Development of an in-situ ß-D-Glucuronidase diagnostic moraxella-based biosensor for potential application in the monitoring of water polluted by faecal material in South Africa
- Authors: Togo, Chamunorwa Aloius
- Date: 2007
- Subjects: Water quality management -- South Africa Water quality bioassay -- South Africa Sewage sludge -- South Africa -- Management Water -- Purification -- Biological treatment -- South Africa Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3947 , http://hdl.handle.net/10962/d1004006
- Description: The prevention of outbreaks of waterborne diseases remains a major challenge to public health service providers globally. One of the major obstacles in this effort is the unavailability of on-line and real-time methods for rapid monitoring of faecal pollution to facilitate early warning of contamination of drinking water. The main objective of this study was to develop a β-glucuronidase (GUD)-based method that could be used for the on-line and real-time monitoring of microbial water quality. GUD is a marker enzyme for the faecal indicator bacteria Escherichia coli. This enzyme breaks down the synthetic substrate p-nitrophenyl-β-D-glucuronide (PNPG) to D-glucuronic acid and p-nitrophenol (PNP), which turns yellow under alkaline pH. The enzymatically produced PNP was used to detect GUD activity. In situ GUD assays were performed using running and stagnant water samples from the Bloukrans River, Grahamstown, South Africa. The physico-chemical properties of environmental GUD were determined, after which a liquid bioprobe and a microbial biosensor modified with Moraxella 1A species for the detection of the enzyme activity were developed. In order to determine the reliability and sensitivity of these methods, regression analyses for each method versus E. coli colony forming units (CFU) were performed. The storage stabilities of the bioprobe and biosensor were also investigated. The physico-chemical properties of in situ GUD were different from those of its commercially available counterpart. The temperature optimum for the former was between 35 and 40 °C while for the latter it was 45 °C. Commercial (reference) GUD had a pH optimum of 8.0 while the environmental counterpart exhibited a broad pH optimum of between pH 5.0 and 8.0. The liquid bioprobe had a limit of detection (LOD) of GUD activity equivalent to 2 CFU/100 ml and a detection time of 24 h. The method was less labour intensive and costly than the culturing method. The liquid bioprobe was stable for at least four weeks at room temperature (20 ± 2 °C). The biosensor was prepared by modifying a glassy carbon electrode with PNP degrading Moraxella 1A cells. The biosensor was 100 times more sensitive and rapid (5-20 min) than the spectrophotometric method (24 h), and was also able to detect GUD activity of viable but non-culturable cells. Thus it was more sensitive than the culturing method. Furthermore, the biosensor was selective and costeffective. The possibility of using a Pseudomonas putida JS444 biosensor was also investigated, but it was not as sensitive and selective as the Moraxella 1A biosensor. The Moraxella biosensor, therefore, offered the best option for on-line and real-time microbial water quality monitoring in South African river waters and drinking water supplies.
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Evaluation and application of electroanalysis for the determination of antioxidants
- Authors: Ragubeer, Nasheen
- Date: 2007
- Subjects: Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3922 , http://hdl.handle.net/10962/d1003981 , Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Description: The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.
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Hydrogenases from sulphate reducing bacteria and their role in the bioremediation of textile effluent
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
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Isolation and characterization of genes encoding heat shock protein 70s (hsp 70s) from two species of the coelacanth, Latimeria chalumnae and Latimeria menadoensis
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
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Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
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Isolation, propagation and rapid molecular detection of the Kalahari truffle, a mycorrhizal fungus occurring in South Africa
- Authors: Adeleke, Rasheed Adegbola
- Date: 2007 , 2013-04-03
- Subjects: Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3889 , http://hdl.handle.net/10962/d1002951 , Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Description: Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
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Localisation of Theiler's Murine Encephalomyelitis virus non-structural proteins 2B, 2C, 2BC and 3A in BHK-21 cells, and the effect of amino acid substitutions in 2C on localisation and virus replication
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
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Molecular characterisation of the chaperone properties of Plasmodium falciparum heat shock protein 70
- Authors: Shonhai, Addmore
- Date: 2007
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Proteins -- Purification Molecular chaperones Malaria -- Prevention
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3977 , http://hdl.handle.net/10962/d1004036
- Description: Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.
