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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 Biofilm distribution in a porous medium environment emulating shallow subsurface conditions(Montana State University - Bozeman, College of Engineering, 2021) Massey, KaeLee Frances; Chairperson, Graduate Committee: Matthew Fields; Heidi J. Smith, Al B. Cunningham, Hannah Dreesbach, Luke J. McKay, Yupeng Fan, Ying Fu, Joy D. Van Nostrand, Jizhong Zhou, Katie F. Walker, Terry C. Hazen and Matthew W. Fields were co-authors of the article, 'Biofilm distribution in a porous medium reactor emulating shallow subsurface conditions' which is contained within this thesis.Microorganisms in the terrestrial subsurface play important roles in nutrient cycling and degradation of anthropogenic contaminants, functions essential to the maintenance of healthy aquifers. Microorganisms have the potential to change the geochemical properties of the shallow terrestrial subsurface, and previous studies have uncovered significant roles microorganisms can play in groundwater processes, such as biogeochemical cycling. Much of the attention given to the shallow terrestrial subsurface has been focused on the effects of contamination and how microorganisms function in these systems, with far less emphasis on understanding how hydraulic properties influence subsurface microbial ecology. To fully understand how environmental factors impact microbial community dynamics, interactions, succession, colonization, and dispersal in the shallow subsurface environment it is essential to understand the link between microbiology and hydrology. In this thesis, an up-flow packed bed reactor (PBR) was designed to emulate select field conditions (i.e., flow rate and particle size) observed at the Oak Ridge National Laboratory-Field Research Center (ORNL-FRC) to observe how environmental factors influences metabolic activity, community establishment, and cell distribution in a micropore environment. Furthermore, we developed methods to visualize the localization of active and non-active cells within the porous medium. The goals of this thesis were to 1) understand how environmental variables impact distribution and metabolic activity of microbial cells in the soil pore microenvironment at the FRC using native sediment bug trap material, 2) evaluate the hydraulic properties of the presented up-flow packed bed reactor (PBR), 3) observe how inert, non-charged particles distribute in a porous media environment, and 4) observe the biofilm distribution a microorganism isolated from the ORNL-FRC using different inoculation strategies. Overall, the data demonstrates that the presented reactor system accurately emulates field conditions and environmental factors (pH, particle size, average pore velocity) and the distribution of cells in ex situ conditions. The results of this thesis have implications for elucidating the impacts of environmental factors on metabolic activity and cell distribution in a field relevant reactor system.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 Investigation of a control strategy for manipulation and prevention of Pseudomonas aeruginosa PAO1 biofilms in metalworking fluids(Montana State University - Bozeman, College of Engineering, 2018) Ozcan, Safiye Selen; Chairperson, Graduate Committee: Christine Foreman; Markus Dieser, Albert E. Parker, Narayanaganesh Balasubramanian and Christine M. Foreman were co-authors of the article, 'Quorum sensing inhibition as a promissing method to control biofilm growth in metalworking fluids' submitted to the journal 'Environmental science & technology' which is contained within this thesis.Microbial contamination in metalworking fluid (MWF) circulation systems is a serious problem. Particularly water based MWFs promote microbial colonization despite the use of biocides. Inhibiting the quorum sensing mechanism (i.e. cell-cell communication) in bacteria is a promising approach to control and prevent biofilm formation. The objective of this study was (i) to determine the microbial community in MWFs from operational machining shops, (ii) to investigate the effect of well-known quorum sensing inhibitors on controlling biofilm formation, and (iii) to implement experimental data from selected enzymes to a computer simulation biofilm accumulation model (BAM). Planktonic and biofilm samples from two local machining shops in Bozeman, MT, were collected to determine the extent of microbial colonization. In both operations, microbial communities were dominated by Pseudomonadales (60.2-99.7%). Rapid recolonization was observed even after dumping spent MWFs and cleaning. Considering the dominance of Pseudomonadales in MWFs, the model organism Pseudomonas aeruginosa PAO1 was selected for testing the effects of quorum sensing inhibitor compounds on biofilm formation. From a variety of enzymes, natural, and chemical compounds screened for quorum sensing inhibition, Patulin (40microns) and Furanone C-30 (75microns), were found to be effective in reducing biofilm formation in MWFs when applied as single compound amendments and in combination with the polysaccharide degrading enzyme alpha-amylase from Bacillus amyloliquefaciens. Particularly Furanone C-30, as a single amendment and in combination with alpha-amylase decreased biofilm formation by 76% and 82% after 48 hours. Putatively identified homoserine lactones in MWFs treated with Furanone C-30 provided evidence for quorum sensing inhibition on biofilm formation. BAM was employed to study the effect of alpha-amylase (3 Units mL -1) on P. aeruginosa PAO1 