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
Investigation of the thermo-chemical behaviour of coal-algae agglomerates
- Authors: Baloyi, Hope
- Date: 2018
- Subjects: Biomass energy , Coal -- South Africa
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/23913 , vital:30642
- Description: There is a growing research interest in the co-processing of biomass and coal, with the aim of addressing the negative attributes associated with the thermal processing of coal alone. Biomass feedstocks are regarded as a clean, renewable source, and the co-utilization of biomass feedstocks with coal is deemed to have a potential to reduce emission of pollutants (i.e. NOx and SOx) and volatile organic compounds (VOC’s). Moreover, biomass are thermally reactive and thus facilitate the conversion of coal during co-processing. Biomass material and coal are two autonomous fuel materials with different chemical characteristics and have a dissimilar thermal behaviour making it difficult to achieve chemical interaction between the two solid fuels to contribute to the formation of products. Coalgae® Technology developed at the Nelson Mandela University, involves the biological treatment of coal fines by adsorbing live microalgae biomass (in slurry form) onto waste coal fines to form coal-microalgae agglomerates. This new innovative approach seeks to integrate bio-based feedstock into coal thermal processing and to improve the utilization and thermal efficiency of coal fines as well as the interaction between the volatile components of biomass and coal during thermal processing (e.g. devolatilization), thereby overcoming some of the challenges that confront the co-processing of coal and biomass. Coal fines are low-ranked coals, generally characterized by high contents of sulphur, high ash yields, low calorific values and poor thermal reactivity, and these attributes limits the thermo-chemical processing of the coal fines. Therefore, this investigation was undertaken to assess the thermo-chemical behaviour of coal-microalgae agglomerates, formed by adsorbing live microalgae slurry at varying ratios onto coal fines. For this purpose, the effects of adsorbing microalgae at varying ratios on the chemical characteristics and thermal behaviour of coal fines under pyrolytic conditions were investigated. The primary aim was to assess whether the thermo-chemical behaviour of coal-microalgae agglomerates, formed by adsorption of live microalgae onto fine coal, is substantively modified compared to a simple additive model of the original coal and pre-dried microalgae biomass samples. Results obtained from the proximate analyses performed on an Eltra Thermo-gravimetric analyzer (TGA) thermostep, have shown that the adsorption of microalgae slurry onto coal fines does not possess greater influence in improving the yield of volatiles and ash in coal fines than can be expected from a simple additive model of the original raw materials. Based on the ultimate analyses results, it was found that the adsorption of microalgae slurry resulted in a systematic reduction in the sulphur content, a notable increase in the hydrogen and oxygen contents, however, no significant disparities were found between the measured ultimate properties of coal-microalgae agglomerates as compared to the theoretically-expected ultimate properties from a simple linear combination of parental coal and microalgae biomass. Assessment of the thermal behaviour of parental samples and coal-microalgae agglomerates involved the use non-isothermal (40-900ºC, 20 K/min) thermogravimetry under inert conditions. It was found that the adsorption of microalgae slurry onto coal fines resulted in an improved thermal reactivity of coal fines, although, did not affect the overall pyrolysis characteristics of the coal fines. Comparison of the thermal profiles (measured and calculated TG/DTG curves), revealed that the yield of volatile products during the pyrolysis of coal-microalgae blends do not exceed the expected volatile yields from a simple combination of coal and microalgae biomass. These results suggest that there was no positive or accelerative synergistic interaction between volatile components of adsorbed microalgae and coal fines during pyrolysis. Mild pyrolysis of raw coal and coal-microalgae performed in a fixed-bed reactor furnace (450ºC), resulted in improved yields of Fossil-Bio crude (FBC) oil (derived from coal-microalgae pyrolysis), at increased biomass ratio compared to coal tar. FBC Oil was found to contain relatively high contents of oxygen, hydrogen, and low sulphur content than coal tar. GC-MS analyses showed the presence of a heterocyclic compounds (i.e. Indole and 2, 6 dimethyl pyridine) in the FBC oil and these were not identified in the coal tar. Furthermore, high boiling compounds such as Flourene, pyrene and pentacosane were identified in the coal tar, however not identified in the FBC oil. Simulated distillation results showed notable differences between the FBC oil and coal tar in terms of the distribution of boiling point fractions particularly, high boing point components. Semi-devolatilized chars derived from coal-microalgae agglomerates showed substantial degree of decarboxylation and dehydrogenation compared to the coal chars.
