Magister Scientiae - MSc (Biotechnology)

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Development of gold nanoparticles based lateral flow assay for detection of food and water-borne pathogens
(Universty of the Western Cape, 2024) Mabhude, Yandiswa; Sibuyi, Nicole
Food and water are two of the necessities required for survival. With increasing global population, urbanization, and climate change, to name a few, the strain on these two precious resources puts pressure on health as well as the economic sector. Microbial contamination of food and water can lead to lethal infections that are detrimental to human health. More than 90% of reported food-borne illnesses were caused by bacterial species such as Staphylococcus, Salmonella, Campylobacter, Listeria, Bacillus etc. In the last decade, Salmonella was the leading cause of bacterial food-borne illnesses; followed by Campylobacter and Listeria. Listeriosis cases were reported in 2017 in Gauteng province, and the number of infected persons rapidly escalated nationwide prompting an investigation wherein a total of 937 cases were identified. Early detection of pathogens in food and water could prevent or slow down the spread of infections and deaths caused by consuming contaminated food and water. The laboratory and on-site approaches that are currently used for quality control technologies are precise and sensitive, but they have drawbacks such as being laborious and time-consuming. Therefore, there is an urgent need for approaches that may be utilized to detect microbial contaminations at a point-of-care (PoC), that are user-friendly, quick, robust, and sensitive. The use of gold nanoparticles (AuNPs)-based lateral flow assay (LFA) for the detection of these pathogens is gaining traction among researchers as an alternative to conventional methods for on-site testing in places where there is no proper infrastructure, particularly low-resourced areas. The current study aimed to develop an aptamer-AuNPs-based LFA for the detection of food and water pathogens at PoC.
Domestic greywater systems: a potential reservoir of antibiotic resistance genes transfer
(University of the Western Cape, 2024) Kariem, Min-ghah; McCullough, Bronwyn Kirby
During the Western Cape drought from 2015 to 2018, the use of greywater to alleviate pressure on the limited potable water available in the province was encouraged. Greywater, however, has the potential to harbour residual antibiotics and pathogenic bacteria, thereby potentially supporting the growth and proliferation of antibiotic resistant bacteria. This study combined traditional microbiology tests with molecular biology to detect resistant microorganisms within domestic greywater systems and their associated biofilms, while Quantitative PCR (qPCR) was used to determine the levels of clinically relevant antibiotic resistance genes (ARGs) (vanA, ampC and aadA), as well as the intI1 gene, which serves as a marker of horizontal gene transfer. Phenotypic resistance was confirmed using the Kirby-Bauer disc diffusion method. Notably, high levels of resistance to ampicillin and kanamycin were found in greywater and biofilm samples, as well as resistance to vancomycin. Bacterial viability was assessed using flow cytometry using the LIVE/DEAD BacLight Bacterial Viability kit, and it was revealed that a large portion of the cell population within greywater was dead or injured. qPCR analysis confirmed the presence of clinically relevant ARGs vanA and ampC, in domestic greywater samples, with abundance fluctuating in response to seasonal change. Additionally, the intI1 gene was detected in all greywater and biofilm samples, which suggests that genetic exchange occurs amongst bacteria in greywater and biofilm samples. Our findings support the hypothesis that greywater systems are colonised by resistant bacteria, which can form stable communities within the water environment which allows for high levels of genetic exchange. These findings support the use of qPCR and flow cytometry, in combination with traditional microbiology tests for monitoring antibiotic resistance in environmental samples. While the use of greywater is a viable method to reduce the demand for potable water, this study highlights the fact that the improper use of greywater poses a risk to the environment and public health. As such, the public should be better educated on how to safely use these systems.
