The immobilization of Microcystis aeruginosa PCC7806 on a membrane nutrient-gradostat bioreacator for the production of the secondary metobolites
- Authors: Strong, Peter James
- Date: 2002
- Subjects: Microcystis aeruginosa , Myrocystins , Bioreactors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11083 , http://hdl.handle.net/10948/283 , Microcystis aeruginosa , Myrocystins , Bioreactors
- Description: A module and an inoculation technique were developed that would allow for the efficient immobilization of Microcystis aeruginosa PCC7806 on a synthetic membrane. A variety of module types, membranes (ceramic, tubular polyethersulfone and externally skinless polyethersulfone capillary membrane), and methods of immobilization (adsorption, pressure filtration and a developed technique that involved drying a cell slurry on a membrane) were assessed. The morphological properties that affected the immobilization of Microcystis aeruginosa PCC7806, as well as the effects of immobilization upon cell morphology were assessed. Cells in the stationary growth phase, which had a well-developed extra-cellular polysaccharide layer and no gas vesicles, were optimal for immobilization. Microcystin production under immobilized conditions was assessed under different nitrate concentrations, light intensities, biofilm thickness and immobilization times. Additional work included assaying for Microcystin production of two airlift-grown cultures under a high light intensity and complete nutrient deprivation and the inoculation of a ceramic membrane. An immunological technique was used to elucidate where toxin production was greatest within a biofilm immobilized upon an externally skinless polyethersulfone capillary membrane. The externally skinless polyethersulfone capillary membrane was evaluated to assess homogeneity and the physical differences between membrane batches that led to the erratic, incomplete biofilm formation, as a biofilm of a constant thickness could not be immobilized. Microcystis aeruginosa PCC7806 was exposed to a variety of solvents in order to permeabilize the cyanobacteria, as that would have enabled a truly continuous extraction process for the metabolite. FDA hydrolysis had to be optimized in order to use it as an indicator of cell viability. In addition a single-step extraction of Microcystin was attempted using live bacteria. A capillary membrane module, containing the externally skinless polyethersulfone capillary membrane, inoculated using pressure filtration, was the most efficient combination to establish a biofilm. Cells that were no longer actively dividing and that lacked buoyancy displayed superior immobilization to cells that were actively dividing and buoyant. The immobilized cells did produce Microcystin but in much lower concentrations to cells grown in an airlift culture. Biofilms grown with a higher nitrate concentration, a lower biofilm thickness and a lower light intensity had a higher specific microcystin content, while biofilms with a higher nitrate concentration a lower light intensity and a longer growth period displayed the a greater toxin production per mm2 of membrane. Microcystin occurred at its highest concentration in cells just above the pore opening. The diffusion of nutrients occurred relatively quickly to the outside layers of the biofilm, with a true gradient being established laterally from these nutrient veins that were above the pores. Permeabilization of the cells proved unsuccessful, as cells that remained viable did not release the intracellular compound into the surrounding medium.
- Full Text:
- Date Issued: 2002
- Authors: Strong, Peter James
- Date: 2002
- Subjects: Microcystis aeruginosa , Myrocystins , Bioreactors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11083 , http://hdl.handle.net/10948/283 , Microcystis aeruginosa , Myrocystins , Bioreactors
- Description: A module and an inoculation technique were developed that would allow for the efficient immobilization of Microcystis aeruginosa PCC7806 on a synthetic membrane. A variety of module types, membranes (ceramic, tubular polyethersulfone and externally skinless polyethersulfone capillary membrane), and methods of immobilization (adsorption, pressure filtration and a developed technique that involved drying a cell slurry on a membrane) were assessed. The morphological properties that affected the immobilization of Microcystis aeruginosa PCC7806, as well as the effects of immobilization upon cell morphology were assessed. Cells in the stationary growth phase, which had a well-developed extra-cellular polysaccharide layer and no gas vesicles, were optimal for immobilization. Microcystin production under immobilized conditions was assessed under different nitrate concentrations, light intensities, biofilm thickness and immobilization times. Additional work included assaying for Microcystin production of two airlift-grown cultures under a high light intensity and complete nutrient deprivation and the inoculation of a ceramic membrane. An immunological technique was used to elucidate where toxin production was greatest within a biofilm immobilized upon an externally skinless polyethersulfone capillary membrane. The externally skinless polyethersulfone capillary membrane was evaluated to assess homogeneity and the physical differences between membrane batches that led to the erratic, incomplete biofilm formation, as a biofilm of a constant thickness could not be immobilized. Microcystis aeruginosa PCC7806 was exposed to a variety of solvents in order to permeabilize the cyanobacteria, as that would have enabled a truly continuous extraction process for the metabolite. FDA hydrolysis had to be optimized in order to use it as an indicator of cell viability. In addition a single-step extraction of Microcystin was attempted using live bacteria. A capillary membrane module, containing the externally skinless polyethersulfone capillary membrane, inoculated using pressure filtration, was the most efficient combination to establish a biofilm. Cells that were no longer actively dividing and that lacked buoyancy displayed superior immobilization to cells that were actively dividing and buoyant. The immobilized cells did produce Microcystin but in much lower concentrations to cells grown in an airlift culture. Biofilms grown with a higher nitrate concentration, a lower biofilm thickness and a lower light intensity had a higher specific microcystin content, while biofilms with a higher nitrate concentration a lower light intensity and a longer growth period displayed the a greater toxin production per mm2 of membrane. Microcystin occurred at its highest concentration in cells just above the pore opening. The diffusion of nutrients occurred relatively quickly to the outside layers of the biofilm, with a true gradient being established laterally from these nutrient veins that were above the pores. Permeabilization of the cells proved unsuccessful, as cells that remained viable did not release the intracellular compound into the surrounding medium.
