Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xi, 209 leaves / Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.

Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry.

Thesis submitted in compliance with the requirements for the Master's Degree in Technology: Industrial Engineering, Department of Industrial Engineering, Durban University of Technology, 2009. / This research focuses on the design and implementation of an intelligent machine vision and sorting system that can be used to sort objects in an industrial environment. Machine vision systems used for sorting are either geometry driven or are based on the textural components of an object’s image. The vision system proposed in this research is based on the textural analysis of pixel content and uses an artificial neural network to perform the recognition task. The neural network has been chosen over other methods such as fuzzy logic and support vector machines because of its relative simplicity. A Bluetooth communication link facilitates the communication between the main computer housing the intelligent recognition system and the remote robot control computer located in a plant environment. Digital images of the workpiece are first compressed before the feature vectors are extracted using principal component analysis. The compressed data containing the feature vectors is transmitted via the Bluetooth channel to the remote control computer for recognition by the neural network. The network performs the recognition function and transmits a control signal to the robot control computer which guides the robot arm to place the object in an allocated position. The performance of the proposed intelligent vision and sorting system is tested under different conditions and the most attractive aspect of the design is its simplicity. The ability of the system to remain relatively immune to noise, its capacity to generalize and its fault tolerance when faced with missing data made the neural network an attractive option over fuzzy logic and support vector machines.

Due to a combination of selection criteria, sludges from upflow anaerobic digesters treating industrial waste waters consist primarily of well-settling, dense agglomerates called granules. Quantification of the component mixed microbial populations of these granules has been severely restricted by the inability of researchers to disrupt them without concomitantly destroying numerous cells. In situ quantification using light and electron microscopy is complicated by the high cell numbers and bacterial diversity; the small cell size; and the destructive nature of electron microscopy preparative techniques preventing the viewing of more than a small percentage of the population at a time. For these reasons, in this investigation, standardization of qualitative electron microscopic techniques was performed prior to their application to granules. Isolation and electron and light microscopic techniques were applied to granules from a fullscale clarigester treating effluent from a maize-processing factory. In addition, a method using montaged transmission electron micrographs (TEMs) taken along a granule radius, and image analysis, was developed for bacterial quantification within granules. This method, together with antibody probe quantification, was applied to granules from an upflow anaerobic sludge blanket (UASB) digester treating a brewery effluent. The clarigester granules contained a metabolically and morphologically diverse population of which many members were not isolated or identified. By contrast, the UASB digester granules consisted primarily of morphotypes resembling Methanothrix, Methanobacterium and Desulfobulbus, in order of predominance. However, only about one-third of the population reacted with antibody probes specific to strains of bacterial species expected to occur within these granules. According to the antibody probe library used, the Methanobacterium-like cells observed in TEMs were probably Methanobrevibacter arboriphilus. From this study it is apparent that different anaerobic digester designs, operational parameters, and the chemical composition of the waste water purified, are factors which influence the formation and maintenance of granules differing with respect to their microbial populations. Until the difficulties associated with quantification are overcome, the processes governing granule formation and/or population selection will remain obscure. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1995.

Interacting microbial associations capable of utilizing thiosulphate as an energy source were enriched/isolated from activated sludge, landfill site [mal covering soil and soil from an acid mine water drainage site. The isolates were designated Lf-I, Ws-2 and Am-3, respectively. Although hydrogen sulphide was the target molecule for gas biofiltration, thiosulphate, which is a key oxidized intermediate, was used in this study due to the difficulty of working with a toxic gas. Together with thiosulphate oxidation, the microbial associations were assessed for their abilities to oxidize dissolved sulphide to elemental sulphur. Physiological analyses (temperature, pH and substrate concentration optimization) were made with closed and open cultures while morphological characterization and species compositional changes were monitored by light and scanning electron microscopy (SEM). To investigate further functional and structural responses to physiological changes, denaturing-gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S DNA gene fragments and Biolog GN microtitre plates were used. The associations were found to be active metabolically between 0 and 35°C, 15 and 50°C, and 15 and 45°C, with optimum temperatures of 25, 40 and 35°C for Lf-l, Ws-2 and Am-3, respectively. The optimum pH range for microbial association Lf-l was between 3 and 4. The maximum specific growth rates of associations Lf-l , Ws-2 and Am-3 were 0.08, 0.06 and 0.03 h~l , respectively. Components of all three Gram negative rod-dominated associations were motile and displayed anaerobiosis. During open culture cultivation the species complement of Lf-l , as determined by morphological analysis, changed. The same association oxidized sulphide (40 ppm) to sulphur although Ws-2 and Am-3 did not have this capacity. Biolog GN plates detected pH-effected species compositional changes in Lf-l and these were confirmed by DGGE. The same technique showed that enrichment had occurred in the Biolog GN wells. Species composition changes also resulted in response to different pH values (2 to 9), temperatures (5 to 40°C) and dilution rates (0.003 to 0.09 h-1 ), but activity changes were not always accompanied by population profile changes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

