Removal of copper and nickel from solution by the non-viable biomass of the water fern Azolla filiculoides in an upscaled fixed-bed column system
- Authors: Thompson, Denis Alan
- Date: 2001
- Subjects: Copper , Nickel , Azolla , Heavy metals -- Absorption and adsorption
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3914 , http://hdl.handle.net/10962/d1003973 , Copper , Nickel , Azolla , Heavy metals -- Absorption and adsorption
- Description: The potential of non-viable Azalia filiculaides for the removal of Cu and Ni from aqueous solutions and the possibility of scaling up existing lab scale Azalia column systems was investigated. The effects of factors such as metal starting concentration, pH and two metals in solution on the removal of Ni and Cu from aqueous solution by dried and crushed Azalia biomass were studied in batch systems. Aqueous solutions of Ni with starting concentrations between 1000 and 2000J.lmolll gave the most efficient Ni removal by Azalla biomass. For Cu the optimum starting concentration for adsorption was 50J.lmol/l. The adsorption capacity of both eu and Ni increased as the starting pH of the sorption media increased. The optimum pH for Ni adsorption was found at pH 7 and for Cu, at pH 5. - Awlla biomass had a higher. maximum binding capacity (qrnax) for Cu than for Ni at pH 5. The removal of both Cu allct Ni showed little or no variation with the presence another metal in solution. Kinetic studies show that both Cu and Ni adsorbed rapidly onto the Azalia biomass. The removal of Cu and Ni from aqueous solutions using non-viable Azalia biomass was investigated in a lab scale fixed-bed column and an upscaled 4L column system. The nonviable Azalla filiculaides biomass when dried and used in a column for adsorption of Cu and Ni showed good physical stability under many different conditions. Preparation of the biomass before it could be used in the columns was very simple and did not involve any significant pretreatment steps. Prolonged exposure to UV light decreases Azalia biomass capacity for Ni and Cu adsorption. Column adsorption of Cu and Ni from aqueous solutions was successfully upscaled approximately 100 times. Relative to the lab scale column, the 4L column performed better for the uptake of Cu and Ni per gram of biomass. The larger column was also able to operate at relatively higher flow rates. The biomass showed good reusability with little change in the amount of Ni adsorbed in 10 consecutive cycles. Electron micrographs showecf little or no change in the physical structure and integrity of the Azolla biomass after exposure to mineral acids, Ni solution and high flow rates over 10 consecutive adsorption and desorption cycles. As much as 80% Ni and 70 % Cu was recovered when desorption profiles were generated using O.lMHCI as a desorption agent. The 4L column system was also tested using a highly concen~rat:~ Ni plating bath solution.(Nicrolyte 1). Only 18 % of the Ni could be removed from the expended Nicrolyte 1 pla~Jng solution after treating only 25L, indicating that Azolla biomass is more suited for removal of metals from more dilute industrial effluents.
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- Date Issued: 2001
The refining of calcium using a sulfate reducing bacterial system
- Authors: Horne, Kerry Allison
- Date: 2001
- Subjects: Calcium carbonate -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3915 , http://hdl.handle.net/10962/d1003974 , Calcium carbonate -- Synthesis
- Description: White lime is used in many industries in South Africa but is not produced locally and must be imported. Many technologies have been suggested for the large-scale manufacture of calcium carbonate but these are not necessarily suitable for application in South Africa. This study investigated a chemical preparation of calcium carbonate combined with biological purification Calcium containing materials from the Pretoria Portland Cement, Lime Division factory at Lime Acres in the Northern Cape were studied as the starting materials for the manufacture. Investigation showed that they contained various impurities, including iron and manganese compounds which were largely responsible for the brown-grey colour of the lime products. Complete dissolution of calcium hydroxide, the purest of the potential starting materials, and subsequent hydroxide precipitation was not successful in removing all iron and manganese. Precipitation with sulfide ions was successfill, decreasing levels of metals to below the detection limit of atomic absorption spectrophotometry. Studies of all potential starting materials revealed that the levels of impurities in the starting material did not have a large effect on levels of impurities in the calcium carbonate produced. It was therefore possible to convert the residual calcium oxide or hydroxide in waste lime dusts to white calcium carbonate, a marketable prciduct Recycling of the water and starting material used in the process served to increase, rather than decrease, the purity of the calcium carbonate product. This allows for water conservation as water is not consumed in the process but merely utilised. When waste lime dust was used as the starting material, sulfate was found in the product. While still a white lime, the calcium carbonate was not chemically pure. Sulfate removal was therefore investigated and the use of sulfate-reducing bacteria was studied as a novel application. A mixed sulfate-reducing bacterial population was isolated and found to be hIghly active at sulfate concentrations between 0.2 and 2 ~~~. They were capable of autotrophic growth and could reduce sulfate in solutions with elevated pH and in calcium carbonate suspensions, although they did not grow readily in these media. A process was designed for the production of bulk quantities of calcium carbonate making use of the facilities and materials available at Lime Acres. This was tested using a small scale bench-top reactor series, with favourable results. The process would allow automatic, continuous production of large quantities of white lime using waste lime dust. Provision was also made for manufacture of smaller quantities of pure calcium carbonate using sulfate-reducing bacteria to remove the sulfate impurity.
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- Date Issued: 2001
Using gene shuffling to increase genetic diversity in genes involved in beta-lactam biosynthesis
- Authors: Tarr, Shahida
- Date: 2001
- Subjects: Beta lactam antibiotics , Genes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4014 , http://hdl.handle.net/10962/d1004074 , Beta lactam antibiotics , Genes
- Description: The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing.
