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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.Item Microgels for single-cell culturing of neurons and chondrocytes(Montana State University - Bozeman, College of Engineering, 2023) Fredrikson, Jacob Preston; Chairperson, Graduate Committee: Abigail Richards; This is a manuscript style paper that includes co-authored chapters.Tissue engineering is a multidisciplinary field that combines engineering and life sciences to restore, improve, or generate biological substitutes to replace damaged tissues or organs. This is often performed using hydrogels that serve as scaffolds for the growth and maintenance of target tissues. Hydrogels, crosslinked polymer networks composed primarily of water, are excellent tissue mimics with highly tunable mechanical and biochemical properties. Hydrogels can be fabricated at the microscale, termed microgels, using drop-based microfluidics, which enables the precise control of cell density within the microgels down to a single cell. Encapsulating cells in microgels allows for the manipulation of microgels after production for single cell analyses. In this dissertation, human articular cartilage (HAC) cells and neurons are cultured within and upon microgel particles that serve as microscale tissue models for the study of chondrocyte matrix production and Herpes Simplex Virus type -1 (HSV-1) infection studies. HAC is the load-bearing tissue that lines the interfaces of joints and is responsible for shock and wear resistance. Chondrocytes, the cells in HAC, are responsible for producing and maintaining HAC. The chondrocyte pericellular matrix (PCM) regulates the metabolism and mechanical strain of the cells, which is critical to cellular function and cartilage homeostasis. However, the PCM is challenging to produce in vitro. The first half of this work applies microgels for PCM formation in chondrocytes. Immunofluorescence and high-performance liquid chromatography-mass spectrometry data demonstrate that chondrocytes grown in alginate microgels form a collagen VI-rich PCM, significantly altering the cells' metabolic response to dynamic compression. Atomic force microscopy data demonstrates that when chondrocytes are grown in alginate microgels for ten days, the elastic modulus of the PCM increases an order of magnitude. HSV-1 is a human pathogen that invades the peripheral nervous system. Understanding the complexities of HSV-1 infection at the single-cell level could lead to better therapeutics and reduced disease outcomes. Drop-based microfluidics (DBM) has recently been adapted for studying single-cell viral infection but has not been applied to neurons and HSV-1. The second half of this work develops a method for growing individual neurons in microgels. These microgel-embedded neurons are isolated, encapsulated with precise inoculating doses of HSV-1 using DBM, and the kinetics of viral gene expression are tracked in individual neurons using a fluorescent-recombinant HSV-1 virus. The data demonstrate that microgels provide a solid scaffold for neuronal development that supports single-cell productive HSV-1 infection within droplets.Item Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels(Wiley, 2023-09) Fredrikson, Jacob P.; Brahmachary, Priyanka P.; June, Ronald K.; Cox, Lewis M.; Chang, Connie B.One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.Item Simulation of catalase-dependent tolerance of microbial biofilm to hydrogen peroxide with a biofilm computer model(Springer Science and Business Media LLC, 2023-08) Stewart, Philip S.; Owkes, MarkHydrogen peroxide (HP) is a common disinfectant and antiseptic. When applied to a biofilm, it may be expected that the top layer of the biofilm would be killed by HP, the HP would penetrate further, and eventually eradicate the entire biofilm. However, using the Biofilm.jl computer model, we demonstrate a mechanism by which the biofilm can persist, and even become thicker, in the indefinite treatment with an HP solution at concentrations that are lethal to planktonic microorganisms. This surprising result is found to be dependent on the neutralization of HP by dead biomass, which provides protection for living biomass deeper within the biofilm. Practically, to control a biofilm, this result leads to the concept of treating with an HP dose exceeding a critical threshold concentration rather than a sustained, lower-concentration treatment.Item Colloids, diagnostics, and 3D-printed hydrogels(Montana State University - Bozeman, College of Engineering, 2021) LeFevre, Thomas Brian; Chairperson, Graduate Committee: James Wilking; This is a manuscript style paper that includes co-authored chapters.Colloidal suspensions are dispersions of microscopic particles in liquid. Their properties have broad impacts in industry, medicine, and biology. In Chapter 2, we focus on measuring the interactions between colloidal particles suspended in water and a glass surface. We measure these interactions using a custom-built fluorescence centrifuge force microscope (F-CFM). This is the first CFM built with fluorescence capability, the first CFM used to measure colloidal interaction forces, and the first CFM capable of operating at speeds above 2000 RPM - and up to 5000 RPM - in a centrifuge. The F-CFM enables colloidal scale objects to be discriminated by fluorescence, which opens potential applications for biological samples that fluoresce under different phenotypic states. In Chapter 3, we focus on designing a point-of-care (POC) saliva collection, metering, and mixing system for detecting viral pathogens. The device was designed for the specific purpose of testing for the presence of SARS-CoV-2 in saliva using molecular amplification methods but could be applied to any pathogen whose constituents can be detected in saliva. The design to prioritizes ease of use, low cost, and scalability in order to facilitate massively widespread testing, which was absent during the first years since the emergence of SARS-CoV-2, In Chapter 4, we describe a method of formulating and printing hydrogel resins with high resolution channels using light-based 3D printing. In Chapter 5, we describe a leak-resistant, pressurized connector platform for connecting modular hydrogels that can be used to create complex assemblies of hydrogel components. In Chapter 6, we describe a microscope sample temperature control platform that fits into standard upright microscope stages in order to heat and cool samples in a controlled manner under the microscope in order to observe temperature dependent reactions like polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). In Chapter 7, we describe a LAMP formulation that can be used to detect the presence of SARS-CoV-2 RNA in saliva despite the inhibitory components present in saliva and demonstrate its comparable accuracy to the gold standard of pathogenic testing: nasopharyngeal PCR testing.