Evaluation of Fungcoal as a bioprocess technology for self-cladding of waste coal dumps
- Authors: Sekhohola, Lerato M
- Date: 2016
- Subjects: Coal mine waste , Fungi -- Biotechnology , Coal -- Biodegradation , Bermuda grass -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5959 , http://hdl.handle.net/10962/d1019992
- Description: Low-grade coal, a poor source of energy, has long been regarded as waste material by the coal mining industry. Biological degradation of this coal material by ligninolytic fungal strains presents a viable strategy towards eliminating this unusable fossil fuel. To this end, a novel and patented bioprocess termed Fungcoal was developed. Fungcoal is a biological process utilised in the in situ treatment of waste coal and is based on the mutualistic relationship between the fungus Neosartorya fischeri and the graminaceous species Cynodon dactylon. The process facilitates the rapid conversion of waste coal into soil-like material that stimulates establishment of vegetation for eventual coal dump rehabilitation. While a number of in vitro studies have identified various fungal strains as efficient coal degraders, the mechanisms involved in the Fungcoal-stimulated degradation process have not been fully elucidated. Furthermore, implementation of Fungcoal at both pilot and commercial scale has not been achieved. Thus the objective of this work was to investigate Fungcoal as a bioprocess via examining the role of coal degrading fungi (CDF) and grasses as biocatalysts in coal biodegradation and for the self-cladding of waste coal dumps. Initially, waste coal degradation by N. fischeri, strain ECCN 84, was investigated, specifically focusing on the mechanisms underpinning the process. In vitro studies showed the addition of waste coal induced active fungal colonisation resulting in increased fungal biomass. Increased extracellular laccase (LAC) activity, occuring concomitantly with an increase in hyphal peroxisome proliferation, was also observed in the coal supplied fungal cultures. Analysis of the colonised waste coal revealed a time dependent reduction in the percentage weight of elemental carbon coupled with an increase in elemental oxygen. The results supported metabolism and degradation of waste coal by N. fischeri strain ECCN 84 and involvement of fungal extracellular laccase. The contribution of C. dactylon, a C4 grass species to in situ biodegradation of waste coal in the presence of coal degrading and mycorrhizal fungi (MF) was also investigated. Enhanced degradation of the waste coal into a humic soil-like material was observed within the rhizosphere. Analysis of the resultant substrate revealed an increased concentration of highly oxidised humic-like substances (HS). Fungi remained viable in the rhizosphere up to 47 weeks post-inoculation and cultivation of C. dactylon, indicating the resultant humic substance-rich rhizosphere provided an environment conducive for microbial proliferation and activity. Furthermore, humic substance enrichment of waste coal substrates supported germination and seedling emergence of several agronomic species including Zea mays (corn), Phaseolus vulgaris (bean), Pisum sativum (pea), and Spinacia oleracea (spinach). Use of various cladding materials to support coal biodegradation, by fungus-grass mutualism and rehabilitation of waste dumps was evaluated at commercial scale. While substantial physico-chemical changes were not evident in the absence of cladding or where waste coal was used as cladding material, successful establishment of grass cover and diversity was achieved within three hydrological cycles on dumps cladded with weathered coal. Work presented in this thesis successfully demonstrates the degradation of waste coal by N. fischeri. The biodegradation process included enhanced extracellular LAC activity coupled with increased 3 waste coal oxidation. Increased HS concentration of waste coal substrate supported germination and early seedling establishment of several agronomic species. At commercial scale a co-substrate in the form of carbon-rich weathered coal was essential to support fungus-grass mutualism and Fungcoal-induced rehabilitation. These findings support the developed Fungcoal concept and the underpinning rationale that the phyto-biodegradation of waste coal indeed depends on the mutualistic interactions between grass root exudates and the ligninolytic and mycorrhizal fungi. Taken together, these findings provide practical evidence of the contribution of fungi and grasses as mutualists in the biodegradation of waste coal and sustainable rehabilitation of waste coal dumps
- Full Text:
- Date Issued: 2016
- Authors: Sekhohola, Lerato M
- Date: 2016
- Subjects: Coal mine waste , Fungi -- Biotechnology , Coal -- Biodegradation , Bermuda grass -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5959 , http://hdl.handle.net/10962/d1019992
