Enzymatic recovery of rhodium(III) from aqueous solution and industrial effluent using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
- Full Text:
- Date Issued: 2005
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
- Full Text:
- Date Issued: 2005
Studies on the ecology and systematics of the diatoms (Bacillariophyta) from some South Africa rivers
- Archibald, Robert Eldred Mostert
- Authors: Archibald, Robert Eldred Mostert
- Date: 1969
- Subjects: Diatoms -- Ecology -- South Africa , Diatoms -- Classification , Aquatic ecology -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4098 , http://hdl.handle.net/10962/d1009494 , Diatoms -- Ecology -- South Africa , Diatoms -- Classification , Aquatic ecology -- South Africa
- Description: This report contains the results of some ecological and systematic studies on the diatoms from the Vaal Dam catchment area in the Transvaal, and the Bloukrans River in the Eastern Cape Province. In Part 1 the effects of high concentrations of nitrogen were studied in relation to the composition of the diatom associations. Water samples from four stations on the Bloukrans River were analysed chemically at certain intervals during the months of April and August 1967. Diatom samples collected from these stations at the beginning and end of each of these sampling periods were subjected to a "Thomasson Analysis" to determine the relative densities of the various species in the diatom associations. A statistical analysis of the results reflected a poor but positive correlation between the two variables, i.e. high numbers of nitrogen heterotrophic Nitzschiae were correlated with high concentrations of nitrogen, while low numbers were correlated with low concentrations. Part 2 presents the results of the ecological studies on the diatom associations of the Vaal Dam Catchment Area. In this section the diatom associations from each sampling point or station were subjected to a "Thomasson Analysis" to determine the relative densities of the different species in the associations. Employing already known correlations between environment and association, the results of this analysis were discussed and the ecological conditions for each sampling station were assessed. The associations were similar in composition over the entire catchment area, and indicated on the whole water of good quality. Points of pollution were detected, but were generally localised and the effects of the pollution were soon removed. Only the Waterval River showed evidence of more constant pollution. The associations provided evidence for some seasonal variation in their composition. Finally in Part 3 the systematics and taxonomy of the diatoms in the Vaal Dam catchment area are discussed. References are made to the original and more recent descriptions of each species found in this study, and a list of synonyms is given wherever 'possible. Comments on the systematics, taxonomy and autecology of each species are given, and the distribution of the species in South Africa and the Vaal Dam catchment area is discussed. A number of species, varieties and forms have been recorded for the first time in South Africa. During the course of this study 20 species have been described as new to science; the descriptions of some have been published, while the descriptions of the others will be published formally in the future. All species described as new or having interesting features are illustrated in the plates.
- Full Text:
- Date Issued: 1969
- Authors: Archibald, Robert Eldred Mostert
- Date: 1969
- Subjects: Diatoms -- Ecology -- South Africa , Diatoms -- Classification , Aquatic ecology -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4098 , http://hdl.handle.net/10962/d1009494 , Diatoms -- Ecology -- South Africa , Diatoms -- Classification , Aquatic ecology -- South Africa
- Description: This report contains the results of some ecological and systematic studies on the diatoms from the Vaal Dam catchment area in the Transvaal, and the Bloukrans River in the Eastern Cape Province. In Part 1 the effects of high concentrations of nitrogen were studied in relation to the composition of the diatom associations. Water samples from four stations on the Bloukrans River were analysed chemically at certain intervals during the months of April and August 1967. Diatom samples collected from these stations at the beginning and end of each of these sampling periods were subjected to a "Thomasson Analysis" to determine the relative densities of the various species in the diatom associations. A statistical analysis of the results reflected a poor but positive correlation between the two variables, i.e. high numbers of nitrogen heterotrophic Nitzschiae were correlated with high concentrations of nitrogen, while low numbers were correlated with low concentrations. Part 2 presents the results of the ecological studies on the diatom associations of the Vaal Dam Catchment Area. In this section the diatom associations from each sampling point or station were subjected to a "Thomasson Analysis" to determine the relative densities of the different species in the associations. Employing already known correlations between environment and association, the results of this analysis were discussed and the ecological conditions for each sampling station were assessed. The associations were similar in composition over the entire catchment area, and indicated on the whole water of good quality. Points of pollution were detected, but were generally localised and the effects of the pollution were soon removed. Only the Waterval River showed evidence of more constant pollution. The associations provided evidence for some seasonal variation in their composition. Finally in Part 3 the systematics and taxonomy of the diatoms in the Vaal Dam catchment area are discussed. References are made to the original and more recent descriptions of each species found in this study, and a list of synonyms is given wherever 'possible. Comments on the systematics, taxonomy and autecology of each species are given, and the distribution of the species in South Africa and the Vaal Dam catchment area is discussed. A number of species, varieties and forms have been recorded for the first time in South Africa. During the course of this study 20 species have been described as new to science; the descriptions of some have been published, while the descriptions of the others will be published formally in the future. All species described as new or having interesting features are illustrated in the plates.
- Full Text:
- Date Issued: 1969
Genetic characterisation of a range of geographically distinct Helicoverpa armigera nucleopolyhedrovirus (HearNPV) isolates and evaluation of biological activity against South African populations of the African bollworm, Helicoverpa armigera (Hu bner) (Lepidoptera: Noctuidae)
- Mtambanengwe, Kudzai Tapiwanashe Esau
- Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
- Date: 2019
- Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/97334 , vital:31426
- Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
- Full Text:
- Date Issued: 2019
- Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
- Date: 2019
- Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/97334 , vital:31426
- Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
- Full Text:
- Date Issued: 2019
Stress manipulation in Dunaliella salina and dual-stage [beta]-carotene production
- Authors: Phillips, Trevor David
- Date: 1994
- Subjects: Dunaliella Carotenes Plants -- Effect of stress on
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4037 , http://hdl.handle.net/10962/d1004097
- Description: The alga Dunaliella salina accumulates large quantities of β-carotene in response to certain environmental and physiological stresses. This hyper-accumulation process has been commercially exploited. However, the currently employed averaging or single-stage process produces β-carotene yields well below the genetic potential of the organism due to the inverse relationship between growth and secondary metabolite production. A dual-stage process, which separates the distinctive growth and secondary metabolite production stages of the alga, has been proposed. The broad aim of the research programme was to evaluate the practicality, scale-up and economic viability of a dual-stage β-carotene production process from D. salina. Preliminary laboratory studies showed that although stress factors such as high salinity and a range of nutrient limitations enhance β-carotene accumulation in D. salina, high light intensity is the single most important factor inducing β-carotene hyper-accumulation in the alga. Furthermore, the preliminary studies indicated that 6-carotene production could be successfully manipulated by the imposition of stress. The stress response of D. salina to high light stress was examined at a fundamental level. The relative partitioning of β-carotene between thylakoid membrane and interthylakoid globular β-carotene has revealed two responses to high light stress. The first is a response in which the alga adapts to the photoinhibitory effects of high light stress by the rapid accumulation and the peripheral localisation of Jl-carotene to the outer extremities of the chloroplast. This is followed by a maintenance response which is characterised by the recovery of the photosynthetic rate and cell growth. A possible interrelationship between the extent of the photo inhibitory response and the amount of β-carotene hyper-accumulation has been noted. An outdoor evaluation of the growth stage of the dual-stage system has demonstrated that D. salina can be grown in a relatively low salinity, nutrient sufficient medium for extended periods without overgrowth by small non-carotenogenic Dunaliella species. In addition, biomass productivities of three times greater than those obtained in the currently employed averaging system were achieved. The role of high light intensity in β-carotene hyper-accumulation was confirmed in outdoor scale-up stress pond studies. The studies demonstrated the feasibility of stress induced ll-carotene production in outdoor cultures of D. salina and β-carotene yields three times greater than those obtained in the currently employed averaging process were achieved. The dual-stage process imposes the specific requirement of viable cell separation on the harvesting system employed. A flocculation-flotation process and an air-displacement crossflow ultrafiltration system were developed and successfully evaluated for the separation of D. salina from the brine solution in a viable form. The extraction of β-carotene from D. salina was evaluated. Supercritical fluid extraction studies showed that the use of a co-solvent mixture of carbon dioxide and propane could effectively reduce the high extraction pressures associated with supercritical carbon dioxide extraction. In addition, a novel hydrophobic membrane assisted hot oil extraction process was developed which separates the complex oil-water emulsions produced during hot oil extraction of 6-carotene from wet D. salina biomass. Process design and economic evaluation studies were undertaken and showed that the economics of the dual-stage process offer significant advantages over the currently employed averaging process.
