The effect of various substrate pretreatment methods on the enzymatic degradability of a Eucalyptus sp. – a potential feedstock for producing fermentable sugars
- Authors: Thoresen, Mariska
- Date: 2021-04
- Subjects: Cellulose , Cellulase , Enzymes , Hydrolysis , Eucalyptus , Biomass energy
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178580 , vital:42952 , DOI 10.21504/10962/178580
- Description: Over the past few years, there has been a global urgency to make the transition from conventional fossil fuels to renewable energy in order to meet the world’s increasing energy demands. Lignocellulosic biomass is currently at the forefront of intensive biofuel research due to its renewable nature. Lignocellulose valorisation into value added products such as bio-ethanol is a multistep process. The first step requires the biomass to go through a recalcitrance-reducing step (pretreatment), after which, enzymatic hydrolysis is required to break down the polysaccharides into simple sugars for fermentation. However, the recalcitrant structure of biomass and the low hydrolytic activities of the enzymes (glycoside hydrolases) on the substrate pose major technical and economic obstacles to the biomass conversion process. Since this process remains more expensive compared to petroleum-based fuels, lignocellulose has been intensively investigated in terms of its cost efficiency and effective decomposition. Although improvements to this process are ongoing, with some of the first commercial facilities producing cellulosic ethanol in 2013 and 2014, there is still a deep sense of urgency to render the facilities more economically feasible. Some important factors that determine the yield and rate of enzymatic hydrolysis include the type of enzymes used, enzyme recognition with the substrate, substrate composition and crystallinity. In this context, the major focus of this study was to develop a deeper understanding of how enzymes co-operate (synergise) at a molecular level using model substrates. This knowledge was then used as a basis for understanding how these enzymes synergise on more natural, complex substrates. This study specifically focused on how different pretreatments affect the chemical and structural properties of Eucalyptus. Lastly, we wanted to develop an effective method of enzyme recycling as a means to reduce the high process costs in biomass saccharification. Enhancing cellulose hydrolysis through enzyme synergy is essential for achieving higher hydrolysis rates, and numerous research efforts have focused on trying to elucidate the enzyme mechanisms required to design optimal enzyme cocktails. Despite the extensive amount of research carried out over the past few years, little is known about the enzymatic machinery underpinning the synergistic interactions between bacterial and fungal cellulases - neither is it understood why only a limited number of Cellobiohydrolases (CBHs) and Endoglucanases (EGs) exhibit synergism. Therefore, the first part of the study evaluated and compared the synergistic relationships between cellulases from different GH families and microbial sources (cross-synergism), i.e. cellobiohydrolase I (CBHI) from Hypocrea jecorina (Cel7A), CBHI from Trichoderma longibrachiatum (Cel7A), CBHI from Clostridium stercorarium (Cel48A), CBHII from a microbial source, CBHII from Clostridium thermocellum (Cel5A), endoglucanases (EG) from Bacillus amyloliquefaciens (Cel5A), EG from Thermotaga maritima (Cel5A), EG from Trichoderma reesei and a β-glucosidase from Aspergillus niger (Novozyme 188). An aim of this study was to provide insights into how the molecular mechanisms of different GH families govern synergism. The results showed that cellulases from different GH families and microbial sources exhibit different substrate specificities, which influence their synergistic interactions with other enzymes. Based on these observations, this study agreed with evidence that not all endo- and exo-cellulase interactions are synergistic, and that the extent of synergism is dependent on the composition of the cellulase systems from various sources and their compatibility in the cellulase cocktail. From the enzymes assessed in this study, an optimal enzyme cocktail (CelMix) was formulated which was composed of Egl 68%, Cel7A 17%, Cel6A 6%, βgl 9%. This method of screening for maximal compatibility between exo- and endo-cellulases from different GH families constituted a critical step towards a better understanding of the specific interactions between the enzymes of interest and how they synergise at the molecular level. Consequently, this information may assist in the design of improved synergistic cellulose-degrading cocktails for industrial-scale biomass degradation. The enzyme synergy studies provided a basis for the second part of this study, where it was assessed how these optimised enzyme cocktails would perform on complex substrates. It is well-known that lignocellulosic substrates are highly recalcitrant to microbial degradation, and although extensive research has been performed to understand biomass recalcitrance, the key features of biomass which hinder enzymatic hydrolysis are yet to be elucidated. In this study, we explored the effect of eight (8) different pretreatment methods on the