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Item An experimental study of drying in porous media in novel 2D micromodels with dual porosity(Montana State University - Bozeman, College of Engineering, 2024) Habib, Md Ahsan; Chairperson, Graduate Committee: Yaofa LiDrying of porous media is pervasive in numerous natural and engineering processes, such as oil recovery, CO 2 storage, and critical zone science. Drying is essentially a multiphase flow process, where the liquid phase evaporates and is displaced/replaced by the gaseous phases, as vapor diffuses out of the porous structure. In terms of pore structure and other physical characteristics like porosity and permeability, many porous matrices exhibit multi-scale heterogeneity. For instance, in critical zone, soil is often viewed as a hierarchical organization: primary particles form aggregates, which in turn form macroaggregates, effectively leading to a dual-porosity medium. Numerous activities, including gases and water transport, are known to be controlled by the resultant multiscale flow dynamics and inter-/intra-aggregate interaction during drying. However, the fundamental physics underlying drying of porous media with dual porosity is not well understood from a fluid mechanics perspective. In this work, a novel 2D microfluidic device fabrication technique has been developed. To study the multi-phase flow of air and water, emphasizing the multi-scale interaction, pore structure, and role of film flows, three distinct types of microfluidic devices have been fabricated, which bear the innovative three-layer glass-silicon- glass architecture, providing precise structural control and excellent optical access from both top and bottom. An innovative dual-magnification imaging technique has been introduced adapted for micro-PIV and epi-fluorescent microscopy which offers insightful information about the flow dynamics at both the micro- and macro-scales concurrently. In this thesis, two distinct types of experiments are outlined, focusing on diffusion-driven drying and flow-through drying, utilizing three diverse micromodels characterized by varying porous structures and distributions. The experimental results have presented the overall drying dynamics observed in different micromodels, each featuring unique porous configurations. The impact of porous geometry and external flow conditions on drying rate and associated pore-scale physics is thoroughly examined. The findings encompass a comprehensive overview of micro-macro pore interactions, as evidenced by separated saturation distribution, displacement rates, and other pertinent flow parameters. The findings have reflected the influence of pore geometry, distribution, hydraulic connectivity, and film flow on the observed effects.Item Experimental characterization of pore-scale capillary pressure and corner film flow in 2D porous micromodels(Montana State University - Bozeman, College of Engineering, 2023) Molla, Razin Sazzad; Chairperson, Graduate Committee: Yaofa LiMultiphase flow in porous media is ubiquitous in natural and engineering processes. A better understanding of the underlying pore-scale physics is crucial to effectively guiding, predicting and improving these applications. Traditional models describe multiphase flows in porous media based on empirical constitutive relations (e.g., capillary pressure vs. saturation), which, however, are known to be hysteretic. It has been theoretically shown that the hysteresis can be mitigated by adding new variables in the functional form. However, experiments are still needed to validate and further develop the theories. In particular, our understanding of capillary pressure characterization and numerous pore-scale mechanisms is still limited. For instance, during capillary pressure measurement, fluid phases become disconnected, making the bulk pressure an inaccurate measure for the actual capillary pressure. In a strongly wetting medium, wetting phase always remains connected by corner films, through which trapped water continues to drain until a capillary equilibrium is reached, but the effects of corner film flow are minimally characterized. In this thesis, two different experiments are presented. In the first experiment, we focused on the capillary pressure characterization and the effect of measurement resolution. Microscopic capillary pressure along with other geometric measures are characterized during drainage and imbibition. By strategically varying the pressure at the boundary, different equilibrium states were achieved and imaged at four different magnifications (i.e., 2, 1.25, 0.5, 0.25 micron/pixel). In the second experiment, we for the first time characterized the corner film flow again during drainage and imbibition condition employing particle image velocimetry. Overall, our results suggest that the calculated macroscale pressure P c and the bulk pressure drop agree reasonably well when only interfaces associated with the connected phases are considered. A spatial resolution of 2 micron/pixel seems to sufficiently resolve the interface, and further increasing the resolution does not have a significant impact on the results. Additionally, corner film flow was found to be an active transport mechanism. During drainage, trapped water is continuously drained over time via thin film, whereas during imbibition snap-off events are enhanced by wetting films. These observations call for future studies to carefully treat corner film flows when developing new predictive models.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 Development of drop-based microfluidic methods for high-throughput biological assays(Montana State University - Bozeman, College of Engineering, 2021) Zath, Geoffrey Kane; Chairperson, Graduate Committee: Connie Chang; This is a manuscript style paper that includes co-authored chapters.Drop-based microfluidics allows single-cell biological assays to be performed by encapsulating samples in picoliter scale drops. Adapting biological assays to drop-based microfluidics requires novel approaches to meet the method requirements of each assay. For example, microtiter plates are a common tool for storing many unique samples in some assays. An equivalent strategy for drops involves labeling samples with a barcode prior to drop encapsulation and storing the barcoded drops in a single mixture, thereby creating a drop library. Other assay adaptions, such as drop-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) require that drops be stabilized during the high temperatures used for thermal cycling. Drop-based RT-qPCR is useful for studying single-cell dynamics in drops, such as influenza A virus (IAV) infection. Conventional methods for measuring IAV output from individual cells are labor intensive and low-throughput. Thus, there is a need to adapt RT-qPCR to drop-based microfluidics for the purpose of high-throughput single cell analysis