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
Ectomycorrhizal characterisation, species diversity and community dynamics in Pinus patula Schelcht. et Cham. plantations
- Authors: Hawley, Greer Leigh
- Date: 2006
- Subjects: Mycorrhizas Ectomycorrhizal fungi Pinus patula -- Irrigation -- South Africa Forests and forestry -- South Africa Forest ecology -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3951 , http://hdl.handle.net/10962/d1004010
- Description: Ectomycorrhizal (ECM) associations are important elements of forest biomes, connecting and transferring nutrients through an intricate and complex system of hyphal networks, ensuring plants of the nutrients they require, in nutrient poor soil. ECM research and particularly investigations into the diversity of the fungal partners has not received much attention in South Africa, hindering the advance of research in this field. This has been attributed to the difficulty of identifying the mycobionts involved in the symbiosis. The objectives of this study were to examine the ECM fungal diversity associating with Pinus patula, in selected forest plantations in Mpumalanga, South Africa. Both morphological and molecular techniques were used to identify specimens of both sporocarp collections and ECM root tip morphotypes. Morphological analysis of the ECM root tips involved characterisation of root morphology such as colour, branching and texture, and anatomical analysis examined hyphal arrangement in the root mantle and rhizomorphs. Molecular analysis involved sequencing of the Internal Transcribed Spacer (ITS) region and comparative BLAST analysis. Twenty-four sporocarp species were identified from 13 genera, namely: Amanita, Boletus, Clavulina, Inocybe, Lactarius, Rhizopogon, Russula, Scleroderma, Suillus, Tricholoma, Thelephora, Tomentella and Xerocomus. ECM root tip analysis led to the characterisation of 7 wild-type morphotypes identified as an Albatrellus sp., 2 Amanita species, a Rhizopogon sp., Thelephora terrestris, a Tomentella sp. and Scleroderma citrinum. A secondary objective was to determine whether fertilisation treatments within the study sites were responsible for differences in fungal species community structure. No evidence of a change in species diversity or shift in species composition was encountered. It is envisaged that these comprehensive ECM descriptions will be used as reference material to stimulate continued research in this field in South Africa.
- Full Text:
- Date Issued: 2006
- Authors: Hawley, Greer Leigh
- Date: 2006
- Subjects: Mycorrhizas Ectomycorrhizal fungi Pinus patula -- Irrigation -- South Africa Forests and forestry -- South Africa Forest ecology -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3951 , http://hdl.handle.net/10962/d1004010
- Description: Ectomycorrhizal (ECM) associations are important elements of forest biomes, connecting and transferring nutrients through an intricate and complex system of hyphal networks, ensuring plants of the nutrients they require, in nutrient poor soil. ECM research and particularly investigations into the diversity of the fungal partners has not received much attention in South Africa, hindering the advance of research in this field. This has been attributed to the difficulty of identifying the mycobionts involved in the symbiosis. The objectives of this study were to examine the ECM fungal diversity associating with Pinus patula, in selected forest plantations in Mpumalanga, South Africa. Both morphological and molecular techniques were used to identify specimens of both sporocarp collections and ECM root tip morphotypes. Morphological analysis of the ECM root tips involved characterisation of root morphology such as colour, branching and texture, and anatomical analysis examined hyphal arrangement in the root mantle and rhizomorphs. Molecular analysis involved sequencing of the Internal Transcribed Spacer (ITS) region and comparative BLAST analysis. Twenty-four sporocarp species were identified from 13 genera, namely: Amanita, Boletus, Clavulina, Inocybe, Lactarius, Rhizopogon, Russula, Scleroderma, Suillus, Tricholoma, Thelephora, Tomentella and Xerocomus. ECM root tip analysis led to the characterisation of 7 wild-type morphotypes identified as an Albatrellus sp., 2 Amanita species, a Rhizopogon sp., Thelephora terrestris, a Tomentella sp. and Scleroderma citrinum. A secondary objective was to determine whether fertilisation treatments within the study sites were responsible for differences in fungal species community structure. No evidence of a change in species diversity or shift in species composition was encountered. It is envisaged that these comprehensive ECM descriptions will be used as reference material to stimulate continued research in this field in South Africa.
- Full Text:
- Date Issued: 2006
Isolation, purification and characterization of a novel glucose oxidase from Penicillium canescens Tt42
- Authors: Simpson, Clinton
- Date: 2006
- Subjects: Penicillium , Glucose , Oxidases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3975 , http://hdl.handle.net/10962/d1004034 , Penicillium , Glucose , Oxidases
- Description: A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.
- Full Text:
- Date Issued: 2006
- Authors: Simpson, Clinton
- Date: 2006
- Subjects: Penicillium , Glucose , Oxidases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3975 , http://hdl.handle.net/10962/d1004034 , Penicillium , Glucose , Oxidases
- Description: A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.
