There is a clinical need for large numbers of phenotypes which are suitable for tissue engineering and cell therapy applications. Pluripotent stem cells (PSCs) are readily expanded in vitro and can differentiate into any somatic phenotype, making them a potential cell source. However, generating clinically-relevant numbers of phenotypes requires culture in stir-based bioreactor systems which expose cells to shear stress. Here we use a parallel plate bioreactor as a surrogate model system to better understand the effects of shear stress on pluripotent embryonic stem cells (ESCs). Initial studies examined the impact of cell deformation by shear stress during early ESC differentiation. Shear-treatment regulated specification into the three germ lineages and promoted mesodermal differentiation. Next we examined the response to shear stress during later specification events. The application of shear stress was found to promote mesodermal differentiation towards both definitive hematopoietic and mature endothelial phenotypes, although delayed applications were less effective at promoting hematopoietic specification. The next studies used low oxygen treatment to study the impact of another differentiation cue in the presence and absence of shear stress. Hypoxia promoted mesodermal phenotypes but the response was highly dependent on the physical microenvironment such as the culture method and the presence of shear stress. The next group of studies examined the impact of shear stress on ESC expansion. ESCs expanded under flow conditions maintained pluripotency but mesodermal specification was regulated in a manner that was dependent on the presence or absence of a ROCK inhibitor. The final studies used small molecule inhibitors to determine the role of specific signaling molecules during the shear-mediated differentiation of ESCs. Although inhibition of ROCK had little effect, inhibition c-SRC, JNK, or ERK modulated the shear-response. These studies highlight the important effects of shear stress during PSC culture and increase the basic science understanding of stem cell regulation by the physical microenvironment. The systematic approach to analyze multiple parameters allows for an improved translation of techniques from the bench-top into large-scale bioprocessing systems. Altogether these studies can inform large-scale differentiation techniques and bioreactor design in order to help establish the cell banks needed for clinical applications. / acase@tulane.edu

The central aim of this thesis was to develop strategies to improve the manufacture of the group B chimeric oncolytic adenovirus, ColoAd1, which rapidly kills and lyses host cells. In attempting to improve the cellular yield of ColoAd1, this thesis therefore sought to identify host infection-related factors that limited ColoAd1 production. In the widely-used manufacturing cell line, HEK293, ColoAd1 replication depleted intracellular ATP earlier than Ad11p and activated the intracellular energy sensor, AMPK. This might have reflected earlier ATP depletion, or possibly the absence of the E4orf4 protein from ColoAd1 compared to Ad11p. Despite this difference in AMPK activation, both viruses appeared able to maintain mTORC1 activity, which may be essential particularly for protein synthesis in the early stages of virus infection. For production purposes, preventing intracellular ATP depletion was seen as an attractive mechanism of maintaining ColoAd1 infected host cell viability and was hypothesised to lead to increased virus yield. A range of strategies were explored to enhance depleting ATP levels. Even though none of these were dramatically successful, they indicated that perhaps the anabolic building blocks required for viral replication were more important than cellular energy levels. Finally, a screening methodology based on siRNA knockdown was used to identify kinases that affected ColoAd1 replication. Many hits were identified, and several candidate kinases indicated a role for intracellular calcium signalling limiting virus particle production. Overall, data presented in this thesis supports the manufacture of ColoAd1 in HEK293 cells and suggest that enhancing glycolysis may increase ColoAd1 yield. It also provides mechanistic insights into the replication of ColoAd1 and Ad11p that may inform the improved design of group B oncolytic adenoviruses.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen Vadlani / Whole stillage and thin stillage from the ethanol production process were evaluated as substrate sources for the production of [beta]-carotenes using Sporobolomyces Roseus (ATCC 28988). This product has the potential to be used as a novel feed ingredient for poultry, swine, or cattle diets. [Beta]-carotenes have been supplemented in animal diets to improve animal health, enhance meat color and quality and increase vitamin A concentrations in milk and meat. Microbial fermentations involving growth and product kinetics were performed in 500 mL baffled shake flasks and in a 5 L fermentation bioreactor. Media optimization was conducted in shake flasks to evaluate two carbon sources: glucose and glycerol, and two nitrogen sources: ammonium sulfate and urea. Final [beta]-carotene concentration of 272.57±4.34 [mu]g [beta]-carotene/g biomass was found to be highest for the whole stillage, with 10 g/L added glucose and 10 g/L nitrogen added through ammonium sulfate supplementation. Glycerol addition yielded no significant increase (P<.05) in [beta]-carotene yield, while urea addition significantly decreased (P<.05) the final [beta]-carotene concentrations. The resulting fermented product can be blended with regular feed using either whole stillage as a dry feed ingredient or thin stillage as a liquid feed additive. The fermentation of whole stillage significantly influenced the physical and flow properties of the material. Even though there was a significant decrease (P<0.05) in bulk density and increase (P<0.05) in tapped density between DDGS and fermented whole stillage, there was a less pronounced difference between the whole stillage and fermented whole stillage. The fermentation of whole stillage significantly influenced the physical and flow properties of the material. This showed that the fermentation process and resulting nutritional profile had a significant effect on the resulting fermented whole stillage. A 50 L bioreactor was specifically designed to evaluate the scalability of the process and to perform subsequent feed production trails. Pilot scale feed pelleting runs were conducted and the resultant product was put in environmental chambers to determine if [beta]-carotene concentration was reduced as a result of storage. There was a significant decrease (P<0.05) in [beta]-carotene levels after pelleting and after 28 d of storage at elevated temperature and humidity. These decreases were consistent with previous research.

