Dissertations / Theses on the topic 'Bioprocess engineering'

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006.
Includes bibliographical references (leaves 109-113).
This thesis presents the design, fabrication, and characterization of a microbioreactor integrated with automated sensors and actuators as a step towards high-throughput bioprocess development. In particular, this thesis demonstrates the feasibility of culturing microbial cells in microliter-volume reactors in batch, continuous, fed-batch operations. The microbioreactor is fabricated out of poly(methylmethacrylate) and poly(dimethylsiloxane). Active mixing is made possible by a miniature magnetic stir bar. Online optical measurements for optical density, pH, and dissolved oxygen are integrated. Oxygenation in the microbioreactor is characterized and reproducible batch fermentation of Escherichia coli and Saccharomyces cerevisiae are demonstrated and benchmarked with benchscale bioreactors. Global gene expression analysis of S. cerevisiae exhibits physiological and molecular characteristics which parallel those of large-scales. A microchemostat, continuous culture of microbial cells, is realized in the microbioreactor. E. coli cells are fed by pressure-driven single phase flow of fresh medium through a microchannel. Chemotaxis, the back growth of bacterial cells into the medium feed channel, is prevented by local heating.
(cont.) Using poly(ethylene glycol) -grafted poly(acrylic acid) copolymer films, PMMA and PDMS surfaces are modified to generate bio-inert surfaces resistant to nonspecific protein adsorption and cell adhesion. These advances enable cell growth kinetics and stoichoimetry to be obtained in the microchemostat consistent with reported phenomena from conventional stirred-tank bioreactors, as indicated by the time profiles of OD600nm, pH, and DO measurements at steady states. Water evaporation from the microbioreactor allows feeding of base and glucose solutions into the small reactor to realize fed-batch operations. Commercial microvalves are integrated to obtain closed-loop pH control. pH value in the microbioreactor is successfully maintained within a physiological scale during the time course of E. coli cell cultivation in rich media. One key issue for high-throughput bioprocessing is the parallel operation of multiple microbial fermentations while keeping each single microbioreactor disposable. Plug-in-and-flow microfluidic connectors and fabricated polymer micro-optical lenses/connectors are integrated in the microbioreactor "cassettes" for fast set-up and easy operation.
(cont.) A protocol multiplexed system for the parallel operation of four microbioreactors is demonstrated. The demonstrated functionality of the microbioreactor with integrated measurements and flexible operations could potentially have a large impact in bioprocess developments.
by Zhiyu Zhang.
Ph.D.

La investigació plasmada en aquesta tesi doctoral tracta l’aplicació dels principis d’enginyeria de reacció i immobilització enzimàtica per a la millora de reaccions d’oxidoreducció biocatalitzades. En una primera part de la tesi, es va estudiar la co-immobilització de la monooxigenasa P450 BM3 juntament amb un enzim regenerador del cofactor NADPH, la glucosa deshidrogenasa (GDH-Tac). Els millors derivats es van obtenir utilitzant dos suports d’agarosa, un funcionalitzat amb grups epoxy (83% i 20% activitats retingudes respectivament) i l’altre amb grups amino (28% i 25% activitats retingudes respectivament). Posteriorment es va provar de re-utilitzar aquests enzims immobilitzats en diferents cicles de reacció utilitzant un dels substrats naturals de la P450 BM3, el laurat de sodi. Un cop demostrat que ambdós enzims immobilitzats podien ser reciclats, es va estudiar l’aplicació d’aquests en dues reaccions d’interès industrial, la hidroxilació de la α-isoforona i la hidroxilació del diclofenac. En el primer cas, va fer falta optimitzar certs paràmetres de la reacció abans d’aplicar els derivats. Un cop es van tenir unes condicions acceptables, es va comprovar que els resultats obtinguts anteriorment amb el laurat de sodi, no podien ser extrapolats. La reutilització no va ser possible. En quant al diclofenac, es van obtenir resultats similars. En ambdós casos però, l’aplicació dels enzims en la seva forma soluble, va permetre obtenir conversions altes: 86.2% per a la α-isoforona (50 mM inicial) i 100% per al diclofenac (3.5 mM inicial). El producte hidroxilat de l’α-isoforona, la 4-hidroxi-isoforona, ha de ser oxidat en un segon pas per arribar a l’intermediari desitjat, la keto-isoforona. Aquesta segona reacció es duu a terme amb una alcohol deshidrogenasa, la qual també necessita un regenerador de cofactor, la NADPH oxidasa. Al aplicar els dos enzims en la seva forma soluble, al cap de 24h, es va aconseguir un 95.7% de rendiment i una productivitat de 6.52 g L-1 dia-1. A part, l’enzim diana, l’alcohol deshidrogenasa, es va poder immobilitzar en epoxy-agarosa obtenint una activitat retinguda del 58.2%. Al intentar re-utilitzar-lo, es va poder operar durant 96h (4 cicles) millorant el rendiment del biocatalitzador fins a 2.5 vegades comparat amb la reacció en soluble. La reacció d’hidrogenació de la α-isoforona, per altre banda, resulta en la 3,3,5- trimetilciclohexanona, un substrat amb interès industrial per a l’obtenció de polímers. En aquest cas, es va utilitzar la Baeyer-Villiger ciclohexanona monooxigenasa juntament amb una glucosa deshidrogenasa comercial (GDH-01) per a realitzar la reacció d’inserció d’un àtom d’oxigen en l’anell de carbonis. Es van optimitzar diferents paràmetres de la reacció com la formulació del biocatalitzador, la velocitat d’adició de substrat i la quantitat d’enzim afegida. Un cop optimitzada la reacció es va escalar primer a 1 litre i finalment a 100 litres. En aquesta última reacció a escala pre-industrial, es va obtenir una conversió del 85%, una productivitat de 2.7 g L- 1 h-1 i un rendiment del biocatalitzador de 0.83 g g-1 cww. Finalment aquesta mateixa reacció es va realitzar utilitzant els enzims immobilitzats i reciclantos. Tant la ciclohexanona monooxigenasa com la glucosa deshidrogenasa (GDH-01) es van poder immobilitzar en agarosa funcionalitzada amb grups amino. En el primer cas es va obtenir una activitat retinguda del 62.4% (mètode obtingut de la bibliografia) i en el segon cas del 62.6%. En reacció, els dos enzims immobilitzats van ser utilitzats tant per separat com conjuntament. Al cap de sis cicles de reacció (132.5 mM de substrat inicial), es va assolir un rendiment de biocatalitzador 3.6 vegades superior per a la monooxigenasa i 1.9 vegades superior per a la GDH- 01 comparat amb la reacció en soluble.
The research performed and disclosed in this thesis deals with the reaction engineering and the enzyme immobilization principles as tools to improve biocatalyzed oxidoreductive reactions. On a first stage, the co-immobilization of the P450 BM3 monooxygenase together with a NADPH cofactor regeneration enzyme, the glucose dehydrogenase (GDH-Tac), was studied. The best derivates were obtained when using two agarose supports, an epoxy functionalized (83% and 20% retained activity respectively) and an amino functionalized (28% and 25% retained activity respectively). Later on, the re-cycling of the immobilized enzymes was tested in reaction cycles using one of the natural substrates of the P450 BM3, the sodium laurate. Once it could be demonstrated that re-cycling of both P450 BM3 and GDH-Tac was possible, both enzymes were studied in two of the project’s target reactions, the hydroxylation of α- isophorone and the hydroxylation of diclofenac. In the first case, the optimization of the reaction conditions had to be performed prior to the reaction cycles. The reactor configuration, the oxygen income or the glucose concentration were adjusted. However, when the reaction was performed using the co-immobilized enzymes, the P450 BM3 was deactivated and it could not be re-used. The same happened with the hydroxylation of diclofenac. On the other hand, the reaction using soluble enzymes, resulted in 86.2% conversion for the α-isophorone (50 mM initial concentration) and 100% for the diclofenac (3.5 mM initial concentration). The product resulting from the hydroxylation of α-isophorone, the 4-hydroxy-isophorone, can be further oxidized to keto-isophorone, an intermediary for the synthesis of carotenoids and vitamin E. In order to enzymatically perform this step, an alcohol dehydrogenase and a NADPH oxidase, as a cofactor regenerator, were employed. When used in their soluble form, after 24 hours, 95.7% yield and a space time yield of 6.52 g L-1 day-1 were achieved. Moreover, the alcohol dehydrogenase was immobilized on epoxy-agarose and 58.2% retained activity was obtained. When re-used, the derivate could operate for 96h (4 cycles) improving the biocatalyst yield 2.5- fold compared with the reaction with soluble enzymes. The hydrogenation of α-isophorone results in 3,3,5-trimethylcyclohexanone, an industrial interesting substrate due to the polymers that can be obtained from its oxidized product, the trimethyl-ε-caprolactone. This compound is obtained by the Baeyer-Villiger insertion of an oxygen atom into the carbon ring. For this purpose, a cyclohexanone monooxygenase together with a commercial glucose dehydrogenase (GDH-01) were used. Different parameters of the reaction were optimized such as the biocatalyst formulation, the substrate addition rate or the biocatalyst loading. Afterwards, the reaction was scaled up to 1 liter first and then up to 100 liters. In this last pre-industrial reaction, 85% conversion, a space time yield of 2.7 g L-1 h-1 and a biocatalyst yield of 0.83 g g-1 cww could be obtained. Finally, this same reaction was performed using both enzymes immobilized and re-cycling was intended. The cyclohexanone monooxygenase could be immobilized following a previously described method and 62.4% retained activity was achieved. In the GDH-01 case, different supports were screened albeit at the end, it was also the amino functionalized agarose that resulted successful. A retained activity of 62.6% was obtained. In the reaction cycles, the immobilized enzymes were used either separately or both together. In the best case scenario, after six cycles of reaction (132.5 mM initial substrate) 3.6-fold and 1.9-fold higher biocatalysts yields were obtained for the monooxygenase and the GDH-01, respectively.

Les factories cel·lulars microbianes poden ser utilitzades per produir un ampli rang de bioproductes d'interès per a la biotecnologia industrial, els quals comprenen principalment la producció de proteïnes recombinants i metabòlits. Pichia pastoris (Komagataela phaffii), emergeix com un hostatger prometedor per a la producció de proteïna recombinant (RPP) ja que comparteix moltes característiques amb Saccharomyces cerevisiae, però, mostra avantatges en relació amb el consum d'oxigen, tenir un patró de glicosilació més simple, i una menor secreció de proteïnes endògenes. Per aquestes raons, s'han realitzat grans esforços amb l'objectiu d'optimitzar l'eficiència d'aquest hostatger, els quals poden agrupar-se en dos principals i complementaris enfocaments: l'enginyeria de soques i bioprocés. La present tesi doctoral es va basar en l'ús de tots dos enfocs per tal de millorar bioprocessos de producció de lipases recombinants amb interès industrial. En primer lloc, es va demostrar la importància del coneixement de les cinètiques de producció com una potent eina per al disseny d'estratègies òptimes en la RPP a través de la caracterització de dos clons amb un diferent comportament, a causa de la seva diferent dosi gènica, expressant la lipasa 1 de Candida rugosa sota la regulació del promotor GAP (PGAP) duent a terme cultius en quimiòstat i fed-batch. Els resultats, també justificats mitjançant anàlisi transcripcional de diversos gens clau com important novetat, van demostrar que la cinètica de producció depèn de les característiques intrínseques de cada clon usat. D'aquesta manera, la selecció d'una μ adequada per a cada cas permet, d'una manera diferent, un desenvolupament racional del procés per poder optimitzar els bioprocessos RPP. Després, es va avaluar la potencial implementació de la deprivació de carboni (carbon-starving) com una innovadora estratègia que millori les velocitats de producció i rendiments de Crl1 en cultius fed-batch amb una prèvia caracterització fisiològica per a cultius en quimiòstat. Els resultats van evidenciar que l'efecte positiu d'utilitzar aquesta estratègia és altament depenent de les particularitats intrínseques del clon utilitzat. Es va realitzar una anàlisi transcripcional addicional (RNAseq) sobre mostres de quimiòstat, ressaltant la diferència en la transcripció per a tots els gens del llevat. A més, el comportament del bioprocés durant l'ús de promotor GAP (PGAP) es va comparar amb la utilització del promotor induïble AOX1 (PAOX1) duent a terme cultius en quimiòstat per a la producció de Crl1. Tot i que en el cas del PAOX1 es va apreciar una major producció, s'hauria de considerar una avaluació econòmica prèvia a l'escalat del bioprocés, tenint en compte els nombrosos inconvenients de l'ús de metanol com a substrat. Finalment, seguint l'enfocament de l'enginyeria de soques, es va caracteritzar l'ús de dos nous promotors independents de l'ús de metanol sobre l'expressió de la lipasa B de Candida antarctica (CalB) com una poderosa eina que permeti explotar el potencial de P. pastoris a la RPP. Tots dos promotors van mostrar molt millors resultats en comparació als obtinguts amb el PGAP tot i que els patrons de producció entre els promotors va ser significativament diferent per a cada cas. En resum, els resultats mostrats al llarg dels diferents capítols de la present tesi reforcen la utilitat de l'enginyeria de bioprocessos i de soques a través dels diferents estudis realitzats, els quals van permetre obtenir millores significatives en l'eficiència de la RPP. El coneixement dels factors clau involucrats en l'expressió recombinant obre una àmplia finestra de noves oportunitats que fan possible que P. pastoris s'estableixi com una plataforma robusta per a la RPP, mostrant-se altament competitiva enfront dels sistemes convencionals.
Las factorías celulares microbianas pueden ser utilizadas para producir un amplio rango de bioproductos de interés para la biotecnología industrial, los cuales comprenden principalmente la producción de proteínas recombinantes y metabolitos. Pichia pastoris (Komagataela phaffii), emerge como un hospedero prometedor para la producción de proteína recombinante (RPP) debido a que comparte muchas características con Saccharomyces cerevisiae, sin embargo, muestra ventajas en relación con el consumo de oxígeno, tener un patrón de glicosilación más simple, y una menor secreción de proteínas endógenas. Por estas razones, se han realizado grandes esfuerzos con el objetivo de optimizar la eficiencia de este hospedero, los cuales pueden agruparse en dos principales y complementarios enfoques: la ingeniería de cepas y bioproceso. La presente tesis doctoral se basó en el uso de ambos enfoques para mejorar bioprocesos de producción de lipasas recombinantes con interés industrial. En primer lugar, se demostró la importancia del conocimiento de las cinéticas de producción como una potente herramienta para el diseño de estrategias óptimas en la RPP a través de la caracterización de dos clones con un diferente comportamiento, debido a su diferente dosis génica, expresando la lipasa 1 de Candida rugosa bajo la regulación del promotor GAP (PGAP) llevando a cabo cultivos en quimiostato y fed-batch. Los resultados, también justificados mediante análisis transcripcional de varios genes clave como importante novedad, demostraron que la cinética de producción depende de las características intrínsecas de cada clon usado. De esta manera, la selección de una µ adecuada para cada caso permite, de una manera diferente, un desarrollo racional del de proceso para poder optimizar los bioprocesos RPP. Después, se evaluó la potencial implementación de la deprivación de carbono (carbon-starving) como una innovadora estrategia que mejore las velocidades de producción y rendimientos de Crl1 en cultivos fed-batch con una previa caracterización fisiológica para cultivos en quimiostato. Los resultados evidenciaron que el efecto positivo de utilizar esta estrategia es altamente dependiente de las particularidades intrínsecas del clon utilizado. Se realizó un análisis transcripcional adicional (RNAseq) sobre muestras de quimiostato, resaltándose la diferencia en la transcripción para todos los genes de la levadura. Además, el comportamiento del bioproceso durante el uso del promotor GAP (PGAP) se comparó con la utilización del promotor inducible AOX1 (PAOX1) llevando a cabo cultivos en quimiostato para la producción de Crl1. Aunque en el caso del PAOX1 se apreció una mayor producción, se debería considerar una evaluación económica previa al escalado del bioproceso, considerando los numerosos inconvenientes del uso de metanol como sustrato. Finalmente, siguiendo el enfoque de la ingeniería de cepas, se caracterizó el uso de dos nuevos promotores independientes del uso de metanol sobre la expresión de la lipasa B de Candida antarctica (CalB) como una poderosa herramienta que permita explotar el potencial de P. pastoris en la RPP. Ambos promotores mostraron mucho mejores resultados en comparación a los obtenidos con el PGAP aunque los patrones de producción entre los promotores fue significativamente diferente para cada caso. En resumen, los resultados mostrados a lo largo de los diferentes capítulos de la presente tesis refuerzan la utilidad de la ingeniería de bioprocesos y de cepas a través de los diferentes estudios realizados, los cuales permitieron obtener mejoras significativas en la eficiencia de la RPP. El conocimiento de los factores clave involucrados en la expresión recombinante abre una amplia ventana de nuevas oportunidades que hacen posible que P. pastoris se establezca como una plataforma robusta para la RPP, mostrándose altamente competitiva frente a los sistemas convencionales.
Microbial cell factories can be used to produce a wide range of bioproducts of interest for the biotechnological industry, which comprises mainly the production of recombinant proteins and metabolites. Pichia pastoris (Komagataela phaffii), emerges as a promising host for recombinant protein production (RPP) due to it shares many features with Saccharomyces cerevisiae, however, displays some advantages in terms of oxygen consumption, simpler glycosylation pattern, and lower endogenous protein secretion. For these reasons, great efforts have been performed with the objective to optimize the efficiency of this host which can be grouped in two main and complementary approaches: the strain and bioprocess engineering. The present PhD thesis was focused in the use of both approaches, in order to improve the production bioprocess of recombinant lipases with industrial interest. At first, it was demonstrated the importance of the knowledge of production kinetics as strong tool to design optimal strategies for RPP through the characterization of two clones with contrasting production performance, due to its different gene dosage, expressing Candida rugosa lipase 1 (Crl1) regulated under GAP promoter (PGAP) using chemostat and fed-batch cultures. The results, also supported by transcriptional analysis of some target genes as marked novelty, demonstrated that production kinetics depends on the intrinsic characteristics of each clone used. Therefore, the selection of adequate µ for each case enables, in a different way, the rational process development to optimize RPP bioprocesses. Later, it was also evaluated the potential implementation of carbon-starving as innovative strategy to enhance the Crl1 production rates and yields on fed-batch cultures with a previous physiological characterization on chemostat cultivation. Results showed that positive effects observed using this strategy are highly dependent on the specific features of the clone used. An additional transcriptomic analysis (RNAseq) was carried out with chemostat samples, pointing out the difference on the transcription of all the genes of the yeast. In addition, bioprocess performance of the GAP promoter (PGAP) was compared with the inducible AOX1 promoter (PAOX1) by carrying out chemostat cultures producing Crl1. Although PAOX1 displayed higher production, an economical evaluation should be necessary before scale-up of the bioprocess, considering the numerous drawbacks of using methanol as substrate. Finally, following the strain engineering approach, it was characterized the use of two alternative methanol free novel promoters on the expression of lipase B from Candida antarctica (CalB) as strong tool that allows to exploit P. pastoris potential on RPP. Both promoters displayed much better production parameters than the observed with PGAP although the production pattern between promoters were significantly different on each case. Overall, the results presented along the different chapters of this current thesis support the usefulness of bioprocess and strain engineering through the different studies performed, which gave significant improvements in RPP efficiency. The knowledge of key factors involved on recombinant expression opens a window of new opportunities that allows P. pastoris to be established as a robust platform for RPP and showing it as highly competitive to conventional systems.

