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.
- Full Text:
- Date Issued: 2005
- 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.
- Full Text:
- Date Issued: 2005
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.
- Full Text:
- Date Issued: 2005
- 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.
- Full Text:
- Date Issued: 2005
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.
- Full Text:
- Date Issued: 2005
- 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.
- Full Text:
- Date Issued: 2005
Development of an experimental system to investigate the interaction between the Helicoverpa armigera stunt virus capsid protein and viral RNA
- Authors: Nel, Andrew James Mascré
- Date: 2005
- Subjects: Helicoverpa armigera , RNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3946 , http://hdl.handle.net/10962/d1004005 , Helicoverpa armigera , RNA viruses
- Description: Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.
- Full Text:
- Date Issued: 2005
- Authors: Nel, Andrew James Mascré
- Date: 2005
- Subjects: Helicoverpa armigera , RNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3946 , http://hdl.handle.net/10962/d1004005 , Helicoverpa armigera , RNA viruses
- Description: Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.
- Full Text:
- Date Issued: 2005
Development of experimental systems for studying the biology of Nudaurelia capensis ß virus
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
Enzymatic recovery of rhodium(III) from aqueous solution and industrial effluent using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
- Full Text:
- Date Issued: 2005
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
- Full Text:
- Date Issued: 2005
Identification of cis-elements and transacting factors involved in the abiotic stress responses of plants
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
Intraspecific comparison of Phanerochaete chrysosporium strains peroxidase production, pollutant degradation and mycelial differentiation
- Authors: Fraser, Sheena Janet
- Date: 2005
- Subjects: Phanerochaete Pollutants -- Biodegradation Lignin -- Biodegradation Bioremediation Peroxidase Fungi -- Differentiation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3964 , http://hdl.handle.net/10962/d1004023
- Description: The wood-degrading basidiomycete, Phanerochaete chrysosporium, has been studied as a model organism in elucidating the mechanisms and pathways enabling this white-rot fungus to degrade recalcitrant lignin. These same mechanisms are implicated in the mineralisation of environmentally persistent, toxic phenolic chemicals. For this reason, P. chrysosporium has been exploited in a number of environmentally sound technologies, including the degradation of the indigestible lignin component in agricultural waste for the generation of digestible animal feedstocks or high sugar content raw materials for ethanol production; brightening processes in the pulp and paper industry; the detoxification and decolourisation of industrial effluents; and the bioremediation of hazardous waste sites. The improvement of these technologies is dependant on ongoing research involving strain selection, strain development using genetic engineering approaches and process development. Strain improvement using non-recombinant methods is beneficial in that it does not limit the inherent robustness observed amongst natural variants. In this research, through a breeding programme, ten P.chrysosporium sibling strains were screened for variable ligninase activities and pollutant degradation capabilities in order to further describe previously identified differences between these organisms. A conventional stationary liquid culture technique was effectively miniaturised from 10 ml flask cultures to a 96-well microtitre plate format, for the assessment of multigenic traits amongst sibling strains. Using the 96-well microtitre plate method, the relationships between P. chrysosporium growth kinetics, peroxidase production, pollutant sensitivity and pollutant degradation was explored. Significant correlations were primarily associated with P. chrysosporium growth [P < 0.05]. Percentage p-cresol removal and tannic acid tolerance were both correlated with a shorter lag phase in growth [tannic acid: r = 0.7698, P < 0.05; p-cresol: r = 0.7584, P < 0.05] and lower stationary phase biomass levels [tannic acid: r = 0.8177, P < 0.05; p-cresol: r = 0.7803, P < 0.05]. A significant correlation (linear relationship) was also detected between percentage Poly-R478 decolourisation and time of onset of MnP [r = 0.9689, P < 0.001]. No correlation was observed between dye decolourisation, p-cresol degradation, lignin degradation and lignin peroxidase (LiP) or manganese peroxidase (MnP) activities [P > 0.05]. These results imply that differences in the biosynthetic pathways for biomass accumulation in sibling strains play a significant role in the intraspecific variation observed in pollutant sensitivity, pollutant degradation, and enzyme production. Categorical analysis of intraspecific differences was assessed according to four criterions. These included growth, extracellular peroxidase activities, tolerance to toxic pollutants and the biodegradation of model pollutants. Sibling strains showing the most variable responses in three or more of the selective criterion were recommended for further studies. These strains include P. chrysosporium ME446, BS 2.52, BS 13, BS 17, BS 18, and BS 24. Interestingly, BS 2.52 (a dikaryotic strain generating from the crossing of two haploid progeny) showed significantly lower degradation capabilities than the wildtype parent strain ME446. The inherited variability observed between sibling strains is to be further explored through proteome and transcriptome analysis and genetic linkage studies aimed at describing the mechanisms or pathways conferring tolerance to or degradation of environmental pollutants. In examining fewer organisms at this next level, the number of replicates examined can be increased and thus the power of detection of experimental procedures improved, enabling the detection of multigenic traits amongst genetically related organisms. Growth was shown to play a significant role in the intraspecific differences detected in pollutant sensitivity and degradation between sibling strains. Little is known about the mechanism of growth and differentiation, or the role of differentiation in regulating the lignolytic activity in this organism. The membrane gradostat bioreactor and a unique plug-flow membrane bioreactor were evaluated as novel tools with which to further explore the relationship between secondary metabolism, pollutant degradation and biofilm development in sibling strains. High yield MnP production at levels as high as 1478.8 U.l-1 was achieved using a laboratory scale membrane gradostat bioreactor. Furthermore, extensive mycelial differentiation and tissue formation are reported for P. chrysosporium in both the membrane gradostat bioreactor and plug-flow membrane bioreactor. Intraspecific differences in the extent of this differentiation were observed in strains ME446, BS 13, BS 17 and BS 26 cultured using the membrane gradostat bioreactor, highlighting the potential of these techniques as a platform for future strain improvement strategies.