Biochemical characterisation of an alpha-amylase with pullulan hydrolase type III characteristics derived from a hot spring metagenomics library
(University of the Western Cape, 2024) Boersma, Bianca; Trindade, Marla
The field of metagenomics has provided biotechnological researchers with new enzymes and insight into previously unknown enzyme families with potential for novel modes of action and being able to function under extreme conditions, such as extremes of temperature, acidity or alkalinity. The enzymes can also often act on multiple substrates and show increased affinity for certain substrates. In this study, the aim was to biochemically characterise a putative type III pullulan hydrolase (PHTIII). A sequence-based metagenomic screen from previous performed by Xiao Ping Hu (2010) was used to identify novel glycosyl hydrolase enzymes in shotgun metagenomic sequence data from the Mphizi hot spring in Malawi. An identified ORF, named Pull3.1, displayed low sequence identity to a characterised α-amylase (62.71%) and 61.66% to a characterised pullulan hydrolase type III, and was, therefore, hypothesized to be an α-amylase (EC 3.2.1.1) with pullulan hydrolase type III (EC 3.2.1.x) characteristics. Further sequence analysis indicated that Pull3.1 had a glycogen binding site and that it belongs to the GH13_20 family as well as having a family 48 carbohydrate-binding module. Pull3.1 displayed the highest substrate conversion rates for starch, pullulan and glycogen. The products released were of the following degrees of polymerisation 3, 2 or 1, in accordance with the products released from the PHTIII from Thermococcus aggregans. Pull3.1 is a thermozyme that shows maximum activity at 75°C and an optimum pH of 7. It was thermostable up to 80°C and had a half-life of 11.6 min at 50°C. Further kinetic characterisation showed that Pull3.1 had a high affinity for starch. The KM value of 0.048 mg/ml is within the range reported for other α-amylase enzymes, and Pull3.1 had a Vmaxof 0,729 (μmol mL−1). All of this data indicates that Pull3.1 is a good candidate for a starch conversion process that requires thermozymes.
South African medicinal plant extracts used in the treatment of fungal infections.
(University of the Western Cape, 2023) Ndlovu, Banele Michelle; Rahaman Fisher, F
The incidence of fungal skin infections is increasing at an alarming rate, especially in people with underlying immunosuppressive conditions. Currently, the most used methods of treatment are in the azole group of synthetic treatments. A major problem with these synthetic treatments’ is the increase in azole-resistant strains spreading worldwide. Additionally, an estimated 65–80% of all fungal infections are biofilm related, with biofilms found in Candida species such as Candida albicans, Candida dubliniensis, Candida glabrata and Candida tropicalis which infect the skin. There is an increased demand for alternative treatments that could be antifungals and medicinal plants could be the potential solution, as some plants possess antifungal properties. The overall aim of this study is to investigate the potential effect of South African medicinal plants used for the treatment of fungal infections against pathogens of dermatological relevance. The objectives were 1) to conduct a comprehensive literature search to identify indigenous medicinal plant extracts used to treat fungal skin infections, 2) to perform a cross-sectional study using an interview-based questionnaire to investigate the knowledge and practices of Cape bush doctors and validate their use of selected medicinal plants for treating dermatophyte infections in the Cape Metropole communities and 3) to investigate the in vitro bioactivity of selected plant extracts identified from the literature search and interviews. Fifteen medicinal plants were found using an in-depth literature search and all 15 plants were validated during the interviews as plants commonly used for fungal skin infections.
Valorisation of brewers’ spent grain: enzymatic hydrolysis in the production of xylooligosaccharides
(University of the Western Cape, 2023) Arries, Chelsey; den Haan, Riaan
The beverage industry constitutes approximately 26% of all food wastes, making it one of the largest contributors in this waste segment. By utilising waste or by-products from agriculture and food production in manufacturing value added compounds, the concepts of waste mitigation and green chemistry can contribute to establishing a circular bio-economy. In a biorefinery, bio-catalytic, thermal, chemical and physical techniques are used to extract valuable compounds from food and agricultural wastes. Brewers’ spent grain (BSG) is an ideal candidate for such a biorefinery approach. This high moisture, nutrient-rich by-product from beer production is either disposed of in landfills or used as an animal feed. However, high value products, such as xylooligosaccharides (XOS) can be extracted from BSG, thereby valorising this brewery waste. Xylooligosaccharides are sought after for their ability to function as a low caloric sweetener while exhibiting prebiotic effects in stimulating the growth of probiotic bacteria in the mammalian gut.
