Department of Microbiology

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Antibiotic resistance in surface waters and biofilm-response to environmental contaminants
(Stellenbosch : Stellenbosch University, 2021-12) Tucker, Keira; Wolfaardt, Gideon M.; Botes, Marelize; Feil, Edward; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Ensuring water security for the future has become important due to rapid urbanisation and diminishing freshwater resources. South Africa’s water resources are scarce and as a result, reclamation of alternative freshwater resources such as treated wastewater is being investigated. There is growing evidence that drinking and wastewater treatment is either non- compliant to quality standards or lacking in certain communities. In areas with no infrastructure for wastewater removal, open sewers create a health risk for humans, animals, and the environment. Poor antimicrobial stewardship, over-use and incorrect disposal has led to increased resistance to antibiotics, rendering some bacterial infections untreatable. There is a concern that sub- inhibitory concentrations of antibiotics create a selection pressure that promotes horizontal gene transfer and emergence of bacterial communities that are resistant to antibiotics. Antibiotics, antibiotic resistant bacteria (ARB), as well as other contaminants that have been shown to promote antimicrobial resistance (AMR) such as heavy metals, enter surface waters and wastewater treatment works (WWTW) in trace concentrations via multiple pathways. As a result, WWTW are deemed hotspots for the emergence and dissemination of AMR. In addition, environmental waters are home to various matrices, including biofilms that are especially problematic in a clinical setting due to their antibiotic resistant and persistent nature. The research presented in this dissertation aimed to contribute to the knowledge surrounding the abundance of ARB in WWTW and surface waters in a South African context. Although ARB and antibiotic resistance genes (ARG) were detected in WWTW effluent, the abundance of both were reduced compared to the influent, suggesting that WWTW played a role in reducing AMR in receiving waters, while exposure to sub-inhibitory concentrations of antibiotics did not result in a significant change in the number of target ARG in isolates selected as representatives of a cultured population. This was emphasised in an expanded study that monitored various regions over a year. In addition, it was shown that surface waters, biofilms and sediments influenced by anthropogenic activities from residential and industrial sectors had higher prevalence of ARB compared to samples influenced by agricultural activity. Metagenomic analysis revealed that ARG relating to efflux pumps were the most common compared to those specific for target antibiotics. Due to heavy-metals and antibiotics being present in the environment in trace concentrations, exposure of mixed-community biofilms to sub-inhibitory concentrations of these contaminants was investigated. AMR in the biofilms did not increase, but it was suggested that the sub-inhibitory exposure promoted the development of persistent mixed community biofilms. Treatment interventions are crucial for removing pollutants and AMR already present in the environment. However, with due recognition of the complexity involved when considering humans, animals, the environment and a diverse pool of contaminants, this dissertation argues the need to expand the approach for mitigation of emergence or dissemination of AMR in the environment by incorporating greater emphasis on antibiotic stewardship, policies around antibiotic usage in all sectors, and overall public awareness.
Diamond based electrodes for water quality applications: disinfection, micropollutant removal and the suppression of biofilm proliferation.