- Full Text:
- Date Issued: 2005
- Authors: Fraser, Sheena Janet
- Date: 2005
- Subjects: Phanerochaete Pollutants -- Biodegradation Lignin -- Biodegradation Bioremediation Peroxidase Fungi -- Differentiation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3964 , http://hdl.handle.net/10962/d1004023
- Description: The wood-degrading basidiomycete, Phanerochaete chrysosporium, has been studied as a model organism in elucidating the mechanisms and pathways enabling this white-rot fungus to degrade recalcitrant lignin. These same mechanisms are implicated in the mineralisation of environmentally persistent, toxic phenolic chemicals. For this reason, P. chrysosporium has been exploited in a number of environmentally sound technologies, including the degradation of the indigestible lignin component in agricultural waste for the generation of digestible animal feedstocks or high sugar content raw materials for ethanol production; brightening processes in the pulp and paper industry; the detoxification and decolourisation of industrial effluents; and the bioremediation of hazardous waste sites. The improvement of these technologies is dependant on ongoing research involving strain selection, strain development using genetic engineering approaches and process development. Strain improvement using non-recombinant methods is beneficial in that it does not limit the inherent robustness observed amongst natural variants. In this research, through a breeding programme, ten P.chrysosporium sibling strains were screened for variable ligninase activities and pollutant degradation capabilities in order to further describe previously identified differences between these organisms. A conventional stationary liquid culture technique was effectively miniaturised from 10 ml flask cultures to a 96-well microtitre plate format, for the assessment of multigenic traits amongst sibling strains. Using the 96-well microtitre plate method, the relationships between P. chrysosporium growth kinetics, peroxidase production, pollutant sensitivity and pollutant degradation was explored. Significant correlations were primarily associated with P. chrysosporium growth [P < 0.05]. Percentage p-cresol removal and tannic acid tolerance were both correlated with a shorter lag phase in growth [tannic acid: r = 0.7698, P < 0.05; p-cresol: r = 0.7584, P < 0.05] and lower stationary phase biomass levels [tannic acid: r = 0.8177, P < 0.05; p-cresol: r = 0.7803, P < 0.05]. A significant correlation (linear relationship) was also detected between percentage Poly-R478 decolourisation and time of onset of MnP [r = 0.9689, P < 0.001]. No correlation was observed between dye decolourisation, p-cresol degradation, lignin degradation and lignin peroxidase (LiP) or manganese peroxidase (MnP) activities [P > 0.05]. These results imply that differences in the biosynthetic pathways for biomass accumulation in sibling strains play a significant role in the intraspecific variation observed in pollutant sensitivity, pollutant degradation, and enzyme production. Categorical analysis of intraspecific differences was assessed according to four criterions. These included growth, extracellular peroxidase activities, tolerance to toxic pollutants and the biodegradation of model pollutants. Sibling strains showing the most variable responses in three or more of the selective criterion were recommended for further studies. These strains include P. chrysosporium ME446, BS 2.52, BS 13, BS 17, BS 18, and BS 24. Interestingly, BS 2.52 (a dikaryotic strain generating from the crossing of two haploid progeny) showed significantly lower degradation capabilities than the wildtype parent strain ME446. The inherited variability observed between sibling strains is to be further explored through proteome and transcriptome analysis and genetic linkage studies aimed at describing the mechanisms or pathways conferring tolerance to or degradation of environmental pollutants. In examining fewer organisms at this next level, the number of replicates examined can be increased and thus the power of detection of experimental procedures improved, enabling the detection of multigenic traits amongst genetically related organisms. Growth was shown to play a significant role in the intraspecific differences detected in pollutant sensitivity and degradation between sibling strains. Little is known about the mechanism of growth and differentiation, or the role of differentiation in regulating the lignolytic activity in this organism. The membrane gradostat bioreactor and a unique plug-flow membrane bioreactor were evaluated as novel tools with which to further explore the relationship between secondary metabolism, pollutant degradation and biofilm development in sibling strains. High yield MnP production at levels as high as 1478.8 U.l-1 was achieved using a laboratory scale membrane gradostat bioreactor. Furthermore, extensive mycelial differentiation and tissue formation are reported for P. chrysosporium in both the membrane gradostat bioreactor and plug-flow membrane bioreactor. Intraspecific differences in the extent of this differentiation were observed in strains ME446, BS 13, BS 17 and BS 26 cultured using the membrane gradostat bioreactor, highlighting the potential of these techniques as a platform for future strain improvement strategies.