- Full Text:
- Date Issued: 2002
The effect of nutrient levels and ratios on the growth of Microcystis aeruginosa and microcystin production
- Authors: Sember, Craig Stewart
- Date: 2002
- Subjects: Microcystis aeruginosa -- Toxicology , Nitrates , Microcystins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11076 , http://hdl.handle.net/10948/287 , Microcystis aeruginosa -- Toxicology , Nitrates , Microcystins
- Description: This study reports the findings on the effect of nitrates and phosphates on the biomass and toxin production of various strains of the unicellular non-nitrogen fixing cyanobacterium, Microcystis aeruginosa. The occurrence of blooms of Microcystis aeruginosa and microcystin in freshwater impoundments across the globe has been on the increase lately due to increased levels of eutrophication, resulting in human and animal deaths and illness, as well as drinking and recreational water foulment. A range of environmental factors have been shown to effect growth and microcystin production. Existing literature however is somewhat contradictory as to the effects of these physical and chemical factors on toxin production. Therefore Microcystis aeruginosa strains were cultured under batch and continuous conditions to determine the effect of nitrate and phosphate concentrations and ratios on biomass and toxin production. Cultures were analysed with regards to internal nutrient stores, biomass production, nutrient depletion, photosynthetic efficiency and microcystin production. Results showed that microcystin production correlated to growth rate, photosynthetic efficiency and internal nitrogen stores and that an optimal N:P ratio was associated with microcystin levels, growth rate and photosynthetic efficiency. Results therefore led to the conclusion that the nitrogen, carbon, and phosphate balance within the cell is closely associated with microcystin production. Whether or not microcystin is produced to maintain this balance or produced as a function of this balance remains to be determined.
- Full Text:
- Date Issued: 2002
- Authors: Sember, Craig Stewart
- Date: 2002
- Subjects: Microcystis aeruginosa -- Toxicology , Nitrates , Microcystins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11076 , http://hdl.handle.net/10948/287 , Microcystis aeruginosa -- Toxicology , Nitrates , Microcystins
- Description: This study reports the findings on the effect of nitrates and phosphates on the biomass and toxin production of various strains of the unicellular non-nitrogen fixing cyanobacterium, Microcystis aeruginosa. The occurrence of blooms of Microcystis aeruginosa and microcystin in freshwater impoundments across the globe has been on the increase lately due to increased levels of eutrophication, resulting in human and animal deaths and illness, as well as drinking and recreational water foulment. A range of environmental factors have been shown to effect growth and microcystin production. Existing literature however is somewhat contradictory as to the effects of these physical and chemical factors on toxin production. Therefore Microcystis aeruginosa strains were cultured under batch and continuous conditions to determine the effect of nitrate and phosphate concentrations and ratios on biomass and toxin production. Cultures were analysed with regards to internal nutrient stores, biomass production, nutrient depletion, photosynthetic efficiency and microcystin production. Results showed that microcystin production correlated to growth rate, photosynthetic efficiency and internal nitrogen stores and that an optimal N:P ratio was associated with microcystin levels, growth rate and photosynthetic efficiency. Results therefore led to the conclusion that the nitrogen, carbon, and phosphate balance within the cell is closely associated with microcystin production. Whether or not microcystin is produced to maintain this balance or produced as a function of this balance remains to be determined.