- Full Text:
- Date Issued: 2018
- Authors: Baloyi, Hope
- Date: 2018
- Subjects: Biomass energy , Coal -- South Africa
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/23913 , vital:30642
- Description: There is a growing research interest in the co-processing of biomass and coal, with the aim of addressing the negative attributes associated with the thermal processing of coal alone. Biomass feedstocks are regarded as a clean, renewable source, and the co-utilization of biomass feedstocks with coal is deemed to have a potential to reduce emission of pollutants (i.e. NOx and SOx) and volatile organic compounds (VOC’s). Moreover, biomass are thermally reactive and thus facilitate the conversion of coal during co-processing. Biomass material and coal are two autonomous fuel materials with different chemical characteristics and have a dissimilar thermal behaviour making it difficult to achieve chemical interaction between the two solid fuels to contribute to the formation of products. Coalgae® Technology developed at the Nelson Mandela University, involves the biological treatment of coal fines by adsorbing live microalgae biomass (in slurry form) onto waste coal fines to form coal-microalgae agglomerates. This new innovative approach seeks to integrate bio-based feedstock into coal thermal processing and to improve the utilization and thermal efficiency of coal fines as well as the interaction between the volatile components of biomass and coal during thermal processing (e.g. devolatilization), thereby overcoming some of the challenges that confront the co-processing of coal and biomass. Coal fines are low-ranked coals, generally characterized by high contents of sulphur, high ash yields, low calorific values and poor thermal reactivity, and these attributes limits the thermo-chemical processing of the coal fines. Therefore, this investigation was undertaken to assess the thermo-chemical behaviour of coal-microalgae agglomerates, formed by adsorbing live microalgae slurry at varying ratios onto coal fines. For this purpose, the effects of adsorbing microalgae at varying ratios on the chemical characteristics and thermal behaviour of coal fines under pyrolytic conditions were investigated. The primary aim was to assess whether the thermo-chemical behaviour of coal-microalgae agglomerates, formed by adsorption of live microalgae onto fine coal, is substantively modified compared to a simple additive model of the original coal and pre-dried microalgae biomass samples. Results obtained from the proximate analyses performed on an Eltra Thermo-gravimetric analyzer (TGA) thermostep, have shown that the adsorption of microalgae slurry onto coal fines does not possess greater influence in improving the yield of volatiles and ash in coal fines than can be expected from a simple additive model of the original raw materials. Based on the ultimate analyses results, it was found that the adsorption of microalgae slurry resulted in a systematic reduction in the sulphur content, a notable increase in the hydrogen and oxygen contents, however, no significant disparities were found between the measured ultimate properties of coal-microalgae agglomerates as compared to the theoretically-expected ultimate properties from a simple linear combination of parental coal and microalgae biomass. Assessment of the thermal behaviour of parental samples and coal-microalgae agglomerates involved the use non-isothermal (40-900ºC, 20 K/min) thermogravimetry under inert conditions. It was found that the adsorption of microalgae slurry onto coal fines resulted in an improved thermal reactivity of coal fines, although, did not affect the overall pyrolysis characteristics of the coal fines. Comparison of the thermal profiles (measured and calculated TG/DTG curves), revealed that the yield of volatile products during the pyrolysis of coal-microalgae blends do not exceed the expected volatile yields from a simple combination of coal and microalgae biomass. These results suggest that there was no positive or accelerative synergistic interaction between volatile components of adsorbed microalgae and coal fines during pyrolysis. Mild pyrolysis of raw coal and coal-microalgae performed in a fixed-bed reactor furnace (450ºC), resulted in improved yields of Fossil-Bio crude (FBC) oil (derived from coal-microalgae pyrolysis), at increased biomass ratio compared to coal tar. FBC Oil was found to contain relatively high contents of oxygen, hydrogen, and low sulphur content than coal tar. GC-MS analyses showed the presence of a heterocyclic compounds (i.e. Indole and 2, 6 dimethyl pyridine) in the FBC oil and these were not identified in the coal tar. Furthermore, high boiling compounds such as Flourene, pyrene and pentacosane were identified in the coal tar, however not identified in the FBC oil. Simulated distillation results showed notable differences between the FBC oil and coal tar in terms of the distribution of boiling point fractions particularly, high boing point components. Semi-devolatilized chars derived from coal-microalgae agglomerates showed substantial degree of decarboxylation and dehydrogenation compared to the coal chars.
