Thesis (MBA (Business Management))--University of Stellenbosch, 2007. / ENGLISH SUMMARY: Fossil fuel has been the preferred source for the production of transportation fuel for many years. However, this is not a renewable resource. Many conflicting reports have been published as to how long this resource will last. One thing is certain: eventually the supply of cheap crude oil will run out. It is therefore crucial to start the search for renewable alternatives now. There are a number of possible candidates vying for replacing fossil fuel as primary transportation fuel. Hydrogen, methanol, biodiesel and bioethanol all have the characteristics required of a good transportation fuel. It is unlikely that only one of these will replace oil. A more likely scenario would be that they all play a role in transportation in the future. Apart from being renewable, these alternatives have the further advantage of being less damaging to the environment, something that will become essential in future. Among the renewable alternatives, bioethanol has the second highest energy density. Currently, ethanol production worldwide almost exclusively uses sugarcane and maize as raw material. However, both these are food crops and using them for ethanol could lead to an increase in food prices. Furthermore, there is not enough agricultural land available to produce sufficient quantities of sugarcane and maize for ethanol to replace fossil fuel. Producing ethanol from plant material has the potential to meet the capacity requirements without impacting directly on food production. Approximately 180 million tons of agricultural biomass are produced in the United States each year, sufficient to produce 75 to 110 billion litres of ethanol. Despite its abundance, the technical challenges in converting cellulose to ethanol are significant. One major obstacle to the production of ethanol out of plant material is that most of the sugar in plant material is unavailable for fermentation by micro-organisms. In order to render the sugars in the cellulose fraction accessible to conversion, it is necessary to treat the plant fibres with a combination of chemical and enzymatic processes. Only when a complex mixture of enzymes is used, does it become possible to break down cellulose to glucose for subsequent fermentation to ethanol. Biomass processing by means of enzymes currently involves four separate biological steps: (i) production of enzymes (cellullases and hemicellulases), (ii) hydrolysis of cellulose and hemicellulose to sugars, (iii) fermentation of hexose sugars and (iv) fermentation of pentose sugars. Consolidated BioProcessing (CBP) will combine all these steps into one. However, CBP is not yet possible and the magnitude of research and developmental advancement required to realize this goal is significant. Both sugar and starch ethanol technologies are well established and major process advances are therefore unlikely. Currently there are no commercial-sized plants for the production of ethanol from lignocellulosics, however this is likely to change in the near future considering the progress made in this field during recent years. This study will focus on the current status of the bioethanol industry, as well as on the potential for future development. / AFRIKAANSE OPSOMMING: Fossielbrandstof was vir baie jare die hoofbron vir die produksie van brandstof vir die vervoerbedryf. Fossielbrandstof is nie ’n hernubare energiebron nie en daar is al baie gespekuleer oor presies hoe lank daar nog goedkoop olie beskikbaar sal wees. Baie min van die gepubliseerde bronne stem ooreen, maar almal is dit eens dat olie op een of ander stadium sal opraak. Om hierdie rede is dit noodsaaklik om nou reeds te soek na alternatiewe. Daar is ’n hele aantal hernubare alternatiewe wat gebruik kan word in die plek van olie. Waterstof, metanol, biodiesel en bioetanol beskik almal oor die nodige eienskappe om ’n effektiewe vervoerbrandstof te wees. Die hoofvoordeel van hierdie brandstowwe is dat hulle minder skadelik is vir die omgewing as olie, ’n eienskap wat baie belangrik sal wees in die toekoms. Die kans is eger skraal dat een van bogenoemde bronne die mark totaal sal oorheers soos wat olie tot op hede oorheers het. ’n Meer waarskynlik uitkoms sou wees dat al hierdie bronne op een of ander manier ’n rol gaan speel in die vervoerbedryf in die toekoms. Etanol het die tweede hoogste energie digtheid van die vier genoemde hernubare brandstowwe. Etanol word tans uitsluitlik van suikerriet en mielies geproduseer. Beide suikerriet en mielies is voedselgewasse en die gebruik daarvan vir brandstof kan lei tot ’n toename in voedselpryse. Daar is ook nie genoeg landbougrond beskikbaar vir die verbouing van suikerriet en mieles sodat genoeg etanol geproduseer kan