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- Date Issued: 2001
Accelerated carbon dioxide deliming of cattle hides and sheepskins
- Authors: Flowers, Karl Bernard
- Date: 2002
- Subjects: Tanning , Hides and skins , Carbon dioxide
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3918 , http://hdl.handle.net/10962/d1003977 , Tanning , Hides and skins , Carbon dioxide
- Description: To avoid environmental pressure from water authorities, specifically regarding nitrogen and sulfate limits in tannery wastewater, modifications to existing deliming processes have been made. Conventional ammonium salt deliming methods contribute to Total Kjeldahl Nitrogen values in the region of 0.5 – 1.0g/L (33-67% of total TKN). Sulfate levels are increased with the use of organic deliming and ammonium sulfate deliming to the extent of 0.9g/L (27% of total sulfate). To understand the dynamics and kinetics of carbon dioxide equilibrium, the movement of carbon dioxide into deliming water, through carbonic acid, bicarbonate and ultimately into carbonates at liming or early deliming pH was studied. It was shown in this study that effective lime removal, at optimum conditions, resulted in fully delimed pelts at highly comparable quality and times compared to conventional ammonium salt deliming
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- Date Issued: 2002
Biological sulphide oxidation in heterotrophic environments
- Authors: Rein, Neil Berthold
- Date: 2002
- Subjects: Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3919 , http://hdl.handle.net/10962/d1003978 , Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Description: Acid mine drainage is a major environmental pollution concern associated with the mining of sulphide-containing ore bodies. Both physicochemical and biological options have been investigated for the treatment of acid mine drainage with recent interest in biological processes targeting low-cost and passive treatment applications. All acid mine drainage biological treatment processes are based to some extent on the activity of sulphate reducing bacteria, and their ability to reduce sulphate to sulphide in the presence of a range of carbon and electron donor sources. A portion of the sulphide produced may be consumed in the precipitation of heavy metals present in the mine drainage. Residual sulphide must be removed, not only due to its toxicity, but especially to prevent its reoxidation to sulphate where salinity reduction is a target of the treatment process. The partial oxidation of sulphide to elemental sulphur is an option that has received considerable attention and both physicochemical and biological options have been investigated. Biological processes have substantial potential cost advantages and run at ambient temperatures and pressures. However, the oxidation of sulphide to elemental sulphur is poised over a narrow redox range and process control to maintain optimum conditions remains a serious problem. In addition little has been reported in the literature on process control of sulphide oxidation to elemental sulphur, in the heterotrophic conditions prevailing in the reaction environment following sulphate reduction. This study undertook an investigation of biological sulphide oxidation under heterotrophic conditions in order to establish the effect of organic compounds on biological sulphide oxidation, and to determine whether the presence of organics, and associated heterotrophic oxygen consumption, may be manipulated to maintain the defined redox conditions required for the production of elemental sulphur. Biological sulphide oxidation under heterotrophic conditions was investigated in a series of flask experiments. Based on these results three different reactor configurations, a Fixed-Film Trickle Filter Reactor, Submerged Fixed-Film Reactor and a Silicone Tubular Reactor were used to investigate sulphur production. The flask studies indicated that organics, and associated heterotrophic metabolism in the presence of excess oxygen in the sulphide oxidation reaction environment, did contribute to the poising of redox conditions and thereby enabling the production of elemental sulphur. While the Fixed-Film Trickle Filter Reactor was found to be redox unstable, probably due to excess oxygen ingress to the system, a reduced oxygen challenge in the Submerged Fixed-Film Reactor configuration was found to be more successful for production of elemental sulphur. However, due to the production of a predominantly filamentous sulphur producing microbial population, recovery of sulphur from the column was intermittent and unpredictable. Extended residence times for produced sulphur on the column increased the likelihood for its eventual oxidation to sulphate. The Silicone Tubular Reactor was found to support a vigorous sulphide oxidising biofilm and produced elemental sulphur effectively. Electron microscopic studies showed that this occurred as both biologically produced sulphur and, probably mainly, as crystalline sulphur in the ortho-rhomic form. Given the linear extension of the sulphur production reaction environment it is was possible to investigate the sequence of the reaction mechanism in grater detail than is possible in mixed systems. Based on these findings a model explaining sulphur production under heterotrophic conditions has been proposed and is presented. The commercial implications of the development have also been noted.
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- Date Issued: 2002
Biosulphidogenic hydrolysis of lignin and lignin model compounds
- Authors: Madikane, Mzekelo
- Date: 2002
- Subjects: Lignin Lignin -- Biodegradation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3917 , http://hdl.handle.net/10962/d1003976
- Description: Lignin degradation under biosulphidogenic conditions has not been extensively reported in the literature. Although aerobic degradation of lignin is well documented, anaerobic biodegradation has focused mainly on methanogenic systems with biosulphidogenic systems receiving less attention. Sulphate reducing bacteria are known to generate moderately high levels of both sulphide and alkalinity at room temperatures, and these conditions draw some comparison with the Kraft pulping process. In the Kraft pulping process, lignin is degraded chemically at ±170°C under high sulphide and alkaline conditions and may provide a model for understanding biosulphidogenic lignin degrading activity. The aim of this study was to investigate the biosulphidogenic hydrolysis of lignin within the context of the chemical and biological conditions generated by a mixed sulphate reducing bacteria consortia. Bioreactor studies with a mixed sulphate reducing consortia and pine wood powder (both untreated and depectinated) resulted in the generation of comparable levels of sulphide and alkalinity used in the chemical hydrolysis studies. Aromatic compound yields were between 20 to 50% of the chemical hydrolysis studies. This fluctuation may have been due to the utilization of these aromatic compounds as electron donors by the sulphate reducing consortia as evidenced by the high rate of sulphate reduction in both the untreated and depectinated wood bioreactors. Biodegradation of lignin model compounds was investigated in order to elucidate lignin degradation mechanisms. Both mono-aromatic and dimeric lignin model compounds were used as electron donors and carbon sources for the mixed sulphate reducing consortia. Biodegradation and mass spectrometer analysis of mono-aromatic compounds, ferulic acid and ferulic acid ethyl ester resulted in the production of intermediates such as catechol, cyclohexane carboxylic acid and adipic acid. These intermediates were also observed in the degradation of dimeric ferulic acid. Biodegradation of salicin resulted in the production of salicyl alcohol, ortho-cresol and acetate. Biodegradation of benzylic ether resulted in the production of vanillin and acetate as end products. The results of these studies provide evidence for a biosulphidogenic hydrolysis of lignin, and also the utilisation of lignin-derived aromatic compounds as electron donor sources, by a mixed sulphate reducing consortia.
