Masters Degrees (Microbiology)

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Adhension of Candida albicans to host cells in culutre.
(1989) Maiter, Aziza Ismail.; Alexander, D. M.; Oellermann, Rolf Alfred.
No abstract available.
Aerobic utilization of selected pharmaceutical and personal care product by estuarine heterotrophic bacteria.
(2014) Bulannga, Rendani Bridghette.; Schmidt, Stefan.
Pharmaceutical and personal care products (PPCPs) constitute a broad class of organic compounds, some of which belong to the list of the OECD high production volume (HPV) chemicals. These compounds have emerged as environmental contaminants with potentially detrimental effects. They have been detected in various environmental compartments typically in a nano- to microgram range and sewage treatment plants represent the major point source for the aquatic environment. Salicylic acid, a monohydroxybenzoic acid, is widely used in cosmetic and therapeutic products and is listed as HPV chemical. Benzyl salicylate and phenyl salicylate are diaryl ester compounds commonly used in pharmaceutical formulations, fragrances and household detergents. Benzyl salicylate is listed as HPV chemical. The fate of salicylic acid in the environment has been reported previously while those of benzyl salicylate and phenyl salicylate are unknown. Although studies are available on the microbial degradation of aromatic compounds, studies exclusive to the catabolism of PPCPs by marine heterotrophic bacterial isolates are rather limited. Therefore, the aim of this thesis was to characterize PPCPs (salicylic acid, benzyl salicylate and phenyl salicylate) utilizing bacteria from an estuarine environment (Durban Harbour, KwaZulu-Natal, South Africa). Selective enrichments were employed using artificial seawater medium typically supplemented with 2 mM of the target compounds (salicylic, benzyl salicylate or phenyl salicylate). After successive subculturing, bacteria capable of utilizing target compounds as sole carbon and energy source were characterized by morphological and physiological features, 16S rRNA gene sequence and MALDI-TOF MS analysis. Growth kinetics were assessed by monitoring the optical density, cell count and protein formation over time. The utilization of salicylic acid and phenyl salicylate was verified using UV spectroscopy and HPLC and the key reactions involved were verified by determining the specific oxygen uptake rates using resting cells and specific activities of representative enzymes. A Gram-negative coccus shaped bacterium belonging to the genus Acinetobacter degrading salicylic acid and phenyl salicylate, a Gram-negative rod shaped marine bacterium belonging to the genus Oceanimonas degrading salicylic acid and phenyl salicylate and a Gram-negative rod shaped bacterium belonging to the genus Pseudomonas utilizing benzyl salicylate in the presence and absence of synthetic surfactants (Tween 80) were isolated. The growth of Acinetobacter and Oceanimonas species was dependent on salicylic acid and phenyl salicylate as carbon source as growth was only observed when the carbon source was present and the compound was degraded almost to completion. Growth of Pseudomonas with benzyl salicylate was enhanced in the presence of surfactant. All three strains did not have an obligate requirement for NaCl. Acinetobacter and Oceanimonas strains were tolerant to high concentrations of salicylic acid and were inhibited at a concentration above 20 mM while phenyl salicylate did not show toxic effects on the strains; instead growth increased with the increase in concentration. Salicylic acid was utilized via catechol by both strains as they showed high specific oxygen uptake rates and catechol-1, 2-dioxygenase activity for this chemical. Phenyl salicylate was hydrolyzed at the ester bond to phenol and salicylic acid, as these were the metabolites that accumulated during growth with phenyl salicylate. The mono-aromatic metabolites resulting from the hydrolysis of diaryl substrate were further metabolized via catechol. Microbial catabolic activities were solely responsible for the loss of contaminant in the medium as confirmed by abiotic controls. Heterotrophic bacteria can therefore play an important role in the removal of contaminants from marine environments.
Analysis of indigenous herbivore faecal matter as a potential source of hydrolytically active microorganisms.
(2014) Ndlela, Luyanda Lindelwa.; Schmidt, Stefan.
Abstract available in PDF file.
Analysis of microbial populations associated with a sorghum-based fermented product used as an infant weaning cereal.
(1999) Kunene, Nokuthula F.; Hastings, John W.; Von Holy, Alexander.
