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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 Naegleria fowleri Detected in Grand Teton National Park Hot Springs(American Chemical Society, 2024-01) Barnhart, Elliot P.; Kinsey, Stacey M.; Wright, Peter R.; Caldwell, Sara L.; Hill, Vince; Kahler, Amy; Mattioli, Mia; Cornman, Robert S.; Iwanowicz, Deborah; Eddy, Zachary; Halonen, Sandra; Mueller, Rebecca; Peyton, Brent M.; Puzon, Geoffrey J.The free-living thermophilic amoeba Naegleria fowleri (N. fowleri) causes the highly fatal disease primary amoebic meningoencephalitis. The environmental conditions that are favorable to the growth and proliferation of N. fowleri are not well-defined, especially in northern regions of the United States. In this study, we used culture-based methods and multiple molecular approaches to detect and analyzeN. fowleri and other Naegleria spp. in water, sediment, and biofilm samples from five hot spring sites in Grand Teton National Park, Wyoming, U.S.A. These results provide the first detections of N. fowleri in Grand Teton National Park and provide new insights into the distribution of pathogenic N. fowleri and other nonpathogenic Naegleria spp. in natural thermal water systems in northern latitudes.Item Development of Martian saline seep models and their implications for planetary protection(Elsevier BV, 2023-12) Mettler, Madelyn K.; Goemann, Hannah M.; Mueller, Rebecca C.; Vanegas, Oscar A.; Lopez, Gabriela; Singh, Nitin; Venkateswaran, Kasthuri; Peyton, Brent M.While life on Mars has not been found, Earth-based microorganisms may contaminate the Red Planet during rover expeditions and human exploration. Due to the survival advantages conferred by the biofilm morphology to microorganisms, such as resistance to UV and osmotic stress, biofilms are particularly concerning from a planetary protection perspective. Modeling and data from the NASA Phoenix mission indicate that temporary liquid water might exist on Mars in the form of high salinity brines. These brines could provide colonization opportunities for terrestrial microorganisms brought by spacecraft or humans. To begin testing for potential establishment of microbes, results are presented from a simplified laboratory model of a Martian saline seep inoculated with sediment from Hailstone Basin, a terrestrial saline seep in Montana (USA). The seep was modeled as a sand-packed drip flow reactor at room temperature fed media with either 1 M MgSO4 or 1 M NaCl. Biofilms were established within the first sampling point of each experiment. Endpoint 16S rRNA gene community analysis showed significant selection of halophilic microorganisms by the media. Additionally, we detected 16S rRNA gene sequences highly similar to microorganisms previously detected in two spacecraft assembly cleanrooms. These experimental models provide an important foundation for identifying microbes that could hitch-hike on spacecraft and may be able to colonize Martian saline seeps. Future model optimization will be vital to informing cleanroom sterilization procedures.Item Longitudinal analysis of the Five Sisters hot springs in Yellowstone National Park reveals a dynamic thermoalkaline environment(Springer Science and Business Media LLC, 2022-11) Peach, Jesse T.; Mueller, Rebecca C.; Skorupa, Dana J.; Mesle, Margaux M.; Kanta, Sutton; Boltinghouse, Eric; Sharon, Bailey; Copie, Valerie; Bothner, Brian; Peyton, Brent M.Research focused on microbial populations of thermoalkaline springs has been driven in a large part by the lure of discovering functional enzymes with industrial applications in high-pH and high temperature environments. While several studies have focused on understanding the fundamental ecology of these springs, the small molecule profiles of thermoalkaline springs have largely been overlooked. To better understand how geochemistry, small molecule composition, and microbial communities are connected, we conducted a three-year study of the Five Sisters (FS) springs that included high-resolution geochemical measurements, 16S rRNA sequencing of the bacterial and archaeal community, and mass spectrometry-based metabolite and extracellular small molecule characterization. Integration of the four datasets facilitated a comprehensive analysis of the interwoven thermoalkaline spring system. Over the course of the study, the microbial population responded to changing environmental conditions, with archaeal populations decreasing in both relative abundance and diversity compared to bacterial populations. Decreases in the relative abundance of Archaea were associated with environmental changes that included decreased availability of specific nitrogen- and sulfur-containing extracellular small molecules and fluctuations in metabolic pathways associated with nitrogen cycling. This multi-factorial analysis demonstrates that the microbial community composition is more closely correlated with pools of extracellular small molecules than with the geochemistry of the thermal springs. This is a novel finding and suggests that a previously overlooked component of thermal springs may have a significant impact on microbial community composition.Item Climate mitigation potential and soil microbial response of cyanobacteria‐fertilized bioenergy crops in a cool semi‐arid cropland(Wiley, 2022-10) Gay, Justin D.; Goemann, Hannah M.; Currey, Bryce; Stoy, Paul