This thesis presents an investigation of novel laser assisted micromachining processes that addresses the limitations of micromachining of hard-to-machine materials. Two different laser assisted approaches are used to machine hard metals and high strength ceramics. For hard metals, the basic approach involves localized thermal softening of the workpiece material by focusing a solid-state continuous wave near infra-red laser beam in front of the micro milling tool (end mills of 0.1 to 0.5 mm diameter). By suitably controlling the laser power, spot size and scan speed, it is possible to produce a sufficiently large reduction in the flow strength of the work material and consequently the cutting forces and tool deflections. A force model is developed to predict the cutting forces in Laser Assisted Micro Milling (LAMM) of hard metals. For high strength ceramics, the approach involves use of a two step process. In the first step, thermal cracks are generated in a confined volume by the steep thermal gradients generated by laser irradiation of the workpiece. In the second step, the weakened region is removed by a micro grinding tool. The characterization and modeling of the process serve as bases for users of the two approaches to select optimal process parameters.

Semi-synthetic β-lactam antibiotics are the most prescribed class of antibiotics in the world. Chemical coupling of a β-lactam moiety with an acyl side chain has dominated the industrial production of semi-synthetic β-lactam antibiotics since their discovery in the early 1960s. Enzymatic coupling of a β-lactam moiety with an acyl side chain can be accomplished in a process that is much more environmentally benign but also results in a much lower yield. The goal of the research presented in this dissertation is to improve the enzymatic synthesis of β-lactam antibiotics via reaction engineering, medium engineering and data-drive protein engineering. Reaction engineering was employed to demonstrate that the hydrolysis of penicillin G to produce the β-lactam nucleus 6-aminopenicillanic acid (6-APA), and the synthesis of ampicillin from 6-APA and (R)-phenylglycine methyl ester ((R)-PGME), can be combined in a cascade conversion. In this work, penicillin G acylase (PGA) was utilized to catalyze the hydrolysis step, and PGA and α-amino ester hydrolase (AEH) were both studied to catalyze the synthesis step. Two different reaction configurations and various relative enzyme loadings were studied. Both configurations present a promising alternative to the current two-pot set-up which requires intermittent isolation of the intermediate, 6-APA. Medium engineering is primarily of interest in β-lactam antibiotic synthesis as a means to suppress the undesired primary and secondary hydrolysis reactions. The synthesis of ampicillin from 6-APA and (R)-PGME in the presence of ethylene glycol was chosen for study after a review of the literature. It was discovered that the transesterification product of (R)-PGME and ethylene glycol, (R)-phenylglycine hydroxyethyl ester, is transiently formed during the synthesis reactions. This never reported side reaction has the ability to positively affect yield by re-directing a portion of the consumption of (R)-PGME to an intermediate that could be used to synthesize ampicillin, rather than to an unusable hydrolysis product. Protein engineering was utilized to alter the selectivity of wild-type PGA with respect to the substituent on the alpha carbon of its substrates. Four residues were identified that had altered selectivity toward the desired product, (R)-ampicillin. Furthermore, the (R)-selective variants improved the yield from pure (R)-PGME up to 2-fold and significantly decreased the amount of secondary hydrolysis present in the reactions. Overall, we have expanded the applicability of PGA and AEH for the synthesis of semi-synthetic β-lactam antibiotics. We have shown the two enzymes can be combined in a novel one-pot cascade, which has the potential to eliminate an isolation step in the current manufacturing process. Furthermore, we have shown that the previously reported ex-situ mixed donor synthesis of ampicillin for PGA can also occur in-situ in the presence of a suitable side chain acyl donor and co-solvent. Finally, we have made significant progress towards obtaining a selective PGA that is capable of synthesizing diastereomerically pure semi-synthetic β-lactam antibiotics from racemic substrates.