Master of Science / Department of Biological and Agricultural Engineering / Mei He / Thermobifida fusca, an aerobic moderately thermophilic, filamentous soil bacterium is capable of producing butyric acid. Butyric acid is a 4-carbon short chain fatty acid that is widely used in the chemical, food, and pharmaceutical industries. Currently, butyric acid is primarily produced through petroleum-based chemical synthesis, but could be a candidate to be produced by fermentation. By producing through a fermentation platform, production of butyric acid can be shifted from a non-renewable to a renewable source. In an effort to make T. fusca produce a high yield of butyric acid, multiple fermentation parameters were explored and optimized. The effect of different carbon sources (mannose, xylose, lactose, cellobiose, glucose, sucrose, and acetates) on butyric acid production was studied, where cellobiose produced the highest yield of 0.67 g/g C (g-butyric acid/g-carbon input). The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. Fermentation was performed on ground corn stover as a substrate to evaluate the production of butyric acid on lignocellulosic biomass, and the optimized conditions resulted in a titer of 2.37 g/L butyric acid. The butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanyol-CoA in T. fusca. A study into the transcriptomics of T. fusca was begun by growing T. fusca under a variety of fermentation conditions, isolating the messenger RNA, and performing a sequence of the mRNA using whole transcriptome shotgun sequencing. The results of sequencing of various samples were plotted to determine correlation across numerous fermentation parameters. This correlation based analysis determined that the carbon to nitrogen ratio has the largest overall impact on gene transcription of T. fusca among all of the fermentation parameters studied. Overall, the work from this study proves that production of butyric acid is possible from a renewable cellulosic feedstock.

Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave-like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built 'g' shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard.