C.; Christiansen, Jesper Riis; Miller, Perry R.; Poulter, Benjamin; Peyton, Brent M.; Brookshire, E. N. JackBioenergy carbon capture and storage (BECCS) systems can serve as decarbonization pathways for climate mitigation. Perennial grasses are a promising second-generation lignocellulosic bioenergy feedstock for BECCS expansion, but optimizing their sustainability, productivity, and climate mitigation potential requires an evaluation of how nitrogen (N) fertilizer strategies interact with greenhouse gas (GHG) and soil organic carbon (SOC) dynamics. Furthermore, crop and fertilizer choice can affect the soil microbiome which is critical to soil organic matter turnover, nutrient cycling, and sustaining crop productivity but these feedbacks are poorly understood due to the paucity of data from certain agroecosystems. Here, we examine the climate mitigation potential and soil microbiome response to establishing two functionally different perennial grasses, switchgrass (Panicum virgatum, C4) and tall wheatgrass (Thinopyrum ponticum, C3), in a cool semi-arid agroecosystem under two fertilizer applications, a novel cyanobacterial biofertilizer (CBF) and urea. We find that in contrast to the C4 grass, the C3 grass achieved 98% greater productivity and had a higher N use efficiency when fertilized. For both crops, the CBF produced the same biomass enhancement as urea. Non-CO2 GHG fluxes across all treatments were low and we observed a 3-year net loss of SOC under the C4 crop and a net gain under the C3 crop at a 0–30 cm soil depth regardless of fertilization. Finally, we detected crop-specific changes in the soil microbiome, including an increased relative abundance of arbuscular mycorrhizal fungi under the C3, and potentially pathogenic fungi in the C4 grass. Taken together, these findings highlight the potential of CBF-fertilized C3 crops as a second-generation bioenergy feedstock in semi-arid regions as a part of a climate mitigation strategy.Item Aboveground and belowground responses to cyanobacterial biofertilizer supplement in a semi-arid, perennial bioenergy cropping system(Wiley, 2021-08) Goemann, Hannah M.; Gay, Justin D.; Mueller, Rebecca C.; Brookshire, E. N. Jack; Miller, Perry; Poulter, Benjamin; Peyton, Brent M.The need for sustainable agricultural practices to meet the food, feed, and fuel demands of a growing global population while reducing detrimental environmental impacts has driven research in multi‐faceted approaches to agricultural sustainability. Perennial cropping systems and microbial biofertilizer supplements are two emerging strategies to increase agricultural sustainability that are studied in tandem for the first time in this study. During the establishment phase of a perennial switchgrass stand in SW Montana, USA, we supplemented synthetic fertilization with a nitrogen‐fixing cyanobacterial biofertilizer (CBF) and were able to maintain aboveground crop productivity in comparison to a synthetic only (urea) fertilizer treatment. Soil chemical analysis conducted at the end of the growing season revealed that late‐season nitrogen availability in CBF‐supplemented field plots increased relative to urea‐only plots. High‐throughput sequencing of bacterial/archaeal and fungal communities suggested fine‐scale responses of the microbial community and sensitivity to fertilization among arbuscular mycorrhizal fungi, Planctomycetes, Proteobacteria, and Actinobacteria. Given their critical role in plant productivity and soil nutrient cycling, soil microbiome monitoring is vital to understand the impacts of implementation of alternative agricultural practices on soil health.Item Antimicrobial Coating Efficacy for Prevention of Pseudomonas aeruginosa Biofilm Growth on ISS Water System Materials(Frontiers Media SA, 2022-04) Mettler, Madelyn K.; Parker, Ceth W.; Venkateswaran, Kasthuri; Peyton, Brent M.Biofilms can lead to biofouling, microbially induced corrosion, physical impediment and eventual loss in function of water systems, and other engineered systems. The remoteness and closed environment of the International Space Station (ISS) make it vulnerable to unchecked biofilm growth; thus, biofilm mitigation strategies are crucial for current ISS operation and future long duration and deep-space crewed missions. In this study, a space flown bacterial strain of Pseudomonas aeruginosa (PA14) was used as a model organism for its ability to form biofilms. Additionally, a novel antimicrobial coating’s ability to reduce biofilm accumulation on stainless steel, Teflon, titanium, and Inconel (all used in the ISS water treatment and handling systems) was analyzed. Coated materials demonstrated reductions of P. aeruginosa biofilm across all materials when tested in a continuous flow system with tryptic soy broth medium. However, the coating lost efficacy in potato dextrose broth medium. These findings were corroborated via scanning electron microscopy. This study illustrates the fundamental importance of using multiple approaches to test antibiofilm strategies, as well as the specificity in which conditions such strategies can be implemented.Item Isolation and Characterization of Lignocellulose-Degrading Geobacillus thermoleovorans from Yellowstone National Park(American Society for Microbiology, 2022-05) Meslé, Margaux M.; Mueller, Rebecca C.; Peach, Jesse; Eilers, Brian; Tripet, Brian