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Item Pseudomonad reverse carbon catabolite repression, interspecies metabolite exchange, and consortial division of labor(Springer Science and Business Media LLC, 2019-11) Park, Heejoon; McGill, S. Lee; Arnold, Adrienne D.; Carlson, Ross P.Microorganisms acquire energy and nutrients from dynamic environments, where substrates vary in both type and abundance. The regulatory system responsible for prioritizing preferred substrates is known as carbon catabolite repression (CCR). Two broad classes of CCR have been documented in the literature. The best described CCR strategy, referred to here as classic CCR (cCCR), has been experimentally and theoretically studied using model organisms such as Escherichia coli. cCCR phenotypes are often used to generalize universal strategies for fitness, sometimes incorrectly. For instance, extremely competitive microorganisms, such as Pseudomonads, which arguably have broader global distributions than E. coli, have achieved their success using metabolic strategies that are nearly opposite of cCCR. These organisms utilize a CCR strategy termed ‘reverse CCR’ (rCCR), because the order of preferred substrates is nearly reverse that of cCCR. rCCR phenotypes prefer organic acids over glucose, may or may not select preferred substrates to optimize growth rates, and do not allocate intracellular resources in a manner that produces an overflow metabolism. cCCR and rCCR have traditionally been interpreted from the perspective of monocultures, even though most microorganisms live in consortia. Here, we review the basic tenets of the two CCR strategies and consider these phenotypes from the perspective of resource acquisition in consortia, a scenario that surely influenced the evolution of cCCR and rCCR. For instance, cCCR and rCCR metabolism are near mirror images of each other; when considered from a consortium basis, the complementary properties of the two strategies can mitigate direct competition for energy and nutrients and instead establish cooperative division of labor.Item Urease immobilization for advancing enzyme-induced calcium carbonate precipitation applications(Montana State University - Bozeman, College of Engineering, 2019) Frieling, Zachary James; Chairperson, Graduate Committee: Robin Gerlach and Adrienne Phillips (co-chair)Microbially induced calcium carbonate precipitation (MICP) is a widely studied field of research exploiting bacterial activity to form a calcium carbonate precipitate that has been used to modify porous media. MICP is an enzymatically driven process and uses the enzyme urease to change solution chemistry to favor calcium carbonate precipitation. An enzyme slurry can be used in lieu of microbial growth and can be applied in a similar fashion and is commonly referred to as enzymatically induced calcium carbonate precipitation (EICP). For some applications temperature can stunt microbial growth and EICP may be the preferred method. However, as the temperature increases further the urease enzyme is thermally inactivated inhibiting calcium carbonate precipitation. Thermal inactivation limits the potential use of EICP in higher temperature environments. To combat thermal inactivation, immobilization of the urease enzyme through entrapment in silica gel and adsorption on an internally porous ceramic proppant was evaluated, and the first order inactivation coefficient (kd) was determined for temperatures between 60°C and 90°C. It was found that immobilization of the urease enzyme drastically reduced the apparent k d when compared to the free, non-immobilized form. Column experiments were performed using the urease immobilized on the ceramic proppant at room temperature (~23°C) and at 60°C. It was found that the immobilized urease retained high activity for the duration of the experiments even when subjected to the elevated temperature condition. The immobilized form of the urease enzyme was indeed protected from thermal degradation. It also seemed that the immobilized form of the urease enzyme was shielded from inactivation from active calcium carbonate precipitation, as observed in previous EICP and MICP experiments, in which ureolytic activity decreased rapidly as calcium carbonate precipitated. As a result, the immobilized form of the urease enzyme showed promise for advancing EICP applications.Item Investigation of field relevant parameters for microbially enhanced coalbed methane scale up(Montana State University - Bozeman, College of Engineering, 2019) Platt, George Addison; Chairperson, Graduate Committee: Robin Gerlach; K. J. Davis, E. P. Barnhart, M. W. Fields and R. Gerlach were co-authors of the article, 'Optimization of 13C-algae amendment concentration for enhanced coal dependent methanogenesis' submitted to the journal 'International journal of coal geology' which is contained within this thesis.; K. J. Davis, H. D. Schweitzer, H. J. Smith, E. P. Barnhart, M. W. Fields, R. Gerlach were co-authors of the article, 'Algal amendment enhances biogenic methane production from coals of different thermal maturity' submitted to the journal 'International journal of coal geology' which is contained within this thesis.Energy production from coal is projected to decline significantly over the next 30 years, due to concerns over anthropogenic carbon emissions, climate change, and