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Item Energy metabolism and mechanotransduction in osteoarthritic chondrocytes: targeted metabolic profiling and flux analysis(Montana State University - Bozeman, College of Engineering, 2023) Erdogan, Ayten Ebru; Chairperson, Graduate Committee: Ronald K. June II; This is a manuscript style paper that includes co-authored chapters.Osteoarthritis (OA) is a prevalent and debilitating disease that affects hundreds of millions people worldwide. One of OAs major consequences is the degradation of articular cartilage, leading to joint pain, stiffness, and loss of function. Currently, there is no treatment for OA. The existing interventions are mostly for suppressing the symptoms: physical therapy, anti-inflammatories, and pain management. The last resort is total joint replacement, which has long-term consequences especially for early-onset OA patients. Thus, researchers are focusing on understanding this complex disease and its molecular components to develop better treatments. Chondrocytes, the sole cell type in articular cartilage, play a crucial role in maintaining tissue homeostasis and responding to mechanical stimuli via synthesis of key structural components like collagen. However, the intracellular pathways underlying chondrocyte mechanotransduction are not fully understood, especially those related to central carbon metabolism. This thesis uses 13C isotopic labeling to trace carbon sources and downstream metabolites related to energy metabolism in vitro. Primary human articular chondrocytes from OA patients exposed to labeled glucose and glutamine, and their global and targeted metabolite profiles are assessed. The results show how both glucose and glutamine utilization as carbon sources flows through the TCA cycle. This work also develops a comprehensive model of mammalian carbon metabolism in OA primary human chondrocytes. The model integrates energy metabolism, amino acid synthesis, and transport reactions contributing to Collagen-II and Collagen-VI production. Using flux balance analysis (FBA), trade-offs between Collagen-II and Collagen-VI synthesis are evaluated based on ATP and carbon source requirements under different oxidative stress conditions. Then, these model predictions are presented with experimental data obtained from OA chondrocytes subjected to shear and compressive mechanical stimulation, which can be integrated in the model later on. These data shed new light on metabolism of primary OA chondrocytes and provides insight into potential therapeutic targets for OA intervention.Item Development and optimization of open-source pumps and drop-based split-and-merge microfluidic devices(Montana State University - Bozeman, College of Engineering, 2023) Sanchez, Humberto Scott; Chairperson, Graduate Committee: Connie B. Chang; This is a manuscript style paper that includes co-authored chapters.Drop-based microfluidics is used to encapsulate small volumes of chemical and biological agents into discrete drops. The contents of these pico-liter sized drops can then be used for biochemical assays. Microfluidic devices are used to manipulate these drops through the removal and addition of drop contents and pumps are used to control the fluid flow within the microfluidic devices. Often, the pumps used to drive fluid flow are prohibitively costly and the fluid dynamics of the devices used to control drop contents are not fully described. This research focuses on de-mystifying both the pump systems and microfluidic devices commonly used in drop-based microfluidics. We focused on developing an open-source pneumatic pressure pump to create microfluidic drops. Pneumatic pressure pumps have been found to be flexible and accurate systems to control fluid flow within microfluidic devices. Our pump system was found to be accurate and fast-responding as well as easy to assemble and control via open-source software. In addition, we developed a design process to develop drop-based split-and-merge (SAM) microfluidic devices. These devices have been commonly used to control drop volumes and add reagents for intra-drop assays. Our design and optimization methodology was then used to develop multiple SAM geometries with various splitting capabilities and the ability to create a broad range of drop volumes. The operating conditions for each SAM device was optimized for different input drop volumes. We also developed an empirical drop model to predict drop volume changes within a SAM device. Our open-source pneumatic pressure pump is a fast responding and accurate system that can be adapted for driving flow in any microfluidic device. Our drop-based SAM devices and the development and modelling process we developed serves as a blueprint for future SAM devices and similar drop manipulation microfluidic devices.Item Biorecovery of rare earth elements and critical minerals via Gluconobacter oxydans(Montana State University - Bozeman, College of Engineering, 2024) Kessler, Kendall Rhea; Co-chairs, Graduate Committee: Ross Carlson and Ryan AndersonThe depletion of high-grade ore deposits, accumulation of electronic waste, and the geopolitical challenges in sourcing critical materials have emphasized the need for sustainable metal recovery methods and recycling efforts in the United States. Conventional