Thesis (M. Eng. and S.B.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2005.
Author received the S.B. degree, June 2005 and the M. Eng. degree, Sept. 2005.
Includes bibliographical references (p. 195-202).
Online monitoring of bioprocesses is essential to expanding the potential of biotechnology. In this thesis, a system to estimate concentrations of chemical components of an Escherichia Coli fermentation growth medium via a remote fiber-optic Raman spectroscopy probe was studied in depth. The system was characterized to determine sources of instability and systematic error. A complete first-order error analysis was conducted to determine the theoretical sensitivity of the instrument. A suite of improvements and new features, including an online estimation of optical density and biomass, a method to correct for wavelength shifts, and a setup to increase repeatability and throughput for offline and calibration methods was developed accordingly. The theoretical and experimental ground work for developing a correction for spectrum distortions caused by elastic scattering, a fundamental problem for many spectroscopic applications, was laid out. In addition, offline Raman spectroscopy was used to estimate concentrations of fructose, glucose, sucrose, and nitrate in an oil palm (Elais guineensis) bioreaction. Finally, an expansion of optical techniques into new scale-up applications in plant cell bioprocesses, such as plant call differentiation was explored.
by Gustavo Adolfo Gil.
M.Eng.and S.B.

Modelling and simulation enhance our insight and understanding of chemical processes and aid in identifying bottlenecks and potential improvements. A simplified simulation package, providing a reasonable estimate of material and energy usage and process emissions is often valuable in very early stages of process development, when temporal and financial limitations do not allow for more detailed estimates. Environmental burdens are an increasing concern in industrial processes and various methodologies and tools have been developed for gathering and analysis of process information to enhance understanding of the process system and inform decision makers. The systems nature of these approaches is aimed at mitigation of environmental burdens through improved technologies, sustainable resource consumption and screening of process alternatives. Ideally, the process design team should bring together these tools in early stages of development when design flexibility is greatest. In the present study, such a simplified approach to bioprocess design is demonstrated using a case study for the large-scale production of citric acid.

This thesis concerns the topic of wastewater biorefineries (WWBR), in which wastewater is not seen simply as a waste stream to be cleaned but as a valuable material flow to be converted into bioproducts, while still meeting discharge limits at the end. To set the scene, similar developing approaches to valorise wastewaters globally are reviewed. Wastewaters in South Africa are reviewed and categorised with regards to their potential to serve as raw material, in terms of their volume, concentration and complexity. Bioproducts possible from wastewater is reviewed and evaluated. The wastewater biorefinery is conceptualised in the context of current wastewater treatment technologies and a set of evaluation criteria is developed. A multi-reactor setup is suggested in which wastewater is used, in series, as substrate by heterotrophic microbes like bacteria, photo-mixotrophic organisms like algae, macrophytes and fungi. Each reactor group is considered in detail and evaluated with regards to its suitability to the wastewater biorefinery, leading to selection of appropriate reactor designs. Stoichiometric mass balances of all unit operations are established, showing the material value flows, and combined to model this multi-bioreactor approach. Subsequently the model is tested against literature data. Finally, the applicability of the wastewater biorefinery concept for certain waste streams is assessed. The thesis contributes to the current body of knowledge in the following ways: 1. Introduction of the concept of the wastewater biorefinery (WWBR) 2. Provision of a potential preliminary guide for classification of wastewaters for use in the WWBR 3. Development of criteria for reactor evaluation for use in the WWBR 4. Development of an integrated model to interrogate bioproduction from wastewater and determine product yields associated with wastewater treatment 5. Creation of new knowledge through the interpretation of the model on different wastewater systems. The wastewater biorefinery is defined as a bioproduction system that integrates multiple unit operations to deliver compliant water as well as a bioproduct or bioproducts. It is approached through the concepts of industrial metabolism and the circular economy. Wastewater biorefineries are shown in this work to be a viable approach to improving resource efficiency while ensuring the better ecological functioning of humans within “greater than human” systems. The work places emphasis on the recovery of bioproducts that conserve molecular complexity but acknowledges that energy production for use on site and in the immediate surroundings is always an important factor in the WWBR. This thesis introduces the need to include a qualitative way to evaluate the complexity of wastewater, in addition to standard classification of volume and concentration of components. Complexity includes both composition of potentially problematic compounds and how unpredictably it changes over time. In this approach, it is preferable to generate three types of products: products of sufficient value to be economically viable; products of variable value with concomitant assimilation of major contaminants; and clean water as a product, typically through multiple unit operations, allowing multi-criteria optimisation. Through this approach, multiple criteria can be met. Function-based products specific to niche industries, particularly those which produced the wastewater of interest, are of substantive interest owing to their streamlined market uptake. This thesis explores the requirements of the products that can be produced from wastewater in a non-sterile context and suggests product groupings that meet these requirements. Products secreted into the bulk volume are difficult to recover, leading preference to biomass associated and intracellular products. The product needs to offer a selective advantage to the organisms producing it to facilitate enrichment through, ecological selection of the microbial consortium with simultaneous cell retention through reactor design and operation. Four groupings of unit operations were reviewed in detail and evaluated with regards to their suitability to the wastewater biorefinery, using a two-part set of evaluation criteria that was developed in this work, considering the reactor design, and its operation. The four unit operations each contribute a specific role to the functioning of the WWBR as a system. It is acknowledged that not all units are commercially important, and that the concept of diminishing returns should be kept in mind. The heterotrophic microbial bioreactor, of which the bacterial biocatalyst is used as a representative example, is helpful for removing a high proportion of the organic carbon. A wide range of commodity products with market potential is known to be produced through heterotrophic microbial systems. Existing heterotrophic microbial reactor systems like the aerobic granular sludge system (AGS) exist that suit the wastewater biorefinery approach particularly well, while activated sludge along with biological nutrient removal (BNR), the most commonly used reactor system in South Africa, is the least suitable to the WWBR. The photo-mixotrophic reactor represented by the algal bioreactor is helpful to scavenge high proportions of nutrients, particularly nitrogen and phosphorus. The algal bioreactor is also known to produce commodity products. Photo-mixotrophic bioreactor systems complement the heterotrophic systems but are unlikely to be the dominant reactor due to land and energy requirements. The macrophytic bioreactor is targeted for polishing the exiting stream in terms of nitrogen, phosphorus and particulates to ensure compliant, fit for purpose water as a product, with a macrophyte-based byproduct. Macrophyte bioreactors, particularly floating wetlands, are promising tertiary systems that should be viewed in conjunction with water sensitive design principles to overcome potential land availability limitations. The solids bioreactor is an emerging beneficiation technology for biotransformation of bio-slurries and the solid phases recovered during WWBR operation to generate products of value, including biosolids. Solids bioreactors have great potential but require more investigation, with key challenges being mass transfer and separation technologies. Operating waste treatment facilities as net income-producing bioprocesses require a mindset change about investment, risk and associated returns. WWBRs require higher capital investment due to the additional process units and downstream processing required and have higher operating costs due to the greater control required during the process and greater number of operators with advanced skillsets. An identification of the relevant product range and comparison between conventional processing routes and those possible from the wastewater is required on a case by case basis, and an overview is given in this thesis. Waste may need to be re-classified to be used as an intermediate by-product or raw material, requiring legal considerations in terms of both the solid waste as per the National Environmental Management Act (NEMA) and liquid waste as per the National Water Act (NWA). The added complexity of reclassifying waste as raw material needs an acknowledgement of institutional challenges such as speaking across department silo’s. In this thesis, a model of these integrated unit operations was developed to generate material inventories across the system. This can be used to evaluate possible scenarios in an integrated context using a generic flowsheet as well as mass balances generated through the model. Three case studies were examined: municipal, abattoir and pulp and paper wastewater. Municipal wastewater was chosenas it represents a complex, dilute, 'suboptimal’ wastewater stream. Abattoir wastewater was chosen as an example of a complex, nutrient-concentrated stream that may be well suited to biological transformation. Pulp and paper wastewater was chosen as an example where the biorefinery concept is already well established, and is a low complexity, low nutrient, high carbon content stream. In considering the above case studies, a number of key learnings resulted. The impact of solids removal was clear and in keeping with existing bioprocessing and wastewater treatment principles of decoupling the hydraulic and solids residence times. Low nitrogen and phosphorus content in the pulp and paper wastewater as compared to the other two case studies indicated the need to conduct integrative studies of the unit operations to determine the most appropriate unit operations across the system. The effect of improving the product conversion yields and product recovery yields were examined, and a surprising result is the amount of nutrients that remain in compliant effluent, due to the large volumes of liquid involved. This leads to the conclusion that while the WWBR is a valuable way to address resource recovery, separation at source and internal process efficiencies are critical to improve overall resource efficiency and environmental protection. With regards to municipal wastewater, which contributes by far the most in terms of volume and nutrients of wastewaters in South Africa from the perspective of reactor design for waste(water) beneficiation, considering the cleaner production principle of separation at source, along with the need to decouple the solid and hydraulic residence times, dry sanitation presents a clear argument for the best WWBR approach. This approach must acknowledge that the transport of the sanitation raw materials is more difficult if hydro-transportation is not available, and needs to ensure operator equity, health and safety, particularly in the handling of the sanitation raw materials. This thesis was developed in conjunction with the Water Research Commission (WRC) project “Introducing the wastewater biorefinery concept: A scoping study of polyglutamic acid production from a Bacillus-rich mixed culture using municipal waste water” (Verster, et al., 2014) and Water Research Commission (WRC) K5/2380 project titled “Towards Wastewater Biorefineries: integrated bioreactor and process design for combined water treatment and resource productivity” (Harrison, et al., 2017). While the project focused on a global and national review on research on wastewater biorefineries and wastewater as a resource, this thesis explores in greater depth the requirements of each of the reactor units and their integration.

Crystal Violet (CV) decolourising deep-sea actinobacteria could provide a great source of novel redox biocatalysts that can be used in various applications such as removal of triphenylmethane dyes from contaminated wastewater and soil, degradation of aromatic environmental pollutants, biotransformation of antimicrobial agents and degradation of xenobiotics. CV is a triphenylmethane dye that has various applications, including use in medical, research and industrial applications, but its release into the environment poses a threat to aquatic life as it has characteristics of a biocide. Only a limited number of microorganisms are able to decolourise and degrade CV, and one of these proposed mechanisms by which they do so is the catalytic effect of oxidoreductase enzymes, including peroxidases, polyphenol oxidases and laccases. Triphenylmethane reductase has also been reported to be involved in decolourising CV, but the reaction involving this enzyme has not been studied systematically. Eleven deep-sea actinobacteria were investigated and found to decolourise CV by either biodegradation or biosorption. Gordonia sp. JC 51 was selected as a candidate for further study as it could decolourise CV efficiently and could tolerate high concentrations (1mM) of CV. A combination of spectral scan studies, dye decolourisation, biodegradation assays, enzymatic assays, SDS-PAGE, Native PAGE, TLC and LC/MS/MS methods revealed the mechanism involved in the decolourisation of CV. Gordonia sp. JC 51 decolourised CV via enzymatic and non-enzymatic mechanisms. However, true decolourisation of CV was performed via biodegrading enzymes. Triphenylmethane reductase and polyphenol oxidase was confirmed to be the enzymes involved. Leucocrystal Violet was identified as the metabolite produced. CV also was sequentially N-demethylated, oxidised and cleaved into smaller compounds such as Michler’s Ketone. In conclusion, Gordonia sp. JC 51 has potential as a whole cell biocatalyst and should be investigated further.

Water pollution is recognized as one of the major environmental problems in the mining industry. This has been compounded with an increase in agriculture activities. Water pollution is a major problem on copper and coal mines throughout the world and Zambia, the focus of this study, is no exception. Worldwide freshwater resources, which provide important ecosystem services to humans, are under threat from rapid population growth, urbanization, industrialization and abandonment of wastelands. There is an urgent need to monitor and assess these resources. In this context, the physical, chemical and ecological water quality of the Munkulungwe Stream located on the Copperbelt of Zambia, was assessed with possible contamination from Bwana Mkubwa TSF, agriculture activities and subsequent impact on the surrounding community. The chemical and physical parameters were assessed at four sampling locations. Sampling site S1 was located on the Munkulungwe stream upstream of Bwana Mkubwa TSF, S2, S3 and S4 were on the main stream downstream of Bwana Mkubwa TSF. In addition, a macroinvertebrate composition analysis was performed to estimate the quality of water using the biotic index score. Finally, the relationship between physiochemical parameters and biotic index score was analysed to interrogate their inter-relationship with respect to water quality. The results showed that the average values of dissolved oxygen (DO) of 4.52 mg/l, turbidity (40.96 NTU), Co (0.24 mg/l), Pb (0.25 mg/l), Fe (0.36 mg/l) and Mn (0.22 mg/l) downstream exceeded international standards for drinking water. Upstream, the values of Co, Pb, Fe and Mn were within acceptable standards for drinking water, DO and turbidity were above acceptable standards. The metal concentration and total dissolved solutes were impacted by closeness to the mine tailings deposit with the heavy metal concentration being highest at S2 and S3. Moreover, high turbidity levels revealed that land erosion induced by agriculture activities is a severe problem in the area. Physical parameters were high in the rainy season due erosion escalated by rains while chemical parameters were high post rainy season. During the rainy season, the chemical contaminants are diluted and thus they are not such a big impact, but they tend to concentrate up during the dry MDNLEE001 III season. The stream at sampling points S2 and S3 was dominated by species tolerant (leech, Isopod and Snail: Pouch) and semi tolerant (Blackfly larvae and Amphipod or Scud) to pollution. The change in season influenced the composition of macroinvertebrates, with the number of species increased post rainy season. The average biotic index score (2.5) showed that the stream condition is not good, it is slightly polluted. The results showed that water quality downstream was substantially affected by Bwana Mkubwa TSF, agriculture activities and is likely to affect human health and food security. It is recommended that groundwater surrounding tailings dams should be monitored in both active and abandoned mines. Curtain boreholes around a tailings dam can be drilled and the water extracted and treated so that it doesn't contaminate other water bodies. To improve the environmental management of mining related impacts in Zambia, mining areas should be completely rehabilitated. There is need for remediation strategies for abandoned mine sites. Constructed wetlands, roughing filtration and phytoremediation are highly promising techniques, as they are reliable, cheap, effective and sustainable.

