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Item Adhension of Candida albicans to host cells in culutre.(1989) Maiter, Aziza Ismail.; Alexander, D. M.; Oellermann, Rolf Alfred.No abstract available.Item 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.Item Alkalic salt-based pretreatment strategies for enhancing sugar recovery from corn cobs and process development for simultaneous saccharification and bioethanol production.(2018) Sewsynker-Sukai, Yeshona.; Gueguim Kana, Evariste Bosco .Abstract available in PDF fileItem 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.Item 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.Item 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.Item 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 Alzheimerās 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 2.395ā3.709 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.Item 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.Item 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.Item 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.Item 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.Item 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.Item Bacterivory of three freshwater ciliates isolated from Blackbarough Spruit in KwaZulu-Natal, South Africa.(2021) Bulannga, Rendani Bridghette.; Schmidt, Stefan.Abstract available in PDF.Item Beta-lactamase mediated resistance in Salmonella spp. at a tertiary hospital in KwaZulu-Natal.(2008) Govinden, Usha.; Essack, Sabiha Yusuf.; Sturm, Adriaan Willem.; Moodley, Prashini.Extended spectrum (3-lactamases (ESBLs) were characterized in Salmonella spp. isolates from a pediatric ward of a hospital in Durban. Forty one Salmonella spp. were subjected to serotyping, antibiotic susceptibility testing, E-Tests for ESBL detection, iso-electric focusing, polymerase chain reaction for detection of genes and sequencing. Isolates were screened for the presence of WaTEM, WaSHV, WaCTX-M, WaOXA , WaCMY, WaDHA and WaACC genes. The most common serotype was Salmonella Typhimurium. Isolates were multi-drug resistant with 100% susceptibility only to meropenem and ciprofloxacin. Tazobactam was the most effective inhibitor. Forty-one percent of the isolates were resistant to ceftriaxone, thus limiting therapeutic options for Salmonella infections.TEM-1 was the most predominant (3-lactamase found in 51% of isolates while SHV-12 found in 39 % was the most common ESBL. TEM-63 was evident in 29 %, TEM-116 in 10 % and TEM-131 was found in one isolate. The high ceftazidime MICs of isolates expressing only TEM-63 were indicative of R164S substitution which widens the binding cavity to accommodate the bulky side chains of oxyiminoaminothiazolyl cephalosporins. The identification of TEM-131 which differs from TEM-63 by 1 amino acid reiterates the evolutionary potential of the TEM-type plactamase. Other ESBLs identified included SHV-2, CTX-M-3, CTX-M-15 and CTX-M-37. CMY-2 and the OXA-1 p-lactamase were also detected. This is the first report of TEM-116, CTX-M-3, -15 and -37 in Salmonella spp. in South Africa. All isolates with nalidixic acid MICs > 48 ug/ml had the mutation D87N, or D87G in the QRDR of the gyrA gene. This study showed that Salmonella spp. may be multi-drug resistant with the propensity to harbour p-lactamases in unique combinations. The diversity of ESBLs and the co-expression of quinolone resistance suggests that their incidence in salmonellae needs to be monitored.Item Biodegradation of pentachlorophenol by Bacillus tropicus isolated from activated sludge of a wastewater treatment plant in Durban, South Africa.(2021) Aregebesola, Oladipupo Abiodun.; Olaniran, Ademola Olufolahan.; Mokoena, Mduduzi Paulos.Pentachlorophenol (PCP) is a persistent organic compound that bio-accumulates in the environment due to its recalcitrant nature and has been listed as a priority pollutant due to its toxicological properties. The recent incidences of xenobiotic at different sites and provinces in South Africa, other African Countries and Europe is worrisome and required a proactive measure. Biodegradation has been projected as one of the best ways to ameliorate recalcitrant impacted sites. This study thus aims to isolate and characterize PCP-degrading microorganism from the environment; degrade PCP and other compounds with the isolate in batch culture; optimize biotransformation processes for effective and efficient transformation; determine biodegradation kinetic parameters; profile metabolites produced; detect and amplify PCPdegrading genes from the selected isolate; map the degradation pathway; clone and overexpress the catabolic genes heterologous; purified and characterized the protein both biologically and structurally and sequence the whole-genome of