- Full Text:
- Date Issued: 1994
- Authors: Phillips, Trevor David
- Date: 1994
- Subjects: Dunaliella Carotenes Plants -- Effect of stress on
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4037 , http://hdl.handle.net/10962/d1004097
- Description: The alga Dunaliella salina accumulates large quantities of β-carotene in response to certain environmental and physiological stresses. This hyper-accumulation process has been commercially exploited. However, the currently employed averaging or single-stage process produces β-carotene yields well below the genetic potential of the organism due to the inverse relationship between growth and secondary metabolite production. A dual-stage process, which separates the distinctive growth and secondary metabolite production stages of the alga, has been proposed. The broad aim of the research programme was to evaluate the practicality, scale-up and economic viability of a dual-stage β-carotene production process from D. salina. Preliminary laboratory studies showed that although stress factors such as high salinity and a range of nutrient limitations enhance β-carotene accumulation in D. salina, high light intensity is the single most important factor inducing β-carotene hyper-accumulation in the alga. Furthermore, the preliminary studies indicated that 6-carotene production could be successfully manipulated by the imposition of stress. The stress response of D. salina to high light stress was examined at a fundamental level. The relative partitioning of β-carotene between thylakoid membrane and interthylakoid globular β-carotene has revealed two responses to high light stress. The first is a response in which the alga adapts to the photoinhibitory effects of high light stress by the rapid accumulation and the peripheral localisation of Jl-carotene to the outer extremities of the chloroplast. This is followed by a maintenance response which is characterised by the recovery of the photosynthetic rate and cell growth. A possible interrelationship between the extent of the photo inhibitory response and the amount of β-carotene hyper-accumulation has been noted. An outdoor evaluation of the growth stage of the dual-stage system has demonstrated that D. salina can be grown in a relatively low salinity, nutrient sufficient medium for extended periods without overgrowth by small non-carotenogenic Dunaliella species. In addition, biomass productivities of three times greater than those obtained in the currently employed averaging system were achieved. The role of high light intensity in β-carotene hyper-accumulation was confirmed in outdoor scale-up stress pond studies. The studies demonstrated the feasibility of stress induced ll-carotene production in outdoor cultures of D. salina and β-carotene yields three times greater than those obtained in the currently employed averaging process were achieved. The dual-stage process imposes the specific requirement of viable cell separation on the harvesting system employed. A flocculation-flotation process and an air-displacement crossflow ultrafiltration system were developed and successfully evaluated for the separation of D. salina from the brine solution in a viable form. The extraction of β-carotene from D. salina was evaluated. Supercritical fluid extraction studies showed that the use of a co-solvent mixture of carbon dioxide and propane could effectively reduce the high extraction pressures associated with supercritical carbon dioxide extraction. In addition, a novel hydrophobic membrane assisted hot oil extraction process was developed which separates the complex oil-water emulsions produced during hot oil extraction of 6-carotene from wet D. salina biomass. Process design and economic evaluation studies were undertaken and showed that the economics of the dual-stage process offer significant advantages over the currently employed averaging process.
- Full Text:
- Date Issued: 1994
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
Removal and recovery of gold and platinum from aqueous solutions utilising the non-viable biomass Asolla filiculoides
- Authors: Antunes, Ana Paula Martins
- Date: 2002
- Subjects: Azolla filiculoides Metal wastes -- Recycling Gold -- Recycling Platinum -- Recycling
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3894 , http://hdl.handle.net/10962/d1003726
- Description: Waste water from the mining industry is generally extremely complex and contains numerous species which influence the adsorption of the metals to any biomass. A variety of factors need to be addressed before treatment is considered viable. It is also beneficial to establish the binding characteristics of the metal of interest to maximise its interaction with the biomass to be utilised. Azalia filiculaides was investigated in the adsorption of gold(III), lead(II), iron(ID), copper(II) and platinum (IV). In batch studies, the optimum biomass and initial gold(III) concentrations were found to be 5 gIL and 8 mgIL respectively. The adsorption of gold(ID) is principally pH-dependent with optimal removal at pH 2. Lead(II), iron(III) and copper(II) did not compete with gold(III) adsorption under equimolar and simulated effluent conditions. Halides, with increasing affinity for gold (chloride < bromide < iodide), can affect gold uptake with the soft base, iodide, exhibiting the most inhibition (25%) and the hard base, chloride, O%. Mercaptoethanol (soft base) showed no interference in gold(III) adsorption while the presence of sulphate (hard base) and sulphite (borderline base) showed that concentrations in excess of 1 0 mM may adversely affect gold(ill) uptake, most likely due to competition for cationic sites on the biomass. Column studies, better suited to high volume treatment, indicated that a flow-rate of 5 mL/min and an initial gold(ill) concentration of 5 mgIL was optimal. Competitive effects between lead, iron, copper and gold again showed little or no interference. The halides, chloride, bromide and iodide, affect gold(ill) uptake similarly to the batch studies, while the bases mercaptoethanol and sulphate minimally affect gold(III) binding with sulphite severely hampering adsorption (70% inhibition). To optimise gold desorption, preliminary batch studies indicated that a ratio of 1:1 of adsorbentdesorbent was optimal, whilst gas purging of thiourea with oxygen, air and nitrogen decreased gold elution in proportion to decreased amounts of oxygen. A series of desorbents were utilised, in column studies, to optimise and determine the speciation of bound gold. The presence of an oxidant with thiourea enhanced desorption greater than 3 fold when compared with thiourea alone. Thiourea desorption studies, aided by the oxidant, suggest that gold is present in the + I and 0 oxidation states. Ultimately thiourea, perchloric acid and hydrochloric acid was found to be the most optimal elutant for gold (J 00% recovery). For selective metal recovery oflead and copper, pre-washing the plant material with water, utilising an acid (0.3 M nitric acid), pumping in an up-flow mode, and recycling the desorbent six times was found to be optimal elutant for gold (J 00% recovery). Cost analysis of utilising elutant versus incinerating the biomass for gold recovery indicated the latter as the most economical. Over a 5 cycle adsorption and desorption series, acid desorption before each adsorption cycle was found to result in greater than 92% desorption for lead and 96% for copper. Gold recovery was 97% with incineration. A preliminary study with gold effluent (Mine C) indicated that nickel and sulphate was removed in batch and column studies. Gold removal was found to be 100% and 4% in batch and column studies respectively. Adsorption of gold in the effluent study was accompanied by the release ofHt. Modifying the plant material with various reagents failed to identify the primary binding sites and the role of polysaccharides, proteins and lipids in gold(ill) uptake. The mode of gold binding is suggested as being initially ionic, this is very rapid, with the interaction of the anionic complex, [AuCI₄]". with the cationic biomass (PH 2). This eventually leads to the displacement of the chloride ligand(s) initiating covalent binding. Spectral studies of the chemical interaction between gold and the representative tannins indicated the protonated hydroxy groups to be responsible. All evidence suggests that the binding mechanisms of gold are not simple. Preliminary adsorption studies of platinum by Azalia filiculaides were conducted. Batch studies indicated that J gIL biomass concentration, initial platinum concentration of 20 mgIL and pH 2 are optimal, while the column studies indicated a flow-rate of! 0 rnL/min and initial platinum concentration of 20 mgIL as optimal. In the platinum effluent study, platinum showed a removal of 23 % and 2 J % for the batch and column studies respectively. Again adsorption was accompanied by //' release. Azalia filiculaides demonstrated its feasibility in the removal of gold and platinum from simulated as well as waste water solutions. Its potential viability as a biosorbent was demonstrated by the high recovery from synthetic solutions of greater than 99% for gold (2-10 mgIL), and greater than 89% for platinum (20 mgIL).