Antibacterial effects of biogenic silver nanoparticles synthesized using manihot esculenta (cassava) leaf aqueous extract
(University of the Western Cape, 2023) Oliver, Toni Charlize Alexia; Madiehe, Abram
Antimicrobial resistance (AMR) is a growing global health concern that poses a serious threat to the health of humans, animals, and plants. The major public health problem of AMR is primarily caused by the incorrect use of antibiotics, which is further compounded by the reduced novel antibiotics discovery rate. AMR bacteria cause infections that are difficult to treat, resulting in prolonged hospital stays and increased healthcare costs. Moreover, AMR is associated with a high risk of morbidity and mortality. Current therapeutic strategies for AMR infections are often inefficacious, associated with side effects, and may further exacerbate AMR. Therefore, there is an urgent need to develop alternative strategies to treat AMR bacterial infections.
Evaluation of the anticancer and molecular effects of green synthesized silver nanoparticles
(University of the Western Cape, 2023) Simon, Sohail; Madiehe, Abram
Globally, cancer is considered the second leading cause of death with a staggering tally of 10 million deaths. Breast, lung, colon, rectum and prostate cancers are amongst the most commonly diagnosed cancers. The current cancer treatment strategies such as chemotherapy, radiotherapy, and surgery; are accompanied with a long list of side effects. Nanotechnology has shown potential in improving biocompatibility of the chemotherapeutic drugs, and also provides a novel approach for the development of novel cancer treatment strategies through the application of the widely investigated nano-scaled materials known as nanoparticles (NPs). These NPs have been used as drug delivery systems and have, therefore, made a mark in the fight against cancer. Furthermore, their anti-cancer properties make them prospective anti-cancer agent.
Biosurfactant screening through metagenomic and culture-based approaches
(University of the Western Cape, 2022) Sithole, Nombuso Slindile; Trindade, Marla
The biosurfactant market is still predominantly represented by the three main products: surfactin, sophorolipids, and rhamnolipids, mainly as a result of the high cost and low yield associated with biosurfactant production. Moreover, only a few novel structures have been discovered in the last decade, due to a lack of interest, low hit rate, and high redundancy, in which the predominant microorganisms producing the same group of biosurfactants are isolated. Hence, there is the obvious need to open up the market with structurally and functionally diverse compounds that have the potential to tailor to specific needs or applications in different industries.
Isolation, identification and characterization of novel actinomycetes from Antarctic soil samples
(University of the Western Cape, 2008) Mavengere, Natasha R; Cowan, Don
Actinomycetes are Gram-positive bacteria that form filamentous branching hyphae with asexual spores. They occur in a variety of environments and are important industrially and pharmaceutically. The aims of this study were to isolate and identify novel actinomycetes from Antarctic soil samples and screen these isolates for the production of secondary metabolites, especially antibiotics. Actinomycetes were successfully isolated from Antarctic Dry Valley soils and optimum growth conditions were determined by investigating the effects of different temperatures and growth media on growth rates. Using phylogenetic (165 rRNA gene sequence), physiological, morphological and biochemical analyses, seven different actinomycete strains were identified, one of which was potentially novel. Isolates were screened for the production of antimicrobial compounds using the disc diffusion and stab culture methods. Antibiotic screening showed that one isolate had antimicrobial activity against eight of the ten bacterial strains tested. Another isolate was observed to inhibit the growth of some contaminating fungi. This observation implied the need to screen isolates for antifungal activity in further studies. Thin layer and high performance liquid chromatography were used in an attempt to characterize the antibiotics.