- Full Text:
- Date Issued: 2006
Voltammetric analysis of pesticides and their degradation: A case study of Amitraz and its degradants
- Authors: Brimecombe, Rory Dennis
- Date: 2006
- Subjects: Hydrolysis , Biodegradation , Voltammetry , Pesticides -- Biodegradation , Pesticides -- Environmental aspects , Acaricides , Acaricides -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4131 , http://hdl.handle.net/10962/d1015724
- Description: Amitraz is a formamide acaricide used predominantly in the control of ectoparasites in livestock and honeybees. Amitraz hydrolysis is rapid and occurs under acidic conditions, exposure to sunlight and biodegradation by microorganisms. The main hydrolysis product of amitraz, 2,4-dimethylaniline, is recalcitrant in the environment and toxic to humans. An electrochemical method for the determination of total amitraz residues and its final breakdown product, 2,4-dimethylaniline, in spent cattle dip, is presented. Cyclic voltammetry at a glassy carbon electrode showed the irreversible oxidation of amitraz and 2,4-dimethylaniline. A limit of detection in the range of 8.5 x 10⁻⁸ M for amitraz and 2 x 10⁻⁸ M for 2,4-dimethylaniline was determined using differential pulse voltammetry. Feasibility studies in which the effect of supporting electrolyte type and pH had on electroanalysis of amitraz and its degradants, showed that pH affects current response as well as the potential at which amitraz and its degradants are oxidised. Britton-Robinson buffer was found to be the most suitable supporting electrolyte for detection of amitraz and its degradants in terms of sensitivity and reproducibility. Studies performed using environmental samples showed that the sensitivity and reproducibility of amitraz and 2,4-dimethylaniline analyses in spent cattle dip were comparable to analyses of amitraz and 2,4-dimethylaniline performed in Britton-Robinson buffer. In addition, the feasibility qf measuring amitraz and 2,4-dimethylaniline in environmental samples was assessed and compared to amitraz and 2,4-dimethylaniline analyses in Britton-Robinson buffer. Amitraz and 2,4-dimethylaniline were readily detectable in milk and honey. Furthermore, it was elucidated that 2,4-dimethylaniline can be metabolised to 3-methylcatechol by Pseudomonas species and the proposed breakdown pathway is presented. The biological degradation of amitraz and subsequent formation of 2,4-dimethylaniline was readily monitored in spent cattle dip. The breakdown of amitraz to 2,4-dimethylaniline and then to 3-MC was monitored using cyclic voltammetry.
- Full Text:
- Date Issued: 2006
- Authors: Brimecombe, Rory Dennis
- Date: 2006
- Subjects: Hydrolysis , Biodegradation , Voltammetry , Pesticides -- Biodegradation , Pesticides -- Environmental aspects , Acaricides , Acaricides -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4131 , http://hdl.handle.net/10962/d1015724
- Description: Amitraz is a formamide acaricide used predominantly in the control of ectoparasites in livestock and honeybees. Amitraz hydrolysis is rapid and occurs under acidic conditions, exposure to sunlight and biodegradation by microorganisms. The main hydrolysis product of amitraz, 2,4-dimethylaniline, is recalcitrant in the environment and toxic to humans. An electrochemical method for the determination of total amitraz residues and its final breakdown product, 2,4-dimethylaniline, in spent cattle dip, is presented. Cyclic voltammetry at a glassy carbon electrode showed the irreversible oxidation of amitraz and 2,4-dimethylaniline. A limit of detection in the range of 8.5 x 10⁻⁸ M for amitraz and 2 x 10⁻⁸ M for 2,4-dimethylaniline was determined using differential pulse voltammetry. Feasibility studies in which the effect of supporting electrolyte type and pH had on electroanalysis of amitraz and its degradants, showed that pH affects current response as well as the potential at which amitraz and its degradants are oxidised. Britton-Robinson buffer was found to be the most suitable supporting electrolyte for detection of amitraz and its degradants in terms of sensitivity and reproducibility. Studies performed using environmental samples showed that the sensitivity and reproducibility of amitraz and 2,4-dimethylaniline analyses in spent cattle dip were comparable to analyses of amitraz and 2,4-dimethylaniline performed in Britton-Robinson buffer. In addition, the feasibility qf measuring amitraz and 2,4-dimethylaniline in environmental samples was assessed and compared to amitraz and 2,4-dimethylaniline analyses in Britton-Robinson buffer. Amitraz and 2,4-dimethylaniline were readily detectable in milk and honey. Furthermore, it was elucidated that 2,4-dimethylaniline can be metabolised to 3-methylcatechol by Pseudomonas species and the proposed breakdown pathway is presented. The biological degradation of amitraz and subsequent formation of 2,4-dimethylaniline was readily monitored in spent cattle dip. The breakdown of amitraz to 2,4-dimethylaniline and then to 3-MC was monitored using cyclic voltammetry.
- Full Text:
- Date Issued: 2006
Characterisation of Human Hsj1a : an HSP40 molecular chaperone similar to Malarial Pfj4
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
- Full Text:
- Date Issued: 2007
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
- Full Text:
- Date Issued: 2007
Determination of the botanical composition of black rhinoceros (Diceros bicornis) dung using the rbcL gene as a molecular marker, and analysis of antioxidant and phenolic content of its browse
- Authors: Bulani, Siyavuya Ishmael
- Date: 2007 , 2013-06-25
- Subjects: Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4070 , http://hdl.handle.net/10962/d1006468 , Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Description: The black rhinoceros remains one of the world's extremely endangered species despite a variety of policies to protect it. The black rhinoceros population at the Great Fish River Reserve (GFRR) in the Eastern Cape in South Africa has increased steadily since their re-introduction in 1986. This megaherbivore is a browser, with a diet obtained largely from the short and medium succulent thicket of the GFRR. Knowledge of the preferential diet of the black rhinoceros on the reserve is an important factor for the effective management of the land and the herbivores that compete for its resources. The dietary preferences of the black rhinoceros at the reserve have been established using backtracking methods. In this study the rbcL gene was used to establish an rbcL gene database of the plants from the GFRR and determine the botanical composition of the black rhinoceros dung from the GFRR. Due to the limited number of rbcL gene plant sequences from the GFRR deposited in the GenBank database, 18 plant species from the GFRR were sequenced. Sequence analyses between the partial rbcL gene sequences generated were able to distinguish between plants down to species level. Plant species from the family Euphorbiaceae and Fabaceae showed sequence variation at intra-specific level compared to those of Tiliaceae which were more conserved. The generated rbcL gene sequences from seasonal dung samples were compared to the rbcL gene sequenced from 18 plant species obtained from the GFRR and those from the GenBank database. A wide range of plant species were identified from the dung samples. There were no major differences in botanical composition between the dung samples, except that Grewia spp. were found to dominate in almost all seasons. The results obtained on the free radical scavenging activity of the extracts against 2,2-Diphenyl-l-picrylhydrazyl (DPPH) increased in the order of methanol > ethyl acetate > chloroform. The DPPH free radical scavenging activity of the methanol plant extracts increased in the order Brachylaena elliptica > Plumbago auriculata > Grewia robusta > Azima tetracantha. Methanol extracts on the TLC plate sprayed with Fe³⁺-2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) showed that the compounds present in the extracts react differently to ferric ion, with most compounds unable to reduce ferric ion. Furthermore the methanol extracts were able to exhibit reduction potentials vs. Ag/AgCl at low concentrations. The compounds in the extracts were shown to be phenolic acids and flavonoid glycosides.
