The cost of deconstructing cellulose into soluble sugars is a key impediment to the commercial production of lignocellulosic biofuels. The use of the quartz crystal microbalance (QCM) to investigate reaction variables critical to enzymatic cellulose hydrolysis is investigated here, extending previous studies of fungal cellulase activity for the first time to whole cell cellulases. Specifically, the activity of the cellulases of Clostridium thermocellum, which are in the form of cellulosomes, was investigated. To clearly differentiate the activity of free cellulosome and cell-bound cellulosome, the distribution of free cellulosome and cell-bound cellulosome in crude cell broth at different growth stages of C. thermocellum (ATCC 27405) was quantified. Throughout growth, greater than 70% of the cellulosome in the crude cell broth was unattached to the cell. The frequency response of the QCM was shown to capture adsorption and hydrolysis of amorphous cellulose films by the whole-cell cellulases. Further, both crude cell broth and free cellulosomes were found to have similar inhibition pattern (within 0 - 10 g/L cellobiose). Thus, kinetic models developed for the cell-free cellulosomes, which allow for more accurate interfacial adsorption analysis by QCM than their cell-attached counterparts, may provide insight into hydrolysis events in both systems.

Recent pharmaceutical developments have investigated using supramolecular nanoparticles in order to increase the bioavailability and solubility of drugs delivered in various methods. Modification of the carbohydrate cyclodextrin increases the ability to encapsulate hydrophobic pharmaceutical molecules by forming a carrier with a hydrophobic core and hydrophilic exterior. Guest molecules are commonly added to these inclusion complexes in order to add stability and further increase targeting abilities of the carriers. One such guest molecule is adamantine combined with a poly(ethylene glycol) chain. Vesicles are formed by hydrating a thin film of amphiphilic cyclodextrin and guest molecules in buffer solution that mimics physiological conditions. The solution is subject to freeze-thaw cycles and extrusion, and the complexes are separated out via size exclusion chromatography. Dynamic Light Scattering instrumentation is used to observe the particle size distribution. Cargo release can be observed in fluorescent dye-loaded vesicles by addition of a membrane-cleaving agent under a fluorimeter instrument. Future work involving this drug delivery technology includes synthesizing a chemically sensitive guest that will cleave in the presence of an intra-cellular anti-oxidant, and finally observing the uptake of these vesicles into live cells and testing the delivery of cargo in vitro under physiological conditions.

ABSTRACT Photobioreactor Design for Improved Energy Efficiency of Microalgae Production Alexander Burns The objective of this research was to investigate a new photobioreactor (PBR) design for microalgae production that retains the typical advantages of existing tubular PBRs while reducing power consumption by providing simultaneous culture circulation and gas exchange with airlift alone and no centrifugal recirculating pump. Traditional tubular PBR designs feature a compressed air supply and a centrifugal pump for culture circulation and gas exchange. Circulation and gas exchange in a closed-system PBR is necessary to keep the algae suspended and to provide sufficient mass transfer (mainly for the exchange of oxygen and carbon dioxide). In a traditional tubular PBR sparged air keeps the culture well mixed and strips out excess dissolved oxygen in an airlift-column unit, while the centrifugal pump circulates the culture in the tubular stage and decreases the amount of air bubbles traveling into this stage; where most of the photosynthesis occurs. The PBR design proposed herein does away with the usual centrifugal pump. The air blower performs both gas exchange in the airlift columns and system-wide circulation. This builds on a previous tubular PBR design that provides circulation and gas exchange by airlift alone, which was patented by Cathcart in 2011. However, the Cathcart patent does not provide data on mixing, gas exchange, energy consumption, flow regime or biomass productivity. The new design described here builds on the Cathcart design, but includes several unique design features, such as larger diffuser columns which provide airlift-induced flow for a series of vertical PBR tubes. To perform a power consumption v analysis, a pilot-scale prototype of the new PBR design was built and operated. The prototype PBR consisted of two airlift columns attached to 9 m of vertical serpentine tubing connected to the top and bottom by standard 90-degree PVC elbows in a U-bend fashion to each column to make a total working volume of 235 L. The airlift columns were about 1.5 m tall and 30.5 cm ID, while the serpentine tubes were about 0.9 m tall and 7.6 cm ID to make a total of five vertical tubes for every airlift column. Data collected for this prototype design suggest an average overall areal productivity (OAP) of 111 g m-2 d-1 (g biomass m-2 total land area with empty space day-1), an average illuminated surface productivity (ISP) of 14.3 g m-2 d-1 (g biomass m-2 reactor photo-stage day-1), an average volumetric productivity (VP) of 0.55 g L-1 d-1 (g biomass L-1 reactor working volume day-1), a specific power input in the range of 330 to 360 W m-3 (W power needed for culture circulation and gas exchange m-3 reactor working volume) and a specific biomass productivity (SBP) in the range of 17.6 to 19.1 mg kJ-1 (mg biomass kJ-1 energy needed for culture circulation and gas exchange) with Chlorella vulgaris as the model algae. The biomass productivity per energy input (SBP) of the new PBR design appears to be higher than that of similar designs currently described in the literature. Elimination of the centrifugal pump in a tubular PBR design is a concept worth further study for potential energy savings.