The incidences of diarrhoeal episodes in infants and children have mostly been associated with the consumption of contaminated weaning foods. This is especially true in developing countries where factors such as the lack of sanitation systems and electricity have been found to contribute to an increase in the incidence of microbiologically contaminated weaning foods. The process of fermentation has been found to reduce the amount of microbiological contamination in such foods as a result of the production of antimicrobial compounds such as organic acids, peroxides, carbon dioxide and bacteriocins. In this study, microbiological surveys were conducted on sorghum powder samples and their corresponding fermented and cooked fermented porridge samples collected from an informal settlement of the Gauteng Province of South Africa. The process of fermentation was found to result in significant decreases (P>0.05) in Gram-negative counts and spore counts, while aerobic plate counts decreased slightly. Lactic acid bacteria counts, however, increased significantly (P>0.05). The cooking process was found to result in further significant decreases (P>0.05) in all counts. Sorghum powder samples and fermented porridge samples were found to be contaminated with potential foodborne pathogens, including Bacillus cereus, Clostridium perfringens and Escherichia coli, however, none of the pathogens tested for were detected in any of the cooked fermented porridge samples. SDS-PAGE and phenotypic analysis of 180 lactic acid bacteria isolated from sorghum powder samples and their corresponding fermented and cooked fermented porridge samples showed that a majority of the isolates were lactobacilli and leuconostocs, however, some isolates were identified as pediococci and lactococci. These results demonstrated the heterogeneity of the lactic acid bacteria isolates that were associated with fermentation processes in this study. Of the lactic acid bacteria identified, Lactobacillus plantarum and Leuconostoc mesenteroides strains were found to have the highest distribution frequencies, being distributed in 87% and 73% of the households, respectively. Analysis of Lactobacillus plantarum (58) and Leuconostoc mesenteroides (46) strains isolated from sorghum powder samples and corresponding fermented and cooked fermented porridge samples by AFLP fingerprinting showed that they originated from a common source, which was sorghum powder. There was, however, evidence of strains that may have been introduced at household level. Antimicrobial activity of selected lactic acid bacteria was found to be mainly due to a decrease in pH in fermented and cooked fermented porridge samples. None of the lactic acid bacteria tested seemed to produce bacteriocins.
Antibiogram and virulence determinants of Pseudomonas and Legionella spp. recovered from treated wastewater effluents and receiving surface water in Durban.
(2015) Ntshobeni, Noyise Babalwa.; Olaniran, Ademola Olufolahan.
No abstract available.
Application of bacterial bioflocculants for wastewater and river water treatment.
(2008) Buthelezi, Simphiwe P.; Pillay, Balakrishna.
Dyes are often recalcitrant organic molecules that produce a colour change and contribute to the organic load and toxicity of textile industrial wastewater. Untreated effluent from such sources is harmful to aquatic life in the rivers and lakes due to reduced light penetration and the presence of highly toxic metal complex dyes. The use of alum as flocculant/coagulant in wastewater treatment is not encouraged as it induces Alzheimer’s disease in humans and results in the production of large amounts of sludge. Therefore, the development of safe and biodegradable flocculating agents that will minimize environmental and health risks may be considered as an important issue in wastewater treatment. Bioflocculants are extracellular polymers synthesized by living cells. In this study, bacterial bioflocculants were assessed for their ability to remove dyes from textile wastewater as well as reducing the microbial load in untreated river water. The bacteria were isolated from a wastewater treatment plant and identified using standard biochemical tests as well as the analysis of their 16S rDNA gene sequences. Six bacterial isolates were identified viz. Staphylococcus aureus, Pseudomonas plecoglossicida, Pseudomonas pseudoalcaligenes, Exiguobacterium acetylicum, Bacillus subtilis, and Klebsiella terrigena. The flocculating activities of the bioflocculants produced by these isolates were characterized. The effect of temperature, pH, cations and bioflocculant concentration on the removal of dyes, kaolin clay and microbial load was also determined. The amount of bioflocculants produced by the bacterial isolates ranged between 5 and 27.66 g/l. According to the findings of the present study, bacterial bioflocculants were composed of carbohydrates, proteins, uronic acid, and hexosamine in varying quantities. The bioflocculants were effective to varying degrees in removing the dyes in aqueous solution, in particular whale dye, medi-blue, fawn dye and mixed dyes, with a decolourization efficiency ranging between 20-99.9%. Decolourization efficiency was influenced by the bioflocculant concentration, pH, temperature, and cations. The bacterial bioflocculants were also capable of reducing both the kaolin clay and the microbial load from river water. The flocculating activity ranged between 2.395–3.709 OD-1 while up to 70.84% of kaolin clay and 99% of the microbial load from the river water was removed. The efficiency of kaolin clay flocculation increased with higher concentration of bacterial bioflocculants. The optimum pH for the flocculating activity was observed between 6 and 9. The best flocculating activity was observed at 28oC. Divalent cations such as Mg2+ and Mn2+ improved the flocculation while salts such as K2HPO4, CH2COONa, and Na2CO3 did not. The findings of this study strongly suggest that microbial bioflocculants could provide a promising alternative to replace or supplement the physical and chemical treatment processes of river water and textile industry effluent.
Application of image analysis in microecophysiology research : methodology development.
(1998) Dudley, B. T.; Wallis, Frederick Michael.