metal recovery approaches, including pyrometallurgy and hydrometallurgy, are not only environmentally unsustainable but also inadequate for the retrieval of metals from low-grade deposits. Biorecovery, defined by microorganism-mediated metal recovery, provides an advantageous alternative to traditional recovery methods due to increased sustainability, lower operational costs, and high efficiencies observed for the recovery of low-grade feedstocks. This study investigates the potential of bioleaching as an eco-friendly alternative in the recycling of two distinct waste feedstocks: magnetic swarf and lithium-ion batteries (LIBs). Cultivation of Gluconobacter oxydans was investigated under varying growth medium compositions, wherein increased concentrations of yeast extract were substituted for KH 2PO 4, to determine the subsequent impact on the base metal and rare earth element recovery through the application of the cell-free biolixiviant. This substitution resulted in increased growth yields and enhanced recovery with respect to magnetic swarf, whereas negligible improvement was observed for LIBs. Biorecovery has also been demonstrated for the recovery of metals from ore, where yields are a function of comminution and concentration efficiencies. Typical compressive comminution practices account for the largest proportion of energy expenditures in a mining process. Transcritical CO 2 (tCO 2) comminution, wherein ore is fractured through overcoming a rock's tensile strength, was examined to determine whether physical differences in particle generation were present as compared to traditional fracture techniques. An ore deposit in British Columbia, rich in a nickel-iron alloy mineral phase called awaruite, was examined through scanning electron microscopy, backscatter electron imaging, and energy dispersive spectroscopy to determine the impact of comminution method on awaruite recovery. Image processing was used to investigate shape factors for the individual particles. Preliminary evidence indicates despite a lack of distinct particle differences, tCO 2 comminution resulted in increased liberation and recovery of awaruite ore. Abiotic leaching studies were conducted to determine whether the method of comminution impacted leaching efficiencies. Although samples could not be quantitatively measured, initial qualitative results indicate tCO 2 comminution provides increased yields.Item Bacterial cultivation in microscale drops and capsules to resolve single-cell growth physiology(Montana State University - Bozeman, College of Engineering, 2023) Pratt, Shawna Leigh; Chairperson, Graduate Committee: Ross Carlson; This is a manuscript style paper that includes co-authored chapters.Single-cell heterogeneity contributes to the complex population dynamics of infectious microbial communities. Improving our understanding of single-cell physiology and heterogeneity may aid in mitigating microbial infections; however, assaying large populations of single cells can be challenging. Despite recent developments in single-cell assaying, tracking the physiology of large numbers of individual cells and their lineages over time is difficult to achieve using current technologies. Here, I apply drop-based microfluidics to develop microscale tools for improving high-throughput single-cell microbial growth assays. Drop-based microfluidics is a technology that generates and manipulates microscale drops. In this work, I create water-in-oil drops and hydrogel-shelled microcapsules using drop-based microfluidics to study the growth of P. aeruginosa bacteria, a key pathogen implicated in chronic lung infections and wounds. The growth of single bacterial cells inside drop microcompartments is observed via time-lapse confocal microscopy. Bacteria were cultured in water-in-oil drops and prepared for long-term storage in a novel microfluidic device environment, which we call a DropSOAC (Drop Stabilization on a Chip) chamber. The DropSOAC method prevents drop destabilization by saturating microfluidic devices with equilibrated water and oil, maintaining phase equilibrium in the drop emulsion. Using DropSOAC, the single-cell growth of starved P. aeruginosa wildtype and hibernation promotion factor mutants were characterized, revealing significant growth heterogeneity in the mutant strain. Finally, we present a method for generating hydrogel-shelled microcapsules that enables the culturing of single cells in microscale environments where nutrients and waste can diffuse in and out of the microculture environment. A 3-D microfluidic device and capsule generation protocol are designed, resulting in an optimized approach for capsule production using phase-separating polymer systems and rapid hydrogel crosslinking. The growth of hundreds of individual P. aeruginosa cells is observed over time with the hydrogel- shelled microcapsules. Due to the permeability of the microcapsules, antibiotics can be introduced at various times during growth to investigate single and biofilm P. aeruginosa physiology. Overall, this work introduces novel approaches for high-throughput, single-cell microbial growth characterization that enables a deeper understanding of the role of heterogeneity in bacterial populations.Item Condensation of chromium vapor, generated in high-temperature (>800°C) environments, and