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- Date Issued: 2002
Elucidation and manipulation of the Hydantoin-Hydrolysing Enzyme System of Agrobacterium tumefaciens RU-OR for the Biocatalytic production of D-amino acids
- Authors: Hartley, Carol Janet
- Date: 2002
- Subjects: Amino acids Agrobacterium tumefaciens
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3916 , http://hdl.handle.net/10962/d1003975
- Description: There is widespread interest in the biocatalytic production of enantiomerically pure D-amino acids for use in the synthesis of antibiotics, insecticides, herbicides, drug carriers and many other pharmaceuticals. Hydantoin-hydrolysing enzyme systems can be successfully utilised to stereoselectively convert racemic hydantoins into enantiomerically pure amino acid products. In fact, the use of microbial D-hydantoinase and D-stereoselective N-carbamoyl amino acid amidohydrolase activity to produce D-p-hydroxyphenylglycine from D,L-5-phydroxyphenylhydantoin has been described as one of the most successful biotechnological applications of enzyme technology developed to date. A need to utilise the novel biodiversity of South African microorganisms for the development of an indigenous process to produce enantiomerically pure amino acids was identified in 1995. Subsequently, the Rhodes Hydantoinase Group was established and several local hydantoin-hydrolysing microorganisms were isolated. The research in this study describes the isolation and selection of Agrobacterium tumefaciens RU-OR, which produced D-stereoselective hydantoinhydrolysing activity. Characterisation of the hydantoin-hydrolysing enzyme system of RU-OR revealed novel biocatalytic properties, and potential for the application of this strain for the biocatalytic production of D-amino acids. A fundamental understanding of the regulation of hydantoin-hydrolysing enzyme activity in A. tumefaciens RU-OR was established, and utilised to produce mutant strains with altered regulation of hydantoin-hydrolysing activity. These strains were used to further elucidate the mechanisms regulating the production of hydantoins-hydrolysing activity in A. tumefaciens RU-OR cells. Overproduction of hydantoinase and N-carbamoyl-D-amino acid amidohydrolase activity in selected mutant strains resulted in efficient conversion of D,L-5-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine. Thus the establishment of a primary understanding of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR could be used to manipulate the hydantoin-hydrolysing activity in RU-OR cells to produce an improved biocatalyst. The isolation of A. tumfecaiens RU-OR genes encoding for hydantoin-hydrolysing activity revealed two separate N-carbamoyl-D-amino acid amidohydrolaseencoding genes (ncaR1 and ncaR2) in this bacterium with distinct chromosomal locations, nucleotide coding sequence and predicted primary amino acid sequence. The novel biocatalytic properties of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR and mutant derivatives present fascinating opportunities for further elucidation of the natural function, regulation and biocatalytic potential of hydantoin-hydrolysing enzymes.
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- Date Issued: 2002
Genetic characterization of conspecific populations of Tilapia Sparrmanii (A.Smith 1840) in the dolomitic sinkholes and springs of the North-West Province (South Africa), and their comparison to Tilapia Guinasana (Trewavas 1936)
- Authors: Nxomani, Clifford David
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4079 , http://hdl.handle.net/10962/d1007452
- Description: This study was undertaken to investigate the genetic relationships of allopatric populations of the cichlid fish, Tilapia sparrmanii (A. Smith 1840) inhabiting the sinkholes and springs of the North West Province, South Africa. It also examined the genetic relationships of T sparrmanii to its polychromatic sister species, Tilapia guinasana (Trewavas 1936) which is endemic to the Guinas sinkhole in Namibia. Finally, the study investigated whether there is a genetic basis for T guinasana's colour polymorphism. The research was prompted by the concern of conservation authorities about the possible loss of unique fauna given the high demand for use of the subterranean waters for agricultural, domestic and industrial purposes. Such demands have the potential to drain these habitats. Further concerns related to habitat destruction and the introduction of alien species in the ecosystems inhabited by both fish species. Three approaches were adopted in attempting to answer the above questions. First was the investigation of Sodium dodecylsulphate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) of total cellular proteins as a fast and relatively inexpensive indicator of genetic relatedness between the fish populations. Secondly, genetic differentiation between the T sparrmanii populations and its relationship to T guinasana were assayed using restriction endonuclease analysis of Polymerase Chain Reaction (PCR)-amplified regions of the cytochrome b gene and the d-Ioop of mitochondrial DNA, coupled with Temperature Gradient Gel Electrophoresis (TGGE) analysis of the same regions. The third approach involved the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting of the populations ofT sparrmanii as an indicator of genetic differentiation between them. RAPD fingerprinting was further used to investigate the genetic relationships between T sparrmanii and T guinasana and to probe the genetic basis of the polychromatism of the latter. SDS-PAGE did not reveal any genetic differentiation between the T sparrmanii populations, nor could the analysis detect variation within them. It however clearly distinguished at a species level between T sparrmanii and T guinasana as well as between these and other fish species, thus indicating its possible utility as an indicator of genetic relatedness at a species level. Mitochondrial studies employing the Restriction Fragment Length Polymorphism (RFLP) of Polymerase Chain Reaction (PCR)-amplified cytochrome b (1.1 kb) and d-Ioop regions (0.9 kb) with six and five restriction enzymes respectively, failed to reveal genetic differences within and between the allopatric populations. TGGE of500 bp of the d-Ioop and 400 bp of the 12sRNA PCR-amplified fragments did not reveal any differences between the populations of T. sparrmanii, nor did the analysis reveal any differences between T. sparrmanii and T. guinasana. The lack of differentiation between the T. sparrmanii populations by these mitochondrial Dna analysis techniques, despite habitat fragmentation, indicated a recent origin of the populations from a common ancestral population. Failure to distinguish between T. sparrmanii and T. guinasana may be related to the sensitivity of the techniques utilized. RAPD fingerprinting analysis indicated that the populations are genetically differentiated from each other. Using a measure of coefficient of variation, the population with the highest variation was the Wondergat population (13.99%), followed by the Klerkskraal popUlation (8.29%), the Malmani and Marico Oog populations (each with 5.88%) and the least variation (4.95 and 4.83%) was with the Amalinda and Molopo Oog populations respectively. This high degree of intra population similarity points to the fact that this differentiation is still confined within the limits of con specificity. The genetic distances between all of the T. sparrmanii populations across all primers ranged from 0.09 to 0.234 and averaged 0.146, a value that falls in the upper end of conspecific population differentiation. Such results indicate populational sub-division below the species level. RAPD fingerprinting therefore proved more sensitive than protein or mitochondrial studies. The differentiation it detected between the populations is a reflection of their adaptation to local conditions of the unique ecosystems they inhabit. A comparison with a subset of primers between T. guinasana and T. sparrmanii confirmed the separate species status of the former from the latter. The mean genetic distance between the T. sparrmanii populations was 0.136, compared to that between T. sparrmanii and T. guinasana which was found to be 0.374. Statistical analysis of the difference between the mean genetic distances indicated significance with 95% confidence. The polychromatism of T guinasana was investigated to determine whether there were significant differences between its five colour morphs. RAPD fingerprinting indicated with 95% confidence that there were significant differences between the colour forms based on the genetic distances computed between them. These genetic differences appeared to correlate with the observed assortative mating between the colour forms of the species. The manifestation of the polychromatism at sexual maturity in T guinasana probably indicates that colouration plays an important role in the breeding process. The genetic uniqueness shown here between the populations of T sparrmanii and the colour forms of T guinasana indicate for protective measures to be put in place if the genetic resources of the isolated fish populations are to be preserved. These must be coupled with a thorough assessment of the temporal and spatial distribution of genetic variability of the populations as a guide to a long-term management strategy for the fish populations and the ecosystems they inhabit. This study therefore has shown that the allopatric populations of T sparrmanii in the sinkholes and springs of the North-West Province are genetically unique, as well as show that the colour forms of T guinasana are genetically distinct.
