Development of an enzyme-synergy based bioreactor system for the beneficiation of apple pomace lignocellulosic waste
- Authors: Abboo, Sagaran
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/315 , vital:19947
- Description: Due to the finite supply of non-renewable fossil fuels, agro-industrial wastes are identified as alternate, renewable sources for energy supply. Large amounts of fruit waste are generated in South Africa due to fruit juice and wine processing from apples, grapes and citrus fruit. Apple pomace is the solid residue that is left over after juice, cider and wine processing and constitutes between 25-30% of the total fruit. On a global scale millions of tonnes of apple pomace are produced; between 2006-2007 over 46 million tonnes were produced. In South Africa a total production of 244 469 tonnes were produced during the 2011- 2012 season. Initially, apple pomace was regarded as a waste by-product used for animal feed and compost in soil, however presently it is considered a source of dietary fiber and natural antioxidants like polyphenols. In addition, apple pomace has a high carbohydrate content and can be enzymatically hydrolysed to produce sugar monomers which, in turn, can be fermented by yeasts to produce bioethanol. The polyphenols present in apple pomace can be used for their health properties, and the bioethanol can be used as a replacement for fossil fuel. Apple pomace is lignocellulosic in nature and consists of hemicellulose, cellulose, lignin and pectin. A combination of enzymes such as cellulases, hemicellulases, pectinases and lignases are required to operate in synergy for the degradation of lignocellulosic biomass. This is due to the recalcitrant nature of lignocellulose. This study investigated the degradation of apple pomace using a combination of commercially obtained enzyme cocktails viz. Viscozyme L , Celluclast 1.5L and Novozyme 188. The commercial enzymes Viscozyme L and Celluclast 1.5L were added in a ratio of 1:1 (50%:50%). The final concentrations of the enzymes were 0.019 mg/ml each. Novozyme 188 was added to provide a final concentration of 0.0024 mg/ml. A novel cost effective 20L bioreactor was designed, constructed and implemented for the degradation of apple pomace to produce value added products. The hydrolysis of the apple pomace was performed initially in 1 L flasks (batch fed) and, once optimized, scaled up to a 20 L bioreactor in batch mode. The bioreactors were operated at room temperature (22 ± 2ºC) and in an unbuffered system. The sugars released were detected and quantified using an optimized validated HPLC method established in this study. The sugars released in the bioreactors were mainly glucose, galactose, arabinose, cellobiose and fructose. The polyphenols released in this study were gallic acid, catechin, epicatechin, chlorogenic acid, rutin and phloridzin, which have a number of health benefits. The simultaneous analyses of the polyphenols were performed using a newly developed and validated HPLC method established in this study. This method was developed to detect nine polyphenols simultaneously. The two HPLC methods developed and validated in this study for the analysis of sugars and polyphenols demonstrated good accuracy, precision, reproducibility, linearity, robustness and sensitivity. Both analytical methods were validated according to the International Convention on Harmonization (ICH). The HPLC parameters for sugar analysis were: refractive index (RI) as the detection mode, the stationary phase was a ligand-exchange sugar column (Shodex SP0810) and an aqueous mobile phase in isocratic mode was used. The HPLC method for polyphenols employed UV diode array detection (DAD) as the detection mode, a reverse phase column as the stationary phase and a mobile phase of consisting of 0.01 M phosphoric acid in water and 100% methanol using gradient elution mode. The highest concentrations of sugars released in the novel 20 L bioreactor with 20% apple pomace (w/v) substrate loading were as follow: glucose (6.5 mg/ml), followed by galactose (2.1 mg/ml), arabinose (1.4 mg/ml), cellobiose (0.7 mg/ml) and fructose (0.5 mg/ml). The amounts of polyphenols released at 20% (w/v) apple pomace substrate were epicatechin (0.01 mg/ml), catechin (0.002 mg/ml), rutin (0.03 mg/ml), chlorogenic acid (0.002 mg/ml) and gallic acid 0.01 (mg/ml). Two mathematical models were developed in this study for kinetic analysis of lignocellulose (apple pomace) hydrolysis in the novel 20 L bioreactor, using the experimental data generated by the above HPLC analyses. The first model, modelling with regression, defines the hydrolysis of the sugars glucose, galactose, cellobiose and arabinose produced in the novel 20 L bioreactor at 5%, 10%, 15% and 20% (w/v) substrate concentrations. The regression model describes the sugars produced in the 20 L bioreactor by minimizing the error of the sugars released by finding a value for K which minimises the function which computes the sum of squares of errors between the solution curves and the data points. The second, more complex, model developed in this study used a system of differential equations model (ODE). This model solved the system by using a numerical method, such as the Runge-Kutta method, then fitted the solution curves to the data. Both models simulated (and had the ability to predict) the production of sugars in the novel 20 L bioreactor for apple pomace hydrolysis. These two models also revealed the time at which the maximum amount of sugars were released, which revealed the optimum time to run the 20 L bioreactor in order to be more cost effective. The optimum time for maximum glucose (the main sugar used in fermentation for biofuel production) release was determined to be around 60 h. The ODE model, in addition, determined the rate at which the substrate became depleted, as well as the rate at which the enzymes became deactivated for the various substrate loadings in the 20 L bioreactor. A third model was developed to determine the optimal running cost of the bioreactor which incorporated the substrate loading and the amount of glucose (g/L) produced. The novel 20 L bioreactor constructed from cost effective materials demonstrated that agro-industrial waste can be converted to value-added products by lignocellolytic enzymes. The sugars released from apple pomace can be used in biofuel production and the polyphenols as food supplements and nutraceuticals for health benefits. This novel study contributes to agro-industrial waste beneficiation via fuel production. In addition, using agro-industrial waste for the generation of value added products (instead of mere disposal) will help prevent environmental pollution.
- Full Text:
- Date Issued: 2016
- Authors: Abboo, Sagaran
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/315 , vital:19947
- Description: Due to the finite supply of non-renewable fossil fuels, agro-industrial wastes are identified as alternate, renewable sources for energy supply. Large amounts of fruit waste are generated in South Africa due to fruit juice and wine processing from apples, grapes and citrus fruit. Apple pomace is the solid residue that is left over after juice, cider and wine processing and constitutes between 25-30% of the total fruit. On a global scale millions of tonnes of apple pomace are produced; between 2006-2007 over 46 million tonnes were produced. In South Africa a total production of 244 469 tonnes were produced during the 2011- 2012 season. Initially, apple pomace was regarded as a waste by-product used for animal feed and compost in soil, however presently it is considered a source of dietary fiber and natural antioxidants like polyphenols. In addition, apple pomace has a high carbohydrate content and can be enzymatically hydrolysed to produce sugar monomers which, in turn, can be fermented by yeasts to produce bioethanol. The polyphenols present in apple pomace can be used for their health properties, and the bioethanol can be used as a replacement for fossil fuel. Apple pomace is lignocellulosic in nature and consists of hemicellulose, cellulose, lignin and pectin. A combination of enzymes such as cellulases, hemicellulases, pectinases and lignases are required to operate in synergy for the degradation of lignocellulosic biomass. This is due to the recalcitrant nature of lignocellulose. This study investigated the degradation of apple pomace using a combination of commercially obtained enzyme cocktails viz. Viscozyme L , Celluclast 1.5L and Novozyme 188. The commercial enzymes Viscozyme L and Celluclast 1.5L were added in a ratio of 1:1 (50%:50%). The final concentrations of the enzymes were 0.019 mg/ml each. Novozyme 188 was added to provide a final concentration of 0.0024 mg/ml. A novel cost effective 20L bioreactor was designed, constructed and implemented for the degradation of apple pomace to produce value