Doctoral Degrees (Microbiology)

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    Functionalised nanofibers for water treatment
    (2023-12) Cloete, Thomas Eugene; Wolfaardt, Gideon M.; Klumperman, Bert ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Access to safe drinking water and adequate sanitation is a pressing global issue with significant implications for health, welfare, and economic development. Nanotechnology holds promise in addressing the challenges associated with water purification. Specifically, functionalized nanofibers have emerged as a potential solution for water treatment. In one study, antimicrobial poly (vinyl alcohol) nanofibers were produced by incorporating AquaQure biocide into the polymer solution. These nanofibers demonstrated strong antimicrobial properties, achieving a substantial reduction in populations of various bacteria strains including E. coli, S. aureus, S. typhimurium, P. aeruginosa and K. pneumoniae bacteria. Additionally, immobilized enzymes were explored as a means of breaking down biofilms and preventing their formation. Non‐woven mats made of Polyacrylonitrile (PAN) were successfully fabricated using different electrospinning processes, retaining over 80% of the enzymes' specific activity. These functionalized membranes offer potential applications in the water filtration industry by preventing biological fouling of membranes. A cartridge filter where activated carbon was encased in electrospun nanofibers was manufactured and evaluated. Culturable methods indicated that the filter could produce 3 l of potable water. However, when using PCR assays, it was unable to eliminate Klebsiella spp., Legionella spp., Pseudomonas spp., and Yersinia spp. from the filtered rainwater harvested tank water. This suggested that these bacteria were in the non‐ culturable but viable state and passed through the filter. Furthermore, adenovirus, being small enough to pass through the membrane's pores, was not effectively removed by the cartridge filter. Overall, this research demonstrates the potential of functionalized nanofibers and immobilized enzymes in developing advanced filter media for water filtration. However, challenges remain in effectively removing certain pathogens and viruses, highlighting the need for further improvements and innovations in the field.
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    Elucidating the duality of biofilms as both a proliferation and survival strategy using novel IoT technology
    (Stellenbosch : Stellenbosch University, 2023-12) Klopper, Kyle Brent; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Microbes rarely exist and proliferate as individual cells, but rather form complex community structures known as biofilms. Biofilm formation ensures that microbial communities are ubiquitous throughout both natural and man-made environments, where the presence of microbial biofilms can be advantageous or detrimental, depending on the context. Biofilm formation is traditionally viewed as a mechanism for microbial survival, since it provides protection from adverse environmental challenges, including starvation, desiccation, predation and antimicrobials. However, a more contemporary perspective also takes the role of biofilms in microbial proliferation into consideration. The reductionist approach, which classifies a biofilm as either a survival or a proliferation mechanism often leads to poor outcomes when the aim is to transition fundamental biofilm research to the real-world and its associated problems. Owing to the inherently complex and dynamic nature of biofilms, a multi-parameter approach is required to better understand its form-function relationship. The development of the novel BioSpec system provides a simple, reliable, and cost-effective tool to monitor biofilm biomass at high- resolution and in real-time, without disturbing the biofilm structure. Coupling of the BioSpec with the carbon dioxide evolution monitoring system (CEMS), facilitates the simultaneous measurement of two important interrelated biofilm metrics; namely biofilm biomass and metabolic activity. The ability to monitor these parameters non-destructively in real-time provides an unprecedented level of insight into biofilm dynamics, as demonstrated here when challenged with biocides at sub-inhibitory and inhibitory concentrations. The inherent complexity of biofilms requires biofouling mitigation strategies that are based on well-informed chemical treatment dosing regimens and the right combination of biocides and dispersants. In industrial applications, the rapid response of microbial respiration rates to chemical treatment offers a strong utility to monitor the success of antimicrobial application, whereas biofilm biomass measurement shows promise as a direct measure of dispersal or removal efficacy. The role of biofilms as ‘planktonic cell production factories’, even when challenged with antimicrobials, was again highlighted in this thesis and emphasises the need for a change in perspective. Microbial proliferation is of critical importance to multiple facets of society ranging from the food, to the medical and industrial sectors. For too long, the emphasis has been placed on the survival attributes of biofilms, thereby neglecting the equally important role of biofilms as mechanism of proliferation and dissemination. The latter has important implications for sanitation regimes in food and beverage processing facilities, where planktonic cell dissemination and secondary biofilm formation at distant locations within a facility, pose serious challenges and risks.