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Regulation of hyu gene expression in Agrobacterium tumefaciens strains RU-AE01 and RU-OR
- Authors: Jiwaji, Meesbah
- Date: 2007
- Subjects: Agrobacterium tumefaciens Amino acids Gene expression Hydrolysis Hydantoin Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3983 , http://hdl.handle.net/10962/d1004042
- Description: Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by deletion analysis. It was proposed that transcription of hyuH was activated by a putative σ[superscript 54]-dependent promoter or a putative σ[superscript 70]-dependent promoter identified upstream of the hyuH gene. The hyuC gene was activated by a putative σ[superscript 70]-dependent promoter identified upstream of the hyuC gene. The regulation of hydantoinase and N-carbamyl amino acid amidohydrolase enzyme activity was compared to the regulation of transcription from the RU-AE01 hyuH-hyuC region. Expression of the hydantoin-hydrolyzing enzymes was regulated by induction which correlated with reporter enzyme expression from the hyuH and hyuC promoter regions. However, the expression of the hydantoin-hydrolyzing enzymes was also regulated by nitrogen catabolite repression (NCR). This did not correlate to the reporter gene expression of the hyuH promoter region but did compare to the reporter gene expression of the hyuC promoter region. This suggested that NCR of hyuH was at the post-translational level whereas NCR of the hyuC promoter was at the transcriptional level. Pathways involved in the regulation of the hyu genes were characterized. The production of the hydantoin-hydrolyzing enzymes in both A. tumefaciens strains RU-AE01 and RU-OR were regulated by proteins involved in the global ntr pathway. The levels of the hydantoin-hydrolyzing enzymes in strain RU-AE01 were elevated in the presence of increased levels of NtrB and NtrC illustrating the importance of the ntr pathway in the regulation of the levels of the hydantoin-hydrolyzing enzymes. Similarly, in RU-OR the presence of exogenous NtrB and NtrC elevated levels of N-carbamyl amino acid amidohydrolase activity. However, the levels of hydantoinase enzyme activity in strain RU-OR were elevated in the presence of NtrC alone. In addition, the presence of a His6-tagged NtrC molecule abolished the elevation in the levels of the hydantoinase but not the N-carbamyl amino acid amidohydrolase enzyme activity in strain RU-OR. This suggests that NtrC has a direct role in the regulation of the expression of hyuH in RU-OR. In addition, it indicates that the hyu genes in the two A. tumefaciens strains RU-AE01 and RU-OR are different. The presence of the RU-AE01 hyuH-hyuC fragment caused a dramatic increase in the hydantoin-hydrolyzing enzyme activity in strain RU-OR but not strain RU-AE01. This implied the incidence of a possible repressor protein in RU-OR, which is titrated out by the presence of the RU-AE01 hyuH-hyuC fragment. Protein-DNA binding assays suggest that this putative repressor may be 38 kDa in RU-OR cells.
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The microbial ecology of sulphidogenic lignocellulose degradation
- Authors: Clarke, Anna Maria
- Date: 2007
- Subjects: Microbial ecology , Lignocellulose , Sulfides , Lignin , Lignocellulose -- Biodegradation , Mines and mineral resources -- Waste disposal , Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4094 , http://hdl.handle.net/10962/d1008181
- Description: Acid mine drainage is a well known environmental pollutant, not only in South Africa, but throughout the world, and the use of microbial processes in the treatment of these wastes has been the subject of investigation over past decades. Lignocellulose packed-bed reactors have been used in passive treatment systems, and, although effective initially, they show early decline in performance while the packing material remains largely un-utilized. Little is known about this phenomenon which remains a severe constraint in the development of efficient passive mine water treatment systems. It has been proposed that the degradation pathways of the complex lignocellulose substrate may be limited in some way in these systems during the manifestation of this effect. This study has addressed the problem using a molecular microbial ecology methodology in an attempt to relate trophic functions of the microbial population to the physico-chemical data of the system. A field-scale lignocellulose packed-bed reactor located at Vryheid Coronation Colliery (Northern Kwa-Zulu Natal province, South Africa) was monitored for six years and the results showed the classic profile of performance decline related to a slowdown in sulphate reduction and alkalinity production. The reactor was decommissioned , comprehensive samples were collected along the depth profile and the microbial populations investigated by means of 16S rRNA gene methodology. The population was found to include cellulolytic Clostridia spp., CytophagaIFlavobacterlBacteroidetes, Sphingomonadaceae and as yet