- Full Text:
- Date Issued: 2002
- Authors: Antunes, Ana Paula Martins
- Date: 2002
- Subjects: Azolla filiculoides Metal wastes -- Recycling Gold -- Recycling Platinum -- Recycling
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3894 , http://hdl.handle.net/10962/d1003726
- Description: Waste water from the mining industry is generally extremely complex and contains numerous species which influence the adsorption of the metals to any biomass. A variety of factors need to be addressed before treatment is considered viable. It is also beneficial to establish the binding characteristics of the metal of interest to maximise its interaction with the biomass to be utilised. Azalia filiculaides was investigated in the adsorption of gold(III), lead(II), iron(ID), copper(II) and platinum (IV). In batch studies, the optimum biomass and initial gold(III) concentrations were found to be 5 gIL and 8 mgIL respectively. The adsorption of gold(ID) is principally pH-dependent with optimal removal at pH 2. Lead(II), iron(III) and copper(II) did not compete with gold(III) adsorption under equimolar and simulated effluent conditions. Halides, with increasing affinity for gold (chloride < bromide < iodide), can affect gold uptake with the soft base, iodide, exhibiting the most inhibition (25%) and the hard base, chloride, O%. Mercaptoethanol (soft base) showed no interference in gold(III) adsorption while the presence of sulphate (hard base) and sulphite (borderline base) showed that concentrations in excess of 1 0 mM may adversely affect gold(ill) uptake, most likely due to competition for cationic sites on the biomass. Column studies, better suited to high volume treatment, indicated that a flow-rate of 5 mL/min and an initial gold(ill) concentration of 5 mgIL was optimal. Competitive effects between lead, iron, copper and gold again showed little or no interference. The halides, chloride, bromide and iodide, affect gold(ill) uptake similarly to the batch studies, while the bases mercaptoethanol and sulphate minimally affect gold(III) binding with sulphite severely hampering adsorption (70% inhibition). To optimise gold desorption, preliminary batch studies indicated that a ratio of 1:1 of adsorbentdesorbent was optimal, whilst gas purging of thiourea with oxygen, air and nitrogen decreased gold elution in proportion to decreased amounts of oxygen. A series of desorbents were utilised, in column studies, to optimise and determine the speciation of bound gold. The presence of an oxidant with thiourea enhanced desorption greater than 3 fold when compared with thiourea alone. Thiourea desorption studies, aided by the oxidant, suggest that gold is present in the + I and 0 oxidation states. Ultimately thiourea, perchloric acid and hydrochloric acid was found to be the most optimal elutant for gold (J 00% recovery). For selective metal recovery oflead and copper, pre-washing the plant material with water, utilising an acid (0.3 M nitric acid), pumping in an up-flow mode, and recycling the desorbent six times was found to be optimal elutant for gold (J 00% recovery). Cost analysis of utilising elutant versus incinerating the biomass for gold recovery indicated the latter as the most economical. Over a 5 cycle adsorption and desorption series, acid desorption before each adsorption cycle was found to result in greater than 92% desorption for lead and 96% for copper. Gold recovery was 97% with incineration. A preliminary study with gold effluent (Mine C) indicated that nickel and sulphate was removed in batch and column studies. Gold removal was found to be 100% and 4% in batch and column studies respectively. Adsorption of gold in the effluent study was accompanied by the release ofHt. Modifying the plant material with various reagents failed to identify the primary binding sites and the role of polysaccharides, proteins and lipids in gold(ill) uptake. The mode of gold binding is suggested as being initially ionic, this is very rapid, with the interaction of the anionic complex, [AuCI₄]". with the cationic biomass (PH 2). This eventually leads to the displacement of the chloride ligand(s) initiating covalent binding. Spectral studies of the chemical interaction between gold and the representative tannins indicated the protonated hydroxy groups to be responsible. All evidence suggests that the binding mechanisms of gold are not simple. Preliminary adsorption studies of platinum by Azalia filiculaides were conducted. Batch studies indicated that J gIL biomass concentration, initial platinum concentration of 20 mgIL and pH 2 are optimal, while the column studies indicated a flow-rate of! 0 rnL/min and initial platinum concentration of 20 mgIL as optimal. In the platinum effluent study, platinum showed a removal of 23 % and 2 J % for the batch and column studies respectively. Again adsorption was accompanied by //' release. Azalia filiculaides demonstrated its feasibility in the removal of gold and platinum from simulated as well as waste water solutions. Its potential viability as a biosorbent was demonstrated by the high recovery from synthetic solutions of greater than 99% for gold (2-10 mgIL), and greater than 89% for platinum (20 mgIL).
- Full Text:
- Date Issued: 2002
Biosorption of precious metals from synthetic and refinery wastewaters by immobilized saccharomyces cerevisiae
- Authors: Mack, Cherie-Lynn
- Date: 2008
- Subjects: Metals -- Refining Metals -- Absorption and adsorption Saccharomyces cerevisiae Factory and trade waste Water reuse Platinum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4071 , http://hdl.handle.net/10962/d1006977
- Description: The process of precious metal refining can be up to 99.99% efficient at best, and although it may seem small, the amount of valuable metal lost to waste streams is appreciable enough to warrant recovery. The method currently used to remove entrained metal ions from refinery wastewaters, chemical precipitation, is not an effective means for selective recovery of precious metals from a wastewater. Biosorption, the ability of certain types of biomass to bind and concentrate metals from even very dilute aqueous solutions, may be an effective point-source metal recovery strategy. The yeast, Saccharomyces cerevisiae, has been found capable of sorbing numerous precious and base metals, and is a cheap and abundant source of biomass. As such, it represents a possible precious metal sorbent for application to refining wastewaters. In this investigation, S. cerevisiae biomass was immobilized, using polyethyleneimine and glutaraldehyde, to produce a suitable sorbent, which was found to be capable of high platinum uptake (150 to 170 mg/g) at low pH (< 2). The sorption mechanism was elucidated and found to be a chemical reaction, which made effective desorption impossible. The sorption process was investigated in a packed bed column conformation, the results of which showed that the diameter and height of the column require further optimization in order to attain the metal uptake values achieved in the batch studies. When applied to a refinery wastewater, two key wastewater characteristics limited the success of the sorption process; the high inorganic ion content and the complex speciation of the platinum ions. The results proved the concept principle of platinum recovery by immobilized yeast biosorption and indicated that a more detailed understanding of the platinum speciation within the wastewater is required before the biosorption process can be applied. Overall, the sorption of platinum by the S. cerevisiae sorbent was demonstrated to be highly effective in principle, but the complexity of the wastewater requires that pretreatment steps be taken before the successful application of this process to an industrial wastewater.
- Full Text:
- Date Issued: 2008
- Authors: Mack, Cherie-Lynn
- Date: 2008
- Subjects: Metals -- Refining Metals -- Absorption and adsorption Saccharomyces cerevisiae Factory and trade waste Water reuse Platinum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4071 , http://hdl.handle.net/10962/d1006977
- Description: The process of precious metal refining can be up to 99.99% efficient at best, and although it may seem small, the amount of valuable metal lost to waste streams is appreciable enough to warrant recovery. The method currently used to remove entrained metal ions from refinery wastewaters, chemical precipitation, is not an effective means for selective recovery of precious metals from a wastewater. Biosorption, the ability of certain types of biomass to bind and concentrate metals from even very dilute aqueous solutions, may be an effective point-source metal recovery strategy. The yeast, Saccharomyces cerevisiae, has been found capable of sorbing numerous precious and base metals, and is a cheap and abundant source of biomass. As such, it represents a possible precious metal sorbent for application to refining wastewaters. In this investigation, S. cerevisiae biomass was immobilized, using polyethyleneimine and glutaraldehyde, to produce a suitable sorbent, which was found to be capable of high platinum uptake (150 to 170 mg/g) at low pH (< 2). The sorption mechanism was elucidated and found to be a chemical reaction, which made effective desorption impossible. The sorption process was investigated in a packed bed column conformation, the results of which showed that the diameter and height of the column require further optimization in order to attain the metal uptake values achieved in the batch studies. When applied to a refinery wastewater, two key wastewater characteristics limited the success of the sorption process; the high inorganic ion content and the complex speciation of the platinum ions. The results proved the concept principle of platinum recovery by immobilized yeast biosorption and indicated that a more detailed understanding of the platinum speciation within the wastewater is required before the biosorption process can be applied. Overall, the sorption of platinum by the S. cerevisiae sorbent was demonstrated to be highly effective in principle, but the complexity of the wastewater requires that pretreatment steps be taken before the successful application of this process to an industrial wastewater.