The implementation of sustainable solvents and processes is critical to new developments in reducing environmental impact, improving net efficiency, and securing economic profitability in the chemical and pharmaceutical industries. In order to address the challenge of sustainability, researchers have used switchable solvents for both reaction and separation by utilizing a built-in switch to undergo a step change in chemical and physical properties. This allows us to facilitate reactions in the solvent then activate the switch to enable separation and facile product recovery. Subsequently, we can recover the solvent for reuse and avoid energy- or waste-intensive separation processes; thus we are developing and using these switchable solvents as sustainable and environmentally benign alternatives to traditional processes. In this research, we enable the sustainable scale-up of a switchable solvent - piperylene sulfone - a "volatile" and recyclable DMSO replacement. In the development of this process, we improved the reaction performances and developed a green purification method. Furthermore, we enable and demonstrate the implementation of a Meerwein-Ponndorf-Verley (MPV) reduction, a pharmaceutically relevant reaction, into a continuous flow platform. The innovation of continuous flow processes can replace traditional batch reaction technology, and is indeed a key research area that has been acknowledged by the pharmaceutical industry. Additionally, we utilize the switchable sulfone solvents, piperylene and butadiene sulfone, for reaction and separation of HMF produced from monosaccharides as an alternative to a process which has been limited by an inefficient separation step.

Cross-linked polyethylene has higher upper use temperature than normal polyethylene and is used as an insulating material for electricity carrying cables and hot water pipes. The most common method of inducing crosslinks is by reaction with silanes. After incorporation of silanes into polyethylene and upon hydrolysis with ambient moisture or with hot water, Si-O-Si crosslinks are formed between the various linear polyethylene chains. Industrially, this reaction is performed routinely. However, the efficiency of this reaction with respect to the silane is low and control of product distribution is difficult. A precise fundamental understanding is necessary to be able to manipulate the reactions and thus, allow for the facile processing of the polymers. Hydrocarbon models of polymers - heptane, dodecane - are being used to study this reaction in the laboratory. For the reaction, vinyltrimethoxysilane is used as the grafting agent along with di-tert-butyl peroxide as the radical initiator. MALDI, a mass spectrometric technique is used for the analysis of the product distribution after work-up. Advanced NMR techniques (COSY, HSQC, DEPT, APT, HMBC) are being conducted on the grafted hydrocarbon compounds to gain an in-depth understanding of the mechanism and regiochemistry of the grafting reaction. Scalable and cost effective methods to capture CO2 are important to counterbalance some of the global impact of the combustion of fossil fuels on climate change. The main options available now include absorption, adsorption and membrane technology. Amines, especially monoethanolamine, have been the most commercialized technology. However, it is not without disadvantages. House et al have investigated the energy penalty involved in the post-combustion CO2 capture and storage from coal-fired power plants and found that 15-20% reduction in the overall electricity usage is necessary to offset the penalty from capturing and storing 80% of United States coal fleet's CO2 emssions1. Novel non-aqueous amine solvents, developed by the Eckert Liotta group, react with CO2 to form ionic liquids. The ionic liquids readily desorb CO2 upon heating, regenerating the reactive amines and this cycle can be carried out multiple times. An iterative procedure is being adopted to develop amine solvents for CO2 capture. Thermodynamic information like reversal temperature and boiling point of the solvents are collected; they are then used to formulate structure property relationships which allow for new molecules to be engineered. On reaction with CO2, there is a sharp increase in viscosity which is unfavorable from a processing standpoint. Many approaches to mitigate and control viscosity are being studied as well. 1House et al, Energy Environ Sci, 2009, 2, 193-205