The environmental impacts of coal processing wastes are a challenge in South Africa as large amounts of coal wastes are produced annually, pegged at 60 million tons per year according to Eberhard (2011). Whilst the fossil fuel-based industry is in decline globally, coal is likely to remain the dominant source of power in South Africa. The major environmental impacts reported in several studies are water pollution and soil quality degradation due to acid rock drainage (ARD) and its associated elevated levels of elements and salts. Several studies have shown the environmental performance of the wastes to be dependent on the geochemical properties of the wastes. Owing to the complex nature of coal wastes, their characterisation using tools developed for hard rock ores is associated with inconsistency and uncertainty. As a result, the South African coal processing wastes are poorly characterized and the associated risks not well understood. This study investigates the reliability of relevant characterisation techniques and interpretation of characterisation data in terms of the environmental risk potential of coal wastes. The outcomes of the study address some of the uncertainties and deficiencies arising from the current characterisation tools and evaluate potential environmental risks posed by coal processing wastes. Laboratory-scale characterisation of the physio-chemical properties and of ARD and elemental risk potential of two ultrafine coal waste and one discard waste sample were conducted. Evaluation of accuracy and repeatability of selected analyses was conducted on a certified coal standard. The selected analyses tested for accuracy and repeatability were total sulphur analysis by Leco and Eschka methods in addition to elemental analysis by wavelength dispersive x-ray fluorescence (WDXRF), inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The ISO 157:1996 and ACARP C15034 protocols for assessment of sulphur forms were also compared and evaluated for precision using the coal standard and coal waste samples. Conversions of the sulphur species under static ARD tests were also studied to understand the sulphur species behaviour and implication on ARD potential. The mineralogy of the coal wastes was evaluated from a quantitative evaluation of minerals by scanning electron microscopy (QEMSCAN) and quantitative x-ray diffraction (QXRD) analysis. In addition, conventional net acid generating (NAG) and acid-base accounting (ABA) static tests were enhanced through extended boil NAG tests to assess the organic acids effect on the NAG capacity. The static tests were validated by theoretical ARD calculated from mineralogy as well as biokinetic shake flask tests which gave the timerelated acid generating behaviour of the coal waste samples. Sequential chemical extractions combined with a simple score and ranking protocol were subsequently used to evaluate the potential water and soil-related risks associated with environmentally available elements and salts in the coal wastes. The results showed both the Leco and Eschka methods to be highly precise (±0.01-0.03 % standard error) but the Leco was more accurate (±3.1 % compared to ±12.5 % relative standard error (RSE)). The total sulphur content of the coal processing waste was less than 2 %. The ISO157:1996 and ACARP C15034 protocols gave comparable and slightly different results but the latter was more precise in sulphate analysis. Furthermore, the ACARP protocol could differentiate the acid forming sulphates from the soluble sulphates giving a better theoretical maximum acid producing potential. The sulphur species from the two chemical methods and QEMSCAN mineralogy showed 52-61 %, 12-26 % and 21-43 % to be sulphide, sulphate and organic/low-risk sulphur respectively. The conversion of the sulphur species showed that partial solubilisation of sulphides in ANC and partial conversion of organic/low-risk sulphur under NAG tests can cause an over or underestimation of ARD potential. The static ARD tests has shown the Witbank coal discards sample to be potentially acid forming (PAF) (9.2-25.9 kg H2SO4/Ton), Waterberg coal slurry to be non-acid forming (NAF) (-68.6 to -46.8 kg H2SO4/Ton) and Witbank coal slurry to be uncertain (-12.1 to 9.9 kg H2SO4/Ton). The extended boil NAG tests showed organic acids effect on the Witbank coal slurry likely caused an overestimation of the NAG capacity. Validation of the static tests by biokinetic tests and ARD calculated from mineralogy classified both Witbank samples as PAF and the Waterberg sample as NAF. The results also showed the net acid producing potential of the coal wastes to depend on the mineralogy of the samples. The elemental results showed WDXRF and LA-ICP-MS analysed most of the elements accurately within ±10 % RSE and that a combination of techniques provides more reliable and accurate results. The analyses showed the coal waste to contain significant amounts of environmentally sensitive elements like Cr, As, Mo, Sb, Se. The ranking and scoring of potentially available elements under oxidising leach conditions evaluated Fe in Waterberg coal slurry and Witbank coal discards to pose high risk in drinking water while S (as sulphate), Pb, Sb, Mn, As, Al and Hg in the three samples pose moderate risk. This case study evaluated the accuracy and precision of commonly used analytical techniques and applicability of risk evaluation protocols for coal processing wastes. The research outcomes underlined some factors that cause uncertainty and inconsistency with the evaluation of ARD potential of coal wastes. The findings highlighted the need to validate and complement the characterisation data using various tools and risk evaluation protocols to overcome specific limitations. The results also indicated the coal wastes have the potential to cause environmental impacts from ARD and elevated concentration of elements and salts, thus providing a basis for designing and implementing waste management strategies which minimise these risks. The mineralogy and elemental composition of coal wastes showed enrichment of elements and presence of potentially usable and economically valuable constituencies for future studies on value recovery. Characterisation of coal processing wastes for air pollution impacts is recommended for future studies as well as a study of ARD behaviour under continuous flow systems to more closely represent the conditions in dump disposal scenario.

The South African economy is an energy-driven economy which relies on coal to meet most of its energy demands. Coal mining has resulted in the generation of coal waste over 60 million tonnes, annually. Apart from the huge footprint of this waste, the sulphide minerals contained in the waste have resulted in the generation of acid rock drainage (ARD). A lot of techniques have been developed to prevent and mitigate ARD, however most of these techniques have fallen short in terms of meeting their desired objectives due to the long-term nature of ARD generation which can persist for hundreds of years after mine closure. This has resulted in emphasis being put on long-term prevention techniques that remove ARD risk over treatment techniques. One prevention technique which has shown good technical potential is the two-stage flotation method developed for desulphurisation of hard rock tailings and coal fines, developed at the University of Cape Town. On desulphurising coal, the first stage produces an upgraded coal product that may be sold, with the second stage used to separate the tailings from the first stage into targeted high-sulphide and low-sulphide fractions which may then be appropriately used or disposed of. An economic assessment of the process showed across a wide range of coal wastes the high cost of oleic acid used in the first stage of the process as a collector was a major contributor to the operating costs. The investigation undertaken in this thesis looked at the potential of algal lipids and their derivatives as biocollectors to replace the oleic acid collector in the desulphurisation process at the laboratory scale. A review of cost was carried out for a process that used raw algal lipids (RALs) or fatty acid methyl esters (FAMEs), which are derived from RALs through transesterification. Batch flotation experiments were used to assess the performance of the two bioflotation reagents in comparison to oleic acid and dodecane, an alternative but less successful chemical collector. The algal lipids cost review was a desktop study which was done by adapting literature data from Davis et al. (2014) which focused on economic evaluation of algal lipid biofuels production pathways. Results from laboratory experiments for two different coal waste feed samples showed that the performance of RALs and FAMEs was similar to that of oleic acid for the sample that was high in ash and sulphur, and better than oleic acid for the sample that was low in ash and sulphur. For example, the product from Site 1 discards from Waterberg had 24.37% ash and 2.76% sulphur using FAMEs, 26.13% ash and 2.56% sulphur with RALs, and 23.48% ash and 2.41% using oleic acid, at a reagent dose of 2.8 kg/t for all reagents. For Site 2 waste tailings from the Witbank area, the product had 23.17% ash and 0.72% sulphur when FAMEs were used as collector, 22.75% ash and 0.75% sulphur with RALs, and 20.18% ash and 0.74% sulphur using oleic acid, at the same reagent dose. Discards from Site 1 had an initial ash and sulphur content of 47.61% and 5.71%, respectively. Site 2 waste tailings had 25.56% ash and 0.91% sulphur before flotation. Increasing biocollector dosage resulted in higher yields with a compromise on the upgraded coal quality. The pH tests showed that the performance of the two bioflotation reagents was best at pH 4 in terms of yield. However, increasing the pH of the process from the natural pH of the sample (pH 2.7) to 7 resulted in collection of more ash and sulphur, thus reducing the product quality. The algal lipids cost review showed that RALs and FAMEs were potentially 20 to 21% cheaper than oleic acid, with more room for improvement. Both the laboratory experiments and the technical evaluation showed that algal lipids and their derivatives have the potential to replace oleic acid in the two-stage desulphurisation process for coal waste to obtain a saleable quality coal product while simultaneously decreasing the impact of ARD from coal waste.

Low solubility of oxygen has resulted in high bioreactor energy requirements in order to supply sufficient oxygen to aerobic bioprocesses. It is desirable to reduce energy consumption in bioreactors to benefit environmental sustainability as well as economic feasibility. This is particularly important with the resurgence of interest in bio-based commodity products. Some research has suggested that venturi aeration of bioreactors will reduce energy consumption by eliminating the need for air compression, while at the same time maintaining or improving oxygen transfer rates. On the other hand, there are findings that suggest venturi aeration has lower energy efficiency than conventional sparging and oxygen transfer rates achieved are too low sustain aerobic biological processes. A comparison of the aeration of geometrically-similar reactors using the same analytical methods to determine kLa is not available in the literature. This comparison should also address analysis of energy input including energy used for compressing gas sparged into a stirred tank reactor; the investigation of mass transfer rates at higher flow rates (vvm) in venturi-aerated reactors and resulting cell response to these higher flow rates. This is the topic of the dissertation presented. In this laboratory scale study, venturi aerators were characterised and energy consumption as a function of oxygen mass transfer rates compared with a geometrically identical aerated stirred tank reactor by evaluating the volumetric mass transfer coefficients (kLa). The kLa was investigated in varying reactor setups using the dynamic gassing-in method.

There is an abundant availability of zinc sulphide (sphalerite) ore (160 million tons at 7.40 % Zn) at Gamsberg, Northern Cape in South Africa. The ore body is South Africa’s greatest unexploited base metal resource. Regardless of its size, the low zinc and high manganese content of the sphalerite combined with the low zinc price prohibits the development of the deposit. Sphalerite is the most common zinc mineral, hence 95 % of the world’s zinc production is from this mineral. Sphalerite is currently processed by crushing-milling-flotation, followed by the roast-leach-electrowinning (RLE) process. This route has major detrimental impacts on the environment, it produces SO2, and cannot treat ores of low grade or higher complexity. Therefore, alternative processes are being sought in order to circumvent the RLE process. This study compares three different process routes in the context of processing ore from the Gamsberg deposit for refining 3.4 million tpa ore in order to produce special high grade (SHG) zinc (>99.995% Zn). These routes include heap leaching and refining locally (route 1), preparing a flotation concentrate and refining it locally (route 2) and lastly, preparing a flotation concentrate and shipping it for toll refining in Europe (route 3). Zinc heap leaching has not yet been commercialised due to the absence of solvent extraction reagents which can selectively extract zinc from a low tenor acidic pregnant leach solution without incorporating the neutralization stage. Therefore, route 1 has higher risk as compared to the other routes. A desktop model which provides a comparison of capital cost, operating cost, NPV, IRR and PVR has been developed. Parameters such as average zinc grade, process recovery and zinc price are provided as inputs. The effects of fluctuations in important parameters such as working capital, and zinc price on NPV are assessed using Matlab.

Eicosapentaenoic acid (EPA; 20:5) is an omega-3 polyunsaturated fatty acid of increasing interest as a nutraceutical. An indigenous microalgal isolate suitable for an EPA bioprocess was selected by screening monoalgal isolates from the Council for Scientific and Industrial Research (CSIR) micro-algal culture collection. A Cymbella diatom (A23.2) was selected for superior EPA production in both growth and stress conditions, using both fluorescent microscopy and flask studies. Studies investigated increasing biomass, improving EPA content, and optimising overall EPA productivity in a multi-stage bioprocess. Growth studies found self-regulatory systems in both phosphate and nitrate metabolism. These mechanisms were absent in silicate and bicarbonate consumption, prompting their optimisation in the bioprocess medium. Cultivation pH was found to have a statistically modelled optimal value of 7.2 and a light intensity at a low range of 60 – 70 ìmol.m-2.s-1 was found to be suitable. Nutrient and physicochemical parameters were assayed individually, and revealed cell productivities of between 2.0 x 108 to 3.0 x 108 cell.L-1.hr-1 in batch culture bioreactor studies. Further studies demonstrated the use of both nutrient stress and physicochemical stress to enhance EPA production. These results informed the choice of operating parameters for a proof of concept, multistage raceway-based EPA bioprocess, consisting of a single growth pond and three stress ponds linked in series. The growth phase EPA productivity data of 0.68 mg.L-1.day-1, was higher than that of the stress phase, supporting its classification as a growth-associated product. Further, the EPA productivity in the raceway was more than twice that of initial batch culture screening. Once experimental limitations are addressed, a re-design of the bioprocess can be undertaken by optimising growth phase residence time, medium flow-rate and partial/complete elimination of the stress phase. The EPA productivity of the diatom used in this work has the potential of reaching commercially viable values. The development of a commercial indigenous EPA producer has a dual impact, as it addresses various nutritional and medicinal market demands and improves the sustainability of the world’s fish stocks.

Electrical and electronic equipment has become an integral part of life in the modern world. When disposed of, it is termed Waste Electrical and Electronic Equipment (WEEE) and is one of the fastest growing waste streams in the world. Disposing the WEEE has associated risks as they contain a high amount of toxic metals (e.g. lead) which can leach into the soil, they place a high load on the land and they contain valuable metals making their recovery beneficial. Printed circuit boards (PCBs) are an essential part of WEEE. Although WEEE forms a small part of the waste stream (3 %), it contains a high concentration of metals. As such, PCBs form the focus of this study. Base metals, especially copper, hamper the recovery of gold and PGMs by cyanidation. Further the copper grades of WEEE exceed those of many low grade ores exploited. Hence copper recovery from PCBs has garnered considerable focus. Bioleaching using the ferric ion and ferrous ion regeneration cycle is applied to the recovery of metals from metal sulphides in virgin ores and there is growing interest in its application to WEEE. The two sub-processes in the ferric regeneration cycle are ferric ion production through microbial oxidation of ferrous ion for growth and metabolic activity; and ferric ion consumption through the reduction of metals leading to metal dissolution. The ferric ion consumption and production rates depend on each other and other factors. The metal dissolution through ferric iron reduction is a function of ferric iron concentration, affected by how fast ferric iron is produced through microbial oxidation. Ferric iron production is a function of both the ferrous iron and ferric iron concentrations and so depends on how fast the ferrous ion substrate is produced through the dissolution of metals which consumes ferric ion in the process. Ferric ion production is also affected by the microbial population and microbial specific rates of oxidation of ferrous ion. Ferric ion consumption is also dependent on the metal dissolution rate which is affected by mass transfer limitations and the type of metal for dissolution. These are two competing subprocesses where the dissolution efficiency of metals is limited by the slower process.

[Page1 is in the PDF] The aim of this study was to investigate and compare various methods to enumerate the number of bacteria in a minerals biooxidation system. In this system most of the bacteria are attached to fine particles of ore and therefore cannot be enumerated by direct cell counting. This has hindered attempts to understand the mechanism by which the bacteria assist in the leaching process. The methods reported in the literature to enumerate both the free and attached bacteria in a biooxidation system can be divided into 2 categories: direct methods and indirect methods. The direct methods involve the quantification of the bacteria by direct observation. It is difficult to enumerate attached bacteria by direct observation but attempts have been made to desorb or dislodge these bacteria. Such experiments have had limited success in achieving dislodgement of all the attached bacteria. However, the results have shown that desorption of the bacteria from the mineral surface is possible. Indirect methods involve the monitoring of a cell component such as protein, nitrogen and carbon. Biomass concentrations have been estimated using its metabolic activity by means of a maximum specific oxygen utilisation rate. The purpose of this study was to compare the various methods and test their suitability to the quantification of biomass in a biooxidation system. In particular the biooxidation system investigated treated an arsenopyrite-pyrite concentrate from Fairview Gold Mine, Barberton, South Africa. The elemental analysis of the concentrate is 5.84% arsenic, 21.71 % sulphur,24.01 % iron and 1.41 % carbon. The dominant bacteria present in the biooxidation system were Leptospirillum ferrooxidans and Thiobacillus thiooxidans as shown by 16S rDNA analysis. The methods investigated are microscopic counting, gravimetric dry weight determination, desorption, determination of chemical oxygen demand, ashing, protein analysis, nitrogen analysis, total organic carbon analysis and measurement of oxygen utilisation rate. The oxygen utilisation rate method differs from the other methods as it uses the metabolic activity of the bacteria to measure the bacterial concentrations.

Bibliography: pages 149-155.
This dissertation presents the results of an investigation into the disruption of microorganisms when agitated in slurries of fine particles in a stirred tank. The most widely used industrial process involving agitation of microorganisms in slurries of particles in stirred tanks is the biooxidation process. Mixed cultures of thiobacilli are used in stirred tank reactors for the biooxidation of sulphide minerals. In addition to operating conditions, the efficiency of biotechnological processes is dependent on the growth and metabolism of the microorganisms. The microorganisms are sensitive to the hydrodynamic conditions generated in the processes. In response to adverse hydrodynamic conditions there may be changes in the growth rate of the microorganisms, the nutrient uptake rate, the product formation rate and morphology of the microorganisms. Under extreme conditions cell damage and disruption may ensue. The presence of particulates in bioprocesses, in the form of solid substrates or support systems for attached growth, further complicate the hydrodynamic conditions. The knowledge of the effect of particulates on microorganisms is an important priority.

