Academic literature 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.

This paper describes the trial of making microcapsules and deformation analysis of a bubble near the curved elastic wall using macro 2d model and plane shock wave. The prototype microcapsules are made by using micromanipulation systems. It is found that by controlling the initial position of a bubble from the wall and the curvature of the wall there is a point to have large deformation, which tends to be collapsed easily. This is one of the results to aid design of DDS or bioprocess for cell-integration.

The analysis of exergy losses of a system is a well-known way to determine the influence of the second law on existing systems. Thermo-economics combines this methodology with economic calculations. Using this methodology engineering becomes an evolutionary process. Since system structures are virtual, reversible system structures are possible and inevitable irreversibility is only caused by its real components. They can be described by their exergetic efficiency. Thus reversible system structures can be used as general valid benchmarks for system engineering. It allows easy comparison or a trade-off between possible solutions. The use of a few basic reversible processes allows the building of larger reversible structures including an effective management of released and demanded entropy within the system, as can be shown in different applications and missions. The effective use of renewable sources can be considered as well however bioprocesses are not investigated yet.