Rehabilitation of landfill sites is important for successful land utilization. Revegetation is one key element of the process since it can overcome aesthetic problems. The inimical challenges of landfill leachate and gas are largely responsible for the difficulties associated with the revegetation of completed sites. Many components of landfill leachate can be catabolized by microbial associations thereby reducing their impacts on the environment. The importance of research on interactions between pollutants, microorganisms and soil is its applicability in environmental risk assessment and impact studies of organic pollutants which enter the soil either accidentally or intentionally. The application of image analysis with microscopy techniques to landfill soil-pollution interactions provides a means to study surface microbiology directly and to investigate microbial cells under highly controlled conditions. This research focused on the development of a method to study the real time processes of attachment, establishment, growth and division of microbial cells/associations in site covering soils. Image analysis provides a powerful tool for differential quantification of microbial number, identification of morphotypes and their respective responses to microenvironment changes. This minimal disturbance technique of examining visually complex images utilizes the spatial distributions and metabolic sensitivities of microbial species. It was, therefore, used to examine hexanoic acid catabolizing species, both free-living and in a biofilm, with respect to obviating the threat of hexanoic acid to reclamation strategies. The three sources of inoculum (soil cover, soil from the landfill base liner and municipal refuse) were compared for their ability to provide associations which catabolized the substrate rapidly. During the enrichment programme the inocula were challenged with different concentrations of hexanoic acid, a common landfill intermediate. From the rates at which the substrate was catabolized conclusions were drawn on which concentration of hexanoate facilitated the fastest enrichment. The results of initial batch culture enrichments confirmed that the soil used contained microbial associations capable of catabolizing hexanoic acid at concentrations < 50mM, a key leachate component. Exposing the landfill top soil microorganisms to a progressive increase in hexanoic acid concentration ensured that catabolic populations developed which, in situ, should reduce the phytotoxic threat to plants subsequently grown on the landfill cover. The analysis of surface colonization was simplified by examining the initial growth on newly-exposed surfaces. The microbial associations generated complex images which were visually difficult to quantify. Nevertheless, the dimensional and morphological exclusions which were incorporated in the image analysis software permitted the quantification of selected components of the associations although morphology alone was inadequate to confirm identification. The effects of increasing the dilution rate and substrate concentration on the growth of surface-attached associations in Continuous Culture Microscopy Units (CCMUs) were examined. Of the five dilution rates examined the most extensive biofilm development (9.88 jum2) during the selected time period (72h) resulted at a dilution rate of 0.5h' (at 10mM hexanoic acid). The highest growth (608 microorganisms.field"1) was recorded in the presence of 50mM hexanoic acid (D = 0.5h"1). To ensure that the different morphotypes of the associations were able to multiply under the defined conditions a detailed investigation of the component morphotypes was made. Numerically, after 60h of open culture cultivation in the presence of 50mM hexanoic acid, rods were the predominant bacterial morphotypes (43.74 field'1) in the biofilms. Both rods and cocci were distributed throughout the CCMUs whereas the less numerous fungal hyphae (0.25 field'1) were concentrated near the effluent port. The specific growth rates of the surface-attached associations and the component morphotypes were determined by area (//m2) colonized and number of microorganisms.field"' and compared to aerobic planktonic landfill associations. From area determinations ( > 0.16 h'1) and the number of microorganisms.field"1 10mM hexanoic acid was found to support the highest specific growth rate ( > 0.05 h"1) of the surfaceattached association isolated from municipal refuse. With optical density determinations, the highest specific growth rate (0.01 h'1) was recorded with 25mM hexanoic acid. The surface-attached microbial associations component species determinations by area and number showed that the hyphae had the highest specific growth rate ( > 0.11 h"1). The surface-attached microbial association specific growth rate determinations from the discriminated phase (0.023 h'1), area colonized (0.023 h"1) and number of microorganisms (0.027 h"1) calculated from the results of the component species rather than the association should give more accurate results. The specific growth rate obtained differed depending on the method of determination. Any one of these may be the "correct" answer under the cultivation conditions. Depending on the state (thickness) of the association (free-living, monolayer or thick biofilm) the different monitoring methods may be employed to determine the growth. As a consequence of the results of this study, the kinetics of microbial colonization of surfaces in situ may be subjected to the same degree of mathematical analysis as the kinetics of homogeneous cultures. This type of analysis is needed if quantitative studies of microbial growth are to be extended to surfaces in various natural and artificial environments.
Assessing the antimicrobial, anti-quorum sensing and anti-biofilm potential of bacteria isolated from pocillopora and acropora corals.
(2021) Buswana, Olona.; Chenia, Hafizah Yousuf.; Pearton, David.