added products. The hydrolysis of the apple pomace was performed initially in 1 L flasks (batch fed) and, once optimized, scaled up to a 20 L bioreactor in batch mode. The bioreactors were operated at room temperature (22 ± 2ºC) and in an unbuffered system. The sugars released were detected and quantified using an optimized validated HPLC method established in this study. The sugars released in the bioreactors were mainly glucose, galactose, arabinose, cellobiose and fructose. The polyphenols released in this study were gallic acid, catechin, epicatechin, chlorogenic acid, rutin and phloridzin, which have a number of health benefits. The simultaneous analyses of the polyphenols were performed using a newly developed and validated HPLC method established in this study. This method was developed to detect nine polyphenols simultaneously. The two HPLC methods developed and validated in this study for the analysis of sugars and polyphenols demonstrated good accuracy, precision, reproducibility, linearity, robustness and sensitivity. Both analytical methods were validated according to the International Convention on Harmonization (ICH). The HPLC parameters for sugar analysis were: refractive index (RI) as the detection mode, the stationary phase was a ligand-exchange sugar column (Shodex SP0810) and an aqueous mobile phase in isocratic mode was used. The HPLC method for polyphenols employed UV diode array detection (DAD) as the detection mode, a reverse phase column as the stationary phase and a mobile phase of consisting of 0.01 M phosphoric acid in water and 100% methanol using gradient elution mode. The highest concentrations of sugars released in the novel 20 L bioreactor with 20% apple pomace (w/v) substrate loading were as follow: glucose (6.5 mg/ml), followed by galactose (2.1 mg/ml), arabinose (1.4 mg/ml), cellobiose (0.7 mg/ml) and fructose (0.5 mg/ml). The amounts of polyphenols released at 20% (w/v) apple pomace substrate were epicatechin (0.01 mg/ml), catechin (0.002 mg/ml), rutin (0.03 mg/ml), chlorogenic acid (0.002 mg/ml) and gallic acid 0.01 (mg/ml). Two mathematical models were developed in this study for kinetic analysis of lignocellulose (apple pomace) hydrolysis in the novel 20 L bioreactor, using the experimental data generated by the above HPLC analyses. The first model, modelling with regression, defines the hydrolysis of the sugars glucose, galactose, cellobiose and arabinose produced in the novel 20 L bioreactor at 5%, 10%, 15% and 20% (w/v) substrate concentrations. The regression model describes the sugars produced in the 20 L bioreactor by minimizing the error of the sugars released by finding a value for K which minimises the function which computes the sum of squares of errors between the solution curves and the data points. The second, more complex, model developed in this study used a system of differential equations model (ODE). This model solved the system by using a numerical method, such as the Runge-Kutta method, then fitted the solution curves to the data. Both models simulated (and had the ability to predict) the production of sugars in the novel 20 L bioreactor for apple pomace hydrolysis. These two models also revealed the time at which the maximum amount of sugars were released, which revealed the optimum time to run the 20 L bioreactor in order to be more cost effective. The optimum time for maximum glucose (the main sugar used in fermentation for biofuel production) release was determined to be around 60 h. The ODE model, in addition, determined the rate at which the substrate became depleted, as well as the rate at which the enzymes became deactivated for the various substrate loadings in the 20 L bioreactor. A third model was developed to determine the optimal running cost of the bioreactor which incorporated the substrate loading and the amount of glucose (g/L) produced. The novel 20 L bioreactor constructed from cost effective materials demonstrated that agro-industrial waste can be converted to value-added products by lignocellolytic enzymes. The sugars released from apple pomace can be used in biofuel production and the polyphenols as food supplements and nutraceuticals for health benefits. This novel study contributes to agro-industrial waste beneficiation via fuel production. In addition, using agro-industrial waste for the generation of value added products (instead of mere disposal) will help prevent environmental pollution.
