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
The role of pacC in Aspergillus flavus
- Authors: Suleman, Essa
- Date: 2007
- Subjects: Fungi -- Biotechnology , Pathogenic microorganisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10332 , http://hdl.handle.net/10948/612 , Fungi -- Biotechnology , Pathogenic microorganisms
- Description: Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.
- Full Text:
- Date Issued: 2007
- Authors: Suleman, Essa
- Date: 2007
- Subjects: Fungi -- Biotechnology , Pathogenic microorganisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10332 , http://hdl.handle.net/10948/612 , Fungi -- Biotechnology , Pathogenic microorganisms
- Description: Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.
- Full Text:
- Date Issued: 2007
The development of an immobilised-enzyme bioprobe for the detection of phenolic pollutants in water
- Authors: Russell, Ingrid Margaret
- Date: 1999
- Subjects: Pollutants -- Biodegradation , Pollutants , Chemical reactors , Membrane reactors , Fungi -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4069 , http://hdl.handle.net/10962/d1006211 , Pollutants -- Biodegradation , Pollutants , Chemical reactors , Membrane reactors , Fungi -- Biotechnology
- Description: The possibility of developing an immobilised-enzyme bioprobe, based on mushroom polyphenol oxidase, for the purely biological detection and quantification of phenolic pollutants in water was investigated. Polyphenol oxidase catalyses the bioconversion of many phenolic compounds into quinone-related coloured products. Thus, in an immobilised form, the enzyme serves as a visible indicator of the presence and concentration of phenolic pollutants in water. The objective of this research was to develop a portable, disposable bioprobe incorporating polyphenol oxidase for this purpose. The intensity of the colour changes produced by the enzyme on reaction with p-cresol, p-chlorophenol and phenol was found to increase proportionally with increasing concentrations of these substrates in solution. Immobilisation of the enzyme on various supports did not appear to significantly affect the catalytic activity of the enzyme. The enzyme was immobilised by adsorption and cross-linking on polyethersulphone, nitrocellulose and nylon membranes with the production of various colour ranges on reaction with the phenolic substrates. The most successful immobilisation of the enzyme, in terms of quantity and distribution of enzyme immobilised and colour production, was obtained with the enzyme immobilised by adsorption on nylon membranes in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme, immobilised using this method, produced ranges of maroon colours in phenolic solutions and orange colours in cresylic solutions. The colour intensities produced were found to increase proportionally with increasing substrate concentration after 5 minutes exposure to the substrates. The bioprobe had a broad substrate specificity and was sensitive to substrate concentrations down to 0.05 mg/L. The enzyme activity of the bioprobe was not significantly affected in a pH range from 4 to 10 and in a temperature range from 5-25⁰C. The bioprobe activity was not affected by various concentrations of salt and metal ions and the bioprobe was able to detect and semi-quantify phenolic substrates in industrial effluent samples. These features of the bioprobe indicate that the commercialisation of such a bioprobe is feasible and this technology has been patented (Patent No. SA 97/0227). , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 1999
- Authors: Russell, Ingrid Margaret
- Date: 1999
- Subjects: Pollutants -- Biodegradation , Pollutants , Chemical reactors , Membrane reactors , Fungi -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4069 , http://hdl.handle.net/10962/d1006211 , Pollutants -- Biodegradation , Pollutants , Chemical reactors , Membrane reactors , Fungi -- Biotechnology
- Description: The possibility of developing an immobilised-enzyme bioprobe, based on mushroom polyphenol oxidase, for the purely biological detection and quantification of phenolic pollutants in water was investigated. Polyphenol oxidase catalyses the bioconversion of many phenolic compounds into quinone-related coloured products. Thus, in an immobilised form, the enzyme serves as a visible indicator of the presence and concentration of phenolic pollutants in water. The objective of this research was to develop a portable, disposable bioprobe incorporating polyphenol oxidase for this purpose. The intensity of the colour changes produced by the enzyme on reaction with p-cresol, p-chlorophenol and phenol was found to increase proportionally with increasing concentrations of these substrates in solution. Immobilisation of the enzyme on various supports did not appear to significantly affect the catalytic activity of the enzyme. The enzyme was immobilised by adsorption and cross-linking on polyethersulphone, nitrocellulose