Coral bleaching, primarily caused by the rise in seawater temperature, is a major threat to the survival and functionality of coral reef ecosystems in the marine environment. The application of probiotics to corals, particularly bacteria, may be a possible solution to test whether the effects of coral bleaching can be reversed to improve coral health. The present study assessed the anti-biofilm and anti-quorum sensing potential of bacteria isolated from Pocillopora and Acropora corals. Intertidal zone corals were collected along the KwaZulu-Natal coast and bacteria were isolated. A total of 210 and 132 bacterial isolates were biobanked from Acropora and Pocillopora spp., respectively. Preliminary characterization of isolated bacteria was carried out, i.e., colony characterization, Gram reaction and cellular morphology. Primary antimicrobial screening was carried against indicator organisms, Pseudomonas aeruginosa ATCC 27853, methicillin-resistant Staphylococcus aureus using the colony drop assay, as well as quorum sensing inhibition screening using Chromobacterium violaceum, Chromobacterium subtsugae and Vibrio harveyi. Based on preliminary data, 16 Pocillopora- and 24 Acroporaassociated bacteria were selected for further investigation. Antimicrobial potential of extracts against P. aeruginosa, S. aureus, Shewanella putrefaciens, Vibrio alginolyticus, Vibrio coralliilyticus, Vibrio parahaemolyticus and Vibrio shilonii were carried out, with Pocilloporaassociated bacterial extracts inhibiting 6.25% (1/16) of clinical indicators and 43.75% (7/16) of marine indicators, while 20.83% (5/24) and 25% (6/24) of Acropora-associated bacterial extracts inhibited clinical and marine indicators, respectively. Qualitative and quantitative antiquorum sensing activity were also assessed against C. violaceum and C. subtsugae using an agar overlay and quantitative assays where 93.75% (15/16) and 100% of Pocilloporaassociated bacterial extracts and 25% (6/24) and 29.16% (7/24) of Acropora-associated bacterial extracts showed activity against C. violaceum and C. subtsugae, respectively. Using the qualitative autoinducer-2 agar overlay inhibition assay with Vibrio harveyi, 68.75% (11/16) and 25% (6/24) of Pocillopora-associated and Acropora-associated bacterial extracts demonstrated autoinducer-2 inhibition, respectively. Biofilm inhibition was assessed against P. aeruginosa ATCC 27853, S. aureus ATCC 43300, S. putrefaciens ATCC 8071, V. coralliilyticus ATCC_BAA 450, V. parahaemolyticus ATCC 17802 and V. shilonii ATCC_BAA 91 with selected extracts demonstrating inhibitory potential against initial adhesion and/or mature biofilm reduction. Coral-associated bacteria have demonstrated antimicrobial and/or anti-virulence properties and could be an important source of novel antimicrobial, anti-QS and anti-biofilm compounds, with potential coral probiotic application to overcome coral bleaching.
Assessing the role of the transcription factor FOXC1 in the expression and regulation of the Adherens junction protein N-Cadherin during corneal endothelium development.
(2011) Govender, Viveshree Shalom.; Sommer, Paula.
The proper organization and differentiation of the anterior segment is pivotal for normal eye development. Neural crest-derived POM cells are key contributors to correct anterior segment formation, differentiating to form the monolayered corneal endothelium. Mice with homozygous null mutations in the forkhead transcription factor gene, Foxc1, fail to develop a proper corneal endothelium stabilized by adherens junctions, with the endothelium adhering to the lens, preventing anterior chamber separation. The aim of this study was to evaluate the interaction between Foxc1 and the adherens junction protein, N-cadherin, as well as an associated gene, Msx1, during key stages in corneal endothelium development. Foxc1 was over-expressed in E12.5 and E13.5 POM cells and qPCR was carried out to determine the effect of Foxc1 on N-cadherin and Msx1 gene expression. Data showed over-expression of Foxc1 in wildtype E12.5 and E13.5 POM cells to cause significant fluctuations in N-cadherin and Msx1 expression (p < 0.05). POM cells were then transfected with a Foxc1 knock-down plasmid or the Foxc1 overexpression plasmid to evaluate the effect of Foxc1 on N-cadherin protein expression by Western blot analysis, however, these results were inconsistent with the gene expression analyses with no significant differences in N-cadherin expression detected. N-cadherin protein expression and localization was then further assessed by means of immunocytochemistry (ICC) and confocal microscopy in monolayer and hanging-drop POM cell cultures. Both qPCR and confocal microscopy data showed consistency, indicating increased amounts of N-cadherin in E12.5 cells relative to E13.5 cells, with membrane-bound N-cadherin showing a clear lattice-work pattern in hanging drop culture. Foxc1 over-expression/knock-down studies on E12.5 and E13.5 POM cells together suggest that N-cadherin is transcriptionally regulated by Foxc1 and that Foxc1 has a threshold level at which it is able to exert control over N-cadherin in POM cells. Foxc1 expression is therefore essential in establishing N-cadherin adhesion junctions in the corneal endothelium. Preliminary data also suggests that Msx1 may directly interact with Foxc1 in POM cells, however, further studies must be undertaken to verify and establish the effects of Foxc1/N-cadherin/ Msx1 interaction in the development of a cohesive, integrated corneal endothelium and functional anterior segment.
Assessment of the diversity of bacteria and methanogenic Archaea in Zebra faeces.
(2013) Naidoo, Kewreshini Kasturi.; Schmidt, Stefan.