(Stellenbosch : Stellenbosch University, 2024-03) Louw, Carli; Wolfaardt, Gideon M. ; Botes, Marelize; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Rivers in most low and middle income countries face a growing burden of pollution in the form of pathogens and persistent micropollutants caused by waste discharge from informal settlements and poorly maintained wastewater treatment plants, operating above capacity. Farming communities are often reliant on the these rivers for irrigation. This study investigated the potential application of diamond based electro-oxidation in decentralized water treatment systems for disinfection and micropollutant abatement. The diamond-electrode based electrochemical in-situ system (DiaDis) forms ozone and hydroxyl radicals via electro-generation. It was hypothesised that the system would possess strong disinfection properties, but that micropollutant abatement would be dependent on the compounds and organic matter present. The disinfection study included the treatment and prevention of biofilms by monitoring the effects on biofilm metabolic activity during DiaDis treatment. A pure culture of Pseudomonas fluorescens sp. was used for the single culture biofilm disinfection study, and for the mixed species biofilm study the culture was sampled from a polluted river. The disinfection capabilities of the DiaDis was found to be comparable or slightly superior to 1:10 dilution sodium hypochlorite in this study for the single culture biofilms, however, the mixed species biofilms were more effectively treated with sodium hypochlorite. Biofilm-forming species have varying resistance to disinfectants and the results suggest the presence of synergistic adaptation. For the single and mixed species biofilms the treatments studied proved more effective when used preventatively than to treat mature biofilms. Removal efficiency of micropollutants by the mixed species biofilms was comparable to published literature, although lower abatement rates were reported for caffeine and acetaminophen, which could be the result of higher organic matter acting as oxidation scavenger in the environmental water. Negative removal rates were obtained for sulfamethoxazole, benzotriazole and efavirenz, likely due to electro- oxidation facilitated reconstitution of breakdown products to the respective parent compounds. The DiaDis was evaluated as a final disinfection step in an aquaponics, as well as a constructed floating wetland system. It performed better in the floating wetland, possibly due to higher levels of organic matter coming from fish-feed in the aquaponics system. Overall the DiaDis showed promising disinfection capabilities, which will benefit from future work to better control pH reduction and to reduce interference of organic matter with micropollutant abatement.
The effect of dissolved oxygen on the development of manganese oxidizing - and reducing biofilms in the lower blyde irrigation system
(Stellenbosch : Stellenbosch University, 2018-12-11) Carstens, Alno; Wolfaardt, Gideon M.; Botes, Marelize; Bryant, Lee; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The Blyderiver dam in Mpumalanga is an important source of water for the irrigation of a variety of produce on farms in Limpopo and Mpumalanga. Water from this dam is used for gravity fed irrigation to farms via the Lower Blyde Irrigation System (LBIS), a network of pipelines of approximately 150 km. Biofilm development in the system causes a reduction in hydraulic capacity, leading to a reduction in water delivery for irrigation. It was hypothesized that high concentrations of manganese (Mn) in the water of the Blyderiver dam could potentially contribute to the development of these biofilms. Dissolved oxygen (DO) and Mn measurements taken during four sampling events from 2015 to 2017 indicated that the water of the Blyderiver dam has a distinct profile, with DO and Mn concentrations showing a strong inverse correlation. DO concentration typically remained constant between 8 and 9 mg l-1 in the upper 30 + meters of the water column, where after it decreased rapidly to below 2 mg l-1 at deeper depths. In contrast, total Mn concentration remained constant between 10 and 100 μg l-1 in the upper regions of the water column, followed by a rapid increase to higher than 8000 μg l-1 near the bottom. The current point of extraction for the LBIS is located near the bottom of the dam in this water with high Mn content. Mn concentration decreased with distance along the LBIS pipeline. For instance, results of the May 2016 sampling showed a decrease in the bulk aqueous phase Mn concentration from 8631 μg l-1 at the extraction point to 134 μg l-1 at 23 km downstream, while a decrease in Mn concentration could also be seen from 30105.4 mg kg-1 biofilm biomass at 4.5 km downstream to 13727.7 mg kg-1 at 28.4 km downstream. This decrease suggests ongoing incorporation of Mn into the biofilm, and thus potentially further loss in hydraulic capacity. Laboratory simulation experiments of the LBIS pipeline were conducted in which biofilms supplied with growth medium with different DO and Mn concentrations were cultivated. These biofilms were analysed by determining cell release into the effluent, Mn measurements, scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), automated ribosomal intergenic spacer analysis (ARISA) as well as confocal laser scanning microscopy (CLSM). Results obtained from colony counts and ARISA indicated that statistically significant differences did exist between biofilms cultivated under different growth conditions in some cases, but that these differences could not be attributed to the effects of different DO and Mn concentrations and were instead due to physical conditions such as shear and flux. These findings were confirmed by CLSM analysis. No statistically significant decrease in Mn concentration could be observed throughout the flow system, indicating that very little to no Mn was incorporated into the biofilm structure under any of the growth conditions tested in the laboratory simulations. SEM/EDS analysis of these biofilms further confirmed that the decreases in Mn concentration observed in both the bulk aqueous phase and biofilms with distance in the LBIS pipeline could not be replicated in these laboratory simulated flow systems. A number of factors were considered to explain this conclusion, including the time period of the experiment, nutrients used in the growth medium and biofilm sorption capacity. Future experimentation should include flow systems in which biofilms will be cultivated for longer time periods, and the use of microfluidic flow channels to provide shear values closer to that in the LBIS.