Spatiotemporal metabolic modeling of microbial metabolism is a step closer to achieving higher dimensionalities in numerical studies (in silico) of biofilm maturation. Dynamic Flux Balance Analysis (DFBA) is an advanced modeling technique because this method incorporates Genome Scale Metabolic Modeling (GSMM) to compute the biomass growth rate and metabolite fluxes. Biofilm thickness is pertinent because this variable of biofilm maturation can be measured in a laboratory (in vitro). Pseudomonas aeruginosa (P. aeruginosa) is the model bacterium used in this computational model based on previous research conducted by Dr. Michael Henson, available GSMMs, and the societal significance of patients suffering from P. aeruginosa airway infections. Spatiotemporal Flux Balance Analysis (SFBA) will be the computational method used in this thesis to simulate biofilm growth. Another level of accuracy will be introduced to SFBA which is a dynamic finite difference grid that will vary relative to the biofilm’s velocity of expansion/contraction. This novel idea is governed by a differential equation that defines the biofilm’s velocity and updates the spatial dependency of the finite difference grid which has never been done while utilizing GSMM. Environmental conditions (bulk concentrations of metabolites) are altered to investigate how varying nutrients (glucose, oxygen, lactate, nitrate) affected biofilm maturation.

<p>Microbiological contamination from bacteria such as <em>Escherichia coli</em> and Salmonella is one of the main reasons for waterborne illness. Real time and accurate monitoring of water is needed in order to alleviate this human health concern. Performing multiple and parallel analysis of biomarkers such as DNA and mRNA that targets different regions of pathogen functionality provides a complete picture of its presence and viability in the shortest possible time. These biomarkers are present inside the cell and need to be extracted for analysis and detection. Hence, lysis of these pathogenic bacteria is an important part in the sample preparation for rapid detection. In addition, collecting a small amount of bacteria present in a large volume of sample and concentrating them before lysing is important as it facilitates the downstream assay. Various techniques, categorized as mechanical, chemical, thermal and electrical, have been used for lysing cells. In the electrical method, cells are lysed by exposure to an external electric field. The advantage of this method, in contrast to other methods, is that it allows lysis without the introduction of any chemical and biological reagents and permits rapid recovery of intercellular organelles. Despite the advantages, issues such as high voltage requirement, bubble generation and Joule heating are associated with the electrical method.</p> <p>To alleviate the issues associated with electrical lysis, a new design and associated fabrication process for a microfluidic cell lysis device is described in this thesis. The device consists of a nanoporous polycarbonate (PCTE) membrane sandwiched between two PDMS microchannels with electrodes embedded at the reservoirs of the microchannels. Microcontact printing is used to attach this PCTE membrane with PDMS.</p> <p>By using this PCTE membrane, it was possible to intensify the electric field at the interface of two channels while maintaining it low in the other sections of the device. Furthermore, the device also allowed electrophoretic trapping of cells before lysis at a lower applied potential. For instance, it could trap bacteria such as <em>E. coli</em> from a continuous flow into the intersection between two channels for lower electric field (308 V/cm) and lyse the cell when electric field was increased more than 1000 V/cm into that section.</p> <p>Application of lower DC voltage with pressure driven flow alleviated adverse effect from Joule heating. Moreover, gas evolution and bubble generation was not observed during the operation of this device.</p> <p>Accumulation and lysis of bacteria were studied under a fluorescence microscope and quantified by using intensity measurement. To observe the accumulation and lysis, LIVE/DEAD BacLight Bacterial Viability Kit consisting of two separate components of SYTO 9 and propidium iodide (PI) into the cell suspension in addition to GFP expressed <em>E. coli</em> were used. Finally, plate counting was done to determine the efficiency of the device and it was observed that the device could lyse 90% of bacteria for an operation voltage of 300V within 3 min.</p> <p>In conclusion, a robust, reliable and flexible microfluidic cell lysis device was proposed and analyzed which is useful for sample pretreatment in a Micro Total Analysis System.</p> / Master of Applied Science (MASc)