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- Date Issued: 2002
Heterologous expression of the helicoverpa armigera stunt virus in Saccharomyces cerevisiae
- Authors: Venter, Philip Arno
- Date: 2002
- Subjects: Helicoverpa armigera Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3895 , http://hdl.handle.net/10962/d1003811
- Description: Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.
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- Date Issued: 2002
Identification of Cowdria ruminantium proteins that induce specific cellular immune responses
- Authors: Van Kleef, Mirinda
- Date: 2002
- Subjects: Ruminants--Pathogens Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4061 , http://hdl.handle.net/10962/d1004373
- Description: Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. , Author: Mirinda van Kleef neé Rossouw
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- Date Issued: 2002
Investigation of the bioconversion of constituents of olive effluents for the production of valuable chemical compounds
- Authors: Notshe, Thandiwe Loretta
- Date: 2002
- Subjects: Phenols , Sewage -- Purification , Effluent quality
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4078 , http://hdl.handle.net/10962/d1007446 , Phenols , Sewage -- Purification , Effluent quality
- Description: Olive mill wastewater is produced in large quantities during the production of olive oil and olive production effluents are produced during the processing of olives. This project was planned to find a use for constituents found in olive production wastewater. The task was carried out by first characterizing the olive effluents, then screening microorganisms for growth in the effluents and reduction of the pollutant properties of the effluents. An investigation into the biotransformation of aromatic compounds present in the effluents into useful chemicals, was carried out. The olive production effluents were collected from different stages in the process for treating olive wastewater, viz, a fermentation tank (FB), the surface of a digester (LV) and an evaporation pond (SO). The three effluents were characterized by investigating their phenolic composition. Protocatechuic acid, vanillic acid, syringic acid, hydroxyphenyl acetic acid, coumaric acid and ferulic acid were identified in an olive effluent, FB, using thin layer chromatography (TLC) and High perfomance liquid chromatography (HPLC). Hydroxyphenyl acetic acid constitutes almost 60% of the organics in olive effluent FB. Five bacteria, namely RU-LV1; RU-FBI and RU-FB2; RU-SOI and RU-S02, were isolated from the olive effluents LV, FB and SO respectively. These isolates were found to be halotolerant and were able to grow over a broad temperature and pH range, with the maximum temperature and pH for growth being 28°C and pH 7 respectively. A range of microorganisms were evaluated for their ability to grow and reduce the total phenolic content of the olive effluents. Among these Neurospora crassa showed the highest potential for the biological reduction of total phenolics in olive effluents. Approximately 70% of the total phenolic content was removed by N. crassa. Trametes verscilor, Pseudomonas putida strains, RU-KMI and RU-KM3s, and the bacteria isolated from olive effluents could also degrade the total phenolic content of olive effluents, but to a lesser extent. The ability of the five bacterial isolates to grow and degrade aromatic compounds was assessed by growing them in medium with standard aromatic compounds. RU-L V1 degraded 96%, 100%, 73% and 100% of caffeic acid, protocatechuic acid, p-coumaric acid and vanillic acid respectively. The other isolates degraded caffeic acid and protocatechuic acid, but their ability to degraded p-coumaric acid and vanillic acid was found to be lesser than the ability of RU-LV1 to degrade the same aromatic compounds. Whole cells of RU-LV1 degraded vanillic acid but no metabolic products were observed on HPLC analysis. Resting cells, French pressed extract, cell free extracts and cell debris from RU-LV1 cells induced with vanillic acid degraded vanillic acid, ferulic acid and vanillin at rates higher than those obtained from non-induced cultures. No products were observed during the degradation of vanillic acid. Ferulic acid was converted into vanillic acid by French pressed extract, cell free extract and cell debris of RU-LV1. The maximum yield of vanillic acid as a product (0 .23 mM, 50 %yield) was obtained when cell free extracts of RU-LVI, grown in glucose and induced by vanillic acid, were used for the degradation of 0.4 mM ferulic acid. Vanillin was rapidly converted into vanillic acid by resting cells, cell free extracts and French pressed extract of RU-LVI. Using molecular techniques, the similarity ranking of the RU-LVI 16S rRNA gene and its clone showed a high similarity to Corynebacterium glutamicum and Corynebacterium acedopltilum. The rapid degradation of vanillin to vanillic acid suggests that extracts from RU-LV1 degrade ferulic acid into vanillin which is immediately oxidized to vanillic acid. Vanillic acid is also considered as a high value chemical. This project has a potential of producing useful chemicals from cheap substrates that can be found in olive effluents. , KMBT_363
- Full Text:
- Date Issued: 2002
Removal and recovery of gold and platinum from aqueous solutions utilising the non-viable biomass Asolla filiculoides
- Authors: Antunes, Ana Paula Martins
- Date: 2002
- Subjects: Azolla filiculoides Metal wastes -- Recycling Gold -- Recycling Platinum -- Recycling
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3894 , http://hdl.handle.net/10962/d1003726
- Description: Waste water from the mining industry is generally extremely complex and contains numerous species which influence the adsorption of the metals to any biomass. A variety of factors need to be addressed before treatment is considered viable. It is also beneficial to establish the binding characteristics of the metal of interest to maximise its interaction with the biomass to be utilised. Azalia filiculaides was investigated in the adsorption of gold(III), lead(II), iron(ID), copper(II) and platinum (IV). In batch studies, the optimum biomass and initial gold(III) concentrations were found to be 5 gIL and 8 mgIL respectively. The adsorption of gold(ID) is principally pH-dependent with optimal removal at pH 2. Lead(II), iron(III) and copper(II) did not compete with gold(III) adsorption under equimolar and simulated effluent conditions. Halides, with increasing affinity for gold (chloride < bromide < iodide), can affect gold uptake with the soft base, iodide, exhibiting the most inhibition (25%) and the hard base, chloride, O%. Mercaptoethanol (soft base) showed no interference in gold(III) adsorption while the presence of sulphate (hard base) and sulphite (borderline base) showed that concentrations in excess of 1 0 mM may adversely affect gold(ill) uptake, most likely due to competition for cationic sites on the biomass. Column studies, better suited to high volume treatment, indicated that a flow-rate of 5 mL/min and an initial gold(ill) concentration of 5 mgIL was optimal. Competitive effects between lead, iron, copper and gold again showed little or no interference. The halides, chloride, bromide and iodide, affect gold(ill) uptake similarly to the batch studies, while the bases mercaptoethanol and sulphate minimally affect gold(III) binding with sulphite severely hampering adsorption (70% inhibition). To optimise gold desorption, preliminary batch studies indicated that a ratio of 1:1 of adsorbentdesorbent was optimal, whilst gas purging of thiourea with oxygen, air and nitrogen decreased gold elution in proportion to decreased amounts of oxygen. A series of desorbents were utilised, in column studies, to optimise and determine the speciation of bound gold. The presence of an oxidant with thiourea enhanced desorption greater than 3 fold when compared with thiourea alone. Thiourea desorption studies, aided by the oxidant, suggest that gold is present in the + I and 0 oxidation states. Ultimately thiourea, perchloric acid and hydrochloric acid was found to be the most optimal elutant for gold (J 00% recovery). For selective metal recovery oflead and copper, pre-washing the plant material with water, utilising an acid (0.3 M nitric acid), pumping in an up-flow mode, and recycling the desorbent six times was found to be optimal elutant for gold (J 00% recovery). Cost analysis of utilising elutant versus incinerating the biomass for gold recovery indicated the latter as the most economical. Over a 5 cycle adsorption and desorption series, acid desorption before each adsorption cycle was found to result in greater than 92% desorption for lead and 96% for copper. Gold recovery was 97% with incineration. A preliminary study with gold effluent (Mine C) indicated that nickel and sulphate was removed in batch and column studies. Gold removal was found to be 100% and 4% in batch and column studies respectively. Adsorption of gold in the effluent study was accompanied by the release ofHt. Modifying the plant material with various reagents failed to identify the primary binding sites and the role of polysaccharides, proteins and lipids in gold(ill) uptake. The mode of gold binding is suggested as being initially ionic, this is very rapid, with the interaction of the anionic complex, [AuCI₄]". with the cationic biomass (PH 2). This eventually leads to the displacement of the chloride ligand(s) initiating covalent binding. Spectral studies of the chemical interaction between gold and the representative tannins indicated the protonated hydroxy groups to be responsible. All evidence suggests that the binding mechanisms of gold are not simple. Preliminary adsorption studies of platinum by Azalia filiculaides were conducted. Batch studies indicated that J gIL biomass concentration, initial platinum concentration of 20 mgIL and pH 2 are optimal, while the column studies indicated a flow-rate of! 0 rnL/min and initial platinum concentration of 20 mgIL as optimal. In the platinum effluent study, platinum showed a removal of 23 % and 2 J % for the batch and column studies respectively. Again adsorption was accompanied by //' release. Azalia filiculaides demonstrated its feasibility in the removal of gold and platinum from simulated as well as waste water solutions. Its potential viability as a biosorbent was demonstrated by the high recovery from synthetic solutions of greater than 99% for gold (2-10 mgIL), and greater than 89% for platinum (20 mgIL).
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- Date Issued: 2002
The biology and molecular ecology of floating sulphur biofilms
- Authors: Bowker, Michelle Louise
- Date: 2002
- Subjects: Biofilms , Microbial ecology , Sulfur
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4056 , http://hdl.handle.net/10962/d1004117 , Biofilms , Microbial ecology , Sulfur
- Description: Floating sulphur biofilms have been observed to occur on sulphate-containing natural systems and waste stabilization ponds. It has been postulated that these biofilms form on the surface of the water because sulphate reducing bacteria present in the bottom layers of the water body reduce sulphate to sulphide which then diffuses upwards and is oxidized under the correct redox conditions to sulphur by sulphide oxidizing bacteria. Very little information exists on these complex floating systems and in order to study them further, model systems were designed. The Baffle Reactor was successfully used to cultivate floating sulphur biofilms. Conditions within the reactor could be closely scrutinized in the laboratory and it was found that sulphate levels decreased, sulphide levels increased and that sulphur was produced over a period of 2 weeks. The success of this system led to it being scaled-up and currently a method to harvest sulphur from the biofilm is under development. It is thought that biofilms are highly complex, heterogeneous structures with different bacteria distributed in different layers. Preliminary work suggested that bacteria were differentially distributed along nutrient and oxygen gradients within the biofilm. Biofilms are very thin structures and therefore difficult to study and Gradient systems were developed in an attempt to spatially separate the biofilm species into functional layers. Gradient Tubes were designed; these provided a gradient of high-sulphide, low oxygen conditions to high-oxygen, low-sulphide conditions. Bacteria were observed to grow in different layers of these systems. The Gradient Tubes could be sectioned and the chemical characteristics of each section as well as the species present could be determined. Silicon Tubular Bioreactors were also developed and these were very efficient at producing large amounts of sulphur under strictly controlled redox conditions. Microscopy and molecular methods including the amplification of a section of Ribosomal Ribonucleic acid by Polymerase Chain Reaction were used in an attempt to characterize the populations present in these biofilm systems. Denaturing Gradient Gel Electrophoresis was used to create band profiles of the populations; individual bands were excised from the gels and sequenced. Identified species included Ectothiorhodospira sp., Dethiosulfovibrio russensis, Pseudomonas geniculata, Thiobacillus baregensis and Halothiobacillus kellyi.