The efficiency of a Swoxid prototype and an antimicrobial nanofiber membrane as POU filters
(Stellenbosch : Stellenbosch University, 2020-04) Gaborone, Mamosete Dorothy; Wolfaardt, Gideon M.; Botes, Marelize; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Clean water is a scarce resource that numerous individuals lack access to. Resultantly, these individuals resort to using surface waters which leaves them exposed to waterborne diseases if they do not clean the water source adequately. Furthermore, these individuals are also exposed to chemical pollutants from agricultural, domestic and industrial waste which drains into rivers as well as downstream from waste water treatment plants (WWTPs). Furthermore, WWTPs which do not remove chemical pollutants efficiently. There are several methods to clean water, one of the most popular being filtration. In this study, the efficiency of two potential point-of-use (POU) filters was determined. The first filter, namely the Swoxid prototype, is comprised of a ceramic membrane functionalised with titanium dioxide (TiO2) and requires ultra violet (UV) light for activation. The mechanism of TiO2 is that when it is activated by UV light, hydroxyl (OH.) radicals are generated, which inactivate bacteria and denature chemical pollutants. To determine the efficiency of the Swoxid prototype to inactivate bacteria, water from a highly polluted source, namely the Plankenburg River, was filtered through the filter in both the absence and the presence of sun exposure. The bacteria tested were the typical indicator organisms namely the coliforms, faecal coliforms, heterotrophic bacteria, Enterococcus spp., Salmonella spp. and Shigella spp. For the first Swoxid prototype, there was complete removal of bacteria under exposure to the sun except for the heterotrophic bacteria and the coliforms. In the case of the coliforms, no bacteria were observed for the first few days; however on the fourth or the sixth day, growth was observed. This suggests that for the first few days, the bacteria are in a stationary phase, likely due to the time required for DNA repair. For most of the bacteria tested, the results of the trials where the first Swoxid prototype was exposed to the sun were comparable to the controlled conditions. This suggests that the removal of bacteria by the Swoxid prototype is mainly due to filtration and not the inactivation of bacteria by the radicals that are generated by the TiO2-UV reaction. The second Swoxid prototype was effective at removing most of the bacteria; however, there was regrowth of the heterotrophic bacteria and coliforms. The results of the Swoxid prototype were comparable to the UV control, suggesting that inactivation was mainly due to UV exposure and not radical degradation. Scanning electron microscopy was used to view the effect of radicals on the membrane of Staphylococcus aureus Xen 36. The results showed that the radicals generated by the TiO2-UV reaction may oxidize the lipid bilayer of bacterial membranes leading to the formation of cracks on the surface of the membrane of bacteria. The Swoxid prototype was also effective in removing several micropollutants, i.e. benzotriazole, codeine, diclofenac, efavirenz and sulfamethoxazole from spiked and river water. However, because the results were comparable to a commercial UV-filter for many of the micropollutants that were tested, it could be that the removal was mainly due to UV bombardment and not removal of the micropollutants by the radicals. Future improvements and amendments to the Swoxid prototype should include an increase in the quantity of TiO2 used to functionalise the membrane. The second filter