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    Interactions between gut microbiota and the central nervous system, with emphasis on quorum sensing between commensal lactic acid bacteria and human cells
    (Stellenbosch : Stellenbosch University, 2023-03) Dicks, Leon Milner Theodore; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The human gut hosts close to 4 trillion microorganisms, which is nearly equivalent to the estimated 3.0×1013 human cells in a 70 kg body. Although the composition of gut microbiota changes with age, variation in diet, medication, hormone levels, stress and other environmental factors, a core group of autochthonous bacteria, between 400 and 500 species, are always present. More than 90% of the gut microbiome is represented by Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, with the latter in the majority. Fusobacteria and Verrucomicrobia make up the remaining 10% of the gut microbiome. The human gut microbiome supersedes the number of cells in our bodies ten-fold. Since lactic acid bacteria (LAB) are the predominant gut microbiota, it is safe to conclude that changes in this group will affect the entire microbiome, ultimately leading to adjustments in the behaviour of intestinal epithelial cells (IECs). Changes in the immune system and quorum sensing (QS) signals instigated by an altering gut environment trigger a cascade of hormonal and neurological reactions. Activation of Toll-like receptors, for instance, induce strong immune and inflammatory reactions, but at the same time stimulate the secretion of hormones such as 5-hydroxytryptamine (5-HT, or serotonin), glucagon-like peptide-1 (GLP-1), peptide tyrosine tyrosine (PYY), glucose-dependent insulinotropic peptide (GIP), cholecystokinin (CCK), ghrelin, leptin, pancreatic polypeptide (PP), oxyntomodulin and neurotensin. Serotonin act as neurotransmitter but also regulates diverse functions such as platelet aggregation, bone development, immune response, cardiac function and gut homeostasis, and control enteric motor and secretory reflex. Gut bacteria also synthesize, or regulate, the production of serotonin and other neurotransmitters such as glutamine (Glu), gamma-amino butyric acid (GABA), dopamine (DA), norepinephrine and histamine. These molecules communicate with the central nervous system (CNS) via afferent fibers in the Vagus nerve (VN), autonomic sympathetic and parasympathetic nervous systems, but also the hypothalamic-pituitary-adrenal axis (HPA). Intermediate compounds such as short-chain fatty acids (SCFAs), tryptophan and secondary bile acids produced by gut bacteria also communicate with the CNS. Signals received from the brain are sent back to entero-epithelial cells (EECs) via the HPA and efferent VN fibers to complete the circle of communication referred to as the gut-brain axis (GBA). The modulation, development, and renewal of neurons in the enteric nervous system (ENS) are controlled by gut microbiota, especially those with the ability to produce and metabolize hormones. Minor activation of the ENS and VN results in drastic changes in the production of neurotransmitters, which also affects digestion, intestinal permeability, gastric motility, and immune regulation. GABA, in addition to other metabolites, play an important role in anti-inflammatory responses and help alleviate psychiatric symptoms stemming from inflammation. Treatment of schizophrenic and bipolar patients with probiotics alleviated symptoms associated with irritable bowel disease (IBD), and autistic children benefitted from probiotic treatment. Obsessive compulsive disorder (OCD)-like behavior could also be controlled by treatment with LAB. Inter- and intra-species signalling systems have been well studied, but far less is known about interkingdom quorum sensing (QS), especially between gut bacteria and intestinal epithelial cells (IECs). Although the auto-inducer 3 (AI-3)/epinephrine (Epi)/norepinephrine (NE) QS signalling system described for pathogenic Escherichia coli, Salmonella typhimurium and Ctirobacter rodentium are widely used by Gram-negative pathogenic bacteria, not all species have receptors that recognize these signals. Instead, they have developed “broad-range” “solo” LuxR-type receptors such as SdiA (a LuxR homolog) and QscR to improve their communication abilities. Despite our knowledge on QS, the effect of these signalling molecules on the CNS is ill-researched. Several QS peptides (QSPs) have the ability to diffuse through the intestinal mucosa and enter the circulatory system, from where they may penetrate the blood-brain barrier (BBB). It may be that LAB communicate with the CNS using small linear or cyclized oligopeptides (QS peptides, QSPs) of 5 to 17 amino acids long, as reported for other Gram-positive bacteria. In our own research we have shown that bacteriocins can indeed transverse epithelial (Caco-2) and endothelial (HUVECs) monolayers without changing the integrity of the membranes and with no toxic effect. Once in the blood stream, bacteriocins may