Bioaccumulation of metal cations by yeast and yeast cell components
- Authors: Brady, Dean
- Date: 1993
- Subjects: Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4046 , http://hdl.handle.net/10962/d1004107 , Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Description: The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.
- Full Text:
- Date Issued: 1993
- Authors: Brady, Dean
- Date: 1993
- Subjects: Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4046 , http://hdl.handle.net/10962/d1004107 , Yeast , Yeast fungi -- Biotechnology , Cations , Metal ions
- Description: The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.
- Full Text:
- Date Issued: 1993
A possible mechanism for enzymic depilation of skins
- Authors: Brady, Dean
- Date: 1989
- Subjects: Chemistry, Technical , Tanning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3877 , http://hdl.handle.net/10962/d1001611
- Description: Streptomyces fradiae is a bacterium which has been previously found to produce extracellular enzymes which are capable of wool degradation and skin depilation. Streptomyces fradiae 3739 and other strains of Streptomyces were found in this study to be able to degrade a keratin source (wool) to a considerable degree. However according to the evidence of SEM micrographs presented here the highly keratinised spindle cells of the paracortex are fairly resistant to protease attack, and it is the cementation material which binds these cells together which is initially degraded by the proteases. A large degree of correlation was found with the strains of Streptomyces studied, between the ability of the individual strains to degrade wool and the ability of their extracellular proteases to reduce the depilation load of sheepskins. With further analysis S. fradiae 3739 was found to produce at least one amylase and four or more proteases. The proteases as a group had maximal proteolytic activity in the 8.0-9.0 pH unit range, and were considerably thermostabilised by the inclusion of calcium ions into the reaction solution. The protease group was found to cause depilation of merino sheepskins. For comparative purposes a protease produced by a strain of Proteus vulgaris isolated from a staling hide with hair slip (natural depilation) was studied. The protease activity was maximal in the alkaline region between 8.0-9.0 pH units. Tbe protease appeared to be a single enzyme with a molecular mass of approximately 44 000 daltons. The protease was maximally active at 40°C, although it was only thermostable to 30°C. The enzyme was ineffectual as a depilant except when the skin was pre-treated with a strong alkali, preferably including sodium sulphite in the protease preparation. One of the most important differences between the extracellular proteases of S. fradiae and P. vulgaris was that the former were greater in variety and caused a greater decrease in the depilation load of sheepskins than the latter. Further research with mixtures of commercial proteases provided evidence that a synergistic depilatory effect occurs when proteases of complementary bond specificities are used in conjunction in enzymic depilatory preparations. Some form of strong alkali treatment of skins was found to be necessary to produce leather of the prerequisite quality when the skin was depilated by proteases, otherwise the skin was found to be depleted and stiff. Calcium hydroxide alone was found to be inadequate for this task, probably owing to the fact that it is less alkaline than the lime-sulphide mixture. The calcium hydroxide (lime) must therefore be used in conjunction with sodium hydroxide (which makes the solution as alkaline as that of the lime-sulphide solution) to produce leather comparable to that produced by the lime sulphide treatment. A combination of the information provided by the present research and that gleaned from the relevent literature allows for the construction of a model to represent the possible mechanism of enzymic depilation of skins, in which depilation is caused by the disruption of the basement membrane at the dermal-epidermal junction by the degradation of its constituent molecular components by general proteases, resulting in the removal of the epidermis and its associated wool or hair
- Full Text:
- Date Issued: 1989
- Authors: Brady, Dean
- Date: 1989
- Subjects: Chemistry, Technical , Tanning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3877 , http://hdl.handle.net/10962/d1001611
- Description: Streptomyces fradiae is a bacterium which has been previously found to produce extracellular enzymes which are capable of wool degradation and skin depilation. Streptomyces fradiae 3739 and other strains of Streptomyces were found in this study to be able to degrade a keratin source (wool) to a considerable degree. However according to the evidence of SEM micrographs presented here the highly keratinised spindle cells of the paracortex are fairly resistant to protease attack, and it is the cementation material which binds these cells together which is initially degraded by the proteases. A large degree of correlation was found with the strains of Streptomyces studied, between the ability of the individual strains to degrade wool and the ability of their extracellular proteases to reduce the depilation load of sheepskins. With further analysis S. fradiae 3739 was found to produce at least one amylase and four or more proteases. The proteases as a group had maximal proteolytic activity in the 8.0-9.0 pH unit range, and were considerably thermostabilised by the inclusion of calcium ions into the reaction solution. The protease group was found to cause depilation of merino sheepskins. For comparative purposes a protease produced by a strain of Proteus vulgaris isolated from a staling hide with hair slip (natural depilation) was studied. The protease activity was maximal in the alkaline region between 8.0-9.0 pH units. Tbe protease appeared to be a single enzyme with a molecular mass of approximately 44 000 daltons. The protease was maximally active at 40°C, although it was only thermostable to 30°C. The enzyme was ineffectual as a depilant except when the skin was pre-treated with a strong alkali, preferably including sodium sulphite in the protease preparation. One of the most important differences between the extracellular proteases of S. fradiae and P. vulgaris was that the former were greater in variety and caused a greater decrease in the depilation load of sheepskins than the latter. Further research with mixtures of commercial proteases provided evidence that a synergistic depilatory effect occurs when proteases of complementary bond specificities are used in conjunction in enzymic depilatory preparations. Some form of strong alkali treatment of skins was found to be necessary to produce leather of the prerequisite quality when the skin was depilated by proteases, otherwise the skin was found to be depleted and stiff. Calcium hydroxide alone was found to be inadequate for this task, probably owing to the fact that it is less alkaline than the lime-sulphide mixture. The calcium hydroxide (lime) must therefore be used in conjunction with sodium hydroxide (which makes the solution as alkaline as that of the lime-sulphide solution) to produce leather comparable to that produced by the lime sulphide treatment. A combination of the information provided by the present research and that gleaned from the relevent literature allows for the construction of a model to represent the possible mechanism of enzymic depilation of skins, in which depilation is caused by the disruption of the basement membrane at the dermal-epidermal junction by the degradation of its constituent molecular components by general proteases, resulting in the removal of the epidermis and its associated wool or hair
- Full Text:
- Date Issued: 1989
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