This thesis reports studies of the biochemical properties of a newly isolated thermostable amidase from Bacillus sp. RAPc8, and development of a continuous reactor process for the production of a target product. The amidase was cloned and over-expressed in E. coli BL21 strain pNH 223 pLySs by Cameron (2001). Earlier work by Cameron (2001) on the production and purification of the recombinant amidase showed that the enzyme could be produced with high levels of expression in shake-flask culture. Furthermore, preliminary studies have also shown that the molecular weight of the amidase was approximately 35kDa, and that it acts optimally at a temperature and pH of 50°C and 7.2 respectively.

The focus of this project is bioconversion of alkanes to dicarboxylic acids. Dicarboxylic acids are versatile chemical intermediates that can be used in the manufacture of perfumes, polymers, adhesives and antibiotics. The use of a hydrocarbon in a biological process, however, introduces several process challenges related to the nature of the substrate. Many of these challenges are common to all hydrocarbon fermentations, regardless of the product formed, and include flammability, volatility and inhibition of cell growth (notably at low carbon chain lengths), insolubility (notably at high carbon chain lengths) and mass transfer limitations, with respect to both oxygen and alkane substrate. In particular, the provision of adequate oxygen transfer to the organism in hydrocarbon-based bioprocesses has been regarded as especially challenging because of the absence of oxygen in the hydrocarbon backbone. In contrast to carbohydrate-based bioprocesses in which the carbohydrate itself supplies about half of the oxygen, the metabolic requirement for oxygen in hydrocarbon-based bioprocesses has to be met entirely by the transfer of oxygen to the broth. This suggests a proportionately higher requirement for oxygen transfer under these conditions. Consequently, the oxygen transfer rate (OTR) has been mooted as a likely major process limitation, leading to a process which is transport, rather than kinetically controlled and correspondingly, a sub-optimal yield and productivity.

In South Africa, the table olive industry is burgeoning and as a consequence, the large volume of fermentation wastewaters that are generated have created environmental concerns, as these wastewaters have the potential to pollute rivers and ground waters. Currently, these wastewaters are disposed of in large evaporation ponds, but this is not considered to be the optimal treatment solution, due to the potential for pollution and environmental damage. This thesis describes an investigation into the bioremediation of black olive fermentation wastewaters. Wastewaters, from both the table olive and olive oil industries, are toxic and this toxicity can be attributed to the phenolic compounds present. These compounds are known to have antimicrobial and phytotoxic effects. Aerobic biological treatments have been extensively investigated in order to reduce the phenolic fraction of olive mill wastewaters (OMW) with relative success. Biological treatment methods are also cheaper than chemical or physical treatment methods. Therefore, it was decided to investigate aerobic biodegradation of black olive fermentation wastewater (olive wastewater) from the production of black (Kalamata) olives.

Includes bibliographical references. / Farmed abalone is a reliable and good quality source of abalone. Cryptococcus species SS1 was isolated from the gut of the South African abalone, Haliotis midae, and has been identified as a potential probiotic for farmed abalone. The implementation of strain SS1 as a probiotic for aquacultured abalone required the design of a fermentation system to produce high concentrations of the yeast strain in order to supply the probiotic to commercial abalone producers. The aim of this project was to assist in the recommendation of a commercial fermentation process that is economically feasible for the production of strain SS1. This involved evaluation of all the main factors that will contribute to the cost of the fermentation; i.e. cultivation medium, fermentation space and time, and productivity.

Includes bibliographical references (leaves 119-125). / This research pertains to bioleaching of copper containing ores with particular reference to the copper sulfide mineral chalcopyrite (CuFeS₂). While it is focused on heap bioleaching, it has applications to stirred tank bioleaching operations. Industrial heap bioleaching offers opportunities for processing of low grade ores but poses process operational challenges. These challenges include ineffective heap inoculation, a lag period before effective leaching commences and poor heap performance. These aspects are attributed to several contributing factors, such as heap construction, engineering and microbial activity. To date little attention has been paid to colonisation as a means of mitigating these challenges and effectively improving process operation. Current literature regarding microbial attachment to sulfide minerals is limited to pure culture studies using iron oxidising mesophiles, and the use of sulfide mineral concentrates. In a heap environment, mineral dissolution is accelerated through the presence of a mixed consortium of microbial species; with the contribution of each not yet fully understood. In addition, gangue minerals comprise the bulk of the minerals present and thus cannot be neglected when attempting to better understand microbial attachment and the role of micro-organisms in a heap environment. The predominant methodology employed to study microbial attachment in a bioleach context has used batch agitated systems (shake flasks). This may not adequately represent attachment under heap-like fluid dynamics. The idea of this project stemmed from a requirement to contribute to the mitigation of challenges faced by industry through addressing the aforementioned gaps prevailing in literature and improving understanding of the role of microbial attachment and colonisation under conditions simulating a heap. The aim of this study was to investigate attachment of three bioleach micro-organisms (A. ferrooxidans, L. ferriphilum and S. metallicus)to complex, sulfide-containing minerals ores in a bioleach environment using methodologies simulating heap-like conditions.