word om fosielbranstof te vergang nie. Die vervaardiging van etanol vanaf lignosellulose het die potensiaal om etanolkapasiteitprobleme te oorkom sonder om direk met voedselproduksie te kompeteer. Ongeveer 180 miljoen ton landbouafval word jaarliks in die Verenigde State geproduseer, genoeg vir die vervaardiging van tussen 75 en 110 biljoen liter etanol. Die tegniese kompleksiteit gekoppel aan die omskakeling van sellulose na etanol is beduidend. Die belangrikste hindernis vir die produksie van etanol vanaf plantmateriaal is die feit dat die meeste van die suiker nie beskibaar is vir fermentasie deur mikroörganismes nie. Plantvesels moet daarom met ’n kombinasie van chemikalieë en ensieme behandel word om sodoende die suiker beskikbaar te maak vir omskakeling. Sellulose kan slegs met ’n komplekse mengsel van ensieme afgebreek word tot glukose wat dan daarna gefermenteer kan word tot etanol. Die verwerking van biomassa met behulp van ensieme behels tans vier afsonderlike biologiese stappe: (i) ensiemproduksie (sellulases en hemisellulases), (ii) hidrolise van sellulose en hemisellulose tot fermenteerbare suikers, (iii) fermentasie van heksose suikers en (iv) fermentasie van pentose suikers. Consolidate BioProcessing (CBP) poog om al vier hierdie stappe te kombineer. Ongelukkig is die CBP proses nog nie moontlik nie en daar moet nog baie navorsing en ontwikkeling gedoen word om dit ’n realiteit te maak. Beide die metodes vir suiker- en styseletanolproduksie is goed gevestig, dus is die kans vir beduidende verbeteringe klein. Daar is tans geen aanlegte van kommersiële grootte vir die produksie van etanol vanaf lignocellulose nie, maar dit gaan waarskynlik binnekort verander as ’n mens die vordering in ag neem wat daar onlangs gemaak is in hierdie veld. Hierdie studie fokus op die huidige stand van sake in die etanolbedryf en die ontwikkelingsmoontlikhede vir die toekoms.

Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.

A gradual depletion of high-grade ores, coupled with the growing demand for mineral commodities across the world has culminated in the increased exploitation of lean-grade ores with complex mineralogy. The mineral processing industry commonly uses an extensive range of inorganic, naturally derived or synthetic organic reagents in the separation of valuable minerals from the ore. Froth flotation is a commonly used separation technique to float or depress different sulfide minerals from the ore, based on their surface properties. In recent times, biological processes have been attracting attention in mineral processing and metal recovery operations due to a number of factors, especially lower operating costs, lesser energy consumption and their environment friendly nature. The use of microorganisms and their direct derivatives in mineral processing, hydrometallurgy and in the bioremediation of mineral industry discharges has led to the emerging area of “Mineral Bioprocessing”. In this study, a family of four microorganisms belonging to the Bacillus species, viz., Paenibacillus polymyxa, Bacillus circulans, Bacillus megaterium and Bacillus subtilis was used to ascertain the selective floatability of sphalerite from a sphalerite-galena mineral mixture. These bacteria are Gram positive, mesophilic, neutrophilic, aerobic and spore forming. The major objectives of the investigation include: a) Identification and characterization of bioreagents derived from Bacillus species for the flotation of sphalerite from a sphalerite-galena mixture b) Optimization of the flotation process for the enhanced recovery of sphalerite using specific bioreagent combinations c) Modes and mechanisms of bacterial adaptation to minerals and their consequent effects on the flotation of sphalerite and galena d) Elucidation of the mechanisms of microbe-mineral interactions and the role of extracellular secretions in sphalerite flotation column and their N-terminal residues were identified using Edmann N-terminal sequencing. Additionally, sequences of several internal peptides from both the proteins were determined using Tandem Mass Spectrometric techniques. A database search revealed that the sequences of these peptides are unique and have not been reported earlier. It was established that the bacterial cells give high flotation recovery of sphalerite under buffered conditions and that it took place only in the presence of anionic buffers. Additionally, the viability of the bacterial cells was not required for the flotation of minerals. A major finding of this study was that other than extracellular DNA (eDNA), none of the other bacterial surface components like teichoic acids, surface proteins, polysaccharides played a positive role in the flotation process. Nucleic acids, more particularly single stranded DNA (ssDNA), facilitated sphalerite flotation relative to double stranded DNA (dsDNA). A probable mechanism of ssDNA -mediated selective flotation of sphalerite has been presented. A negative role for non-DNA surface components was also observed. This led to the realization of the need for an optimum ratio of DNA to non-DNA components in the selective flotation of sphalerite from a sphalerite-galena mixture. It was found that the surface physiochemical properties of the mineral adapted bacteria differed significantly from that of the unadapted bacteria. Adaptation enhanced the flotation recoveries of the corresponding mineral vis-à-vis the unadapted bacteria. Sphalerite adapted bacteria secreted more extracellular proteins while the galena adapted bacteria secreted more polysaccharides compared to the unadapted bacteria. Sphalerite adapted bacteria selectively floats more sphalerite from the mineral mixture than the galena adapted as well as the unadapted bacteria. It was evident from the electrokinetic studies that the surface charge of the chosen sulfide mineral adapted bacteria was less negative relative to the unadapted bacteria. This phenomenon was observed with all the four bacterial species used in this study. A noteworthy finding was that the bacteria especially B.circulans induce a change in morphology from rod to sphere as a strategy during adaptation to a toxic mineral such as galena. This phenomenon has been shown to involve changes in crucial cell wall components as well as changes in the levels of expression of bacterial cytoskeleton elements involved in the maintenance of the rod shape. This aspect of the study involved the partial sequencing of the B.circulans homolog of the key cytoskeleton gene, mreB (B gene in murien cluster e), using the Polymerase Chain Reaction (PCR) followed by DNA sequencing. A Genbank search indicated that this is the first report of the sequence of B.circulans mreB gene. This was followed by measuring the hypothesized downward changes in the levels of expression of the mreB gene by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The possible mechanisms of the adaptive morphological changes and of the interaction of the chosen sulfide minerals with the family of microorganisms studied have been discussed with respect to their bioflotation efficiency.

<p>Municipal wastewater treatment plants treat wastewater such as domestic and industrial sewage and recirculates the clean water back into nature’s waterways. However, the wastewater treatment process is costly and complex. The cost of running a municipal wastewater treatment plant is funded via ratepayer fee dollars from customers and therefore receives a fixed budget for which to run the plant according to environmental standards. A local initiative was established to upgrade a Midwestern municipal wastewater facility to utilize biomass renewable energy to a greater extent than what is used by the wastewater facility. The first phase of the initiative tested the suitability of utilizing organic substrates from local industrial plants with the potential to produce larger amounts of biogas via anaerobic digestion. The analysis evaluated the technical and financial viability of utilizing biomass technologies to help power the facility efficiently and economically. The financial and technical analysis will include a cost-benefit analysis by comparing current and forecasted natural gas demand and costs for running heating the WWTP to biogas produced by the anerobic digesters. The results of the research study found that the industrial waste substrates are suitable for anaerobic digestion and yield a higher biogas potential than what is currently used for anaerobic digestion by the WWTP. The initial financial analysis found it is feasible and economical, for at least certain months of the year, for the WWTP to refrain from purchasing natural gas and instead utilize the produced biogas.</p>

Micro-scale processing (MSP) techniques are miniaturized version of upstream and downstream conventional unit operations that are designed to accelerate the pace of bioprocess design and development. Previous ‘dead end’ filtration studies have demonstrated the usefulness of this concept for membrane filtration processes. However, these experiments were performed without stirring which is the most common strategy to control the effects of concentration polarization and fouling on filtration performance. In this work, the pressure-driven stirred conditions of a conventional stirred-cell module were integrated with a 96-well filter plate to develop a high throughput technique called ‘stirred-well filtration’ (SWF). The design allowed for up to eight constant flux filtration experiments to be conducted at once using a multi-rack programmable