This thesis concerns the development of a method to quantify the morphology of the bacterium Bacillus thuringiensis, and to automatically count the bacteria. The need to quantify the bacterial morphology arose out of the possibility of controlling a fermentation based on the morphology of the observed bacteria. Automatic counting of bacteria was considered necessary to reduce the inaccuracies that resulted in manual counts performed by different people. Bacillus thuringiensis is a spore forming, gram-positive bacterium, which produces both intracellular spores and insecticidal protein crystals. The production of the insecticidal protein crystal makes Bacillus thuringiensis important as a producer of biological insecticides. Automatic counting was developed in a Thoma counting chamber (Webber Scientific) at 200x magnification under dark field illumination. It was found that at this magnification the problem of out of focus cells was eliminated. The use of a thick coverslip, which reduces variability in slide preparation, was also possible at 200xmagnification as the focal depth of the 20x objective lens was considerably larger than the 1 00x objective lens and thus the 20x objective lens could focus through the thick coverslip (20x objective lens with 1 Ox magnification in eyepiece = 200xmagnification). An automatic algorithm to acquire images was developed and 5images per sample were acquired. Processing of the images involved automatically thresholding and then counting the number of bright objects in the image. Processing was thus rapid and the processing of the five images took no more than a few seconds. Results showed that the correlation between the automatic and manual counts was good and that the use of a thick coverslip reduced variability in slide preparation. It was shown that the manual -counting procedure, which necessarily used a thin coverslip at 1000x magnification, underestimated the volume of the Thoma counting chamber. This was a result of warping in the thin coverslip.

Bibliography: pages 117-123.
Within the aquaculture industry a potential has been identified for the pigment astaxanthin. Astaxanthin is the carotenoid responsible for the distinctive coloration of salmonids, crustaceans and certain birds. Due to the fact that animals cannot synthesize carotenoids themselves, it is necessary for these pigments to be present in their food source. In the case of farm-raised salmonids and crustaceans, supplementation of their food with astaxanthin is required. The chemical synthesis of astaxanthin is very costly and complicated. As a result natural, microbial sources of astaxanthin are being investigated. Phaffia rhodozyma is the only yeast known to synthesize astaxanthin as its principle carotenoid. The aim of this dissertation is to present a study investigating the growth and pigmentation of P. rhodozyma, with a view to its commercial production. A P. rhodozyma mutant (UCT-1 N-3693) with a 50% increased total carotenoid content was selected after NTG mutagenesis of the wild strain.

The aim of this project is to establish and understand the production of glucose oxidase and gluconic acid using the Aspergillus niger bioprocess and predict its response to operating conditions. Glucose oxidase and gluconic acid are produced by a wide range of microbes and have a variety of applications. In this study Aspergillus niger was chosen as the microorganism as it has the "generally accepted as safe" (gras) status in the U.S.A. It is also the major industrial producer. Glucose oxidase catalyses the conversion of glucose, oxygen and water to hydrogen peroxide and gluconic acid. This enzyme is used as a glucose and oxygen scavenger in the food industry and as a diagnostic tool in medicine for glucose determination. Gluconic acid is an organic acid used as a sequestering agent with a broad spectrum of applications. The world market for gluconic acid and its various salts was 45 000 metric tonnes in 1985 (Bigelis cited by Markwell et al. 1989). Gluconic acid and its derivatives can be produced using three technologies: electrolysis, mild chemical oxidation and bioprocess. The first two technologies have not been proven to be comnercially viable. The bioprocess offers diversity of feed and produces other products such as glucose oxidase. Literature has shown that the production of gluconic acid involves two kinetic areas. Firstly, the glucose oxidase enzyme must be induced. Secondly, glucose is converted to gluconic acid by the enzyme glucose oxidase. The factors affecting the kinetics associated with the induction of glucose oxidase have only been described qualitatively. Glucose, oxygen and pH have been shown to affect the induction of glucose oxidase. The effect of pH has been studied by Roukas and Harvey (1989) who found that induction, is maximal at a pH of between 5 and 6. The effect of glucose and oxygen have not been quantified. The kinetics of glucose oxidase conversion of glucose to gluconic acid have been well described by Atkinson and Lester (1974).

Cyanide (CN) is used in the gold mining industry to dissolve gold from free milling, complex and refractory gold containing ores. Processing sulphide containing refractory ores using biooxidation as a pre-treatment has become increasingly important due to the depletion of free milling ores. The reaction of CN with reduced sulphur species during the cyanidation process results in the formation of thiocyanate (SCN), often at relatively high concentrations (> 5 000 mg/L). The SCN and residual free CN are deported with the tailings as components of the liquid fraction. The concentration of SCN often exceeds the legislated discharge specification, necessitating on-site treatment, while water would also require treatment before on-site recycling and reuse. Biological degradation of CN and particularly SCN in these effluents provides an alternative to the more traditional processes such as SO2 treatment or UV destruction. The traditional destruction processes focus on breaking the chemical bonds, through physical or chemical means, thereby converting the toxic CN and SCN species to less toxic compounds. These processes generally suffer from high reagent cost, incomplete removal of CN and particularly SCN species and the generation of by-products which require further treatment. A number of microorganisms are capable of utilising CN and SCN as a source of sulphur, nitrogen and carbon, as well as generating energy from their oxidation. Additional removal of metal-CN complexes may be achieved by adsorption to the cell surface or extracellular polymeric substances secreted by the cells. The activated sludge tailings effluent remediation (ASTERTM) process was developed for the biological treatment of especially SCN, but also free CN and metal-cyanide complexes, such as CuCN and Zn(CN)2. The basic ASTERTM technology consists of an aerated reactor, in which SCN and CN species are oxidised and a settler to facilitate the recovery of water and potentially biomass. The desire to expand the commercial application of the technology necessitated a more complete, fundamental understanding of the ASTERTM process and required focused, in-depth research. This research aimed to define the viable operating window for SCN destruction, as well as optimising practical SCN and CN destruction process conditions. The ASTERTM process relies on a complex microbial community, so understanding the community structure and metabolic potential for SCN and CN destruction, further enhanced the fundamental and mechanistic understanding of this bioprocess. The research contributed to the fundamental understanding of this technology and enhanced the commercial application thereof. The first step in defining the operating window was to investigate the effect of feed SCN concentration on the SCN destruction ability of the mixed microbial community. Experiments were conducted at feed SCN concentrations ranging from 60- 1 800 mg/L. Complete SCN destruction was achieved across the range at ambient temperature. The maximum SCN destruction rate was 15.7 mg/L.h at an initial SCN concentration of 1 400 mg/L. Temperature was investigated in the range of 10-45°C with an initial SCN concentration range of 60-180 mg/L. A maximum SCN destruction rate of 17.4 mg/L.h was measured at 35°C, with an initial SCN concentration of 180 mg/L. A wide pH range (pH 5.0-10.0) was tolerated, with optimal performance recorded at pH 7.0. This evaluation identified not only the optimum operating pH, but also highlighted the negative impact of a sudden pH change on the efficiency of SCN destruction. Residual SCN concentrations below 1 mg/L were achieved in all cases, which would allow for discharge or recycling of treated water. Floc (sludge) formation was observed in experiments with high initial SCN concentrations and indicated a possible stress response during these batch experiments. Floc (sludge) formation were taken as microbial cells imbedded within extracellular polymeric substances and not only an aggregate of cells. Evaluating the maximum potential for SCN destruction and optimising the operating conditions and system configuration was investigated using continuous reactor experiments. A maximum SCN destruction rate of 87.4 mg/L.h (2 098 mg/L.d) was achieved at a feed SCN concentration of 1 000 mg/L and eight hour hydraulic retention time (HRT) during these experiments. The formation of substantial amounts of sludge was observed, with attachment to the reactor surfaces. The maximum feed SCN concentration, where substantial destruction was measured, was at 2 500 mg/L, achieving a practical SCN destruction rate of 972 mg/L.d. Significant inhibition of microbial inactivity was observed beyond this feed SCN concentration. The microbial community was able recover performance, within six days, after an extended period (54 days) of inactivity when the feed concentration was reduced from 3 500 mg/L SCN to 1 000 mg/L. The nature of the accumulated biofilm did not appear to change during the period of limited SCN destruction activity. Calculation of specific SCN destruction rates was not possible due to the nature of the sludge and heterogeneous dispersion of microbial members. Biomass (cells embedded in the EPS sludge) loading experiments showed SCN destruction rates increased with an increase in biomass loading, but this relationship was not proportional. A 25-fold increased biomass concentration resulted in only a 2-fold increase in destruction rate, suggesting a mass transfer limitation. The sludge most likely offers protection against unfavourable conditions, such as high residual SCN concentrations, by presenting a mass transfer barrier, resulting in an SCN concentration gradient across the sludge matrix. This enhances the robustness of the process and would facilitate rapid recovery in the case of a system upset at commercial scale. This research is the first to demonstrate the effective removal of SCN in the presence of suspended tailing solids, under conditions well suited for commercial application. The maximum SCN destruction rate achieved was 57 mg/L.h in the presence of 5.5% (m/v) solids. Sludge formation was not observed in the reactors containing solids, despite substantial sludge formation under similar operating conditions in the absence of solids, most likely due to shear-related effects. Fluctuations in pH, due to the nature of the solid material, were identified to negatively impact reactor performance and pH control was required. Moreover, the type of solid particle was found to influence the SCN destruction rate showing a need for adaptation not only to the presence of solids but also to various types of solids that are to be treated. Treatment of residual CN in solution is critical to ensure safe disposal or recycling of water. Treatment of SCN and CN was successfully demonstrated at feed concentrations up to 2 000 and 50 mg/L, respectively. The presence of residual CN (0.5 mg/L) prevented complete destruction of SCN, while complete SCN destruction was measured in the absence of CN under identical conditions. A range of reactor configurations were investigated and the optimum system required biomass retention, by means of attached biomass and complete destruction of any residual CN prior to SCN destruction. Conversion of SCN-S to SO4-S was stoichiometrically proportional in solution, while the majority of the liberated nitrogen appeared to be assimilated. Pre-colonisation of the reactor with attached biomass is beneficial and removed the need for a solid-liquid separation unit, reducing the potential footprint of the process. Additional treatment capacity could be created by operation of reactors in series. The diversity of the microbial community responsible for destruction of especially SCN were shown to be far more extensive than initially expected. Initial molecular characterisation of the microbial community selected for 185 representatives of bacterial 16S rRNA genes, of which 106 non-identical genotypes were sequenced. In contrast, for the reactor containing solids, only 48 representatives were selected and 30 genotypes were sequenced. Bacteria implicated in SCN destruction in the reactor containing suspended solids were members from the genera Bosea, Microbacterium and Thiobacillus. In the absence of solids, members capable of SCN destruction were identified from genera including Thiobacillus and Fusarium. High-throughput genome sequencing, followed by sequence assembly confirmed the dominance of Thiobacillus spp. Metabolic predictions indicated the autotrophs, gaining energy from the oxidation of reduced sulphur intermediates produced during SCN destruction were the dominant community members. The potential for ammonium oxidation and denitrification within the microbial community was identified during analysis of the metabolic potential, based on the metagenomic sequence data. These would be required for complete remediation of wastewater. The data generated during the research led to the development of a conceptual model to describe the evolution of system performance. Following inoculation with planktonic culture the SCN destruction is performed by the planktonic microbial community. An increased residual SCN concentration results in floc formation and the colonisation of reactor surfaces by attached biofilm. A concomitant decrease in planktonic cell concentration was observed, while SCN destruction rates increased. The extracellular material provided a matrix for biomass retention, resulting in high cell concentrations, and provided some protection against high SCN concentrations by providing a barrier to mass transfer. The attached biofilm developed to the point where overall SCN degradation rates may become limited by reduced oxygen penetration. The research presented in this thesis has been used to inform the design and operation of the ASTERTM process at commercial scale, specifically with respect to the benefits of attached biomass and the demonstration that the process can be used in the presence of suspended solids. The latter has been particularly important in applications where the available footprint is constrained.

Bioethanol plays a significant role in the world of liquid biofuel. However, majority of bioethanol is produced from edible food crops such as corn and sugarcane that causes an increase in demand for vacant lands for food production and, subsequently, increase in the cost of food manufacturing. Therefore, alternative raw materials for bioethanol production are sought after, such as sugarcane bagasse which is a waste material from the sugar industry. South Africa, a net sugar exporter, has a large potential to produce bioethanol from sugarcane bagasse. This research focuses on the study of the production of bioethanol from glucose and xylose which are the two most abundant sugars in hydrolysed sugarcane bagasse. To date, no suitable wild type organisms can concomitantly ferment both glucose and xylose to ethanol efficiently. Options to address the co-fermentation of glucose and xylose include genetic modification of the selected microorganism to include both pathways - limitation in the understanding of the metabolic pathways regulations - or utilization of two microorganisms in co-culture or sequential culture e.g. Zymomonas mobilis and Pichia stipitis for efficient fermentation of glucose and xylose respectively. In this study, the dual micro-organism route is explored. There are numerous problems associated with co-culturing. Xylose, a non-preferred carbon source is only converted if the glucose concentration is adequately low due to catabolite repression. In order to increase xylose conversion, a low glucose concentration is required. Therefore, two stage sequential fermentation either in one or two reactors was tested. A high inoculum of suspended or immobilized Z. mobilis was inoculated in the first stage to convert the glucose rapidly. Varying reactor configuration, including the continuous fluidized bed, continuous stirred tank reactor (CSTR) and stirred batch reactor were considered. The products and residual substrate from this fermentation was then directed to a second stage, using either a CSTR or stirred batch configuration, with a high inoculum of P. stipitis in suspension culture for conversion of xylose. When immobilized, Z. mobilis was entrapped in calcium alginate beads. On the issue of ethanol tolerance, P. stipitis is generally more easily inhibited by ethanol (threshold ethanol concentration of 35 g L-1) compared to other ethanol producing strains such as Z. mobilis (threshold ethanol concentration of 127 g L-1) and Saccharomyces cerevisiae (threshold ethanol concentration of 118.2 g L-1). In order to overcome this, a continuous bioprocess was investigated to keep ethanol concentrations in Stage II below 35 g L-1 to prevent inhibition of metabolic reactions in P. stipitis. Further, ethanol fermentation by Z. mobilis requires obligate anaerobic conditions while xylose conversion by P. stipitis is optimum under microaerobic conditions. Therefore, oxygen was sparged into the second P. stipitis stage only. The following components were carried out in this project to improve the kinetic model and to find accurate kinetic data in the selected process of the two stage sequential fermentation. Firstly, where kinetic parameters were not available in literature, the kinetic parameter relationships of glucose and xylose utilization between different constructs of the same species were examined, for example, a wild type and engineered strain. This approach was used for glucose conversion using wild type Z. mobilis, owing to the ill-fit of available kinetic parameters with experimental results. In this study, the correction factors on estimated kinetic parameters from linear and non-linear regression when a xylose fermentation route was inserted recombinantly (S. cerevisiae RWB 217) into the native culture (S. cerevisiae CEN.PK 113-7D) were determined. From kinetic parameters of an engineered strain with the xylose-fermenting pathway (Z. mobilis ZM4 (pZB5)) and the correction factors, kinetic parameters of the wild-type Z. mobilis ZM4 were determined. Predicted rates of Z. mobilis ZM4 were then validated with experimental data generated in this study. Then, the optimum initial biomass concentration required to provide a faster volumetric rate of sugar utilisation and ethanol production, as well as the optimum oxygenation level for xylose conversion using P. stipitis achieved through appropriate aeration were investigated through experimental observation and using a MATLAB mathematical model developed through combination of the Andrews and Levenspiel's models, with oxygen, substrate, cell and product terms. Experiments were carried out to validate the kinetic model and data under anaerobic and microaerobic growth conditions in a batch process. The results showed that both increasing the initial biomass concentration (3 g L-1) and operating under optimum oxygenation levels (0.1 vvm) benefitted the ethanol production and yield by P. stipitis from xylose. It was also concluded that the addition of the oxygen effective factors in the developed model allowed for optimization of aeration in the fermentation system. Next, the custom kinetic model for fermentation process of bioethanol production was developed in Aspen Custom Modeller (ACM) and embedded in Aspen Plus. The model includes equations of vapour-liquid equilibrium (VLE), mass balance, and energy balance (e.g. molecular weight, thermodynamic phase equilibria, kinetic equation). The obtained results showed better agreement between industrial data and kinetic model (1% differences) than a stoichiometric model (9% differences). The simulation showed that ACM integrated into Aspen Plus allowed for complex biological processes to be accurately predicted for biomass growth, ethanol production and sugar consumption. Finding suitable microorganisms and process conditions for efficient glucose and xylose conversion is still currently a challenge and requires optimization. Therefore, this research focusses on improving the conversion of glucose and xylose to bioethanol, with specific emphasis on the fermentation systems used to maximize biomass efficiency, and ethanol yields and productivities. Manipulation of process conditions ranging from operation conditions (e.g. batch, fed-batch, continuous), process parameters (aeration, temperature, pH), immobilization technique and type of microorganism initially using kinetic models and thereafter validating with experimental data, therefore, offers a quick and strong foundation in improving bioethanol yields and productivities.