- Description: Low-grade coal, a poor source of energy, has long been regarded as waste material by the coal mining industry. Biological degradation of this coal material by ligninolytic fungal strains presents a viable strategy towards eliminating this unusable fossil fuel. To this end, a novel and patented bioprocess termed Fungcoal was developed. Fungcoal is a biological process utilised in the in situ treatment of waste coal and is based on the mutualistic relationship between the fungus Neosartorya fischeri and the graminaceous species Cynodon dactylon. The process facilitates the rapid conversion of waste coal into soil-like material that stimulates establishment of vegetation for eventual coal dump rehabilitation. While a number of in vitro studies have identified various fungal strains as efficient coal degraders, the mechanisms involved in the Fungcoal-stimulated degradation process have not been fully elucidated. Furthermore, implementation of Fungcoal at both pilot and commercial scale has not been achieved. Thus the objective of this work was to investigate Fungcoal as a bioprocess via examining the role of coal degrading fungi (CDF) and grasses as biocatalysts in coal biodegradation and for the self-cladding of waste coal dumps. Initially, waste coal degradation by N. fischeri, strain ECCN 84, was investigated, specifically focusing on the mechanisms underpinning the process. In vitro studies showed the addition of waste coal induced active fungal colonisation resulting in increased fungal biomass. Increased extracellular laccase (LAC) activity, occuring concomitantly with an increase in hyphal peroxisome proliferation, was also observed in the coal supplied fungal cultures. Analysis of the colonised waste coal revealed a time dependent reduction in the percentage weight of elemental carbon coupled with an increase in elemental oxygen. The results supported metabolism and degradation of waste coal by N. fischeri strain ECCN 84 and involvement of fungal extracellular laccase. The contribution of C. dactylon, a C4 grass species to in situ biodegradation of waste coal in the presence of coal degrading and mycorrhizal fungi (MF) was also investigated. Enhanced degradation of the waste coal into a humic soil-like material was observed within the rhizosphere. Analysis of the resultant substrate revealed an increased concentration of highly oxidised humic-like substances (HS). Fungi remained viable in the rhizosphere up to 47 weeks post-inoculation and cultivation of C. dactylon, indicating the resultant humic substance-rich rhizosphere provided an environment conducive for microbial proliferation and activity. Furthermore, humic substance enrichment of waste coal substrates supported germination and seedling emergence of several agronomic species including Zea mays (corn), Phaseolus vulgaris (bean), Pisum sativum (pea), and Spinacia oleracea (spinach). Use of various cladding materials to support coal biodegradation, by fungus-grass mutualism and rehabilitation of waste dumps was evaluated at commercial scale. While substantial physico-chemical changes were not evident in the absence of cladding or where waste coal was used as cladding material, successful establishment of grass cover and diversity was achieved within three hydrological cycles on dumps cladded with weathered coal. Work presented in this thesis successfully demonstrates the degradation of waste coal by N. fischeri. The biodegradation process included enhanced extracellular LAC activity coupled with increased 3 waste coal oxidation. Increased HS concentration of waste coal substrate supported germination and early seedling establishment of several agronomic species. At commercial scale a co-substrate in the form of carbon-rich weathered coal was essential to support fungus-grass mutualism and Fungcoal-induced rehabilitation. These findings support the developed Fungcoal concept and the underpinning rationale that the phyto-biodegradation of waste coal indeed depends on the mutualistic interactions between grass root exudates and the ligninolytic and mycorrhizal fungi. Taken together, these findings provide practical evidence of the contribution of fungi and grasses as mutualists in the biodegradation of waste coal and sustainable rehabilitation of waste coal dumps
- Full Text:
- Date Issued: 2016
The antifungal activity of an aqueous Tulbaghia violacea plant extract against Aspergillus flavus
- Authors: Belewa, Xoliswa Vuyokazi
- Date: 2015
- Subjects: Medicinal plants , Antifungal agents , Fungi -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/5858 , vital:21001
- Description: Phytochemical analysis of both HEA1 and the crude plant extract showed the presence of phenolics, tannins and saponins. Saponins were the predominant secondary metabolites and were mostly abundant in the plant extract and to a lesser extent in the active compound. Steroidal saponins, tannins and phenolics were also detected in the plant extract, but only the phenolics were detected in the active compound. The results of the phytochemical analysis showed that those compounds that were not present in the active compound could be removed from the crude extract during the TLC purification process. Investigation on the mechanism of action of the crude plant extract on the sterol production by A. flavus showed that the plant extract affected ergosterol biosynthesis by causing an accumulation of oxidosqualene in the ergosterol biosynthetic pathway resulting in a decline in ergosterol production. An oscillatory response in lanosterol production was observed in the presence of the plant extract, which may be an adaptation mechanism of A. flavus to unfavourable conditions and compensation for the