Department of Biochemistry

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Browsing Department of Biochemistry by browse.metadata.advisor "Cloete, Thomas Eugene"

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    Biofilms as multifunctional surface coatings and adaptive systems: a biomimetic approach
    (Stellenbosch : Stellenbosch University, 2016-12) Loots, Ruenda; Cloete, Thomas Eugene; Swart, Pieter; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Biomimicry is an emerging scientific discipline that promotes nature-inspired innovation for sustainable solutions. Several patterns and survival strategies are repeated in Nature and these have been extrapolated into a hierarchical set of biomimetic principles that can be used to investigate the complexity of natural systems. A biomimetic approach was used to review biofilm literature and create a novel framework based on these principles to describe microbial biofilms on a molecular, structural and systems level. By reinterpreting current biofilm knowledge within a biomimetic framework, this study demonstrates that microorganisms use life-friendly chemistry to integrate biofilm development with growth, giving rise to resource-efficient systems. Furthermore, these structured microbial communities are responsive to their local environment, adapt to changes and, ultimately, evolve to survive. Subsequently, the application of biomimetic principles to biofilms was investigated using various analytical techniques. Two gfp-labelled Pseudomonas strains and an environmental multi-species community were selected for this study. Microscopic and spectroscopic techniques were used for biochemical investigations of single-species biofilm composition and structure. The distribution of biomolecules in Pseudomonas biofilms was investigated using protein- and glycoconjugate-specific fluorescent stains and confocal laser scanning microscopy (CLSM). CLSM was also used to investigate structural adaptations of Pseudomonas biofilms to changes in nutrient availability and hydrodynamic conditions. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to explore biochemical adaptations of single- and multi-species biofilms cultivated in different nutrient media. ATR-FTIR spectra, visual observations and the quantification of biofilm parameters by digital image analysis of CLSM images support the hypothesis that biofilms are resource-efficient, self-organised systems that are built from the bottom up using life-friendly chemical principles. Both Pseudomonas strains adapted to environmental conditions by changing the three-dimensional structure of their biofilms, specifically in terms of biomass, substratum area coverage, average thickness and the surface area of biofilms exposed to the bulk liquid. In order to study biofilms as a system and investigate the responsiveness of a biofilm community as a whole, a relatively new approach was used to monitor biofilm responses in real time by measuring CO2 production as an indication of whole-biofilm metabolism. A CO2 evolution measurement system (CEMS) was combined with metabolic assays and direct plate count methods to monitor biofilm metabolism and biofilm-derived planktonic cell yield in response to environmental changes, i.e. changes in nutrient source and concentration or exposure to antimicrobial compounds (either streptomycin or a solution containing isothiazolone). The metabolic responses of biofilms, measured as CO2 production rates, showed that both single- and multi-species biofilms are able to respond rapidly to changes in nutrient availability or exposure to biocides and antibiotics. Multi-species biofilms generally recover faster after environmental changes or antimicrobial exposures, indicating that diversity adds to biofilm resilience and adaptability. Regardless of the conditions, single- and multi-species biofilms are able to maintain some level of metabolic activity, as well as release high numbers of planktonic cells into the effluent. The maintenance of biofilm-derived planktonic cell yield supports the hypothesis that biofilms are active proliferation sites in order to ensure survival – a feature of biofilms that is often overlooked in biofilm research. This study contributes to the growing field of biomimicry by applying biomimetic principles in biofilm research for the first time. A biomimetic approach can inform novel anti-biofilm strategies, promote biofilm-inspired innovation and explain complex microbial ecological phenomena. Within a biomimetic framework, the increasing degrees of complexity in biofilms are organised in a new way, demonstrating that the biochemical, structural and functional complexity of microbial communities are interconnected and need to be considered together in biofilm studies. To this end, the usefulness of CEMS as a non-destructive technique to study real-time biofilm responses is demonstrated.