P.; Bothner, Brian; Copié, Valérie; Peyton, Brent M.The microbial degradation of lignocellulose in natural ecosystems presents numerous biotechnological opportunities, including biofuel production from agricultural waste and feedstock biomass. To explore the degradation potential of specific thermophiles, we have identified and characterized extremophilic microorganisms isolated from hot springs environments that are capable of biodegrading lignin and cellulose substrates under thermoalkaline conditions, using a combination of culturing, genomics, and metabolomics techniques. Organisms that can use lignin and cellulose as a sole carbon source at 60 to 75°C were isolated from sediment slurry of thermoalkaline hot springs (71 to 81°C and pH 8 to 9) of Yellowstone National Park. Full-length 16S rRNA gene sequencing indicated that these isolates were closely related to Geobacillus thermoleovorans. Interestingly, most of these isolates demonstrated biofilm formation on lignin, a phenotype that is correlated with increased bioconversion. Assessment of metabolite level changes in two Geobacillus isolates from two representative springs were undertaken to characterize the metabolic responses associated with growth on glucose versus lignin carbon source as a function of pH and temperature. Overall, results from this study support that thermoalkaline springs harbor G. thermoleovorans microorganisms with lignocellulosic biomass degradation capabilities and potential downstream biotechnological applications.Item Potential biofilm control strategies for extended spaceflight missions(Elsevier BV, 2020-12) Zea, Luis; McLean, Robert J. C.; Rook, Tony A.; Angle, Geoffrey; Carter, D. Layne; Delegard, Angela; Denvir, Adrian; Gerlach, Robin; Gorti, Sridhar; McIlwaine, Doug; Nur, Mononita; Peyton, Brent M.; Stewart, Philip S.; Sturman, Paul; Justiniano, Yo Ann VelezBiofilms, surface-adherent microbial communities, are associated with microbial fouling and corrosion in terrestrial water-distribution systems. Biofilms are also present in human spaceflight, particularly in the Water Recovery System (WRS) on the International Space Station (ISS). The WRS is comprised of the Urine Processor Assembly (UPA) and the Water Processor Assembly (WPA) which together recycles wastewater from human urine and recovered humidity from the ISS atmosphere. These wastewaters and various process streams are continually inoculated with microorganisms primarily arising from the space crew microbiome. Biofilm-related fouling has been encountered and addressed in spacecraft in low Earth orbit, including ISS and the Russian Mir Space Station. However, planned future missions beyond low Earth orbit to the Moon and Mars present additional challenges, as resupplying spare parts or support materials would be impractical and the mission timeline would be in the order of years in the case of a mission to Mars. In addition, future missions are expected to include a period of dormancy in which the WRS would be unused for an extended duration. The concepts developed in this review arose from a workshop including NASA personnel and representatives with biofilm expertise from a wide range of industrial and academic backgrounds. Here, we address current strategies that are employed on Earth for biofilm control, including antifouling coatings and biocides and mechanisms for mitigating biofilm growth and damage. These ideas are presented in the context of their applicability to spaceflight and identify proposed new topics of biofilm control that need to be addressed in order to facilitate future extended, crewed, spaceflight missions.Item In Silico Analysis of Functionalized Hydrocarbon Production Using Ehrlich Pathway and Fatty Acid Derivatives in an Endophytic Fungus(MDPI, 2021-05) Hunt, Kristopher A.; Mallette, Natasha D.; Peyton, Brent M.; Carlson, Ross P.Functionalized hydrocarbons have various ecological and industrial uses, from signaling molecules and antifungal/antibacterial agents to fuels and specialty chemicals. The potential to produce functionalized hydrocarbons using the cellulolytic, endophytic fungus, Ascocoryne sarcoides, was quantified using genome-enabled, stoichiometric modeling. In silico analysis identified available routes to produce these hydrocarbons, including both anabolic- and catabolic-associated strategies, and determined correlations between the type and size of the hydrocarbons and culturing conditions. The analysis quantified the limits of the wild-type metabolic network to produce functionalized hydrocarbons from cellulose-based substrates and identified metabolic engineering targets, including cellobiose phosphorylase (CP) and cytosolic pyruvate dehydrogenase complex (PDHcyt). CP and PDHcyt activity increased the theoretical production limits under anoxic conditions where less energy was extracted from the substrate. The incorporation of both engineering targets resulted in near-complete conservation of substrate electrons in functionalized hydrocarbons. The in silico framework was integrated with in vitro fungal batch growth experiments to support O2 limitation and functionalized hydrocarbon production predictions. The metabolic reconstruction of this endo-phytic filamentous fungus describes pathways for both specific and general production strategies of 161 functionalized hydrocarbons applicable to many eukaryotic hosts.