uncultured microorganisms related to microbiota identified in the rumen and termite gut. These are all known to be involved as primary fermenters of cellulose. Oesulphosporosinus was present as sulphate reducer. A comparison of substrata sampling and population distribution suggested that spatial and temporal gradients within the system may become established over the course of its operation. Based on these findings, a laboratory-scale reactor was constructed to simulate the performance of the packed-bed reactor under controlled experimental conditions. The laboratory-scale reactor was operated for 273 days and showed comparable performance to that in the field in both biomolecular and physicochemical data. Clearly defined trophic niches were observed. These results suggested that a sequence of events does occur in lignocellulose degradation over time. Based on the spatial and temporal column studies, a descriptive model was proposed to account for these events. It was found that fermentative organisms predominate in the inlet zone of the system using easily extractable compounds from the wood, thus providing feedstock for sulphate reduction occurring in the succeeding compartments. Production of sulphide and alkalinity appears to be involved in the enhancement of lignin degradation and this, in turn, appears to enhance access to the cellulose fraction. However, once the readily extractables are exhausted, the decline in sulphide and alkalinity production leads inexorably to a decline in the overall performance of the system as a sulphate reducing unit operation. These observations led to the proposal that with the addition of a limited amount of a readily available carbon source, such as molasses, in the initial zone of the the reactor, the ongoing generation of sulphide would be sustained and this in turn would sustain the microbial attack on the lignocellulose complex. This proposal was tested in scale-up studies and positive results indicate that the descriptive model may, to some extent, provide an account of events occurring in these systems. The work on sustaining lignocellulose degradation through the maintenance of sulphate reduction in the initial stages of the reactor flow path has led to the development of the Degrading Packed-bed Reactor concept and that, has subsequently been successfully evaluated in the field.
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The Rhodes BioSURE process and the use of sustainability indicators in the development of biological mine water treatment
- Authors: Neba, Alphonsus
- Date: 2007
- Subjects: Acid mine drainage Water -- Purification -- Biological treatment Mine water Mine water -- Purification Sewage -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3984 , http://hdl.handle.net/10962/d1004043
- Description: Polluted waters, arising from extensive past and ongoing mining operations in South Africa, pose serious environmental threats to the limited fresh water resource. The long time periods, of decades to centuries, over which decanting mine waters may be expected to flow raises additional concerns about the sustainability of these resources. Responses to the problem have thus increasingly been directed towards the long-term sustainability of mine water treatment technologies (MWTT) as a critical indicator in both their research and development, and application. Bioprocess treatments have been considered in this regard and, among these, the Rhodes BioSURE Process has been investigated in preliminary studies using complex organic carbon wastes as the carbon source and electron donor for the central sulphate reduction unit operation. Although both the mining industry and the related statutory/regulatory authority in South Africa share public commitment to sustainability in the treatment of mine waters, no systematic mechanism has emerged to enable the application of sustainability thinking as a guiding principle in the selection and application of MWTTs, nor in the research and development undertaking. This study undertook the development of a Sustainability Indicator Framework in order to provide a systematic basis for the incorporation of sustainability objectives in MWTT bioprocess development, and specifically to use this framework as an input to the investigation of the scaleup development of the Rhodes BioSURE Process. In the development of the MWTT Sustainability Indicator Framework, an initial survey of industry thinking in this area was undertaken and, based on these outcomes, a detailed questionnaire methodology was developed in order to identify and quantify critical sustainability indicators. These included analysis of environmental, economic, social and technical indicators used in sustainability accounting practice in the industry. Statutory/regulatory sustainability targets in the same categories were derived from State of the Environment Reports (SoER) from Provincial authorities where mining is undertaken in South Africa. A synthesis of industry and SoER values was derived from