Acid rock drainage (ARD) is defined as acidic waste-water contaminated with sulphate and heavy metals which is generated through the oxidation of sulphidic ores in the presence of water and oxygen. Mining activities accelerate this process by bringing these ores to the surface where they are further crushed and, eventually end up in waste rock dumps and tailing impoundments where they continue to generate ARD into perpetuity. Active mining operations are mandated to prevent the discharge of ARD into the environment. This ARD is commonly remediated by expensive yet highly effective active treatment strategies such as high-density sludge processes and reverse osmosis. South Africa has an extensive history of gold and coal mining which has left abandoned mine workings with associated waste rock dumps throughout northern and eastern parts of the country. As many of these mines have long been abandoned, the responsibility to mitigate the environmental impact of the generated ARD lies solely with government. Although these diffuse sites often generate smaller volumes of less aggressive ARD compared to that generated through mine water rebound, the sheer number and the continual ARD generation from these sites is a severe threat to South Africa's already poor water security. Biological sulphate reduction (BSR) has long been considered an attractive option for the longterm remediation of these low-volume sources of ARD – but its implementation has shown mixed success. BSR is a process catalysed through the innate metabolism of sulphate-reducing bacteria (SRB) which coexist within complex microbial communities. SRB themselves are a highly diverse group of anaerobic microorganisms which use sulphate as a terminal electron acceptor. The sulphide and bicarbonate produced during BSR can be used to precipitate heavy metals and aid in the neutralisation of the ARD, respectively. The implementation of BSR is, therefore, a comprehensive remediation strategy for diffuse sources of ARD. The study of BSR, using various reactor configurations and operating conditions shows much promise. However, the microbial ecology of the complex communities within BSR systems, and their links to the performance of BSR processes, has received far less attention in published literature. This is not a result of underappreciation of the role microbial communities but rather a historical lack of tools, specifically high-throughput techniques, available to assess complex microbial consortia. It is asserted that the success of a sustainable BSR process developed for the long-term remediation of ARD requires an in-depth understanding the microbial communities associated with this process. The identification of the microorganisms which are key to the process, thosewhich threaten the stability of the community and the optimal growth conditions of these microorganisms, can be used to inform how these bioreactors are designed and operated. This study investigated the performance and microbial ecology of several continuous BSR reactors using culture-independent metagenomic sequencing approaches. The performance and microbial ecology of these reactors were evaluated at a range of hydraulic residence times (HRT) over the course of approximately 1000 days of continuous operation, from five- through to one-day(s). The tested reactor configurations included a continuous stirred tank reactor (CSTR), an up-flow anaerobic packed bed reactor (UAPBR) and a linear flow channel reactor (LFCR) that were each operated in duplicate and supplemented with either lactate or acetate as an electron donor. The different reactor configurations and supplied electron donors, as well as the varied applied HRT, generated a range of microenvironments which were hypothesised to lead to the divergence of the initial microbial community of the inoculum and generate numerous distinct microbial communities throughout and across the reactor systems. 16S rRNA gene amplicon sequencing was used to assess the microbial community structure of the numerous populations across the reactor systems and monitor how these communities responded to the change in the applied HRT. Genome-resolved metagenomics was employed in parallel to recover the genomes of all predominant microorganisms identified through gene amplicon sequencing. This allowed the interrogation of the composition of the respective microbial communities as well as the genetic potential of each microorganism and encompassing the communities represented within specific reactor environments. The CSTRs were selected as these systems are characterised as well-mixed, support solely suspended biomass and kinetic equilibriums are achieved rapidly. This allows the performance of these reactors to be predictable and provides a benchmark to which the LFCRs and UAPBRs could be compared. The lactate-supplemented CSTR performed largely as anticipated based on available literature, demonstrating a maintained sulphate conversion of approximately 55% over the course of the study. The reactor achieved a maximum observed volumetric sulphate reduction rate (VSRR) of 17 mg/ℓ.h at a one-day HRT. The system supported a low SRB diversity, constituted almost entirely by a Desulfomicrobium and two Desulfovibrio operational taxonomic units (OTUs). The acetate-supplemented CSTR was able to maintain sulphate reducing performance at HRT where complete washout of SRB had been predicted based on literature. This reactor exhibited a maximum VSRR of 10.8 mg/ℓ.h at a 1.5-day HRT and was dominated by the same Desulfovibrio and Desulfomicrobium observed in the lactate-supplemented CSTR, along with several other SRB genera at lower abundance. The LFCRs demonstrated an approximately ten-fold greater biomass retention than the corresponding CSTRs. This was facilitated through the incorporation of carbon microfibres, whichfacilitated microbial colonisation and biofilm formation within the reactors. Surprisingly, the lactate-supplemented LFCR, underperformed compared to the lactate-supplemented CSTR, achieving a maximum VSRR of 14.8 mg/ℓ.h at a one-day HRT. This reduced performance, in spite of the enhanced biomass retention, was concluded to result from the out-competition of lactateoxidising SRB in the reactor by Veillonella and Enterobacter OTUs. The acetate-supplemented LFCR exhibited a period of underperformance before recovering and subsequently demonstrated a maximum VSRR of 17.1 mg/ℓ.h at a one-day HRT. Evaluations of the microbial communities of this system during the HRT study revealed a dramatic shift in the SRB communities from being dominated by Desulfatitalea and Desulfovibrio to being dominated predominantly by Desulfomicrobium and Desulfobacter. The UAPBRs are governed by plug-flow which resulted in the generation of gradients of decreasing substrates and increasing products throughout the height of the reactors. This, as hypothesised, resulted in the stratification of the microbial communities throughout the height of these reactors. This allowed many associations to be made between specific microorganisms and their ideal growth environments. Both UAPBRs demonstrated competitive sulphate reducing performance. The lactate-supplemented UAPBR proved especially successful as this system was able to maintain >95% sulphate conversion at one-day HRT, corresponding with a VSRR of 40.1 mg/ℓ.h. The performance of this reactor was attributed to the significant quantity of retained biomass and the successful harbouring of lactate-oxidising SRB towards the inlet zone of the reactor as well as propionate- and acetate-oxidising SRB towards the effluent zones of the reactor. The acetatesupplemented UAPBR exhibited a maximum VSRR of 23.2 mg/ℓ.h at a one-day HRT and a maximum sulphate conversion of 79% at a 2.3-day HRT. The stratification of the microbial communities within the acetate-supplemented UAPBR was less pronounced than the lactatesupplemented UAPBR, as a result of the fewer available volatile fatty acid species. However, the stratification which was observed in this system could be used to postulate the growth kinetics associated with the identified SRB – a Desulfobulbus was associated with rapid acetate oxidation in the inlet zone while a Desulfatitalea and a Desulfosarcina could be implicated in sulphate scavenging in the effluent zone of this reactor. This proved particularly valuable for elucidating the roles of these same SRB in the well-mixed reactor systems. Genome-resolved metagenomics was employed to recover the genomes of the microorganisms identified in these systems and determine the metabolic potential of these microorganisms. Hydrogen-evolving hydrogenase genes were found to be widespread in genomes not capable of sulphate reduction. In contrast, hydrogen-consuming hydrogenases as well as autotrophic gene pathways were common amongst SRB genomes. The ubiquity of hydrogenase genes in these environments indicated that inter-species hydrogen transfer was an important feature within thesemicrobial communities. The dual consumption of both acetate and hydrogen was concluded to have facilitated the maintained sulphate reducing performance of the acetate-supplemented reactor systems at short HRT where system failure had been predicted. Indices of replication (iRep) were used to estimate the instantaneous growth rates of the microorganisms from metagenomic shotgun sequencing datasets. This revealed that, at a four-day HRT, the microorganisms within the biofilms were comparably active to planktonic microorganisms. This, together with the dynamic changes in the composition of these biofilms during the HRT study, suggests these biofilms are even more active and competitive than previously thought. The combined use of next-generation gene amplicon sequencing and genome-resolved metagenomics has given unprecedented insights into the microbial communities of BSR reactor systems. Using this approach, it was possible to uncover a seldom discussed form of hydrogen cycling within BSR systems and has shown that there is no ‘one-size-fits-all' approach when inoculating BSR reactors. The SRB within these systems were often highly specialised to particular environments, specific electron donors and each showed differing growth kinetics. The success of long-term, semi-passive BSR reactor systems would benefit greatly from the tailoring of SRB inoculums informed by the chosen reactor configuration and operating conditions. The outcomes of the kinetic reactor experiments have led to several recommendations for the design and operation of these systems.

Colonisation of mineral surfaces by acidophilic microorganisms during bioleaching is important for accelerating the extraction of valuable metals from mineral sulfide ores of varying grades through biohydrometallurgy. It also influences acid formation and mineral deportment from sulfidic waste rock generated in mining processes and is key to its comprehensive waste rock characterisation for acid forming potential. This study assesses mixed mesophilic microbial interactions with, and colonisation of, pyrite concentrates and pyrite bearing waste rocks. The assessment of these interactions was carried out in this study in a synergistic qualitative as well as quantitative manner, with a particular focus on heap bioleaching for metal extraction and on disposal of waste rock, the latter through the case of characterisation of ARD generation potential. Using the tools developed, both the course of colonisation and development of metabolic activity with time of colonisation, as well as their correlation with leaching performance were studied. Furthermore, specific operating parameters such as ore grade and irrigation rates were explored. Finally, the application of this knowledge in a characterisation study was explored. To achieve the set of tools required for this study, two quantitative techniques were refined to characterise these microbial-mineral interactions. In the first, an isothermal microcalorimetric (IMC) method was developed and optimised to determine microbial colonisation of mineral surfaces quantitatively as a function of surface area (m-2 ). Three IMC configurations were considered: colonised pyrite-coated beads submerged in fresh media; beads submerged in cell free leachate; and beads in an unsaturated bed, each in the IMC vial. The highest heat output was measured in the unsaturated bed (263.3 mW m-2 ). The consistency of heat produced by the colonising microorganisms was determined through reproducibility studies. Using IMC, chemically and microbially facilitated pyrite oxidation rate studies were performed on unsaturated beds with varying surface area loadings, correlating to varying bead number. Results obtained showed similar normalised oxidation rates per surface area across the surface loadings. However, with more microbially colonised surface area loaded, the maximum heat generated was reached more quickly. This suggested that there was reagent (possibly O2) limitation in the system, which restricted microbial activity and its associated heat generation. Reagent limitation in the system was tested and validated through varying the O2 availability in the IMC vial by air displacement with CO2 and N2 gas, with the systems containing less O2 showing limited activity. Collectively the data showed that high activity, facilitated microbially, was achieved in unsaturated systems in a reproducible manner. Secondly, oxidation rates were determined and O2 limitation in the system was overcome. This then fundamentally informed the determination of activity from microbial-mineral interaction, using IMC, as a function of surface area. Secondly, a detachment protocol developed at UCT to recover microbial cells from surfaces of crushed and agglomerated ore to assess microbial growth rates and distribution in the ore bed, including cells in the interstitial phase and those weakly and strongly attached to the ore surface, was refined to assess colonisation of the finely milled pyrite-bearing concentrate or waste rock coated onto glass beads in continuous flow assays. The detachment protocol was assessed quantitatively by measuring initial and residual microbial activity, as a function of wash number, using IMC, thus providing a new level of confidence in the method. Mineral surfaces were visualised using scanning electron microscopy (SEM) following detachment for qualitative assessment. These data, together with microscopic enumeration of detached cells with increased number of washes, allow refinement of the assay and showed that six washes provided reliable estimation of mineral associated microbial cells. Extracellular polymeric substances (EPS) produced in this process were extracted using crown ether and the capsular bound components analysed. The analysed components included lipids (4.2 %), iron (16.4 %), DNA (26.8 %), and total carbohydrates (28.5 %), which are typical components of EPS. The carbohydrate fraction was further resolved to trehalose (26.2 %), fructose (36.5 %) and galactose (37.3 %) sugar monomers. The analysed EPS components confirmed presence of the EPS secreted by cells colonising the mineral ore or waste rock surface in a flow-through system, and visualised via SEM. The microcalorimetric approach developed together with the refined detachment method were applied to samples from a flow-through mini-column system, used to simulate microbe-mineral contacting in a heap. Here, the colonisation of pyrite concentrate by a mixed mesophilic culture of iron and sulfur oxidising microorganisms was assessed progressively over 30 days. The progression of mineral colonisation in the mini-column system was monitored using a combination of IMC, scanning electron microscopy, detachment method and conventional wet chemistry measurements. We observed an increase in the heat output from the colonised surfaces of pyrite mineral concentrate caused by oxidative reactions facilitated by mineral-microbial biofilm. This confirmed that the attached microorganisms were metabolically active and facilitated ongoing mineral leaching through regeneration of lixiviants. Correlation was shown between number of cells detached from the mineral surface and the heat generated, with a constant heat output per cell observed until day 15 of operation. Thereafter, the measured heat generated per cell increased, suggesting reduced efficiency of cell detachment owing to increasing firm attachment, or the lack in separation of single cells embedded within EPS matrix (clumps observed under light microscope after detachment). Using IMC to quantify the activity of the residual microorganisms on the mineral surface following detachment, it was confirmed that >95% of activity was detached through this protocol, hence the lower detached cell numbers determined following EPS formation were attributed to clumping of the detached cells. This correlated to an increased presence of EPS and was supported by SEM observation. Following the study of pyrite concentrate, colonisation of two pyrite bearing waste rock samples was assessed, with simultaneous establishment of the flow-through mini column biokinetic test configuration that resembles open flow in the waste rock dump. The flowthrough configuration was run alongside the refined UCT-developed batch biokinetic test using suspended mineral. In this study, two pyritic waste rock samples, liberated by milling, were characterised using three biokinetic test approaches: the slurry batch test (BT), the batch test using mineral-coated beads (BT-CB) and flow-through column test with mineral-coated beads (FT-CB). Our results have shown through static tests, solution redox potential and pH analysis that both waste rocks were acid forming. Furthermore, it was demonstrated in the FT-CB system that microbial proliferation on the waste rock surfaces progressed with time such that oxidative exothermic reactions facilitated by the increasing microbial presence on the surfaces were demonstrated using Isothermal microcalorimetry. This study presents and informs the on-going refinement of the biokinetic test through establishment of a flow-through test for ARD characterisation while providing insight into the role of the microbial phase in ARD generation. Microbial-mineral association was assessed under various operating conditions, including two solution flow rates (60 and 4 ml h -1 ) and minerals of varying sulfide content, including a pyrite concentrate (96 % pyrite), a high sulfide waste rock (33 % pyrite) and a low sulfide waste rock (14 % pyrite). Mineral grade impacted the activity of mineral associated microorganisms with higher activities observed on a mineral surface with high sulfide content. The activity measured from microorganisms that were associated with the pyrite concentrate was 827 mW m-2 at a 60 ml h -1 flow rate, whereas activity measured on low and high sulfide waste rock (PEL-LS and PEL-HS) were 293 mW m-2 and 157 mW m-2 respectively operated on the same flow rate. On decreasing the flow rate to 4 ml h -1 , the activity of microbial cells on PEL-LS and PEL-HS were 153 mW m-2 and 146 mW m-2 respectively. This study showed that the growth of microbial cell numbers coupled with metabolic activity is important to facilitate accelerated dissolution of sulfidic mineral surfaces. The rate of oxidation increased in the presence of EPS and thus EPS was further analysed, and its composition was confirmed. Overall, this study contributed to the understanding of microbial colonisation of mineral surfaces in a non-destructive quantitative manner. This study thus demonstrates the ability to measure and track both the growth and activity of microorganisms that are associated with mineral surfaces. This is important as it provides an approach to understanding microbe mineral surface interactions and, therefore, potential strategies to increase microbial colonisation of low-grade minerals that house valuable metals, during commercial heap bioleach processes. Furthermore, the ability to monitor progressive growth and activity of mineral associated microbial communities within a flow-through biokinetic test, as successfully demonstrated in this study, has the potential to significantly enhance current management of mine waste materials and ARD mitigation strategies. Therefore, on-going investigations of progressive microbe-mineral interactions will continue to be valuable both in terms of bioleaching for metal recovery and the mitigation of ARD through effective characterisation of mine waste material.