Includes bibliographical references. / In this thesis it was demonstrated that the current L-lysine fermentation technology can be enhanced by continuously withdrawing spent medium while recycling the biomass in the culture suspension to the bioreactor. The biomass in the reactor outlet stream is separated from the spent medium using cross-flow filtration. The objective of this thesis was to study, understand, model and optimise the performance of the L-Lysine fermentation with biomass retention using cross flow filtration. Following a review of the factors affecting cross-flow filtration and modelling approaches available, the most suitable filtration flux estimation equation was selected. The impact of filtration on microbial performance was assessed and approaches to modelling the lysine fermentation overviewed, leading to the selection of an appropriate model. Thereafter a rigorous approach to the optimisation of the biomass recycling system for lysine production was conducted and experimentally validated. A generic form of Hermia's blocking laws was found to be well suited to the description of the initial stages of cross-flow microfiltration. A constant term (the pseudo steady state flux) has been included to provide a semi-empirical correlation of the cross flow filtration flux. The pseudo steady state flux is based on Darcy's law and a combination of the shear induced diffusion and surface transport models. The presented model adequately described the experimental data. The qualitative effects of the increased hydrodynamic shear stress experienced in the filtration recycling loop on the growth, metabolism and morphology of Corynebacterium glutamicum cells have been investigated. It was found that the cell volume increases under increased hydrodynamic shear although increased shear does not alter the cell shape. The apparent specific growth rate, the yield of biomass from threonine and the specific lysine productivity of the cells exposed to hydrodynamic shear in the filtration system decreases at increased hydrodynamic shear. Using a bioreaction network (BRN) model, it was postulated that increased hydrodynamic shear causes a shift in cellular metabolism from oxidative phosphorylation to substrate level phosphorylation and glycolysis. Furthermore it is postulated that increased hydrodynamic shear causes an increase in the flux of carbon towards the cell wall to either repair or strengthen the cell wall. Fermentation models were developed based on mass and volume balances coupled to either a set of empirical correlations of the cellular metabolism developed from experimental data or a bioreaction network. The impact of filtration-associated hydrodynamic stress on the cellular metabolism was modelled based on a linear relationship between the metabolic impact and the average energy dissipation rate per unit cell mass. A critical average energy dissipation rate was identified below which no impact on the fermentation performance relative to conventional batch fermentations was detected. The fed batch fermentation with biomass recycling using cross flow filtration was optimised using an equation-based dynamic simulation package (gPROMS). The predicted optimum represented a 26% reduction in variable cost of production compared to the conventional fed-batch fermentation technology (R14.50/kg vs. R10.65/kg). The predicted optimum was physically achievable and the experimental results obtained when a fermentation was conducted at the optimal conditions corresponded well with that predicted by the proposed model. The model parameters were re-established for the industrial lysine producing strain (AEC94). At the optimum conditions the model predicted a 12% improvement in variable cost of production while a 14% improvement was realised from experimental data.

Includes bibliographical references (leaves 143-153). / The aim of this study is to investigate the economic and technical feasibility of processing platinum group metals (PGMs) and base metals (BMs) from a low-grade ore concentrate produced in the concentrator plant at Lonmin Pic. The PGMs of particular interest are platinum, palladium, ruthenium and rhodium, while the BMs of interest are copper and nickel. The ore concentrate, as a by-product, represents only 5 % of the total PGM value but as much as 70 % of the total tonnage of material processed in the concentrator plant. Further upgrading this material is not considered a viable route. However, even this low PGM content in the concentrate material accumulates to appreciable value on an annual basis motivating the need to develop alternative methods of extracting value from it. Initial estimates indicate that extraction levels of at least 50 % of the PGMs and 50 % of the BMs would need to be achieved, using low cost hydrometallurgical processes, to make the venture economically viable. These methods would exclude treatment via the smelter and pressure leaching: which are costly. energy intensive and result in leaching of large quantities of non-valuable elements. Previous studies revealed that organic acids had the potential to economically extract the PGMs under alkaline conditions, and BMs under acidic conditions, from various ores and concentrate materials. A literature survey confirmed that certain organic acids can be used to leach metals from ores and concentrates via chemical complexation. It further revealed that other chemical agents. namely cyanide, thiosulphate and bisulphide, were similarly capable of strongly complexing PGMs under various conditions of pH and temperature. The survey also revealed industrially established methods for extracting BMs from low-grade ores and concentrates. Based on this material, this study experimentally evaluated these options with the intent to propose a flowsheet to treat the concentrate material. This was conducted in two phases of experimental work.