- Full Text:
- Date Issued: 2007
- Authors: Bulani, Siyavuya Ishmael
- Date: 2007 , 2013-06-25
- Subjects: Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4070 , http://hdl.handle.net/10962/d1006468 , Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Description: The black rhinoceros remains one of the world's extremely endangered species despite a variety of policies to protect it. The black rhinoceros population at the Great Fish River Reserve (GFRR) in the Eastern Cape in South Africa has increased steadily since their re-introduction in 1986. This megaherbivore is a browser, with a diet obtained largely from the short and medium succulent thicket of the GFRR. Knowledge of the preferential diet of the black rhinoceros on the reserve is an important factor for the effective management of the land and the herbivores that compete for its resources. The dietary preferences of the black rhinoceros at the reserve have been established using backtracking methods. In this study the rbcL gene was used to establish an rbcL gene database of the plants from the GFRR and determine the botanical composition of the black rhinoceros dung from the GFRR. Due to the limited number of rbcL gene plant sequences from the GFRR deposited in the GenBank database, 18 plant species from the GFRR were sequenced. Sequence analyses between the partial rbcL gene sequences generated were able to distinguish between plants down to species level. Plant species from the family Euphorbiaceae and Fabaceae showed sequence variation at intra-specific level compared to those of Tiliaceae which were more conserved. The generated rbcL gene sequences from seasonal dung samples were compared to the rbcL gene sequenced from 18 plant species obtained from the GFRR and those from the GenBank database. A wide range of plant species were identified from the dung samples. There were no major differences in botanical composition between the dung samples, except that Grewia spp. were found to dominate in almost all seasons. The results obtained on the free radical scavenging activity of the extracts against 2,2-Diphenyl-l-picrylhydrazyl (DPPH) increased in the order of methanol > ethyl acetate > chloroform. The DPPH free radical scavenging activity of the methanol plant extracts increased in the order Brachylaena elliptica > Plumbago auriculata > Grewia robusta > Azima tetracantha. Methanol extracts on the TLC plate sprayed with Fe³⁺-2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) showed that the compounds present in the extracts react differently to ferric ion, with most compounds unable to reduce ferric ion. Furthermore the methanol extracts were able to exhibit reduction potentials vs. Ag/AgCl at low concentrations. The compounds in the extracts were shown to be phenolic acids and flavonoid glycosides.
- Full Text:
- Date Issued: 2007
Development of an in-situ ß-D-Glucuronidase diagnostic moraxella-based biosensor for potential application in the monitoring of water polluted by faecal material in South Africa
- Authors: Togo, Chamunorwa Aloius
- Date: 2007
- Subjects: Water quality management -- South Africa Water quality bioassay -- South Africa Sewage sludge -- South Africa -- Management Water -- Purification -- Biological treatment -- South Africa Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3947 , http://hdl.handle.net/10962/d1004006
- Description: The prevention of outbreaks of waterborne diseases remains a major challenge to public health service providers globally. One of the major obstacles in this effort is the unavailability of on-line and real-time methods for rapid monitoring of faecal pollution to facilitate early warning of contamination of drinking water. The main objective of this study was to develop a β-glucuronidase (GUD)-based method that could be used for the on-line and real-time monitoring of microbial water quality. GUD is a marker enzyme for the faecal indicator bacteria Escherichia coli. This enzyme breaks down the synthetic substrate p-nitrophenyl-β-D-glucuronide (PNPG) to D-glucuronic acid and p-nitrophenol (PNP), which turns yellow under alkaline pH. The enzymatically produced PNP was used to detect GUD activity. In situ GUD assays were performed using running and stagnant water samples from the Bloukrans River, Grahamstown, South Africa. The physico-chemical properties of environmental GUD were determined, after which a liquid bioprobe and a microbial biosensor modified with Moraxella 1A species for the detection of the enzyme activity were developed. In order to determine the reliability and sensitivity of these methods, regression analyses for each method versus E. coli colony forming units (CFU) were performed. The storage stabilities of the bioprobe and biosensor were also investigated. The physico-chemical properties of in situ GUD were different from those of its commercially available counterpart. The temperature optimum for the former was between 35 and 40 °C while for the latter it was 45 °C. Commercial (reference) GUD had a pH optimum of 8.0 while the environmental counterpart exhibited a broad pH optimum of between pH 5.0 and 8.0. The liquid bioprobe had a limit of detection (LOD) of GUD activity equivalent to 2 CFU/100 ml and a detection time of 24 h. The method was less labour intensive and costly than the culturing method. The liquid bioprobe was stable for at least four weeks at room temperature (20 ± 2 °C). The biosensor was prepared by modifying a glassy carbon electrode with PNP degrading Moraxella 1A cells. The biosensor was 100 times more sensitive and rapid (5-20 min) than the spectrophotometric method (24 h), and was also able to detect GUD activity of viable but non-culturable cells. Thus it was more sensitive than the culturing method. Furthermore, the biosensor was selective and costeffective. The possibility of using a Pseudomonas putida JS444 biosensor was also investigated, but it was not as sensitive and selective as the Moraxella 1A biosensor. The Moraxella biosensor, therefore, offered the best option for on-line and real-time microbial water quality monitoring in South African river waters and drinking water supplies.
