Theses and dissertations (Applied Sciences)
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Item Development of chitosan biopolymer films by fungal fermentation of waste substrates(2024-05) Naidoo, Krinolen Krishna Rajahrathanum; Permaul, Kugen; Govender, Algasan; Puri, Adarsh KumarZygomycetes are known for their relatively high chitosan content (approximately 10% m/m) in comparison with other fungal genera. In this study, Mucor circinelloides was grown on the following industrial waste substrates: corn steep liquor (CSL); soft drink overflow spillage waste (DBW); and sugarcane molasses (MOL). Biomass production on waste substrates was statistically optimized by Plackett-Burman design in conjunction with Response Surface Methodology, followed by validation of the model. DBW hindered fungal biomass growth and was found to be a statistically insignificant variable and therefore omitted from further optimizations. The validated model produced a biomass of 77.87 g/L, a 2.65-fold increase over the highest-yielding unoptimized medium. Fungal biomass obtained after batch fermentation was subjected to acid-alkaline treatment for chitin extraction from the cell wall and deacetylation of the chitin to chitosan. A yield of 8-9% chitosan was obtained from the fungal biomass. FTIR spectroscopic analysis was conducted on the extracted fungal chitosan to compare extracted chitosan against commercial chitosan and chitosan monomer. The waste-grown, fungal-derived chitosan profiles were similar to those of commercial crustacean chitosan. The extracted chitosan was used in conjunction with additives and solvent systems to create biopolymer variants with differing properties. A library of data from the chitosan biopolymer variants was generated with considerable differences in characteristics based on their composition. Improvements in sample #11 (the most modified formulation) in contrast to the most common chitosan biopolymer film composition used in literature (sample #9), included a 3.37-fold improvement in the static force required to break the film. There was a 3.39-fold increase in tensile strength and an 11-fold reduction in elongation (%) and elongation rates. The creation of these variants will allow the use of these chitosan biopolymers for specific industrial applications.Item Optimization of biomass and lipids production from microalgae using wastewater in a pilot scale raceway pond(2021) Rawat, Ismail; Bux, FaizalMicroalgae provide a sustainable renewable solution for the production of commodity products such as liquid biofuels. There are numerous benefits to using algae for the production of biofuels, however, the cost of production is a major hurdle to commercial-scale development. Major factors influencing the production of algae are the cost of nutrients, availability of water, contamination, and grazers. Research into algal biomass for biofuels production at laboratory scale does not translate directly to cultivation at large scale due to the change in cultivation conditions and the constant flux of environmental factors. This study focuses on the upstream processes of cultivation of biomass in a ~ 1146 m2 raceway pond. It demonstrates biomass productivity under different climatic conditions and utilisation of post-chlorinated wastewater as a water and nutrient source. The study further elucidates the population dynamics of the system and provides insight into the challenges faced during the cultivation of algae at large scale. An indigenous Scenedesmus sp. gave biomass productivity of 31.23 g/m2 /d with lipid production of 29.6 % lipid/g DCW in a 10 m2 raceway pond in a greenhouse using BG11. Biomass productivity was reduced to 13.09 g/m2 /d with a lipid content of 22.9 % lipid/g DCW under 3-fold higher irradiance. Biomass productivity of circular 3000L ponds at the large scale site resulted in the highest biomass and acceptable lipid content using 250mg/L NaNO3 although significantly lower than the 10 m2 raceway ponds. Wastewater has shown potential to replace conventional media. Post-chlorinated wastewater was found to have low levels of nitrogen and phosphorus but contained metals that act as micronutrients for algae. Supplemented wastewater proved to be an effective growth. Six individual runs of a covered 1146 m2 raceway pond driven by paddlewheel were conducted over 15 months. The average water temperature ranged from 20.61±0.68°C during mid-winter to 31.03±2.22°C in late summer. Daylight ranges from 10.25 to 14 hours in winter and summer respectively. The highest average light intensity was 359.00±212.71 µmol/m2 /s from Mid-winter to early spring and 645.44±330.58 µmol/m2 /s in late summer. Biomass productivities were low ranging from 2.7 to 7.34 g/m2 /d for most runs of the raceway pond, mainly due to the long periods of cultivation. Average productivity at day 7 for all raceway runs was 7.25 g/m2 /d. Adaptive Neuro-Fuzzy Inference System (ANFIS) modelling of the system elicited that the major factors affecting biomass productivity in the raceway pond were light intensity, pH, and depth for the raceway pond. The model showed that maximum biomass productivity is possible at a depth between 20 and 22 cm at light intensities between 200 and 400 