The use of laboratory scale Microbial Fuel Cells (MFCs) for the combined generation of electricity and the treatment of wastewater has been well documented in literature. In addition to this the integration of MFCs into wastewater treatment reactors has also been shown to have several benefits. These include the improved treatment of wastewater, reduced solid waste and the potential to offset the energy costs of the process through the generation of electricity (Du et al., 2007). The treatment of sulphate-rich wastewater, and in particular Acid Rock Drainage (ARD), has become of increasing importance in water sparse countries like South Africa where mining is currently and has taken place. A semi-passive method of continuous ARD waste treatment is currently being investigated within the Centre for Bioprocess Engineering Research (CeBER) (van Hille et al., 2015). This research involves the use of a Linear Flow Channel Reactor (LFCR) designed for combined biological sulphide reduction and sulphide oxidation to yield a sulphur product. Sulphate Reducing Bacteria (SRB) mediate the biological sulphide reduction. Chemical and biological sulphide oxidation takes place in a Floating Sulphur Biofilm (FSB) on the surface of the reactor and is mediated by Sulphide Oxidising Bacteria (SOB). Sulphate-rich wastewater can therefore be remediated through total sulphur species removal.

Includes bibliographical references.
The mining and beneficiation of gold generates large tonnages of waste, with up to 99% of mined gold ore discharged as waste. The waste generated contains unoxidized sulfides that when exposed to air and water react to form acid, which results in acid rock drainage (ARD). ARD is usually associated with low pH, high sulfate content and elevated concentrations of toxic elements. The mobility of ARD affects our scarce water resources, land and aquatic species. Methods applied to treat ARD do not provide a walk-away solution and they are either expensive or difficult to maintain. The best solution to completely eradicate ARD is to prevent it from the source. However, the effectiveness of ARD prevention depends on the accuracy of predicting future drainage quality. This can be done by using ARD prediction tests, which are generally classified as either static (acid base accounting, ABA, net acid generation, NAG) or kinetic (column leach, humidity cell, biokinetic test). There is no single test capable enough to accurately predict acid generating potential. It is therefore usual practise to conduct more than one test and cross-check results to ensure that the appropriate conclusions are made. In doing so, the reliability of the tests is improved but in some cases the different test results do not correlate. Mineralogy is an analytical technique that can be used to understand the nature of the errors and to better understand the leaching behaviour of minerals in the different tests. This study uses mineralogy to analyse both static and biokinetic test results of a Witwatersrand gold sample in order to improve the understanding of behaviour of mine wastes under different ARD prediction test conditions. A run-of-mine gold sample from the Witwatersrand region in South Africa was used as a case study to explore the mineral leaching behaviour for different ARD prediction tests.

In South Africa, over 10 million tons of ultrafine coal wastes are discarded every year, typically in the form of ultrafine slurries. These fines have a high calorific value, and contain sulfur minerals, particularly pyrite. The high calorific value of these discards leads to a waste of energy that could be harnessed and used, while the high sulfur content contributes to adverse environmental effects such as acid rock drainage (ARD). The University of Cape Town (UCT) has developed a two-stage flotation process, which involves coal flotation in the first stage and pyrite flotation of the tailings in the second stage, for mitigating the ARD potential of ultrafine wastes. Research has shown that this two stage froth flotation process was sufficient to render the tailings non-acid forming. At the same time, North West University (NWU) has been carrying out research on coal fines using the recently invented reflux classifier. The reflux classifier is claimed to be capable of separating particles down to 38 ìm in size; however, no work has been done using the reflux classifier to separate pyrite from coal. This dissertation investigates the effectiveness of combining flotation and reflux classification for removing sulfide minerals from two South African coal ultrafines, whilst recovering valuable coal, and compares the results to those obtained using the UCT two-stage flotation process. As no previous work has been done using reflux classification to remove sulfide minerals from coal, this is the first time that the reflux classifier will be investigated for this purpose. Two process routes were investigated: (i) froth flotation followed by reflux classification of the tailings (process route 1), and (ii) reflux classification followed by froth flotation of the overflow (process route 2). Coal flotation, sulfide flotation and reflux classification were conducted on samples of Waterberg and Witbank coals, using a 3 L Leeds-type flotation cell and a 10 L batch reflux classifier constructed at NWU. Acid base accounting (ABA) and net acid generating (NAG) static characterization tests were performed on the products and feeds from all three process routes.

Includes bibliography.
This study has been encouraged by the successful recovery of useful energy from brewery wastewater using anaerobic digestion technology. It aims to evaluate the environmental benefits or burden of improving energy production by using organic brewery by-products as additional feedstock into the SABWTP. An environmental impact assessment on the SABWTP and its associated process was carried out using life cycle assessment (LCA) tools. Anaerobic digestibility of the two major organic brewery by-products, brewer’s spent grain and brewer’s spent yeast, was evaluated experimentally using laboratory bench scale reactors. The results were used to postulate the feasibility of adding these feedstocks into the SABWTP. Based on these findings, three viable processing scenarios were synthesised and assessed in terms of environmental impact analysis. In the environmental impact analysis, the three scenarios were compared using average process conditions and the main contributing factors to environmental burdens associated with each scenario were identified.

Microalgae are recognised as a source of lipids for bioenergy, nutrients and pharmaceuticals. Photobioreactors, closed vessels for microalgal cultivation, are known to have high energy consumption due to mixing and aeration. Sparging is commonly used for mixing and gas-liquid mass transfer in photobioreactors, but is energy intensive. The aim of this work was to reduce these energy requirements by optimising conventional sparging and considering surface aeration coupled with mechanical agitation as an alternative. An airlift photobioreactor was used as a base for comparison with two novel, surface aerated reactors: oscillatory baffled and wave photobioreactors. The three bioreactors were compared in terms of power input, mixing, CO2 mass transfer, algal growth and lipid production. Prior to comparison, each photobioreactor was optimised based on these parameters. To calculate power input, isothermal gas expansion equations were used for sparged systems and calorimetry was used for mechanically agitation systems. Mixing was investigated using a salt tracer and phenolphthalein indicator and mass transfer was measured using the gassing-in method. Scenedesmus sp., a high lipid-producer, was cultivated in low nitrate media across a range of mixing rates in each photobioreactor.In the airlift photobioreactor a critical minimum CO2 supply rate (of 2.7×10-5 m s-1) was found, below which carbon was limiting and above which energy was spent on sparging without increased productivity (0.20 g L-1 d-1 biomass; 0.03 g L-1 d-1 lipid). In the oscillatory baffled reactor, insufficient mass transfer limited algal productivity (0.11 g L-1 d-1 biomass; 0.02 g L-1 d-1 lipid). The wave reactor had high CO2 mass transfer coefficients (10 – 140 h-1) in comparison to the airlift (2.7 – 40 h-1) and oscillatory baffled reactors (6.3 – 37 h-1). Sufficient biomass productivity (0.18 g L- -1 d-1) and higher lipid productivity (0.045 g L-1 d-1) at lower power input in the wave reactor resulted in higher energy efficiency compared to the airlift reactor. Life cycle analysis of simulated algal biodiesel production showed that bioreactor energy contributed 99% of total energy consumption. Therefore, the global warming potential was reduced by 73% when the airlift reactor was operated at the critical minimum CO2 supply (with gas compression to 2 bar) and a further 19% when the wave reactor was used. This work offers an energy efficient alternative to sparging, through the generation of a well-mixed wave in a surface aerated bioreactor. It also offers methods for optimisation of energy usage with respect to mixing and aeration. Reducing bioreactor energy consumption is key to feasibility, and was demonstrated here to reduce energy-related environmental burdens.

Amylases are hydrolytic enzymes that cause the breakdown of starch and related polysaccharides to simple sugars. Amylases are applied in brewing, food, detergent and textile industries. Most commercial amylases are derived from fungi or bacteria. Bacterial amylases are desired for commercial use, due to their thermo-stability and faster production rates. Bacteria of the genus, Bacillus, are considered to be a good source of extracellular proteins because they have high growth rates and have a naturally high capacity for secretion of extracellular proteins. Bacillus halodurans Alk36 is an alkaliphilic, thermotolerant isolate that can grow over a wide pH and temperature range. Preliminary studies have shown that B. halodurans Alk36 can grown in EnBase® medium (at pH 8.5) containing starch as the carbon source, without the addition of a commercial amylase. The ability to grow on starch, in the absence of an external amylase, indicated that this strain produces endogenous alkaliphilic amylases, which may be exploited for a number of industrial applications. In the present study, the physiological and biochemical characterisation of B. halodurans Alk36 and its endogenous amylases were investigated.

The growth of microalgae has the potential to be extremely useful for the production of a wide range of products or for specific processes, such as capture and cycling of CO₂. The fast micro-algal growth rates and ability to grow on agriculturally poor land and in waste water means that bio-production using algae has many advantages over traditional agricultural processes for certain applications. The raceway pond is the most common reactor used for the growth of microalgae, due to low capital costs, low operating costs, higher energy efficiency, improved net energy recovery and ease of installation. Low carbon dioxide mass transfer, which limits algal growth and productivity, is currently one of the largest issues in photo bioreactors of all forms. The microalgae within these systems only obtain carbon from the dissolved inorganic carbon and hence sufficient carbon dioxide mass transfer is one of the most important design parameters for any photobioreactor. This is particularly evident in raceway ponds as they have a lower volumetric mass transfer rate than other photobioreactors and are typically mass transfer limited. [Please note: the full text of this thesis has been deferred until 30 September 2017]

Resistance to antibiotics by microbial pathogens continues to be a major global health problem. Treatment of bacterial infections is becoming increasingly complex and expensive. Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is affected by antibiotic resistance. In South Africa, the Western Province is the worst affected, with an increasing incidence of both multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis. Both resistant forms of TB increase the length of treatment to almost 24 months and cost by as much as 1400 times that of regular anti-tubercular chemotherapy. A potential solution to this problem is the discovery of new drugs, which can be obtained from natural sources. Actinomycetes are good sources of these drugs, with over 45% of current medicines derived from these bacteria. The actinobacterium Streptomyces polyantibioticus SPRT (SPRT) was locally isolated and first described by Le Roes (2006). It has been shown to produce bioactive molecules active against a range of bacteria, including compounds (drugs) that have anti-tubercular properties. One of the anti-tubercular molecules was identified as 2,5-diphenyloxazole (DPO). DPO is currently used as a component of scintillation fluid for its luminescent properties and is synthesised chemically in industry. SPRT is the only reported biological source of DPO, it is however not yet produced commercially via a biological route. The present study was performed to inform future process development of DPO production from SPRT. An investigation into the growth and production of antimicrobial compounds from submerged cultures of SPRT in shake flasks, and scale-up of the process into a laboratory stirred tank bioreactor (STR) was done in the present study. The work focused on obtaining growth kinetics and suitable operating conditions for cultivation. Characterisation of the growth profile of SPRT and determination of the kinetic growth parameters was carried out. Additionally, the antimicrobial production phases, and factors influencing their production was investigated. It was determined that the most reliable method of measuring biomass concentration was by dry cell gravimetric measurement of whole shake flasks following vacuum filtration, as it best suited the non-homogenous filamentous nature of SPRT.

A transition is needed to shift the global economy to a more sustainable and clean economy to counteract the depletion of abiotic resources, generation of emissions and waste. Replacing petroleum plastics by bio-plastics is key to this move. This study identifies the bio-based and bio-degradable plastics, polyhydroyalkanoates (PHAs) as promising alternatives to petroleum plastics. PHAs are biologically synthesised polyesters which accumulate intracellularly in the presence of excess carbon from renewable resources and, limited nutrients in the form of nitrogen, phosphorus or sulphur. Different PHA polyesters can be synthesised by varying the growth conditions of the microbial culture. The flexible nature of PHA synthesis results in a large group of PHA polyesters with a variety of properties that span those of petroleum plastics in terms of strength, rigidity, durability and mechanical properties. Due to their versatility, PHAs have a wide range of applications in packaging and coating, health care and hygiene products, fibres, adhesives, components of toner and developer fluids and medical sutures and implant materials. Waste products of PHA production are mostly carbon dioxide and water. PHAs are biodegradable in all living systems including marine environment. PHAs are also bio-compatible and thus are not toxic to living organisms. In spite of the numerous advantages that PHAs offer, they have not yet achieved substantial commercial success due to poor market penetration arising from their high costs of production. The typical PHA sale price ranged from $ 4 to 9/kg in 2014. Factors that have been reported to affect the production costs of PHAs are the PHA content and concentration achieved by the microorganism, the purification and recovery process of PHAs together with the type of carbon substrate used. Additionally, the efficiency of the cell disruption and energy required for the washing steps can also influence the production system and are addressed in this study along with the other factors. The overall PHA production system must also be sustainable minimising its energy, water usage and carbon dioxide emissions. When faced with limited time and resources, insight is needed to identify which of the aforementioned factors or combination of factors are more crucial to optimise in order to lower production costs and environmental burden. Economic assessment is a powerful tool to identify promising economically viable process options and discard unfavourable ones. Since PHA production is not an established technology, process conditions and pilot scale data are not easily available. This study proposes a generic large scale PHA production system that can be simulated using minimum inputs to deliver material and energy inventories which can further be used to investigate the production costs of PHAs. The PHA monomer that was simulated is the most characterised PHA, polyhydroxybutyrate (PHB).

The South African coal mining industry generates large volumes of coal ultrafine waste (< 150 microns) each year, with a significant amount being dumped in tailing slurry dams. These slurry dams have been associated with prolonged pollution and loss of valuable resources. In the two stage flotation process developed at the University of Cape Town, froth flotation is used to both recover coal (stage 1) and remove pyritic sulfur (stage 2) from ultrafine coal waste, resulting in three outputs streams: a saleable coal product, a small volume sulfide-rich stream, and a reduced volume sulfide lean tailings stream. Pre-disposal removal of sulfide sulfur and coal recovery by means of froth flotation is aimed at effectively removing the acid rock drainage (ARD) risk associated with sulfide bearing waste s and at recovering valuable resources respectively. Previous studies have demonstrated the technical feasibility of this process for a number of coal waste types on a laboratory-scale, with results indicating that it is possible to recover large quantities of useable coal whilst generating a tailings waste stream with a reduced sulfur content and negligible ARD risk. An order of magnitude financial model for a fictitious plant has also been developed, and applied to demonstrate the economic viability for s elected case studies. To date, however, studies on the environmental viability of the process have only focused on the ARD mitigating potential of the two-stage flotation process and little attention has been given to the systemic environmental implication s of the process such as the energy and reagent usage. The research study therefore aims to evaluate the environmental burdens and benefits of the two-stage flotation process, particularly from a South African context, and to compare the environmental performance to the conventional disposal of untreated coal ultrafines. Furthermore, this project aims to establish which stages along the process contribute the most to the environmental burdens of the process and how the variations of the input parameters affect the overall environmental performance of the proposed process. To this end, a life cycle inventory of inputs and outputs was compiled on the basis of the empirical results derived from a previous laboratory-scale case study conducted on a sample of an acid generating ultrafine coal waste from the Waterberg region. Experimental results from the case study, which entailed two-stage flotation (using Naflote 9858 as a coal collector and xanthate (SIBX) as a sulfide collector in stages 1 and 2), and detailed characterisation of the feed and desulfurised tailings, was supplemented with literature information and data from mass and energy balance calculations for a fictitious plant. An environmental impact analysis was subsequently conducted using a combination of Life Cycle Impact Assessment and risk-based impact assessment techniques and criteria. The impact categories selected included climate change, terrestrial acidification, fossil fuel depletion, natural land transformation, aquatic water pollution risk, drinking water quality risk, aqueous acidification, salinity and consumptive water footprint. Aquatic water pollution risk, drinking water quality risk and aqueous acidification impact indicators were calculated by summing up risk potential factors for the constituents of the final disposed waste streams. The rest of the impact categories were calculated by multiplying the inventory result with a characterisation factor developed from impact assessment models The case study results indicated that the simple mentation of the two-stage flotation process results in a notable decrease in eco-toxicity, salinity, consumptive water footprint, metal toxicity, aqueous acidification, fossil fuel depletion and natural land transformation impacts. However, the results al so indicated an increase in atmospheric related impacts (climate change and terrestrial acidification impacts), which has been attributed to the additional energy consumption associated with the two-stage flotation process and the production processes associated with the flotation reagents. Analyses of the process contributions to the individual impact categories for the two-stage flotation process revealed the climate change and terrestrial acidification impact categories to be dominated by the electricity production process and the flotation reagents production process. The sensitivity analyses revealed a higher dependence of the fossil fuel depletion impact category on the percentage coal yield than the electricity consumption of the foreground process. Furthermore the sensitivity analyses indicated a strong dependence of the climate change and terrestrial acidification impacts on the electricity consumption and the SIBX dosage in the foreground process. In the South African context, implementation the two-stage flotation process would result in a significant recovery of coal (approximately 1.2 million tonnes for every 4 million tonnes dry coal ultrafines lost per annum) and a sulfide-rich product which can be utilised for electricity production and sulfuric acid production respectively, hence promoting resource efficiency. Although higher than in the case of conventional land disposal, the energy used in the two-stage flotation process is infinitesimal compared to the energy recovered in the process through the generation of additional coal, and results in only a 0.025 % increase in the annual greenhouse gas emissions. The implementation of the two-stage flotation would also result in reduced water losses in comparison to conventional land disposal, which is beneficial in the South African context as South Africa is a water scarce region. Lastly whilst the implementation of the two-stage flotation process would result in the reduction of water related impacts associated with acidification, salinization and metal pollution, it might pose a further threat to aquatic life if the xanthate salt reagents are emitted to local water sources. The limitations of the study were mainly associated with the quality of the input and output data, the impact categories and the system boundary and scenario development. The multiple sources of information and the variations in literature of the energy input estimates were noted as a source of uncertainty. The lack of characterisation factors for some of the substances in the system as well as the exclusion of the possibility of utilization of the sulfide-lean stream were also part of the limitations associated with the study. Recommendations for future work include improving the environmental assessment by incorporating various case studies and by incorporating downstream processing as well as optimizing the two-stage flotation process by using less energy and by using less toxic flotation reagents.