- Full Text:
- Date Issued: 2018
The extraction, quantification and application of high-value biological compounds from olive oil processing waste
- Authors: Postma-Botha, Marthie
- Date: 2018
- Subjects: Organic compounds , Biochemistry , Biomass energy , Olive oil industry
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/34383 , vital:33371
- Description: Olive oil processing waste (pomace) as a by-product of the olive oil industry is regarded as a rich source of high-value biological compounds exhibiting antioxidant potential. The objective of the present work was to obtain a concentrated extract of high-value biological antioxidants from the pomace. The effect of extraction conditions on the concentration of the bioactive compounds in the extracts was investigated. The simultaneous recovery of both hydrophilic and lipophilic high-value biological compounds exhibiting antioxidant potential was achieved through a one-step extraction method under reduced pressure using a non-toxic solvent blend. A multilevel experimental design was implemented with the aim of optimising the recovery of selected compounds, namely, hydroxytyrosol, tyrosol, oleuropein, α-tocopherol and squalene from olive pomace by using solvent blends of n-heptane, d-limonene, ethanol and water. The factors considered were: extraction time, percentage composition of solvent blends and extraction temperature. The results suggested that a good recovery of the hydrophilic polyphenolic compounds, namely, hydroxytyrosol, tyrosol and oleuropein, as well as the lipophilic compounds, α-tocopherol and squalene may be achieved at a solvent temperature of 60°C at 400 mbar with a solvent blend of 30% n-heptane, 50% ethanol and 20% water and an extraction time of two hours. It was found that freeze-drying the pomace before extraction minimised production of artefacts, avoided degradation of biophenols, ensured long term stability of a reproducible sample and achieved better recovery of important hydrophilic and lipophilic bioactive compounds. Since the bioactive compounds are temperature sensitive, the extraction was performed under reduced pressure in order to reduce solvent reflux temperature and to improve extraction efficiency. The quantitative and qualitative determinations of the aforementioned high-value compounds were performed by high-performance liquid chromatography (HPLC), which revealed that the hydrophilic polyphenolic as well as the lipophilic α-tocopherol and squalene were present. In this study hydroxytyrosol, tyrosol, oleuropein, α-tocopherol and squalene were extracted from the pomace of two olive cultivars (Frantoio and Coratina). A comparison among the two cultivars showed quantitative differences between the two cultivars in all five high-value biological compounds and in the antioxidant capacity of the extracts evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical. Coratina cultivar was found to have a significantly higher antioxidant capacity than Frantoio due to the much greater oleuropein content in the Coratina compared to the Frantoio although Frantoio had a significantly greater amount of hydroxytyrosol. The stability of olive waste extracts stored at four temperatures was also investigated and the results show that increased temperatures caused greater extent of degradation of both the hydrophilic polyphenolic and lipophilic compounds. The proposed optimum storage condition for the olive pomace extracts was found to be at 5°C in the absence of light. The extracts were incorporated into two cosmetic formulations and were found, from a stability study, to be stable at room temperature and optimally stable at 5°C in the absence of light.
- Full Text:
- Date Issued: 2018
- Authors: Postma-Botha, Marthie
- Date: 2018
- Subjects: Organic compounds , Biochemistry , Biomass energy , Olive oil industry
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/34383 , vital:33371
- Description: Olive oil processing waste (pomace) as a by-product of the olive oil industry is regarded as a rich source of high-value biological compounds exhibiting antioxidant potential. The objective of the present work was to obtain a concentrated extract of high-value biological antioxidants from the pomace. The effect of extraction conditions on the concentration of the bioactive compounds in the extracts was investigated. The simultaneous recovery of both hydrophilic and lipophilic high-value biological compounds exhibiting antioxidant potential was achieved through a one-step extraction method under reduced pressure using a non-toxic solvent blend. A multilevel experimental design was implemented with the aim of optimising the recovery of selected compounds, namely, hydroxytyrosol, tyrosol, oleuropein, α-tocopherol and squalene from olive pomace by using solvent blends of n-heptane, d-limonene, ethanol and water. The factors considered were: extraction time, percentage composition of solvent blends and extraction temperature. The results suggested that a good recovery of the hydrophilic polyphenolic compounds, namely, hydroxytyrosol, tyrosol and oleuropein, as well as the lipophilic compounds, α-tocopherol and squalene may be achieved at a solvent temperature of 60°C at 400 mbar with a solvent blend of 30% n-heptane, 50% ethanol and 20% water and an extraction time of two hours. It was found that freeze-drying the pomace before extraction minimised production of artefacts, avoided degradation of biophenols, ensured long term stability of a reproducible sample and achieved better recovery of important hydrophilic and lipophilic bioactive compounds. Since the bioactive compounds are temperature sensitive, the extraction was performed under reduced pressure in order to reduce solvent reflux temperature and to improve extraction efficiency. The quantitative and qualitative determinations of the aforementioned high-value compounds were performed by high-performance liquid chromatography (HPLC), which revealed that the hydrophilic polyphenolic as well as the lipophilic α-tocopherol and squalene were present. In this study hydroxytyrosol, tyrosol, oleuropein, α-tocopherol and squalene were extracted from the pomace of two olive cultivars (Frantoio and Coratina). A comparison among the two cultivars showed quantitative differences between the two cultivars in all five high-value biological compounds and in the antioxidant capacity of the extracts evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical. Coratina cultivar was found to have a significantly higher antioxidant capacity than Frantoio due to the much greater oleuropein content in the Coratina compared to the Frantoio although Frantoio had a significantly greater amount of hydroxytyrosol. The stability of olive waste extracts stored at four temperatures was also investigated and the results show that increased temperatures caused greater extent of degradation of both the hydrophilic polyphenolic and lipophilic compounds. The proposed optimum storage condition for the olive pomace extracts was found to be at 5°C in the absence of light. The extracts were incorporated into two cosmetic formulations and were found, from a stability study, to be stable at room temperature and optimally stable at 5°C in the absence of light.
- Full Text:
- Date Issued: 2018
A lignocellulolytic enzyme system for fruit waste degradation : commercial enzyme mixture synergy and bioreactor design
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
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