Effect of alkaline pre-treatments on the synergistic enzymatic hydrolysis of sugarcane (Saccharum officinarum) bagasse by Clostridium cellulovorans XynA, ManA and ArfA
- Authors: Beukes, Natasha
- Date: 2011
- Subjects: Sugarcane -- Biotechnology Lignocellulose -- Biotechnology Renewable energy sources Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3952 , http://hdl.handle.net/10962/d1004011
- Description: The continual increase in industrialization and global population has increased the dependency and demand on traditional fossil fuels for energy; however, there are limited amounts of fossil fuels available. The slow depletion of fossil fuels has sparked a fresh interest in renewable sources such as lignocellulose to produce a variety of biofuels, such as biogases (e.g. methane), bioethanol, biodiesel and a variety of other solvents and economically valuable by-products. Agricultural crop wastes produced in surplus are typically lignocellulosic in composition and thus partially recalcitrant to enzymatic degradation. The recalcitrant nature of plant biomass and the inability to obtain complete enzymatic hydrolysis has led to the establishment of various pre-treatment strategies. Alkaline pre-treatments increase the accessibility of the exposed surface to enzymatic hydrolysis through the removal of acetyl and uronic acid substituents on hemicellulose. Unlike the use of steam and acid pre-treatments, alkaline pre-treatments solubilize lignin and a small percentage of the hemicellulose, increasing enzyme accessibility and thus the hydrolysis of lignocellulose. The majority of Clostridium cellulovorans associated enzyme synergy studies have been devoted to an understanding of the cellulolytic and hemi-cellulolytic degradation of plant cell walls. However, little is known about the effect of various physical and chemical pre-treatments on the synergistic enzymatic degradation of plant biomass and possible depolymerization of plant cell walls. This study investigates the use of slake lime, sodium hydroxide and ammonium hydroxide to pre-treat sugarcane bagasse under mild conditions and elucidates potentially important synergistic associations between the C. cellulovorans enzymes for the enhanced degradation of lignocellulose. The primary aims of the study were addressed using of a variety of techniques. This included suitable vector constructs for the expression and purification of recombinant C. cellulovorans enzymes, identification of the effects of various pre-treatments on enzyme synergy, and identification of the resultant reducing sugars and phenolic compounds (released during the pre-treatment of the bagasse). This study also made use of physical and chemical pre-treatment methods, protein purification using affinity, high performance liquid and thin layer chromatography, mass spectrometry, sodium dodecyl sulphate and fluorophore-assisted polyacrylamide gel electrophoresis (FACE) , enzymatic degradation and synergy studies with various substrates indirectly using the 3, 4-dinitrosalicylic acid (DNS) reducing sugar assay. From this investigation, the following conclusions were made: alkaline pre-treatment successfully solublised, redistributed and removed lignin from the bagasse, increasing the digestibility of the substrates. In summary, the most effective pre-treatment employed 0.114 M ammonium hydroxide / gram bagasse at 70°C for 36 hours, followed by hydrolysis with an enzyme cocktail containing 25% ManA and 75% XynA. This increased the production of sugars approximately 13-fold. Analysis of the sugars produced by the synergistic hydrolysis of sugarcane bagasse (SCB) indicated the presence of xylose, indicating that the enzymes are potentially bifunctional under certain conditions. This study indicated that the use of mild pre-treatment conditions sufficiently removed a large portion of lignin without affecting the hemicellulose moiety of the SCB. This facilitated the potential use of the hemicellulose component for the production of valuable products (e.g. xylitol) in addition to the production of bioethanol. Thus, the potential use of additional components of holocellulose may generate an additional biotechnological benefit and allow a certain degree of flexibility in the biofuel industry, depending on consumer and industrial needs.
- Full Text:
- Date Issued: 2011
- Authors: Beukes, Natasha
- Date: 2011
- Subjects: Sugarcane -- Biotechnology Lignocellulose -- Biotechnology Renewable energy sources Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3952 , http://hdl.handle.net/10962/d1004011
- Description: The continual increase in industrialization and global population has increased the dependency and demand on traditional fossil fuels for energy; however, there are limited amounts of fossil fuels available. The slow depletion of fossil fuels has sparked a fresh interest in renewable sources such as lignocellulose to produce a variety of biofuels, such as biogases (e.g. methane), bioethanol, biodiesel and a variety of other solvents and economically valuable by-products. Agricultural crop wastes produced in surplus are typically lignocellulosic in composition and thus partially recalcitrant to enzymatic degradation. The recalcitrant nature of plant biomass and the inability to obtain complete enzymatic hydrolysis has led to the establishment of various pre-treatment strategies. Alkaline pre-treatments increase the accessibility of the exposed surface to enzymatic hydrolysis through the removal of acetyl and uronic acid substituents on hemicellulose. Unlike the use of steam and acid pre-treatments, alkaline pre-treatments solubilize lignin and a small percentage of the hemicellulose, increasing enzyme accessibility and thus the hydrolysis of lignocellulose. The majority of Clostridium cellulovorans associated enzyme synergy studies have been devoted to an understanding of the cellulolytic and hemi-cellulolytic degradation of plant cell walls. However, little is known about the effect of various physical and chemical pre-treatments on the synergistic enzymatic degradation of plant biomass and possible depolymerization of plant cell walls. This study investigates the use of slake lime, sodium hydroxide and ammonium hydroxide to pre-treat sugarcane bagasse under mild conditions and elucidates potentially important synergistic associations between the C. cellulovorans enzymes for the enhanced degradation of lignocellulose. The primary aims of the study were addressed using of a variety of techniques. This included suitable vector constructs for the expression and purification of recombinant C. cellulovorans enzymes, identification of the effects of various pre-treatments on enzyme synergy, and identification of the resultant reducing sugars and phenolic compounds (released during the pre-treatment of the bagasse). This study also made use of physical and chemical pre-treatment methods, protein purification using affinity, high performance liquid and thin layer chromatography, mass spectrometry, sodium dodecyl sulphate and fluorophore-assisted polyacrylamide gel electrophoresis (FACE) , enzymatic degradation and synergy studies with various substrates indirectly using the 3, 4-dinitrosalicylic acid (DNS) reducing sugar assay. From this investigation, the following conclusions were made: alkaline pre-treatment successfully solublised, redistributed and removed lignin from the bagasse, increasing the digestibility of the substrates. In summary, the most effective pre-treatment employed 0.114 M ammonium hydroxide / gram bagasse at 70°C for 36 hours, followed by hydrolysis with an enzyme cocktail containing 25% ManA and 75% XynA. This increased the production of sugars approximately 13-fold. Analysis of the sugars produced by the synergistic hydrolysis of sugarcane bagasse (SCB) indicated the presence of xylose, indicating that the enzymes are potentially bifunctional under certain conditions. This study indicated that the use of mild pre-treatment conditions sufficiently removed a large portion of lignin without affecting the hemicellulose moiety of the SCB. This facilitated the potential use of the hemicellulose component for the production of valuable products (e.g. xylitol) in addition to the production of bioethanol. Thus, the potential use of additional components of holocellulose may generate an additional biotechnological benefit and allow a certain degree of flexibility in the biofuel industry, depending on consumer and industrial needs.
- Full Text:
- Date Issued: 2011
An investigation into the synergistic association between the major Clostridium cellulovorans cellulosomal endoglucanase and two hemicellulases on plant cell wall degradation
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
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