The need to develop a renewable, environmentally friendly source of energy has become a primary focus in modern science, with bio gas showing considerable potential. Interest in the methanogenic Archaea has therefore grown in recent years and extensive studies have been carried out to investigate the population diversity in various habitats. Presently, there are only a few studies that have evaluated the microbial communities inhabiting the gastrointestinal tract of wildlife native to southern Africa. This study aimed to investigate the microbial diversity, in particular the bacterial and methanogen communities involved in fermentative digestion in the gastrointestinal tract of zebra. Assessment of the microbial diversity in zebra faeces included both culture-based techniques and nucleic acid targeting analysis via 16S rRNA gene sequencing. Quantitative analysis using selected solid media revealed high counts for aerobic and anaerobic Bacteria (7.51x108 and 2.45x109/gram of faecal sample respectively). The majority of aerobic colonies that were detected exhibited Bacillus-like morphology. Nucleic acid based analysis of the diversity of both Bacteria and methanogenic Archaea in zebra faecal material was performed. Both manual and kit based extractions were used for DNA isolation in order to compare the efficiency of the two methods. Results show that a vigorous mechanical treatment was best for the release of DNA from the faecal matter. Amplification of target gene regions was carried out using established primer pairs (ARCH69F/ARCH915R and EUB338F/EUB907R) for methanogen and bacterial DNA respectively. Amplified 16S rRNA gene regions were cloned into a high copy number vector and random clones were selected for evaluation. Clones containing the target gene were further analysed by ARDRA and were assigned to a specific phylotype. Two bacterial (105 clones in total) and three methanogen (178 clones in total) clone libraries were constructed, of which 24 phylotypes were established for Bacteria and 25 for methanogenic Archaea. A representative of each phylotype was analysed by sequencing and further phylogenetic analysis was conducted. Six bacterial phylotypes, which represented 56% of all bacterial clones, exhibited 99% sequence similarity to Bacillus species. Six methanogen phylotypes, which exhibited 99% sequence similarity to the hydrogenotrophic species Methanobrevibacter gottschalkii strain PG, were established to be predominant in zebra faeces. These phylotypes represented 71% of all archaeal clones selected for analysis in this study.
Bacterial degradation of 2,4-dichlorophenol : catabolic genes detection and enzyme characterization.
(2017) Setlhare, Boitumelo.; Mokoena, Mduduzi Paulos.; Olaniran, Ademola Olufolahan.
Abstract available in PDF file.
Bioprocess development for hydrogen production by dark fermentation using waste sugarcane leaves.
(2015) Moodley, Preshanthan.; Gueguim Kana, Evariste Bosco.
Abstract available in PDF file.
Characterization of antimicrobial resistance genes of Aeromonas spp. isolated from fish and investigation of phytochemical treatment efficacy against resistant isolates.
(2015) Okolie, Chinenye Adaku.; Chenia, Hafizah Yousuf.
Abstract available from print copy.
Characterization of factors involved in and affecting biofilm formation by Aeromonas spp. Isolates.
(2012) Duma, Sphumelele Thuledu.; Chenia, Hafizah Yousuf.
Aeromonas spp. isolates, which are fish and opportunistic human pathogens, form biofilms, however the factors involved in and affecting biofilm formation have not been fully elucidated. Biofilm formation is affected by motility, cell surface characteristics, and/or metabolism, thus it is important to identify factors potentially contributing to initial attachment and/or biofilm formation and their correlation with biofilm formation by Aeromonas spp. isolates. With knowledge of the stages of biofilm formation, mechanisms involved in biofilm formation and its physiology, various strategies may be applied to control aeromonad biofilms. Factors potentially involved in initial attachment and/or biofilm formation were investigated for 99 Aeromonas isolates obtained from seawater and cultured fish. Aeromonad biofilm formation was assessed using microtiter plate assays under varying physicochemical conditions. The disk diffusion method was used to determine the antimicrobial susceptibility profiles of isolates, for comparison to clinical and aquaculture isolates reported in other studies. The MICs and MBICs for antimicrobial agents (azithromycin, ceftazidime, ciprofloxacin, gentamicin and tetracycline) of planktonic cells and biofilm cells, respectively, were investigated using the broth microdilution and modified microtiter plate assays. The effect of sub-MIC (0.5 × MIC) and supra-MIC (2 × MIC) exposures on biofilm-forming cells was also determined using microtiter plate assays. The presence of efflux pump-mediated resistance in 45 Aeromonas spp. isolates was determined using the disk diffusion assay incorporating efflux pump inhibitors (EPIs) [carbonyl cyanide 3-chlorophenylhydrazone (CCCP), phenylalanine arginine β-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP)]. Modified microtite plate assays were used to determine the effect of EPIs [CCCP, PAβN, and NMP], matrix-degrading