the isolate with the view to determine the evolution and arrangements of PCP-catabolic genes in the genome of the isolate as well as exploring other potentials of the isolates. A PCP-degrading strain was isolated and characterized using a PCR amplification and analysis of the 16S rRNA. Biodegradation process parameters were optimized using response surface methodology. Degradation kinetics were determined via substrate inhibition models, while PCP transformation was evaluated by spectrophotometric and GC-MS analysis. Catabolic genes were detected and amplified via PCR. Genes were cloned via heat-shock technique using chemically competent cells. Proteins purification, digestion and sequencing were done using affinity chromatography, tryptic digestion, and Liquid ChromatographyāMass Spectrometry (LC-MS) techniques respectively. Pacific Biosciences RS II sequencer with the Single Molecule, Real-Time (SMRT) Link was used to sequence the whole genome of the isolate. Cotingās were assembled and analysed using the HGAP4-de-novo assembly application. Genes were annotated on the Rapid Annotation using Subsystem Technology tool kit (RASTtk) and ab initio prediction (PROKKA) using the prokaryotic genome annotation pipelines. Metabolic model pathways of the bacterium was reconstructed using the RAST SEED Viewer. Primarily, the isolate was identified as Bacillus cereus strain AOA-CPS1 (BcAOA) based on the 16S rDNA sequence analysis. However, a quality control test by NCBI for the submitted whole genome sequence of the strain, using an average nucleotide identity (which compares the submitted genome sequence against the whole genomes of the type strains that are already in GenBank) resulted in the renaming of BcAOA as Bacillus tropicus strain AOA-CPS1 (BtAOA). BcAOA was renamed as BtAOA (based on the whole genome data submitted at NCBI under accession number CP049019). The bacterium degraded 74% of PCP (within 9 days at initial PCP concentration of 350 mg l-1 in a batch culture) and other chlorophenolic compounds in co-metabolism. The degradation followed first and zero-order kinetics at low and high PCP concentration, respectively with biokinetic constants: maximum degradation rate (0.0996 mg l-1 h-1); substrate inhibition constant (723.75 mg l-1) and a halfsaturation constant (171.198 mg l-1) and R2 (0.98). The genes (pcpABCDE, cytochrome P450) encoding the enzymes involved in the biodegradation of PCP were amplified from the genomic DNA of BcAOA. Further, depending upon the genes amplified and identified metabolites using GC-MS, there are two different PCP transformation pathways were proposed in this study. At optimized conditions, BtAOA transformed 98.2% of 500 mg L-1 of PCP in 6 days which represent a significant 59.2% increase in PCP transformation compared to the unoptimized conditions. The kinetic parameters for PCP transformation at optimized conditions were: 1.064 Ā± 0.114 mg l-1 h-1 (maximum biodegradation rate); 229 Ā± 19.5 mg l-1 (half-saturation constant); 535 mg l-1 (inhibition constant); and R2 = 0.96. Each of the catabolic genes shared >99% sequences homologies with the corresponding genes in the genomes of their ancestors, however, their biological functions remain putative to date. The optimum temperature and pH of CpsB were 30oC and 7.0. CpsB showed functional stability between pH 6.0-7.5 and temperature 25oC- 30oC. CpsB activity was stimulated by Fe2+, Ca2+, EDTA (0.5-1.5 mM) and Dithiothreitol (0.5- 1.0 mM) but inhibited by sodium azide and sodium dodecyl sulphate (>0.5 mM). CpsB enzyme substrate reaction kinetics studies showed allosteric nature of the enzyme and followed presteady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465 Ī¼mol.s-1, 140 Ī¼mol, 0.099 s-1 and 7.07 x 10-4 Ī¼mol-1.s-1, respectively, for the substrate PCP and 0.259 Ī¼mol.s-1, 224 Ī¼mol, 0.055 s-1 and 2.47 x 10-4 Ī¼mol-1.s-1, respectively, for co-substrate NADH. The Hill plots and M-W-C model reveal CpsB allosteric nature and belong to K-System. CpsB shared 100% sequence homology with aromatic amino acid hydroxylase and is classified as aromatic amino acid hydroxylase superfamily with multiple putative conserved domains and metal ion binding sites confirming its allosteric nature. Bacillus tropicus AOA-CPS1 Cytochrome P450 monooxygenase (P450CPS1) exhibited optimum activity at 40oC and pH 7.5. The P450CPS1 was stable between 25oC-30oC retaining 100% of its residual activity after 240 min of incubation. The activity of P450CPS1 was stimulated by Mn2+, Fe2+, and Fe3+ typical of an oxidoreductase but inhibited by 2.0 mM piperonyl butoxide and sodium dodecyl sulphate. The reaction kinetics studies showed allosteric nature of P450CPS1 showing apparent vmax, Km, kcat and kcat/Km values of 0.069 Ī¼mol.s-1, 200 Ī¼mol, 0.011 s-1 