Theses and Dissertations at Montana State University (MSU)
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Item Removal of PFAS from synthetic wastewater using aerobic granular sludge(Montana State University - Bozeman, College of Engineering, 2023) Ritu, Tasnim Sultana; Co-chairs, Graduate Committee: Catherine KirklandThe project assesses the performance of the aerobic granular sludge (AGS) to remove poly- and per-fluoroalkyl substances (PFAS) and conventional nutrients like carbon, nitrogen, and phosphorus from synthetic wastewater in a sequencing batch reactor (SBR). AGS is an emerging wastewater treatment biofilm that may be effective in reducing the PFAS concentration in wastewater via sorption. PFAS are a class of man-made chemicals used as surfactants, fire retardants, and coating materials. PFAS compounds are very persistent in the environment and can lead to adverse health outcomes in humans. PFAS can migrate from consumer products and enter the influent of wastewater treatment plant (WWTP). PFAS compounds are poorly removed by conventional wastewater treatment methods making effluent from WWTP a significant source of PFAS in the environment. The project uses two specific PFAS which are perfluorooctanoic acid (PFOA) and perfluoro octane sulfonate acid (PFOS). Other objectives of this project are to monitor how PFAS influences the treatment of conventional wastewater constituents and the granules' structure and morphology. Two SBRs were started with floccular sludge from seed granules and continued for 402 days. Some standard laboratory analytical methods for nitrogen, phosphorus, and organic carbon were used to monitor the removal efficiencies of the granules. Solid phase extraction (SPE) and liquid chromatography with mass spectrometry (UPLC with ESI Q-TOF-MS) were used to assess the removal of PFOA and PFOS both from liquid and sludge phases. Maximum removal of 33% for PFOS and 28% for PFOA was achieved by AGS in the test SBR. PFOS/PFOA exposure affected the granule's physical properties, and the properties recovered within approximately 34 days of dosing. PFOS/PFOA contamination produced no significant effect on conventional nutrient removal except nitrification. Thus, the treatment of PFAS by AGS is economical, since AGS can treat several pollutants simultaneously in a single reactor. More research should be done on the disposal of PFAS-contaminated sludge and to increase the treatment efficiency.Item Assessing a novel approach to pharmaceutical removal from wastewater: aerobic granular sludge(Montana State University - Bozeman, College of Engineering, 2024) Bodle, Kylie Brigitta; Chairperson, Graduate Committee: Catherine Kirkland; This is a manuscript style paper that includes co-authored chapters.Pharmaceutical concentrations in various environmental matrices are increasing across the globe. Effluent discharge from wastewater treatment plants is a major vector by which pharmaceuticals enter the environment, as many of these compounds are not biodegradable under conventional wastewater treatment conditions. Although concentrations are currently low (ng/L to ?g/L levels), pharmaceutical contamination poses risks to both human and animal health, as many pharmaceuticals can have toxic effects on fish, birds, and small mammals, as well as contribute to the proliferation of antibiotic resistance genes in bacteria. Aerobic granular sludge (AGS), an emerging biofilm-based wastewater treatment biotechnology and the subject of this dissertation, may be capable of enhancing pharmaceutical removal from wastewater. Scientific literature indicates that AGS uses a mixture of both biodegradation and adsorption to remove pharmaceuticals, but thus far, studies on this topic are limited. The research detailed herein investigated how AGS was affected by a mixture of three common, but relatively unstudied, pharmaceuticals: diclofenac (anti-inflammatory), erythromycin (antibiotic), and gemfibrozil (lipid regulator). Studies described herein examined how AGS grown in two different environments--the lab versus a full-scale wastewater treatment plant--responded to pharmaceuticals. Pharmaceutical effects on wastewater treatment efficacy, active microbial populations, and biofilm structures were investigated. Pharmaceutical fates in both the aqueous and solid phases were also tracked. In general, lab-grown AGS was more negatively impacted by pharmaceutical exposure, evidenced by reduced wastewater treatment efficacy, declines in key wastewater-treating microbial populations, and reductions in biofilm lipid content. Pharmaceuticals were also poorly removed by lab-grown granules. In contrast, key microbial populations and biofilm structures remained stable throughout dosing in environmentally-grown AGS, and gemfibrozil was completely biodegraded. An important caveat to comparison of the two studies, however, is that the pharmaceutical dose to lab-grown AGS was approximately double that to environmental granules. Altogether, the research described herein demonstrates the promise of AGS as a dual wastewater and pharmaceutical treatment technology, but illustrates the importance of conducting experiments under conditions as environmentally relevant as possible.