerequisite to this approach is the knowledge of the genetic diversity of this fungal pathogen. This study assessed the genetic diversity of 25 South African isolates of A. bataticola from naturally infected leaves and stems collected from different sweetpotato growing regions in South Africa by (i) characterising the isolates based on their morphology (ii) pathogenicity tests (iii) random amplified polymorphic DNA (RAPD) (iv) variation of the ITS2 sequences and (v) prediction of the ITS2 secondary structures. The isolates revealed some variation in colony colour pigments after culturing but Koch’s postulates were confirmed by their pathogenicity tests. The analysis of RAPD and variation of the ITS2 sequences showed high levels of variation (100%) among the isolates. Dendrograms generated from these analyses had many subclusters and did not cluster the isolates according to their geographic origins. The ITS2 secondary structures were predicted and can be used to identify and distinguish the isolates. This information in addition to the genetic diversity of the A. bataticola isolates will aid plant breeders in the development of resistant sweetpotato cultivars and early management of blight disease in South Africa.
Characterization and identification of microbial communities in pigeon droppings using Culture-Independent techniques
(Vaal University of Technology, 2015-08) Leareng, Samuel Keeng; Feto, Naser Aliye, Dr.; Pillay, M., Prof.
Pigeon droppings, found in abundance in most cities and towns where pigeons are found, are a source of potential yeast and molds into the environment. Invasive fungal infections are a cause of morbidity and often mortality in immunocompromised individuals. The objective of this study was to the identification of bacterial and mold agents from pigeon droppings. Pigeon droppings samples were collected from three locations during the winter and summer months and studied for the occurrence of bacteria, yeast and molds by utilising culture-independent techniques. Amplification of the 16S rDNA gene and the internal transcribed spacer (ITS) region, cloning and ARDRA and DGGE were used for the characterisation of the microbial populations followed by sequencing. Several mold and yeasts, as well as bacteria were found to be present in pigeon droppings, which can spread into the environment and be transmitted to immunocompromised individuals and children. DGGE analysis of the bacterial communities revealed banding patterns that clustered all but one winter samples and all summer samples, showing a high similarity among the microbial members in both seasons and sample locations. Fungal DGGE analysis revealed clusters that grouped summer and winter samples from Johannesburg and Pretoria while VUT samples were clustered on their own. From the identification of fungal and bacterial DNA, Cryptococcus species was the majority of fungi isolated from the dropping samples. Geotrichum, Kazachstania and Fusarium species were isolated from phylotypes obtained from ITS amplicons analysed by ARDRA. Lactobacillus and Enteroccoccus species, organisms usually found in the gastrointestinal tract were the common bacterial members identified. The results showed no difference in microbial communities across all sample locations, while seasonal changes also had no impact in microbial community patterns.
Development of a HPLC method for the detection of Levetiracetam in blood of patients with epilepsy
(Vaal University of Technology, 2016-05) Engelbrecht, Lynette; Rheeders, M.; Grobler, C. J., Dr.
Approximately 1% of the world’s population has epilepsy, the second most common neurological disorder after stroke. In South Africa almost 1 in every 100 people has epilepsy, affecting all ages. Levetiracetam (LEV), marketed as Keppra® is an anticonvulsant drug used in the treatment of epilepsy. The daily dosage is 500 mg twice daily with a maximum of 3000 mg. The therapeutic range of LEV is between 12-46 μg/ml. Therapeutic drug monitoring (TDM) should be considered for LEV in patients with poor seizure control or long term treatment. TDM depends on accurate drug concentration measurements. In order to provide an accurate measurement, the High performance liquid chromatography (HPLC) method was developed, compared with a commercially available kit, and the stability of the samples was investigated. Ethical approval was obtained from the Human Research Ethics Committee (Medical), VUT (Ethics reference number: 2015024.4). The study was conducted from January to October 2015. This study involved three groups of volunteers who gave written consent. The first group were fifteen healthy MTech students in the Biomedical Technology Department at the Vaal University of Technology (VUT). Their blood samples were used for the analytical validation of the method and for the stability studies over a 4 weeks period. The second group were six patients from Pathcare Laboratories in Potchefstroom, Klerksdorp and Vereeniging who used Levetiracetam. Their blood samples were used to investigate the influence of different collection tubes as well as the handling and storage of samples on the LEV concentration. The third group were forty four patients from Pathcare Laboratories, Cape Town. Their blood samples were transported to Clinical Pharmacokinetic Laboratory (CPL) for routine therapeutic drug monitoring analysis of LEV and used to compare the newly developed HPLC method and the Commercial kit. The HPLC method was successfully developed and validated to determine LEV in human plasma/serum samples. The calibration curves showed good linearity (r2 =0,999) over the concentration range of 1 – 60 μg/ml. Accuracy, mean extraction recovery, lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 98-112%, 97,15% (±1,57), 0,5 and 1,0 μg/ml respectively, in plasma standards. The method was shown to be simple and fast, reproducible and effective for routine laboratory analyses in the future. The agreement between the newly developed method and the ClinRep® HPLC complete commercial kit was the same and there was a statistical significant correlation between the two methods (average r=0.999; p-value > 0.0001, F-test with a true value =0). The method was much cheaper than the commercial kit, used less sample (100 μl) and had a longer running time (15 minutes) to ensure no endogenous interference. The costs of the developed method was 71-82% lower than the three commercial kits available in South Africa. Stability experiments were performed to evaluate the stability of LEV in human plasma/serum, simulating the same conditions which occurred during study samples’ analyses. The % RSD was lower than 5% under all the conditions: freeze, fridge, room temperature and auto sampler over the 4 week period. The results showed that both LEV and the I.S (internal standard) were stable in human serum/plasma under all these conditions. The influence of five different collection tubes, Gold (SST Gel), Red, Purple (EDTA)Green (Heparin) and Blue (Sodium Citrate) was investigated. In two patients, decreased levels were observed in tubes containing blue (sodium citrate) and Green (Heparin). The decrease was not statistically significant. This is an important observation and is an indication that anticoagulants may cause some problems due to drug-protein binding and interference in the matrix effect. A cost effective and reliable HPLC-method with minimal sample preparation time for the routine determination of LEV in plasma/serum samples was developed. It was also shown that the plasma/serum samples were stable at different temperatures over a time period. The only collection tubes that may interfere with the concentrations were the Green (Heparin) and Blue (Sodium Citrate) tubes.
