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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 Treatment performance and microbial community structure in an aerobic granular sludge sequencing batch reactor amended with diclofenac, erythromycin, and gemfibrozil(Frontiers Media SA, 2023-09) Bodle, Kylie B.; Mueller, Rebecca C.; Pernat, Madeline R.; Kirkland, Catherine M.This study characterizes the effects of three commonly detected pharmaceuticals—diclofenac, erythromycin, and gemfibrozil—on aerobic granular sludge. Approximately 150 µg/L of each pharmaceutical was fed in the influent to a sequencing batch reactor for 80 days, and the performance of the test reactor was compared with that of a control reactor. Wastewater treatment efficacy in the test reactor dropped by approximately 30-40%, and ammonia oxidation was particularly inhibited. The relative abundance of active Rhodocyclaceae, Nitrosomonadaceae, and Nitrospiraceae families declined throughout exposure, likely explaining reductions in wastewater treatment performance. Pharmaceuticals were temporarily removed in the first 12 days of the test via both sorption and degradation; both removal processes declined sharply thereafter. This study demonstrates that aerobic granular sludge may successfully remove pharmaceuticals in the short term, but long-term tests are necessary to confirm if pharmaceutical removal is sustainable.Item Chickensplash! Exploring the health concerns of washing raw chicken(AIP Publishing, 2022-03) Carmody, Caitlin D.; Mueller, Rebecca C.; Grodner, Benjamin Michael; Chlumsky, Ondrej; Wilking, James N.; McCalla, Scott G.The Food and Drug Administration recommends against washing raw chicken due to the risk of transferring dangerous food-borne pathogens through splashed drops of water. Many cooks continue to wash raw chicken despite this warning, however, and there is a lack of scientific research assessing the extent of microbial transmission in splashed droplets. Here, we use large agar plates to confirm that bacteria can be transferred from the surface of raw chicken through splashing. We also identify and create a phylogenetic tree of the bacteria present on the chicken and the bacteria transferred during splashing. While no food-borne pathogens were identified, we note that organisms in the same genera as pathogens were transferred from the chicken surface through these droplets. Additionally, we show that faucet height, flow type, and surface stiffness play a role in splash height and distance. Using high-speed imaging to explore splashing causes, we find that increasing faucet height leads to a flow instability that can increase splashing. Furthermore, splashing from soft materials such as chicken can create a divot in the surface, leading to splashing under flow conditions that would not splash on a curved, hard surface. Thus, we conclude that washing raw chicken does risk pathogen transfer and cross-contamination through droplet ejection, and that changing washing conditions can increase or decrease the risk of splashing.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 Root exudate composition reflects drought severity gradient in blue grama (Bouteloua gracilis)(Springer Nature, 2022-07) Ulrich, Danielle E. M.; Clendinen, Chaevien S.; Alongi, Franklin; Mueller, Rebecca C.; Chu, Rosalie K.; Toyoda, Jason; Gallegos‑Graves, La Verne; Goemann, Hannah M.; Peyton, Brent; Sevanto, Sanna; Dunbar, JohnPlant survival during environmental stress greatly affects ecosystem carbon (C) cycling, and plant–microbe interactions are central to plant stress survival. The release of C-rich root exudates is a key mechanism plants use to manage their microbiome, attracting beneficial microbes and/or suppressing harmful microbes to help plants withstand environmental stress. However, a critical knowledge gap is how plants alter root exudate concentration and composition under varying stress levels. In a greenhouse study, we imposed three drought treatments (control, mild, severe) on blue grama (Bouteloua gracilis Kunth Lag. Ex Griffiths), and measured plant physiology and root exudate concentration and composition using GC–MS, NMR, and FTICR. With increasing drought severity, root exudate total C and organic C increased concurrently with declining predawn leaf water potential and photosynthesis. Root exudate composition mirrored the physiological gradient of drought severity treatments. Specific compounds that are known to alter plant drought responses and the rhizosphere microbiome mirrored the drought severity-induced root exudate compositional gradient. Despite reducing C uptake, these plants actively invested C to root exudates with increasing drought severity. Patterns of plant physiology and root exudate concentration and composition co-varied along a gradient of drought severity.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 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.