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 Electrochmical impedance spectroscopy biosensor platform for evaluation of biofilm(Montana State University - Bozeman, College of Engineering, 2023) McGlennen, Matthew Connor Dusenbery; Co-chairs, Graduate Committee: Christine Foreman and Stephan Warnat; This is a manuscript style paper that includes co-authored chapters.Microbial biofilms are organized communities of surface-attached microorganisms encased in a self-produced extracellular matrix that pose significant challenges in medicine, the environment, and industry. Biofilms can cause chronic infections, biofouling, and equipment failure, while existing methods for biofilm detection are slow, costly, and labor-intensive. Recently, the use of microfabricated electrochemical impedance spectroscopy (EIS) biosensors has emerged as a promising technique for evaluating biofilm growth in real-time with advantages of small-size, adaptability, low-cost, and high-sensitivity. In this work, EIS biosensors featuring gold micro-interdigitated electrodes were produced using standard microfabrication techniques. Sensors were integrated into a custom 3D-printable flow cell system, enabling EIS measurements and confocal laser scanning microscopy (CLSM) imaging simultaneously. Green fluorescently labeled Pseudomonas aeruginosa PA01, a model biofilm forming bacteria, was introduced into flow chambers and subsequent growth was monitored by EIS, CLSM, and biomass enumeration. Using the system, biofilm growth, dispersal, and the effects of cell-signaling suppression were evaluated. The sensors were also tested in an oil-water emulsion and field-tested on an alpine snow-patch and pond. Improved stability of EIS measurements was achieved by coating the sensors' counter and reference electrodes with an electrically conductive polymer. Biofilm growth was successfully detected using EIS biosensors at an optimized single-frequency, with average decreases in impedance of ~22% by 24 hours. Likewise, biofilm dispersal via chemical treatments were successfully detected with average increases in impedance of ~14% over the ensuing 12 hours. When cells were exposed to a quorum sensing inhibition agent, impedance did not decrease for 18 hours. Impedance changes due to biofilm growth, dispersal, and effects of quorum sensing inhibition were validated by CLSM images and biofilm enumeration. Similarly, in an oil-water emulsion the biosensors successfully detected biofilm growth, dispersal, and effects of quorum sensing inhibition. In an alpine field-test, samples containing varying concentrations of microbes could be detected using the EIS biosensors. This work demonstrates that EIS biosensors are a promising tool for real-time monitoring of biofilm dynamics in a variety of aqueous environments. Overall, EIS biosensing holds great potential for in situ and real-time data regarding biofilm colonization that is not possible with existing techniques.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 A biofilm model that avoids a tragedy of the commons(Montana State University - Bozeman, College of Letters & Science, 2021) Dayutis, Seth Aaron; Chairperson, Graduate Committee: Jack D. DockeryThe study of competition between multiple species is of great significance in biology. Competitive behavior is often observed to occur in biofilms and understanding cooperation between multiple species in a single biofilm is the center of much research. The species that grow in biofilms are frequently studied in chemostats, which have a rich history in mathematical modeling. In this thesis, a review of a mathematical chemostat model is presented in which a tragedy of the commons occurs. The chemostat model is then developed into a biofilm model to see if a tragedy occurs in a biofilm under similar conditions. The biofilm and chemostat model consist of two species, a cooperator and a cheater. The cooperator produces an enzyme that combines with a substrate to produce a nutrient. The nutrient is then consumed by the cooperator and cheater. The cooperator is at a disadvantage since it must allocate some of its nutrient uptake towards enzyme production. A one dimensional biofilm model is developed with reaction advection equations governing the behavior of the species and reaction-diffusion equations governing the behavior of the substrate, nutrient ,and enzyme. A set of numerical methods is then outlined on how to solve the system of equations. It is found that a tragedy of the commons is avoided in the biofilm and both species can persist when numerical simulations are run for a finite amount of time. It is then argued that the cooperative behavior exhibited by the two species is a stable equilibrium by approximating the steady state solutions. Further evidence is provided for the existence of a stable equilibrium by perturbing the system and finding that the perturbed system tends back to the equilibrium. Finally, the eigenvalues of the discretized linear system are computed and the results suggest that either the equilibrium is stable or moves away from the equilibrium slowly.Item The biofilm matrix in sulfate-reducing bacterial biofilms: potential roles for electron mediators and large proteins(Montana State University - Bozeman, College of Letters & Science, 2019) Krantz, Gregory Peter; Chairperson, Graduate Committee: Matthew Fields; Kilean Lucas, Erica L.