syringe pump and a magnetic lateral tumble stirrer. An array of pressure transducers was used to monitor the transmembrane pressure (TMP) in each well. The protein sieving behavior and fouling propensity of Omega™ ultrafiltration membranes were assessed via a combination of hydraulic permeability measurements and protein sieving tests in constant filtrate flux mode. The TMP profile during filtration of bovine serum albumin (BSA) solution was strongly dependent on the stirring conditions – for example the maximum TMP in the stirred wells were an average of 7.5, 3.8, and 2.6 times lower than those in the unstirred wells at filtrate fluxes of 12, 36, and 60 LMH (5, 15, and 25 μL/min) respectively. The consistency of the data across different wells for the same stirring condition was very good. To demonstrate the effectiveness of the SWF technique, the eight tests for a simple 2^2 factorial design-of-experiments (DOE) test with duplicates was run to evaluate the effect of solution pH and salt concentration on protein filtration. The combination of SWF with statistical methods such as DOE is shown to be an effective strategy for high-throughput optimization of membrane filtration processes. / Dissertation / Master of Applied Science (MASc)

No description available.

<p dir="ltr">Biopharmaceutical manufacturing efforts are gradually shifting toward single-use bioreactors (SUB) because of their lower cost and modular scalability. One challenge that hinders further adoption of SUBs is the lack of sensors that can be integrated into sterile equipment, while being cost-effective for disposal with the SUB at the end of its operational life.</p><p dir="ltr">In this dissertation I discuss the development of γ-irradiated thin-film sensors that provide accurate pH monitoring in sterile media, with a focus on the reference electrode (RE). Thin-film Ag/AgCl REs exhibit excellent stability across a pH range of 4.5–9.0 and are inert to various organic species, for up to 60 days post irradiation. Notably, γ-irradiation of a thin-film pH sensor (pH electrode plus RE) produces a logarithmic voltage decay that can be readily corrected by a single-point calibration function. These sensors can accurately report pH changes in sterile cell culture media over several weeks.</p><p dir="ltr">The scalable production of thin-film pH sensors can be achieved by optimizing a semi-automated fabrication process for membrane deposition. Improvements in process control are fortified by a comprehensive uncertainty analysis to determine the relative contributions of error from manufacturing, measurement, and data analysis. Identifying key sources of uncertainty enables further optimization of the fabrication process as well as sensor performance.</p><p dir="ltr">This dissertation also includes a discussion on novel approaches for reducing the conditioning time of thin-film REs, a current impediment for their rapid deployment. Studies include the effects of autoclaving on membrane permeability and tuning the porosity of prefabricated membranes, and their impacts on ion flux across a high-impedance barrier. Changes in permeability produces a trade-off between conditioning speed and potentiometric inertness of the RE, but also reveals insights for future efforts to improve electrode performance.</p>

Lignocellulosic biomass is an abundant renewable resource targeted for biofuel production. Cellulose and hemicellulose from biomass both contain fermentable sugars and other moieties that can be converted to biofuels or other commodity chemicals. Enzymatic hydrolysis of these biopolymers is a critical step in the liberation of sugars for fermentation into desired products. In nature, anaerobic microbes produce protein nanostructures called cellulosomes that efficiently degrade cellulose substrates by combining multiple enzyme activities onto a scaffolding protein. However, current enzyme cocktails used in industry contain secretomes of aerobic microbes and are not efficient enough to be highly economical. Furthermore, most bio-processes focus on cellulose, rendering hemicellulose under-utilized. The three main objectives of this dissertation are to 1) develop multi-functional, self-assembling protein nanostructures for hemicellulose degradation using the architecture provided by cellulosomes, 2) understand the self-assembly mechanism at conditions for consolidated bioprocessing applications, and 3) compare the effectiveness of structured to non-structured hemicellulases in the hydrolysis of biomass. Xylan is a major type of hemicellulose in biomass feedstocks targeted for biofuel production. Six different xylanosomes were designed for hydrolysis of xylan within multiple biomass substrates using the cohesin-dockerin domain systems from Clostridium