weighted averages and the Sustainability Indicator Framework based on these outcomes. A Conceptual Decision-Support System, to guide the selection and development of MWTTs, was proposed and also based on these results. In the development of the Rhodes BioSURE Process the use of primary sludge (PS) had been investigated as a potential complex carbon and electron donor source. In this regard the utility operator, and sewage treatment process infrastructure, was identified as potentially meeting aspects of the sustainability objectives identified for MWTT application development. Both the Sustainability Indicator Framework and the Conceptual Decision-Support System provided inputs in the formulation of the experimental programme relating to the scale-up development of the Rhodes BioSURE Process. Based on these outcomes, a series of single- and multi-stage reactor configuration, optimisation and enzymology studies were undertaken at bench-, pilot- and technical-scale operations. These units were operated at hydraulic retention times (HRT) ranging between 22 to 72 hours and at chemical oxygen demand to sulphate ratios (COD:SO[subscript 4]) ranging between 1:1 to 2:1. Studies undertaken in fed-batch, bench-scale reactors confirmed the preliminary feasibility of using established sewage treatment infrastructure as a replacement for novel reactor configurations that had been used in the initial studies. The results further indicated that the hydrolysis of PS occurred at different rates under biosulphidogenic conditions in the different reactor configurations investigated. Scale-up of these findings in multi-stage pilot- (7.4m[superscript 3]) and technical-scale plants (680m[superscript 3]) showed comparable performances between the unit operations in terms of SO[subscript 4] and COD removal. These results indicated no apparent advantages in the uncoupling of hydrolysis and sulphate reduction in separate unit operations as had been suggested in previous studies. Scale-down/scale-up studies were undertaken in a continuously fed single-stage reactor configuration and showed that the process could be effectively operated in this way. Previous proposals that chemical and biological gradients established in the sludge bed of the Recycling Sludge Bed Reactor (RSBR) exercised an influence on the rates of substrate hydrolysis were investigated and the relative activity of α- and β-glucosidase and protease enzymes was measured. Results provided additional support for this hypothesis and it was shown that enzyme assay may also provide a useful tool in process development and monitoring studies. While sulphide recovery, following the sulphate reduction step in the BioSURE Process, was not investigated as a component of this study, the treatment of final effluent or waste spills was identified as an important sustainability requirement given the toxicity of sulphide to human and ecosystem environments. A conventional trickle filter reactor system was evaluated for this purpose and showed close to 100% oxidation to sulphate in a short contact time operating regime. Although residual COD removal was low at ~20% of influent, it is considered that high rate recycle biofilter operation could achieve the COD discharge standard of 75 mg/l. The results of the above studies provided inputs into the design, construction and commissioning of the first full-scale commercial application of the Rhodes BioSURE Process for mine wastewater treatment using sewage sludge as the carbon and electron donor source. An adjacent mine and sewage works have been linked by pipeline and an operational capacity of 10 Ml/day water treated has been established with sulphate reduced from ~1300mg/l to <200mg/l. These developments constitute a novel contribution in the mine waste water treatment field.
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The role of arbuscular mycorrhizal fungi in the biotransformation of coal and application in dump rehabilitation
- Authors: Mukasa-Mugerwa, Thomas Tendo
- Date: 2007
- Subjects: Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3999 , http://hdl.handle.net/10962/d1004059 , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Description: Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.
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Treatment of wine distillery wastewaters by high rate anaerobic digestion and submerged membrane systems
- Authors: Melamane, Xolisa Lorraine
- Date: 2007
- Subjects: Wine and wine making -- Waste disposal Sewage -- Purification -- Anaerobic treatment Membrane reactors Bioreactors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3963 , http://hdl.handle.net/10962/d1004022