- Full Text:
- Date Issued: 2016
The inhibitory effects of various substrate pre-treatment by-products and wash liquors on mannanolytic enzymes
- Malgas, Samkelo, Van Dyk, J Susan, Abboo, Sagaran, Pletschke, Brett I
- Authors: Malgas, Samkelo , Van Dyk, J Susan , Abboo, Sagaran , Pletschke, Brett I
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66156 , vital:28911 , https://doi.org/10.1016/j.molcatb.2015.11.014
- Description: publisher version , Biomass pre-treatment is essential for achieving high levels of bioconversion through increased accessibility of hydrolytic enzymes to hydrolysable carbohydrates. However, pre-treatment by-products, such as sugar and lignin degradation products, can negatively affect the performance of hydrolytic (mannanolytic) enzymes. In this study, two monomeric sugars, five sugar degradation products, five lignin derivatives and four liquors from biomass feedstocks pre-treated by different technologies, were evaluated for their inhibitory effects on mannanolytic enzymes (α-galactosidases, β-mannanases and β-mannosidases). Lignin derivatives elicited the greatest inhibitory effect on the mannanolytic enzymes, followed by organic acids and furan derivatives derived from sugar degradation. Lignin derivative inhibition appeared to be as a result of protein–phenolic complexation, leading to protein precipitating out of solution. The functional groups on the phenolic lignin derivatives appeared to be directly related to the ability of the phenolic to interfere with enzyme activity, with the phenolic containing the highest hydroxyl group content exhibiting the greatest inhibition. It was also demonstrated that various pre-treatment technologies render different pre-treatment soluble by-products which interact in various ways with the mannanolytic enzymes. The different types of biomass (i.e. different plant species) were also shown to release different by-products that interacted with the mannanolytic enzymes in a diverse manner even when the biomass was pre-treated using the same technology. Enzyme inhibition by pre-treatment by-products can be alleviated through the removal of these compounds prior to enzymatic hydrolysis to maximize enzyme activity.
- Full Text: false
- Date Issued: 2016
- Authors: Malgas, Samkelo , Van Dyk, J Susan , Abboo, Sagaran , Pletschke, Brett I
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66156 , vital:28911 , https://doi.org/10.1016/j.molcatb.2015.11.014
- Description: publisher version , Biomass pre-treatment is essential for achieving high levels of bioconversion through increased accessibility of hydrolytic enzymes to hydrolysable carbohydrates. However, pre-treatment by-products, such as sugar and lignin degradation products, can negatively affect the performance of hydrolytic (mannanolytic) enzymes. In this study, two monomeric sugars, five sugar degradation products, five lignin derivatives and four liquors from biomass feedstocks pre-treated by different technologies, were evaluated for their inhibitory effects on mannanolytic enzymes (α-galactosidases, β-mannanases and β-mannosidases). Lignin derivatives elicited the greatest inhibitory effect on the mannanolytic enzymes, followed by organic acids and furan derivatives derived from sugar degradation. Lignin derivative inhibition appeared to be as a result of protein–phenolic complexation, leading to protein precipitating out of solution. The functional groups on the phenolic lignin derivatives appeared to be directly related to the ability of the phenolic to interfere with enzyme activity, with the phenolic containing the highest hydroxyl group content exhibiting the greatest inhibition. It was also demonstrated that various pre-treatment technologies render different pre-treatment soluble by-products which interact in various ways with the mannanolytic enzymes. The different types of biomass (i.e. different plant species) were also shown to release different by-products that interacted with the mannanolytic enzymes in a diverse manner even when the biomass was pre-treated using the same technology. Enzyme inhibition by pre-treatment by-products can be alleviated through the removal of these compounds prior to enzymatic hydrolysis to maximize enzyme activity.
- Full Text: false
- Date Issued: 2016
Phenolic compounds in water and the implications for rapid detection of indicator micro-organisms using ß-D-Galactosidase and ß-D-Glucuronidase
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
- «
- ‹
- 1
- ›
- »