-Wunderlich, Linh T. Hoang, Recep Avci, Gary Siuzdak and Matthew W. Fields were co-authors of the article, 'Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm' in the journal 'Biofouling' which is contained within this dissertation.; Peter J. Walian, Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Large extracellular proteins sense hydrodynamic force and drive biofilm formation in Desulfovibrio vulgaris' which is contained within this dissertation.; Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Characterization of extracellular biofilm mutants cultivated on 1018 carbon steel in Desulfovibrio vulgaris Hildenborough' which is contained within this dissertation.Sulfate-reducing bacteria grow and form biofilms in soil and benthic environments across much of the Earth's surface. Formation of these prevalent biofilms requires the secretion of an extracellular polymeric substance (EPS) to allow the cells to stick together, as well as adhere to a surface. The specific interactions that occur between EPS components of an SRB biofilm are poorly understood. The data presented in this dissertation suggest the presence of two extracellular mechanisms utilized in these communities. The first mechanism was observed in a study altering the lactate (electron donor) and sulfate (electron acceptor) ratios to create limiting nutrient conditions in Desulfovibrio alaskensis G20 (G20) biofilms. G20 was grown under two conditions: electron donor limited (EDL) and electron acceptor limited (EAL) conditions. When grown on a 1018 carbon steel substrate, the G20 consumes all of the available lactate, and once limited, it turns to the high energy electrons in the Fe 0 for growth. Corrosion rates in the steel increased two fold compared to the EAL condition. Global metabolomic analysis revealed increased lumichrome levels under the EDL condition, which suggested higher flux through the riboflavin/FAD biosynthetic pathway. Previous research showed that synthetically adding riboflavin and FAD increases the corrosion rate of a SRB biofilm on 1018 carbon steel, and paired with these results, suggest G20 produces a flavin-based extracellular electron transfer molecule endogenously, and uses it to harvest high energy electrons from Fe 0 when limited for electron donor. The second mechanism was observed in Desulfovibrio vulgaris Hildenborough (DvH) biofilms grown on glass. Two proteins, DVU1012 and DVU1545 were found to be the most abundant extracellular peptides in a DvH biofilm. Single deletion strains for these proteins grew biofilm similar to the wild type strain, but a double deletion strain had decreased ability to form biofilm, demonstrating that at least one of the peptides must be present in order to form a biofilm. Exposure to increased shear force caused an large increase in wild-type biofilm biomass, yet eliminated the double mutant biofilm. These proteins are required for a DvH biofilm to respond to shear force.Item Metabolic interactions and activity partitioning in a methanogenic, interdomain biofilm(Montana State University - Bozeman, College of Letters & Science, 2019) Camilleri, Laura Beth; Chairperson, Graduate Committee: Matthew Fields; Kristopher A. Hunt, Aurelien Mazurie, Jennifer Kuehl, Alex Michaud, James Connolly, Egan Lohman, Zack Miller, Adam M. Deutschbauer and Matthew W. Fields were co-authors of the article, 'Differential gene expression of a bacterial-archaeal interdomain biofilm producing methane' submitted to the journal 'Biofilms' which is contained within this dissertation.; B.P. Bowen, C.J. Petzold, T.R. Northen and M.W. Fields were co-authors of the article, 'Activity partitioning in an archaeal-bacterial biofilm' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methanococcus maripaludis factor causes slowed growth in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Growth effects of sulfopyruvate and sulfoacetate on the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and the methanogenic archaeon Methanococcus maripaludis S2' submitted to the journal 'Scientific reports' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methane production in Pelosinus fermentans JBW45' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.Biofilms are an ancient survival strategy in which communities of organisms can grow as a cohesive unit, generally attached to a surface and/or at interfaces. Despite the paradigm that 99% of microorganisms grow as a biofilm in the environment, current research methods are largely limited to monoculture planktonic studies. Although more investigations are trying to improve culture complexity by evaluating interactions between two or more populations, experiments are still more readily performed with microorganisms in the planktonic growth mode. The research presented here aims to elucidate the complexity of interactions between two microorganisms from different domains of life that results in enhanced metabolism due to localization of cells in close proximity within an anaerobic biofilm. Desulfovibrio vulgaris Hildenborough (DvH) and Methanococcus maripaludis S2 (Mmp) form a syntrophic mutualism when grown in sulfate-limited media that requires electron flux from DvH to Mmp through what is commonly assumed to be interspecies hydrogen transfer, thereby establishing cross-feeding. The biofilm has been shown to promote a stable and more even carrying capacity for both populations that is likely linked to improved hydrogen transfer (and/or other potential carbon and electron co-metabolites) as compared to planktonic populations. Transcriptomic and proteomic analyses, utilizing RNA-seq and deuterated water respectively, were used to elucidate genes and proteins that contribute to the biofilm growth mode that results in a more efficient metabolism