µmol/m2 /s. pH in the range of 9 to 9.5 correlated positively with light intensity ranging from 200 to 1000 µmol/m2 /s with maximum biomass expected in the region of 400 to 500 µmol/m2 /s. The main algal constituents for the raceway ponds were Scenedesmus obliquus, Scenedesmus dimorphus, Chlorella, Keratococcus, and species of unidentified cyanobacteria. Either Scenedesmus or Chlorella was dominant for extended periods. Bacteria in open systems can have a positive or negative effect on the growth of microalgae but is dependent on the strains of microalgae and bacteria as well as prevailing conditions making these systems highly complex. Rhodobacteraceae, Plactomycetaceae, Xanthomonadaceae, Flavobacteriaceae, Phycisphaeraceae, Comamonadaceae, and Cyclobacteriaceae were found to be the major families of bacteria that proliferate at different levels during the cultivation period in the circular ponds and the raceway pond. These families of bacteria have several beneficial traits to algae cultivation however further investigation is required. Modelling the system revealed that pH, depth, and light intensity were factors having a substantial effect on biomass productivity. As the system was carbon limited addition of CO2 (preferably a waste stream) could significantly enhance the overall biomass productivity. A major factor negatively affecting biomass productivity was the size of the pond. Inadequate mixing impacts biomass productivity in terms of access to nutrients and gaseous exchange. Shorter periods of cultivation resulted in higher productivities. For the scale of the system, semi-continuous harvesting would be required to achieve shorter residence time. This must be balanced against the energy utilization and cost of harvesting potentially lower culture densitiesItem Valorisation of bambara and cowpea haulms for bioethanol production(2020) Okuofu, Somiame Itseme; Pillai, Santhosh Kumar KuttanBambara and cowpea are important pulses grown in semi-arid South Africa due to their balanced nutrient profile and drought resilient capacity. The haulm is the lignocellulosic residue obtained after grain harvest and are rich in carbohydrates. However, these haulms are underutilised and under researched. The aim of the study, therefore, was to investigate the potential to valorise bambara haulms (BGH) and cowpea haulms (CH) to bioethanol which is the most promising biofuel with commercial prospects currently. The structural and chemical composition of BGH and CH was elucidated using techniques such as compositional analysis, XRD, FTIR, ICP-AES, and SEM. Results indicated a volatile matter and fixed carbon mass fraction of 77.70% and 13.15% (w/w) in BGH and 76.16% and 16.26% (w/w) in CH respectively. The polysaccharides make up the largest fraction (51%), followed by extractives (> 20%), while the lignin in BGH (12%) and CH (10%) was low. X-ray diffraction pattern showed a higher percentage of amorphous regions in BGH (78%) than CH (56%). CH was then subjected to dilute acid pretreatment (DAP) to enhance biosugar production for bioethanol fermentation. The effects of operational factors for DAP including temperature, time, and acid concentration on sugar yield and inhibitor formation was investigated and optimised using response surface methodology (RSM). The solid recovered after DAP was subjected to prehydrolysis with simultaneous saccharification and fermentation (PSSF). In addition, the pretreatment hydrolysate was detoxified and fermented to ethanol using cocultures of Saccharomyces cerevisiae BY4743 and Scheffersomyces stipitis wild type (PsY633). A total ethanol titre of 15.67 g/L was obtained corresponding to 75% conversion efficiency. On the other hand, BGH was subjected to deep eutectic solvent (DES) pretreatment. Five deep eutectic solvents were prepared and screened for their effectiveness in improving enzymatic sugar yield. This was achieved by pretreating BGH with each DES followed by a 48 h enzymatic saccharification. Choline chloride – lactic acid (ChCl-LA) treatment provided the most promising result and was further optimised by investigating the effect of different temperatures and time on cellulose loss and enzymatic sugar yield. ChCl-LA pretreatment at 100°C for 1 h was observed to be the best condition for maximum sugar recovery. The hydrolysate thus obtained was concentrated and fermented for 72 h with S. cerevisiae BY4743. A maximum ethanol yield of 11.57 g/L was obtained. From the results, it is evident that bambara and cowpea haulm are promising substrates for bioethanol production. Dilute acid hydrolysis was shown to be effective in the pretreatment of CH with over 85% of the theoretical sugar recoverable for conversion to bioethanol. In addition, deep eutectic solvents are effective media for breaking the recalcitrance in BGH to achieve high sugar yield for conversion to bioethanol. However, further studies are required to reduce cellulose loss during pretreatment to improve bioethanol yield.Item Enhancement of biohydrogen production from the aquatic weed Pistia stratiotes through a dark fermentation process(2019) Mthethwa, Nonsikelelo Precios; Pillai, Santhosh Kumar Kuttan; Bux, Faizal; Kiambi, Sammy LewisAquatic weeds are well known for