and nylon membranes with the production of various colour ranges on reaction with the phenolic substrates. The most successful immobilisation of the enzyme, in terms of quantity and distribution of enzyme immobilised and colour production, was obtained with the enzyme immobilised by adsorption on nylon membranes in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme, immobilised using this method, produced ranges of maroon colours in phenolic solutions and orange colours in cresylic solutions. The colour intensities produced were found to increase proportionally with increasing substrate concentration after 5 minutes exposure to the substrates. The bioprobe had a broad substrate specificity and was sensitive to substrate concentrations down to 0.05 mg/L. The enzyme activity of the bioprobe was not significantly affected in a pH range from 4 to 10 and in a temperature range from 5-25⁰C. The bioprobe activity was not affected by various concentrations of salt and metal ions and the bioprobe was able to detect and semi-quantify phenolic substrates in industrial effluent samples. These features of the bioprobe indicate that the commercialisation of such a bioprobe is feasible and this technology has been patented (Patent No. SA 97/0227). , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 1999
Fungal and substrate-associated factors affecting lignocellulolytic mushroom cultivation on wood sources available in South African [i.e. Africa]
- Authors: Da Serra, Maria Fatima
- Date: 1997
- Subjects: Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4020 , http://hdl.handle.net/10962/d1004080 , Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Description: Vast- quantities of lignocellulosic materials, representing potential substrates for the cultivation of speciality mushrooms, are produced annually in South Africa. A number of these materials are derived as waste products of the timber and agricultural industries, e.g. Maranti (Shorea spp.) and Port Jackson Willow (Acacia longifolia) respectively. The screening of various wood-degrading fungi, which are cultivated worldwide for their production of speciality mushrooms, indicated that under the environmental conditions considered, certain species were adapted to cultivation on these lignocellulosic wastes (Pleurotus species) whereas others were not (Lentinus edodes and Flammulina velutipes). Furthermore, intra- and interspecies specific differences in the growth and production potential of the various lignocellulolytic fungi investigated on synthetic and natural medium were discovered. Biochemical and genetical investigations of these strains indicated differences between and within species which were often significant. Species varied qualitatively and quantitatively in the lignocellulolytic enzymes produced, which was loosely correlated with productivity on the different media investigated. Genetical studies, using RAPD fingerprinting, indicated that the Pleurotus genus is highly variable which supports the observed differences in growth, yield and enzymatic activity between different strains and species.
- Full Text:
- Date Issued: 1997
- Authors: Da Serra, Maria Fatima
- Date: 1997
- Subjects: Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4020 , http://hdl.handle.net/10962/d1004080 , Lignocellulose , Mushroom culture , Cultivated mushroom , Fungi -- Cultures and culture media , Fungi -- Biotechnology , Mushroom culture -- South Africa
- Description: Vast- quantities of lignocellulosic materials, representing potential substrates for the cultivation of speciality mushrooms, are produced annually in South Africa. A number of these materials are derived as waste products of the timber and agricultural industries, e.g. Maranti (Shorea spp.) and Port Jackson Willow (Acacia longifolia) respectively. The screening of various wood-degrading fungi, which are cultivated worldwide for their production of speciality mushrooms, indicated that under the environmental conditions considered, certain species were adapted to cultivation on these lignocellulosic wastes (Pleurotus species) whereas others were not (Lentinus edodes and Flammulina velutipes). Furthermore, intra- and interspecies specific differences in the growth and production potential of the various lignocellulolytic fungi investigated on synthetic and natural medium were discovered. Biochemical and genetical investigations of these strains indicated differences between and within species which were often significant. Species varied qualitatively and quantitatively in the lignocellulolytic enzymes produced, which was loosely correlated with productivity on the different media investigated. Genetical studies, using RAPD fingerprinting, indicated that the Pleurotus genus is highly variable which supports the observed differences in growth, yield and enzymatic activity between different strains and species.
- Full Text:
- Date Issued: 1997
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