- Full Text:
- Date Issued: 2007
- Authors: Togo, Chamunorwa Aloius
- Date: 2007
- Subjects: Water quality management -- South Africa Water quality bioassay -- South Africa Sewage sludge -- South Africa -- Management Water -- Purification -- Biological treatment -- South Africa Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3947 , http://hdl.handle.net/10962/d1004006
- Description: The prevention of outbreaks of waterborne diseases remains a major challenge to public health service providers globally. One of the major obstacles in this effort is the unavailability of on-line and real-time methods for rapid monitoring of faecal pollution to facilitate early warning of contamination of drinking water. The main objective of this study was to develop a β-glucuronidase (GUD)-based method that could be used for the on-line and real-time monitoring of microbial water quality. GUD is a marker enzyme for the faecal indicator bacteria Escherichia coli. This enzyme breaks down the synthetic substrate p-nitrophenyl-β-D-glucuronide (PNPG) to D-glucuronic acid and p-nitrophenol (PNP), which turns yellow under alkaline pH. The enzymatically produced PNP was used to detect GUD activity. In situ GUD assays were performed using running and stagnant water samples from the Bloukrans River, Grahamstown, South Africa. The physico-chemical properties of environmental GUD were determined, after which a liquid bioprobe and a microbial biosensor modified with Moraxella 1A species for the detection of the enzyme activity were developed. In order to determine the reliability and sensitivity of these methods, regression analyses for each method versus E. coli colony forming units (CFU) were performed. The storage stabilities of the bioprobe and biosensor were also investigated. The physico-chemical properties of in situ GUD were different from those of its commercially available counterpart. The temperature optimum for the former was between 35 and 40 °C while for the latter it was 45 °C. Commercial (reference) GUD had a pH optimum of 8.0 while the environmental counterpart exhibited a broad pH optimum of between pH 5.0 and 8.0. The liquid bioprobe had a limit of detection (LOD) of GUD activity equivalent to 2 CFU/100 ml and a detection time of 24 h. The method was less labour intensive and costly than the culturing method. The liquid bioprobe was stable for at least four weeks at room temperature (20 ± 2 °C). The biosensor was prepared by modifying a glassy carbon electrode with PNP degrading Moraxella 1A cells. The biosensor was 100 times more sensitive and rapid (5-20 min) than the spectrophotometric method (24 h), and was also able to detect GUD activity of viable but non-culturable cells. Thus it was more sensitive than the culturing method. Furthermore, the biosensor was selective and costeffective. The possibility of using a Pseudomonas putida JS444 biosensor was also investigated, but it was not as sensitive and selective as the Moraxella 1A biosensor. The Moraxella biosensor, therefore, offered the best option for on-line and real-time microbial water quality monitoring in South African river waters and drinking water supplies.
- Full Text:
- Date Issued: 2007
Evaluation and application of electroanalysis for the determination of antioxidants
- Authors: Ragubeer, Nasheen
- Date: 2007
- Subjects: Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3922 , http://hdl.handle.net/10962/d1003981 , Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Description: The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.
- Full Text:
- Date Issued: 2007
- Authors: Ragubeer, Nasheen
- Date: 2007
- Subjects: Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3922 , http://hdl.handle.net/10962/d1003981 , Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Description: The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.
- Full Text:
- Date Issued: 2007
Hydrogenases from sulphate reducing bacteria and their role in the bioremediation of textile effluent
- Mutambanengwe, Cecil Clifford Zvandada
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
Isolation and characterization of genes encoding heat shock protein 70s (hsp 70s) from two species of the coelacanth, Latimeria chalumnae and Latimeria menadoensis
- Modisakeng, Keoagile William
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
- Full Text:
- Date Issued: 2007
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
- Full Text:
- Date Issued: 2007
Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
Isolation, propagation and rapid molecular detection of the Kalahari truffle, a mycorrhizal fungus occurring in South Africa
- Authors: Adeleke, Rasheed Adegbola
- Date: 2007 , 2013-04-03
- Subjects: Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3889 , http://hdl.handle.net/10962/d1002951 , Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Description: Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2007
- Authors: Adeleke, Rasheed Adegbola
- Date: 2007 , 2013-04-03
- Subjects: Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3889 , http://hdl.handle.net/10962/d1002951 , Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Description: Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2007
Localisation of Theiler's Murine Encephalomyelitis virus non-structural proteins 2B, 2C, 2BC and 3A in BHK-21 cells, and the effect of amino acid substitutions in 2C on localisation and virus replication
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
- Full Text:
- Date Issued: 2007
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
- Full Text:
- Date Issued: 2007
Molecular characterisation of the chaperone properties of Plasmodium falciparum heat shock protein 70
- Authors: Shonhai, Addmore
- Date: 2007
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Proteins -- Purification Molecular chaperones Malaria -- Prevention
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3977 , http://hdl.handle.net/10962/d1004036
- Description: Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.