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- Date Issued: 2002
The biotransformation of phenolic pollutants using polyphenol oxidase
- Authors: Boshoff, Aileen
- Date: 2002
- Subjects: Polyphenol oxidase Sewage -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3976 , http://hdl.handle.net/10962/d1004035
- Description: The potential of using mushroom polyphenol oxidase (EC 1.14.18.1) as a biocatalyst for the biotransformation of phenols to produce catechols in an aqueous medium was investigated. Polyphenol oxidase is characterised by two distinct reactions i.e., the ortho-hydroxylation of phenols to catechols (cresolase activity) and the subsequent oxidation of catechols to orthoquinones (catecholase activity). In order to facilitate the development of a process to produce catechols, the accumulation of catechol as a true intermediate product released in the reaction system needed to be investigated, as its release had been disputed due to the oxidation of catechols to o-quinones. Using LC-MS, catechol products were successfully identified as true intermediate products formed during biocatalytic reactions in water.
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- Date Issued: 2002
The characterisation of a South African isolate of Cryptophlebia leucotreta Granulovirus (CIGV)
- Authors: Singh, Shalene
- Date: 2002
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4067 , http://hdl.handle.net/10962/d1004929 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Description: The false codling moth (FCM), Cryptophlehia Leucatreta, causes widespread damage to economically important fruit crops throughout sub-Saharan Africa. Fruit are rendered unfit for consumption once they have been stung by FCM larvae. Larval infestation of fruit can lead to significant pre-harvest losses or post-harvest waste, posing a major problem to the citrus industry. Current control of the pest includes the use of chemical pesticides. The larval form of FCM is known to be infected by a granulovirus called Cryptophlebia leucotreta granulovirus (CIGV). Granuloviruses are highly specific against their hosts and are harmless to vertebrates, plants and the environment. The development of CIGV into a biological control agent would offer an attractive and safer alternative for the control of this pest. A full characterisation of CIGV is required prior to the virus being disseminated into the environment. In this project, the characteristics of CIGV will be examined. Viral DNA was extracted from infected larvae and the DNA analysed by restriction fragment length polymorphism (RFLP). Fragmentation profiles of the South African and Cape Verde (CV3) isolates of the virus were compared, revealing distinct differences between them. The size of the CIGV-SA genome was calculated to be 112 kbp, identical to the size of the CV3 isolate. Physical maps for five restriction enzymes were constructed for the CIGV-SA genome. The alignment of these maps with maps the CV3 isolate (for the same enzymes) further highlighted the differences between the isolates. The genetic engineering of granuloviruses could significantly improve the speed of kill of these viruses. Therefore essential genes like egt and granulin were isolated (by PCR) and their position located in the genome. Both genes were sequenced and their phylogeny with other granulin and egt genes investigated. Finally, tbe incidence of CIGV in natural populations of FCM larvae was investigated, by screening field-collected larvae for the presence of the virus. CIGV was successfully detected from dot blots of larval DNA using both radiolabelled and non-radiolabelled probes and by PCR. Trends regarding the incidence of CIGV in natural populations of larvae were also determined.
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- Date Issued: 2002
The molecular microbial ecology of sulfate reduction in the Rhodes BioSURE process
- Authors: Chauke, Chesa Gift
- Date: 2002
- Subjects: Water -- Purification -- Biological treatment , Acid mine drainage , Water -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4082 , http://hdl.handle.net/10962/d1007475 , Water -- Purification -- Biological treatment , Acid mine drainage , Water -- Microbiology
- Description: The research reported here investigated the use of a Baffle Reactor in order to study aspects of the biological sulfur cycle, where a floating sulfur biofilm formation occurs and where complex organic compounds provide electron donor sources. The development of a laboratory-scale Baffle Reactor model system satisfied the requirements for sulfate reducing bacterial biomass growth and sulfur biofilm formation. Since relatively little is known about the microbial ecology of floating sulfur biofilm systems, this study was undertaken to describe the sulfate reducing sludge population of the system together with its performance. A combination of culture- and molecular-based techniques were applied in this study in order to investigate the microbial ecology of the sulfate-reducing bacteria component of the system. These techniques enabled the identification and the analysis of the distribution of different sulfate reducing bacterial strains found within the sludge bioreactors. Strains isolated from the sludge were characterised based on culture appearance, gram staining and scanning electron microscopy morphology. Molecular methods based on the PCR-amplified 16S rRNA including denaturing gradient gel electrophoresis were employed in order to characterise sulfate-reducing bacteria within the reactors. Three novel Gram negative sulfate-reducing bacteria strains were isolated from the sludge population. Strains isolated were tentatively named Desulfomonas rhodensis, Desulfomonas makanaiensis, and Clostridium sulforhodensis. Results obtained from the Baffle Reactor showed that three dominant species were isolated from the DNA extracted from the whole bacterial population by peR. Three of these were similar to those mentioned above. The presence of these three novel unidentified species suggest that there are a range of other novel organisms involved in sulfate reduction processes.