- Full Text:
- Date Issued: 2008
Purification and characterization of fructosyltransferase for the synthesis of short-chain fructo-oligosaccharides and investigation into thier anti-carcinogenic properties
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
An investigation into the bacterial diversity associated with South African latrunculid sponges that produce bioactive secondary metabolites
- Authors: Walmsley, Tara Aisling
- Date: 2014
- Subjects: Sponges -- South Africa -- Algoa Bay , Sponges -- Classification , Metabolites -- South Africa -- Algoa Bay , Marine metabolites -- South Africa -- Algoa Bay , PQQ (Biochemistry) , Bacterial diversity
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4109 , http://hdl.handle.net/10962/d1012943
- Description: Algoa Bay Latrunculid sponges are well known for their production of cytotoxic pyrroloiminoquinones with speculation that these secondary metabolites may have a microbial origin. This study describes a thorough investigation into the bacterial community associated with Tsitsikamma favus, Tsitsikamma scurra a newly described Latrunculia sp. and a yellow encrusting sponge associated with T. scurra. Molecular and chemical characterisation were used in conjunction with traditional taxonomy in identification of the sponge specimens. The 28S rRNA and COX1 analysis confirmed the traditional taxonomy with T. favus and T. scurra being very closely related. Chemical analysis revealed that T. favus and T. scurra shared the discorhabdins 2,4-debromo-3-dihydrodiscorhabdin C, 7,8-dehydro-3-dihydrodiscorhabdin C and 14-bromo-1-hydroxy-discorhabdin V in common with each other and Tsitsikamma pedunculata indicating that these pyrroloiminoquinones are common to Tsitsikamma sponges in general. The bacterial community associated with T. favus was explored using 16S rRNA molecular techniques including DGGE, clonal libraries of full length 16S rRNA genes, as well as 454 pyrosequencing. DGGE analysis revealed that the bacterial community associated with T. favus appeared to be highly conserved, which was confirmed by both the clone library and 454 pyrosequencing, with the Betaproteobacteria as the most dominant class. Further exploration into T. favus, as well as T. scurra, Latrunculia sp. and the yellow encrusting sponge indicated that the bacterial populations associated with each of these sponge species were conserved and species specific. OTU analysis to the species level revealed that T. favus and T. scurra shared an abundant Spirochaete species in common while the most abundant species in the Latrunculia sp. and the yellow encrusting sponge belonged to the class Betaproteobacteria. The exclusivity of the tsitsikammamines to T. favus precipitated attempts to culture the T. favus associated bacteria, with a focus on the dominant betaproteobacterium as indicated by the 16S rRNA clone library. Actinobacteria associated with the Algoa Bay sponge specimens were also cultured and the actinobacterial isolates were sent for screening against Mycobacterium aurum with two Kocuria kristinae isolates and a Streptomyces albdioflavus isolate showing good antimycobacterial activity.
- Full Text:
- Date Issued: 2014
- Authors: Walmsley, Tara Aisling
- Date: 2014
- Subjects: Sponges -- South Africa -- Algoa Bay , Sponges -- Classification , Metabolites -- South Africa -- Algoa Bay , Marine metabolites -- South Africa -- Algoa Bay , PQQ (Biochemistry) , Bacterial diversity
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4109 , http://hdl.handle.net/10962/d1012943
- Description: Algoa Bay Latrunculid sponges are well known for their production of cytotoxic pyrroloiminoquinones with speculation that these secondary metabolites may have a microbial origin. This study describes a thorough investigation into the bacterial community associated with Tsitsikamma favus, Tsitsikamma scurra a newly described Latrunculia sp. and a yellow encrusting sponge associated with T. scurra. Molecular and chemical characterisation were used in conjunction with traditional taxonomy in identification of the sponge specimens. The 28S rRNA and COX1 analysis confirmed the traditional taxonomy with T. favus and T. scurra being very closely related. Chemical analysis revealed that T. favus and T. scurra shared the discorhabdins 2,4-debromo-3-dihydrodiscorhabdin C, 7,8-dehydro-3-dihydrodiscorhabdin C and 14-bromo-1-hydroxy-discorhabdin V in common with each other and Tsitsikamma pedunculata indicating that these pyrroloiminoquinones are common to Tsitsikamma sponges in general. The bacterial community associated with T. favus was explored using 16S rRNA molecular techniques including DGGE, clonal libraries of full length 16S rRNA genes, as well as 454 pyrosequencing. DGGE analysis revealed that the bacterial community associated with T. favus appeared to be highly conserved, which was confirmed by both the clone library and 454 pyrosequencing, with the Betaproteobacteria as the most dominant class. Further exploration into T. favus, as well as T. scurra, Latrunculia sp. and the yellow encrusting sponge indicated that the bacterial populations associated with each of these sponge species were conserved and species specific. OTU analysis to the species level revealed that T. favus and T. scurra shared an abundant Spirochaete species in common while the most abundant species in the Latrunculia sp. and the yellow encrusting sponge belonged to the class Betaproteobacteria. The exclusivity of the tsitsikammamines to T. favus precipitated attempts to culture the T. favus associated bacteria, with a focus on the dominant betaproteobacterium as indicated by the 16S rRNA clone library. Actinobacteria associated with the Algoa Bay sponge specimens were also cultured and the actinobacterial isolates were sent for screening against Mycobacterium aurum with two Kocuria kristinae isolates and a Streptomyces albdioflavus isolate showing good antimycobacterial activity.
- Full Text:
- Date Issued: 2014
The role of arbuscular mycorrhizal fungi in the biotransformation of coal and application in dump rehabilitation
- Mukasa-Mugerwa, Thomas Tendo
- Authors: Mukasa-Mugerwa, Thomas Tendo
- Date: 2007
- Subjects: Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3999 , http://hdl.handle.net/10962/d1004059 , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Description: Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.
- Full Text:
- Date Issued: 2007
- Authors: Mukasa-Mugerwa, Thomas Tendo
- Date: 2007
- Subjects: Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3999 , http://hdl.handle.net/10962/d1004059 , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Description: Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.
- Full Text:
- Date Issued: 2007
Molecular chaperone expression and function in breast cancer and breast cancer stem cells
- Authors: Sterrenberg, Jason Neville
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells , Cancer cells
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4141 , http://hdl.handle.net/10962/d1016238
- Description: The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.
- Full Text:
- Date Issued: 2012
- Authors: Sterrenberg, Jason Neville
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells , Cancer cells
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4141 , http://hdl.handle.net/10962/d1016238
- Description: The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.
- Full Text:
- Date Issued: 2012
Studies on the gastric proteases in three South African snake species
- Robertson, Sirion Sholto Douglas
- Authors: Robertson, Sirion Sholto Douglas
- Date: 1987
- Subjects: Snakes -- South Africa Proteolytic enzymes Pepsin Pepsinogen
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4063 , http://hdl.handle.net/10962/d1004639
- Description: The pepsinogens and pepsins of cobra, mole snake and puff adder have been studied. The pepsinogens of all three species fall into two distinct groups, here designated PI and PII. At least the latter group, in all cases, shows substantial microheterogeneity. Physicochemical studies suggest that the cobra and puff adder PII groups are more similar to each other than either is to the mole snake PII group. Kinetic studies indicate that, in the cobra and mole snake, the PI and PII pepsins differ in their Arrhenius activation energies. Such difference is smaller, or absent, in the case of the puff adder PI and PII pepsins. These characteristics of the pepsins are assessed in the context of the differences between the oral secretions of the three species studied. The suggestion is advanced that the puff adder's strongly proteolytic venom has influenced certain properties of its gastric proteases.
- Full Text:
- Date Issued: 1987
- Authors: Robertson, Sirion Sholto Douglas
- Date: 1987
- Subjects: Snakes -- South Africa Proteolytic enzymes Pepsin Pepsinogen
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4063 , http://hdl.handle.net/10962/d1004639
- Description: The pepsinogens and pepsins of cobra, mole snake and puff adder have been studied. The pepsinogens of all three species fall into two distinct groups, here designated PI and PII. At least the latter group, in all cases, shows substantial microheterogeneity. Physicochemical studies suggest that the cobra and puff adder PII groups are more similar to each other than either is to the mole snake PII group. Kinetic studies indicate that, in the cobra and mole snake, the PI and PII pepsins differ in their Arrhenius activation energies. Such difference is smaller, or absent, in the case of the puff adder PI and PII pepsins. These characteristics of the pepsins are assessed in the context of the differences between the oral secretions of the three species studied. The suggestion is advanced that the puff adder's strongly proteolytic venom has influenced certain properties of its gastric proteases.
