Scholarly Work - Center for Biofilm Engineering
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Item Diversity and evolution of nitric oxide reduction in bacteria and archaea(Proceedings of the National Academy of Sciences, 2024-06) Murali, Ranjani; Pace, Laura A.; Sanford, Robert A.; Ward, L. M.; Lynes, Mackenzie M.; Hatzenpichler, Roland; Lingappa, Usha F.; Fischer, Woodward W.; Gennis, Robert B.; Hemp, JamesWith the advent of culture-independent techniques for studying environmental microbes, our knowledge of their diversity has exploded, uncovering unique organisms, pathways, and proteins carrying out important processes in the biosphere. Novel biochemical reactions are often proposed based on sequence data, but experimental validation is difficult and rare. In this work, we used environmental sequence data to find enzymes that produce the greenhouse gas N2O from NO and validated our hypothesis with experiments. These new enzymes likely contribute to global N2O fluxes and expand the breadth of nitrogen cycling. We also demonstrated that these enzymes evolved multiple times from oxygen reductases, indicating that the evolutionary histories of aerobic respiration and denitrification—and more broadly the oxygen and nitrogen cycles—are tightly connected.Item Beyond the Surface: Non-Invasive Low-Field NMR Analysis of Microbially-Induced Calcium Carbonate Precipitation in Shale Fractures(Springer Science and Business Media LLC, 2024-07) Willet, Matthew R.; Bedey, Kayla; Crandall, Dustin; Seymour, Joseph D.; Rutqvist, Jonny; Cunningham, Alfred B.; Phillips, Adrienne J.; Kirkland, Catherine M.Microbially-induced calcium carbonate precipitation (MICP) is a biological process in which microbially-produced urease enzymes convert urea and calcium into solid calcium carbonate (CaCO3) deposits. MICP has been demonstrated to reduce permeability in shale fractures under elevated pressures, raising the possibility of applying this technology to enhance shale reservoir storage safety. For this and other applications to become a reality, non-invasive tools are needed to determine how effectively MICP seals shale fractures at subsurface temperatures. In this study, two different MICP strategies were tested on 2.54 cm diameter and 5.08 cm long shale cores with a single fracture at 60 ℃. Flow-through, pulsed-flow MICP-treatment was repeatedly applied to Marcellus shale fractures with and without sand (“proppant”) until reaching approximately four orders of magnitude reduction in apparent permeability, while a single application of polymer-based “immersion” MICP-treatment was applied to an Eagle Ford shale fracture with proppant. Low-field nuclear magnetic resonance (LF-NMR) and X-Ray computed microtomography (micro-CT) techniques were used to assess the degree of biomineralization. With the flow-through approach, these tools revealed that while CaCO3 precipitation occurred throughout the fracture, there was preferential precipitation around proppant. Without proppant, the same approach led to premature sealing at the inlet side of the core. In contrast, immersion MICP-treatment sealed off the fracture edges and showed less mineral precipitation overall. This study highlights the use of LF-NMR relaxometry in characterizing fracture sealing and can help guide NMR logging tools in subsurface remediation efforts.Item Aging alters the subchondral bone response 7 days after noninvasive traumatic joint injury in C57BL/6JN mice(Wiley, 2024) Dauenhauer, Lexia A.; Hislop, Brady D.; Brahmachary, Priyanka; Devine, Connor; Gibbs, Dustin; June, Ronald K.; Heveran, Chelsea M.Posttraumatic osteoarthritis (PTOA) commonly develops following anterior cruciate ligament (ACL) injuries, affecting around 50% of individuals within 10–20 years. Recent studies have highlighted early changes in subchondral bone structure after ACL injury in adolescent or young adult mice, which could contribute to the development of PTOA. However, ACL injuries do not only occur early in life. Middle-aged and older patients also experience ACL injuries and PTOA, but whether the aged subchondral bone also responds rapidly to injury is unknown. This study utilized a noninvasive, single overload mouse injury model to assess subchondral bone microarchitecture, turnover, and material properties in both young adults (5 months) and early old age (22 months) female C57BL/6JN mice at 7 days after