- Full Text:
- Date Issued: 2005
Investigation into the biological removal of sulphate from ethanol distillery wastewater using sulphate-reducing prokaryotes
- Authors: Smuts, Lizl
- Date: 2005
- Subjects: Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3941 , http://hdl.handle.net/10962/d1004000 , Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Description: Ethanol production wastewater is known to be toxic, and is not easily biodegradable. It also consists of a variety of coloured components adding to the complex composition of this wastewater. Disposal of this wastewater into water courses is not recommended and yet is performed all over the world. Investigation of this wastewater found that there was a high concentration of sulphate which, in the presence of sulphate-reducing prokaryotes can cause sulphide corrosion of cement. The concentration of sulphate in the wastewater was approximately 2770 mg/L. It was also found that the wastewater pH was very low and discharge of the wastewater into the wastewater treatment works caused a negative impact on the overall quality of the final wastewater discharged to sea. It was found using FISH techniques that there were no sulphate-reducing prokaryotes present in the wastewaters but that a sulphate-reducing population existed on the sewer wall. An anaerobic contact process was designed to treat this wastewater targeting sulphate reduction to sulphide, to be converted into elemental sulphur and to increase the wastewater pH. The process did not achieve this aim and only approximately 20-30 % reduction in sulphate from the wastewater was achieved with little to no change in the pH. A 95 % reduction in sulphate concentration was needed in order to reach acceptable discharge limits. Sulphate reduction could not be carried out, even under ideal laboratory conditions. It was found that the barrier causing the digester failure was the high concentration of phenols present in the wastewater (3.3 g/L) together with the production of high concentrations of volatile fatty acids (on average 13 g acetic/L). These two components are known to cause digester failure, especially phenols, and phenols are usually only degraded by fungal species. It was concluded that the wastewater itself was not amenable to this method of biological treatment.
- Full Text:
- Date Issued: 2005
- Authors: Smuts, Lizl
- Date: 2005
- Subjects: Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3941 , http://hdl.handle.net/10962/d1004000 , Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Description: Ethanol production wastewater is known to be toxic, and is not easily biodegradable. It also consists of a variety of coloured components adding to the complex composition of this wastewater. Disposal of this wastewater into water courses is not recommended and yet is performed all over the world. Investigation of this wastewater found that there was a high concentration of sulphate which, in the presence of sulphate-reducing prokaryotes can cause sulphide corrosion of cement. The concentration of sulphate in the wastewater was approximately 2770 mg/L. It was also found that the wastewater pH was very low and discharge of the wastewater into the wastewater treatment works caused a negative impact on the overall quality of the final wastewater discharged to sea. It was found using FISH techniques that there were no sulphate-reducing prokaryotes present in the wastewaters but that a sulphate-reducing population existed on the sewer wall. An anaerobic contact process was designed to treat this wastewater targeting sulphate reduction to sulphide, to be converted into elemental sulphur and to increase the wastewater pH. The process did not achieve this aim and only approximately 20-30 % reduction in sulphate from the wastewater was achieved with little to no change in the pH. A 95 % reduction in sulphate concentration was needed in order to reach acceptable discharge limits. Sulphate reduction could not be carried out, even under ideal laboratory conditions. It was found that the barrier causing the digester failure was the high concentration of phenols present in the wastewater (3.3 g/L) together with the production of high concentrations of volatile fatty acids (on average 13 g acetic/L). These two components are known to cause digester failure, especially phenols, and phenols are usually only degraded by fungal species. It was concluded that the wastewater itself was not amenable to this method of biological treatment.
- Full Text:
- Date Issued: 2005
Isolation and characterization of a novel thermostable and catalytically efficient laccase from Peniophora sp. strain UD4
- Authors: Jordaan, Justin
- Date: 2005
- Subjects: Enzymes Enzymes -- Industrial applications Peniophora Laccase
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3970 , http://hdl.handle.net/10962/d1004029
- Description: Enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as chirally selective biocatalysis. The ligninolytic enzymes have recently received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. This group of enzymes was therefore identified as the target group for this study. Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi. Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50. Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production. The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-α-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-α-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability. This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date.