DNase I and quorum-sensing inhibitors (QSIs; vanillin, 2(5H)-furanone, S-adenosylhomocysteine and cinnamaldehyde) on initial attachment and mature biofilm. Majority of isolates were motile by swimming and swarming and displayed caseinase, gelatinase, and DNase activities, as well as an A-layer phenotype. Majority of isolates displayed high levels of autoaggregation and were hydrophilic. Isolates showed varying levels of adherence, but majority were strongly adherent in nutrient-rich media at 30 ºC. Motility appeared to be a significant characteristic for biofilm formation. Majority of Aeromonas isolates spp. showed high levels of resistance to β-lactams, trimethoprim and sulphamethoxazole, and were susceptible to augmentin, piperacillin-tazobactam, aztreonam, 2nd and 3rd generation cephalosporins, carbapenems, macrolides, fluoroquinolones and aminoglycosides . High levels of resistance towards ceftazidime (MIC > 32 μg/ml) were observed for isolates, while levels of resistance towards remaining antimicrobial agents tested (tetracycline, azithromycin, ciprofloxacin, and gentamicin) were ≤ 32 μg/ml. There was a ≥16-fold increase in MBICs (4096 μg/ml) compared to the MICs for all the antimicrobial agents. The sub-MIC, MIC, and supra-MIC exposures of all antimicrobial agents had an inhibitory effect on both initial attachment and pre-formed biofilms by Aeromonas spp. isolates. Majority of isolates were more susceptible to tetracycline, norfloxacin, and azithromycin due to CCCP and NMP inhibition of the efflux pumps eliminating these antimicrobial agents. Susceptibility to erythromycin was observed for 51% and 47% of isolates, respectively, due to NMP and PAβN inhibition of the efflux pump/s eliminating erythromycin. In the microtiter plate assays, CCCP, NMP and PABN exposures resulted in significant reduction of biofilm formation by majority of Aeromonas spp. isolates in both initial attachment and mature biofilm assays, with CCCP being more effective. DNase I was more effective in reducing mature biofilm, causing reduction for 60% of isolates, compared to its effect on initial attachment. QSIs were also more effective in reducing mature biofilm compared to inhibiting initial attachment. Although increased biofilm dispersal was observed with all QSIs, vanillin and 2(5H)-furanone were more effective compared to S-adenosylhomocysteine and cinnamaldehyde. Based on data obtained in this study, antimicrobial agents, EPIs and QSIs can be used as potential biofilm-inhibiting compounds in aquaculture to control aeromonad infections and may not only prevent disease outbreaks but they could also increase the effectiveness of existing therapeutic agents.
Characterization of selected Bacillus isolates exhibiting broad spectrum antifungal activity.
(2004) Tewelde, Teklehaimanot Weldeslasie.; Hunter, Charles Haig.; Beukes, Mervyn.
The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized.
Complex soil-microorganism-pollutant interactions underpinning bioremediation of hydrocarbon/heavy metal contaminated soil.
(1996) Phaal, Clinton B.; Senior, Eric.; Du Plessis, Chris Andre.
This study evaluated the efficacy of bioremediation as a treatment option for a hydrocarbon and heavy metal contaminated soil. Microbial degradation of hydrocarbons under aerobic, nitrate-reducing and sulphate-reducing conditions was examined. Nutrient supplementation with nitrogen and phosphate as well as aeration seemed to be the most important factors for enhancing biodegradation. From initial batch studies, a carbon: nitrogen ratio of 50: 1 was found to be optimal for biodegradation. However, very low carbon to nitrogen ratios were undesirable since these inhibited microbial activity. Manipulation of the pH did not seem to be beneficial with regard to hydrocarbon biodegradation. However, low pH values induced elevated concentrations of leachate heavy metals. Aerobic conditions provided optimal conditions for hydrocarbon catabolism with up to 54% of the original contaminant degraded after 2 months of treatment. Further treatment for up to 20 months did not significantly increase hydrocarbon biodegradation. Under nitrate- and sulphatereducing conditions, 6% and 31 % respectively of the initial contaminant was degraded after 2 months while after a further 20 months, 50% and 42%, respectively were degraded. The addition of soil bulking agents and the use of sparging did not significantly increase biodegradation. Similarly, the addition of inoculum did not influence biodegradation rates to any great degree. The presence of heavy metals up to concentrations of 400 mgt1 Mn, 176 mgt1 Zn and 94 mgt1 Ni did not reduce microbial activity within the soil. During the treatment phase, heavy metal and hydrocarbon migration were limited even under water saturation and low pH conditions. A Biodegradation Index was developed and evaluated and may, potentially, find use as an in situ assessment technique for microbial hydrocarbon catabolism. The iodonitrophenyltetrazolium salt assay was also found to be an effective and rapid alternative assay for monitoring bioremediation progress.
Cost-benefit analysis of the environmental impacts of Darvill Wastewater Works, Pietermaritzburg, KwaZulu-Natal.
(2002) Sikhakhane, Sindisiwe S.; Ahmed, Fethi B.; Darroch, Mark Andrew Gower.