thermocellum, Clostridium cellulovorans, and Clostridium cellulolyticum. Each two-unit structure contained a xylanase for internal cleavage of the xylan backbone and one side-chain acting enzyme, either a ferulic acid esterase or bi-functional arabinofuranosidase/xylosidase. Expansion to three-unit xylanosomes included a family 10 or 11 xylanase, a bi-functional arabinofuranosidase/xylosidase, and bi-functional ferulic acid esterase/acetylxylan esterase. These multi-functional biocatalysts were used to degrade hemicellulose-rich wheat arabinoxylan and cellulose-containing destarched corn bran. Synergistic release of soluble sugars and ferulic acid was observed with select xylanosomes and in some cases required addition of an endoglucanase and cellobiohydrolase for enhanced hydrolysis. Furthermore, a putative ferulic acid esterase gene from the soil bacterium Cellvibrio japonicus was characterized and its role in xylan hydrolysis investigated. Information for the development of stable and functional cellulosome-like biocatalysts in metabolically-engineered microbes was collected using surface plasmon resonance. The protein-protein interaction of cohesin and dockerin domains for xylanosome self-assembly was examined at various temperatures and in the presence of ethanol to mimic different hydrolysis and fermentation processes and found to retain high affinities at the selected conditions. Moreover, the high-affinity interaction of cohesin and dockerin domains in the presence of non-specific proteins eliminated the need for protein purification for xylanosome construction. In addition to development of the first cellulosome-like biocatalysts targeted for hemicellulose degradation, this dissertation provides insight on possible improvements for the enzymatic hydrolysis of biomass, as well as the applicability of xylanosomes in consolidated bioprocessing.

Data Science plays an essential role in many organizations and industries to become data-driven in their decision-making and workflow, as models can provide relevant input in areas such as social media, the stock market, and manufacturing industries. To train models of quality, data preparation methods such as feature extraction are used to extract relevant features. However, global features are often ignored when feature extraction is performed on time-series datasets. This thesis aims to investigate how state-of-the-art tools and methods in data preparation and analytics can be used to extract global features and evaluate if such data could improve the performance of ML models. Global features refer to information that summarizes a full dataset such as the mean and median values from a numeric dataset. They could be used as inputs to make models understand the dataset and generalize better towards new data. The thesis went through a literature study to analyze feature extraction methods, time-series data, the definition of global features, and their benefits in bioprocessing. An effort was conducted to analyze and extract global features using tools and methods for data manipulation and feature extraction. The data used in the study consists of bioprocessing measurements of E. Coli cell growth as time-series data. The global features were evaluated through a performance comparison between models trained on a combined set of the dataset and global features, and models trained only on the full dataset. The study presents a method to extract global features with open-source tools and libraries, namely the Python language and the Numpy, Pandas, Matplot, and Scikit libraries. The quality of the global features depends on the experience in data science, data structure complexity, and domain area knowledge. The results show that the best models, trained on the dataset and global features combined, perform on average 15-18% better than models trained only on the dataset. The performance depends on the type and the number of global features combined with the dataset. Global features could be useful in manufacturing industries such as pharmaceutical and chemical, by helping models predict the inputs that lead to the desired trends and output. This could help promote sustainable production in various industries. / Datavetenskap spelar en stor roll inom många organsationer och industrier för att bli data-drivna inom beslutsfattande och arbetsflöde, varav maskininlärningsmodeller kan ge relevanta förslag inom områden som social media, aktiemarknaden samt tillverkningsindustrin. För att träna kvalitativa modeller används dataförberedande verktyg som funktionsextraktion för att utvinna relevanta egenskaper från data. Dock tar man ej hänsyn till globala egenskaper när funktionsextraktion utförs på tidsserie data. Denna examensarbete undersöker hur nuvarande verktyg inom