- Full Text:
- Date Issued: 2002
The role of cellulases and glucohydrolases in the solubilisation of primary sewage sludge
- Authors: Ngesi, Nosisa
- Date: 2002 , 2013-05-09
- Subjects: Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4080 , http://hdl.handle.net/10962/d1007454 , Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Description: Biological sulph ate reduction has been identi fied as a potentially valuable process for removing sulphate and heavy metals from indllstrial effluents. The role of sulphate reducing bacteria (SRB) in this process has attracted the attention of biotechnologists and recently of enzymologists due to its fundamental properties and possible role in AMD bioremediation. These obligatory anaerobic sulphate-reducing bacteria are commonly known to dissimilate sulphate for energy. Under anaerobic conditions SRB oxidize simple organic compounds such as lactic acid with the sulphate and thereby generate hydrogen sulphide (a stTong reducing agent) and bicarbonate ions. The hydrogen sulphide in turn reacts with contaminant metals contained in AMD and precipitates them out of solution as metal sulphides. Bicarbonate ions neutralize AMD by reaction with protons to form carbon dioxide and water. Organic matter in the municipal sewage sludge has been identified as a potential source of electron donors for su lphate reduction. However, this organic matter is in the polymeric form that cannot be util ised by SRB. The latter depend on the activities of other hydrolytic bacteria for the degradation of complex polymers. Hence the availability of these monomeric substrates is a major factor, which may constrain further process development and is considered a rate-limiting step. Thi s study is therefore undertaken to investigate the bacterial glucohydrolase enzymes involved in the digestion of the polysaccharides present in the sewage sludge with specific interest in cellulases and/or p-glucosidase enzymes. The goals of the research are to: isolate, identify, purify and quantify these enzymes; study their distribution with respect to time, pH, and temperature; maximize and quantify the hydrol ys is products; study whether sulphide and sulphate have an enhancing or an inhibitory effect on the activity of enzymes; optimize the enzyme activity against substrate and/or product inhibition and soluble heavy metal salts. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
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- Date Issued: 2002
An investigation into the biological treatment of platinum refinery effluent using the plant Azolla Filiculoides
- Authors: Marran, Vernon Edward
- Date: 2003
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193453 , vital:45333
- Description: In order to understand the effects of metals contained in effluent and to define effluent quality suitable for safe discharge to natural water streams, it is essential to understand the effects of the interaction of metal ions with plants. The availability of metal ions and their ability to bind to plants are dependent on the chemical speciation of metals and on the biological factors governing the availability of metals within the plant cells. This thesis will address both aspects and thereby propose a combination of an appropriate chemical and biological approach to the investigation of bioaccumulation of the plant Azolla Filiculoides. Laboratory studies have shown that varying concentrations of free metal ions in solution determine efficiency of metal uptake and that metal toxicity can also be detrimental to plant life and efficiency of metal recovery from solution. Many questions however, remain unanswered with regard to the application of a biological treatment for effluent discharge. This thesis includes the determination of metal speciation combined with the study of bioaccumulation of metals in plants and their effects from test- work utilising effluent generated from a Precious Metals Refinery (PMR). Plant species are known to differ widely in their tolerance to metals, however despite an abundant knowledge on molecular, biochemical and physiological effects of metals to plants, only a few general principles have been proposed to guide the prediction of tolerance differences. The properties of protective cellular responses as well as of the molecular target sites are important components in determining the intrinsic tolerance of a particular species to a metal. The role of the whole assembly of cellular ligands in buffering metal ions within the cells will be evaluated. Standard preparation methods combined with use of Inductively Coupled Plasma Emission Spectrophotometer (1CP) used for analytical analysis will be included to reflect analytical data in providing evidence to support a conclusion. The outcome of the test work utilising the aquatic plant Azoila has proven that it can be used as a process step to re-mediate effluent generated from Precious Metal Refining operations. This process offers an alternative to the classical chemical methods widely used in the Precious Metals Refining industry proving economically viable and ensuring environmental sustainability in comparison to the current known methods of effluent treatment. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2003
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- Date Issued: 2003
An investigation of the isolation, characterisation and application of hydantoinases for the industrial production of amino acids
- Authors: Kirchmann, Shaun
- Date: 2003
- Subjects: Hydantoin Amino acids Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3969 , http://hdl.handle.net/10962/d1004028
- Description: This thesis describes a series of investigations into the hydantoin-hydrolysing activity of bacterial strains RU-KM1 and RU-OR, which were previously isolated for their ability to hydrolyse hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids using a bioreactor based system utilising the hydantoin hydrolysing enzymes of the two isolated microorganisms. Different substituted hydantoins may be used as substrates by these enzymes for the production of a variety of amino acids. These are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as semisynthetic penicillin/ampicillin, L-aspartame (sweetener), Fluvalinate (insecticide), Enalapril (ACE inhibitor). Thus, the ability of the above-mentioned strains to hydrolyse these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an N-carbamylamino acid intermediate, and subsequently, an amino acid. The hydantoin-hydrolysing enzymes of a Pseudomonas sp. RU-KM1, and an Agrobacterium sp. RU-OR were characterised as whole cells and in a crude extract preparation, and reaction conditions for its biocatalytic application were optimised. The optimum conditions for conversion of hydantoin to glycine were found to be 1 hour at 40 °C, with conversion yields greater than 30 % achieved. The enzymes of RU-KM1 demonstrated considerable stability, retaining 80 % of their activity after storage for 2 weeks at 4 °C. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamoylase of RU-KM1 suggested that these enzymes required metal ions for activity, with metal ions such as Cu[superscript (2+)], Fe[superscript (2+)], and Co[superscript (2+)] resulting in no significant change in enzyme activity, however there was an activation of the enzymes when Mn[superscript (2+)] was added to the enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was D-selective, whereas the N-carbamoylase was shown to be L-selective by other researchers. The hydantoin substrate selectivity of RU-KM1 and RU-OR was investigated, and the organisms were shown to be able to hydrolyse all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing slightly lower activity than whole cells in RU-KM1, and the whole cells or RU-OR showing the lower activity than its crude extract. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolysed p-hydroxyphenylhydantoin, achieving 57 % and 52 % conversions respectively. RU-OR whole cells preferred methylhydantoin where as the crude extract preferred isopropylhydantoin, and showed 49 % and 51 % conversions respectively. The enzymes were characterised in terms of their temperature and pH optima, inducer requirements, and product inhibition studies. The hydantoinase of RU-KM1 was shown to be inducible with low levels of hydantoin, and thermostable upto 75 °C with its optima between 60 and 70 °C. The N-carbamoylase was shown to have its optima at 50 °C. The addition of ATP (0.5 mM), DTT (1 mM) and a protease inhibitor (2 mg.mL[superscript (-1)]) all increased the hydantoinase activity of RU-KM1 crude extract, however they had very little effect on the N-carbamoylase activity. The hydantoinase enzyme from extracts of RU-KM1 was partially purified by development of cell disruption methods using mechanical and lysing enzymes, followed by precipitation and chromatographic resolution. The results obtained showed a hydantoinase enzyme of between 48 and 66 kDa. RU-KM1 was grown under fermentation conditions using different minimal media. The activity and yields under these conditions were low. Previous attempt to grow the organism in a rich medium had resulted in an increase in biomass but no hydantoinase activity. A rich medium was developed by carbon and nitrogen optimisation and yielded biomass up to 30 g.L[superscript (-1)] dry cell weight. The hydantoinase activity was restored by nitrogen starvation in stationary phase. This resulted in high biomass with increased activity. This data is currently in press. Crude extract and whole cells were immobilised on flat sheet membranes, hollow fibre membranes and in alginate beads. Low hydantoinase activity was measured in bioreactors using membranes in different configurations. A significant increase in hydantoinase activity was measured when the crude extract was immobilised in sodium alginate, as a result of stabilisation of the N-carbamoylase. Temperature and pH optima were unaffected by the immobilisation procedure, however the durability of the enzymes increased 2-fold. Different configurations of the bioreactor were investigated, as well as a hydroxyphenylhydantoin as an alternative substrate in this study. The bioreactors showed a near 95 % conversion of the hydantoin to glycine, and a 99 % conversion using HPG. In conclusion, the hydantoin-hydrolysing enzymes of RU-KM1 have been shown to be possibly membrane associated, which is a novel finding. This study has shown that the hydantoinase of RU-KM1 is D-stereoselective, with high temperature stability. A growth medium was developed for the rapid production of active biomass. A bioreactor was developed using a single and a dual biocatalyst configuration, which was capable of hydrolysing hydantoin and monosubstituted hydantoins to produce amino acids. To our knowledge this system is the first such dual biocatalyst system reported for the production of amino acids.