cost. As coal-based energy production decreases, the demand for natural gas is expected to increase. Coalbed methane (CBM), a biogenic natural gas resource found in subsurface coal beds, may aid in meeting the projected increase in demand. However, costs associated with traditional CBM extraction currently make utilizing this resource economically prohibitive due to slow coal-to-methane conversion rates and the necessity to treat co-produced water. Algae can be cultivated in co-produced formation water and the addition of very small amounts of this algal biomass can increase coal-to-methane conversion rates. The goal of this work was to determine the optimal algae amendment concentration for the enhancement of microbial coal-to-methane conversion to maximize return on investment. Concentrations of 13C-labeled algae amendment ranging from 0.01-0.50 g/L (equivalent to 0.0001-0.005 g per g of coal) were tested in coal-containing batch microcosms. Enhanced methane production was observed in all amended microcosms and maximum methane production occurred between 169-203 days earlier than in unamended microcosms. When as little as 0.01 g/L algae amendment was added, 13CH 4 and 12CH 4 tracking revealed that the improvement in coal-to-methane conversion kinetics was due to enhanced coal degradation. Increasing amendment concentrations to 0.05-0.50 g/L improved coal-to-methane conversion rates further, but improvements from amendment concentrations above 0.05 g/L were insignificant. The geologic scope of this CBM enhancement strategy was investigated by studying methane production from five coals ranging in thermal maturity. Biogenic methane was produced from all coals, with subbituminous coals generally producing more methane than thermally mature bituminous coals. The addition of algae amendment to thermally mature coal microcosms resulted in methane production that was comparable to production from unamended, thermally immature coals. This improvement was associated with an increased relative abundance of coal degrading microorganisms. Collectively, this work demonstrates that algae amendment concentrations can be reduced further (to 0.01-0.05 g/L) relative to the previously investigated concentrations (ranging from 0.1-0.5 g/L) and still improve coal-to-methane conversion rates for a range of coal sources.Item Spatiotemporal mapping of oxygen in model porous media biofilms using 19 F magnetic resonance oximetry(Montana State University - Bozeman, College of Engineering, 2019) Simkins, Jeffrey William; Chairperson, Graduate Committee: Philip S. Stewart and Joseph D. Seymour (co-chair); Philip S. Stewart and Joseph D. Seymour were co-authors of the article, 'Spatiotemporal mapping of oxygen in a microbially-impacted packed bed using 19 F nuclear magnetic resonance oximetry' in the journal 'The journal of magnetic resonance' which is contained within this dissertation.; Philip S. Stewart, Sarah L. Codd and Joseph D. Seymour were co-authors of the article, 'Non-invasive imaging of oxygen concentration in a complex in vitro biofilm infection model using 19 F MRI: persistence of an oxygen sink despite prolonged antibiotic therapy' submitted to the journal 'Magnetic resonance in medicine' which is contained within this dissertation.; Philip S. Stewart and Joseph D. Seymour were co-authors of the article, 'Microbial growth rates and local external mass transfer resistance in a porous bed biofilm system measured by 19 F magnetic resonance imaging of structure, oxygen concentration, and flow velocity' submitted to the journal 'Biotechnology and bioengineering' which is contained within this dissertation.Biofilms, microbial aggregates anchored to a surface using a sticky matrix of metabolic products called extracellular polymeric substances (EPS), are the dominant form of bacterial life and are widespread in nature, from glaciers to hot springs. The transition from the planktonic state to a biofilm is associated with striking changes to microbial phenotype which confer unique, biofilm-specific properties to resident cells that have important implications for medicine, industry, and environmental study. Many of these properties are caused in large part by oxygen transport limitation, which arises due to restriction of fluid flow in cell aggregates and consumption of oxygen for respiration. The balance of reactive and diffusive processes establishes strong spatial gradients in oxygen concentration which lead to profound spatial heterogeneity in bacterial species composition, growth yield, antimicrobial susceptibility, and reaction kinetics, among other traits. However, despite the importance of oxygen gradients in a host of highly-relevant biofilm phenomena, quantification of oxygen profiles in biofilms is difficult, both in the field and the lab, with the gold standard of measurement, the microelectrode, having significant limitations. 