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A new reactor and a novel in-situ sampling technique were developed for the study of the synthesis of CeO2 powders produced from dissolved cerium nitrate salts. The reactor minimized particle recirculation and provided a highly symmetric and undisturbed plasma flow suitable for the analysis of the phenomena affecting the formation of CeO2 powders. The sampling probe provided in-situ sampling of in-flight CeO2 particles and allowed continuous sampling without cross contamination. The sampled particles were collected using a wet collection system composed of a mist atomizer acting as a scrubber and a custom-made spray chamber. The entire collection system is portable and it was tested in the particle range of 20 nm to 100 mum. This information provided a picture of how CeO2 particles were formed. A numerical simulation of different plasma operating parameters using Fluent was presented. A comprehensive droplet-to-particle formation mechanism was deduced based on calorimetry, thermodynamics of CeO2 formation, numerical simulations and collected particles. The effect of adding water soluble fuels (alanine and glycine) to the original cerium nitrate solutions was investigated. Fuel addition decreased the temperature of CeO2 formation by acting as a local heat source as a result of fuel auto-ignition. The addition of fuel caused "particle size discrimination", and a single mode particle size distribution centered between 50-140 nm was achieved along the centerline of the reactor. / Also, heat and mass transfer effects were numerically investigated in evaporating solution droplets (20-40 mum in diameter) containing dissolved hexahydrated cerium nitrate in a stationary rf Ar-O2 thermal plasma. This model was developed to study the evaporation of a solution droplet surrounded by a porous crust in a stagnant rf Ar-O2 thermal plasma under reduced pressure. It considered a three phase system: a liquid core of dissolved Ce(NO 3)3.6H2O in water, a dry porous crust of homogeneously precipitated spherical crystals of equal size, and an Ar-O2 plasma under reduced pressure. The impact of different plasma operating parameters on the temperature and dissolved solid content profiles in the droplet was studied, i.e. surrounding plasma temperature, initial salt content and droplet size, plasma gas composition, and system pressure. Temperature and composition dependant thermophysical properties were used. The model was solved in a moving boundary frame using an ALE approach and considering Stefan flow. It provided the necessary information to understand the droplet to particle transformation steps in regions where in-flight probing was unfeasible, i.e. torch zone.

The research undertaken in this thesis includes microwave-assisted extraction (MAE), synthesis, and the investigation of the scale-up of the microwave-assisted processes with the numerical aid. / The main goal of this research is to study the various problems associated with the scale-up of the microwave-assisted extraction and synthesis processes. Laboratory studies were carried out to investigate the microwave-assisted extraction of known components from peppermint leaves and American ginseng. Various factors that influence the extraction processes were studied. Microwave-assisted extraction method was compared with conventional heating and room temperature extraction methods on the extraction of ginsenosides from American ginseng. Microwave-assisted extraction method was determined to have higher extraction rate than both room temperature extraction and reflux temperature extraction using hotplate heating indicating that there is acceleration factor in enhancing the extraction rate beyond the temperature influence. / In the study of synthesizing n-butyl paraben, microwave-assisted synthesis was observed to greatly increase the yield of n-butyl paraben in much shorter period of time compared to the classic synthesis method. A transition state theory was proposed to explain this rate enhancement. The study of the synthesis of parabens with different alcohol and the influencing factors on the synthesis of n-butyl paraben yield were also studied. / A visualization method was developed to determine the microwave distribution in a domestic microwave cavity. The method uses gypsum plate as carrier and cobalt chloride as indictor. A simulation program was developed using the finite difference time domain (FDTD) approach and written in C programming language. The program was proved to be very versatile in different type of cavity simulation. Not only cavities with different dimensions and geometrical designs can be simulated, multiple magnetrons and various ways of magnetron placement can also be integrated into the simulation program. The detailed power distribution can be visualized in a 3-D plot, and the power distribution in each layer can be analyzed using the simulation result. The power distribution information will be very useful and necessary before any real equipment development.