for the syntrophic co-culture (defined by biomass per substrate flux). The results demonstrate the expression of many genes with unknown functions, and others that contribute to cell-cell interactions as well as active proteins in electron processing (e.g., lactate oxidation) in DvH and CO2 reduction (e.g., methanogenesis) in Mmp. A metabolic model of the coculture provided reinforcement for transcriptomic assumptions and aided in the identification of a sulfonate and other amino acids as important syntrophic metabolites. Assessment of biofilm co-culture activity utilizing a new method, Biorthogonal Noncanonical Amino Acid Tagging (BONCAT), showed Mmp was less active in the uptake of a methionine analog as compared to DvH. Alternate assessments confirmed that Mmp was in fact active (based upon methane generation) although translational activity was below the detection limit. Further investigation of the system under sulfate stress showed that the metabolic pairing is more stable than previously thought and could indicate survival strategies that drive the seemingly 'mutualistic' relationship as a forced cooperation. The sulfate stress response coincided with observed lags in DvH growth when grown in Mmp spent medium that was associated with a decoupling of lactate-oxidation and sulfate-reduction. Together the results demonstrate metabolic interactions and activity partitioning within a methanogenic archaeal-bacterial biofilm. The dogma of mutualism being synonymous with equal reciprocity is challenged as it pertains to this model biofilm system. Moreover, this unique bacterial-archaeal biofilm represents interdomain interactions that could represent systems that contributed shared metabolic processes that lead to the development of eukaryotic life.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 Design, synthesis, and evaluation of novel antimicrobials for the eradication of biofilms(Montana State University - Bozeman, College of Letters & Science, 2020) Walsh, Danica Jade; Chairperson, Graduate Committee: Thomas S. Livinghouse; Thomas Livinghouse was a co-author and corresponding author and Darla M. Goeres, Madelyn Mettler, and Philip S. Stewart were co-authors of the article, 'Antimicrobial activity of naturally occurring phenols and derivatives against biofilm and planktonic bacteria' in the journal 'Frontiers in chemistry' which is contained within this dissertation.; Thomas Livinghouse was a co-author and corresponding author and Greg M. Durling, Yenny Chase-Bayless, Adrienne D. Arnold and Philip S. Stewart were co-authors of the article, 'Sulfenate esters of simple phenols exhibit enhanced activity against biofilms' submitted to the journal 'ACS Omega' which is contained within this dissertation.; Thomas Livinghouse was a co-author and corresponding author and Greg Durling, Adrienne Arnold, Whitney Braiser, Luke Berry, Darla M. Goeres and Philip S. Stewart were co-authors of the article, 'Enhanced antimicrobial activity of prodrug phenols against biofilms and planktonic bacteria' which is contained within this dissertation.The majority of microorganisms live in association with surfaces as biofilms. Biofilm communities are encased in a robust, extracellular matrix that reduces their susceptibility to antimicrobial agents. This poses a health concern due to the potential for pathogenic bacteria to cause serious infections. For example, hospital-acquired infections are among the top ten leading causes of death in the U.S. and are responsible for nearly 23,000 deaths per year. The goal of my research is to develop efficient antimicrobial agents capable of eradicating biofilms. In this project, I have focused on three different derivatizations of small, phenolic compounds in effort to increase efficacy towards biofilms. An initial study compared the potency of small, naturally occurring phenols and their corresponding allyl, propyl, and methallyl derivatives against bacteria. This study showed that in parent and derivative pairs potency increased towards free floating cells but decreased towards biofilms. This illustrated the importance of evaluating antimicrobial efficacy toward biofilms when the bacteria they intend to treat has the propensity to form biofilms. This was in contrast to a second studyishowing that trichloromethylsulfenate ester derivatives generally increased potency towards both biofilms and planktonic cells. In a third study, we found that iminodiacetoxy-methylester (AM) appendages increase potency towards planktonic cells and biofilms. AM appendages are ester groups that are employed as part of a prodrug design. Prodrugs are biologically inactive compounds until metabolized. Ester groups are commonly used in prodrug intracellular dyes, where, once inside the cell, ester groups are cleaved enzymatically, resulting in a negatively charged dye that is retained in the cell. Similarly, after the cleavage event, the AM antimicrobial compound will concentrate within the cell. This design serves two functions to increase potency: increasing permeability towards the biofilm matrix and achieving cellular retention. We have shown that the efficacy of antimicrobial agents towards biofilms can be increased through this strategic design. This class of prodrugs presents a wide array of potential applications, from controlling hospital-acquired infections to incorporation into household cleaning products and addresses the need for novel treatments of pathogenic bacteria.