- Full Text:
- Date Issued: 2007
- Authors: Shonhai, Addmore
- Date: 2007
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Proteins -- Purification Molecular chaperones Malaria -- Prevention
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3977 , http://hdl.handle.net/10962/d1004036
- Description: Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.
- Full Text:
- Date Issued: 2007
Regulation of hyu gene expression in Agrobacterium tumefaciens strains RU-AE01 and RU-OR
- Authors: Jiwaji, Meesbah
- Date: 2007
- Subjects: Agrobacterium tumefaciens Amino acids Gene expression Hydrolysis Hydantoin Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3983 , http://hdl.handle.net/10962/d1004042
- Description: Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by deletion analysis. It was proposed that transcription of hyuH was activated by a putative σ[superscript 54]-dependent promoter or a putative σ[superscript 70]-dependent promoter identified upstream of the hyuH gene. The hyuC gene was activated by a putative σ[superscript 70]-dependent promoter identified upstream of the hyuC gene. The regulation of hydantoinase and N-carbamyl amino acid amidohydrolase enzyme activity was compared to the regulation of transcription from the RU-AE01 hyuH-hyuC region. Expression of the hydantoin-hydrolyzing enzymes was regulated by induction which correlated with reporter enzyme expression from the hyuH and hyuC promoter regions. However, the expression of the hydantoin-hydrolyzing enzymes was also regulated by nitrogen catabolite repression (NCR). This did not correlate to the reporter gene expression of the hyuH promoter region but did compare to the reporter gene expression of the hyuC promoter region. This suggested that NCR of hyuH was at the post-translational level whereas NCR of the hyuC promoter was at the transcriptional level. Pathways involved in the regulation of the hyu genes were characterized. The production of the hydantoin-hydrolyzing enzymes in both A. tumefaciens strains RU-AE01 and RU-OR were regulated by proteins involved in the global ntr pathway. The levels of the hydantoin-hydrolyzing enzymes in strain RU-AE01 were elevated in the presence of increased levels of NtrB and NtrC illustrating the importance of the ntr pathway in the regulation of the levels of the hydantoin-hydrolyzing enzymes. Similarly, in RU-OR the presence of exogenous NtrB and NtrC elevated levels of N-carbamyl amino acid amidohydrolase activity. However, the levels of hydantoinase enzyme activity in strain RU-OR were elevated in the presence of NtrC alone. In addition, the presence of a His6-tagged NtrC molecule abolished the elevation in the levels of the hydantoinase but not the N-carbamyl amino acid amidohydrolase enzyme activity in strain RU-OR. This suggests that NtrC has a direct role in the regulation of the expression of hyuH in RU-OR. In addition, it indicates that the hyu genes in the two A. tumefaciens strains RU-AE01 and RU-OR are different. The presence of the RU-AE01 hyuH-hyuC fragment caused a dramatic increase in the hydantoin-hydrolyzing enzyme activity in strain RU-OR but not strain RU-AE01. This implied the incidence of a possible repressor protein in RU-OR, which is titrated out by the presence of the RU-AE01 hyuH-hyuC fragment. Protein-DNA binding assays suggest that this putative repressor may be 38 kDa in RU-OR cells.
- Full Text:
- Date Issued: 2007
- Authors: Jiwaji, Meesbah
- Date: 2007
- Subjects: Agrobacterium tumefaciens Amino acids Gene expression Hydrolysis Hydantoin Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3983 , http://hdl.handle.net/10962/d1004042
- Description: Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by deletion analysis. It was proposed that transcription of hyuH was activated by a putative σ[superscript 54]-dependent promoter or a putative σ[superscript 70]-dependent promoter identified upstream of the hyuH gene. The hyuC gene was activated by a putative σ[superscript 70]-dependent promoter identified upstream of the hyuC gene. The regulation of hydantoinase and N-carbamyl amino acid amidohydrolase enzyme activity was compared to the regulation of transcription from the RU-AE01 hyuH-hyuC region. Expression of the hydantoin-hydrolyzing enzymes was regulated by induction which correlated with reporter enzyme expression from the hyuH and hyuC promoter regions. However, the expression of the hydantoin-hydrolyzing enzymes was also regulated by nitrogen catabolite repression (NCR). This did not correlate to the reporter gene expression of the hyuH promoter region but did compare to the reporter gene expression of the hyuC promoter region. This suggested that NCR of hyuH was at the post-translational level whereas NCR of the hyuC promoter was at the transcriptional level. Pathways involved in the regulation of the hyu genes were characterized. The production of the hydantoin-hydrolyzing enzymes in both A. tumefaciens strains RU-AE01 and RU-OR were regulated by proteins involved in the global ntr pathway. The levels of the hydantoin-hydrolyzing enzymes in strain RU-AE01 were elevated in the presence of increased levels of NtrB and NtrC illustrating the importance of the ntr pathway in the regulation of the levels of the hydantoin-hydrolyzing enzymes. Similarly, in RU-OR the presence of exogenous NtrB and NtrC elevated levels of N-carbamyl amino acid amidohydrolase activity. However, the levels of hydantoinase enzyme activity in strain RU-OR were elevated in the presence of NtrC alone. In addition, the presence of a His6-tagged NtrC molecule abolished the elevation in the levels of the hydantoinase but not the N-carbamyl amino acid amidohydrolase enzyme activity in strain RU-OR. This suggests that NtrC has a direct role in the regulation of the expression of hyuH in RU-OR. In addition, it indicates that the hyu genes in the two A. tumefaciens strains RU-AE01 and RU-OR are different. The presence of the RU-AE01 hyuH-hyuC fragment caused a dramatic increase in the hydantoin-hydrolyzing enzyme activity in strain RU-OR but not strain RU-AE01. This implied the incidence of a possible repressor protein in RU-OR, which is titrated out by the presence of the RU-AE01 hyuH-hyuC fragment. Protein-DNA binding assays suggest that this putative repressor may be 38 kDa in RU-OR cells.