membrane prototype comprised of a poly (D,L-lactic acid) (PLA) nanofiber membrane functionalised with an antimicrobial solution (biocide) containing copper (Cu(II)) and zinc (Zn(II)) ions. The proposed mechanisms of Cu(II) and Zn(II) entail disrupting the membrane of microorganisms, decreasing the membrane potential and binding to sulfur-containing proteins and DNA. Ten varieties were fabricated that included low, medium and high biocide loading and low and high density membranes. As part of the initial experiments, leaching experiments were performed using inductively coupled mass spectrometry (ICPMS) to determine whether or not the quantity of Cu(II) and Zn(II) that leach is below the limit set United States Environmental Protection Agency (USEPA) and South African standards for drinking water. The results showed that the concentration of Cu(II) and Zn(II), which the maximum that leached were 33.6 μg/L and 100.5 μg/L, respectively were indeed below the limits set in the drinking standards of both the EPA and South Africa. The results also showed that membranes with a higher density leached more Cu(II) and Zn(II) than their lower density counterparts. Moreover, the results showed that more Zn(II) leached from the membranes than the Cu(II) which suggests that the Cu(II) is more tightly retained by the PLA fibres. The membranes were then exposed to Escherichia coli Xen 14 and S. aureus Xen 36 and the decrease in bioluminescence was observed over time using the XENOGEN VIVO VISION In Vivo Imaging Lumina System (IVIS). The experiment showed that the membranes with the highest biocide and higher density were most effective at inactivating the metabolism of bacteria. Therefore subsequent experiments entailed filtering reverse osmosis (RO) water spiked with 107 Escherichia coli Xen 14 and S. aureus Xen 36 through the antimicrobial nanofiber membranes to determine the log reductions of the bacteria. However, the membrane with the highest biocide, density and fibre diameter only led to a 1.5 log reduction. The design of the nanofiber membrane was modified to consist of one layer of PLA nanofibers and a second layer containing PLA and the biocide. Three varieties were fabricated consisting of three different densities and controls containing no biocide. Bacteria were only removed when the high planar density membranes were stacked on top of one another with the highest log reduction being 5 when three membranes were used. The nanofiber membranes were also characterized before and after filtration using scanning electron microscopy (SEM). It was determined that filtration does not have a significant effect on the structure of the membrane as the nanofibers were still intact. The pores were, however, larger than bacteria in size and that explains the poor filtration results when just one membrane is used. Moreover, it was discovered through electron dispersive spectroscopy (EDS) that the copper binds more tightly to the membrane than the Zn(II) as the difference in the content of copper before and after filtration was very little. In addition to the structure of the antimicrobial nanofiber membrane, the structure of the bacteria after filtration was also viewed. There were several bacteria with damaged outer surfaces, particularly those that were located near what appeared to be the biocide. The design of the antimicrobial nanofiber membrane can be improved by increasing the density of the membrane and ensuring smaller pore sizes. Additionally, the membranes should be designed in such a way that the Cu(II) leaches more readily at concentrations that are still below the limits set in the EPA and South African Drinking Water Standards.