- Full Text:
- Date Issued: 2005
- Authors: Jordaan, Justin
- Date: 2005
- Subjects: Enzymes Enzymes -- Industrial applications Peniophora Laccase
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3970 , http://hdl.handle.net/10962/d1004029
- Description: Enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as chirally selective biocatalysis. The ligninolytic enzymes have recently received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. This group of enzymes was therefore identified as the target group for this study. Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi. Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50. Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production. The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-α-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-α-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability. This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date.
- Full Text:
- Date Issued: 2005
Isolation, purification and characterization of inulin and fructooligosaccharides from chicorium intybus and inulinase from aspergillus niger
- Mavumengwana, Vuyo Bhongelethu
- Authors: Mavumengwana, Vuyo Bhongelethu
- Date: 2005
- Subjects: Aspergillus , Inulin , Chicory -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3954 , http://hdl.handle.net/10962/d1004013 , Aspergillus , Inulin , Chicory -- South Africa
- Description: Inulin is a non-digestible carbohydrate fructan polymer consisting mainly of β (1→2) fructosyl fructose links. Enzymatic hydrolysis of inulin by inulinase results in the production of low D.P (degree of polymerization) oligosaccharides also called fructooligosaccharides(FOS). Isolation of inulin from chicory root (Chicorium intybus) was achieved by first, extraction using deionized water (600C), followed by carbonation (0.1 M Ca(OH)2 and CO2 gas). This was filtered in order to remove the non sugars, thereafter, treated successfully with polyamide 6 powder. A cation exchanger and an anion exchanger were used to further exclude other components such as tannins and pigments. The extracted inulin was quantified using the Somogyi-Nelson colourimetric assay. Chicory root (207 g, 30 % being water) yielded 30 g of the raw extract. A 100 mg of the raw extract was assayed and found to contain 11 % yield of inulin which was 80.2 % in purity and 4 % free fructose. Analysis of the crude and purified inulin extracts on the MALDI TOF spectrometry showed the samples to have a DP of 2 to 22 and 2 to 27 respectively. Maximum inulinase production from Aspergillus niger grown on inulin was observed after 60 hours. The enzyme activity was found to be 1.168 U/ml with a temperature and pH optimum of 30 °C and 7.7 respectively. The enzyme proved to be unstable as it progressively lost its total activity during attempts at purification.
- Full Text:
- Date Issued: 2005
- Authors: Mavumengwana, Vuyo Bhongelethu
- Date: 2005
- Subjects: Aspergillus , Inulin , Chicory -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3954 , http://hdl.handle.net/10962/d1004013 , Aspergillus , Inulin , Chicory -- South Africa
- Description: Inulin is a non-digestible carbohydrate fructan polymer consisting mainly of β (1→2) fructosyl fructose links. Enzymatic hydrolysis of inulin by inulinase results in the production of low D.P (degree of polymerization) oligosaccharides also called fructooligosaccharides(FOS). Isolation of inulin from chicory root (Chicorium intybus) was achieved by first, extraction using deionized water (600C), followed by carbonation (0.1 M Ca(OH)2 and CO2 gas). This was filtered in order to remove the non sugars, thereafter, treated successfully with polyamide 6 powder. A cation exchanger and an anion exchanger were used to further exclude other components such as tannins and pigments. The extracted inulin was quantified using the Somogyi-Nelson colourimetric assay. Chicory root (207 g, 30 % being water) yielded 30 g of the raw extract. A 100 mg of the raw extract was assayed and found to contain 11 % yield of inulin which was 80.2 % in purity and 4 % free fructose. Analysis of the crude and purified inulin extracts on the MALDI TOF spectrometry showed the samples to have a DP of 2 to 22 and 2 to 27 respectively. Maximum inulinase production from Aspergillus niger grown on inulin was observed after 60 hours. The enzyme activity was found to be 1.168 U/ml with a temperature and pH optimum of 30 °C and 7.7 respectively. The enzyme proved to be unstable as it progressively lost its total activity during attempts at purification.