biofilms in batch reactors for 24 and 48 hours. In the absence of alpha-amylase, biofilm thickness was predicted to be 23.11 and 31.37 microns, while its presence reduced thickness to 10.47 and 13.07 microns after 24 and 48 hours, respectively. The results presented herein highlight the potential effectiveness of quorum sensing inhibition as a strategy to reduce biofilms in MWFs.Item Nuclear magnetic resonance studies of biofilm - porous media systems(Montana State University - Bozeman, College of Engineering, 2017) Kirkland, Catherine Mullinnix; Chairperson, Graduate Committee: Sarah L. Codd; Joseph D. Seymour (co-chair); Sarah L. Codd was a co-author of the article, 'Low-field borehole NMR applications in the near subsurface environment' submitted to the journal 'Vadose zone journal' which is contained within this thesis.; Randy Hiebert, Adrienne Phillips, Elliot Grunewald, David O. Walsh, Joseph D. Seymour and Sarah L. Codd were co-authors of the article, 'Biofilm detection in a model well-bore environment using low-field NMR' in the journal 'Groundwater monitoring and remediation' which is contained within this thesis.; Maria P. Herrling, Randy Hiebert, Andrew T. Bender, Elliot Grunewald, David O. Walsh and Sarah L. Codd were co-authors of the article, 'In-situ detection of subsurface biofilm using low-field NMR - a field study' in the journal 'Environmental science and technology' which is contained within this thesis.; Sam Zanetti, Elliot Grunewald, David O. Walsh, Sarah L. Codd and Adrienne J. Phillips were co-authors of the article, 'Detecting microbially-induced calcite precipitation (MICP) in a model well-bore using downhole low-field NMR' in the journal 'Environmental science and technology' which is contained within this thesis.; Jessica Weisbrodt, Catherine M. Kirkland, Nathan H. Williamson, Susanne Lackner, Sarah L. Codd, Joseph D. Seymour, Gisela Guthausen and Harald Horn were co-authors of the article, 'NMR investigation of water diffusion in different biofilm structures' submitted to the journal 'Biotechnology and bioengineering' which is contained within this thesis.Nuclear magnetic resonance (NMR) allows for in-situ non-invasive studies of opaque systems over a wide range of length and time scales, making the method uniquely suited to studies of biofilms and porous media. The research comprising this thesis uses NMR to explore biophysical, chemical, and transport properties within heterogeneous porous media systems at both a macro- and micro-scale. The macro-scale projects validate a low-field borehole NMR instrument to monitor field-scale environmental engineering applications like subsurface biofilms and microbially-induced calcite precipitation (MICP). Subsurface biofilms are central to bioremediation of chemical contaminants in soil and groundwater whereby micro-organisms degrade or sequester environmental pollutants like nitrate, hydrocarbons, chlorinated solvents and heavy metals. When composed of ureolytic microbes, subsurface biofilms can also induce calcite precipitation. MICP has engineering applications that include soil stabilization and subsurface barriers, as well as sealing of cap rocks and well-bore regions for carbon dioxide sequestration. To meet the design goals of these beneficial applications, subsurface biofilms and MICP must be monitored over space and time - a challenging task with traditional methods. The low-field borehole NMR tool recorded changes in the T 2 relaxation distribution where enhanced relaxation indicated biofilm accumulation in a sand bioreactor and in subsurface soil. Additionally, the tool was able to detect MICP in a sand bioreactor. The changed mineral surface of the sand lead to an increase in T 2 relaxation times. The complementary high-field NMR project investigated micro-scale internal structures and mass transport within biofilm granules used for wastewater treatment. Granular sludge, composed of spherical aggregates of biofilm grown without a carrier, is an innovative biological treatment method with the potential to vastly reduce the cost of wastewater treatment without sacrificing efficiency. Large gaps remain, however, in our understanding of the fundamental formation mechanisms and the factors that control granule activity and stability. Magnetic resonance imaging (MRI) identified heterogeneous internal structures within aerobic granular sludge where relaxation rates and diffusion coefficients vary. Ultimately, these results will help improve modeling for optimization of granular sludge wastewater treatment process design.Item Electric current and magnetic field effects on bacterial biofilms(Montana State University - Bozeman, College of Engineering, 2014) Sandvik, Elizabeth Louise; Chairperson, Graduate Committee: Phillip S. Stewart; Bruce R. McLeod, Albert E. Parker, Philip S. Stewart were co-authors of the article, 'Direct electric current treatment under physiologic saline conditions kills Staphylococcus epidermidis biofilms via electrolytic generation of hypochlorous acid' in the journal 'PLoS ONE' which is contained within this thesis.The ability of bacteria to form and grow as biofilm presents a major challenge in clinical medicine. Through this work, two alternative electromagnetic treatment strategies were investigated to combat bacterial biofilms like those that cause chronic infections on indwelling medical devices. Direct electric current (DC) was applied at current densities of 0.7 to 1.8 mA/cm 2 alone and in conjunction with antibiotic. Unlike most previous studies, chloride ions were included in the treatment solution at a