Exploring a rumen-modelled carboxylate platform for the conversion of agricultural wastes to green chemicals and fuels in South Africa
(Stellenbosch : Stellenbosch University, 2021-03) Njokweni, Sesethu Gift; Van Zyl, Willem Heber; Botes, Marelize; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The notion of a “biobased economy” in the context of a developing country such as South Africa (SA) necessitates research and development of technologies that i) utilize sustainable feedstocks ii) have simple and robust operation iii) could work at small scale and iv) produces a variety of valuable bioproducts, fitting the biorefinery concept. One of the technologies that have been gaining increased global interest is the carboxylate platform based on ruminants. This platform uses mixed/undefined anaerobic co-cultures of rumen microorganisms to convert a variety of organic biomass to volatile fatty acids (VFA). VFA are short-chain fatty acids with two to four carbon atoms, which include acetic acid (C2), propionic acid (C3) and butyric acid (C4). These fatty acids have a wide variety of applications including the production of hydrocarbon fuels through electrolysis. Technological modelling has shown that each VFA corresponds to the production of a mixture of alkane hydrocarbons when using Kolbe electrolysis that may range from methane to octane depending on the average chain length and yield of alkyl groups in the produced VFA. Therefore, in order to produce higher alkanes, there is a need to produce VFA with higher yields of alkyl groups. In turn, these yields depend on a variety of operational aspects and input feedstocks used that need to be optimized to make the process economically feasible. Some of the operational challenges that need to be addressed include directing the microbiome into the desired products; Optimization of fermentation parameters and developing strategies for the preservation of the core microbiome involved in the production of the VFA. This study aimed to assess the feasibility of utilizing the ruminal carboxylate platform in SA to convert locally abundant sustainable organic feedstocks to VFA towards hydrocarbon fuel production. The study was separated into two research chapters: 1) Optimisation of the in-vitro anaerobic rumen fermentation conditions and selection of ideal SA organic waste products including fruit wastes (apple, grape and citrus pomace) and invasive plants (Prosopis juliflora) in terms of degradability, VFA yield and utility to serve as substrates for electrochemical conversion to hydrocarbons. 2) Examine the potential of preserving the core ruminal microbiome involved in VFA production using the two most common cryogenic agents (glycerol and dimethyl sulfoxide) and analyze population dynamics to determine any shifts using β-diversity distance metrics. It was shown that all organic waste products have the potential to serve as substrates for electrochemical conversion with citrus pomace showing the highest utility with 136 mM concentration of VFA (fractional yield of 0.52 out of a possible 0.75) and 12.16 mmol alkyl.g-1 present for conversion. Comparison of fresh rumen with glycerol preserved rumen showed Similarity index (β-diversity scale of 0 to 0.8, with 0.8 being the most diverse) with unweighted unifrac between the communities was 0.359 while the comparison of fresh rumen and DMSO cryo-preserved rumen was 0.250. This showed that both glycerol and DMSO cryo-preserved the rumen fluid without dramatic shifts in the community but the DMSO cryo-preserved community better resembled the core microbiome on the fresh rumen inoculum. This was further supported by analysis of VFA production and digestibility of pomace substrates with the preserved microbiome showing significantly similar yields to the fresh rumen inoculum. The results of this study further highlighted the potential of using a rumen-based carboxylate platform as a small-scale alternative towards biofuels production.
Selection of probiotic lactic acid bacteria for horses based on in vitro and in vivo studies
(Stellenbosch : Stellenbosch University, 2011-12) Botha, Marlie; Dicks, Leon Milner Theodore; Botes, Marelize; Stellenbosch University. Faculty of Sciences. Dept. of Microbiology.
ENGLISH ABSTRACT: The equine gastro-intestinal tract (GIT) is a relatively unexplored niche concerning the presence of natural microbiota. Studies have shown that disruption of the microbial population naturally present in the GIT leads to the onset of several forms of gastro-intestinal disorders. To maintain a balanced microbiota, probiotic bacteria need to be administered at specific levels. Beneficial microorganisms assist with digestion of the feed, absorption of nutrients from the GIT, strengthens the immune system and improves the animal‟s growth. Various combinations of lactic acid bacteria (LAB) have been administered to horses, but have failed to benefit the host in any of the latter criteria. The screening for alternative strains with probiotic properties is thus necessary. Two strains (Lactobacillus equigenerosi Le1 and Lactobacillus reuteri Lr1) were originally isolated from horse faeces. Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, both bacteriocin-producing strains, were isolated from sorghum beer and soy beans, respectively. All four strains survived growth at acidic conditions (pH 3) and the presence of 0.5%, 1.0% and 1.5% (w/v) bile salts. L. reuteri Lr1 was the most resistant to these conditions. All strains adhered to buccal (cheek) epithelium cells sampled from horses. L. equigenerosi Le1 and E. mundtii ST4SA, however, invaded the cells, but without visible signs of disrupting the cells. None of the strains contained genes encoding adhesion to collagen (Ace), resistance to vancomycin A, B and C, or, production of aggregation substance (AS), cytolysin (Cyl) and, non-cytolysin (β hemolysin III), suggesting that they are non-virulent. Of all strains, L. equigenerosi Le1 competed the best with Clostridium sp. C6 for adherence to epithelial cells. L. equigenerosi Le1 and L. reuteri Lr1, showed the highest level of co-aggregation with Clostridium sp. C6. When the four strains were administered to horses over a period of 10 days, L. reuteri Lr1 was retained the longest (8 days) in the GIT. The numbers of viable cells of Clostridium spp. and Salmonella spp. remained constant during administration of the four strains. Blood analyses showed no negative effects from administering the strains. Total white blood cell counts remained unchanged. However, a small but tentative increase in neutrophil and eosinophil cell numbers has been recorded, suggesting that the LAB may have elicited a mild, transient, intolerance reaction. The glucose, lactate and urea levels decreased during administration with the four LAB strains.