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- Date Issued: 2003
Binding and transcriptional activation by Uga3p, a zinc binuclear cluster protein of Saccharomyces cerevisiae redefining the UAS [subscript GABA] and the Uga3p binding site
- Authors: Idicula, Anu Mary
- Date: 2003
- Subjects: Saccharomyces cerevisiae GABA
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3933 , http://hdl.handle.net/10962/d1003992
- Description: Uga3p, a member of the zinc binuclear cluster transcription factor family, is required for [gamma]-aminobutyric acid-dependent transcription of the UGA genes in Saccharomyces cerevisiae. Crystallographic data of some of the protein-DNA complexes of this family reveal that members of this family bind to CGG triplets. A conserved 19-nucleotide activation element in certain UGA gene promoter regions contains a CCG-N4-CGG everted repeat, proposed to be the binding site of Uga3p, UAS[subscript GABA]. The spacer region (N4) between the CGG triplets has been suggested to be the specificity determinant for binding to UAS[subscript GABA]. The data available from the Saccharomyces genome database indicates that there are multiple repeats of -CCG-N4-CGG- regions within the genome. These transcription factors are involved in the activation of specific pathways and the question arises as to how their specificity of binding is determined. The aim of this study was to understand the binding characteristics of Uga3p to UAS[subscript GABA] and to determine the affinity and specificity of this interaction. In this study, full-length (tagged and untagged) and truncated (1-124 a.a.) Uga3p was produced in a heterologous expression system (E. coli). The interaction of Uga3p with UAS[subscript GABA] in Saccharomyces cerevisiae was characterized in terms of binding in vitro and the transcriptional activation of lacZ reporter genes in vivo. The Uga3p was capable of binding to these sites in vitro independent of exogenous GABA. Electrophoretic mobility shift assays (EMSA) of the full-length Uga3p with the wild type UAS[subscript GABA] sequences produced two distinct mobility complexes. The complexes formed in the EMSA of the full-length Uga3p were those specific to the interaction of the Uga3p to UAS[subscript GABA]. The truncated Uga3p(1-124 a.a.), which has the DNA-binding zinc cluster domain, the linker region and the putative coiled-coil domain was not functionally equivalent to the full-length protein with respect to binding in vitro because the EMSAs of the UAS[subscript GABA] with the truncated Uga3p produced indistinct complexes. EMSAs using mutant UAS[subscript GABA] sequences and heterologously-produced full-length Uga3p, demonstrated that UAS[subscript GABA] consists of two, independent Uga3p-binding sites. This work presents evidence that the two Uga3p molecules bound to UAS[subscript GABA] most likely interact with each other. Unlike other zinc cluster binding sites the Uga3p-binding site is an asymmetric site of 5’-SGCGGNWWT-3’ (S= G or C, W = A or T and N = no nucleotide or G or C). UAS[subscript GABA] is a palindrome containing the two asymmetric Uga3p-binding sites. The two-site consensus sequence required for the binding of Uga3p to the UAS[subscript GABA] is present upstream of UGA1 (region -387 to -370) and UGA4 (region -403 to -387). Furthermore, a single Uga3p-binding site was identified in the 5’ untranslated regions of UGA2 (region -219 to -211). GABA-dependent transcriptional activation by UAS[subscript GABA] in vivo could be directly correlated to a high affinity, specific interaction of two Uga3p molecules to this UAS. Binding with high affinity required the conserved sequences flanking the everted repeat. This study provided evidence that the binding pattern of Uga3p is novel compared to other zinc cluster motifs investigated, as the sequences flanking the everted repeat are important regions for recognition by Uga3p. The studies with the truncated Uga3p (1 –124 a.a.), also suggested that the regions C-terminal to the DNA-binding motif and putative coiled-coil area of this protein are important for Uga3p-specific interactions with UAS[subscript GABA]. Investigation of regions C-terminal to the zinc cluster, linker and putative coiledcoil revealed an eight-motif regulatory region similar to that in other zinc cluster proteins. This indicated that the regions C-terminal to these domains are important for the regulation and activity of these proteins. A putative seven repeat WD40-like motif was identified within this region. This putative domain has been speculated to be important for protein-protein interactions. Phosphorylation and dephosphorylation in other proteins of this class have been indicated to be important for the regulation of the activity of these proteins. The bioinformatic analysis of Uga3p revealed two possible cAMP/cGMP-dependent protein kinase phosphorylation sites, four putative protein kinase C phosphorylation motifs and four putative casein kinase II phosphorylation motifs. This study has contributed to the understanding of the nature of interactions between Uga3p and its specific UAS [subscript GABA] and how the regions flanking the everted repeat determine its specificity. The comparison of the nature of the binding of truncated and full-length Uga3p in vitro provided evidence for the role played by the full-length protein in determining this specific interaction. This evidence suggested that the in vitro binding evidence for other proteins of this family, using truncated peptides that carry the DNA-binding domain, might not reflect the true nature of interactions between the proteins of this class and their specific UASs in vivo.
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- Date Issued: 2003