- Full Text:
- Date Issued: 2002
The effect of selenium in the detoxification of the microcystin hepatotoxins
- Authors: Downs, Kerry
- Date: 2002
- Subjects: Cynaobacterial toxins , Microcystins , Selenium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11070 , http://hdl.handle.net/10948/284 , Cynaobacterial toxins , Microcystins , Selenium
- Description: Blooms of cyanobacteria have been known to cause illness in humans and death in wild and domestic animals. One of the toxins produced by cyanobacteria is microcystin, which is a potent hepatotoxin. Microcystin is taken up by bile acid transporters in the intestine and transported into the liver. After exposure to acute doses of microcystin, severe haemorrhage has been observed along with apoptotic and necrotic hepatocytes. The cytoskeletal structure of the hepatocytes is disrupted and oxidative stress is induced. Selenium, a known anti-oxidant, has been shown to induce increased activity of glutathione peroxidase. Glutathione peroxidase removes peroxides from cells protecting them from oxidative stress. This study set out to determine if selenium could play a role in preventing the damage to mice livers due to microcystin toxin. The protective role of selenium was explored in three main studies: in the first study, the ability of selenium to increase the survival time of mice exposed to a lethal dose of toxin was determined. In the second study the mice were exposed to sublethal chronic doses of toxin over 30 days. The ability of selenium to minimise liver damage under these conditions was determined. The final study investigated the mechanism of the protective effect of selenium. The results of the first study suggested that selenium could extend survival time. In the second study the selenium supplemented mice showed a reduction in the extent of the increase in liver weight and a decrease in the amount of lipid peroxidation induced compared to the mice that received only toxin. The histology of the selenium supplemented mice also showed a decrease in the severity and amount of morphological changes in the liver. The third study indicated that the protection shown by selenium might be mediated by an increase in the glutathione peroxidase (GPX) activity in selenium supplemented mice. This increase in GPX activity would increase the removal of the lipid hydroperoxides and prevent the damage they would cause in the cell. A further result indicated an increase in glutathione S-transferase in only the toxin control mice when compared to the selenium supplemented and control mice. ii In conclusion selenium offers protection against microcystin but further studies need to be done to provide statistically valid results to clarify the level of protection.
- Full Text:
- Date Issued: 2002
- Authors: Downs, Kerry
- Date: 2002
- Subjects: Cynaobacterial toxins , Microcystins , Selenium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11070 , http://hdl.handle.net/10948/284 , Cynaobacterial toxins , Microcystins , Selenium
- Description: Blooms of cyanobacteria have been known to cause illness in humans and death in wild and domestic animals. One of the toxins produced by cyanobacteria is microcystin, which is a potent hepatotoxin. Microcystin is taken up by bile acid transporters in the intestine and transported into the liver. After exposure to acute doses of microcystin, severe haemorrhage has been observed along with apoptotic and necrotic hepatocytes. The cytoskeletal structure of the hepatocytes is disrupted and oxidative stress is induced. Selenium, a known anti-oxidant, has been shown to induce increased activity of glutathione peroxidase. Glutathione peroxidase removes peroxides from cells protecting them from oxidative stress. This study set out to determine if selenium could play a role in preventing the damage to mice livers due to microcystin toxin. The protective role of selenium was explored in three main studies: in the first study, the ability of selenium to increase the survival time of mice exposed to a lethal dose of toxin was determined. In the second study the mice were exposed to sublethal chronic doses of toxin over 30 days. The ability of selenium to minimise liver damage under these conditions was determined. The final study investigated the mechanism of the protective effect of selenium. The results of the first study suggested that selenium could extend survival time. In the second study the selenium supplemented mice showed a reduction in the extent of the increase in liver weight and a decrease in the amount of lipid peroxidation induced compared to the mice that received only toxin. The histology of the selenium supplemented mice also showed a decrease in the severity and amount of morphological changes in the liver. The third study indicated that the protection shown by selenium might be mediated by an increase in the glutathione peroxidase (GPX) activity in selenium supplemented mice. This increase in GPX activity would increase the removal of the lipid hydroperoxides and prevent the damage they would cause in the cell. A further result indicated an increase in glutathione S-transferase in only the toxin control mice when compared to the selenium supplemented and control mice. ii In conclusion selenium offers protection against microcystin but further studies need to be done to provide statistically valid results to clarify the level of protection.
- Full Text:
- Date Issued: 2002
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