and 5.42 Ć 10-5 Ī¼mol-1.s-1, respectively, for the substrate PCP and 0.385 Ī¼mol.s-1, 56.46 Ī¼mol, 0.06 s-1 and 1.77 Ć 10-3 Ī¼mol- 1.s-1, respectively, for co-substrate NADH. CpsD showed optimal activity at pH 7.5 and temperature between 30oCā40oC. The enzyme was stable between pH 7.0 ā 7.5 and temperature between 30oC and 35oC. CpsD activity was enhanced by Fe2+ ion and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 Ī¼mol s-1, 94 Ī¼mol, 0.029 s-1 and 3.13x10-4s-1Ī¼mol-1, respectively, for substrate tetrachloro-1,4-benzoquinone. CpsD belongs to the pterin-4Ī±-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF-1 (DCoH) superfamily, with specific conserved protein domains of pterin-4Ī± dehydratase (PCD), validated Pterin-4Ī±-carbinolamine dehydratase (DCoH), and coenzyme transport and metabolism proteins. CpsA showed optimum activity at 30oC and pH 9.0. CpsA was stable between 20oC-40oC, and also retained about 90% of its activity at 60oC. The enzyme retained about 90% activity between pH 9.0 and 11.5 and 60% activity at pH 13.0. CpsA was found to be Fe2+ dependent as about 90% increased activity was observed in the presence of FeSO4. CpsA showed apparent Vmax, Km, Kcat and Kcat/Km of 27.77Ā±0.9 Ī¼M s-1, 0.990Ā±0.03 mM, 4.20Ā±0.04 s-1 and 4.24Ā±0.03 s-1 mM-1, respectively at pH 9.0. CpsA 3D structure revealed a conserved 2-His-1-carboxylate facial triad motif (His 9, His 244 and Thr 11), with Fe3+ at the centre. The whole genome of the isolate comprises one chromosome and one plasmid. The metabolic reconstruction for Bacillus tropicus strain AOACPS1 showed that the organism has been exposed to various chlorophenolic compounds including 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane, 1,2-dichloroethane, 1,4- dichlorobenzene, 2,4-dichlorobenzene, atrazine, and other xenobiotics previously and it has recruited enzymes for their degradation. PCP degradation by the isolate was independent of substrate concentration but highly dependent on the maximum specific growth and degradation rates. The low-affinity coefficient and high inhibition constant obtained in this study showed that the bacterium has a high affinity and tolerance to PCP, which could be explored for bulk remediation of PCP. The combination of the recombinant plasmidās vector harbouring the PCP catabolic genes can be used for direct bioremediation of PCP in a bioreactor optimized for the growth of the hosts for overexpression of the proteins. Alternatively, concoction of the enzymes can be produced and immobilised for direct enzymatic bioremediation of PCP and other related recalcitrant xenobiotics. The study proposed that CpsD catalysed the reduction of tetrachlorobenzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine-hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in Bacillus tropicus AOA-CPS1 PheOHS using a probable Yin-Yang mechanism. CpsD may play a catalytic and regulatory role in Bacillus cereus PheOHS and PCP degradation pathway. Findings from this study provide new insights into the biological role of CpsA in PCP degradation and suggest alternate possible mechanism of ring-cleavage by dioxygenases. The study also provides the first experimental evidence of the involvement of a putative cytochrome p450 from Bacillus tropicus group in PCP transformation. Sequence mining and comparative analysis of the metabolic reconstruction of BtAOA with the closest strain and other closely related strains suggests that the operon encoding the first two enzymes in the PCP pathway were acquired from a pre-existing pterin-carbinolamine dehydratase subsystem. The next two enzymes were recruited (via horizontal gene transfer) from the pool of hypothetical proteins with no previous specific function while the last enzyme was recruited from pre-existing enzymes from the tricarboxylic acid cycle or serine-glyoxalase cycle via horizontal gene transfer events.Item Bioprocess development for hydrogen production by dark fermentation using waste sugarcane leaves.(2015) Moodley, Preshanthan.; Gueguim Kana, Evariste Bosco.Abstract available in PDF file.Item Bioremediation of chemically contaminated soil : extraction/analysis methodology development.