Includes bibliographical references / Metabolic flux analysis is commonly used in the modelling of biochemical reactions. The use of MFA models has gained large amounts of interest due to the simplicity of the computational procedures required for the model, and the exclusion of difficult to measure intracellular reaction data. There are many examples of the use of MFA models in literature studies in a number of applications, ranging from the medical industry through to the development of novel biochemical processes. Little to no mention is provided in literature studies regarding the applicability of the MFA model to a specified set of reaction data. Furthermore, the techniques and routines used to compute the flux models are not well described in these studies. The objectives of this research were to determine the sensitivity of the MFA models to various operating and kinetic parameters and to highlight the considerations required when setting up the computational routine used to solve the flux balances. The study was conducted using a model pathway populated with a set of hypothetical elemental reactions and branch points. The model pathway was used in this study to negate the affects of complex regulatory biochemical architectures which are not well described in literature. The use of the model pathway ensured that the reaction system was thermodynamically feasible and there was consistency in the mass balances. The exclusion of the complex regulatory reactions did not affect the accuracy of the results generated in this study. A set of reaction mechanisms were used to describe each reaction step and were populated with parameters reference from literature. The cellular and reactor mass balances were generated using correlations presented in literature.

Includes bibliographical references. / Large stockpiles of linear hydrocarbons have arisen as by-products from the global expansion of gas-to-liquid refining processes. Furthermore, these linear alkanes feature one of the strongest chemical bonds in nature and typically are of a low value due to their inertness. In an effort to valorise this resource, catalytic routes are being sought in order to improve their value by introducing functional groups into the inert carbon backbone of such linear alkanes. Biocatalytic approaches have thus far provided the most feasible route for industrial applications of this chemistry as they feature a uniquely high selectivity for a vast range of products and operate under mild processing conditions. This study focuses on the biological hydroxylation of n-octane to 1-octanol using a previously developed cytochrome P450 monooxygenase, CYP153A6, enzymatic system. The biocatalyst was expressed in Escherichia coli BL21(DE3) by using a pET28b-PFR1500 plasmid encoding the complete operon from Mycobacterium sp. HXN-1500 which included the ferrodoxin reductase (FdR) and ferrodoxin (Fdx) redox partner proteins. CYP153A6 offers several benefits over other biocatalysts such as a notable !95 regioselectivity for terminal carbon hydroxylation and lack of product degradation through overoxidation and by-product formation. Studies to date focussing on understanding interactions between physiological, molecular and bioprocess conditions have yielded maximum specific biocatalyst activities and biocatalyst concentrations in the range of 4.0-5.5 μmoloctanol•gDCW−1•min−1 and 0.18 μmolP450•gDCW−1 respectively. Thus far, this particular P450-based biocatalytic system for n-octane hydroxylation has only been applied in small-scale vials. The objective of this work was to scale-up the experimental apparatus of this system from 1 ml (working volume) vials to bioreactors with a working volume in excess of 1 l because reactors afford improved process stability, control and easier analyses than small-scale experimental setups. The scope of this study focussed on aspects of process configuration and the scale-up for this process. More specifically, optimal timings with respect to induction of gene expression and alkane addition were ascertained with a focus on process stability. Molecular changes to this system were not considered.

Includes bibliographies. / Bioleaching is a biohydrometallurgical process whereby mineral sulphides are metabolically oxidised by microorganisms, releasing precious metals encapsulated in them. This pre-treatment is based on the action of microorganisms affecting oxidation of reduced sulphur species and ferrous iron to sulphate and ferric iron respectively. Conventionally Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans are implemented in this process in the region of 40-45°C and pH 1.8. A high temperature (65- 800C) process, utiltising thermophilic archaea such as Sulfolobus spp. can be considered as an alternative to current bioleaching practice. Literature indicates that there is an overall increase, 6 fold on average, in the rate of leaching due to the use thermophilic organisms. Bioleaching. involves nutrient transfer to microorganisms and interactions between several ionic species, including iron and sulphate. Thus, it is necessary to investigate the effect of the increased temperature on the gas-liquid mass transfer as well as ionic speciation of the system. Hence, the objectives of the present research were established as follows: to elucidate the effect of temperature on mass transfer from a theoretical point of view to establish whether ionic speciation is a contributing factor in thermophilic bioleaching to develop a generic and flexible means of representing ionic species