Identification of the dominant bacteria associated with the spoilage of UHT full cream milk
(Vaal University of Technology, 2016-11) Moloto, Phuti Gladys; Ssemakalu, C., Dr.; Feto, N., Dr.; Pillay, M., Prof.
The Organization for Economic Co-operation and Development (OECD) and the Food and Agriculture Organization (FAO) of the United Nations predict that milk production and the dairy sector will remain one of the fastest-growing agricultural subsectors over the coming decade. The global milk production is projected to expand over the 2011-2020 period at an annual rate of 2%. In South Africa alone, approximately 14 – 15 million litres of milk are wasted annually due to microbial spoilage. Therefore, the identification of the spoilage microorganisms in the milk products is necessary. This will contribute towards the design of appropriate measures to prevent wastage due to spoilage and in turn contribute towards sustainability of the sector. Accordingly, one hundred samples of spoiled full cream UHT milk were collected from two plants of each of the two largest milk processors. These samples were examined visually, and the pH was measured. A presumptive identification up to genus level was conducted by examining morphological features and conducting Gram-stain, catalase and oxidase tests. Species-specific identification was done by using the Analytical Profile Index and Biolog system. Molecular profiling was done by sequencing the rDNA genes. The main spoilage organisms identified in the samples were Pseudomonas, Micrococcus, Bacillus, Enterococcus and Lactobacillus. All organisms belonging to the five genera were psychrotrophs, which are commonly found in biofilms in UHT milk processing equipment. Therefore, according to the study, the spoilage bacteria apparently entered into the milk due to inadequate cleaning-in-place (CIP) processes. More importantly, further studies should be conducted in order to identify the spoilage microbes and how CIP processes can be improved.
Assessing the morphological variation and characterising the proteins of bambara groundnut (Vigna Subterranea L. Verdc)
(Vaal University of Technology, 2016-12) Evangeline, Unigwe Amara; Adebola, P., Dr.; Pillay, M., Prof.
Bambara groundnut (Vigna subterranea L. Verdc) is an underutilized crop in the African continent. It is a drought tolerant crop and fixes atmospheric nitrogen. Bambara groundnut is primarily grown for the protein content of its seeds and is mainly produced by small scale farmers at the subsistence level. However, despite its importance as a subsistence crop in many African countries, only local landraces of bambara groundnut are still cultivated. Mass selection of a few local varieties for the main agronomic characteristics has been carried out. All the bambara groundnut germplasm in South Africa has not been morphologically characterized. Although the protein of bambara groundnut is of good quality and is rich in lysine, there is no information on the characterisation of these proteins. The presence of antinutritional factors in the crop has also received little attention. This study focused on three major objectives including: (I) to assess the extent of morphological variations among thirty selected landraces of bambara groundnut, (II) to characterize the major seed proteins in these accessions using one dimensional gel electrophoresis, and (III) to determine the presence of any anti-nutritional factors in the seeds of the selected bambara groundnut landraces. 30 accessions of bambara groundnut were evaluated for their variability in agronomic and morphological traits. The field experiment was conducted at ARC-VOPI in Roodeplaat research farm during the 2014/2015 summer cropping season. The field trial was arranged as a complete randomized block design with 3 replications. 18 quantitative traits were recorded to estimate the level of genetic variability among accessions. 4 different methods were employed to extract seed proteins from 30 bambara groundnut accessions in order to ascertain the best method for protein extraction. These methods included: 10%-80% isopropanol, 10% trichloroacetic acid (TCA) in acetone solution, sonication and 2x Lammeli buffer extraction methods. The quick start Qubit® fluorometer protein kit was used to determine the protein concentration in each sample. The samples were then subjected to one dimensional gel electrophoresis. For antinutritional analysis, 5 factors (condensed tannins, free and phytic acid phosphate, polyphenol and trypsin contents) were used to determine the amount of antinutrient in 30 bambara seeds that were ground to a fine powdery flour. 