- Full Text:
- Date Issued: 2005
Screening of technologies for the recovery of rhodium (III) metal ions from a precious metal refinery wastewater
- Authors: Mack, Cherie-Lynn
- Date: 2005
- Subjects: Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3987 , http://hdl.handle.net/10962/d1004046 , Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Description: The selective recovery of rhodium from wastewaters, in which the metal would be otherwise lost, would be highly profitable if the process were suitably low-cost. Current recovery processes are generally high maintenance and high-cost, whereas biological processes can be engineered to run with little external input in terms of cost and maintenance. Three emerging technologies were chosen based on their reported efficiency when removing base metals from wastewaters. The first technology screened, the sulphide-extraction membrane bioreactor (SEMB), consists of a sulphate-reducing prokaryote (SRP) anaerobic digester, in which a silicone membrane is submerged. Wastewater is passed through the membrane and metal ions are precipitated as metal sulphides by the hydrogen sulphide gas, which is capable of permeating the membrane. The second technology screened was a fluidized sand bed reactor in which metal ions are removed from solution via induction of nucleated precipitation by sodium carbonate onto the sand grains. The third, and most well established removal technology screened was a biosorption system using immobilized Saccharomyces cerevisiae biomass as the biosorbent. Experimental trials with each technology highlighted drawbacks with each; the SEMB system proved to be largely ineffective when challenged with the removal of rhodium from the wastewater as the rhodium precipitate fouled the membrane within hours, the fluidized bed system seemed unable to overcome the acidity of the wastewater and thus could not precipitate out the rhodium metal, and the efficiency of the biosorption process was hampered by the diversity of rhodium species present in the wastewater, which reduced the amount recovered. The outcomes of the trials with each technology indicated that further optimization of the technology or pretreatment of the wastewater is necessary before any of these options can be implemented. It could be concluded, however, that despite further optimization, both the SEMB and the fluidized bed system were not applicable in this case as precipitation would be non-specific, resulting in the necessity for further steps in order to purify the rhodium ions. Hence, the biosorption system was shown to be most applicable, and further optimization of the system could yield a highly efficient rhodium recovery process.
- Full Text:
- Date Issued: 2005
- Authors: Mack, Cherie-Lynn
- Date: 2005
- Subjects: Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3987 , http://hdl.handle.net/10962/d1004046 , Rhodium , Metal ions , Sewage -- Purification -- Metals removal , Platinum group
- Description: The selective recovery of rhodium from wastewaters, in which the metal would be otherwise lost, would be highly profitable if the process were suitably low-cost. Current recovery processes are generally high maintenance and high-cost, whereas biological processes can be engineered to run with little external input in terms of cost and maintenance. Three emerging technologies were chosen based on their reported efficiency when removing base metals from wastewaters. The first technology screened, the sulphide-extraction membrane bioreactor (SEMB), consists of a sulphate-reducing prokaryote (SRP) anaerobic digester, in which a silicone membrane is submerged. Wastewater is passed through the membrane and metal ions are precipitated as metal sulphides by the hydrogen sulphide gas, which is capable of permeating the membrane. The second technology screened was a fluidized sand bed reactor in which metal ions are removed from solution via induction of nucleated precipitation by sodium carbonate onto the sand grains. The third, and most well established removal technology screened was a biosorption system using immobilized Saccharomyces cerevisiae biomass as the biosorbent. Experimental trials with each technology highlighted drawbacks with each; the SEMB system proved to be largely ineffective when challenged with the removal of rhodium from the wastewater as the rhodium precipitate fouled the membrane within hours, the fluidized bed system seemed unable to overcome the acidity of the wastewater and thus could not precipitate out the rhodium metal, and the efficiency of the biosorption process was hampered by the diversity of rhodium species present in the wastewater, which reduced the amount recovered. The outcomes of the trials with each technology indicated that further optimization of the technology or pretreatment of the wastewater is necessary before any of these options can be implemented. It could be concluded, however, that despite further optimization, both the SEMB and the fluidized bed system were not applicable in this case as precipitation would be non-specific, resulting in the necessity for further steps in order to purify the rhodium ions. Hence, the biosorption system was shown to be most applicable, and further optimization of the system could yield a highly efficient rhodium recovery process.
- Full Text:
- Date Issued: 2005
Stress-inducible protein 1: a bioinformatic analysis of the human, mouse and yeast STI1 gene structure
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
Tertiary treatment in integrated algal ponding systems
- Authors: Wells, Charles Digby
- Date: 2005
- Subjects: Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4068 , http://hdl.handle.net/10962/d1006162 , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Description: Inadequate sanitation is one of the leading causes of water pollution and consequently illness in many underdeveloped countries, including South Africa and, specifically, the Eastern Cape Province, where cholera has become endemic. As modern wastewater treatment processes are often energy intensive and expensive, they are not suitable for use in these areas. There is thus a need to develop more sustainable wastewater treatment technologies for application in smaller communities. The integrated algal ponding system (IAPS) was identified as a possible solution to this wastewater management problem and was investigated for adaptation to local conditions, at the Rhodes University Environmental Experimental Field Station in Grahamstown, South Africa. The system was monitored over a period of nine years, with various configuration adjustments of the high rate algal pond (HRAP) unit operation investigated. Under standard operating conditions, the system was able to achieve levels of nutrient and organic removal comparable with conventional wastewater treatment works. The mean nitrate level achieved in the effluent was below the 15mg.l-1 South African discharge standard, however, nitrate removal in the IAPS was found to be inconsistent. Although the system was unable to sustain chemical oxygen demand (COD) removal to below the 75mg.l-1 South African discharge standard, a removal rate of 87% was recorded, with the residual COD remaining in the form of algal biomass. Previous studies in the Eastern Cape Province have shown that few small wastewater treatment works produce effluent that meets the microbial count specification. Therefore, in addition to the collation of IAPS data from the entire nine year monitoring period, this study also investigated the use of the HRAP as an independent unit operation for disinfection of effluent from small sewage plants. It was demonstrated that the independent high rate algal pond (IHRAP) as a free standing unit operation could consistently produce water with Escherichia coli counts of 0cfu.100ml-1. The observed effect was related to a number of possible conditions prevailing in the system, including elevated pH, sunlight and dissolved oxygen. It was also found that the IHRAP greatly enhanced the nutrient removal capabilities of the conventional IAPS, making it possible to reliably and consistently maintain phosphate and ammonium levels in the final effluent to below 5mg.l-1 and 2mg.l-1 respectively (South African discharge standards are 10mg.l-1 and 3mg.l-1 in each case). The quality of the final effluent produced by the optimisation of the IAPS would allow it to be used for irrigation, thereby providing an alternative water source in water stressed areas. The system also proved to be exceptionally robust and data collected during periods of intensive and low management regimes were broadly comparable. Results of the 9 year study have demonstrated reliable performance of the IAPS and its use an appropriate, sustainable wastewater treatment option for small communities.