their fast growth rate and high carbohydrate content that can be easily hydrolysed into fermentable sugars. This study was aimed at the utilization of an indigenous aquatic weed, Pistia stratiotes for biohydrogen production through the dark fermentation process. Characterization of the biomass, effect of pre–treatment methods on biomass hydrolysis, effect of reactor operational conditions and type of inoculum on enhancing hydrogen production potential of P. stratiotes was assessed. Physical and chemical pre–treatments were employed on P. stratiotes biomass to increase digestibility and to achieve high conversion rates of fermentable sugars. The highest sugar yield of 139± 0.8 mg/g was obtained when the oven dried biomass was subjected to H2SO4 (2.5%) pre– treatment followed by autoclaving at 121°C for 30 min. Biohydrogen production under different operational conditions was thereafter optimized using One–factor–at–a–time (OFAT) batch experiments in 120 mL serum bottles. A maximum hydrogen yield (HY) of 2.46 ± 0.14 mol-H2/mol-glucose (3.51 ± 0.20 mg-H2/g-dry weight) and 2.75 ± 0.07 mL h-1 hydrogen production rate was observed under optimized conditions (pH 5.5, Temp 35°C, S/X: 1.0 g-COD/g-VSS and HRT 8 h). The organic mass balance (92 – 96%) and electron– equivalent balance (92 – 98%) further indicated the reliability of the obtained fermentation data. Assessment of microbial activity was achieved using molecular techniques such as quantitative polymerase chain reaction (qPCR) targeting both 16s rRNA (of Clostridium spp., Bacillus spp., and Enterobacter spp.) and the functional hydrogenase gene (hydA). The highest gene activity of hydrogenase was noted at pH of 5.5 with 2.53×104 copies/ng-DNA compared to low pH: 4.5 (6.95 × 103 copies/ng-DNA) and high pH: 8.5 (7.77×103 copies/ng- DNA). A similar trend was also observed for the species containing a highly active hydrogenase (i.e. Clostridium spp., Bacillus spp., and Enterobacter spp.). During the optimum reactor conditions, three hydrogen producing bacterial strains Bacillus cereus and Enterobacter cloacae were successfully isolated. These isolates were used as inoculums for the pure culture studies and achieved HYs of 2.2, 1.10 and 1.97 mol-H2/mol-glucose respectively under optimized fermentation conditions. However, the thermally treated mixed culture displayed a marginally higher HY (2.46 mol-H2/mol-glucose) compared to the pure culture used alone. Furthermore, the cost estimation indicated a potential and economically feasible for biotransformation of P. stratiotes to hydrogen energy. In conclusion, the results from this study has revealed the potential of employing P. stratiotes biomass for biohydrogen production. The results also indicated the importance of employing suitable pre–treatment methods, operating conditions as well as inoculum types for enhanced hydrogen production using P. stratiotes.Item Evaluating the sustainability of waste substrates for microalgal biomass production using different modes of cultivation(2018) Ramsundar, Prathana; Bux, Faizal; Guldhe, AbhishekThe utilization of wastewater as a substrate for microalgal biomass cultivation is one of the few potentially viable routes for fuel and feed applications. In this study, the suitability of various liquid wastewater streams and waste biosolids from a domestic wastewater treatment plant was assessed for microalgal cultivation. The wastewater substrates were analyzed for nutrient content as a potential growth medium. For liquid waste substrates, physical, thermal and biological pre-treatment methods were evaluated to minimize the bacterial load. Biomass, physiology, nutrient removal efficiencies and biochemical constituents of Chlorella sorokiniana were investigated in influent (INF) and anaerobic tank centrate (AC) under mixotrophic (Mixo) and heterotrophic (Hetero) cultivation modes. Mixotrophic cultivation conditions demonstrated efficient ammonium (94.29%) and phosphate (83.30%) removal with promising biomass (77.14 mgL-1d-1), lipid (24.91 mgL-1d-1), protein (22.36 mgL-1d-1) and carbohydrate (20.10 mgL-1d-1) productivities. Urea supplementation (1500 mgL-1) further enhanced biomass (162.50 mgL-1d-1), lipid (24.91 mgL-1d-1), protein (22.36 mgL-1d-1) and carbohydrate (20.10 mgL-1d-1) productivities in Mixo AC. Therefore, the urea supplemented Mixo AC approach for microalgal cultivation was developed as a suitable biomass production strategy. This work also elucidated a novel algae cultivation strategy for utilisation of waste biosolids, where nutrient-rich waste activated sludge (WAS) and final effluent (FE) from the wastewater treatment process was used for microalgal biomass generation. This strategy reduced the use of synthetic nutrients, fertilizers and freshwater which contribute significantly towards the overall cost of biomass production. Strategy development included the investigation of physical, thermal and chemical pre-treatment methods to assist in effective nutrient release and bacterial load reduction. Evaluation of growth kinetics, photosynthetic performance, nutrient removal efficiencies and biochemical composition of microalgae under mixotrophic and heterotrophic modes of cultivation were performed. Furthermore, urea