Call number: LD2668 .T4 1984 D93 / Master of Science

Call number: LD2668 .T4 1984 G666 / Master of Science

Call number: LD2668 .T4 1984 M37 / Master of Science

A dissertation submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy in Engineering, October 2017 / The dwindling fossil reserves coupled with environmental pollution necessitate the search for clean and sustainable energy resources. Biohydrogen is emerging as a suitable alternative to fossil fuels and has received considerable attention in recent years due to its economic, social, and environmental benefits. However, the industrial application of biohydrogen has been hindered by low yield. Therefore, development of novel techniques to enhance the yield is of immense importance towards large-scale production of biohydrogen. Thus, this research effort explored various options to enhance the yield of biohydrogen during dark fermentation process. Some options explored included (i) the utilization of feedstocks from the agricultural, industrial and municipal sectors, (ii) parametric optimization of biohydrogen production, (iii) investigation of biohydrogen production using metal ions and nitrogen gas sparging, and (iv) assessing the feasibility of biohydrogen scale-up study to pave the way for pilot-scale development. Solid biowaste feedstocks consisting of apple, bread, brewery residue, cabbage, corn-cob, mango, mealie-pap, pear, potato, and sugarcane were investigated for dark fermentative biohydrogen production using anaerobic mixed sludge. The experimental results showed that substrates which are rich in carbohydrates are suitable for dark fermentative biohydrogen-producing bacteria. Consequently, a maximum biohydrogen fraction of 43.98, 40.32 and 38.12% with a corresponding cumulative biohydrogen yield of 278.36, 238.32 and 215.69 mL H2/g total volatile solids (TVS) was obtained using potato, cabbage, and brewery wastes, respectively. Based on these results, potato waste was chosen as a suitable substrate for subsequent biohydrogen production studies. Parametric optimization was carried out on biohydrogen production via dark fermentation using potato waste as the substrate. Effects of operating variables such as pH, temperature, fermentation time, and substrate concentration were investigated via response surface methodology (RSM) approach using a two-level-four factor (24) central composite design (CCD). The obtained predictive model (statistical model) was used to explain the main and interaction effects of the considered variables on biohydrogen production. In addition, the model was employed in the optimization of the operating conditions. Consequently, a secondorder polynomial regression with a coefficient of determination (R2) of 0.99 was obtained and used in the explanation and optimization of operating variables. The optimum operating conditions for biohydrogen production were 39.56 g/L, 5.56, 37.87 oC and 82.58 h for potato waste concentration, pH, temperature and fermentation time, respectively, with a corresponding biohydrogen yield of 68.54 mL H2/g TVS. These results were then validated experimentally and a high biohydrogen yield of 79.43 mL H2/g TVS indicating a 15.9% increase was obtained. Furthermore, the optimized fermentation conditions were applied in the scale-up study of biohydrogen production that employed anaerobic mixed bacteria (sludge) which was immobilized in calcium alginate beads. A biohydrogen fraction of 56.38% with a concomitant yield of 298.11 mL H2/g TVS was achieved from the scale-up study. The research also investigated the influence of metal ions (Fe2+, Ca2+, Mg2+ and Ni2+) on biohydrogen production from suspended and immobilized cells of anaerobic mixed sludge using the established optimal operating conditions. A maximum biohydrogen fraction of 45.21% and a corresponding yield of 292.8 mL H2/g TVS was achieved in fermentation using Fe2+ (1000 mg/L) and immobilized cells. The yield was 1.3 times higher than that of suspended cultures. The effect of nitrogen gas sparging on biohydrogen conversion efficiency (via suspended and immobilized cells) was studied as well. Cell immobilization and nitrogen gas sparging were effective for biohydrogen production enhancement. A maximum biohydrogen fraction of 56.98% corresponding to a biohydrogen yield of 294.83 mL H2/g TVS was obtained in a batch process using nitrogen gas sparging with immobilized cultures. The yield was 1.8 and 2.5 times higher than that of nitrogen gas sparged and non-sparged suspended cell system, respectively. Understanding the functional role of microorganisms that actively participate in dark fermentation process could provide in-depth information for the metabolic enhancement of biohydrogen-producing pathways. Therefore, the microbial composition in the fermentation medium of the optimal substrate (potato waste) was examined using PCR-based 16S rRNA approach. Microbial inventory analysis confirmed the presence of Clostridium species which are the dominant biohydrogen-producing bacteria. The results obtained from this research demonstrated the potential of producing biohydrogen using South African solid biowaste effluents. These feedstocks are advantageous in biohydrogen production because they are highly accessible, rich in nutritional content, and cause huge environmental concerns. Furthermore, optimization techniques using these feedstocks will play a pivotal role towards large-scale production of biohydrogen by increasing throughput and reducing the substrate costs which accounts for approximately 60% of the overall costs. The findings from this research also provide a solid basis for further scale-up and techno-economic studies. Such studies are necessary to evaluate the competitiveness of this technology with the traditional processes of hydrogen production. In summary, the findings from this research effort have been communicated to researchers in the area of biohydrogen process development in the form of peer-reviewed international scientific publications and conference proceedings, and could provide a platform for developing an economic biohydrogen scaled-up process. / CK2018

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 26, 2010). Thesis advisor: Dr. Wyatt Thompson. Includes bibliographical references.

Title from PDF of title page (University of Missouri--Columbia, viewed on March 2, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dr. Sunggyu Lee, Dissertation Advisor. Vita. Includes bibliographical references.

M.Sc. / Biofuels have the potential to reduce a country’s dependence on imported oil, to ensure diversity of energy sources, to increase the availability of renewable energy sources and to address global environmental issues. In recognition of the potential benefits of the production and use of biofuels, the Department of Minerals and Energy released the Draft Biofuels Industrial Strategy in December 2006 with the aim to increase the use of biofuels in South Africa to replace 4.5% of conventional transport fuels by 2013. However, there are several barriers that need to be overcome before South Africa can establish a large-scale biofuel industry to achieve the DME’s biofuel target. This includes environmental barriers, such as the availability of land for the cultivation of biofuel feedstocks and potential threats to food security. This study focuses on these environmental barriers and aims to determine the potential for bioethanol production from maize in South Africa to 2013. To this purpose, a bioethanol potential model is developed to simulate the potential for bioethanol production from maize in South Africa between 2008 and 2013. The model incorporates four key elements that all impact on the availability of maize for bioethanol production, namely: maize demand; maize supply; the demand for maize as biomaterial; and the available land area for the cultivation of maize. The study makes further use of the scenario planning method to determine the potential for bioethanol production from maize in South Africa. Four unique bioethanol potential scenarios are designed and simulated within the bioethanol potential model developed for this purpose. Each scenario plays out a differentstoryline for the future social, economic and natural environment that will impact on the availability of maize for bioethanol production. The results of the bioethanol potential scenario simulations show that South Africa will be able to produce enough maize to meet the DME’s biofuel target of 1.2 billion liters of bioethanol for all scenarios between 2009 and 2010. From 2011 onwards, the bioethanol potential decreases below the DME’s target value in both the worst case and rapid change scenarios. The study concludes that the production of bioethanol from maize in South Africa will have various social, economic and environmental consequences for the country’s agricultural sector. The depletion of domestic maize supplies will seriously threaten food security and consequently, increase the country’s dependence on maize imports. This will not only affect the country’s maize producing regions, but spread throughout South Africa as the demand for agriculturally productive land for maize production increases. Domestic food security is therefore at risk and South Africa will have to resort to other energy technologies to achieve a sustainable and renewable energy future for road transport.

The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.

Thesis (M. App. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 130-133). Also available in electronic format on the Internet.

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.