Synthetic domestic wastewater sludge as electron donor in the reduction of sulphate and treatment of acid mine drainage
(Stellenbosch : Stellenbosch University, 2015-04) Van den Berg, Francis; Cloete, T. Eugene; Botes, Marelize; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Acid mine drainage (AMD) is wastewater generated by mine and industrial activity with typically high heavy metal and sulphur content potentially resulting in toxic wastewater upon exposure to dissolved oxygen, water and micro-organisms. Due to the hazardous consequences of untreated AMD, treatment methods such as semi-passive biotic treatments, including constructive wetlands and microbial bioreactors were developed. Microbial bioreactors rely on suitable carbon sources such as ethanol, grasses and manure and the creation of anaerobic conditions for the reduction of sulphate, chemical oxidizable organic matter (COD) and to neutralise pH. Domestic wastewater sludge has also been identified as an economical and readily available carbon source that allows the treatment of both AMD and domestic wastewater. A synthetic medium simulating the COD and the biological degradable organic matter (BOD) of domestic wastewater sludge was formulated to exclude variations in the evaluation of domestic wastewater sludge as carbon source in the treatment of AMD. Firstly the BOD and COD of anaerobic domestic wastewater sludge was determined and used as parameters in the formulation of the synthetic medium. A ratio of 1:1 AMD: synthetic domestic wastewater sludge (SDWWS) was the optimum ratio in terms of sulphate and COD removal. Secondly, medical drip bags were used as anaerobic bioreactors to determine the microbial diversity in AMD treated with SDWWS using different variables. Data analyses from next generation sequencing showed that Chlorobium spp. dominated the 90 d pioneer trials at relative percentages of 68 % and 76 %. Transmission electron microscopy (TEM) images and the bright green colour of the liquid contents confirmed the data analyses. Sulphates and COD were removed at > 98 % and > 85 %, respectively. A shorter incubation time was investigated in the 30 d pioneer trial. Chlorobium spp. was dominant, followed by Magnetospirillum spp. and Ornithobacterium spp. The liquid content changed to a dark brown colour. COD and sulphate concentrations were reduced by 60.8 % and 96 %, respectively, within 26 d, after which a plateau was reached. The effect of an established biofilm in the bioreactors showed that Chlorobium spp. also dominated approximately 62 %, in comparison to the 36 % in the 30 d pioneer trial. A sulphate and COD reduction of 96 % and 58 %, respectively, was obtained within 26 d and the liquid content was the same colour as in the 30 d pioneer trial. It is possible that brown Green sulphur bacteria were present. Therefore, although Chlorobium spp. was present at a higher percentage as in the 30 d pioneer trial, the removal of COD and sulphate was similar. During the 30 d trials a white precipitant formed at the top of the bioreactors, consisting primarily of sulphate and carbon that was also indicative of the presence of Chlorobium spp. Incubation at reduced temperature reduced sulphates by only 10 % and COD by 12 % after 17 d, followed by a plateau. Ornithobacterium spp. dominated in the first trial and Magnetospirillum spp. in the second trial.

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