Darvill Wastewater Works (DWWW) receives and treats both domestic and industrial wastewater from the city of Pietermaritzburg, in KwaZulu-Natal. Sludge from the wastewater treatment is sprayed onto surrounding lands, causing odour and fly problems. The plant also discharges treated effluent into the Msunduzi River, compromising water quality. This study uses several economic valuation techniques to estimate the value of the benefits of improving air and water quality to overcome these problems caused by DWWW. The benefits. are then compared with the costs of upgrading DWWW to see whether or not upgrading DWWW to improve air and water quality would be worthwhile. The Contingent Valuation Method (CVM) was used to elicit people's willingness to pay (WTP) for improvements in air quality due to the elimination of odours and flies caused by sludge deposited by DWWW. The WTP estimates reflect individual's preferences for improvements in air quality. The stated WTP amounts were positively related to household income, but negatively related to the age and gender of the respondent and the number of dependants in the household. The mean monthly WTP for the surveyed households is higher for those that are closer to the pollution source (R23.00 and R29.00 for Zones land 2) and less for those further away (RI4.00 for Zone 3). Sobantu residential area had the lowest mean monthly WTP (R18.00), followed by Lincoln Meade (R27.00) and Hayfields (R54.00). This is expected, as Sobantu has relatively high levels of unemployment and lower household incomes. Strategic, hypothetical and free rider bias may have led to the unexpected signs of some estimated regression coefficients in linear regression models used to estimate WTP. The mean WTP was estimated as R307.20 per annum per household, and when this is aggregated over the total population in the residential areas impacted by odours and flies (37192 households), the benefits of eliminating odours and flies are estimated as R11 425 382.00 per annum. A hedonic price method was used to quantify the decline in property values as a result of odours and flies caused by sludge deposited by DWWW. Properties experienced a R6650.08 decline in selling price if the distance from them to DWWW is decreased by one kilometre. Properties that are closer to DWWW were worth RI5 953.90 less than those further away from DWWW. Aggregating these values over all estimated impacted households in the study, gives an estimated benefit of improving air quality of R28 480 518.00 per annum. The impact of water pollution was quantified by estimating the revenue (R3 744 975.00) that would be lost by Pietermaritzburg if the Duzi Canoe Marathon were to be cancelled due to incidences of diarrheoa reported during the race. A cost of illness procedure was adopted to quantify the effect of water pollution on the health of communities that use the Msunduzi River as a source of potable water supply. A value of R1 243 372.50 was estimated as the annual cost of water-related illnesses in these rural areas. This value represents the costs of the river pollution to those communities. Both of these exercises indicated that improving water quality of the Msunduzi River would be beneficial to society. The effect of nutrient enrichment of the Msunduzi River was quantified by estimating the cost of removing water hyacinth from the Inanda Dam, treatment cost at Wiggins water treatment works and the value of recreation at Mahlabathini Park (Inanda Dam). The annual cost of removing water hyacinth was estimated from the direct costs of chemicals and labour as R47 202.15. The increased treatment costs at Wiggins attributable to DWWW were estimated as R1 104 999.20 and R956 924.15 per annum for removal of algae, and tastes and odours, respectively. The value of R706.90 per annum was estimated as the consumer surplus accruing to recreationists, and, therefore, the value of recreation at Mahlabathini Park to an individual. These annual benefits, when aggregated over the total study population (296 590) were over two hundred million rands (R209 659 470.00). The estimated total benefits (R256 662 840.00) of eliminating odours and flies and effluent problems were compared to the actual costs of two alternative methods of upgrading DWWW using cost-benefit analysis. These alternatives were co-disposal option (R170 473 320) and a land disposal option (R168 809377). Benefit-cost ratios of 1.51 and 1.52 suggest that from society's standpoint, it would be beneficial to upgrade the plant in order to eliminate its adverse environmental impacts. The study results have important implications for policy makers, both the DWWW management and the Pietermaritzburg-TLC municipality. At present DWWW is operating beyond its design capacity, and this problem, together with the poor status of Pietermaritzburg's reticulation system, causes overflow of untreated or compromised final effluent into the Msunduzi River during rainy seasons. These problems also impact on the efficient operation of the plant as the sludge is not properly digested before being sprayed onto surrounding land. Thus to prevent further environmental degradation, a fundamental basis of the National Environmental Management Act, DWWW would need to address these issues. Upgrading DWWW would be a short-term solution if the problems with the storm-runoff into the plant is not addressed.
Design, implementation and assessment of a novel bioreactor for dark fermentative biohydrogen production.
(2020) Khan, Mariam Bibi Hassan.; Gueguim Kana, Evariste Bosco.