supplementation was studied to improve biomass productivity. Microalgae cultivation in acid pre-treated (pH 2) WAS + FE with urea supplementation (1500 mgL-1) showed enhanced biomass productivity of 298.75 mgL-1d-1. Microalgal biomass grown with WAS + FE using the developed strategy exhibited greater lipid (72.95 mgL-1d-1) and protein (72.84 mgL-1d-1) productivities and comparable carbohydrate yields (73.07 mgL-1d-1) to that of synthetic media. Thus mixotrophic mode of cultivation coupled with urea supplementation to WAS + FE proved to be a suitable cultivation strategy for C. sorokiniana. The study developed an efficient strategy to utilize AC and WAS + FE as a growth medium for microalgae. Furthermore, findings from this study have demonstrated the potential of waste streams and waste solids from domestic wastewater treatment plants for microalgal biomass generationItem Thermophysical properties of biofuel components derived from biomass(2016) Nduli, Mbalenhle B.; Deenadayalu, NirmalaThe thermophysical properties of the binary mixtures containing biofuel components derived from biomass were determined. Experimental densities, speed of sound, and refractive indices for the binary mixtures (methanol or 1-ethyl-3-methylimidazolium acetate [EMIM][OAc] + furfural or furfuryl alcohol ) were measured at T = (298.15, 303.15, 308.15, 313.15 and 318.15) K. From the experimental data, excess molar volume, E m V , isentropic compressibility, s , molar refractions, R, and deviation in refractive index, Δn, were calculated. The excess molar volumes were found to be negative for all systems studied. The isentropic compressibility were found to be both positive for the whole composition and temperature range and increases slightly with increasing temperature. The deviation in refractive index was positive over the whole composition range. The obtained values of excess molar volumes and changes of refractive index on mixing were satisfactorily correlated by the Redlich–Kister equation. The Lorentz–Lorenz equation was applied to predict the density and calculate the excess molar volume of the binary mixtures.Item Design and operation of a laboratory scale photobioreactor for the cultivation of microalgae(2011) Bhola, Virthie; Bux, FaizalDue to greenhouse gas emissions from fossil fuel usage, the impending threat of global climate change has increased. The need for an alternative energy feedstock that is not in direct competition to food production has drawn the focus to microalgae. Research suggests that future advances in microalgal mass culture will require closed systems as most microalgal species of interest thrive in highly selective environments. A high lipid producing microalga, identified as Chlorella vulgaris was isolated from a freshwater pond. To appraise the biofuel potential of the isolated strain, the growth kinetics, pyroletic characteristics and photosynthetic efficiency of the Chlorella sp was evaluated in vitro. The optimised preliminary conditions for higher biomass yield of the selected strain were at 4% CO2, 0.5 g l-1 NaNO3 and 0.04 g l-1 PO4, respectively. Pulse amplitude modulation results indicated that C. vulgaris could withstand a light intensity ranging from 150-350 μmol photons m-2s-1. The pyrolitic studies under inert atmosphere at different heating rates of 15, 30, 40 and 50 ºC min-1 from ambient temperature to 800 oC showed that the overall final weight loss recorded for the four different heating rates was in the range of 78.9 to 81%. A tubular photobioreactor was then designed and utilised for biomass and lipid optimisation. The suspension of microalgae was circulated by a pump and propelled to give a sufficiently turbulent flow periodically through the illuminated part and the dark part of the photobioreactor. Microalgal density was determined daily using a Spectrophotometer. Spectrophotometric determinations of biomass were periodically verified by dry cell weight measurements. Results suggest that the optimal NaNO3 concentration for cell growth in the reactor was around 7.5 g l-1, yielding maximum biomass of 2.09 g l-1 on day 16. This was a significant 2.2 fold increase in biomass (p < 0.005) when compared to results achieved at the lowest NaNO3 cycle (of 3.8 g l-1), which yielded a biomass value of 0.95 g l-1 at an OD of 1.178. Lipid accumulation experiments revealed that the microalga did not accumulate significant amounts of lipids when NaNO3 concentrations in the reactor were beyond 1.5 g l-1 (p > 0.005). The largest lipid fraction occurred when the NaNO3 concentration in the medium was 0.5 g l-1. Results suggest that the optimal trade-off between maximising biomass and lipid content occurs at 0.9 g l-1 NaNO3 among the tested conditions within the photobioreactor. Gas chromatograms showed that even though a greater number of known lipids were produced in Run 8, the total lipid percentage was much lower when compared to Runs 9-13. For maximal biomass and lipid from C. vulgaris, it is therefore crucial to optimise nutritional parameters such as NaNO3. However, suitable growth conditions for C. vulgaris in a tubular photobioreactor calls for innovative technological breakthroughs and therefore work is ongoing globally to address this.