Research into the use of 'algal' biomass for human consumption is receiving increased attention due to their favourable nutritional value, photosynthetic efficiency, and lower requirement of land and fresh water as compared to terrestrial crops. The Spirulina species, also known as Arthrospira, is of particular interest due to its high protein content and nutritional value. Open raceway pond systems are popularly used for commercial industrial scale cultivation of microalgae due to their economic feasibility. These open cultivation systems are, however, susceptible to contamination by other microorganisms. This raises concerns relating to suitability for human ingestion and the need to control bacterial growth to prevent contamination by pathogens and to minimise the overall bacterial load. Further, bacterial contamination in processed (harvested and dried) Spirulina biomass has been reported, suggesting that some of these contaminants may end up in the market ready product where appropriate processing approaches are not used. This study sought to identify the microorganisms that typically contaminate Spirulina cultivation ponds, to understand their interaction with Spirulina biomass during cultivation and to evaluate the vulnerabilities of these contaminants, in order to generate strategies for controlling their populations during open pond cultivation. The main objectives of this study were therefore: • To quantify the bacterial load in processed Spirulina powder from a single pilot facility to ascertain the presence of the contaminant in the final product derived from the outdoor pond system used as a case study, and to quantify the bacterial load in the outdoor cultivation cultures. • To identify and characterize the bacteria associated with these Spirulina cultures and processed powder from a pilot operation carried out in Franschhoek, South Africa, with a particular focus on evaluating the likelihood for pathogens. • To establish the dynamics of the relationship between Spirulina and bacterial growth under different environmental conditions including pH, salinity and temperature. • To develop practical methods to control and minimize contamination.

Maximising performance of microbial processes, including yeast-based processes, in an industrial setting requires understanding of the impact of process stresses. These may be the result of process configuration, dilution, temperature changes, hydrodynamic conditions or process perturbations. Methods to determine the microbial metabolic response to such stresses have long been sought, but are typically limited, often requiring the use of a suite of methods to assess the physiological status and state. The recent technical advances in microcalorimetry suggest potential for the use of isothermal microcalorimetry (IMC) to determine yeast viability and vitality and is investigated here. IMC is a laboratory method whereby the real-time heat produced by a chemical, biological or physical process is measured in the micro to nano watt range. It is proposed that this heat production may be correlated to the physiological state of the microbial catalyst and can be used to measure the impact of different stresses. In this study, the potential of IMC as a method for exploring process stress is investigated using Saccharomyces cerevisiae and its application in the beer brewing industry as a case study. Here, it is well known that yeast viability and vitality have commercial significance. IMC is sufficiently sensitive to detect the heat given off by 1000 yeast cells. However, IMC cannot distinguish between different heat flows within a system i.e. it is non-specific. The literature demonstrates how IMC has been used in the study of numerous microbiological fields, including the growth and metabolism of yeast. Previous studies have successfully derived the specific growth rate and cell numbers of a growing yeast population from analysing power and heat curves. The specific growth activity and specific growth retardation of yeast and how these parameters relate to bactericidal and bacteriostatic effects has also been examined by a number of authors. The key objectives of this study were to determine the viability and vitality of Saccharomyces cerevisiae using IMC and to assess the impact of stresses on yeast viability and vitality. This was achieved by measuring the thermal power produced by a growing yeast suspension as a function of its overall growth and metabolism. Two industrially relevant stresses were examined: cold shock and ethanol shock. The effect of these stresses has yet to be studied using microcalorimetry. The growth of Saccharomyces cerevisiae under ethanol stress was used as an inhibition study to isolate its effects on the growth thermogram. Following the generation of thermograms under control and stress conditions using IMC, a method for their quantitative analysis was developed. Curves were fitted to the heat data using an exponential growth equation and the time for the heat flow curve to peak was determined. From the exponential curve, the specific growth rate of the yeast was determined with a high degree of repeatability. The coefficient of the exponential term in the growth equation gave highly reproducible and distinguishable results relating to the viability and vitality of the initial yeast population. The time of peak heat flow was also affected by the initial viability and vitality of the yeast and was used to estimate the initial active cell population size.

Cyanide heap leaching had been proposed as an alternative to the classic crush-mill-loatsmelt-refine route for processing platinum group metals (PGMs) from the Platreef ore body. Overall the process includes two stages of leaching. The first stage involves the thermophile bioleaching of the base metal (BM) sulphide minerals and acts as a form of pre-treatment to oxidise sulphur compounds and recovery valuable metals such as Cu, Ni and Co. The second stage focuses on cyanide-based heap leaching for the recovery of precious metals (PGMs +gold) from the solid residue of the first stage. Exploration and optimisation of this second stage in the context of a whole ore Platreef material is the focus of the present study. The first part of the study used a series of laboratory tests simulating heap leaching, conducted on coarse ore. The initial tests showed high recoveries of base metals (Cu, Ni and Co) could be achieved in a pre-treatment bioleach process, while in the second stage cyanide leach high levels of Pd and Au were extracted, but only 58% of the Pt after 60 days from the whole ore. It was observed that during the 60 day leaching period the rate of Pt leaching decreased considerably after 35 days. From the trajectory of the Pt leach curve from the 35 day mark onwards, it was observed that the leaching would not cease even after 60 days but would likely proceed but at that slow pace which indicated further Pt extraction would not be commercially viable in the long run. Mineralogical analysis has indicated that a significant component of the Pt in the ore is in the form a mineral sperrylite (PtAs2), which appears to leach slowly in cyanide as compared to other mineral forms such as certain tellurides and sulphides in the ore. Subsequently, efforts were made to investigate methods to improve the second stage leach process, in terms of Pt leaching from sperrylite, through further work on a pure mineral sample. The key focus was on finding a suitable oxidant that can be used in cyanide solutions, from among air, oxygen and ferricyanide, to facilitate the dissolution. Various tests using sperrylite mineral samples micronized to 5 μm in batch stirred tank reactors (BSTR) at 50°C were conducted. It was found that a combination of ferricyanide with cyanide extracted as much as 16 times more Pt than tests using only cyanide. The presence of air or pure oxygen did not contribute significantly to the amount of Pt leached in this system and made no difference at all in the leach tests using only cyanide. Further bench-scale studies focused on characterising the leaching mechanism of sperrylite in cyanide-ferricyanide solutions. It was found that the reaction, after proceeding at appreciable rates initially, tended to cease after 1 day, indicating some form of surface passivation, tentatively related to some form of solution equilibrium being achieved. However after re-leaching the sample with fresh solution, the Pt dissolution improved tremendously. This was further investigated in continuous leaching of a sample of the mineral using a small bed of sperrylite fixed in mini-columns. The results from the minicolumns showed the same leaching pattern as the experiments using BSTRs. It was eventually revealed that a suitable wash of the sperrylite sample using water removes the inhibiting layer and facilitates further and improved leaching. Unlike the cyanide-only system where the passivation was attributed to As build-up at the surface, in the cyanide-ferricyanide system it was attributed to adsorption of unknown reaction products on the mineral surface. Residual samples from batch leach experiments were analysed using X-ray photoelectron spectroscopy and showed samples from the cyanide-ferricyanide tests had less As on the surface than the untreated sample and the sample leached in cyanide. To some degree this supported the hypothesis that Pt leaching is eventually hindered by As passivation in a cyanide system. The presence of ferricyanide serves to oxidise As and thereby release more Pt in solution. Additionally, electrochemical techniques using a sperrylite electrode were employed to further understand the redox reaction under varying oxidation conditions. While the tests indicated a weak current under mildly oxidising conditions in cyanide solutions, this became rapidly limiting at potentials expected in a ferricyanide solution, indicating a form of surface passivation. An attempt was made to determine the number of electrons transferred during Pt dissolution to indicate the primary reaction mechanism through a long-term test held at constant potential, but dissolution rates were too small to be conclusive. Hence the study has shown that the cyanide-based heap leaching of PGMs from Platreef type ores is feasible in principle, but the dissolution of PtAs2 remains limited. While the study has given valuable pointers to understanding this observation, the conclusion is that PtAs2 is refractory in the given context and further development of this process remains promising through further investigation into the use of the cyanide-ferricyanide combination.

Mineral beneficiation processes such as base metal and coal mining produce large amounts of waste rock and coal discards that contain significant quantities of sulphide minerals with Acid Rock Drainage (ARD) generating potential. ARD is caused by the exposure of sulphide minerals, primarily pyrite (FeS2), to both water and oxygen, and microorganisms. This is a naturally occurring process, but the exposure of the sulphide containing mining wastes greatly accelerates ARD formation. Thus, ARD is a major issue associated with inactive mines, waste rock dumps and tailings impoundments, which over time presents a major environmental risk. The desulphurisation of coal discards, mine tailings and finely divided waste rock prior to their disposal has been proposed as a method of preventing ARD formation. This involves the selective separation of residual values from the waste rock, followed by selective separation of sulphide minerals – especially pyrite – from the residual waste material using a two-stage froth flotation to obtain a values stream, a low volume sulphide-rich concentrate that can be easily contained, and a high volume benign tailings fraction that can be safely disposed of. The technical feasibility of this two-stage process has been demonstrated; however, the cost of the flotation reagents used in this process are particularly high in comparison to the other operating costs, contributing as much as 75% of the operating costs for desulphurisation of coal fines. Furthermore, apart from being expensive, many of the inorganic flotation reagents are relatively toxic and could be hazardous to the environment due to their slow degradation rate. Microorganisms and their metabolic products have been identified in literature as potential reagents that can be used in the selective separation of sulphide minerals using froth flotation. Just like conventional chemical flotation reagents, the microorganisms assist separation through surface chemical alterations that modify a mineral’s hydrophobic properties, thus facilitating bioflotation. The aim of this study was to investigate the prevention of ARD formation through the desulphurisation of pyrite-containing coal discards and base metal hard rock samples using microbial cultures as alternative bioflotation reagents. In this study the feasibility of using P. polymyxa, R. palustris, R. opacus, B. subtilis, and B. licheniformis as biocollectors for the removal of pyritic sulphur in the second stage of the two-stage desulphurisation froth flotation process was investigated. Microbial screening tests were performed using a pyrite concentrate to assess each microbial culture’s affinity to pyrite and their ability to float the mineral in a batch flotation cell. Attachment experiments and batch bioflotation tests were carried out to screen for a microbial culture that showed potential. Following attachment experiments at pH 4 and pH 7, all microorganisms except B. licheniformis exhibited attachment to pyrite. The level of attachment was different for each microbial culture. P. polymyxa had the highest percentage attachment of 95.6 ± 1.0 % at pH 4 and 97.1 ± 0.7 % at pH 7 after 20 minutes of interaction. Subsequent results from the pyrite-only bioflotation tests revealed that R. opacus, R. palustris and B. subtilis did not affect the floatability of pyrite. P. polymyxa, however, showed a significant effect on the floatability of pyrite, achieving a cumulative mass recovery of 7.0 ± 0.42 % at pH 4 and 81.3 ± 0.4 % at pH 7. Zeta-potential tests revealed that P. polymyxa had the most neutral net surface charge across the pH range tested, while the other microorganisms had a large net positive or negative charge. Based on this result, it was deduced that the hydrophobicity of P. polymyxa as a consequence of its near neutral surface strongly made it seek out a surface to attach to rather than remaining suspended in water. Hence, P. polymyxa was chosen as the bio-collector candidate for the bioflotation separation of pyritic sulphur from coal discard and base metal hard rock samples. Despite the positive batch pyrite bioflotation tests, P. polymyxa was not successful for the flotation of pyrite from the coal discards nor did it upgrade pyritic sulphur to the concentrate, with the bioflotation results not significantly different from the negative control without collector. P. polymyxa did affect the floatability of the base metal hard rock, achieving cumulative mass recoveries comparable with the chemical control using PAX. However, there was no significant upgrade of pyritic sulphur content, with the biofloat achieving 22.6 % total sulphur in the concentrate which was significantly less than the 66.4 % total sulphur recovered with PAX. The study thus yielded positive results from fundamental studies of P. polymyxa’s ability to enhance the flotability of pyrite. However, tests using actual samples were less successful. Although P. polymyxa enhanced the floatability of the base metal hard rock, it did not achieve the aim of obtaining a low volume sulphide-rich concentrate as the PAX did. Recommendations for the continuation of this work include contact angle measurements and FT-IR spectroscopy to better understand the effects of P. polymyxa attachment, as well as performing a kinetic study on the growth of P. polymyxa alongside adaptation of the microbial culture to a pyrite mineral concentrate in order to test if this can improve selective flotation of the desired mineral owing to modified surface properties.

The world sugar price is constantly changing in response to supply and demand and is currently very low as compared to the prices it is sold at domestically in South Africa. The drop in the worldwide price of sugar is due to its oversupply as yields of sugar production have increased in recent years and subsidies and protection measures in other producing countries. The low prices also mean imports are cheaper than local sugar. This pushes down the average sugar price and leads to a low profit margin. Further, sugar production in South Africa is facing a number of challenges. The industrialization of the sugar belt in KwaZulu-Natal has resulted in less plantations and challenging topography for these. Incentivisation of small, medium and micro-scale commercial operations has increased the number of smaller scaled operations, with less economy of scale and less capital backing. Climatic factors have impacted crop yields. Production costs have increased in accordance with South Africa’s consumer price index whereas selling price has moved with the less inflationary global platform. Together, these have made the industry less economically viable. This has led to a need for value addition to sucrose and to eliminate the dependency on a single commodity. Re-positioning of sugar into value-added products has potential to boost the country’s economy by introducing other sources of revenue. Moreover there is a worldwide need to find alternative means to produce petroleum-based fuels and chemicals and bio-based products are being targeted to meet some of this need. A review of the global status shows that there has been value addition in the sugar industry producing mostly ethanol and other commodity chemicals such as surfactants, organic acids and polyols. It is therefore imperative to find sustainable ways of generating value added platform chemicals from sucrose. The quantitative and qualitative study of this project looks at determining the chemicals that should be considered as having the highest potential for value addition from sucrose in a South African context. The project was scoped to focus on chemicals and fuels that can be produced by biological conversions of sucrose. For the quantitative study, a set of 39 chemicals was selected from major studies performed globally on potential bio-based platform chemicals and these catalogued according to a set of criteria. The decision of the chemical/fuel to be studied was based on the gap in the chemical industry. This list comprised of chemicals that were selected in the US department of energy top 10 list in 2004 and 2010 and top 15 chemicals in the EU list in 2015. In addition to these, chemicals that are currently of interest (which were mostly chemicals that can be used as polymers and biofuels) were included to make up the list of 39 chemicals. The selected chemicals then went through a knock out selection where chemicals that cannot be produced with current technology from sugar or via a biological route were eliminated from the list. A quantitative analysis was then done on the remaining chemicals from the knock out stage. A weighting method which considered a series of factors was used to determine the top platform chemicals. The factors used were to identify platform chemicals that are at a high demand (both in South Africa and internationally), chemicals that showed great potential for profitability based on cost, technology readiness level and product yield. The quantitative analysis allowed seven chemicals to be selected. Finally a qualitative study based on interviews with experts in the field was done. Most of this information provided by the experts was supported by several literatures (Taylor, et al., 2015; Villadsen, et al., 2011; Choi, et al., 2015; Jansen & van Gulik, 2014). The qualitative study identified Succinic acid, Lactic acid and Citric acid as the top three chemicals. A techno-economic study was done on succinic acid, one of the most promising platform chemicals identified. The reasons for its selection was because it has a higher performance and it generates less carbon footprint than petroleum based succinic acid, competiveness for niche market, multiple application via BDO and PBS and its overall favourable environmental process that uses up carbon dioxide from the environment. Firstly, the succinic acid process was designed to be produced using Saccharomyces cerevisiae in a dual phase fed batch fermentation process. The overall design for the succinic acid process was based on the design proposed by Efe, et al (2013). A cost evaluation was then done on the design for an economic analysis. The economic analysis was done on the process to ascertain that there is indeed value addition of sucrose to the platform chemicals chosen. This was done in the form of profitability analysis of the process. An economic analysis of the design shows that the plant is profitable after the first year of operation. The total investment on the plant is R 22.3 billion and the start-up expense is R 1.05 billion. This project serves as a preliminary paper based overview of the general background for the selected platform chemicals that will be researched further in subsequent research.