The majority of the world’s energy consumption and electricity generation is derived from fossil fuel sources. Their consumption has a negative environmental impact, thus the need for renewable energies. Hydrogen being a high energy zero carbon fuel source presents a profound appeal. Hydrogen may be produced biologically via various methods, this work involves dark fermentative hydrogen production (DFHP). A review of literature on the physicochemical parameters affecting fermentative hydrogen bioprocess was conducted. Bioreactor design was identified as a fundamental component that regulates the overall process outcome and was therefore analysed at length. The review highlighted that existing reactor configurations are unable to sustain a comprehensive criteria of efficient DFHP. A consolidation of biomass retention and non-invasive agitation were distinguished as crucial. The need for a novel reactor configuration possessing these attributes was consequently accentuated. This study focuses on the design, implementation and assessment of novel bioreactor configuration for DFHP. The vessel was formed from a 2L glass and fitted with ports. Three 3D-printed permeable cartridges enclosed immobilized microbial cells and functioned as baffles. The localization and motion of the cartridges promoted improved exposure between microbial cells and substrate. Agitation was accomplished by rocking the vessel at 180°. All the control set points were adjustable, presenting the option of evaluating diverse control regimes. The implemented reactor showed a 35% increase in the peak hydrogen fraction and a 58% reduction in lag time compared to the control shake flask reactor. These findings showed that the novel reactor configuration, by means of the cartridge structure supporting the immobilized cells, enhanced the biohydrogen production process. Subsequently, a preliminary scale up of the cartridge concept was implemented and incorporated into a continuous stirred tank reactor (CSTR). The cartridge (46x40x300mm) consisted of perforated hollow rectangular tubes, joined to form a single amalgamation. This unit was used as substitute for the standard impellers of the CSTR and aligned at 120° laterally to the agitating shaft. The modified reactor prepared with Immobilized cells in cartridge (ICC) was comparatively assessed with the standard CSTR operated with suspended cells in reactor (SCR) and immobilized cells in reactor (ICR). ICC reduced fermentation time by 52 and 65% compared to SCR and ICR respectively. Gompertz model coefficients indicated a 98 and 37% increase in the maximum hydrogen production rate (Rm) using the ICC compared to the SCR and ICR fermentations respectively. ICC also showed better pH buffering capacity and complete glucose degradation. These findings further demonstrated that the scale up reactor configuration with the cartridge structure improved biohydrogen productivity, yield and process economics. The novel configuration reduced process time, improved Hydrogen yield and ensured complete substrate degradation. Furthermore, the structural integrity of immobilized cells was maintained. These findings demonstrated that the novel bioreactor design improved biohydrogen production and showed potential for further DFHP research and development.
The develolpment of a rapid diagnostic test for the detection of haemophilus ducrey.
(2010) Pillay, Mona.; Sturm, Adriaan Willem.
Aim: To develop an antigen detection test that would quickly exclude H. ducreyi infection in individuals with genital ulcers. Materials and Methods: H. ducreyi strains A54 and A68 were grown on Modified Bieling (MB) agar plates and in MB broth under microaerophilic conditions. The 58.5 kDa GroEL Heat Shock Protein (HSP) was extracted from H. ducreyi strain A54 by means of sonication. The purified HSP was used to raise antibodies in rabbits. HSP determination and separation was done on SDS PAGE gels and protein was eluted by means of a passive elution process. Antibody was purified by affinity chromatography and a fraction of the antibody was conjugated to a chromogen to be used as a detection antibody. An ELISA was developed to evaluate the antibody response to the HSP. A second ELISA was developed to evaluate test parameters. Results: A good immune response was achieved with the crude serum of one of the three rabbits when tested against the antigen by means of ELISA. However, after purification of the IgG from the serum of the same rabbit no antigen-antibody binding was observed. Anti-rabbit IgG was able to recognise the antibodies. Discussion and Conclusion: While the Fc portion of the purified IgG remained active, the Fab portion of the antibody had lost biological activity. This loss of biological activity of antibody can be attributed to the low pH of the elution buffers used during the purification steps. Alternative antibody purification systems need to be explored. The use of monoclonal antibodies also needs to be considered.
Development of a laboratory river model to determine the environmental impacts of key xenobiotic compounds.
(1996) Hunter, Charles Haig.; Senior, Eric.; Howard, John.; Bailey, Ian.
Microorganisms are increasingly used in toxicological studies to determine potential environmental impacts of xenobiotic compounds. A multi-stage laboratory model was developed to facilitate the examination of environmental impacts of selected pollutants on fundamental cycling processes inherent to aquatic ecosystems, namely, the degradation of organic substances and nitrogen transformations under aerobic conditions. A microbial association representative of riverine ecosystems was enriched for, isolated and cultured within the model. Characterisation of the microbial association were undertaken. Scanning electron microscopy and bright field microscopy revealed that a diverse heterogenous community of microorganisms had established within the model. Successional metabolic events, namely organic carbon catabolism, ammonification of organic nitrogen and the process of nitrification were differentiated in time and space with the microbial association integrity still being retained. The establishment of a microbial association within the model was primarily dependent on: dilution rates, specific growth rates and interactions between microorganisms and the prevailing environmental conditions. Growth-rate independent populations of microorganisms established within the model and were thought to contribute significantly to the metabolic processes within the model. Nitrifying activity was identified as a rate-limiting process within the model. Following separation of metabolic events, the ecotoxicological impacts of phenol and 2,4-dichlorophenol on the association were assessed. The biological oxidation of ammonia through to nitrate (nitrification) was found to be a sensitive indicator of perturbation. The model was found to be suitable for testing both acute and chronic intoxication by pollutant compounds as well as for biodegradation testing and the possible evaluation of ecotoxicological impacts of wastewater treatment plants. The main disadvantages of the model arose from its operational complexity, its empirical nature and its impracticality for screening large numbers of compounds. A bioassay based on the inhibition of ammonium oxidation was developed in order to fulfil the requirements for a simple and rapid test protocol for the initial screening of perturbant compounds.

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