- Description: Experiences in treating wine distillery wastewaters (WDWs) contribute to the field of oenology as many oenologists are concerned with the selection, efficiency and economy of their wastewaters. Wine distillery wastewaters are strongly acidic, have high chemical oxygen demand (COD), high polyphenol content and are highly variable. Primary attention was focussed on sustainable biological treatment of raw wine distillery wastewater (RWDW) and fungally pre-treated wine distillery wastewater (FTWDW) by energy-efficient high rate anaerobic digestion (AD). This study also explored the development of a novel dual-stage anaerobic digestion ultrafiltration (ADUF) process, using a ceramic submerged membrane bioreactor (SMBR) in the treatment of both RWDW and FTWDW. The first stage was for the selection of microorganisms that were able to treat the toxic pollutants from WDWs. It was operated at a high feed-to-microorganism ratio. The second stage, a secondary digester, was operated like a typical membrane bioreactor at a low feed-to-microorganism ratio to sustain a stable efficient population for a long period. The characteristics of RWDW were as follows: pH 3.83, 15 000 mg/l soluble COD (CODs) and 5229 mg/l of phenols. After pre-treatment of RWDW with Trametes pubescens, starting parameters for FTWDW were as follows: pH 6.7, 7000 mg/l soluble COD (CODS) and 1440 mg/l of phenols. During operation of a high rate anaerobic digester for RWDW treatment, K2HPO4 was required for buffering the digester. Volatile fatty acid concentrations were <300 mg/l throughout the study, indicating degradation of organic acids present. Mean CODS removal efficiency for the 130 day study was 87 %, while the mean polyphenol removal efficiency was 85 %. Addition of 50 mg/l Fe3+ increased the removal efficiencies of CODS to 97 % and of polyphenols to 99 %. High removal efficiencies of CODS and polyphenols were attributed to the addition of macronutrients and micronutrients that caused pH stability and stimulated microbial activity. The CODS removal efficiency of high rate anaerobic digestion of FTWDW reached 99.5%. During FTWDW digestion, pH buffering was achieved using K2HPO4. A combination of a SMBR and a secondary digester was tested for the treatment of RWDW and FTWDW during a 30 day study. Results for RWDW showed that pH buffering was achieved by dosing the feed stream with CaCO3 and K2HPO4. Buffering proved to be significant for optimum performance of the system in removal of soluble CODS, and volatile fatty acids (VFAs). Different batches of RWDW used for feeding the reactor had variable compositions with respect to concentrations of nitrates, ammonium and total phenolic compounds. Ammonium accumulated in the secondary digester after 14 days of system operation, indicated the time required for the establishment of anaerobic conditions in the system. Dosing of the SMBR treating FTWDW with CaCO3 and K2HPO4 buffered the pH; iii this proved significant for optimum performance of the system in removal of CODS. The system eliminated an average of 86 (± 4) % of CODS present in the FTWDW. The residual CODS levels in the effluent were approximately 400 mg/l, significantly lower than the concentrations observed when treating RWDW, indicating that fungal pre-treatment might have provided additional nutrients for removal of recalcitrant components of the wastewater. The resulting effluent was rich in nitrates and phosphates and might be used as a fertiliser. Alternatively, a membrane process, such as reverse osmosis (RO) or nanofiltration (NF) could be applied to raise the water quality to meet the levels required for reuse. Biomass samples were obtained from the four treatment systems and population shifts characterization using phospholipids fatty acids (PLFA) and 16S rRNA analysis to provide an indication of limitations within the microbial population. The values of the concentrations of the individual PLFAs detected in the samples indicated that ten bacterial species were present, with the GC content of the 16S rRNA increasing from 1 to 10. Analysis of denaturing gradient gel electrophoresis DGGE data indicated that the composition of the archeal community changed the consortia used for both RWDW and FTWDW treatment. Changes in band intensities indicated the presence of different components of the archeal communities. The results were not conclusive in terms of species identity as cloning, sequencing and phylogenetic analyses were not performed, but they did indicate microbial population shifts and species diversity for high rate anaerobic digestion. The results also confirmed prevalence of relatively few species during operation of SMBRs for treatment of RWDW and FTWDW, which suggested that the microorganisms that survived were either tolerant of toxic components of RWDW and FTWDW or they were able to remove polyphenols.
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Wheat stress responses during Russian wheat aphid and Bird Cherry Oat aphid infestation: an analysis of differential protein regulation during plant biotic stress responses
- Authors: Louw, Cassandra Alexandrovna
- Date: 2007
- Subjects: Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3995 , http://hdl.handle.net/10962/d1004055 , Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Description: Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.