physiologically-relevant concentration. Using this approach, low levels of DC alone were demonstrated to have a dose-responsive, biocidal effect against Staphylococcus epidermidis and Pseudomonas aeruginosa biofilms with no synergistic enhancement of antibiotic activity. Through a series of experiments using chemical measures, cell viability, and global gene expression, electrolytic generation of chlorine, a potent disinfectant, was identified as the predominant mechanism by which DC kills bacteria in biofilm. The second treatment strategy investigated weak, extremely low-frequency magnetic fields (ELF-MFs) as a noninvasive approach, involving an extension of concepts from well-studied ELF-MF effects observed in eukaryotic systems to bacterial biofilm. S. epidermidis biofilms grown in weak, extremely low-frequency magnetic fields (ELF-MFs) at Ca 2+ and K + ion resonance frequencies were assessed using global gene expression to determine if S. epidermidis in biofilm detect and respond to ELF-MFs. Frequency-dependent changes in gene expression were observed with upregulation of genes involved in transposase activity, signal transduction systems, and membrane transport processes indicating possible effects consistent with theories of ELF-MF induced changes in ion transport reported in eukaryotic cells. This is the first transcriptome study to indentify ELF-MF effects in bacteria. While no direct biocidal effect was observed with ELF-MF treatment, alteration of membrane transport processes could potentially modify biofilm susceptibility to certain antibiotics. The ELF-MF responses identified in this work provide a platform for future study.Item Biofilm-induced carbonate precipitation at the pore-scale(Montana State University - Bozeman, College of Engineering, 2015) Connolly, James Martin; Chairperson, Graduate Committee: Robin Gerlach; Robin Gerlach was a co-author of the article, 'Microbially induced carbonate precipitation in the subsurface: fundamental reaction and transport processes' in the book 'Handbook of Porous Media, 3rd Ed.' which is contained within this thesis.; Megan Kaufman, Adam Rothman, Rashmi Gupta, George Redden, Martin Schuster, Frederick Colwell and Robin Gerlach were co-authors of the article, 'Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation' in the journal 'Journal of microbiological methods' which is contained within this thesis.; Benjamin Jackson, Adam P. Rothman, Isaac Klapper and Robin Gerlach were co-authors of the article, 'Estimation of a biofilm-specific reaction rate: kinetics of bacterial urea hydrolysis in a biofilm' submitted to the journal 'NPJ biofilms and microbiomes' which is contained within this thesis.; Johannes Hommel and Robin Gerlach were co-authors of the article, 'Reactive transport and permeability reduction in a synthetic 2D porous medium with biofilm-induced carbonate precipitation' which is contained within this thesis.There are many methods available to decrease permeability in the subsurface but one that has been the subject of much research over the last decade is microbially-induced carbonate precipitation (MICP). In this process, microbial activity is promoted that increases pore water alkalinity. When calcium or other divalent cations are supplied to the system, solid carbonate minerals can form which occupy pore space and can decrease permeability. Permeability reduction can also come from microbial biofilms forming in the pore space. The goal of the work presented in this dissertation is to understand how pore space is affected, both physically and chemically, by biofilms and the precipitates that they can form. Fundamental research presented here is intended to inform ongoing application-based research and development. Previously it has been a challenge to image MICP at high resolution without the use of destructive techniques. To overcome that obstacle, a fluorescently-tagged bacterium capable of urea hydrolysis-driven MICP was constructed. Biofilms were grown in two-dimensional microscale porous media reactors and allowed to precipitate calcium carbonate under varied conditions. These reactors were imaged noninvasively using confocal microscopy so that both biofilms and carbonate minerals could be resolved at micrometer resolution. Image analysis was utilized to quantify how much pore space was occupied by the biofilm and minerals in order to estimate porosity reduction. Finally, pore-scale reactive transport modeling was utilized in order to estimate local concentrations within the reactors. The results show that the extent to which the porosity and permeability of the porous medium was decreased depended on when the calcium was added to the system. Also, periods of low flow were found to decrease porosity and permeability to a greater extent. This result adds to the evidence that a pulsed flow injection strategy may be most effective for permeability reduction via MICP in the subsurface. Additionally, reactive transport modeling predicts a heterogeneous mineral saturation environment at the pore-scale which highlights the challenge of predicting precipitation behavior in Darcy-scale reactive transport models.Item Effects of substratum topography on bacterial adhesion(Montana State University - Bozeman, College of Engineering, 1996) Scheuerman, Teresa RushItem Chemically induced biofilm detachment(Montana State University - Bozeman, College of Engineering, 1998) Chen, XiaoItem Corrosion of 316L stainless steel influenced by manganese oxidizing bacteria(Montana State University - Bozeman, College of Engineering, 2001) Geiser, Michael Joseph