- Full Text:
- Date Issued: 2007
The microbial ecology of sulphidogenic lignocellulose degradation
- Authors: Clarke, Anna Maria
- Date: 2007
- Subjects: Microbial ecology , Lignocellulose , Sulfides , Lignin , Lignocellulose -- Biodegradation , Mines and mineral resources -- Waste disposal , Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4094 , http://hdl.handle.net/10962/d1008181
- Description: Acid mine drainage is a well known environmental pollutant, not only in South Africa, but throughout the world, and the use of microbial processes in the treatment of these wastes has been the subject of investigation over past decades. Lignocellulose packed-bed reactors have been used in passive treatment systems, and, although effective initially, they show early decline in performance while the packing material remains largely un-utilized. Little is known about this phenomenon which remains a severe constraint in the development of efficient passive mine water treatment systems. It has been proposed that the degradation pathways of the complex lignocellulose substrate may be limited in some way in these systems during the manifestation of this effect. This study has addressed the problem using a molecular microbial ecology methodology in an attempt to relate trophic functions of the microbial population to the physico-chemical data of the system. A field-scale lignocellulose packed-bed reactor located at Vryheid Coronation Colliery (Northern Kwa-Zulu Natal province, South Africa) was monitored for six years and the results showed the classic profile of performance decline related to a slowdown in sulphate reduction and alkalinity production. The reactor was decommissioned , comprehensive samples were collected along the depth profile and the microbial populations investigated by means of 16S rRNA gene methodology. The population was found to include cellulolytic Clostridia spp., CytophagaIFlavobacterlBacteroidetes, Sphingomonadaceae and as yet uncultured microorganisms related to microbiota identified in the rumen and termite gut. These are all known to be involved as primary fermenters of cellulose. Oesulphosporosinus was present as sulphate reducer. A comparison of substrata sampling and population distribution suggested that spatial and temporal gradients within the system may become established over the course of its operation. Based on these findings, a laboratory-scale reactor was constructed to simulate the performance of the packed-bed reactor under controlled experimental conditions. The laboratory-scale reactor was operated for 273 days and showed comparable performance to that in the field in both biomolecular and physicochemical data. Clearly defined trophic niches were observed. These results suggested that a sequence of events does occur in lignocellulose degradation over time. Based on the spatial and temporal column studies, a descriptive model was proposed to account for these events. It was found that fermentative organisms predominate in the inlet zone of the system using easily extractable compounds from the wood, thus providing feedstock for sulphate reduction occurring in the succeeding compartments. Production of sulphide and alkalinity appears to be involved in the enhancement of lignin degradation and this, in turn, appears to enhance access to the cellulose fraction. However, once the readily extractables are exhausted, the decline in sulphide and alkalinity production leads inexorably to a decline in the overall performance of the system as a sulphate reducing unit operation. These observations led to the proposal that with the addition of a limited amount of a readily available carbon source, such as molasses, in the initial zone of the the reactor, the ongoing generation of sulphide would be sustained and this in turn would sustain the microbial attack on the lignocellulose complex. This proposal was tested in scale-up studies and positive results indicate that the descriptive model may, to some extent, provide an account of events occurring in these systems. The work on sustaining lignocellulose degradation through the maintenance of sulphate reduction in the initial stages of the reactor flow path has led to the development of the Degrading Packed-bed Reactor concept and that, has subsequently been successfully evaluated in the field.
- Full Text:
- Date Issued: 2007
- Authors: Clarke, Anna Maria
- Date: 2007
- Subjects: Microbial ecology , Lignocellulose , Sulfides , Lignin , Lignocellulose -- Biodegradation , Mines and mineral resources -- Waste disposal , Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4094 , http://hdl.handle.net/10962/d1008181
- Description: Acid mine drainage is a well known environmental pollutant, not only in South Africa, but throughout the world, and the use of microbial processes in the treatment of these wastes has been the subject of investigation over past decades. Lignocellulose packed-bed reactors have been used in passive treatment systems, and, although effective initially, they show early decline in performance while the packing material remains largely un-utilized. Little is known about this phenomenon which remains a severe constraint in the development of efficient passive mine water treatment systems. It has been proposed that the degradation pathways of the complex lignocellulose substrate may be limited in some way in these systems during the manifestation of this effect. This study has addressed the problem using a molecular microbial ecology methodology in an attempt to relate trophic functions of the microbial population to the physico-chemical data of the system. A field-scale lignocellulose packed-bed reactor located at Vryheid Coronation Colliery (Northern Kwa-Zulu Natal province, South Africa) was monitored for six years and the results showed the classic profile of performance decline related to a slowdown in sulphate reduction and alkalinity production. The reactor was decommissioned , comprehensive samples were collected along the depth profile and the microbial populations investigated by means of 16S rRNA gene methodology. The population was found to include cellulolytic Clostridia spp., CytophagaIFlavobacterlBacteroidetes, Sphingomonadaceae and as yet uncultured microorganisms related to microbiota identified in the rumen and termite gut. These are all known to be involved as primary fermenters of cellulose. Oesulphosporosinus was present as sulphate reducer. A comparison of substrata sampling and population distribution suggested that spatial and temporal gradients within the system may become established over the course of its operation. Based on these findings, a laboratory-scale reactor was constructed to simulate the performance of the packed-bed reactor under controlled experimental conditions. The laboratory-scale reactor was operated for 273 days and showed comparable performance to that in the field in both biomolecular and physicochemical data. Clearly defined trophic niches were observed. These results suggested that a sequence of events does occur in lignocellulose degradation over time. Based on the spatial and temporal column studies, a descriptive model was proposed to account for these events. It was found that fermentative organisms predominate in the inlet zone of the system using easily extractable compounds from the wood, thus providing feedstock for sulphate reduction occurring in the succeeding compartments. Production of sulphide and alkalinity appears to be involved in the enhancement of lignin degradation and this, in turn, appears to enhance access to the cellulose fraction. However, once the readily extractables are exhausted, the decline in sulphide and alkalinity production leads inexorably to a decline in the overall performance of the system as a sulphate reducing unit operation. These observations led to the proposal that with the addition of a limited amount of a readily available carbon source, such as molasses, in the initial zone of the the reactor, the ongoing generation of sulphide would be sustained and this in turn would sustain the microbial attack on the lignocellulose complex. This proposal was tested in scale-up studies and positive results indicate that the descriptive model may, to some extent, provide an account of events occurring in these systems. The work on sustaining lignocellulose degradation through the maintenance of sulphate reduction in the initial stages of the reactor flow path has led to the development of the Degrading Packed-bed Reactor concept and that, has subsequently been successfully evaluated in the field.