- Full Text:
- Date Issued: 1987
The measurement of genetic diversity in mycobacterium tuberculosis using random amplified polymorphic DNA profiling
- Authors: Richner, Sharon M
- Date: 2000
- Subjects: Tuberculosis -- History -- 20th century , Tuberculosis -- Africa, Southern , Tuberculosis -- Treatment , Tuberculosis -- Africa , Tuberculosis -- Prevention , Tuberculosis -- Pathogenesis , Mycobacterium tuberculosis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4008 , http://hdl.handle.net/10962/d1004068 , Tuberculosis -- History -- 20th century , Tuberculosis -- Africa, Southern , Tuberculosis -- Treatment , Tuberculosis -- Africa , Tuberculosis -- Prevention , Tuberculosis -- Pathogenesis , Mycobacterium tuberculosis
- Description: Mycobacterium tuberculosis has caused a resurgence in pulmonary disease in both developed and developing countries in recent times, particularly amongst people infected with the human immunodeficiency virus. The disease has assumed epidemic proportions in South Africa and in the Eastern Cape Province in particular. Of further concern is the isolation of increasing numbers of multiply drug resistant strains. Knowledge of the genetic capability of this organism is essential for the successful development of novel antibiotics and vaccines in an attempt to bring the global pandemic under control. Measurement of the genetic diversity of the organism may significantly contribute to such knowledge, and is of vital importance in monitoring epidemics and in improving treatment and control of the disease. This will entail answering a number of questions related to the degree of genetic diversity amongst strains, to the difference between urban and rural strains, and between drug resistant and drug sensitive strains, and to the geographical distribution of strains. In order to establish such baseline information, RAPD profiling of a large population of isolates from the western and central regions of the Eastern Cape Province was undertaken. A smaller number of drug resistant strains from a small area of KwaZulu-Natal were also analysed, with a view to establishing the genetic difference between strains from the two provinces. Cluster analysis, analysis of molecular variance and Geographical Information Systems technology were used to analyse the RAPD profiles generated. An unexpectedly high degree of genetic diversity was detected in strains from both provinces. While no correlation was seen between genetic diversity and either urban-rural situation or geographical location, a small degree of population structure could be correlated with drug resistance in the Eastern Cape. Furthermore, a significant degree of population structure was detected between strains from the two provinces, although this was still within the parameters for conspecific populations. Future work is necessary to further characterise strains from rural areas of both provinces, as well as from the eastern region of the Eastern Cape in an attempt to pinpoint the cause of the separation of the provincial populations.
- Full Text:
- Date Issued: 2000
- Authors: Richner, Sharon M
- Date: 2000
- Subjects: Tuberculosis -- History -- 20th century , Tuberculosis -- Africa, Southern , Tuberculosis -- Treatment , Tuberculosis -- Africa , Tuberculosis -- Prevention , Tuberculosis -- Pathogenesis , Mycobacterium tuberculosis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4008 , http://hdl.handle.net/10962/d1004068 , Tuberculosis -- History -- 20th century , Tuberculosis -- Africa, Southern , Tuberculosis -- Treatment , Tuberculosis -- Africa , Tuberculosis -- Prevention , Tuberculosis -- Pathogenesis , Mycobacterium tuberculosis
- Description: Mycobacterium tuberculosis has caused a resurgence in pulmonary disease in both developed and developing countries in recent times, particularly amongst people infected with the human immunodeficiency virus. The disease has assumed epidemic proportions in South Africa and in the Eastern Cape Province in particular. Of further concern is the isolation of increasing numbers of multiply drug resistant strains. Knowledge of the genetic capability of this organism is essential for the successful development of novel antibiotics and vaccines in an attempt to bring the global pandemic under control. Measurement of the genetic diversity of the organism may significantly contribute to such knowledge, and is of vital importance in monitoring epidemics and in improving treatment and control of the disease. This will entail answering a number of questions related to the degree of genetic diversity amongst strains, to the difference between urban and rural strains, and between drug resistant and drug sensitive strains, and to the geographical distribution of strains. In order to establish such baseline information, RAPD profiling of a large population of isolates from the western and central regions of the Eastern Cape Province was undertaken. A smaller number of drug resistant strains from a small area of KwaZulu-Natal were also analysed, with a view to establishing the genetic difference between strains from the two provinces. Cluster analysis, analysis of molecular variance and Geographical Information Systems technology were used to analyse the RAPD profiles generated. An unexpectedly high degree of genetic diversity was detected in strains from both provinces. While no correlation was seen between genetic diversity and either urban-rural situation or geographical location, a small degree of population structure could be correlated with drug resistance in the Eastern Cape. Furthermore, a significant degree of population structure was detected between strains from the two provinces, although this was still within the parameters for conspecific populations. Future work is necessary to further characterise strains from rural areas of both provinces, as well as from the eastern region of the Eastern Cape in an attempt to pinpoint the cause of the separation of the provincial populations.
- Full Text:
- Date Issued: 2000
Vitamin E supplementation and secondary metabolites interactions and effects on melanoma growth
- Authors: Ottino, Paulo
- Date: 1997
- Subjects: Vitamin E Melanoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4016 , http://hdl.handle.net/10962/d1004076
- Description: The present study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate on malignant murine melanoma (BL6) and non-malignant monkey kidney (LLCMK) cell growth in vitro. Studies revealed that supplementation of 5, 7 and lOJLg/ml vitamin E succinate significantly inhibited BL6 cell growth, while in LLCMK cells no significant increase or decrease in growth was observed. The actual mechanism by which vitamin E succinate inhibits BL6 cell growth is at present unclear. Studies have suggested a radical or oxidant involvement in a number of degenerative diseases such as cancer, and that supplementation of antioxidant vitamins such as vitamin E may function to reduce cancer cell growth by quenching free radical species and preventing lipid peroxidation. In addition to its antioxidant role in a cell, vitamin E is believed to modulate the activities of various enzymes and metabolites in the eicosanoid pathway. Hence, this study investigated the effects of vitamin E succinate supplementation on free radical and lipid peroxidation levels, as well as the activities of various enzymes and metabolites ill the eicosanoid pathway. Throughout this study, emphasis was placed on BL6 melanoma cells since the magnitude of the relationship between LLCMK growth and the levels of various enzymes and metabolites in the eicosanoid pathway varied considerably from one experiment to another and did not show the consistent trend found with the BL6 cells. A decrease in cell growth was found to be accompanied by a concomitant increase rather than a decrease in the levels of free radicals and lipid peroxidation, suggesting that the growth inhibitory effects of vitamin E succinate on BL6 cells in vitro was not due to its antioxidant properties associated with the vitamin E component, but rather due to one or more of its other potential roles within the cell. This proposal was further strengthened by findings that vitamin E succinate, a non-physiological antioxidant in its esterified form, did not undergo significant cleavage to free vitamin E in the BL6 cells. Vitamin E succinate is believed to modulate membrane bound enzyme activities through physicochemical interactions with membrane lipids and changes in membrane fluidity. Hence, this study investigated the role of vitamin E succinate in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation of l-lOjLg/ml vitamin E succinate resulted in an overall increase in phospholipase A2 activity while cyclooxygenase and adenyl ate cyclase activities were found to be significantly increased at vitamin E succinate concentrations of 7 and WjLg/ml respectively. A significant increase in" 5-LOX activity was observed a! 10jLg/mi supplementation. The suggestion that vitamin E succinate modulates membrane bound enzyme activities was further strengthened by uptake and cellular distribution studies, which showed significantly higher levels of vitamin E succinate in membrane fractions of BL6 cells when compared with stroma fractions. Another factor which could account for elevated PLA2,-5-LOX and COX activities in BL6 cells as a result of vitamin E succinate supplementation, was that of intracellular calcium levels. Supplementation of BL6 cells with 1-7 jLg/ml vitamin E succinate resulted in an overall increase in intracellular calcium levels. These changes in calcium levels however were positively correlated with changes in PLA2 activity only. Since the rate of prostaglandin synthesis is controlled by phospholipase A2 activity, and net prostagiandin production is dependant on cyclooxygenase activity, the effects of vitamin E succinate supplementation on prostaglandin levels in BL6 cells was determined. Vitamin E succinate supplementation resulted in a significant decrease in prostaglandin D2 levels at vitamin E succinate concentrations of 3, 5, 7 and lOjLg/ml respectively, while prostaglandin F2a levels were significantly decreased at 1-10jLg/ml vitamin E succinate. The increases in prostaglandin E2 and 12 levels were inversely related to BL6 cell growth suggesting that both prostaglandins may act as negative regulators of BL6 cell growth. When comparing prostaglandin E2 levels to prostaglandin 12 levels in BL6 cells, significantly higher levels of prostaglandin E2 were found, suggesting that vitamin E succinate effects were mediated primarily through an increase in prostaglandin E2 levels. Furthermore, prostaglandin E2 levels are believed to modulate adenylate cyclase activity. It is therefore reasonable to conclude that the increased adenyl ate cyclase activity found in BL6 cells was dependant on prostaglandin E2 levels, since increases in prostaglandin E2 levels at 7 and lOjLg/ml vitamin E succinate correlated with an increase in adenylate cyclase activity and cyclic adenosine monophosphate levels. Thus it appeared that the observed inhibitory effects of vitamin E succinate supplementation on BL6 cell growth was not due to the antioxidant properties associated with the vitamin E component of the vitamin E succinate molecule, but was rather mediated in part through a cascade effect initiated by phospholipase A2 activation and archidonic acid release. This initial effect then appeared to result in an increase in cyclooxygenase activity and activation of a prostaglandin E2-adenylate cyclase-cyclic adenosine monophosphate linked system, ultimately altering cyclic adenosine monophosphate levels and inhibiting BL6 cell growth. This was confirmed when BL6 cells were supplemented with indomethacin, a cyclooxygenase inhibitor. Supplementation with the inhibitor resulted in vitamin E succinate having no inhibitory effects on BL6 cell growth. Furthermore, when comparing the levels of prostaglandin ~, adenylate cyclase activity and cyclIC adenosine monophosphate in the indomethacin treated cultures to non-indomethacin treated cultures, markedly lower levels of these metabolites were found in the indomethacin treated cultures. The cause of the increase in free radical and lipid peroxidation levels in BL6 cells following vitamin E succinate supplementation was further investigated. Cyclooxygenase enzymes are believed to generate free radical species and contribute to lipid peroxidation levels during catalytic activity. Markedly lower levels of free radicals and lipid peroxidation in indomethacin treated cultures were found when compared with vitamin E succinate treated cultures alone, suggesting that the increases in free radical and lipid peroxidation levels in BL6 cells supplemented with vitamin E succinate were indirectly due to an increase in cyclooxygenase activity in these cells.