- Full Text:
- Date Issued: 2005
- Authors: Wells, Charles Digby
- Date: 2005
- Subjects: Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4068 , http://hdl.handle.net/10962/d1006162 , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Description: Inadequate sanitation is one of the leading causes of water pollution and consequently illness in many underdeveloped countries, including South Africa and, specifically, the Eastern Cape Province, where cholera has become endemic. As modern wastewater treatment processes are often energy intensive and expensive, they are not suitable for use in these areas. There is thus a need to develop more sustainable wastewater treatment technologies for application in smaller communities. The integrated algal ponding system (IAPS) was identified as a possible solution to this wastewater management problem and was investigated for adaptation to local conditions, at the Rhodes University Environmental Experimental Field Station in Grahamstown, South Africa. The system was monitored over a period of nine years, with various configuration adjustments of the high rate algal pond (HRAP) unit operation investigated. Under standard operating conditions, the system was able to achieve levels of nutrient and organic removal comparable with conventional wastewater treatment works. The mean nitrate level achieved in the effluent was below the 15mg.l-1 South African discharge standard, however, nitrate removal in the IAPS was found to be inconsistent. Although the system was unable to sustain chemical oxygen demand (COD) removal to below the 75mg.l-1 South African discharge standard, a removal rate of 87% was recorded, with the residual COD remaining in the form of algal biomass. Previous studies in the Eastern Cape Province have shown that few small wastewater treatment works produce effluent that meets the microbial count specification. Therefore, in addition to the collation of IAPS data from the entire nine year monitoring period, this study also investigated the use of the HRAP as an independent unit operation for disinfection of effluent from small sewage plants. It was demonstrated that the independent high rate algal pond (IHRAP) as a free standing unit operation could consistently produce water with Escherichia coli counts of 0cfu.100ml-1. The observed effect was related to a number of possible conditions prevailing in the system, including elevated pH, sunlight and dissolved oxygen. It was also found that the IHRAP greatly enhanced the nutrient removal capabilities of the conventional IAPS, making it possible to reliably and consistently maintain phosphate and ammonium levels in the final effluent to below 5mg.l-1 and 2mg.l-1 respectively (South African discharge standards are 10mg.l-1 and 3mg.l-1 in each case). The quality of the final effluent produced by the optimisation of the IAPS would allow it to be used for irrigation, thereby providing an alternative water source in water stressed areas. The system also proved to be exceptionally robust and data collected during periods of intensive and low management regimes were broadly comparable. Results of the 9 year study have demonstrated reliable performance of the IAPS and its use an appropriate, sustainable wastewater treatment option for small communities.
- Full Text:
- Date Issued: 2005
The degradation of lignocellulose in a biologically-generated sulphidic environment
- Authors: Roman, Henry James
- Date: 2005
- Subjects: Lignocellulose Sulfides Lignin Lignocellulose -- Biodegradation Mines and mineral resources -- Waste disposal Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3997 , http://hdl.handle.net/10962/d1004057
- Description: South Africa is renowned for its mining industry. The period over which the polluted waters from the existing and abandoned mines will require treatment has driven research into the development of passive treatment systems. These waters are characterised by a low pH, high concentrations of heavy metals, high levels of sulphate salts and low concentrations of organic material. The biological treatment of these waters has been a subject of increasing focus as an alternative to physicochemical treatment. The utilisation of lignocellulose as a carbon source has been restricted by the amount of reducing equivalents available within the lignocellulose matrix. After a few months of near 100% sulphate reduction, it was found that although there was a large fraction of lignin and cellulose remaining, sulphate reduction was reduced to less than 20%. The present study demonstrated that lignocellulose can be utilised as a carbon source for sulphate reduction. It was established that lignocellulose degradation was enhanced under biosulphidogenic conditions and that lignin could be degraded by a sulphate reducing microbial consortium. It was established using lignin model compounds synthesized in our laboratory, that the bonds within the lignin polymer can be cleaved within the sulphidic environment. The presence of cellulolytic enzymes, using CMCase as a marker enzyme, was detected within the sulphate reducing microbial consortium. Based on the results obtained a descriptive model was formulated for the degradation of lignocellulose under biosulphidogenic conditions. It was determined that the initial reduction in sulphate observed using lignocellulose as a carbon source was due to the easily extractable components. The degradation of which resulted in the production of sulphide, which aided in the degradation of lignin, allowing greater access to cellulose. Once the easily extractable material is exhausted, the cycle is halted, unless the sulphide production can be maintained. This is the focus of an ongoing project, testing the hypothesis that an easy to assimilate carbon source added after exhaustion of the easily extractable material, can maintain the sulphide production.