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Ectomycorrhizal characterisation, species diversity and community dynamics in Pinus patula Schelcht. et Cham. plantations
- Authors: Hawley, Greer Leigh
- Date: 2006
- Subjects: Mycorrhizas Ectomycorrhizal fungi Pinus patula -- Irrigation -- South Africa Forests and forestry -- South Africa Forest ecology -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3951 , http://hdl.handle.net/10962/d1004010
- Description: Ectomycorrhizal (ECM) associations are important elements of forest biomes, connecting and transferring nutrients through an intricate and complex system of hyphal networks, ensuring plants of the nutrients they require, in nutrient poor soil. ECM research and particularly investigations into the diversity of the fungal partners has not received much attention in South Africa, hindering the advance of research in this field. This has been attributed to the difficulty of identifying the mycobionts involved in the symbiosis. The objectives of this study were to examine the ECM fungal diversity associating with Pinus patula, in selected forest plantations in Mpumalanga, South Africa. Both morphological and molecular techniques were used to identify specimens of both sporocarp collections and ECM root tip morphotypes. Morphological analysis of the ECM root tips involved characterisation of root morphology such as colour, branching and texture, and anatomical analysis examined hyphal arrangement in the root mantle and rhizomorphs. Molecular analysis involved sequencing of the Internal Transcribed Spacer (ITS) region and comparative BLAST analysis. Twenty-four sporocarp species were identified from 13 genera, namely: Amanita, Boletus, Clavulina, Inocybe, Lactarius, Rhizopogon, Russula, Scleroderma, Suillus, Tricholoma, Thelephora, Tomentella and Xerocomus. ECM root tip analysis led to the characterisation of 7 wild-type morphotypes identified as an Albatrellus sp., 2 Amanita species, a Rhizopogon sp., Thelephora terrestris, a Tomentella sp. and Scleroderma citrinum. A secondary objective was to determine whether fertilisation treatments within the study sites were responsible for differences in fungal species community structure. No evidence of a change in species diversity or shift in species composition was encountered. It is envisaged that these comprehensive ECM descriptions will be used as reference material to stimulate continued research in this field in South Africa.
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Isolation, purification and characterization of a novel glucose oxidase from Penicillium canescens Tt42
- Authors: Simpson, Clinton
- Date: 2006
- Subjects: Penicillium , Glucose , Oxidases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3975 , http://hdl.handle.net/10962/d1004034 , Penicillium , Glucose , Oxidases
- Description: A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.
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Voltammetric analysis of pesticides and their degradation: A case study of Amitraz and its degradants
- Authors: Brimecombe, Rory Dennis
- Date: 2006
- Subjects: Hydrolysis , Biodegradation , Voltammetry , Pesticides -- Biodegradation , Pesticides -- Environmental aspects , Acaricides , Acaricides -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4131 , http://hdl.handle.net/10962/d1015724
- Description: Amitraz is a formamide acaricide used predominantly in the control of ectoparasites in livestock and honeybees. Amitraz hydrolysis is rapid and occurs under acidic conditions, exposure to sunlight and biodegradation by microorganisms. The main hydrolysis product of amitraz, 2,4-dimethylaniline, is recalcitrant in the environment and toxic to humans. An electrochemical method for the determination of total amitraz residues and its final breakdown product, 2,4-dimethylaniline, in spent cattle dip, is presented. Cyclic voltammetry at a glassy carbon electrode showed the irreversible oxidation of amitraz and 2,4-dimethylaniline. A limit of detection in the range of 8.5 x 10⁻⁸ M for amitraz and 2 x 10⁻⁸ M for 2,4-dimethylaniline was determined using differential pulse voltammetry. Feasibility studies in which the effect of supporting electrolyte type and pH had on electroanalysis of amitraz and its degradants, showed that pH affects current response as well as the potential at which amitraz and its degradants are oxidised. Britton-Robinson buffer was found to be the most suitable supporting electrolyte for detection of amitraz and its degradants in terms of sensitivity and reproducibility. Studies performed using environmental samples showed that the sensitivity and reproducibility of amitraz and 2,4-dimethylaniline analyses in spent cattle dip were comparable to analyses of amitraz and 2,4-dimethylaniline performed in Britton-Robinson buffer. In addition, the feasibility qf measuring amitraz and 2,4-dimethylaniline in environmental samples was assessed and compared to amitraz and 2,4-dimethylaniline analyses in Britton-Robinson buffer. Amitraz and 2,4-dimethylaniline were readily detectable in milk and honey. Furthermore, it was elucidated that 2,4-dimethylaniline can be metabolised to 3-methylcatechol by Pseudomonas species and the proposed breakdown pathway is presented. The biological degradation of amitraz and subsequent formation of 2,4-dimethylaniline was readily monitored in spent cattle dip. The breakdown of amitraz to 2,4-dimethylaniline and then to 3-MC was monitored using cyclic voltammetry.