Reconstruction of major male and female lineages of the kensington Muslim community
(University of the Western Cape, 2010) Isaacs, Shafieka; Benjeddou, Mongi
The earliest Cape Muslims were brought to the Cape from Africa and Asia from 1652 to 1834. They were part of an involuntary migration of slaves, political prisoners and convicts, and they contributed in the ethnic diversity of the present Cape Muslim population. In addition to migrations, this population has grown by combined and continuous multiplex processes of intermarriage, natural increase, conversion and blending with other communities. Previous studies on the Cape Muslim population and local Cape Muslim communities have taken advantage of the huge amount of archival material covering the past three hundred years of Cape Muslim history in South Africa. The aim of this study, however was to investigate the genetic diversity and origins of one of these local Muslim communities, by taking advantage of the availability of modern molecular genetics tools. A pilot study conducted with Muslim volunteers residing all over the Cape Metropolitan area was used as a point of reference for the Kensington Muslim community study. Samples were investigated for DNA polymorphisms from both maternal (mitochondrial DNA) and paternal (Y-chromosome) in eages. Variations in these two types of DNA are grouped into continent-specific haplogroups or lineages. Six mtDNA and eight Y- chromosome SNP markers were screened using polymerase chain reaction- restriction fragment length olymorphisms (PCR-RFLP). Maternal lineages of Cape General Muslims were 34% African, 33% Asian and 33% European. The highest frequency for African lineages was observed in Cape Coloured Muslims (47%) and Cape Malay Muslims (45%). Cape Indian Muslims however displayed a higher frequency for Asian lineages (45%), while Cape Other Muslims for European lineages (50%). Paternal lineages indicated that 5% were of African descent, 75% Asian, 13% European and 7% of lineages remained undefined. Asian lineages were the most dominant paternal lineage with the highest frequency observed in Cape Indian Muslims (97%). In the second part of the study, contributions of major maternal African, Asian and European haplogroups to the Kensinglon Muslim community gene pool was investigated. This was achieved by examining mtDNA variations found within this community using twelve mtDNA coding region SNPs genotyped by the SNaPshot Mini sequencing assay. Maternal lineages of Kensington General Muslims indicated that 55% were of African descent, 34% Asiart, lYo European and 100% were of Eurasian descent. African lineages were the most frequent lineages of Kensington Coloured Muslims (56%), Kensington Malay Muslims (67%) and Kensington Other Muslims (100%). Kensington Indian Muslims maternal lineages were mainly of Eurasian origin (43%). The last part of the study investigated the contribution of the major male continent-specific lineages in the Y-chromosome diversity of the Kensington Muslim community by typing thirteen Y-chromosome SNP markers. Patemal lineages of Kensington General Muslims indicated that 4o/o were of African origin, 51% Asian and 45o% European. The highest frequency for Asian lineages was observed in Kensington Malay Muslims (56%), Kensington Indian Muslims (71%) and Kensington Other Muslims 11% (100%). However, Kensington Coloured Muslims paternal lineages were mainly of European origin (49%). The data obtained for Cape Coloured Muslims and Cape Malay Muslims and their Kensington Muslim counterpart's maternal and paternal gene pool were in agreement with historical findings concerning the origins of earlier Cape Muslims. Cape Indian Muslims and Kensington Indian Muslims maternal and paternal gene pool however demonstrated a strong accordance to mtDNA and Y-chromosome lineages observed in indigenous Indian populations.
identification of Quantitative Tralt Locl Controlling the Requirement for chilling ln Vegetative budbreak in apple (malus x domestica Borkh')
(University of the Western Cape, 2007) Van Dyk, Maria Magdalena; Rees, D. J. G; Labuschagn6, I. F
The domesticated apple (Malus x domestica Borkh.) has been distributed into diverse climatic conditions worldwide for commercial production of fruit. Apple trees need exposure to cold temperatures, referred to as chill unit (cu) accumulation during winter, in order for budbreak to occur promptly and uniformly after winter. ln warmer production areas the application of dormancy breaking chemicals has enabled successful production of high chilling requiring apple cultivars in suboptimal environmental conditions. ln the Western Cape region of South Africa it is common orchard practice to apply dormancy breaking chemicals after winter in order to stimulate vegetative growth. lf this is not done prolonged dormancy symptoms (PDS) are experienced which include extended rest' less synchronised breaking of buds and reduced branching' An increasing awareness of both global warming and the negative effects associated with the use of chemical sprays (for both pest and disease resistance and growth regulation) has resulted in the need to breed cultivars better adapted to current and future environmental conditions' The breeding of new cultivars using conventional breeding methods is a time-consuming process, especially in perennial tree species with a long juvenile phase such as apple. The implementation of marker-assisted breeding
An investigation of the plants used medicinally In self-care in the Bredasdorp / Elim region
(University of the Western Cape, 2004) Thring, T; Weitz, F
Much of the traditional medicinal plant knowledge is to a large extent known by the older generations. This knowledge is at risk of disappearing due to not being passed down to younger members of the respective families and communities. The Bredasdorp / Elim area in the Southern Overberg has many individuals who possess such knowledge. The aims of this study were to identify what plants were in use in the area, to document this knowledge and to choose certain plants to test in antimicrobial bioassays.