cross the BBB, similar to that reported for the heptapeptide PapRIV produced by Bacillus. Our understanding of exactly how gut microorganisms control cognitive behavior, mood, and neuropsychiatric disorders remains limited. However, the more we discover about the gut microbiome, QS, neurotransmitters and the GBA, the greater the chance of developing novel therapeutics, probiotics and psychobiotics to treat gastro-intestinal disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), but also improve cognitive functions and prevent or treat mental disorders. This calls for in-depth deciphering of the complex, everchanging network between cells and neurons. Research on the quenching of QS signals need to be prioritised. We need to understand how quorum quenching (QQ) therapy will affect beneficial gut microbiota. Biomarkers need to be developed to identify differences in the gut microbiome of individuals suffering from psychological disorders. Interactions between drugs used in treatment and gut microbiota need to be studied in greater depth. We need to understand the effect psychiatric medication may have on the composition of the gut microbiome. Are intestinal microbiota able to metabolise these drugs? Studies should include multi-omics of gut and oral microbiota to have a better understanding of the mutual interplay between phyla. Will it be possible to develop probiotics to treat dysbiosis and neuropsychiatric abnormalities?
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    A comparative assessment of water quality of two reservoirs in an ephemeral river in the desert climate of Namibia: Measures to control, and factors affecting growth of toxic cyanobacteria
    (Stellenbosch : Stellenbosch University, 2022-12) Sirunda, Jaime Johannes; Wolfaardt, Gideon M.; Oberholster, Paul; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: Globally, there is an increasing demand for freshwater of suitable quality. Satisfying the water requirement for ecosystem services and anthropogenic activities is challenging and may prove difficult in the context of climate change, growing population, and an increase in pollution from land use activities. With the rapid increase in human population in catchment areas, sustaining the associated human needs will often result in the generation of waste which will end up in water sources downstream causing eutrophication resulting in the proliferation of toxin-producing phytoplankton. The overall aim of the research was to carry out a comparative assessment of the water quality deterioration of ephemeral river connected dams with desert climate conditions, found in the Omaruru-Swakop catchment associated with a mixture of land use activities. The Swakoppoort (SWP) and Von Bach (VB) dams as the major dams in the Omaruru-Swakop catchment found on the Swakop River were selected and assessed seasonally for a number of years. Water samples were analyzed for various constituents, firstly to assess the water quality status of the two dams receiving a mixture of pollutants over space and time using the combination Water Quality Index (WQI). Secondly, to describe the vertical and temporal dynamics of phytoplankton communities in the two dams. Thirdly to investigate the effect of prolonged drought on phytoplankton biomass measured as Chlorophyll a (Chl a) and cyanobacteria. Lastly, a case study to compare the effectiveness of two phytoplankton control measures employed in the SWP Dam was conducted. The two dams were found to be impacted by nutrient, salinity, and particulate matter pollution. The WQIs showed poor water quality conditions in both dams for 17 years. Given the poor water quality of the two dams, Microcystis dominated the vertical and temporal dynamics, followed by Dolichospermum. In the dry seasons, higher cyanobacteria cell numbers were observed in comparison to the rainy season in both dams. In the SWP Dam, the preferable depth ranges for toxic cyanobacteria species were 5 to 10 m while in the VB Dam at 0 to 5 m range. Higher frequencies of prolonged drought years were recorded in the VB Dam in comparison to SWP Dam. The influence of a decrease in vol % on the phytoplankton biomass was observed in the SWP Dam but not in the VB Dam. However, the pattern and magnitude of the statistically significant responses (t-test, p < 0.05) varied among the drought and rainy years. Furthermore, the results showed that, the Solar Powered Circulation (SPC) and Phoslock® as the two phytoplankton control measures employed in the SWP Dam had no effect on cyanobacteria cells. It was evident that the two control measures were ineffective in reducing cyanobacterial cells. Incorporation of the current study research outputs in the utility surface water quality assessment and control program, water withdrawal for treatment and transfer plans, wastewater discharge regulations, and the revision of the integrated water resources management plan in Namibia, may ultimately safeguard the scarce water resources to improve resilience to climate change.