3 replicates of all the samples were ground for each assay evaluated. The flour was then immediately extracted and used for the different assays. The analysis of variance revealed significant differences only in 10 of the 18 phenotypic traits that were evaluated. The UPGMA cluster analysis based on the quantitative traits produced four distinct groups of genotypes and a singleton. Genotypes SB11-1A, SB19-1A, SB12-3B and Bambara-12 were found to possess good vegetative characters and are recommended for use as suitable parents when breeding cultivars for fodder production. Desirable yield and yield-related traits were identified in B7-1, SB4-4C, SB19-1A, Bambara-12 and SB16-5A and are recommended as suitable parental lines for bambara groundnut grain production improvement. The quantitative characters therefore provided a useful measure of genetic variability among bambara genotypes and will enable the identification of potential parental materials for future breeding programmes in South Africa. Out of the 4 different seed protein extraction methods exploited for this study, the 2x Laemmli buffer extraction method produced the best result with clear protein bands. A unique feature from all extraction methods was the presence of a common protein band at ̴ 75 kDa. All extraction methods except 10 % TCA-Acetone resolved common banding patterns in all the bambara groundnut samples. This data suggests that there is very little or no intraspecific genetic diversity among the seed proteins of bambara groundnut accessions studied. There was wide variation in the content of the five antinutritional compounds among the thirty bambara groundnut accessions. The mean values for condensed tannin content ranged between 0.20 - 6.20 mg/g. Free phosphate recorded an overall mean of 1.71 mg/g while a range of 1.35 - 4.93 mg/g was observed by phytic acid phosphate (PAP). The polyphenol content had an overall mean of 0.39 mg/g and trypsin inhibitor (TIA) was quite variable among the bambara groundnut accessions ranging from 5.30 - 73.40 TIA/mg. Generally, higher levels of antinutrients were observed in this study compared to the other studies. The results obtained in this study led to a conclusion that although variations exits among the accessions studied, further research is required to verify the extent of morphological variations, the efficiency of protein extractions methods evaluated and the effects of these antinutrients in human and animal feeds.
Correlation of saliva and serum antibody titre response to pneumococcal capsular polysaccharides
(Vaal University of Technology, 2017-10) Madimabe, Metsekae Richard; Adrian, P. V., Dr.; Grobler, C. J., Dr.
Background: Infants and young children under the age of 2 years are vulnerable to Streptococcus pneumoniae infections, especially those who are born in developing countries. Antibiotic therapy was an effective treatment until resistance to antibiotics emerged, and then vaccines were developed to assistant with the treatment. The first successful conjugate vaccine was the 7-valent pneumococcal conjugate which resulted in a decrease in vaccine serotype IPD in infants and children below the age of two years. Objectives: The main aim of this study was to assess the saliva and serum antibody concentration response to pneumococcal capsular polysaccharides in children vaccinated and unvaccinated. Design: This is a sub-study within a retrospective analysis of a prospective cohort study on the safety and immunogenicity of 7-valent pneumococcal polysaccharide-protein conjugate vaccine (PncCV) and the immunogenicity of a H. influenzae type b conjugate vaccine (HibCV). Setting and participation: Infants aged between ≥ 4 and ≤ 10 weeks were enrolled, who only received BCG and Polio vaccine following birth. Infants were enrolled according to HIV status during routine antenatal screening in the obstetrics wards of the two hospitals, in Johannesburg and Cape Town. Measurements: Saliva IgG and IgA concentrations against pneumococcal capsular polysaccharides serotype 4, 6B, 7F, 9V, 14, 18C, 19F and 23F were quantified a by multiplex bead-based assay using the Luminex technology. Serum IgG against polysaccharides serotype 4, 6B, 7F, 9V, 14, 18C, 19F and 23F were measured by a competitive Enzyme Linked Immuno-Sorbent Assay (ELISA). Results: Post three primary vaccine doses, both serum IgG and saliva IgG and IgA antibody concentrations to vaccine serotypes were protective in children who received the vaccine. The antibody concentration in children whom did not receive the vaccine was much lower in comparison to the vaccine group in both serum and saliva to vaccine serotypes (P = 0.0001). And also high positive correlation (>0.6) was observed of IgG in serum and in saliva following vaccination. Conclusion: Pneumococcal conjugate vaccines induce pneumococcal capsular polysaccharide specific antibodies in both serum and saliva. However, there are differences between the vaccines’ ability to induce mucosal immune response and there are also serotype specific differences in the antibody concentrations and in the proportion of positive samples after a series of vaccinations. The pneumococcal conjugate vaccine in this study was able to induce mucosal immune memory: the anti-pneumococcal IgA concentrations also increased with age in the saliva of unvaccinated children.

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