Isolation and identification of PNP-A homologues from Xerophyta viscosa Baker
(University of the Western Cape, 2004) Mabuto, S; Gehring, C
Maintenance of growth, development and normal physiological processes remains crucial for the survival of plants. Plant hormones are described as integrators of external signal and internal developmental process in plants. They also attribute as mediators of physiological and developmental adaptation throughout the plant life cycle (Wilkinson et al.,1997). It has been well over 50 years since Went and Thimann (1937) published their classical book Phytohorrnones. At that time, the term phytohorrnone was synonymous to auxin, although compounds like cell division factors were anticipated to be phytohormones based on physiological experiments. Since then a number of plant hormones were discovered and a series of studies concluded that plant hormones are at least partly responsible for plant growth, development, cell elongation, cell division, differentiation and stomatal movement in response to environmental stimuli. Plant hormones are categorized into three groups, known as classical, non-classical and peptide hormones. Here, Plant Natriuretic Peptide (PNP) will be reviewed as a proposed additional peptide hormone.
Investigation of the anti-obesity effects of citrate-capped gold nanoparticles on 3T3-L1 cells and their genetic profiling
(University of the Western Cape, 2023) Mbengashe, Anelisiwe; Sibuyi, Nicole
The incidence and prevalence of obesity and its related diseases has nearly tripled and is increasing at an alarming rate worldwide. Several modern methods for the management and treatment of obesity are available, with anti-obesity drugs being the preferred choice. Despite the availability of anti-obesity drugs, their therapeutic capabilities are hampered by drug toxicity and undesirable side effects, implying the need for alternative therapies. Thus, nanotechnology-based strategies may provide a novel beneficial mediator in the management and treatment of obesity.
Using CRISPR/Cas9 to construct consolidated bioprocessing strains from natural isolates of Saccharomyces cerevisiae
(University of the Western Cape, 2022) Minnaar, Letitia; den Haan, Riaan
Saccharomyces cerevisiae has gained much attention as a host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high ethanol producing titres, heterologous protein producing capabilities, and tolerance to various industry-relevant stresses. Since the secretion profiles of heterologous proteins are relatively low in industrial and laboratory strains of S. cerevisiae, natural isolates may offer a more diverse genetic background with increased robustness to allow for improved heterologous protein secretion. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core cellulase enzyme complex (CBHI, CBHII, EG and BGL), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated.
Biological and enzymatic activity of actinobacteria associated with aloe ferox
(University of the Western Cape, 2023) Isaacs, Nasreen; Kirby-McCullough, Bronwyn
Antimicrobial resistance is a global crisis that has been on the rise for decades, threatening the health and safety of many nations. This highlights the need to discover novel antimicrobial compounds to combat already resistant pathogens, as well as newly emerging pathogens. Historically soil bacteria, particularly actinobacteria, have been a source of novel bioactive compound, however, the constant re-isolation of known actinobacterial strains led to the need to explore unique environments. Higher plants, especially medicinal plants, represent a unique niche for the isolation of rare, bioactive actinobacterial species.
Developing a lateral flow device for the detection of novel breast cancer biomarkers
(University of the Western Cape, 2023) Tejada, Aaron; Meyer, M
Breast cancer is the most prevalent malignancy and one of the leading causes of death in women worldwide. Delayed diagnosis of breast cancer greatly reduces the effectiveness of treatment and is considered one of the major contributing factors to its high mortality rate. Cancer treatment is most effective during the early stages of the disease, therefore the earlier that cancer is detected, the greater the chance for survival. Currently, breast cancer screening relies largely on imaging techniques such as mammography and magnetic resonance imaging (MRI) which lack sensitivity and specificity; and are both invasive, cost-intensive and are often not easily accessible to patients in low-income countries.