- Full Text:
- Date Issued: 2005
- Authors: Roman, Henry James
- Date: 2005
- Subjects: Lignocellulose Sulfides Lignin Lignocellulose -- Biodegradation Mines and mineral resources -- Waste disposal Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3997 , http://hdl.handle.net/10962/d1004057
- Description: South Africa is renowned for its mining industry. The period over which the polluted waters from the existing and abandoned mines will require treatment has driven research into the development of passive treatment systems. These waters are characterised by a low pH, high concentrations of heavy metals, high levels of sulphate salts and low concentrations of organic material. The biological treatment of these waters has been a subject of increasing focus as an alternative to physicochemical treatment. The utilisation of lignocellulose as a carbon source has been restricted by the amount of reducing equivalents available within the lignocellulose matrix. After a few months of near 100% sulphate reduction, it was found that although there was a large fraction of lignin and cellulose remaining, sulphate reduction was reduced to less than 20%. The present study demonstrated that lignocellulose can be utilised as a carbon source for sulphate reduction. It was established that lignocellulose degradation was enhanced under biosulphidogenic conditions and that lignin could be degraded by a sulphate reducing microbial consortium. It was established using lignin model compounds synthesized in our laboratory, that the bonds within the lignin polymer can be cleaved within the sulphidic environment. The presence of cellulolytic enzymes, using CMCase as a marker enzyme, was detected within the sulphate reducing microbial consortium. Based on the results obtained a descriptive model was formulated for the degradation of lignocellulose under biosulphidogenic conditions. It was determined that the initial reduction in sulphate observed using lignocellulose as a carbon source was due to the easily extractable components. The degradation of which resulted in the production of sulphide, which aided in the degradation of lignin, allowing greater access to cellulose. Once the easily extractable material is exhausted, the cycle is halted, unless the sulphide production can be maintained. This is the focus of an ongoing project, testing the hypothesis that an easy to assimilate carbon source added after exhaustion of the easily extractable material, can maintain the sulphide production.
- Full Text:
- Date Issued: 2005
The development of the emerging technologies sustainability assessment (ETSA) and its application in the design of a bioprocess for the treatment of wine distillery effluent
- Authors: Khan, Nuraan
- Date: 2005
- Subjects: Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3953 , http://hdl.handle.net/10962/d1004012 , Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Description: Emerging Technologies Sustainability Assessment (ETSA) is a new technology assessment tool that was developed in order to compare emerging processes or technologies to existing alternatives. It utilizes infoIDlation modules, with the minimum use of resources such as time and money, in order to deteIDline if the process under development is comparatively favourable and should be developed beyond the early conceptual phase. The preliminary ETSA is vital in order to identify the gaps in the existing information and the specific methodologies to be used for data capture and analysis. The use of experimental design tools, such as Design-Expert, can facilitate rapid and efficient collection of necessary data and fits in well with the rationale for the ETSA. Wine distillery effluent (vinasse) is the residue left after alcohol has been distilled from fennented grape juice. It is an acidic, darkly coloured effluent, with a high COD and polyphenol content. The most popular method of disposal of this effluent, land application, is no longer viable due to stricter legislation and pressure on the industry to better manage its wastes. Although the ability of whiterot fungi to degrade a number of pollutants is well-known, fungal treatment of wine distillery effluent is still in the conceptual phase. The perfoIDlance of the fungal remediation system was assessed experimentally in terms of COD removal and laccase production using Design-Expert. Although Pycnoporus sanguine us was found to be most efficient at COD removal (85%) from 30% vinasse, laccase production was low (0.021 U/I). The optimum design for economically viable fungal treatment used Trametespubescens. This fungus was able to remove over 50% of the COD from undiluted vinasse while producing almost 800U/l of the valuable laccase enzyme within three days. Since the effluent from the fungal system did not meet the legal limits for wastewater disposal, a two-stage aerobicanaerobic system is suggested to improve the quality of the effluent prior to disposal. The ETSA was used to assess the fungal technology in relation to the two current methods of vinasse treatment and disposal, namely land application and anaerobic digestion. Based on the ETSA, which considered environmental, social and economic impacts, the fungal system proved to be potentially competitive and further development of the technology is suggested.