injury. Mice underwent either joint injury (i.e., produces ACL tears) or sham injury procedures on both the loaded and contralateral limbs, allowing evaluation of the impacts of injury versus loading. The subchondral bone response to ACL injury is distinct for young adult and aged mice. While 5-month mice show subchondral bone loss and increased bone resorption postinjury, 22-month mice did not show loss of bone structure and had lower bone resorption. Subchondral bone plate modulus increased with age, but not with injury. Both ages of mice showed several bone measures were altered in the contralateral limb, demonstrating the systemic skeletal response to joint injury. These data motivate further investigation to discern how osteochondral tissues differently respond to injury in aging, such that diagnostics and treatments can be refined for these demographics.Item Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle(Elsevier BV, 2024-06) Barbosa, Ana; Azevedo, Nuno F.; Goeres, Darla M.; Cerqueira, LauraThere has been considerable discussion regarding the environmental life cycle of Legionella pneumophila and its virulence potential in natural and man-made water systems. On the other hand, the bacterium's morphogenetic mechanisms within host cells (amoeba and macrophages) have been well documented and are linked to its ability to transition from a non-virulent, replicative state to an infectious, transmissive state. Although the morphogenetic mechanisms associated with the formation and detachment of the L. pneumophila biofilm have also been described, the capacity of the bacteria to multiply extracellularly is not generally accepted. However, several studies have shown genetic pathways within the biofilm that resemble intracellular mechanisms. Understanding the functionality of L. pneumophila cells within a biofilm is fundamental for assessing the ecology and evaluating how the biofilm architecture influences L. pneumophila survival and persistence in water systems. This manuscript provides an overview of the biphasic cycle of L. pneumophila and its implications in associated intracellular mechanisms in amoeba. It also examines the molecular pathways and gene regulation involved in L. pneumophila biofilm formation and dissemination. A holistic analysis of the transcriptional activities in L. pneumophila biofilms is provided, combining the information of intracellular mechanisms in a comprehensive outline. Furthermore, this review discusses the techniques that can be used to study the morphogenetic states of the bacteria within biofilms, at the single cell and population levels.Item Critical analysis of methods to determine growth, control and analysis of biofilms for potential non-submerged antibiofilm surfaces and coatings(Elsevier BV, 2024-06) Redfern, J.; Cunliffe, A.J.; Goeres, D.M.; Azevedo, N.F.; Verran, J.The potential uses for antibiofilm surfaces reach across different sectors with significant resultant economic, societal and health impact. For those interested in using antibiofilm surfaces in the built environment, it is important that efficacy testing methods are relevant, reproducible and standardised where possible, to ensure data outputs are applicable to end-use, and comparable across the literature. Using pre-defined keywords, a review of literature reporting on antimicrobial surfaces (78 articles), within which a potential application was described as non-submerged/non-medical surface or coating with antibiofilm action, was undertaken. The most used methods utilized the growth of biofilm in submerged and static systems. Quantification varied (from most to least commonly used) across colony forming unit counts, non-microscopy fluorescence or spectroscopy, microscopy analysis, direct agar-contact, sequencing, and ELISA. Selection of growth media, microbial species, and incubation temperature also varied. In many cases, definitions of biofilm and attempts to quantify antibiofilm activity were absent or vague. Assessing a surface after biofilm recovery or assessing potential regrowth of a biofilm after initial analysis was almost entirely absent. It is clear the field would benefit from widely agreed and adopted approaches or guidance on how to select and incorporate end-use specific conditions, alongside minimum reporting guidelines may benefit the literature.Item Greenhouse gas production from an intermittently