Enabling the production of black soldier fly larval protein from lignocellulosic biomass through pre-treatment and enzymatic hydrolyse
(University of the Western Cape, 2023) Bothma, Michaela; Den Haan, Riaan
Due to the rapid increase in the world’s population, alternative food resources are crucial to sustain the human diet. Insects can produce valuable proteins and nutrients that can be used as high-quality animal feedstuff and help to reduce organic waste. Hermetia illucens, also known as the Black soldier fly, has attracted the attention of researchers due to the veracious nature of the fly larvae (BSFL). These insects can be reared on any organic waste, consuming large quantities of raw organic material more efficiently and faster compared to other known fly species. While consuming organic waste, BSFL build up their body composition to approximately 30% fat and 40% protein, which can be a substitute for nutritionally comparable fishmeal in pet foods and animal feed. Although BSFL are mainly used for animal feed, they represent a promising source of nutrients for human consumption, although limited knowledge regarding the use of BSFL for human consumption is available. Lignocellulose could serve as an alternative, “clean” feedstock consisting of carbohydrates, that can be fed to Black soldier fly larvae.
Analysis of the proteome of red and green leaf phenotypes of 'Bon Rouge' pear trees \trus communrls L. by 2-dimensional gel electrophoresis
(University of the Western Cape, 2007) Sehata, Majimi James; du Preez, Marlene; Ndimba, Bongani; Rees, Jasper
The 'Bon Rouge' pear is a red pear cultivar derived from a rare, spontaneous bud mutation which occurred on the green pear 'William's Bon Chretien' (Bartlett). 'Bon Rouge' pear cultivar was observed to be reverting back to its original phenotypic green colour. To study the cellular changes occurring within the cells both the mutated red phenotype and the wild type green phenotype of 'Bon Rouge' were investigated using proteomics approach. The proteins from the tissues of interest were precipitated in the presence of l0o/o TCA and solubilized in urea/thiourea lysis buffer. The proteins were separated onl2Yo SDS-PAGE and2D PAGE in order to compare their protein expression profiles. The results from SDS-PAGE and 2D PAGE profiles show an elevated level of 53 kDa protein in green pears which was either absent or less expressed in red phenotype and this protein appears to be the only differentiating factor between the red and green 'Bon Rouge' phenotypes. This protein was subsequently identified by the MALDI-TOFMS to be large subunit of RuBisCO. Expression proteomics is a preferred method due to its reproducibility and it has a potential to provide direct identification of cultivars related proteins. Additionally, proteomics approaches could help the fruit crop gtowers andbreeders, to select and determine types of different cultivars at various growth stages.
A study of the problems and possibilities of using the marine intertidal zone for teaching principles of ecology in senior secondary schools: A survey of biology teachers in the western cape.
(University of the Western Cape, 1994) Reddy, Chris; Keats, D.W.
In this study I investigate the attitudes of a group biology teachers in the Western Cape, to using the marine intertidal zone for teaching principles of ecology in senior secondary schools, by exploring the problems experienced and solutions envisaged. The study investigates the problems perceived/experienced by means of a semistructured interview and seeks solutions via teacher workshops and an excursion to the seashore. Teachers mentioned many constraints and school based problems such as time-tab1ing, teacher attitudes, lack of ethos lack of funds, Iarge numbers in classes and collectively found useful answers which reduced the emphasis of many of the problems mentioned. Problems such ds, the lack of knowledge of the marine environment, limited experience of fieldwork technique and management, could only be solved by pre and in-service teacher education programmes. The workshops produced useful solutions and suggestions for implementation by teachers, education departments and governmental and non-governmental agencies that would assist in making this a reality. These include resource development, teacher networking, peer teaching, in-service and pre-service programmes with a marine emphasis, and funding of appropriate programmes

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