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 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.Item Ecosystem engineering at the streambed: how net-spinning caddisflies influence substrate flow dynamics(Montana State University - Bozeman, College of Letters & Science, 2020) MacDonald, Michael Joseph; Chairperson, Graduate Committee: Lindsey Albertson; Lindsey K. Albertson and Geoffrey C. Poole were co-authors of the article, 'Ecosystem engineering at the streambed: how net-spinning caddisflies influence substrate flow dynamics' submitted to the journal 'Ecohydrology' which is contained within this thesis.The streambed is an ecotone between surface waters and underlying hyporheic systems. Identifying the controls on advective flow through this ecotone is critical to understanding the movement of energy and matter in streams. Hydropsychids (net-spinning caddisflies) are aquatic macroinvertebrate ecosystem engineers that influence streambed cohesion, yet evidence of direct influence on hydrologic processes is lacking. Utilizing a novel downward flow permeameter, we demonstrate how net-spinning caddisfly colonization of the streambed interstitia at moderate but common densities (2,000 m^-2) can reduce the vertical hydraulic conductivity (KV) by up to 55% in coarse sand and gravels (median diameter = 12.91 mm). Sediment columns incubated in artificial stream water occupied by caddisflies showed greater reductions in KV relative to those without caddisflies. Additionally, organic matter content within sediment columns showed that occupation by caddisflies resulted in nearly two-fold increases in organic matter AFDM. Our research shows that the ubiquitous and numerous net-spinning caddisflies are likely to modulate the exchange of channel and hyporheic water by constructing nets in open pore spaces, increasing flow resistance, and decreasing flow velocities, as well as stimulating organic matter deposition with potential consequences for biofilm growth. These results suggest that caddisfly induced reductions to flow may influence transfer processes occurring at the streambed ecotone, altering biogeochemical processes in streams.