- Full Text:
- Date Issued: 2007
The Rhodes BioSURE process and the use of sustainability indicators in the development of biological mine water treatment
- Authors: Neba, Alphonsus
- Date: 2007
- Subjects: Acid mine drainage Water -- Purification -- Biological treatment Mine water Mine water -- Purification Sewage -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3984 , http://hdl.handle.net/10962/d1004043
- Description: Polluted waters, arising from extensive past and ongoing mining operations in South Africa, pose serious environmental threats to the limited fresh water resource. The long time periods, of decades to centuries, over which decanting mine waters may be expected to flow raises additional concerns about the sustainability of these resources. Responses to the problem have thus increasingly been directed towards the long-term sustainability of mine water treatment technologies (MWTT) as a critical indicator in both their research and development, and application. Bioprocess treatments have been considered in this regard and, among these, the Rhodes BioSURE Process has been investigated in preliminary studies using complex organic carbon wastes as the carbon source and electron donor for the central sulphate reduction unit operation. Although both the mining industry and the related statutory/regulatory authority in South Africa share public commitment to sustainability in the treatment of mine waters, no systematic mechanism has emerged to enable the application of sustainability thinking as a guiding principle in the selection and application of MWTTs, nor in the research and development undertaking. This study undertook the development of a Sustainability Indicator Framework in order to provide a systematic basis for the incorporation of sustainability objectives in MWTT bioprocess development, and specifically to use this framework as an input to the investigation of the scaleup development of the Rhodes BioSURE Process. In the development of the MWTT Sustainability Indicator Framework, an initial survey of industry thinking in this area was undertaken and, based on these outcomes, a detailed questionnaire methodology was developed in order to identify and quantify critical sustainability indicators. These included analysis of environmental, economic, social and technical indicators used in sustainability accounting practice in the industry. Statutory/regulatory sustainability targets in the same categories were derived from State of the Environment Reports (SoER) from Provincial authorities where mining is undertaken in South Africa. A synthesis of industry and SoER values was derived from weighted averages and the Sustainability Indicator Framework based on these outcomes. A Conceptual Decision-Support System, to guide the selection and development of MWTTs, was proposed and also based on these results. In the development of the Rhodes BioSURE Process the use of primary sludge (PS) had been investigated as a potential complex carbon and electron donor source. In this regard the utility operator, and sewage treatment process infrastructure, was identified as potentially meeting aspects of the sustainability objectives identified for MWTT application development. Both the Sustainability Indicator Framework and the Conceptual Decision-Support System provided inputs in the formulation of the experimental programme relating to the scale-up development of the Rhodes BioSURE Process. Based on these outcomes, a series of single- and multi-stage reactor configuration, optimisation and enzymology studies were undertaken at bench-, pilot- and technical-scale operations. These units were operated at hydraulic retention times (HRT) ranging between 22 to 72 hours and at chemical oxygen demand to sulphate ratios (COD:SO[subscript 4]) ranging between 1:1 to 2:1. Studies undertaken in fed-batch, bench-scale reactors confirmed the preliminary feasibility of using established sewage treatment infrastructure as a replacement for novel reactor configurations that had been used in the initial studies. The results further indicated that the hydrolysis of PS occurred at different rates under biosulphidogenic conditions in the different reactor configurations investigated. Scale-up of these findings in multi-stage pilot- (7.4m[superscript 3]) and technical-scale plants (680m[superscript 3]) showed comparable performances between the unit operations in terms of SO[subscript 4] and COD removal. These results indicated no apparent advantages in the uncoupling of hydrolysis and sulphate reduction in separate unit operations as had been suggested in previous studies. Scale-down/scale-up studies were undertaken in a continuously fed single-stage reactor configuration and showed that the process could be effectively operated in this way. Previous proposals that chemical and biological gradients established in the sludge bed of the Recycling Sludge Bed Reactor (RSBR) exercised an influence on the rates of substrate hydrolysis were investigated and the relative activity of α- and β-glucosidase and protease enzymes was measured. Results provided additional support for this hypothesis and it was shown that enzyme assay may also provide a useful tool in process development and monitoring studies. While sulphide recovery, following the sulphate reduction step in the BioSURE Process, was not investigated as a component of this study, the treatment of final effluent or waste spills was identified as an important sustainability requirement given the toxicity of sulphide to human and ecosystem environments. A conventional trickle filter reactor system was evaluated for this purpose and showed close to 100% oxidation to sulphate in a short contact time operating regime. Although residual COD removal was low at ~20% of influent, it is considered that high rate recycle biofilter operation could achieve the COD discharge standard of 75 mg/l. The results of the above studies provided inputs into the design, construction and commissioning of the first full-scale commercial application of the Rhodes BioSURE Process for mine wastewater treatment using sewage sludge as the carbon and electron donor source. An adjacent mine and sewage works have been linked by pipeline and an operational capacity of 10 Ml/day water treated has been established with sulphate reduced from ~1300mg/l to <200mg/l. These developments constitute a novel contribution in the mine waste water treatment field.