(2002) Khan, Fatima.; Wallis, Frederick Michael.The efficacies of soil extraction methods, namely, Soxhlet, sonication, agitation, alkaline digestion and the ethyl acetate micro-method, for monitoring soil bioremediation were evaluated using three soil types, Swartland, Rensburg and Hutton, encompassing the mineralogical range prevalent in Kwa Zulu Natal. Phenol, atrazine and the BTEX component of petrol were the molecules used in this study and were extracted under different spiking concentrations, after prolonged ageing times up to 21 days and after changing the composition of the spiking solution. It was concluded that extraction methods must be validated for the specific conditions under which they would be used, taking into consideration, soil type, spiking solutions, moisture content, weathering times and the analyte(s) in question. A preliminary appraisal of atrazine degradation in a Hutton soil was then made under the conditions of sterilized, fertilized/non-fertilized and non-sterilized, fertilized/nonfertilized soils. The predominant pathway of atrazine degradation was deemed to be chemically/abiotically mediated due to the soil pH and the presence of iron and aluminium oxides as well as the high levels of manganese in the soil. The results obtained prompted further study into atrazinecatabolism using soil-slurry reactors, under the conditions of carbon-limitation, nitrogen limitation, carbon/nitrogen non-limitation and carbon/nitrogen limitation. A comparison was made between inoculated and non-inoculated bioreactors. The ability of the indigenous microbial population to return the Hutton soil to its original pristine state was confirmed. The expense of inoculation and culture maintenance could be avoided since carbon and nitrogen supplementation would be as equally effective as inoculation.Item Bioremediation of creosote-contaminated soil by microbial intervention..(2002) Atagana, Harrison Ifeanyichukwu.; Haynes, Richard John.No abstract available.Item Bioremediation of heavy metal polluted waters.(1995) Meyer, Angela.; Wallis, Frederick Michael.Microorganisms have the potential to remove heavy metals from polluted waters and effluents and may be used in clean-up processes. Microbial associations were enriched for and adapted to grow in nutrient solutions containing various concentrations of different metals. As immobilised cells are known to be more stable and more efficient in metal uptake than are corresponding planktonic or free-living cells the attachment of the microbial associations was investigated using a model stream and it was found that biofilm development was better on rough surfaces such as ground glass and polystyrene than on smooth surfaces such as unetched glass plates and glass beads. When comparing metal uptake by planktonic and attached microorganisms, attached populations were found to have a greater metal-uptake capacity. The uptake of individual metals from various metal combinations was tested with various proportions of pregrown metal-adapted microbial populations as inoculum and it was found that a particular metal was taken up more readily by microbial associations which had previously been exposed to that metal. Lead (Pb2+) appeared to be taken up more readily than copper (Cu2+) or cadmium (Cd2+) while Cd2+ was more actively removed than Cu2+ from solution. pH also affected metal uptake and the optimum range for Cu2+ uptake by the Cu2+ -adapted microbial association was found to be between 5.8 and 7.0. Dead microbial biomass was investigated and found to have efficient metal uptake capacity. Living mycelium from an isolated Aspergillus species showed poor uptake of Cu2+ initially, but when this fungus was pregrown and subsequently killed by moist heat treatment the non-living mycelium was efficient in removal of Pb2+ and Cu2+ ions. The optimum mycelial biomass concentration for metal uptake was also determined. The mechanism of metal uptake by this Aspergillus species was determined, using electron microscopy and EDX techniques, to be metabolism-independent biosorption onto the hyphal surface. Thus the microbial associations and fungal cultures used in this study were shown to have the potential for use in the removal of heavy metals from polluted waters.Item Bioremediation of oil-contaminated soil : a South African case study.(1996) Lees, ZoĆ« Marie.; Senior, Eric.; Hughes, Jeffrey Colin.In 1990, an oil recycling plant situated in Hammarsdale, South Africa, was decommissioned and a decision was taken by management to rehabilitate the site in preparation for resale. The heavily impacted area covered over two hecatares and oil contamination penetrated soil to depths in excess of three metres, making excavation and removal of the soil very expensive. The options for remediation of the site were limited. No facility for incineration of contaminated soil exists in South Africa, and landfilling was not permitted. The emphasis in developing a remediation strategy, therefore, focussed upon the possibility of in situ remediation with minimal excavation of soil. This study, the first of its kind in South Africa, was subsequently initiated to assess the feasibility of this approach, the results of which would underpin a full-scale cleanup programme. The development of such a strategy involved four key stages of work : (1) a comprehensive site investigation to evaluate