- Full Text:
- Date Issued: 1997
- Authors: Ottino, Paulo
- Date: 1997
- Subjects: Vitamin E Melanoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4016 , http://hdl.handle.net/10962/d1004076
- Description: The present study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate on malignant murine melanoma (BL6) and non-malignant monkey kidney (LLCMK) cell growth in vitro. Studies revealed that supplementation of 5, 7 and lOJLg/ml vitamin E succinate significantly inhibited BL6 cell growth, while in LLCMK cells no significant increase or decrease in growth was observed. The actual mechanism by which vitamin E succinate inhibits BL6 cell growth is at present unclear. Studies have suggested a radical or oxidant involvement in a number of degenerative diseases such as cancer, and that supplementation of antioxidant vitamins such as vitamin E may function to reduce cancer cell growth by quenching free radical species and preventing lipid peroxidation. In addition to its antioxidant role in a cell, vitamin E is believed to modulate the activities of various enzymes and metabolites in the eicosanoid pathway. Hence, this study investigated the effects of vitamin E succinate supplementation on free radical and lipid peroxidation levels, as well as the activities of various enzymes and metabolites ill the eicosanoid pathway. Throughout this study, emphasis was placed on BL6 melanoma cells since the magnitude of the relationship between LLCMK growth and the levels of various enzymes and metabolites in the eicosanoid pathway varied considerably from one experiment to another and did not show the consistent trend found with the BL6 cells. A decrease in cell growth was found to be accompanied by a concomitant increase rather than a decrease in the levels of free radicals and lipid peroxidation, suggesting that the growth inhibitory effects of vitamin E succinate on BL6 cells in vitro was not due to its antioxidant properties associated with the vitamin E component, but rather due to one or more of its other potential roles within the cell. This proposal was further strengthened by findings that vitamin E succinate, a non-physiological antioxidant in its esterified form, did not undergo significant cleavage to free vitamin E in the BL6 cells. Vitamin E succinate is believed to modulate membrane bound enzyme activities through physicochemical interactions with membrane lipids and changes in membrane fluidity. Hence, this study investigated the role of vitamin E succinate in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation of l-lOjLg/ml vitamin E succinate resulted in an overall increase in phospholipase A2 activity while cyclooxygenase and adenyl ate cyclase activities were found to be significantly increased at vitamin E succinate concentrations of 7 and WjLg/ml respectively. A significant increase in" 5-LOX activity was observed a! 10jLg/mi supplementation. The suggestion that vitamin E succinate modulates membrane bound enzyme activities was further strengthened by uptake and cellular distribution studies, which showed significantly higher levels of vitamin E succinate in membrane fractions of BL6 cells when compared with stroma fractions. Another factor which could account for elevated PLA2,-5-LOX and COX activities in BL6 cells as a result of vitamin E succinate supplementation, was that of intracellular calcium levels. Supplementation of BL6 cells with 1-7 jLg/ml vitamin E succinate resulted in an overall increase in intracellular calcium levels. These changes in calcium levels however were positively correlated with changes in PLA2 activity only. Since the rate of prostaglandin synthesis is controlled by phospholipase A2 activity, and net prostagiandin production is dependant on cyclooxygenase activity, the effects of vitamin E succinate supplementation on prostaglandin levels in BL6 cells was determined. Vitamin E succinate supplementation resulted in a significant decrease in prostaglandin D2 levels at vitamin E succinate concentrations of 3, 5, 7 and lOjLg/ml respectively, while prostaglandin F2a levels were significantly decreased at 1-10jLg/ml vitamin E succinate. The increases in prostaglandin E2 and 12 levels were inversely related to BL6 cell growth suggesting that both prostaglandins may act as negative regulators of BL6 cell growth. When comparing prostaglandin E2 levels to prostaglandin 12 levels in BL6 cells, significantly higher levels of prostaglandin E2 were found, suggesting that vitamin E succinate effects were mediated primarily through an increase in prostaglandin E2 levels. Furthermore, prostaglandin E2 levels are believed to modulate adenylate cyclase activity. It is therefore reasonable to conclude that the increased adenyl ate cyclase activity found in BL6 cells was dependant on prostaglandin E2 levels, since increases in prostaglandin E2 levels at 7 and lOjLg/ml vitamin E succinate correlated with an increase in adenylate cyclase activity and cyclic adenosine monophosphate levels. Thus it appeared that the observed inhibitory effects of vitamin E succinate supplementation on BL6 cell growth was not due to the antioxidant properties associated with the vitamin E component of the vitamin E succinate molecule, but was rather mediated in part through a cascade effect initiated by phospholipase A2 activation and archidonic acid release. This initial effect then appeared to result in an increase in cyclooxygenase activity and activation of a prostaglandin E2-adenylate cyclase-cyclic adenosine monophosphate linked system, ultimately altering cyclic adenosine monophosphate levels and inhibiting BL6 cell growth. This was confirmed when BL6 cells were supplemented with indomethacin, a cyclooxygenase inhibitor. Supplementation with the inhibitor resulted in vitamin E succinate having no inhibitory effects on BL6 cell growth. Furthermore, when comparing the levels of prostaglandin ~, adenylate cyclase activity and cyclIC adenosine monophosphate in the indomethacin treated cultures to non-indomethacin treated cultures, markedly lower levels of these metabolites were found in the indomethacin treated cultures. The cause of the increase in free radical and lipid peroxidation levels in BL6 cells following vitamin E succinate supplementation was further investigated. Cyclooxygenase enzymes are believed to generate free radical species and contribute to lipid peroxidation levels during catalytic activity. Markedly lower levels of free radicals and lipid peroxidation in indomethacin treated cultures were found when compared with vitamin E succinate treated cultures alone, suggesting that the increases in free radical and lipid peroxidation levels in BL6 cells supplemented with vitamin E succinate were indirectly due to an increase in cyclooxygenase activity in these cells.
- Full Text:
- Date Issued: 1997
The biology and molecular ecology of floating sulphur biofilms
- Authors: Bowker, Michelle Louise
- Date: 2002
- Subjects: Biofilms , Microbial ecology , Sulfur
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4056 , http://hdl.handle.net/10962/d1004117 , Biofilms , Microbial ecology , Sulfur
- Description: Floating sulphur biofilms have been observed to occur on sulphate-containing natural systems and waste stabilization ponds. It has been postulated that these biofilms form on the surface of the water because sulphate reducing bacteria present in the bottom layers of the water body reduce sulphate to sulphide which then diffuses upwards and is oxidized under the correct redox conditions to sulphur by sulphide oxidizing bacteria. Very little information exists on these complex floating systems and in order to study them further, model systems were designed. The Baffle Reactor was successfully used to cultivate floating sulphur biofilms. Conditions within the reactor could be closely scrutinized in the laboratory and it was found that sulphate levels decreased, sulphide levels increased and that sulphur was produced over a period of 2 weeks. The success of this system led to it being scaled-up and currently a method to harvest sulphur from the biofilm is under development. It is thought that biofilms are highly complex, heterogeneous structures with different bacteria distributed in different layers. Preliminary work suggested that bacteria were differentially distributed along nutrient and oxygen gradients within the biofilm. Biofilms are very thin structures and therefore difficult to study and Gradient systems were developed in an attempt to spatially separate the biofilm species into functional layers. Gradient Tubes were designed; these provided a gradient of high-sulphide, low oxygen conditions to high-oxygen, low-sulphide conditions. Bacteria were observed to grow in different layers of these systems. The Gradient Tubes could be sectioned and the chemical characteristics of each section as well as the species present could be determined. Silicon Tubular Bioreactors were also developed and these were very efficient at producing large amounts of sulphur under strictly controlled redox conditions. Microscopy and molecular methods including the amplification of a section of Ribosomal Ribonucleic acid by Polymerase Chain Reaction were used in an attempt to characterize the populations present in these biofilm systems. Denaturing Gradient Gel Electrophoresis was used to create band profiles of the populations; individual bands were excised from the gels and sequenced. Identified species included Ectothiorhodospira sp., Dethiosulfovibrio russensis, Pseudomonas geniculata, Thiobacillus baregensis and Halothiobacillus kellyi.