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    Fabrication and characterization of anti-microbial and biofouling resistant nanofibers with silver nanoparticles and immobilized enzymes for application in water filtration
    (Stellenbosch : University of Stellenbosch, 2011-03) Du Plessis, Danielle Marguerite; Cloete, Thomas Eugene; Dicks, Leon Milner Theodore; Swart, Pieter; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Due to a global lack of access to potable water, a problem particularly affecting people in developing countries and the poor, improvement on existing water purification methods are necessary to provide more cost effective, accessible and efficient methods of water purification. In drinking water systems, biofilms are a potential source of contamination, which can affect the biological stability and hygienic safety of water. In industrial water systems, biofilms can cause corrosion, resistance in flow systems and a decrease in efficiency of membranes. Nanotechnology has been identified as a technology to utilize in water purification problem solving. Alternatives to the use of chemical biocides and antibiotics need to be investigated therefore; the focus of this study was the fabrication and characterization of polymer nanofibers containing silver nanoparticles as biocide and anti-biofouling nanofibers with hydrolytic enzymes immobilized on the surface. The aim of this study was to synthesize and compare poly (vinyl alcohol) (PVA) nanofibers and poly (acrylonitrile) (PAN) nanofibers with silver nanoparticles to determine which type of fiber will be the most appropriate for application in water sanitation. The two types of fibers were to be compared based on morphology, silver nanoparticle content, physical distribution of silver nanoparticles, levels of silver leaching from the fibers in water, which could imply toxicity, and most importantly, anti-microbial efficacy. Back scattering electron images revealed that silver nanoparticles in PVA nanofibers were more evenly dispersed than in PAN nanofibers, but that PAN nanofibers had higher silver nanoparticle content. This was confirmed by energy dispersive X-ray (EDX) analysis. Both PVA and PAN nanofibers containing silver nanoparticles had excellent anti-microbial activity, with PVA nanofibers killing between 91% and 99% of bacteria in a contaminated water sample and PAN nanofibers killed 100%. When investigated by SEM, the biocidal effect of PAN nanofibers containing silver nanoparticles can be observed as morphological changes in the cell walls. Neither PVA nor PAN nanofibers leached silver into water. PVA is a non-toxic and biodegradable synthetic polymer, and PVA-silver nanofibers have excellent anti-microbial activity, making it applicable in water sanitation in an environmental conscious milieu. PAN nanofibers are more conductive to the formation of silver nanoparticles, have higher silver nanoparticle content, allowing the complete sanitation of pathogenically contaminated water samples. PAN nanofibers also have better longevity and strength in water, making it ideal for water filtration and sanitation in higher throughput systems. Furthermore, immobilized enzymes are being investigated as possible alternatives to inefficient conventional methods of controlling and removing biofilms from filtration systems. This study demonstrates the covalent immobilization of two industrial proteases and an amylase enzyme onto polymer nanofibers widely used in filtration membranes. Confirmed by FTIR, these nanofibers were successfully activated by amidination, allowing the covalent immobilization of respectively two serine proteases and an α-amylase onto the fibers. When inspected visually, fibers largely retained their original morphology after activation and enzyme immobilization. Immobilized enzymes were, however visible as aggregated particles on the nanofiber surfaces. The large surface area to volume ratio provided by the nanofibers as immobilization surface, allowed sufficient amounts of enzymes to be immobilized onto the fibers so that all enzymes retained above 80% of the specific activity of the free enzymes. For each of the immobilized enzymes, just below 30% of initial activity was retained after 10 repeated cycles of use. Fibers with immobilized enzymes on their surface did not support the growth of biofilms, as opposed to plain nanofibers, which did support the growth of biofilms. When considering the combined advantages of this effective immobilization process, the robustness of the enzymes used in this study, and their effectiveness against biofilms in their immobilized state, a valuable addition has been made to technology available for the control of biofilm formation on filtration membranes, and could potentially be employed to control biofilm formation in water filtration systems. A combination of anti-microbial and anti-biofouling nanofibers into a single nanofiltration product may prove to be highly applicable in water sanitation systems.
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    A high rate biofilm contact reactor for winery wastewater treatment
    (Stellenbosch : Stellenbosch University, 2016-12) De Beer, Danielle Marguerite; Cloete, Thomas Eugene; Swart, Pieter; Botes, Marelize; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Winemaking produces variable volumes wastewater rich in biodegradable organic material, with fluctuating chemical composition and pH values according to the seasonal activities of the cellar. Releasing untreated winery wastewater into the environment can cause eutrophication and toxicity in surface water and has detrimental effects on soil condition and ground water quality. Rising costs of effluent disposal, limited availability of freshwater resources and increasingly stringent water use regulations imposed on wineries are enthusing interest in low cost, sustainable, and robust wastewater treatment solutions for wineries. The objective of this study was to design, construct and implement an easily pre-assembled, energy efficient pilot scale biofilm reactor with a small footprint for winery wastewater treatment. A commercial cooling tower as a trickling filter reactor unit was central to the design. The system was tested at a winery in Stellenbosch and after proving to be effective, was up-scaled by adding a second cooling tower to the system as a secondary reactor, treating the effluent from the first subunit, contributing to the overall waste removal efficiency of the system. The double-unit pilot system was tested in six trials over three years. The system showed effective, robust treatment of winery wastewater of varying strengths with minimal solid waste production, consistently reducing chemical oxygen demand (COD) (average 93% reduction), total nitrogen, sulfate, phosphate and suspended solids (average 90% reduction) to meet prescribed regulations for irrigation. The system performed at its peak when treating highly concentrated wastewater during harvest season. The pH of treated wastewater was consistently buffered from highly acidic and basic values to close to neutral. To understand how the biofilm worked to remove contaminants within the system, and how the additional cooling tower unit expanded the treatment scope of the system, a three-tiered investigation of the microbial community structure, distribution of microorganisms and collective metabolic capabilities of biofilm samples from each cooling tower subunit was investigated. Next generation sequencing revealed that the biofilm populations of the two reactor subunits were phylogenetically distinct, with only 12% of operational taxonomic units (OTUs) overlapping between the two biofilms. Taxonomic data indicated that carbohydrate reducing bacteria dominated the population of the first cooling tower, while nitrifying and denitrifying bacteria dominated the second. Fluorescent in situ hybridization coupled with confocal laser scanning microscopy (FISH-CLSM) revealed the stratified distribution of aerobic Gammaproteobacteria across the depth of the biofilm from the first cooling tower unit, and showed distinct distribution patterns of Nitrosomonas and Nitrospirae in biofilm samples from the first and second cooling tower units. Substrate utilization analyses using the Biolog system revealed that the majority of the carbon substrates that were tested were utilized in the biofilm samples from both cooling towers, but that important metabolic utilization capabilities fell exclusively either within the consortium of the biofilm from tower 1 or tower 2. Collectively, the data from each of the three analytical approaches indicated that by adding a second subunit to the bioreactor, the treatment capacity of the system was not merely expanded, but that the second reactor subunit added to the microbial and metabolic diversity of the system.