and fully understand the particular problems at the site; (2) treatability studies to determine the potential for biological treatment of the contaminated soil and the optimisation of such treatments, particularly in terms of time and cost; (3) the testing of some of the more effective treatments on a pilot-scale; and (4) recommendations for full-scale bioremediation of the contaminated site. various conditions unique to South Africa had to be considered at each stage viz. the lack of funds and remediation experience, which created numerous problems and emphasised the requirement for a simple, "low-tech" approach. Site investigations revealed that in situ remediation may be possible due to the high permeability of the sandy soils and low concentrations of heavy metals. Laboratory experiments also showed that a mixed association of indigenous microorganisms was present which, once stimulated by nutrient supplementation at C:N:P, ratios of between 10:1:1 and 20:1:1, was capable of degrading total petroleum hydrocarbons at an average rate of 11% week -1. Further experimentation, aimed at reducing the cost of remediation and improving the soil quality, focussed on the efficacy of oil solubilisers, a soil ameliorant (composted pine-bark), indigenous fungi and higher plants in the remedial process. Three commercial surfactants (Arkopal N-050, N-060 and E2491) and one natural solubiliser (soybean lecithin) were tested for their biotoxicity, solubilisation and biodegradability at various concentrations (0.01 - 1.0%). Formulation E2491 was able to support a microbial population and was selected as the preferred commercial surfactant if soil washing was to be recommended; however, lecithin was considered to be more useful in situ because of its localised solubilising effect, biological origin and nutritional contribution. The use of fungi was of particular interest in addressing the persistent organic compounds, such as the heavy fractions of oil, for which bacterial remediation methods have been slow or ineffective. While it was not possible, however, to demonstrate in the laboratory that the indigenous fungi contributed significantly towards the degradation of the contaminating oil, the basic trends revealed that the fungal component of the indigenous microbial population was readily stimulated by the addition of nutrient supplements. The bulking-up process was also a success and additional exploratory work was proposed in the form of a larger scale composting design. Finally, the potential for using higher plants and 20% (v / v) composted pinebark (in addition to nutrients) to increase the microbial degradation of the contamination was investigated in both greenhouse and field plot studies. Greenhouse investigations employed soybeans which were postulated to have soil quality and cost benefits. However, although the soybeans were found to significantly enhance the remedial process, the complex soil-contaminant- plant interactions gave rise to strange nutritional effects and, in some cases, severe stunting. In contrast, the field studies employed grasses that had previously established on the site and which ultimately demonstrated a better tolerance for the contaminated conditions. Scanning electron microscopy revealed that there were considerable differences between the root tips of soybean plants which had been grown in contaminated soil and those which had been grown in uncontaminated soil. It was concluded that toxicity symptoms, which are readily observed in the root, could be used as an early indicator for determining the suitability of vegetation for remediation purposes. In both instances, despite the differences, the addition of composted pine-bark and nutrients (nitrogen and phosphorus) resulted in total petroleum hydrocarbon reductions of >85%, illustrating the benefits of plant establishment and oxygen availability. The need to link results from laboratory or pilot-scale experiments to achieve reliable predictions of field-scale behaviour was an essential component of this research. The results of the field study provided evidence, similar to that found in the pot trial, of the accelerated disappearance of organic compounds in the rhizosphere. All experiments incorporated parallel measurements of hydrocarbon residues, microbial activity and pH changes in the contaminated soil, the results of which strongly supported the argument that biodegradation was the dominant component of the remediation process. Thus, after consideration of the significant interactions which dominated the study (time-contaminant-nutrient; time-contaminant-pine-bark; and time-contaminant- pine-bark-plant), it was clear that, aside from these limiting factors, little should preclude the in situ bioremediation of the impacted soil.