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A new reactor and a novel in-situ sampling technique were developed for the study of the synthesis of Ce02 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 Ce02 powders. The sampling probe provided in-situ sampling of in-flight CeCb 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 jam. This information provided a picture of how Ce02 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 Ce02 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 CeC>2 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. [...] / Un nouveau reacteur et une technique innovatrice d’echantillonnage in situ furent developpes pour etudier la synthese de poudres de Ce02 produites a partir de sels de nitrate de cerium dissous. Le reacteur minimise la recirculation des particules et fournit un plasma non perturbe et grandement symetrique approprie pour l’analyse des phenomenes affectant la formation de poudres de Ce02. De plus, une sonde permet un echantillonnage in situ et en vol des particules de Ce02 et ce, en continu et sans contamination croisee. Les particules ainsi captees sont recueillies grace a un systeme de collecte par voie humide qui est compose d’un atomiseur de brume (mist atomizer) qui joue le role d’un recureur (scrubber) et d’une chambre d’atomisation (spray chamber) maison. Le systeme d’echantillonnage est mobile et fut teste sur des particules ayant des tailles de 20 nm a 100 jam. C’est grace a l’analyse des particules ainsi recueillies que nous sommes en mesure de comprendre comment les particules de Ce02 sont formees. Nous presentons aussi une simulation numerique, effectuee avec le logiciel Fluent, qui utilise les differents parametres d’experimentation. Le mecanisme detaille de la formation des particules a partir de gouttelettes fut deduit grace a des etudes calorimetriques, a une etude thermodynamique de la formation du Ce02, a des simulations numeriques et a l’analyse des particules recueillies. Nous avons aussi etudie l’effet d’un ajout de combustibles hydrosolubles (l’alanine et la glycine) a la solution originale de nitrate de cerium. Cette addition de combustibles diminue la temperature de formation du Ce02 en agissant comme une source locale de chaleur resultant de 1’auto-ignition du combustible. Aussi, le combustible cause une « segregation des particules » selon leur taille.

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Optimal utilization of microwave energy requires more basic knowledge of the dielectric properties of the material under investigation. The dielectric properties of materials subjected to microwaves are known to depend on moisture content, temperature and density of the material as well as the frequency of the applied microwave field. This thesis is focussed on the development and evaluation of the new Permittivity Analyser to measure the dielectric properties of agri-food materials at 915 and 2450 MHz using cavity perturbation concept. / In this study, the dielectric properties measuring system was designed and developed using cavity perturbation concept to measure the essential and fundamental parameters of microwave-material interaction; dielectric constant and dielectric loss factor of selected agri-food substances and organic solvents. The TM010 mode of electromagnetic energy was selected and configured to operate at the peakresonant condition for both ISM (Industrial, Scientific and Medical) approved frequencies of .915 and 2450 MHz frequencies. The resonant perturbation cavities were designed, fabricated and tested using the network analyser and the permittivity analyser. High Q (ratio of energy supplied to absorbed) values were reported for both frequencies. Basic calibration of the measurement system was performed using standard media such as air, water and alcohol. Necessary mathematical steps and algorithms were written and integrated into a user-friendly software program (Visual basic 6.0) to carry out the entire measurement. / The dielectric properties (epsilon' & epsilon ″) of materials such as; edible oils---canola, soya and sunflower oils, neem oil/pulp, homogenized milk (1, 2 and 3.25% fat), organic solvents such as ethanol, hexane and their mixtures were determined at various temperatures and frequency (915, 2450 MHz) combinations, using cavity perturbation technique. Linear relationships between the dielectric properties and temperature found in the literature were confirmed to be valid for certain ranges in case of edible oils, organic solvents and milk samples tested with the cavity perturbation method. Repeatibility and variability aspects of the permittivity analyzer at both the frequencies are presented.

Much research has proven that computer controlled vegetable storage can achieve better storage conditions than traditional control systems. During the last 10 years, the use of microcomputer-based environmental control systems has become commonplace. However, to take full advantage of this computerization of the control process, it is not enough only to program the control functions that are performed by normal analog equipment. New and better control strategies must be developed. Recent advances in computer technology have made possible the development of expert systems; a branch of artificial intelligence. One of the advantages of developing such a system is that it provides a reasoning tool which approaches the level of proficiency human experts exhibit in that field. The application of new control methods using expert systems has been extensively demonstrated for greenhouse environments. However, the application of expert systems for the control of vegetable storage is still to be investigated. In the first phase of this project, the development and implementation of a sophisticated control software, using a conventional algorithm-based programming language, were achieved. Throughout the three years of experimentation in an industrial potato storage, the software proved to be appropriate for the control of storage environmental parameters (temperature and relative humidity). During the second phase, the application of an expert system for the on-line control of potato storage was explored. The development of a rule-based expert system, that could replace the conventional algorithm-based control routines was achieved. The integration of the expert system into the control software will result in a highly efficient control software, which can easily be maintained and improved as new knowledge emerges. The use of an expert system will also render possible the representation of heuristic knowledge in the form of rules, which was not possible with the use of conven

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.