Hydrogels are popular materials for biological applications since they exhibit properties like that of natural soft tissue and have tunable properties. Biodegradable hydrogels provide an added advantage in that they degrade in an aqueous environment thereby avoiding the need for removal after the useful lifetime. In this work, we investigated poly(β-amino ester) (PBAE) biodegradable hydrogel systems. To begin, the factors affecting the macromer synthesis procedure were studied to optimize the reproducibility of the resulting hydrogels made and create new methods of tuning the properties. Hydrogel behavior was then tuned by altering the hydrophilic/hydrophobic balance of the chemicals used in the synthesis to develop systems with linear and two-phase degradation profiles. The goal of the research was to better understand methods of controlling hydrogel properties to develop systems for several biomedical applications. Several systems with a range of properties were synthesized, and their in vitro behavior was characterized (degradation, mechanical properties, cellular response, etc.). From these studies, materials were chosen to serve as porogen materials and an outer matrix material to create a composite scaffold for tissue engineering. In most cases, a porous three dimensional scaffold is ideal for cellular growth and infiltration. In this work, a composite with a slow degrading outer matrix PBAE with fast degrading PBAE microparticles was created. First, a procedure for developing porogen particles of controlled size from a fast-degrading hydrogel material was developed. Porogen particles were then entrapped in the outer hydrogel matrix during polymerization. The resulting composite systems were degraded and the viability of these systems as tissue engineering scaffolds was studied. In a second area of work, two polymer systems, one PBAE hydrogel and one sol-gel material were altered through the addition of iron oxide nanoparticles to create materials with remote controlled properties. Iron oxide nanoparticles have the ability to heat in an alternating magnetic field due to the relaxation processes. The incorporation of these nanoscale heating sources into thermosensitive polymer systems allowed remote actuation of the physical properties. These materials would be ideal for use in applications where the system can be changed externally such as in remote controlled drug delivery.

Biomolecules on cell surfaces play critical roles in diverse biological and physiological processes. However, conventional bulk scale techniques are unable to clarify the density and distribution of specific biomolecules in situ on single, living cell surfaces at the micro or nanoscale. In this work, a single cell analysis technique based on Atomic Force Microscopy (AFM) is developed to spatially identify biomolecules and characterize nanomechanical properties on single cell surfaces. The unique advantage of these AFM-based techniques lies in the ability to operate in situ (in a non-destructive fashion) and in real time, under physiological conditions or controlled micro-environments. First, AFM-based force spectroscopy was developed to study the fundamental biophysics of the heparin/thrombin interaction at the molecular level. Based on force spectroscopy, a force recognition mapping strategy was developed and optimized to spatially detect single protein targets on non-biological surfaces. This platform was then translated to the study of complex living cell surfaces. Specific carbohydrate compositions and changes in their distribution, as well as elasticity change were obtained by monitoring Bacillus cells sporulation process. The AFM-based force mapping technique was applied to different cellular systems to develop a cell surface biomolecule library. Nanoscale imaging combined with carbohydrate mapping was used to evaluate inactivation methods and growth temperatures effects on Yersinia pestis surface. A strategy to image cells in real time was coupled with hydrophobicity mapping technique to monitor the effect of antimicrobials (antimicrobial polymer and copper) on Escherichia coli and study their killing mechanisms. The single spore hydrophobicity mapping was used to localize the exosporium structure and potentially reconstruct culture media. The descriptions of cell surface DNA on single human epithelial cells potentially form a novel tool for forensic identification. Overall, these nanoscale tools to detect and assess changes in cell behavior and function over time, either as a result of natural state changes or when perturbed, will further our understanding of fundamental biological processes and lead to novel, robust methods for the analysis of individual cells. Real time analysis of cells can be used for the development of lab-on-chip type assays for drug design and testing or to test the efficacy of antimicrobials.