dataförberededning och analys can användas för att utvinna global funktioner och utvärderar om sådan data kan förbättra prestandan hos maskinlärningsmodeller. Globla funktioner beskriver information som sammanfattar hel data, till exempel medelvärdet och medianen. De kan användas som indata för att få modeller förstå data och generalizera bättre mot ny data. Först utfördes en litteraturstudie inom metoder för funktionsextraktion, tidsserie data, definition av globala egenskaper samt möjligheter inom bioutvinning. Därefter utfördes en analys och utvinning av globala egenskaper med verktyg och metoder för data manipulation och funktionsutvinning. Den data som användes i arbetet består av mätningar från bioutvinning av E. Coli bakterier i form av tidsserie data. De globala funktionerna utvärderades genom en jämnförelse mellan modeller tränade på kombination av hel data och globala funktioner, och modeller tränade enbart på hel data. Studien presenterar en metod för att extrahera globala funktioner med öppet tillgänglig verktyg och bibliotek, som Python språket och Numpy, Pandas, Matplot och Scikit bibloteken. Kvaliteten på de globala funktionerna baseras på erfarenheten inom datavetenskap, datas komplexitet samt förståelse för domänområdet. Resultat visar att de bästa modellerna, tränade på data och globala funktioner, presterar i genomsnitt 15-18% bättre än modeller som tränats enbart på hel data. Prestandan detta beror på typen och antalet globala funktioner som kobineras med ursprungliga datat. Globala funktioner kan vara till nytta inom tillverkningsindustrier som farmaceutisk eller kemiska, genom att hjälpa modeller att förutsäga ingångsparametrar som leder till önskad produktion. Detta kan bidra till en hållbar produktion imon flera industrier.

<p><a href="" target="_blank">Synthetic microbial communities (SynComs) are an important focus in modern microbiology. SynComs are used for studying the dynamics of naturally occurring microbial communities ranging from soil to the human gut. Synthetic refers to the assemblage of some or all the members of these communities in a laboratory setting. SynComs allow for communities difficult to study <i>in situ</i> to be studied in a controlled environment, or they may be used to create beneficial products like biofertilizers. When SynComs are used as products, the focus shifts to optimizing a desired outcome of a culture. For biofertilizers, a high diversity is key to producing a functionally redundant product for stimulating plant growth. Media manipulation is a common approach for inducing community changes in a SynCom. Amino acids (AA) are a media supplement that soil microbes (often the components of biofertilizers) are particularly fond of to support their metabolic activities. This study took a scale-up approach to assess the changes in growth dynamics of a SynCom (Environoc© 401) when supplemented with different concentrations of commercially available AA products from plant and animal sources. Expanding from microplates, to shake flasks, then into a 4L bioreactor, Environoc© 401 cultures were compared for their maximum growth rate, time in lag phase, and final growth (optical density or viable cell density) when supplemented with these AA products at various concentrations. Furthermore, Illumina sequencing of the 16S rRNA gene was used to evaluate community-level changes from these treatments based on taxonomic, alpha (Chao 1 and Shannon indices), and beta diversity (Generalized UniFrac) of shake flask and bioreactor samples. Quantitative PCR was also used to assess the relative change of three select species of the SynCom in each AA treatment. Shake flask data revealed significant changes in the growth dynamics of the SynComs within AA treatment groups. Whether the AA was animal or plant derived, generally as the concentration of AA increased, the maximum growth rate decreased, lag time increased, and final growth readings increased. The best AA supplement and rate according to the growth metrics and <i>Curveball </i>analysis was Stimtide at a 33% supplementation rate. This was compared with the control at the bioreactor scale where it showed higher overall final growth and a higher taxonomic and alpha diversity. The scale-up approach to this study was successful at selecting the best amino acid supplement type and rate despite having less sophisticated control and monitoring compared to larger scales (i.e. the bioreactor). Use of the <i>Curveball</i> modeling program was useful for treatment group selection but did not always predict the outcomes seen in the live cultures. The use of AA as a media supplement can increase growth and diversity of a SynCom, though not all AA supplements or rates affect growth and community dynamics in the same way</a>.</p>