- Full Text:
- Date Issued: 2002
- Authors: Bowker, Michelle Louise
- Date: 2002
- Subjects: Biofilms , Microbial ecology , Sulfur
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4056 , http://hdl.handle.net/10962/d1004117 , Biofilms , Microbial ecology , Sulfur
- Description: Floating sulphur biofilms have been observed to occur on sulphate-containing natural systems and waste stabilization ponds. It has been postulated that these biofilms form on the surface of the water because sulphate reducing bacteria present in the bottom layers of the water body reduce sulphate to sulphide which then diffuses upwards and is oxidized under the correct redox conditions to sulphur by sulphide oxidizing bacteria. Very little information exists on these complex floating systems and in order to study them further, model systems were designed. The Baffle Reactor was successfully used to cultivate floating sulphur biofilms. Conditions within the reactor could be closely scrutinized in the laboratory and it was found that sulphate levels decreased, sulphide levels increased and that sulphur was produced over a period of 2 weeks. The success of this system led to it being scaled-up and currently a method to harvest sulphur from the biofilm is under development. It is thought that biofilms are highly complex, heterogeneous structures with different bacteria distributed in different layers. Preliminary work suggested that bacteria were differentially distributed along nutrient and oxygen gradients within the biofilm. Biofilms are very thin structures and therefore difficult to study and Gradient systems were developed in an attempt to spatially separate the biofilm species into functional layers. Gradient Tubes were designed; these provided a gradient of high-sulphide, low oxygen conditions to high-oxygen, low-sulphide conditions. Bacteria were observed to grow in different layers of these systems. The Gradient Tubes could be sectioned and the chemical characteristics of each section as well as the species present could be determined. Silicon Tubular Bioreactors were also developed and these were very efficient at producing large amounts of sulphur under strictly controlled redox conditions. Microscopy and molecular methods including the amplification of a section of Ribosomal Ribonucleic acid by Polymerase Chain Reaction were used in an attempt to characterize the populations present in these biofilm systems. Denaturing Gradient Gel Electrophoresis was used to create band profiles of the populations; individual bands were excised from the gels and sequenced. Identified species included Ectothiorhodospira sp., Dethiosulfovibrio russensis, Pseudomonas geniculata, Thiobacillus baregensis and Halothiobacillus kellyi.
- Full Text:
- Date Issued: 2002
Phenolic compounds in water and the implications for rapid detection of indicator micro-organisms using ß-D-Galactosidase and ß-D-Glucuronidase
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
The involvement of TRAP1 in the mitochondrial localization of STAT3 in mammalian cells
- Authors: Kadye, Rose
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55760 , vital:26731
- Description: STAT3 (signal transducer and activator of transcription 3), an oncogene and transcription factor of genes involved in cellular differentiation, proliferation and immune function, that classically localizes in the cytosol and nucleus has also been found in the mitochondria. However, STAT3 does not have a mitochondrial transit peptide, and its mechanism for mitochondrial localization is unknown. Cytosolic Hsp90s chaperone STAT3 to the nucleus therefore we investigated the involvement of the nuclear-encoded mitochondrial Hsp90 molecular chaperone tumor necrosis receptor associated protein 1 (TRAP1) in STAT3’s mitochondrial localization. Using TRAP1 transient over-expression, STAT3 inhibitor S3I- 201 and Hsp90 inhibitor geldanamycin, we demonstrate that TRAP1 and STAT3 co-localize and co-immunoprecipitates in mammalian systems. Taken together with the observation that STAT3 potentially directly interacts with TRAP1, these data suggest that TRAP1 plays a role in the mitochondrial localization of STAT3.
- Full Text:
- Date Issued: 2014
- Authors: Kadye, Rose
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55760 , vital:26731
- Description: STAT3 (signal transducer and activator of transcription 3), an oncogene and transcription factor of genes involved in cellular differentiation, proliferation and immune function, that classically localizes in the cytosol and nucleus has also been found in the mitochondria. However, STAT3 does not have a mitochondrial transit peptide, and its mechanism for mitochondrial localization is unknown. Cytosolic Hsp90s chaperone STAT3 to the nucleus therefore we investigated the involvement of the nuclear-encoded mitochondrial Hsp90 molecular chaperone tumor necrosis receptor associated protein 1 (TRAP1) in STAT3’s mitochondrial localization. Using TRAP1 transient over-expression, STAT3 inhibitor S3I- 201 and Hsp90 inhibitor geldanamycin, we demonstrate that TRAP1 and STAT3 co-localize and co-immunoprecipitates in mammalian systems. Taken together with the observation that STAT3 potentially directly interacts with TRAP1, these data suggest that TRAP1 plays a role in the mitochondrial localization of STAT3.
- Full Text:
- Date Issued: 2014
Enzymology of activated sewage sludge during anaerobic treatment of wastewaters : identification, characterisation, isolation and partial purification of proteases
- Tshivhunge, Azwiedziswi Sylvia
- Authors: Tshivhunge, Azwiedziswi Sylvia
- Date: 2001
- Subjects: Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4012 , http://hdl.handle.net/10962/d1004072 , Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Description: During anaerobic digestion bacteria inside the digester require a carbon source for their growth and metabolism, sewage sludge was used as a carbon source in this study. The COD content was used to measure the disappearance of the substrate. COD content was reduced by 48.3% and 49% in the methanogenic and sulphidogenic bioreactors, respectively, while sulphate concentration was reduced by 40%, producing 70mg/L of hydrogen sulphide as the end product over the first 5-7 days. Sulphate (which is used as a terminal electron acceptor of sulphur reducing bacteria) has little or no effect on the sulphidogenic and methanogenic proteases. Sulphite and sulphide (the intermediate and end product of sulphate reduction) increased protease activity by 20% and 40%-80%, respectively. Maximum protease activity occurred on day 21 in the methanogenic reactor and on day 9 in the sulphidogenic reactor. The absorbance, which indicates the level of amino acid increased to 2 and 9 for methanogenic and sulphidogenic bioreactors, respectively. Proteases that were active during anaerobic digestion were associated with the pellet (organic particulate matter) of the sewage. These enzymes have an optimum activity at pH 10 and at temperature of 50°C. The proteases that were active at pH 5 and 7, had optimum temperatures at 30°C and 60°C, respectively. Due to their association with organic particulate matter, these enzymes were stable at their optimum temperatures for at least five hours at their respective pH. Inhibition by PMSF, TPCK and 1.10-phenanthroline suggested that proteases inside the anaerobic digester are a mixture of cysteine, serine and metalloproteases. At pH 5, however, EDTA appeared to enhance protease activity by 368% (three-fold). Acetic acid decreased protease activity by 21%, while both propionic and butyric acid at 200 mg/L cause total inhibition of protease activity while these acids at higher pH (where they exist as their corresponding salts) exerted little effect. Copper, iron and zinc inhibited protease activity by 85% at pH 5 with concentrations ranging between 200 and 600 mg/L. On the other hand, nickel, showed an increase in protease activity of nearly 250%. At pH 7 and 10, copper had no effect on protease activity while iron, nickel and zinc inhibited these enzymes by 20-40%. Proteases at pH 7 were extracted from the pellet by sonication, releasing 50% of the total enzymes into the solution. The enzymes were precipitated by ammonium sulphate precipitation, and further purified by ion exchange chromatography and gel filtration. Ion exchange chromatography revealed that most of the enzymes that hydrolyse proteins are negatively charged while gel filtration showed that their molecular weight is approximately 500 kDa.