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A comparative bioinformatic analysis of zinc binuclear cluster proteins
- Authors: Mthombeni, Jabulani S
- Date: 2005
- Subjects: Bioinformatics , Zinc proteins , GABA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4004 , http://hdl.handle.net/10962/d1004064 , Bioinformatics , Zinc proteins , GABA
- Description: Members of the zinc binuclear cluster family are important fungal transcriptional regulators sharing a common DNA binding domain. Da181p is a pleotropic zinc binuclear cluster protein involved in the induction of the UGA genes required for the γ-aminobutyrate nitrogen catabolic pathway in Saccharomyces cerevisiae. The zinc binuclear cluster domain is indispensable for function in Da181p and little is known about other domains in this protein. The aim of the study was to explore the zinc binuclear cluster protein family using comparative bioinformatics as a complement to biochemical and structural approaches. A database of all zinc binuclear cluster proteins was composed. A total of 118 zinc binuclear proteins are reported in this work. Thirty nine previously unidentified zinc binuclear cluster proteins were found. Four homologues of Da181p were identified by homology searching. Important sequence motifs were identified in the aligned sequences of Da181p and its homologues. The coiled coil motif found in the Ga14p zinc binuclear cluster protein could not be identified in Da181p and its homologues. This suggested that Da181p did not dimerise through this structural motif as other zinc binuclear cluster proteins. Solvent accessible site that could be phosphorylated by protein kinase C or casein kinase II and the role of such sites in the possible regulation of Da181p function were discussed.
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African mead biotechnology and indigenous knowledge systems in iQhilika process development
- Authors: Cambray, Garth Anton
- Date: 2005
- Subjects: Biotechnology Indigenous peoples -- Africa Ethnoscience -- Africa Mead -- Africa Brewing -- Microbiology Honeybee Honey
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3929 , http://hdl.handle.net/10962/d1003988
- Description: While the production of mead, a fermented honey beverage, has declined in popularity around the world in recent centuries, a substantial mead industry continues to exist in Africa with an estimated annual production of 1 to 1.7 billion litres. This is largely an ‘invisible industry’, and has functioned outside the formal economy due to proscription of indigenous beverages during colonial times. The traditional African mead industry is, however, also now under pressure due to the environmental degradation of scarce natural ingredients, urbanisation and loss of indigenous knowledge systems (IKS) and, with time, the beverage will likely follow the declining trend of mead consumption observed elsewhere. An analysis of early reports of African mead production suggested that the Khoi-San, among the earliest inhabitants of the continent, are the originators of the mead making techniques which use fibrous plant materials derived from specific plant species, to facilitate mead fermentation in some way. The Eastern Cape represents a region with a large body of Khoi-San IKS preserved in their descendants among the Afrikaans and Xhosa populations. A survey to establish a baseline of mead-making technology in the Eastern Cape was undertaken, and involved interviewing traditional mead makers across an area of roughly 100 000 km2, showing that the mead, iQhilika(Xhosa) Kari (Khoi-San/Afrikaans), is produced using a very similar process throughout the region. This involves the roots of a Trichodiadema sp. plant (imoela – Xhosa, karimoer – Afrikaans/Khoi-San), honey, extract of brood and/or pollen and water. Various other fruit sugar sources were also found to be added at times producing seasonal beverages with unique organoleptic properties. A model traditional iQhilika production operation was investigated in order to describe the main features of the process. Biomass immobilised on Trichodiadema root segments was found to be distributed evenly through the profile of the bioreactor resulting in a well mixed fermentation and a productivity of 0.74 g EtOH/l/h. In the initial stages of fermentation, the ethanol yield was highest in the mid-regions of the bioreactor, but with time the regions closer to the surface, which had atmospheric contact had a higher yield. This phenomenon was attributed to aerobic fatty acid synthesis which allowed the yeast close to the surface to function more efficiently despite rising ethanol concentrations. The mead contained 44.25 g/l (7 % volume) ethanol produced in a fermentation time of 43.5 h. Yeast biomass in the traditional process was either immobilised in the form of flocs or attached to the Trichodiadema intonsum support. Electron microscopy revealed that the cells were covered in a layer of extra-cellular polymeric substance apparently assisting the immobilization, and which was populated by a consortium of yeasts and bacteria. Yeasts isolated from iQhilika brewed in two regions separated by 350 km were found to be very closely related Saccharomyces cerevisiae strains as determined by molecular genetic