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    Producing organic acids from pomace wastes – a biorefinery concept
    (Stellenbosch : Stellenbosch University, 2022-04) Steyn, Annica; Van Zyl, Willem Heber; Viljoen-Bloom, Marinda; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: The production of energy and valuable products via petroleum refineries could be partially replaced by biorefineries that are utilising renewable biomass as substrate. For example, an important metabolite such as malic acid in fruit wastes, like apple and grape pomace, could serve as a substrate for microbial conversion to organic acids and other green chemicals. Specific waste sources were investigated to isolate native yeast strains that may have acquired improved malic acid degradation abilities. The 98 new isolates from grape, apple and plum waste were screened for their ability to degrade extracellular malic acid relative to 50 known strains. Most (94%) of the new isolates degraded more than 50% of the malic acid in both the presence and absence of glucose, whereas only 14% of the known strains could do so, thus confirming the value of exploring and exploiting natural biodiversity for new candidates. The eight best isolates were evaluated in synthetic media, with two strains showing potential for the production of ethanol and acetic acid during aerobic and oxygen-limited growth on apple and grape pomace. The screening of yeasts led to the identification of Saccharomyces cerevisiae strain 61 that showed stronger malic acid-degrading capabilities than expected for this species. Preliminary characterisation revealed that strain 61 degraded malic acid in synthetic media more efficiently (potential for application in the wine industry), produced significantly higher biomass (potential for recombinant protein expression in S. cerevisiae) and is more heat-resistant (potential for consolidated bioprocessing) than commercial wine yeasts. Recombinant S. cerevisiae strains were constructed to express either the Candida krusei (Ckr_fum) or the codon-optimised Escherichia coli (Eco_fum) fumarase gene with or without the XYNSEC secretion signal of Trichoderma reesei xylanase 2. These strains were further engineered to co-express the Schizosaccharomyces pombe transporter (mae1) gene for the active uptake of malic acid. Both strains 5A and 31B(p1), expressing the Ckr_fum and mae1 genes [31B(p1) also contains the XYNSEC secretion signal], produced fumaric acid from extracellular malic acid. Our findings illustrate that disruption of the natural FUM1 gene in S. cerevisiae strains is beneficial when using malic acid as a substrate for fumaric acid production. The inclusion of a transporter allowed for better malic acid degradation and quicker fumaric acid production, and the yeast fumarase was more effective than the bacterial fumarase for fumaric acid production. Strain 5A was able to produce 0.065 g/L fumaric and 2.55 g/L ethanol after 72 h on grape pomace, suggesting that this strain may have potential application for a fruit waste biorefinery following optimisations. This study resulted in the isolation of a number of yeast strains with the ability to utilise malic acid, including S. cerevisiae strain 61. After unravelling the reason(s) for its enhanced malic acid utilisation, this strain could be a promising candidate for the development of a fumaric acid-producing biorefinery host strain. However, this would require the construction of a recombinant strain expressing an effective fumarase and dicarboxylic acid transporter, with the Ckr_fum and mae1 genes as potential candidates.