- Full Text:
- Date Issued: 2001
- Authors: Tshivhunge, Azwiedziswi Sylvia
- Date: 2001
- Subjects: Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4012 , http://hdl.handle.net/10962/d1004072 , Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Description: During anaerobic digestion bacteria inside the digester require a carbon source for their growth and metabolism, sewage sludge was used as a carbon source in this study. The COD content was used to measure the disappearance of the substrate. COD content was reduced by 48.3% and 49% in the methanogenic and sulphidogenic bioreactors, respectively, while sulphate concentration was reduced by 40%, producing 70mg/L of hydrogen sulphide as the end product over the first 5-7 days. Sulphate (which is used as a terminal electron acceptor of sulphur reducing bacteria) has little or no effect on the sulphidogenic and methanogenic proteases. Sulphite and sulphide (the intermediate and end product of sulphate reduction) increased protease activity by 20% and 40%-80%, respectively. Maximum protease activity occurred on day 21 in the methanogenic reactor and on day 9 in the sulphidogenic reactor. The absorbance, which indicates the level of amino acid increased to 2 and 9 for methanogenic and sulphidogenic bioreactors, respectively. Proteases that were active during anaerobic digestion were associated with the pellet (organic particulate matter) of the sewage. These enzymes have an optimum activity at pH 10 and at temperature of 50°C. The proteases that were active at pH 5 and 7, had optimum temperatures at 30°C and 60°C, respectively. Due to their association with organic particulate matter, these enzymes were stable at their optimum temperatures for at least five hours at their respective pH. Inhibition by PMSF, TPCK and 1.10-phenanthroline suggested that proteases inside the anaerobic digester are a mixture of cysteine, serine and metalloproteases. At pH 5, however, EDTA appeared to enhance protease activity by 368% (three-fold). Acetic acid decreased protease activity by 21%, while both propionic and butyric acid at 200 mg/L cause total inhibition of protease activity while these acids at higher pH (where they exist as their corresponding salts) exerted little effect. Copper, iron and zinc inhibited protease activity by 85% at pH 5 with concentrations ranging between 200 and 600 mg/L. On the other hand, nickel, showed an increase in protease activity of nearly 250%. At pH 7 and 10, copper had no effect on protease activity while iron, nickel and zinc inhibited these enzymes by 20-40%. Proteases at pH 7 were extracted from the pellet by sonication, releasing 50% of the total enzymes into the solution. The enzymes were precipitated by ammonium sulphate precipitation, and further purified by ion exchange chromatography and gel filtration. Ion exchange chromatography revealed that most of the enzymes that hydrolyse proteins are negatively charged while gel filtration showed that their molecular weight is approximately 500 kDa.
- Full Text:
- Date Issued: 2001
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 development and evaluation of Cryptophlebia Leucotreta granulovirus (CrleGV) as a biological control agent for the management of false codling moth, Cryptophlebia Leucotreta, on citrus
- Authors: Moore, Sean Douglas
- Date: 2003
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- Control Pests -- Biological control Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3942 , http://hdl.handle.net/10962/d1004001
- Description: A granulovirus isolated from Cryptophlebia leucotreta larvae was shown through restriction endonuclease analysis to be a novel strain (CrleGV-SA). No more than one isolate could be identified from a laboratory culture of C. leucotreta. However, a preliminary examination of restricted DNA profiles of isolates from different geographical regions indicated some minor differences. In surface dose bioassays on artificial diet, LC50 and LC90 values with neonate larvae were estimated to be 4.095 x 103 OBs/ml and 1.185 x 105 OBs/ml respectively. LT50 and LT90 values with neonate larvae were estimated to be 4 days 22 h and 7 days 8 h, respectively. Detached fruit (navel orange) bioassays with neonate larvae indicated that virus concentrations that are likely to be effective in the field range from 1.08 x 107 to 3.819 x 1010 OBs/ml. In surface dose bioassays with fifth instar larvae LC50 and LC90 values were estimated to be 2.678 x 107 OBs/ml and 9.118 x 109 OBs/ml respectively. LT50 and LT90 values were estimated to be 7 days 17 h and 9 days 8 h, respectively. A new artificial diet for mass rearing the host was developed. Microbial contamination of diet was significantly reduced by adding nipagin and sorbic acid to the diet and by surface sterilising C. leucotreta eggs with Sporekill. Almost 20 % more eggs were produced from moths reared on the new diet compared to moths reared on the old diet. A further 9 % improvement in egg production and a reduction in the labour required to produce eggs, was made with the development of a new oviposition cage attached to the moth eclosion box. Virus was mass produced in fifth instar C. leucotreta larvae by surface inoculating diet with the LC90. When 300 individuals were placed onto inoculated diet, 56 % of them were recovered six to 11 days later as infected larvae. Mean larval equivalents was 1.158 x 1011 OBs/larva. When larvae and diet were harvested together, highest yields of virus were achieved at eight days after inoculation. Microbial contamination in semi-purified preparations of CrleGV ranged from 176211 to 433594 (OB:CFU ratio). Half-life of CrleGV in the field was estimated to be less than 1 day on the northern aspect of trees and between 3 - 6 days on the southern aspect. Original activity remaining (OAR) of the virus dropped below 50 % after 5 days on the northern aspect of trees and was still at 69 % on the southern aspect of trees after 3 weeks. In field trials, CrleGV reduced C. leucotreta infestation of navel oranges by up to 60 % for a period of 39 days. CrleGV in combination with augmentation of the C. leucotreta egg parasitoid, Trichogrammatoidea cryptophlebiae, reduced infestation by 70 %. The integration of CrleGV into an integrated pest management (IPM) system for the management of C. leucotreta on citrus is proposed.
- Full Text:
- Date Issued: 2003
- Authors: Moore, Sean Douglas
- Date: 2003
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- Control Pests -- Biological control Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3942 , http://hdl.handle.net/10962/d1004001
- Description: A granulovirus isolated from Cryptophlebia leucotreta larvae was shown through restriction endonuclease analysis to be a novel strain (CrleGV-SA). No more than one isolate could be identified from a laboratory culture of C. leucotreta. However, a preliminary examination of restricted DNA profiles of isolates from different geographical regions indicated some minor differences. In surface dose bioassays on artificial diet, LC50 and LC90 values with neonate larvae were estimated to be 4.095 x 103 OBs/ml and 1.185 x 105 OBs/ml respectively. LT50 and LT90 values with neonate larvae were estimated to be 4 days 22 h and 7 days 8 h, respectively. Detached fruit (navel orange) bioassays with neonate larvae indicated that virus concentrations that are likely to be effective in the field range from 1.08 x 107 to 3.819 x 1010 OBs/ml. In surface dose bioassays with fifth instar larvae LC50 and LC90 values were estimated to be 2.678 x 107 OBs/ml and 9.118 x 109 OBs/ml respectively. LT50 and LT90 values were estimated to be 7 days 17 h and 9 days 8 h, respectively. A new artificial diet for mass rearing the host was developed. Microbial contamination of diet was significantly reduced by adding nipagin and sorbic acid to the diet and by surface sterilising C. leucotreta eggs with Sporekill. Almost 20 % more eggs were produced from moths reared on the new diet compared to moths reared on the old diet. A further 9 % improvement in egg production and a reduction in the labour required to produce eggs, was made with the development of a new oviposition cage attached to the moth eclosion box. Virus was mass produced in fifth instar C. leucotreta larvae by surface inoculating diet with the LC90. When 300 individuals were placed onto inoculated diet, 56 % of them were recovered six to 11 days later as infected larvae. Mean larval equivalents was 1.158 x 1011 OBs/larva. When larvae and diet were harvested together, highest yields of virus were achieved at eight days after inoculation. Microbial contamination in semi-purified preparations of CrleGV ranged from 176211 to 433594 (OB:CFU ratio). Half-life of CrleGV in the field was estimated to be less than 1 day on the northern aspect of trees and between 3 - 6 days on the southern aspect. Original activity remaining (OAR) of the virus dropped below 50 % after 5 days on the northern aspect of trees and was still at 69 % on the southern aspect of trees after 3 weeks. In field trials, CrleGV reduced C. leucotreta infestation of navel oranges by up to 60 % for a period of 39 days. CrleGV in combination with augmentation of the C. leucotreta egg parasitoid, Trichogrammatoidea cryptophlebiae, reduced infestation by 70 %. The integration of CrleGV into an integrated pest management (IPM) system for the management of C. leucotreta on citrus is proposed.
- Full Text:
- Date Issued: 2003