interactions with aluminosilicate surfaces(Montana State University - Bozeman, College of Engineering, 2024) van Leeuwen, Travis Kent; Chairperson, Graduate Committee: Paul E. Gannon; This is a manuscript style paper that includes co-authored chapters.This work represents a collection of research and reporting with the goal of improving fundamental understanding of chromium (Cr) vapor reactive condensation, relevant in many high-temperature (>800°C) process environments where Cr-containing alloys are used. While reactive evaporation of Cr from stainless steels used in high-temperature solid oxide electrochemical systems is well-documented, the dynamics of Cr condensation onto surrounding interfaces during complex and dynamic system operation is less understood. Understanding these interactions during operation is critical for improving system performance and safeguarding environmental, health and safety, as some condensed species contain hexavalent chromium (Cr(VI)), a known carcinogen. A series of studies were designed and conducted to investigate the condensation pathways of Cr vapors within representative high-temperature system environments, simulating extreme conditions for Cr evaporation and downstream aluminosilicate fibers used in high-temperature insulation. The first study focuses on the influence of water vapor concentration in the gaseous environment on reactive Cr condensation and speciation onto aluminosilicate fibers. The second study explores the effects of alkaline oxide additives in aluminosilicate fibers on Cr condensation and speciation. The third study investigates the effects of presence of alkaline oxides within the Cr vapor source on reactive evaporation and condensation of Cr vapors onto downstream aluminosilicate fibers. To accomplish the specific objectives of these studies, Cr vapors, produced by high-temperature (>800°C) air exposures of trivalent chromium (Cr(III)) oxide (Cr 2O 3) (chromia) powder with variable moisture content, were condensed onto various ceramic materials (aluminosilicate fibers) downstream at lower temperatures (100-500°C). Total condensed Cr and ratios of oxidation states were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES) and diphenyl carbazide (DPC) colorimetric/direct UV-VIS spectrophotometric analyses, respectively. Results indicate presence of both Cr(III) and Cr(VI) species condensed on all samples investigated. Total Cr and ratio of Cr(VI) to total Cr detected was significantly more on those containing alkaline oxides and at higher atmospheric water vapor concentration, while the presence of alkaline oxides in the Cr vapor source (Cr 2O 3) decreased the evaporation and amount of Cr/Cr(VI) condensed on the samples downstream. Computational thermodynamic equilibrium modelling helps explain experimental results showing the relative stability of alkaline-chromate compounds.Item Biocorrosion of copper by Oleidesulfovibrio alaskensis G20 biofilms in static and dynamic environments(Montana State University - Bozeman, College of Engineering, 2024) Keskin, Yagmur; Chairperson, Graduate Committee: Brent M. Peyton; Matthew Fields (co-chair); This is a manuscript style paper that includes co-authored chapters.This study presents a detailed examination of the intricate relationships between Oleidesulfovibrio alaskensis G20 and copper (101), emphasizing three interconnected perspectives: the kinetics of copper toxicity in three distinct media, the impact of surface finishing on microbiologically influenced corrosion (MIC), and the interaction of G20 biofilms and copper in CDC biofilm reactors. Initially, the study concentrates on the kinetic effects of copper toxicity on the growth of G20. The research meticulously quantifies the detrimental impact of different copper (II) concentrations (6, 12, 16, and 24 micron) on bacterial growth kinetics in three media: LS4D balanced (BAL), electron acceptor-limited (EAL), and electron donor-limited (EDL). Using a non-competitive inhibition model, I50 (concentrations of copper causing 50% inhibition of bacterial growth) values were calculated to be 13.1, 13.87, and 11.31 micron for LS4D BAL, EAL, and EDL media, respectively. The second part of the study shifts its focus to the effect of surface finishing on MIC of copper 101 by G20. The biofilm and corrosion pit depths were measured through a series of sophisticated analyses employing 3D optical profilometry, Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX), and X-ray Diffraction Analysis (XRD). The research investigates how different levels of surface roughness, applied through metallographic grinding and polishing, influence corrosion. The findings demonstrate a clear pattern of both uniform and pitting corrosion across all surface finishes. Notably, a statistically significant decrease in corrosion rates was observed when the surface roughness of copper was altered from approximately 13?m to about 0.06?m. Finally, the study explores the interaction between G20 biofilms and copper (101) into CDC reactors to understand biofilm development on copper surfaces and its subsequent impact on copper corrosion in a dynamic environment over periods of 7, 9, and 14 days. The results showed robust biofilm formation through hexose and protein analyses and SEM images displaying progressive increases