dosed cold-climate wastewater treatment wetland(Elsevier BV, 2024) Ayotte, S. H.; Allen, C. R.; Parker, A.; Stein, O. R.; Lauchnor, E. G.This study explores the greenhouse gas (GHG) fluxes of nitrous oxide (N2O), methane (CH4) and carbon dioxide (CO2) from a two-stage, cold-climate vertical-flow treatment wetland (TW) treating ski area wastewater at 3 °C average water temperature. The system is designed like a modified Ludzack-Ettinger process with the first stage a partially saturated, denitrifying TW followed by an unsaturated nitrifying TW and recycle of nitrified effluent. An intermittent wastewater dosing scheme was established for both stages, with alternating carbon-rich wastewater and nitrate-rich recycle to the first stage. The system has demonstrated effective chemical oxygen demand (COD) and total inorganic nitrogen (TIN) removal in high-strength wastewater over seven years of winter operation. Following two closed-loop, intensive GHG winter sampling campaigns at the TW, the magnitude of N2O flux was 2.2 times higher for denitrification than nitrification. CH4 and N2O emissions were strongly correlated with hydraulic loading, whereas CO2 was correlated with surface temperature. GHG fluxes from each stage were related to both microbial activity and off-gassing of dissolved species during wastewater dosing, thus the time of sampling relative to dosing strongly influenced observed fluxes. These results suggest that estimates of GHG fluxes from TWs may be biased if mass transfer and mechanisms of wastewater application are not considered. Emission factors for N2O and CH4 were 0.27 % as kg-N2O-N/kg-TINremoved and 0.04 % kg-CH4-C/kg-CODremoved, respectively. The system had observed seasonal emissions of 600.5 kg CO2 equivalent of GHGs estimated over 130-days of operation. These results indicate a need for wastewater treatment processes to mitigate GHGs.Item Anti-Biofilm Efficacy of Commonly Used Wound Care Products in In Vitro Settings(MDPI AG, 2023-03) Regulski, Matthew; Myntti, Matthew F.; James, Garth A.Considering the prevalence and pathogenicity of biofilms in wounds, this study was designed to evaluate the anti-biofilm capabilities of eight commercially available wound care products using established in vitro assays for biofilms. The products evaluated included dressings with multiple delivery formats for ionic silver including nanocrystalline, gelling fibers, polyurethane (PU) foam, and polymer matrix. Additionally, non-silver-based products including an extracellular polymeric substance (EPS)-dissolving antimicrobial wound gel (BDWG), a collagenase-based debriding ointment and a fish skin-based skin substitute were also evaluated. The products were evaluated on Staphylococcus aureus and Pseudomonas aeruginosa mixed-species biofilms grown using colony drip flow reactor (CDFR) and standard drip flow reactor (DFR) methodologies. Anti-biofilm efficacy was measured by viable plate counts and confocal scanning laser microscopy (CSLM). Four of the eight wound care products tested were efficacious in inhibiting growth of new biofilm when compared with untreated controls. These four products were further evaluated against mature biofilms. BDWG was the only product that achieved greater than 2-log growth reduction (5.88 and 6.58 for S. aureus and P. aeruginosa, respectively) of a mature biofilm. Evaluating both biofilm prevention and mature biofilm disruption capacity is important to a comprehensive understanding of the anti-biofilm efficacy of wound care products.Item Biofilm reactors for the treatment of used water in space:potential, challenges, and future perspectives(Elsevier BV, 2023-12) Espinosa-Ortiz, Erika J.; Gerlach, Robin; Peyton, Brent M.; Roberson, Luke; Yeh, Daniel H.Water is not only essential to sustain life on Earth, but also is a crucial resource for long-duration deep space exploration and habitation. Current systems in space rely on the resupply of water from Earth, however, as missions get longer and move farther away from Earth, resupply will no longer be a sustainable option. Thus, the development of regenerative reclamation water systems through which useable water can be recovered from “waste streams” (i.e., used waters) is sorely needed to further close the loop in space life support systems. This review presents the origin and characteristics of different used waters generated in space and discusses