Heap bioleaching is a hydrometallurgical technology, used to facilitate the extraction of valuable metals such as copper, gold, nickel and uranium from low-grade, typically sulphidic, ores. The process is highly complex as it is influenced by interactions of different sub-processes including flow of leaching solution around the ore particles, mass and heat transfer within and around the particles, chemical reactions, microbially-mediated reactions and microbial growth. Contact of leaching solution with mineral grains is necessary for oxidation of the sulphide minerals. However, a large fraction of the mineral grains is positioned below the surface of the ore particles and so contact with the liquid occurs through cracks and pores in the ore connected to the surface. Long extraction times and low metal recoveries typical of heap systems can be attributed to the slow leaching rate of these non-surface mineral grains as well as constraints on their accessibility. Most of the valuable grains that remain in the residue ores are non-surface grains. Therefore, investigation of the mechanism and behaviour of non-surface grain leaching and quantification of the factors contributing to their leaching is expected to be highly beneficial in the optimisation of leach conditions and recoveries. Non-surface grain leaching within large particles cannot be investigated via traditional experimental methods reliant on bulk measurements, 2D or destructive methodologies. However, it can be studied using high resolution, non-destructive 3D X-ray micro-Computed Tomography (μCT), an imaging technique for investigation of internal structure of opaque objects. X-ray μCT has previously been developed and used for investigation of different aspects of heap leaching. In the current study, the viability of using X-ray μCT to study heap bioleaching systems and affecting variables is assessed. This required establishment of procedures for measurement and analysis of sulphide and oxide mineral recoveries and leaching penetration distances. The feasibility of studying biotic heap leaching by X-ray μCT was explored through investigation of the relative energies required for high mineral resolution and avoidance of microbial inactivation. Specific bioleaching operating variables that were subsequently considered included: the accuracy and representivity of the X-ray μCT images, the influence of agglomeration pre-treatment, operating temperature, and type of ore on non-surface grain leaching. Addition of surfactants to the leaching solution was explored with the aim of changing surface activity to influence the penetration of the leach agent into pores and cracks in the ore. The effects of operating conditions on non-surface mineral grain leaching was studied using mini-column experiments. Three different low-grade ores, namely a chalcopyrite-rich ore, a malachite ore and a waste rock containing pyrite were prepared for the leaching experiment. The ores were crushed using a jaw crusher and comminuted down to 100% passing 16 mm. The products were sieved into six fractions (<0.25 mm, 0.25 - 1 mm, 1 - 2 mm, 2 - 5.6 mm, 5.6 - 8 mm, 8 - 16 mm) and each fraction then representatively split into smaller portions using a rotary splitter. One portion of each size fraction was taken for XRD, AAS and QEMSCAN analyses. Mini leaching columns were designed and constructed based on the target mineral grain distribution in the ores to ensure that the mineral grains were detectable using X-ray µCT, given its resolution limitations. The columns were charged with 50 g of agglomerated or non-agglomerated ore and lixiviant was provided at a flow rate of 2.55 mL h -1 for a period of 5.5 months for chalcopyrite and pyrite and 26 days for malachite in incubators at 30 °C, 37 °C and 65 °C. In order to select a surfactant suitable for use in a biological leach experiment, the effect of five different types and concentration of non-ionic surfactants on bioleaching microorganisms was studied in terms of microbial growth, ability for ferrous ion oxidation and chalcopyrite bioleaching. This was done in shake flask experiments using mineral concentrate. Based on the results of these experiments, Tween® 20 (10 mg L -1 ) was selected to study the effect of surfactant on non-surface mineral grain leaching in the mini-columns. Each column was scanned by X-ray μCT at 100 kV and 150 mA using a 0.38 mm copper filter and at a distance of 59.40 mm between X-ray gun and specimen. The advanced 3D analysis software Avizo® 9 was used to visualize and analyse image data. The Interactive Thresholding function in Avizo® 9 software was used for segmentation of ore particles from air and sulphide minerals from air and gangue minerals, to measure the target minerals' volume reduction during leaching. The Distance Map Algorithm was applied on a binary (segmented) image to calculate the distance of the sulphide mineral from the ore particle surface. Imaging of the whole mini-column was done before leaching and at the end of each experiment and imaging of certain sections was done at select time points during leaching to track temporal leaching dynamics. Good agreement was seen between the bulk mineral recovery data, determined using standard chemical assays, and the leaching curves generated using the X-ray µCT images for all the ores, confirming that the X-ray µCT images were a good quantitative measurement of the sulphide and oxide mineral leaching. Liquid microbial culture experiments were used to confirm that exposure to X-ray does not affect microbial activity for energy doses between 35 and 90 kV at 200-280 μA. However, X-ray exposure was found to have a slight negative influence at higher voltages of 120 and 150 kV, temporarily reducing the specific ferrous ion oxidation and suppressing the specific growth rate of the bioleaching microorganisms. The X-ray exposure thus negatively affected both the total microbial population available for leaching (population viability) as well as the metabolic activity of the individual microorganisms (population vitality). The effect of X-ray exposure on bioleaching cultures attached to a mineral surface was examined using pyrite-coated glass beads packed into mini-columns. The energy dosage limits identified in the liquid culture experiments were found to be compatible with the X-ray μCT imaging conditions (minimum energy dosage and sample position) required for acquisition of complete and accurate images of the columns at a resolution that allows identification of individual mineral grains. Following X-ray exposure, the performance of the exposed bioleaching mini-columns was equivalent to the unexposed control column. Similarly, the microbial activity and presence on the mineral surface appeared unchanged. Finally, the experiment was performed on the chalcopyrite ore and the microorganisms were found to still be able to convert Fe2+ to Fe3+ after 2 scanning runs. Thus, all sets of results confirm that X-ray μCT can be compatible with heap bioleaching experiments, while still permitting appropriate resolution of the mineral grains to make an X-ray μCT investigation worthwhile. However, cognisance that an upper limit of tolerable X-ray exposure exists must be taken. This may present a challenge if it is desired to image larger or denser ore samples which require a greater X-ray energy level for sufficient penetration of the sample by the X-rays and hence accurate imaging. In chalcopyrite leaching, increasing temperature from 37 °C to 65 °C resulted in clear enhancement of leaching based on both analysis methods, with the copper recovery increasing from 20% to 64% by the end of the leaching period, and the overall sulphide mineral dissolution increasing from 24% to 67%. Increasing temperature from 37 °C to 65 °C resulted in an increased leaching penetration distance and crack development in the particles, and thus an enhancement in copper recovery and sulphide mineral dissolution. This was in addition to the thermodynamically expected increased leaching rate. The maximum leaching penetration distance, beyond which no mineral volume change is observed, at 37 °C was 1.7 mm. This increased to 2.5 mm at 65 °C. As a result of addition of 10 mg L-1 Tween® 20 into the leaching solution, the final copper recovery was improved by 4% to 68% and the maximum penetration distance increased to 2.9 mm. However, when the availability of sulphide mineral was not rate limiting, the copper recovery and sulphide mineral volume reduction in the mini-column with surfactant was lower than the system without surfactant. This may have been due to depression of diffusion of ferric ion to the ore surface as a result of the formation of an adsorbed surfactant layer on the mineral surface. The performance with surfactant became superior as the amount of readily leachable mineral became limiting. In the pyrite waste rock, an increase in temperature did not have any effect on the maximum penetration distance and any increase in iron recovery was only for thermodynamic reasons. Similarly to the chalcopyrite ore, during the later period of leaching when readily exposed mineral grains have been depleted, the system performed better in the presence of surfactant. The addition of surfactant increased the maximum penetration distance from 2.7 to 2.9 mm. The cumulative copper recovery of 86% was obtained for malachite ore in 26 days of acid leaching and the maximum penetration distance was 2.2 mm. This study thus demonstrates the value of the X-ray µCT technique for quantitative investigation of non-surface mineral grain leaching and confirms that the maximum penetration distance can be affected with changing operation conditions or ore type. This study thus demonstrates the X-ray µCT technique for quantitative investigation of non-surface mineral grain bioleaching and confirms that the maximum penetration distance can be affected with changing operation conditions. Critically, the results confirm that X-ray μCT can be compatible with bioleaching microorganisms, while still permitting appropriate resolution of the mineral grains to make an X-ray μCT investigation worthwhile.

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In this thesis, it is hypothesized that in bioleaching flow reactor systems, high reaction rate regions exist that can be maintained by application of biological stress trajectories. Reactor models are developed for the purpose of optimising plant operation, understood here as maximising the production rate. Complicating this attempt are a) the non-linear dynamics associated with the kinetics and b) the primary reaction's being multiphase. Mathematical models are developed to establish which particle parameters are necessary to describe reactor performance using the method of segregation. The models are distinguished by the combination of either particle residence time or age and/or particle size distributions. The models evaluated at steady state are validated against pilot plant data obtained from the Fairview Mine in South Africa and were found to be in good agreement with the data. As the model was developed using a segregation approach and thus incorporates age distributions in the model formulation, the model could be extended to unsteady state operation.

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Namakwa Sands is a heavy mineral mining and beneficiation business within Tronox, and produces two major products, zircon (zr02.Si02 99.9%) and rutile (Ti02 99.9%) at a combined annual rate of 140kt. The heavy mineral concentrates are exported to international markets to make specialist coatings for the paints and ceramics industries. The ceramic industry is very strict on the purity of the minerals used. Namakwa Sands prides itself in being able to produce zircon and rutile at these requirements; however, strict requirements, especially in terms of Fe impurities (Fe203 content in zircon concentrate must be < 600ppm), limit the productivity and come at a cost to recovery. The concentration and separation of heavy minerals is a complex process, which utilizes conductivity differences between minerals. Zircon coated with iron oxides (Fe203, FeOOH) reports as more conductive during electrostatic separation, which can result in a zircon particle to behave like a rutile particle and in this way cause both products (rutile and zircon) to become off specification.

Includes bibliographical references (p. 122-129).
Efficient intracellular product release from yeast is required for the recovery of many bioproducts, recombinant or other. Traditionally such product release is achieved by non-selective, energy demanding mechanical disruption. The fine debris resulting from mechanical disruption is also challenging in the solid-liquid separation in downstream process. This study investigates the effect of the pretreatment on the energy efficiency of cell disruption, the extent of product release and its selective product release. Saccharomyces cerevisiae and Kluyveromyces lactis were used as the model microorganisms while disruption following pretreatment was achieved on exposure to ultrasound or passing through the high pressure homogenisation (HPH). Pretreatments were selected for their ability to weaken the yeast cell wall, rather than to permeabilise the cell. This allowed product release to be concentrated into the disruption step only, not distributed between the disruption and pretreatments steps. Rapid temperature treatment at 40 to 60CC, pH shock across the range pH 9 to 11 and osmotic pressure between 0.5 MPa and 5 MPa were used as single pretreatment. Combined pretreatments were also considered. These were affected by diluting the yeast suspension into a pre-warmed pH or high osmolarity buffer. On dilution, the temperature was increased rapidly to 40CC, while the pH or osmotic pressure was increased to pH 10 or 1 MPa.

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Hydrometallurgical methods are increasingly considered as alternatives to conventional pyrometallurgical techniques for extraction of metals from mineral ores. Chalcopyrite is a key copper mineral due to its abundance on earth. However, due to its refractory nature in sulfate leaching systems, a viable hydrometallurgical route for its extraction remains elusive...The work in this present thesis is presented as an initial study to commission the potentiostat instrument, investigate the common trends that are observed in running potential controlled (voltammetric) studies of chalcopyrite and compare them to those reported in literature, and investigate the electrochemical behaviour under different reaction conditions.

An electrochemical study was conducted on a stationary, synthetically produced, covellite electrode in acidic, chloride solutions at ambient conditions to investigate the dissolution behaviour of the mineral over a surface potential range from the open circuit potential (OCP) to about 0.62 V (vs. SHE). The electrode was mounted in an apparatus, which was designed to resemble leaching of the mineral under conditions applicable to heap leaching of whole ores, where the mineral occurs in cracks or pores in the gangue matrix or is covered (or partially covered) by reaction products.

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Rising global energy demands and global warming concerns from fossil derived fuels are two major problems affecting future generations. Bioenergy from algae offers a part solution that is both attractive and sustainable, by supplementing energy demands from a renewable energy source (the sun) and consuming carbon dioxide in the process. Bioenergy from algae is a proven concept (e.g algal biodiesel), yet the low productivities and high costs of existing processes limit their ability to make a significant contribution. Algae production occurs in specially designed photobioreactors, which are typically light limited. Hence, optimization of light supply to algae is key. A mathematical model of a photobioreactor is useful to aid in the design and optimization process. A model enables the prediction of productivities as a function of changing model inputs and hence allows optima to be predicted. While these are typically validated experimentally, this greatly reduces the number of experiments required, thereby saving cost and time. For this work, the production of algal biodiesel using airlift photobioreactors was used as the case study for the model development. Scenedesmus sp. was chosen as the model species owing to its comparatively high lipid productivity, a desirable trait for the production of biofuels. Although many parameters affect algal growth and lipid productivity, this project focused on one critical parameter, that of light provision.

World-wide, acid rock drainage (ARD) is one of the biggest environmental challenges facing environments with current or previously active mining activities. Formed from the exposure of sulphide mineral to both water and air, and catalyzed by naturally occurring iron- and sulphur-oxidizing micro-organisms, ARD pollution is predominantly associated with the mining of sulphidic ores and coal. Of particular concern are the large volumes of mining wastes from which the generation of ARD and the associated pollution effects often persist over tens to hundreds of years after mining operations have ceased. Current ARD management strategies focus on the prevention of ARD through mineral waste deposition or remediation options once ARD has formed. These strategies, however, do not remove the risk of ARD generation in the future. The aim of this study was to investigate the removal of the potential for ARD generation from a low-grade copper waste rock through the accelerated removal of the sulphur components via reaction. The three waste rock samples used in this investigation had total sulphur grades of between 2.20 and 3.20 % with the majority of the sulphide present as pyrite, chalcopyrite and galena. Significant quantities of non-sulphide associated iron minerals, predominantly magnetite, were also present in the three samples. The waste rock samples were sourced from mining operations in Chile and South Africa and had a D80 of approximately 0.8 cm. All three waste rock samples were potentially ARD generating.

Production of biofuel from microalgae is an attractive and sustainable option for meeting rising global energy demands and mitigating global warming. However, for commercial production of microalgae to be economically feasible, high biomass productivities and low auxiliary energy inputs must be achieved in large photobioreactors. According to literature, one of the main factors limiting growth is the inefficiency of light utilization (Posten, 2009; Janssen et al., 2003; Carvalho et al., 2006). In a photobioreactor, as biomass concentration and depth of culture increase, the amount of light that is able to penetrate the culture decreases exponentially. This occurs because of mutual shading of algal cells via adsorption of pigments or via scattering of cells. The purpose of this study was to optimize biomass productivity and biomass concentration by developing a thorough understanding of the microalgal response to light. In particular, the effects of light source, light intensity, configuration (internal and external), reactor design and the related variation in light/dark cycling were investigated.

Amidases are hydrolytic enzymes that catalyze the hydrolysis of amides to their corresponding carboxylic acids and ammonia. Amidases are ubiquitous in nature, and they have been isolated from a wide range of microorganisms, the most common source being bacteria. Amidases are recognized as potential industrial biocatalysts in processes that involve the synthesis of chiral compounds, mostly used in the pharmaceutical, agrochemical and food industries. The discovery of amidases from extremophiles has increased the potential for application of these enzymes for the development of new processes. In nonaqueous media, amidases have the ability to synthesize enantiopure amides due to the shift in thermodynamic equilibrium towards synthesis. For synthesis to occur, an acyl donor and an acyl acceptor are required, in which the acyl acceptor acts as a nucleophile. The applicability of amidases in non-aqueous media opens new possibilities for processes in which the enzyme can be used for the industrial synthesis of commercially relevant new products. A novel amidase was previously isOlated from a thermophilic Geobacillus species, and the amidase was cloned and expressed in an Escherichia coli BL21 strain. Also in previous studies, it was shown that the enzyme exhibits both amide hydrolysis and acyl transfer activities. The highest activity of the G. pallidus RAPc8 amidase was observed at 50°C in the presence of acetamide and substrate preference was towards aliphatic, short chain amides. Furthermore, the enzyme displayed enantioselectivity towards lactamide, which is a chiral compound. The amidase compound showed selectivity towards the D-isomer of lactamide and no detectable activity on the L-isomer. This study presents the investigation and development of a novel biocatalytic process that involves the synthesis of enantiopure amides in non-aqueous media, using the G. pal/idus RAPc8 amidase. The amidase was produced and expressed in E. coli BL21.