- Title
- Development of an enzyme-synergy based bioreactor system for the beneficiation of apple pomace lignocellulosic waste
- Creator
- Abboo, Sagaran
- Date Issued
- 2016
- Date
- 2016
- Type
- Thesis
- Type
- Doctoral
- Type
- PhD
- Identifier
- http://hdl.handle.net/10962/315
- Identifier
- 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.
- Format
- 259 leaves
- Format
- Publisher
- Rhodes University
- Publisher
- Faculty of Science, Biochemistry and Microbiology
- Language
- English
- Rights
- Abboo, Sagaran
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Abboo, Sagaran (Dr)
RU Department of Biochemistry and Microbiology (2015-)
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