enzymatic hydrolysis of a Eucalyptus sp. – a potential feedstock for biofuel production. This study was performed to increase our understanding of the relationship between biomass architecture and hydrolysis yield potential. Our results demonstrated that pretreatments induce changes at a micro- and macro-level in the cell walls of Eucalyptus, and that cellulose accessibility, cellulose crystallinity and the changes in the lignin S/G ratio played an important role in the enzymatic activity on the biomass. Thus, this study provided insight into important cellulose structural features related to biomass recalcitrance arising from various pretreatment methods, which may ultimately be used for the development of more efficient conversion technologies for better, more competitive bio-refineries. Lastly, a simple and yet effective method for desorbing the adsorbed cellulases on lignocellulosic substrates was established for better understanding cellulase adsorption and desorption in order to develop an effective enzyme recycling strategy. Various reagents were assessed to determine how effective they were in promoting enzyme desorption. Tris-HCl buffer (pH 9.0; 0.05 M) was the most effective method for promoting enzyme desorption and retained a substantial amount of hydrolytic activity after elution. However, minor activity loss was observed due to irreversible binding, which was further confirmed by SDS-PAGE analysis. With this information available, the feasibility of recovering the enzymes from the solid fraction after enzymatic hydrolysis of steam pretreated Eucalyptus was evaluated by two different approaches, i.e.: i) re-adsorption of the entire hydrolysed insoluble biomass fraction (no desorption) to fresh biomass (recycling approach 1 - RA1) and ii) re-adsorption of alkaline elution desorbed enzymes from hydrolysed biomass to fresh biomass (recycling approach 2 - RA2). The recycling performance of RA1 and RA2 achieved > 95% of the initial sugar liberation for three continuous rounds, whilst successfully reducing enzyme loadings by 50% and 40% for RA1 and RA2, respectively. This study presented a simple and effective pathway for improving the economic feasibility of fermentable sugar production for biofuels. In conclusion, this study has contributed to expanding our knowledge and providing new insights into factors relating to the biomass conversion process, including enzyme synergism, pretreatment methods and enzyme recycling strategies. Ultimately, the knowledge and information gained from this study can be used as a platform for the development of more efficient conversion technologies for better, more competitive bio-refineries. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Thoresen, Mariska
- Date: 2021-04
- Subjects: Cellulose , Cellulase , Enzymes , Hydrolysis , Eucalyptus , Biomass energy
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178580 , vital:42952 , DOI 10.21504/10962/178580
- Description: Over the past few years, there has been a global urgency to make the transition from conventional fossil fuels to renewable energy in order to meet the world’s increasing energy demands. Lignocellulosic biomass is currently at the forefront of intensive biofuel research due to its renewable nature. Lignocellulose valorisation into value added products such as bio-ethanol is a multistep process. The first step requires the biomass to go through a recalcitrance-reducing step (pretreatment), after which, enzymatic hydrolysis is required to break down the polysaccharides into simple sugars for fermentation. However, the recalcitrant structure of biomass and the low hydrolytic activities of the enzymes (glycoside hydrolases) on the substrate pose major technical and economic obstacles to the biomass conversion process. Since this process remains more expensive compared to petroleum-based fuels, lignocellulose has been intensively investigated in terms of its cost efficiency and effective decomposition. Although improvements to this process are ongoing, with some of the first commercial facilities producing cellulosic ethanol in 2013 and 2014, there is still a deep sense of urgency to render the facilities more economically feasible. Some important factors that determine the yield and rate of enzymatic hydrolysis include the type of enzymes used, enzyme recognition with the substrate, substrate composition and crystallinity. In this context, the major focus of this study was to develop a deeper understanding of how enzymes co-operate (synergise) at a molecular level using model substrates. This knowledge was then used as a basis for understanding how these enzymes synergise on more natural, complex substrates. This study specifically focused on how different pretreatments affect the chemical and structural properties of Eucalyptus. Lastly, we wanted to develop an effective method of enzyme recycling as a means to reduce the high process costs in biomass saccharification. Enhancing cellulose hydrolysis through enzyme synergy is essential for achieving higher hydrolysis rates, and numerous research efforts have focused on trying to elucidate the enzyme mechanisms required to design optimal enzyme cocktails. Despite the extensive amount of research carried out over the past few years, little is