in SRB cell accumulation over time. Localized pit depths were measured and compared to static conditions, and pits showed only a 20% increase in a dynamic environment. These findings offer an improved understanding of the complex interactions between G20 and MIC of copper.Item Material properties of ureolytically induced calcium carbonate adhesives(Montana State University - Bozeman, College of Engineering, 2023) Anjum, Sobia; Chairperson, Graduate Committee: Robin Gerlach; This is a manuscript style paper that includes co-authored chapters.Polymers used in adhesive applications are often petrochemical-based and release volatile organic compounds (VOCs) during application. These VOCs can accumulate indoors to the detriment of human health. Biopolymers potentially offer a non-toxic and sustainable alternative to synthetic polymers but generally have limited physical stability and low mechanical performance. One of the methods of improving the stability and adhesive performance of biopolymers is the addition of a mineral phase to reinforce biopolymer adhesives. In this work, biomineral-reinforced biopolymer adhesives were produced by ureolytically induced precipitation of calcium carbonate in the presence of guar gum and soy protein. The microbially and enzymatically induced ureolysis was carried out by the ureolytic bacterium, Sporosarcina pasteurii, or by jack bean urease. The resulting adhesives were referred to as ureolytically induced calcium carbonate precipitation (UICP)-reinforced adhesives and specifically microbially and enzymatically induced calcium carbonate (MICP and EICP)- reinforced adhesives. The adhesive strength of these composite adhesives was optimized by varying calcium and cell (or enzyme) concentrations. The adhesive strength of biomineral reinforced guar gum and soy protein biopolymers was up to 2.5 and 6 times higher than the adhesive strength of the biopolymers alone, respectively. The durability of the MICP-reinforced adhesives was tested after varying immersions (24 h and 7 days), relative humidities (50 and 80% RH), and temperatures (-20, 100, and 300?C). The durability of the MICP-reinforced adhesives, upon immersion, was significantly improved compared to biopolymer alone, and maintained their adhesive strength at moderate humidities and from below-freezing to room temperatures after 7- day exposures. To determine the effect of biopolymers on the nanoscale material properties of biomineral aggregates, enzymatically induced calcium carbonate precipitation was induced in the presence of a standard protein, Bovine Serum Albumin (BSA). Nanoindentation and Atomic Force Microscopy show that the moduli of the mineral precipitates were significantly lowered in the presence of BSA. Atomic force microscopy also showed that BSA introduced structural variations and moduli gradation in biominerals. These results demonstrate that the presence of a protein additive, specifically BSA, can alter the nanoscale structure and material properties of calcium carbonate precipitates. Using an organic additive to manipulate microscale material properties of biominerals offers possibilities for advanced control at the microscale and enhanced toughness at the macroscale for engineering applications such as in construction, binder, and adhesive applications.Item Algal biofilms and lipids: bicarbonate amendment and nitrate stress to stimulate lipid accumulation in algal biofilms(Montana State University - Bozeman, College of Engineering, 2022) Rathore, Muneeb Soban; Chairperson, Graduate Committee: Brent M. Peyton; This is a manuscript style paper that includes co-authored chapters.Algal biofuels are compounds obtained by transesterification of algal lipids to fatty acid methyl esters (FAMEs) which can be used as biodiesel. Algal biofilms have a potential for commercial applications of algal biomass for biofuel production and provide concentrated biomass requiring less water removal to reduce biofuel production costs. Lipid production in algal biofilms is low as compared to planktonic algal growth systems and strategies for enhancing lipid content in algal biofilms need to be developed. The overarching goal of the studies presented herein was to develop lipid accumulation strategies in algal biofilms using nutrient stresses to increase triacylglycerides (TAGs) and FAMEs. First, a reactor was designed for photoautotrophic biofilm growth incorporating a novel algal biomass harvesting mechanism. Chlorella vulgaris biofilm growth was demonstrated to establish the reactor characteristics under three different inorganic carbon regimes and the presence of excess calcium to facilitate biofilm attachment and accumulation. Excess calcium resulted in precipitate formation and increasing ash content in biomass and caused difficulty in biofilm detachment. However, the highest biomass accumulation was observed in the bicarbonate and the bicarbonate with calcium treatments. Second, two different algal strains were tested for lipid accumulation under two nutrient conditions: nitrate limitation and bicarbonate addition. Algal strains included, an extremophilic freshwater diatom RGd-1, a Yellowstone National Park (YNP) isolate, and oleaginous chlorophyte C. vulgaris. High bicarbonate content at low nitrate concentration in the bulk medium provided the highest