analysis. The traditional beverage was found to contain populations of Lactic acid bacteria (LAB), which are known spoilage organisms in other beverages. Spoilage characteristics of these organisms matched descriptions of spoilage provided by the IKS survey. Other possibly beneficial LAB, which may contribute useful flavour compounds, were also found to be present in the system. The basic functional aspects of the traditional process were used to design a continuous bench-scale tower bioreactor and process development was based on the IKS survey. This consisted of a packed bed bioreactor, consisting of 2 mm3 T. intonsum root segments, immobilising a novel Saccharomyces cerevisiae strain isolated from a traditional batch of iQhilika. The bioreactor performed well with a yield of close to the theoretical maximum and an ethanol productivity of 3.45 g EtOH/l/h. The parameters of the 5.6 l/d bench-scale bioreactor were used to design a full-scale production bioreactor with a planned maximum output of 330 l/d. This bioreactor had a productivity of 0.19 g EtOH/l/h. The organoleptic properties of the product produced were considered by a taste panel to be better than those of the product of the bench-scale tower bioreactor. This research was based on the development of IKS which imposed a number of constraints and obligations on the project to ensure environmental, and social, in addition to financial viability of the scale-up operation. Makana Meadery was established in partnership with Rhodes University as an empowerment company which, in addition to undertaking the commercialisation of the iQhilika process, would also develop methods for the production of scarce ingredients traditionally unsustainably sourced from fragile ecosystems, provide beekeeping training and the manufacture of beehives.
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Characterization of the hydantoin-hydrolysing system of Pseudomonas putida RU-KM3s
- Authors: Matcher, Gwynneth Felicity
- Date: 2005
- Subjects: Hydantoin Hydrolysis Pseudomonas Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3939 , http://hdl.handle.net/10962/d1003998
- Description: The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps: the hydrolysis of hydantoin to N-carbamylamino acid by an hydantoinase or dihydropyrimidinase enzyme, followed by conversion of the Ncarbamylamino acid to the corresponding amino acid by an N-carbamoylase enzyme. This biocatalytic process has been successfully applied in several industrial processes for the production of enantiomerically pure amino acids used in the synthesis of pharmaceuticals, insecticides, hormones, and food additives. P. putida RU-KM3S was selected for study based on inherent high levels of hydantoinase and N-carbamoylase activity. Subsequent biocatalytic analysis of the enzyme activity within this strain revealed unique properties thus prompting further characterization. The main focus of this research was the isolation of the genes encoding the hydantoin-hydrolysing pathway in RU-KM3S. A genomic library was constructed and screened for heterologous expression of the hydantoin-hydrolysing enzymes. However, this approach was unsuccessful prompting the use of transposon mutagenesis in order to circumvent the drawbacks associated with complementation studies. The enzymes responsible for hydantoin-hydrolysis were identified by insertional inactivation as a dihydropyrimidinase and b-ureidopropionase encoded by dhp and bup respectively. A third open reading frame, encoding a putative transport protein, was identified between the dhp and bup genes and appeared to share a promoter with bup. Analysis of the amino acid sequence deduced from bup and dhp substantiated the distinctive properties and potential industrial application of the L-enantioselective b-ureidopropionase and provided targets for potential optimisation of the substrate-selectivity and activity of the dihydropyrimidinase by site directed mutagenesis. Several transposon-generated mutants with an altered phenotype for growth on minimal medium with hydantoin as the sole source of nitrogen were also isolated. Analysis of the insertion events in these mutants revealed disruptions of genes encoding key elements of the Ntr global regulatory pathway. However, inactivation of these genes had no effect on the dihydropyrimidinase and b-ureidopropionase activity levels. An additional mutant in which the gene coding for the dihydrolipoamide succinyltransferase, which is involved in the TCA cycle, was isolated with reduced levels of both dihydropyrimidinase and b-ureidopropionase activities. These results indicated that the hydantoin-hydrolysis pathway in RU-KM3S is regulated by carbon rather than nitrogen catabolite repression. This was confirmed by the reduction of hydantoin-hydrolysis in cells grown in excess carbon as opposed to nitrogen. Identification of a putative CRP-binding site within the promoter region of these enzymes further supported the regulatory role of carbon catabolite repression (CCR). As CCR in Pseudomonads is poorly understood, elucidation of the mechanism by which the hydantoinhydrolysing pathway in RU-KM3S is regulated would provide valuable insight into this complex process.
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