known about the enzymatic machinery underpinning the synergistic interactions between bacterial and fungal cellulases - neither is it understood why only a limited number of Cellobiohydrolases (CBHs) and Endoglucanases (EGs) exhibit synergism. Therefore, the first part of the study evaluated and compared the synergistic relationships between cellulases from different GH families and microbial sources (cross-synergism), i.e. cellobiohydrolase I (CBHI) from Hypocrea jecorina (Cel7A), CBHI from Trichoderma longibrachiatum (Cel7A), CBHI from Clostridium stercorarium (Cel48A), CBHII from a microbial source, CBHII from Clostridium thermocellum (Cel5A), endoglucanases (EG) from Bacillus amyloliquefaciens (Cel5A), EG from Thermotaga maritima (Cel5A), EG from Trichoderma reesei and a β-glucosidase from Aspergillus niger (Novozyme 188). An aim of this study was to provide insights into how the molecular mechanisms of different GH families govern synergism. The results showed that cellulases from different GH families and microbial sources exhibit different substrate specificities, which influence their synergistic interactions with other enzymes. Based on these observations, this study agreed with evidence that not all endo- and exo-cellulase interactions are synergistic, and that the extent of synergism is dependent on the composition of the cellulase systems from various sources and their compatibility in the cellulase cocktail. From the enzymes assessed in this study, an optimal enzyme cocktail (CelMix) was formulated which was composed of Egl 68%, Cel7A 17%, Cel6A 6%, βgl 9%. This method of screening for maximal compatibility between exo- and endo-cellulases from different GH families constituted a critical step towards a better understanding of the specific interactions between the enzymes of interest and how they synergise at the molecular level. Consequently, this information may assist in the design of improved synergistic cellulose-degrading cocktails for industrial-scale biomass degradation. The enzyme synergy studies provided a basis for the second part of this study, where it was assessed how these optimised enzyme cocktails would perform on complex substrates. It is well-known that lignocellulosic substrates are highly recalcitrant to microbial degradation, and although extensive research has been performed to understand biomass recalcitrance, the key features of biomass which hinder enzymatic hydrolysis are yet to be elucidated. In this study, we explored the effect of eight (8) different pretreatment methods on the enzymatic hydrolysis of a Eucalyptus sp. – a potential feedstock for biofuel production. This study was performed to increase our understanding of the relationship between biomass architecture and hydrolysis yield potential. Our results demonstrated that pretreatments induce changes at a micro- and macro-level in the cell walls of Eucalyptus, and that cellulose accessibility, cellulose crystallinity and the changes in the lignin S/G ratio played an important role in the enzymatic activity on the biomass. Thus, this study provided insight into important cellulose structural features related to biomass recalcitrance arising from various pretreatment methods, which may ultimately be used for the development of more efficient conversion technologies for better, more competitive bio-refineries. Lastly, a simple and yet effective method for desorbing the adsorbed cellulases on lignocellulosic substrates was established for better understanding cellulase adsorption and desorption in order to develop an effective enzyme recycling strategy. Various reagents were assessed to determine how effective they were in promoting enzyme desorption. Tris-HCl buffer (pH 9.0; 0.05 M) was the most effective method for promoting enzyme desorption and retained a substantial amount of hydrolytic activity after elution. However, minor activity loss was observed due to irreversible binding, which was further confirmed by SDS-PAGE analysis. With this information available, the feasibility of recovering the enzymes from the solid fraction after enzymatic hydrolysis of steam pretreated Eucalyptus was evaluated by two different approaches, i.e.: i) re-adsorption of the entire hydrolysed insoluble biomass fraction (no desorption) to fresh biomass (recycling approach 1 - RA1) and ii) re-adsorption of alkaline elution desorbed enzymes from hydrolysed biomass to fresh biomass (recycling approach 2 - RA2). The recycling performance of RA1 and RA2 achieved > 95% of the initial sugar liberation for three continuous rounds, whilst successfully reducing enzyme loadings by 50% and 40% for RA1 and RA2, respectively. This study presented a simple and effective pathway for improving the economic feasibility of fermentable sugar production for biofuels. In conclusion, this study has contributed to expanding our knowledge and providing new insights into factors relating to the biomass conversion process, including enzyme synergism, pretreatment methods and enzyme recycling strategies. Ultimately, the knowledge and information gained from this study can be used as a platform for the development of more efficient conversion technologies for better, more competitive bio-refineries. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