lipid accumulation as determined by Nile Red fluorescence and Gas Chromatography Mass Spectrometry (GCMS) analysis of extracted FAMEs (7-22 % wt/wt). For prevention of biomass loss and quick response to nutrient stresses to stimulate lipid accumulation, the growth medium was exchanged after initial biofilm accumulation and operated in batch mode. This was implemented to quickly introduce nutrient stresses using fresh medium to vary bicarbonate and nitrate concentrations as needed. Thus, the work presented here demonstrated enhanced lipid production in algal biofilms with nitrate stress and bicarbonate amendment is a viable strategy to increase lipid accumulation. Increased lipid content may help offset the cost for biodiesel production with more lipid product and lower processing requirements for water removal.Item NMR characterization of unfrozen brine vein distribution and structure in frozen systems(Montana State University - Bozeman, College of Engineering, 2022) Lei, Peng; Chairperson, Graduate Committee: Sarah L. Codd; This is a manuscript style paper that includes co-authored chapters.The liquid vein network (LVN) that forms in the interface of ice crystals or particles exists in frozen porous media due to the freezing point depression. The distribution and structure of the LVNs are dynamic due to the ice recrystallization phenomenon. In ice alone, the LVNs formed by the ice crystal interfaces can be characterized as a porous medium in terms of surface to volume ratio (SV /) and the tortuosity (alpha).The presence of solid particles or ice-binding proteins (IBPs) make the frozen system much more complex. The research presented uses nuclear magnetic resonance (NMR) experimental techniques, including magnetic resonance imaging (MRI), relaxation and self-diffusion measurements, to study the development of the LVNs in complex frozen systems containing solid particles or IBPs. Poly-methyl methacrylate (PMMA) particles of diameters 0.4, 9.9, and 102.2 microns are used with brine solution concentrations of 15, 30, and 60 mM Magnesium chloride (MgCl 2) to simulate complex frozen systems. The dynamic rearrangement with time of LVNs can be studied as a function of temperature, MgCl 2 concentration, and PMMA particle size. The results indicate that small solid particles dominate the structure dynamics while in larger solid particle packed beds the solute effect dominates. This behavior is quantified by determination of SV / and alpha from NMR relaxation and diffusion data. Additionally, IBP produced from the V3519-10 organism isolated from the Vostok ice core in Antarctica is added to ice samples frozen from 30, 60 and 120 mM MgCl 2 solution to investigate its influence on LVNs over months of aging. The interplay of the solute and biological effects is complicated but it appears the biological effect is more pronounced at lower salt concentrations. The data provide a basis for eventual combination of salt, IBP and solid particulate studies. The result of MRI, relaxation and self-diffusion measurements indicate the inhibition of ice recrystallization as a function of particle size, MgCl 2 concentration and the presence of IBP. The non-invasive data presented along with calibration of the relaxation experiments with self-diffusion experiments, demonstrate the continued extension of NMR techniques developed from porous media to frozen porous media and ice LVN structure.Item Single cell encapsulation, detection, and sorting of Pseudomonas syringae using drop-based microfluidics(Montana State University - Bozeman, College of Engineering, 2023) Lindsay, Travis Carson; Chairperson, Graduate Committee: Abigail Richards; Connie Chang (co-chair)Bacteria can survive antibiotic or bactericidal treatment through genetic mutations. Even within bacterial populations that are fully susceptible to treatment, a small proportion of cells can have enhanced survival capacity in a phenomenon called persistence. Traditional microbiology methods can fail to identify or isolate these persister cells present within the population. A novel method for high-throughput single cell analyses of microbial populations is that of drop-based microfluidics, in which individual cells can be isolated within picoliter-sized drops. In this work, fluorescent detection and dielectrophoresis-based sorting of drops was developed for isolating Pseudomonas syringae persister cells following antimicrobial treatment. We demonstrate: (1) the dielectrophoresis-based sorting of dye-filled 25 micron drops based upon two colors, (2) differences between laser-induced fluorescent detection of dyes compared to single bacterial cells, (3) single-cell isolation of P. syringae into 25 micron droplets with ~10% of droplets containing singlecells, and (4) the treatment, staining, and fluorescent characterization of P. syringae at 0.5x, 5x, and 50x the minimum inhibitory concentration of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an antibiotic which resulted in 6.2%, 10.2%, and 88.6% cell death of the population, respectively. These results provide the groundwork for studying antibiotic-treated P. syringae and the isolation of surviving cells that will lend insight into the molecular basis of persistence for preventing recurrent infections and decreasing the likelihood of antibiotic resistance.