- Full Text:
- Date Issued: 2007
- Authors: Neba, Alphonsus
- Date: 2007
- Subjects: Acid mine drainage Water -- Purification -- Biological treatment Mine water Mine water -- Purification Sewage -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3984 , http://hdl.handle.net/10962/d1004043
- Description: Polluted waters, arising from extensive past and ongoing mining operations in South Africa, pose serious environmental threats to the limited fresh water resource. The long time periods, of decades to centuries, over which decanting mine waters may be expected to flow raises additional concerns about the sustainability of these resources. Responses to the problem have thus increasingly been directed towards the long-term sustainability of mine water treatment technologies (MWTT) as a critical indicator in both their research and development, and application. Bioprocess treatments have been considered in this regard and, among these, the Rhodes BioSURE Process has been investigated in preliminary studies using complex organic carbon wastes as the carbon source and electron donor for the central sulphate reduction unit operation. Although both the mining industry and the related statutory/regulatory authority in South Africa share public commitment to sustainability in the treatment of mine waters, no systematic mechanism has emerged to enable the application of sustainability thinking as a guiding principle in the selection and application of MWTTs, nor in the research and development undertaking. This study undertook the development of a Sustainability Indicator Framework in order to provide a systematic basis for the incorporation of sustainability objectives in MWTT bioprocess development, and specifically to use this framework as an input to the investigation of the scaleup development of the Rhodes BioSURE Process. In the development of the MWTT Sustainability Indicator Framework, an initial survey of industry thinking in this area was undertaken and, based on these outcomes, a detailed questionnaire methodology was developed in order to identify and quantify critical sustainability indicators. These included analysis of environmental, economic, social and technical indicators used in sustainability accounting practice in the industry. Statutory/regulatory sustainability targets in the same categories were derived from State of the Environment Reports (SoER) from Provincial authorities where mining is undertaken in South Africa. A synthesis of industry and SoER values was derived from weighted averages and the Sustainability Indicator Framework based on these outcomes. A Conceptual Decision-Support System, to guide the selection and development of MWTTs, was proposed and also based on these results. In the development of the Rhodes BioSURE Process the use of primary sludge (PS) had been investigated as a potential complex carbon and electron donor source. In this regard the utility operator, and sewage treatment process infrastructure, was identified as potentially meeting aspects of the sustainability objectives identified for MWTT application development. Both the Sustainability Indicator Framework and the Conceptual Decision-Support System provided inputs in the formulation of the experimental programme relating to the scale-up development of the Rhodes BioSURE Process. Based on these outcomes, a series of single- and multi-stage reactor configuration, optimisation and enzymology studies were undertaken at bench-, pilot- and technical-scale operations. These units were operated at hydraulic retention times (HRT) ranging between 22 to 72 hours and at chemical oxygen demand to sulphate ratios (COD:SO[subscript 4]) ranging between 1:1 to 2:1. Studies undertaken in fed-batch, bench-scale reactors confirmed the preliminary feasibility of using established sewage treatment infrastructure as a replacement for novel reactor configurations that had been used in the initial studies. The results further indicated that the hydrolysis of PS occurred at different rates under biosulphidogenic conditions in the different reactor configurations investigated. Scale-up of these findings in multi-stage pilot- (7.4m[superscript 3]) and technical-scale plants (680m[superscript 3]) showed comparable performances between the unit operations in terms of SO[subscript 4] and COD removal. These results indicated no apparent advantages in the uncoupling of hydrolysis and sulphate reduction in separate unit operations as had been suggested in previous studies. Scale-down/scale-up studies were undertaken in a continuously fed single-stage reactor configuration and showed that the process could be effectively operated in this way. Previous proposals that chemical and biological gradients established in the sludge bed of the Recycling Sludge Bed Reactor (RSBR) exercised an influence on the rates of substrate hydrolysis were investigated and the relative activity of α- and β-glucosidase and protease enzymes was measured. Results provided additional support for this hypothesis and it was shown that enzyme assay may also provide a useful tool in process development and monitoring studies. While sulphide recovery, following the sulphate reduction step in the BioSURE Process, was not investigated as a component of this study, the treatment of final effluent or waste spills was identified as an important sustainability requirement given the toxicity of sulphide to human and ecosystem environments. A conventional trickle filter reactor system was evaluated for this purpose and showed close to 100% oxidation to sulphate in a short contact time operating regime. Although residual COD removal was low at ~20% of influent, it is considered that high rate recycle biofilter operation could achieve the COD discharge standard of 75 mg/l. The results of the above studies provided inputs into the design, construction and commissioning of the first full-scale commercial application of the Rhodes BioSURE Process for mine wastewater treatment using sewage sludge as the carbon and electron donor source. An adjacent mine and sewage works have been linked by pipeline and an operational capacity of 10 Ml/day water treated has been established with sulphate reduced from ~1300mg/l to <200mg/l. These developments constitute a novel contribution in the mine waste water treatment field.
- Full Text:
- Date Issued: 2007