loss of enzyme activity which may have occurred as a result of the accumulation of oxidosqualene. The antifungal activity of the plant extract on ergosterol production by A. flavus may also be due to saponins which target the cell membrane and ergosterol production in fungi. The effect of the plant extract on the fungal cell wall of A. flavus also showed that the plant extract caused a decline in β-(1, 3) glucan production by inhibiting β-glucan synthase. The plant extract also affected the chitin synthesis pathway of A. flavus, by causing a decline in chitin production, which was due to the inhibition of chitin synthase. Investigation of chitinase production using 4MU substrates showed that the plant extract caused an accumulation of chitobioses, by activating chitobiosidases and endochitinases. A decline in N-acetylglucosaminidase activity in the presence of the plant extract was observed and this prevented the formation of N-acetylglucosamine. The accumulation of chitobiosidase and endochitinase may be as a result of autolysis that may be triggered by A. flavus as a survival mechanism in the presence of the plant extract and as a compensatory mechanism for the loss of β-glucans and chitin. The antifungal effect of the plant extract on various components of the cell wall of A. flavus, makes T. violacea aqueous plant extract an ideal chemotherapeutic agent against both human and plant pathogens of Aspergillus. The broad spectrum of antifungal activity of T. violacea against A. flavus also eliminates any chances of the fungus developing resistance towards it and would make it a candidate for use as a potential antifungal agent. Further identification and possible chemical synthesis is needed to shed light on the safety and efficacy of the active compound for further development as a chemotherapeutic agent.
- Full Text:
- Date Issued: 2015
- Authors: Belewa, Xoliswa Vuyokazi
- Date: 2015
- Subjects: Medicinal plants , Antifungal agents , Fungi -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/5858 , vital:21001
- Description: Phytochemical analysis of both HEA1 and the crude plant extract showed the presence of phenolics, tannins and saponins. Saponins were the predominant secondary metabolites and were mostly abundant in the plant extract and to a lesser extent in the active compound. Steroidal saponins, tannins and phenolics were also detected in the plant extract, but only the phenolics were detected in the active compound. The results of the phytochemical analysis showed that those compounds that were not present in the active compound could be removed from the crude extract during the TLC purification process. Investigation on the mechanism of action of the crude plant extract on the sterol production by A. flavus showed that the plant extract affected ergosterol biosynthesis by causing an accumulation of oxidosqualene in the ergosterol biosynthetic pathway resulting in a decline in ergosterol production. An oscillatory response in lanosterol production was observed in the presence of the plant extract, which may be an adaptation mechanism of A. flavus to unfavourable conditions and compensation for the loss of enzyme activity which may have occurred as a result of the accumulation of oxidosqualene. The antifungal activity of the plant extract on ergosterol production by A. flavus may also be due to saponins which target the cell membrane and ergosterol production in fungi. The effect of the plant extract on the fungal cell wall of A. flavus also showed that the plant extract caused a decline in β-(1, 3) glucan production by inhibiting β-glucan synthase. The plant extract also affected the chitin synthesis pathway of A. flavus, by causing a decline in chitin production, which was due to the inhibition of chitin synthase. Investigation of chitinase production using 4MU substrates showed that the plant extract caused an accumulation of chitobioses, by activating chitobiosidases and endochitinases. A decline in N-acetylglucosaminidase activity in the presence of the plant extract was observed and this prevented the formation of N-acetylglucosamine. The accumulation of chitobiosidase and endochitinase may be as a result of autolysis that may be triggered by A. flavus as a survival mechanism in the presence of the plant extract and as a compensatory mechanism for the loss of β-glucans and chitin. The antifungal effect of the plant extract on various components of the cell wall of A. flavus, makes T. violacea aqueous plant extract an ideal chemotherapeutic agent against both human and plant pathogens of Aspergillus. The broad spectrum of antifungal activity of T. violacea against A. flavus also eliminates any chances of the fungus developing resistance towards it and would make it a candidate for use as a potential antifungal agent. Further identification and possible chemical synthesis is needed to shed light on the safety and efficacy of the active compound for further development as a chemotherapeutic agent.
- Full Text:
- Date Issued: 2015
Bioethanol production from waste paper through fungal biotechnology
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
- Date Issued: 2010
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
- Date Issued: 2010
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
- «
- ‹
- 1
- ›
- »