The hardwood Eucalyptus grandis has been shown to be an important commodity for forestry-related industries as it has significantly faster specific growth rates per annum when compared with other types of tree species. It has therefore been suggested that residues from E. grandis may be a useful source of biomass for use in the production of biofuels for the transportation industry. Notably, E. grandis plantations within the Southern Hemisphere have some of the fastest growth rates worldwide. Due to the inherent nature of biomasses, such as lignocellulosic types having a significant amount of oxygen present, upgrading of biofuels produced from E. grandis is necessary. Several approaches were therefore evaluated to upgrade pyrolysis oils produced from E. grandis so as to increase their calorific values by decreasing oxygen content and subsequently increasing the hydrogen ratio. The hydrogen-to-carbon (H/C) and oxygen-to-carbon (O/C) ratios may be used successfully to evaluate the performances of catalyst-based upgrading techniques for either in situ or ex situ pyrolysis. In this regard the van Krevelen diagram, in which biofuels can be compared for their suitability as transportation fuels, along with their respective calorific values, is useful. The pyro-gas chromatography/mass spectroscopy (GC/MS) equipment is useful for the rapid and accurate evaluation of different catalysts for fast pyrolysis applications, and it was used here to evaluate the performances of the catalysts bentonite and ZSM-5 zeolite for upgrading pyrolysis oil produced from E. grandis biomass. A van Krevelen diagram was used to evaluate the performance of these catalysts, in conjunction with calorific values, based on the higher heating values v for the pyrolysis oils. Further studies were completed for microwave pyrolysis as it is a less harsh form of pyrolysis based on energy-transfer mechanisms. Mass balances were done and demonstrated good repeatability, with more stable pyrolysis oils being produced. This stability may be attributed to similarities between microwave pyrolysis and hydrothermal liquefaction as microwave pyrolysis induces conditions comparable to those of hydrothermal liquefaction within the wood cells, and both methods produce a stable product called bio-crude. Furthermore, it was found that these pyrolysis oils could be distilled so as to remove some of the water content and improve the higher heating value (HHV) from 13.80 to 23.30 MJ/kg. However, this was not as high as the theoretical yield of 26.70 MJ/kg, and better performance was obtained for fast pyrolysis catalysed with ZSM-5 zeolite at 300 °C, which achieved an HHV of 34.54 MJ/kg. It is recommended that ZSM-5 zeolite catalysis be used in microwave-assisted vacuum pyrolysis to determine whether a similar improvement may be realised. Microwave-assisted pyrolysis should also be investigated as a possible technology for inducing conditions similar to hydrothermal liquefaction processes within the cells that make up the biomass. / Dissertation (MEng)--University of Pretoria, 2015. / Chemical Engineering / Unrestricted

Carbon dioxide is a viable resource, if used as a raw material for bioprocessing. It is abundant and can be collected as a byproduct from industrial processes. Globally, photosynthetic organisms utilize around 6’000 TW (terawatt) of solar energy to fix ca. 800 Gt (gigaton) of CO2 in the planets largest carbon-capture process. Photosynthesis combines light harvesting, charge separation, catalytic water splitting, generation of reduction equivalents (NADH), energy (ATP) production and CO2 fixation into one highly interconnected and regulated process. While this simplicity makes photosynthetic production of commodity interesting, yet photosynthesis suffers from low energy efficiency, which translates in an extensive footprint for solar biofuels production conditions that store < 2% of solar energy. Electron transfer processes form the core of photosynthesis. At moderate light intensity, the electron transport chains reach maximum transfer rates and only work when photons are at appropriate wavelengths, rendering the process susceptible to oxidative damage, which leads to photo-inhibition and loss of efficiency. Based on our fundamental analysis of the specialized tasks in photosynthesis, we aimed to optimize the efficiency of these processes separately, then combine them in an artificial photosynthesis (AP) process that surpasses the low efficiency of natural photosynthesis. Therefore, by combining photovoltaic light harvesting with electrolytic water splitting or CO2 reduction in combination with microbiological conversion of electrochemical products to higher valuable compounds, we developed an electro-microbial carbon capture and conversion setups that capture CO2 into the targeted bioplastic; polyhydroxybutyrate (PHB). Based on the type of the electrochemical products, and the microorganism that either (i) convert products formed by electrochemical reduction of CO2, e.g. formate (using inorganic cathodes), or (ii) use electrochemically produced H2 to reduce CO2 into higher compounds (autotrophy), three AP setups were designed: one-pot, two-pot, and three-pot setups. We evaluated the kinetic (microbial uptake and conversion, electrochemical reduction) and thermodynamics (efficiencies) of the separate processes, and the overall process efficiency of AP compared to photosynthesis. We address the influence of several parameters on efficiencies and time-space yields, e.g. salinity, pH, electrodes, media, partial pressures of H2 and CO2. These data provide a valuable basis to establish a highly efficient and continuous AP process in the future.