- Full Text:
- Date Issued: 2005
- Authors: Khan, Nuraan
- Date: 2005
- Subjects: Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3953 , http://hdl.handle.net/10962/d1004012 , Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Description: Emerging Technologies Sustainability Assessment (ETSA) is a new technology assessment tool that was developed in order to compare emerging processes or technologies to existing alternatives. It utilizes infoIDlation modules, with the minimum use of resources such as time and money, in order to deteIDline if the process under development is comparatively favourable and should be developed beyond the early conceptual phase. The preliminary ETSA is vital in order to identify the gaps in the existing information and the specific methodologies to be used for data capture and analysis. The use of experimental design tools, such as Design-Expert, can facilitate rapid and efficient collection of necessary data and fits in well with the rationale for the ETSA. Wine distillery effluent (vinasse) is the residue left after alcohol has been distilled from fennented grape juice. It is an acidic, darkly coloured effluent, with a high COD and polyphenol content. The most popular method of disposal of this effluent, land application, is no longer viable due to stricter legislation and pressure on the industry to better manage its wastes. Although the ability of whiterot fungi to degrade a number of pollutants is well-known, fungal treatment of wine distillery effluent is still in the conceptual phase. The perfoIDlance of the fungal remediation system was assessed experimentally in terms of COD removal and laccase production using Design-Expert. Although Pycnoporus sanguine us was found to be most efficient at COD removal (85%) from 30% vinasse, laccase production was low (0.021 U/I). The optimum design for economically viable fungal treatment used Trametespubescens. This fungus was able to remove over 50% of the COD from undiluted vinasse while producing almost 800U/l of the valuable laccase enzyme within three days. Since the effluent from the fungal system did not meet the legal limits for wastewater disposal, a two-stage aerobicanaerobic system is suggested to improve the quality of the effluent prior to disposal. The ETSA was used to assess the fungal technology in relation to the two current methods of vinasse treatment and disposal, namely land application and anaerobic digestion. Based on the ETSA, which considered environmental, social and economic impacts, the fungal system proved to be potentially competitive and further development of the technology is suggested.
- Full Text:
- Date Issued: 2005
The role of parallel computing in bioinformatics
- Authors: Akhurst, Timothy John
- Date: 2005
- Subjects: Bioinformatics , Parallel programming (Computer science) , LINDA (Computer system) , Java (Computer program language) , Parallel processing (Electronic computers) , Genomics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3986 , http://hdl.handle.net/10962/d1004045 , Bioinformatics , Parallel programming (Computer science) , LINDA (Computer system) , Java (Computer program language) , Parallel processing (Electronic computers) , Genomics -- Data processing
- Description: The need to intelligibly capture, manage and analyse the ever-increasing amount of publicly available genomic data is one of the challenges facing bioinformaticians today. Such analyses are in fact impractical using uniprocessor machines, which has led to an increasing reliance on clusters of commodity-priced computers. An existing network of cheap, commodity PCs was utilised as a single computational resource for parallel computing. The performance of the cluster was investigated using a whole genome-scanning program written in the Java programming language. The TSpaces framework, based on the Linda parallel programming model, was used to parallelise the application. Maximum speedup was achieved at between 30 and 50 processors, depending on the size of the genome being scanned. Together with this, the associated significant reductions in wall-clock time suggest that both parallel computing and Java have a significant role to play in the field of bioinformatics.
- Full Text:
- Date Issued: 2005
- Authors: Akhurst, Timothy John
- Date: 2005
- Subjects: Bioinformatics , Parallel programming (Computer science) , LINDA (Computer system) , Java (Computer program language) , Parallel processing (Electronic computers) , Genomics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3986 , http://hdl.handle.net/10962/d1004045 , Bioinformatics , Parallel programming (Computer science) , LINDA (Computer system) , Java (Computer program language) , Parallel processing (Electronic computers) , Genomics -- Data processing
- Description: The need to intelligibly capture, manage and analyse the ever-increasing amount of publicly available genomic data is one of the challenges facing bioinformaticians today. Such analyses are in fact impractical using uniprocessor machines, which has led to an increasing reliance on clusters of commodity-priced computers. An existing network of cheap, commodity PCs was utilised as a single computational resource for parallel computing. The performance of the cluster was investigated using a whole genome-scanning program written in the Java programming language. The TSpaces framework, based on the Linda parallel programming model, was used to parallelise the application. Maximum speedup was achieved at between 30 and 50 processors, depending on the size of the genome being scanned. Together with this, the associated significant reductions in wall-clock time suggest that both parallel computing and Java have a significant role to play in the field of bioinformatics.
- Full Text:
- Date Issued: 2005
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