Formulation of an enzyme cocktail, HoloMix, using cellulolytic and xylanolytic enzyme core-sets for effective degradation of various pre-treated hardwoods
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
Nanofiber immobilized cellulases and hemicellulases for fruit waste beneficiation
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
- Date Issued: 2015
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
- Date Issued: 2015
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
Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
The role of cellulases and glucohydrolases in the solubilisation of primary sewage sludge
- Authors: Ngesi, Nosisa
- Date: 2002 , 2013-05-09
- Subjects: Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4080 , http://hdl.handle.net/10962/d1007454 , Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Description: Biological sulph ate reduction has been identi fied as a potentially valuable process for removing sulphate and heavy metals from indllstrial effluents. The role of sulphate reducing bacteria (SRB) in this process has attracted the attention of biotechnologists and recently of enzymologists due to its fundamental properties and possible role in AMD bioremediation. These obligatory anaerobic sulphate-reducing bacteria are commonly known to dissimilate sulphate for energy. Under anaerobic conditions SRB oxidize simple organic compounds such as lactic acid with the sulphate and thereby generate hydrogen sulphide (a stTong reducing agent) and bicarbonate ions. The hydrogen sulphide in turn reacts with contaminant metals contained in AMD and precipitates them out of solution as metal sulphides. Bicarbonate ions neutralize AMD by reaction with protons to form carbon dioxide and water. Organic matter in the municipal sewage sludge has been identified as a potential source of electron donors for su lphate reduction. However, this organic matter is in the polymeric form that cannot be util ised by SRB. The latter depend on the activities of other hydrolytic bacteria for the degradation of complex polymers. Hence the availability of these monomeric substrates is a major factor, which may constrain further process development and is considered a rate-limiting step. Thi s study is therefore undertaken to investigate the bacterial glucohydrolase enzymes involved in the digestion of the polysaccharides present in the sewage sludge with specific interest in cellulases and/or p-glucosidase enzymes. The goals of the research are to: isolate, identify, purify and quantify these enzymes; study their distribution with respect to time, pH, and temperature; maximize and quantify the hydrol ys is products; study whether sulphide and sulphate have an enhancing or an inhibitory effect on the activity of enzymes; optimize the enzyme activity against substrate and/or product inhibition and soluble heavy metal salts. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2002
- Authors: Ngesi, Nosisa
- Date: 2002 , 2013-05-09
- Subjects: Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4080 , http://hdl.handle.net/10962/d1007454 , Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Description: Biological sulph ate reduction has been identi fied as a potentially valuable process for removing sulphate and heavy metals from indllstrial effluents. The role of sulphate reducing bacteria (SRB) in this process has attracted the attention of biotechnologists and recently of enzymologists due to its fundamental properties and possible role in AMD bioremediation. These obligatory anaerobic sulphate-reducing bacteria are commonly known to dissimilate sulphate for energy. Under anaerobic conditions SRB oxidize simple organic compounds such as lactic acid with the sulphate and thereby generate hydrogen sulphide (a stTong reducing agent) and bicarbonate ions. The hydrogen sulphide in turn reacts with contaminant metals contained in AMD and precipitates them out of solution as metal sulphides. Bicarbonate ions neutralize AMD by reaction with protons to form carbon dioxide and water. Organic matter in the municipal sewage sludge has been identified as a potential source of electron donors for su lphate reduction. However, this organic matter is in the polymeric form that cannot be util ised by SRB. The latter depend on the activities of other hydrolytic bacteria for the degradation of complex polymers. Hence the availability of these monomeric substrates is a major factor, which may constrain further process development and is considered a rate-limiting step. Thi s study is therefore undertaken to investigate the bacterial glucohydrolase enzymes involved in the digestion of the polysaccharides present in the sewage sludge with specific interest in cellulases and/or p-glucosidase enzymes. The goals of the research are to: isolate, identify, purify and quantify these enzymes; study their distribution with respect to time, pH, and temperature; maximize and quantify the hydrol ys is products; study whether sulphide and sulphate have an enhancing or an inhibitory effect on the activity of enzymes; optimize the enzyme activity against substrate and/or product inhibition and soluble heavy metal salts. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2002
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