19 F Nuclear Magnetic Resonance (NMR) oximetry, a magnetic resonance-based technique for oxygen quantification that has been used to characterize oxygen usage in blood tissues and tumors, exploits the linear dependence of spin-lattice relaxation rate R 1 on local oxygen partial pressure for fluorine nuclei in perfluorocarbon (PFC) phases. In the current work, we apply 19 F NMR oximetry to a model packed bed biofilm system to generate novel insights into microbial oxygen usage and to introduce a complimentary oximetry tool for biofilm experimenters. We develop methodology for the introduction and fixation of a fluorinated oxygen sensor to facilitate long-term oxygen monitoring. We use 19 F oxygen distribution measurements in compliment to traditional NMR methods to correlate fluid flow with growth rate, generate spatial maps of oxygen utilization rate, identify differences in oxygen utilization behavior between different species, characterize infection persistence during antibiotic therapy, mathematically model macroscale oxygen sink development, and quantify local mass transfer phenomena.Item Gastrointestinal organoid structure and transport(Montana State University - Bozeman, College of Engineering, 2019) Sidar, Barkan; Chairperson, Graduate Committee: James Wilking; Thomas A. Sebrell was an author and Rachel Bruns, Royce A. Wilkinson, Blake Wiedenheft, Paul J. Taylor, Brian A. Perrino, Linda C. Samuelson, James N. Wilking and Diane Bimczok were co-authors of the article, 'Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation, and fusion events' in the journal 'Cell and tissue research' which is contained within this dissertation.; Thomas A. Sebrell, Bengisu Kilic, David Brown, Mert Aytac, Brian A. Perrino, Linda C. Samuelson, Henry Fu, Diane Bimzcok, James N. Wilking were co-authors of the article, 'Rupturing of human gastric organoids' which is contained within this dissertation.; Brittany R. Jenkins, Sha Huang, Jason R. Spence, Seth T. Walk and James N. Wilking were co-authors of the article, 'Flow through human intestinal organoids with the gut organoid flow chip (GOFlowChip)' submitted to the journal 'Lab on a Chip' which is contained within this dissertation.; Dissertation contains two articles of which Barkan Sidar is not the main author.Organoids are three-dimensional (3D) self-assembled, mammalian tissue cultures derived from stem cells that differentiate to contain multiple cell types. These cells spatially organize within the 3D structure and are capable of recapitulating the structure and function of a particular organ. Organoids offer a variety of existing and potential applications in medicine and biotechnology, including drug formulation testing, regenerative medicine, and microbiome research. Despite their value, knowledge of how organoid structure impacts dynamics, mechanics, and transport is lacking. This is particularly true for gastrointestinal organoids, which are composed of a monolayer-thick epithelial sheet wrapped into a closed sphere. The primary goals of this dissertation are to understand the impact of gastrointestinal organoid structure on organoid function, develop a millifluidic chip platform to improve their viability and reliability as a model system and to explore their uses as model co-culture systems. To achieve this, we use a combination of time-lapse microscopy, image analysis, modeling, and fluidics fabrication techniques to develop an understanding of organoid growth and development in addition to expanding current uses as model systems. Our observations revealed that human gastric organoid growth was associated with cyclic rupture of the epithelial shell, rotational movement around their axes within the Matrigel matrix and luminal fusion by adjacent organoids. Furthermore, the rupture events are an indirect result of osmotic swelling carried out by the diffusion of water due to osmolyte concentration regulation by the epithelial shell. To overcome the advection limitation due to the topologically closed spherical structure of the organoids, we developed a millifluidic device called the Gut Organoid Flow Chip (GOFlowChip). This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid. Given that organoids show great potential as model systems, established co-culture system consisting of dendritic cells (DC) with infected human gastric organoids shows the gastric epithelium actively recruits DCs for immunosurveillance with increased recruitment upon active Helicobacter pylori infection. Finally, investigation on CD103 attachment protein in gastric DCs revealed that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin but is not a significant driver of DC adhesion to gastrointestinal epithelia.Item The effect of solvent polarity on autocatalytic furfural production confirmed by multivariate statistical analysis(2019-10) Romo, Joelle E.; Miller, Kyle C.; Sundsted, Tara L.; Job, Adam L.; Hoo, Karlene A.; Wettstein, Stephanie G.Autocatalytic dehydration of xylose to furfural was studied in pure aqueous and monophasic organic/water mixtures to determine the effect reaction media and conditions have on conversion and yield. This study identified that the severity (Ro) of the reaction and polarity, as determined by the Hansen Solubility Parameter, δP, strongly correlate with xylose conversion and furfural yield. Increasing the Ro and δP increased both conversion and yield in pure aqueous and organic/water mixtures of sulfolane, γ‐butyrolactone, γ‐valerolactone, γ‐hexalactone, and tetrahydrofuran. Additionally, it was found that at a specified Ro and δP, similar conversions and yields were achieved using different combinations of time, temperature, and solvent mixture. Using principal component analysis and projection to latent structures, a semi‐empirical model was developed that provided estimates of xylose conversion and furfural yield over a range of experimental Ro and δP values.Item Nisin penetration and efficacy against Staphylococcus aureus biofilms under continuous-flow conditions(2019-07) Godoy-Santos, Fernanda; Pitts, Betsey; Stewart, Philip S.; Mantovani, Hilario C.Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus . Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.Item DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology(2019-09) Pratt, Shawna L.; Zath, Geoffrey K.; Williamson, Kelly S.; Franklin, Michael J.; Chang, Connie B.The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 μm diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (DropStabilization On AChip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Δhpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.Item Janthinobacterium CG23_2: comparative genome analysis reveals enhanced environmental sensing and transcriptional regulation for adaptation to life in an Antarctic supraglacial stream(2019-10) Dieser, Markus; Smith, Heidi J.; Ramaraj, Thiruvarangan; Foreman, Christine M.As many bacteria detected in Antarctic environments are neither true psychrophiles nor endemic species, their proliferation in spite of environmental extremes gives rise to genome adaptations. Janthinobacterium sp. CG23_2 is a bacterial isolate from the Cotton Glacier stream, Antarctica. To understand how Janthinobacterium sp. CG23_2 has adapted to its environment, we investigated its genomic traits in comparison to genomes of 35 published Janthinobacterium species. While we hypothesized that genome shrinkage and specialization to narrow ecological niches would be energetically favorable for dwelling in an ephemeral Antarctic stream, the genome of Janthinobacterium sp. CG23_2 was on average 1.7 ± 0.6 Mb larger and predicted 1411 ± 499 more coding sequences compared to the other Janthinobacterium spp. Putatively identified horizontal gene transfer events contributed 0.92 Mb to the genome size expansion of Janthinobacterium sp. CG23_2. Genes with high copy numbers in the species-specific accessory genome of Janthinobacterium sp. CG23_2 were associated with environmental sensing, locomotion, response and transcriptional regulation, stress response, and mobile elements—functional categories which also showed molecular adaptation to cold. Our data suggest that genome plasticity and the abundant complementary genes for sensing and responding to the extracellular environment supported the adaptation of Janthinobacterium sp. CG23_2 to this extreme environment.Item Sodium bicarbonate amendment for enhanced astaxanthin production from Haematococcus pluvialis(Montana State University - Bozeman, College of Engineering, 2019) Erturk, Berrak; Chairperson, Graduate Committee: Brent M. Peyton; Christian Lewis and Brent M. Peyton were co-authors of the article, 'Sodium bicarbonate amendment for enhanced astaxanthin production from Haematococcus pluvialis' submitted to the journal 'Algal research' which is contained within this thesis.Haematococcus pluvialis is a freshwater green microalga that is widely considered to be the richest natural source of the high value carotenoid astaxanthin. The use of bicarbonate salts as a means of efficiently delivering inorganic carbon in microalgal cultivation is a relatively new concept and its application is continuously growing. Previous studies have largely focused on increasing the lipid content in microalgae via the use of high concentrations of sodium bicarbonate under nitrogen deplete culture conditions. Lipid accumulation is directly related to astaxanthin production as astaxanthin is dissolved and stored in lipid bodies in H. pluvialis. Because of this relationship in H. pluvialis, the effects of sodium bicarbonate addition on astaxanthin production was investigated in this study. Due to its complex life cycle, H. pluvialis is commonly cultivated in two stages called the 'green' and 'red' stage. Different approaches have been proposed in each stage to increase the astaxanthin production, namely by growing microalgae under nutrient-limited conditions or resuspending the cells into nutrient deplete conditions. In this study, H. pluvialis (UTEX 2505) was cultivated in stirred (120 rpm) batch reactors containing MES-Volvox medium with a 12 h:12 h light/dark cycle. Sodium bicarbonate (2.5 mM) was used as an additional inorganic carbon source in the green stage and 50 mM of sodium bicarbonate was used as a trigger mechanism to induce astaxanthin production in the red stage. Following the trigger, the astaxanthin accumulation rate increased from 0.13 mg L ^-1 day ^-1 to 0.64 mg L ^-1 day ^-1 with an astaxanthin concentration of 1.56 + or - 0.01 mg L ^-1 and 3.95 + or - 1.25 mg L ^-1 respectively. Whereas, an addition of 2.5 mM sodium bicarbonate at the green stage increased the final astaxanthin accumulation rate up to 2.12 mg L ^-1 day ^-1 and the astaxanthin concentration to 11.2 + or - 0.56 mg L ^-1. Increasing biomass in the green stage resulted in higher astaxanthin content at the end of the red stage. In addition to increasing the total astaxanthin content, 2.5 mM of sodium bicarbonate led to faster nitrogen utilization during the green stage. With this faster utilization of nitrogen, the cultures were grown with a one-stage cultivation approach, where the astaxanthin production occurred in continuous mode.