>Magister Scientiae - MSc / Consolidated bioprocessing (CBP), in which a single organism in a single reactor is responsible for the conversion of pretreated lignocellulosic biomass to bioethanol, remains an attractive option for production of commodity products if an organism fit for this process can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second generation bioethanol. Current recombinant yeast strains engineered for this purpose must overcome the drawback of generally low secretion titres. A promising strategy for directly converting lignocellulose to ethanol is by displaying heterologous cellulolytic enzymes on the cell surface by means of the glycosylphosphatidylinositol (GPI) or similar anchoring systems. Recently, a strain producing cell-adhered enzymes in a ratio-optimized manner was created that showed significant crystalline cellulose hydrolysis.

Two possible ways for increasing the rate of separation for magnetic bead separation has been observed. Increasing NP concentration by 2.5x gave a slight increase in rate of separation while 1.5x and 2.0x concentration increase resulted in a slight decrease in rate of separation. Synthesizing the magnetic beads under the influence of an external magnetic field also showed promising results. In a literature review, several types of magnetic beads and technologies are discussed, and how there is a great future potential for magnetic beads in the isolation of several types of biomolecules. It is concluded that the market for magnetic beads for cell isolation is expanding greatly with many different applications and expects to be worth 14.64 billion USD by 2025.

Therapeutic viruses are a class of biotherapeutic which have enabled new treatments and medical advances in the areas of vaccines, cancer treatment, gene therapy, and more. In the production and purification of these products, the sterile filtration unit operation is known to have poor yields and contribute to the high cost of the final product, significantly hampering the large-scale production of some therapeutic viruses. Thus, this thesis seeks to explore various aspects of process development and fundamental understanding in the sterile filtration of therapeutic viruses. This thesis explores the mechanisms and membrane properties which govern how bacteria are retained during filtration, and applies these insights to improve the sterile filtration recovery of a therapeutic virus through proper membrane selection. To better understand the factors which cause membrane fouling and loss of virus during sterile filtration, the effect of host cell impurities on filtration performance was investigated. This revealed that small amounts of host cell protein are a major factor in both membrane fouling and reduced virus yield, and that there is a synergistic effect between the virus and the host cell protein adsorbing to the membrane surface. Recognizing that conventional polymeric membranes have many limitations, a novel ultrathin, isoporous, microfabricated silicon nitride membrane was tested for suitability as a sterile filter. Finally, the application of nanoparticles as model virus particles in filtration testing was examined, and a process was developed through which nanoparticles could be fused together to create controlled amounts of particle aggregates, similar to how viruses can be prone to aggregation. The work described here will help enable the development of next generation sterile filtration membranes and provides both insights and methodologies for improving sterile filtration performance. / Thesis / Doctor of Philosophy (PhD) / While many people are aware that viruses can be used in medicine as vaccines, there are even more new and developing ways they can be used, such as in fighting cancer or treating previously uncurable diseases. However, testing of and patient access to these new treatments is often limited due to the challenges in producing and purifying enough of the virus. Viruses are highly complex and large relative to other products, and so many of the common methods and manufacturing processes which are standard in the industry need to be significantly adapted or improved to suit the production of viruses. This study investigates one step of the purification process, sterile filtration, and considers how a variety of factors from the materials used to the properties of the virus solution can be optimized to improve performance. With a deeper understanding of the sterile filtration process, recommendations can be made to help improve the production of future virus-based therapies.