the intrinsic challenges of developing suitable technologies to treat such streams given the unique constrains of space exploration and habitation (e.g., different gravity conditions, size and weight limitations, compatibility with other systems, etc.). In this review, we discuss the potential use of biological systems, particularly biofilms, as possible alternatives or additions to current technologies for water reclamation and waste treatment in space. The fundamentals of biofilm reactors, their advantages and disadvantages, as well as different reactor configurations and their potential for use and challenges to be incorporated in self-sustaining and regenerative life support systems in long-duration space missions are also discussed. Furthermore, we discuss the possibility to recover value-added products (e.g., biomass, nutrients, water) from used waters and the opportunity to recycle and reuse such products as resources in other life support subsystems (e.g., habitation, waste, air, etc.).Item Antimicrobial effects of an acidified nitrite foam on drip flow reactor biofilm(European Wound Management Association, 2024-04) Miller, C. Michael; James, Garth; Bell, David; Schultz, GregBackground. Nitric oxide (NO) plays critical roles in wound healing, including stimulating vasodilation, angiogenesis and broad antimicrobial activity. Aim. To measure the effect of an acidified nitrite foam (ANF) on biofilms created by six different microbes. Methods. A novel method of generating, delivering and topically applying NO gas at the point of care was developed using ANF in a mixed bubble foam and was tested in vitro against six common microbial wound pathogens. Results. A single 5-minute topical exposure of the NO bubble gas formulation generated a 5.8-log10 reduction of mature biofilm of Pseudomonas aeruginosa, a 5.1-log10 reduction of Staphylococcus aureus biofilm, a 4.0-log10 reduction of Staphylococcus epidermidis biofilm, a 3.2-log10 reduction of Proteus mirabilis biofilm, a 2.7-log10 reduction of Acinetobacter baumannii biofilm, and a 1.5-log10 reduction of Candida albicans biofilm. Conclusion. The efficacy of a 5-minute treatment of ANF used on biofilms of P. aeruginosa, A. baumannii, S. aureus, C. albicans, P. mirabilis and S. epidermidis was confirmed. The treatment resulted in a significant reduction in colony-forming units per square centimetre (CFU/cm2) comparable to or surpassing other methods of NO gas application, suggesting NO containing foam’s utility as a point of care solution for chronic wounds with elevated bioburden and biofilms where levels of endogenously produced NO may be insufficient for wound healing completion.Item Critical analysis of methods to determine growth, control and analysis of biofilms for potential non-submerged antibiofilm surfaces and coatings(Elsevier BV, 2024-06) Redfern, J.; Cunliffe, A. J.; Goeres, D. M.; Azevedo, N. F.; Verran, J.The potential uses for antibiofilm surfaces reach across different sectors with significant resultant economic, societal and health impact. For those interested in using antibiofilm surfaces in the built environment, it is important that efficacy testing methods are relevant, reproducible and standardised where possible, to ensure data outputs are applicable to end-use, and comparable across the literature. Using pre-defined keywords, a review of literature reporting on antimicrobial surfaces (78 articles), within which a potential application was described as non-submerged/non-medical surface or coating with antibiofilm action, was undertaken. The most used methods utilized the growth of biofilm in submerged and static systems. Quantification varied (from most to least commonly used) across colony forming unit counts, non-microscopy fluorescence or spectroscopy, microscopy analysis, direct agar-contact, sequencing, and ELISA. Selection of growth media, microbial species, and incubation temperature also varied. In many cases, definitions of biofilm and attempts to quantify antibiofilm activity were absent or vague. Assessing a surface after biofilm recovery or assessing potential regrowth of a biofilm after initial analysis was almost entirely absent. It is clear the field would benefit from widely agreed and adopted approaches or guidance on how to select and incorporate end-use specific conditions, alongside minimum reporting guidelines may benefit the literature.