Non-catalytic protein-carbohydrate interactions are an essential element of various biological events. This dissertation presents the work on understanding carbohydrate recognition mechanisms and their physical significance in two groups of non-catalytic proteins, also called lectins, which play key roles in major applications such as cellulosic biofuel production and drug delivery pathways. A computational approach using molecular modeling, molecular dynamic simulations and free energy calculations was used to study molecular-level protein-carbohydrate and protein-protein interactions. Various microorganisms like bacteria and fungi secret multi-modular enzymes to deconstruct cellulosic biomass into fermentable sugars. The carbohydrate binding modules (CBM) are non-catalytic domains of such enzymes that assist the catalytic domains to recognize the target substrate and keep it in proximity. Understanding the protein-carbohydrate recognition mechanisms by which CBMs selectively bind substrate is critical to development of enhanced biomass conversion technology. We focus on CBMs that target both oligomeric and non-crystalline cellulose while exhibiting various similarities and differences in binding specificity and structural properties; such CBMs are classified as Type B CBMs. We show that all six cellulose-specific Type B CBMs studied in this dissertation can recognize the cello-oligomeric ligands in bi-directional fashion, meaning there was no preference towards reducing or non-reducing end of ligand for the cleft/groove like binding sites. Out of the two sandwich and twisted forms of binding site architectures, twisted platform turned out to facilitate tighter binding also exhibiting longer binding sites. The exterior loops of such binding sites were specifically identified by modeling the CBMs with non-crystalline cellulose showing that high- and low-affinity binding site may arise based on orientation of CBM while interacting with non-crystalline substrate. These findings provide various insights that can be used for further understanding of tandem CBMs and for various CBM based biotechnological applications. The later part of this dissertation reports the identification of a physiological ligand for a mammalian glycoprotein YKL-40 that has been only known as a biomarker in various inflammatory diseases and cancers. It has been shown to bind to oligomers of chitin, but there is no known function of YKL-40, as chitin production in the human body has never been reported. Possible alternative ligands include proteoglycans, polysaccharides, and fibers such as collagen, all of which make up the mesh comprising the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. We considered the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. Our simulation results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, as the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non- specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to the associated increases in hyaluronan.

Metal-organic frameworks (MOFs) are a new class of nanoporous materials that have received great interest since they were first synthesized in the late 1990s. Practical applications of MOFs are continuously being discovered as a better understanding of the properties of materials adsorbed within the nanopores of MOFs emerges. One such potential application is as a component of an explosive-sensing system. Another potential application is for hydrogen storage. This work is focused on tailoring MOFs to adsorb/desorb the explosive, RDX. Classical grand canonical Monte Carlo (GCMC) and molecular dynamic (MD) simulations have been performed to calculate adsorption isotherms and self-diffusivities of RDX in several IRMOFs. Because gathering experimental data on explosive compounds is dangerous, data is limited. Simulation can in part fill the gap of missing information. Through these simulations, many of the key issues associated with MOFs preconcentrating RDX have been resolved. The issues include both theoretical issues associated with the computational generation of properties and practical issues associated with the use of MOFs in explosive-sensing system. Theoretically, we evaluate the method for generating partial charges for MOFs and the impact of this choice on the adsorption isotherm and diffusivity. Practically, we show that the tailoring of an MOF with a polar group like an amine can lead to an adsorbent that (i) concentrates RDX from the bulk by as much as a factor of 3000, (ii) is highly selective for RDX, and (iii) retains sufficient RDX mobility allowing for rapid, real time sensing. Many of the impediments to the effective explosive detection can be framed as shortcomings in the understanding of molecule surface interactions. A fundamental, molecular-level understanding of the interaction between explosives and functionalized MOFs would provide the necessary guidance that allows the next generation of sensors to be developed. This is one of the main driving forces behind this dissertation. Another important achievement in this work is the demonstration of a new direction for tailoring MOFs. A new class of tailored MOFs containing porphyrins has been proposed. These tailored MOFs show greater capability for hydrogen storage, which also demonstrated the great functionalization of MOFs and great potential to serve as preconcentrators. The use of a novel multiscale modeling technique to develop equations of state for inhomogeneous fluids is included as a supplement to this dissertation.

Oral mucositis is a painful and debilitating chronic inflammatory condition that can result from chemo and/or radiotherapy. While current treatment strategies which provide temporary relief exist, there is still an unmet clinical need for a robust long active barrier strategy which can simultaneously provide protection and release drug to enhance the wound healing response. It is proposed that an affinity based layer-by-layer self-assembled barrier administered as a series of mouth rinses can allow for wound specific drug delivery, providing an effective regenerative therapy. In this work, biotinylated poly(acrylic acid) is used to develop LBL assemblies based upon biotin-streptavidin affinity interactions. To explore the ability of developed LBL assemblies to resist the harsh intraoral environment, in vitro chemical and ex vivo mechanical tests are performed. The stability results demonstrate significant LBL barrier stability with wear resistance. From principal component regression analysis, factors such as polymer MW and number of layers in assemblies contributed significantly to chemical barrier stability. Also it is observed that the extent of biotin conjugation plays a significant role in LBL development and in mechanical stability. Thus, the proposed affinity based multilayered assemblies with their excellent barrier properties offer a modular treatment approach in oral mucosal injuries.