of infected cells. The research presented here focuses on the characterization of the Pressure Cooker Chip (PCC) to rapidly encapsulate drop libraries and the development of a drop-based RT-qPCR method to measure IAV output from infected cells. The PCC was used to make drop libraries by rapidly generating drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard microtiter well plate. The drop library was optically barcoded using a two-color combination of fluorescent microbeads or quantum dots with 24 or 192 unique combinations, respectively. To adapt RT-qPCR in drops, known PCR additives were systematically tested to optimize drop stability and limit dye diffusion during thermocycling. A novel qPCR data analysis method was developed to convert drop fluorescence data collected at a single thermocycle to an initial RNA template concentration. Together, the additive screening and novel qPCR data analsyis method enabled the use of drop-based RT-qPCR to quantify the highly heterogeneous IAV burst size from single cells in thousands of drops. Our method is the first to measure single cell IAV burst size using a high-throughput, drop-based RT-qPCR assay.Item Design and fabrication of membrane-based pressure sensor for capillary pressure measurement in micromodels(Montana State University - Bozeman, College of Engineering, 2021) Raventhiran, Nishagar; Chairperson, Graduate Committee: Yaofa LiPressure is a fundamental quantity in virtually all problems in fluid dynamics from macro-scale to micro/nano scale flows. Although technologies are well developed for its measurement at the macro-scale, pressure quantification at the microscale is still not trivial. Yet, precise pressure mapping at microscale such as in microfluidics is imperative in a variety of applications, including porous media flows and biomedical engineering. In particular, pore-scale capillary pressure is a defining variable in multiphase flow in porous media and has rarely been directly measured. To that end, this study aims to design and fabricate an on-chip sensor that enables quantification of capillary pressure in microfluidic porous media, called micromodels. The micromodel is fabricated in polydimethylsiloxane (PDMS) using soft lithography with a thin membrane incorporated that deflects with pressure variations in the fluid flow. Employing a microscope coupled with a high-speed camera and the astigmatism particle tracking principle, precise pressure measurement is achieved with an accuracy of ~ 60Pa. This sensor is then applied to characterize the viscous pressure drop in single phase flows, and the capillary pressure in a water-air multiphase in microchannels, and good agreement is obtained between the sensor measurement, theoretical values and measurements employing a commercial pressure transducer. This thesis provides a novel method for in-situ quantification of local pressure and potentially 2D pressure field in microfluidics and thus opens the door to a renewed understanding of pore-scale physics of multiphase flow in porous media.Item Microbially induced calcium carbonate precipitation: meso-scale optimization and micro-scale characterization(Montana State University - Bozeman, College of Engineering, 2020) Zambare, Neerja Milind; Chairperson, Graduate Committee: Robin Gerlach and Ellen G. Lauchnor (co-chair); Ellen Lauchnor and Robin Gerlach were co-authors of the article, 'Controlling the distribution of microbially precipitated calcium carbonate in radial flow environments' in the journal 'Environmental science and technology' which is contained within this dissertation.; Robin Gerlach and Ellen Lauchnor were co-authors of the article, 'Spatio-temporal dynamics of strontium partitioning with microbially induced calcium carbonate precipitation in porous media flow cells' submitted to the journal 'Environmental science & technology' which is contained within this dissertation.; Robin Gerlach and Ellen Lauchnor were co-authors of the article, 'Co-precipitation of strontium and barium' submitted to the journal 'Environmental science & technology' which is contained within this dissertation.; Nada Naser, Robin Gerlach and Connie Chang were co-authors of the article, 'Visualizing microbially induced mineral precipitation from single cells using drop-based microfluidics' submitted to the journal 'Nature methods' which is contained within this dissertation.Microorganisms have the potential to impact processes on a scale orders of magnitude larger than their size. For example, microbe-mineral interactions at the micro-scale can drive macro-scale processes such as rock formation and weathering. Many bioremediation technologies derive inspiration from microbial mineralization processes. Microbially induced calcium carbonate precipitation (MICP) can produce calcium carbonate (CaCO 3) precipitates which can be utilized as a biological cement to strengthen porous media by reducing fluid permeability in subsurface fractures or as an immobilization matrix to remove metal contaminants dissolved in groundwater. To make MICP a feasible and successful bioremediation technology in the world outside the lab, it is necessary to bridge the gap between the meso-scale research studies and macro-scale applications. This thesis focuses on such meso-scale studies but also contributes to bridging the gap in the other direction, i.e., meso-scale to micro-scale to gain a fundamental understanding of the cellular level processes behind MICP. The research presented here investigates two applications of MICP with a focus on controlling precipitate distribution and process efficiency in target environments. Subsurface precipitate distribution and metal partitioning during MICP were studied in novel reactive transport systems that mimic application-environment conditions. A radial flow reactor was used to study the spatial distribution of precipitates in conditions similar to subsurface injection well environments. The distribution and degree of metal partitioning during MICP was investigated in batch reactors and porous media flow cells to study kinetics and reactive transport effects on kinetics. In the radial flow environment, more precipitates formed away from the center injection zone. Results showed that longer reactant residence times and an equimolar ratio of calcium to urea were able to maximize precipitation efficiency. Metal partitioning could be maximized at low reactant flow rates and low metal concentrations. The novel flow cell set up used revealed a spatial decoupling between ureolysis and precipitation. A micro-scale investigation of the fundamental MICP process itself is presented wherein microbe-mineral interactions are observed at the cell level. A semi-correlative approach to investigating individual precipitates in microdroplets is presented, using a multitude of microscopy and microanalysis techniques. The presented studies capture MICP across a range of scales.