Publications by Colleges and Departments (MSU - Bozeman)
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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 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.Item Integrating CUREs in Ongoing Research: Undergraduates as Active Participants in the Discovery of Biodegrading Thermophiles(2021-09) Peyton, Brent M.; Skorupa, Dana J.Research-based courses are a powerful way to engage undergraduates in the scientific process while simultaneously teaching participants relevant laboratory, analysis, and scientific communication skills. In most programs, students conduct a simulated project which effectively improves student conceptions of scientific thinking but does not produce research-quality data. The course described here delivered an authentic research experience by assigning undergraduates an objective from an active grant-funded project. Participants contributed to research aimed at culturing biodegrading thermophiles from hot springs in Yellowstone National Park. Students participated in a backcountry field experience, collecting environmental samples of their choosing and determining appropriate culturing conditions. Following high-temperature incubations, 16S rRNA gene sequencing identified enriched microbial populations, with analytical and microscopy methods tracking degradation and growth. Importantly, several teams successfully cultivated thermophilic plastic-degrading consortia. Student learning was assessed using several methods, including grade distributions on assignments and statistical comparisons of pre- and posttests. A consistent and, in most cases, statistically significant increase was observed in the students’ posttest scores. The grade distribution on summative assessments also suggests that students achieved the desired learning outcomes. Student perceptions of their learning and experience gains were high, with participants reporting improvements in components emphasized in the research activities. Overall, the findings highlight how involving undergraduates in real-world research projects can enhance student interest and ownership of scientific research, along with contributing quality data that inform active studies.Item Validating an Automated Nucleic Acid Extraction Device for Omics in Space Using Whole Cell Microbial Reference Standards(2020-08) Urbaniak, Camilla; Wong, Season; Tighe, Scott; Arumugam, Arunkumar; Liu, Bo; Parker, Ceth W.; Wood, Jason M.; Singh, Nitin K.; Skorupa, Dana J.; Peyton, Brent M.; Jenson, Ryan; Karouia, Fathi; Dragon, Julie; Venkateswaran, KasthuriNASA has made great strides in the past five years to develop a suite of instruments for the International Space Station in order to perform molecular biology in space. However, a key piece of equipment that has been lacking is an instrument that can extract nucleic acids from an array of complex human and environmental samples. The Omics in Space team has developed the μTitan (simulated micro(μ) gravity tested instrument for automated nucleic acid) system capable of automated, streamlined, nucleic acid extraction that is adapted for use under microgravity. The μTitan system was validated using a whole cell microbial reference (WCMR) standard comprised of a suspension of nine bacterial strains, titrated to concentrations that would challenge the performance of the instrument, as well as to determine the detection limits for isolating DNA. Quantitative assessment of system performance was measured by comparing instrument input challenge dose vs recovery by Qubit spectrofluorometry, qPCR, Bioanalyzer, and Next Generation Sequencing. Overall, results indicate that the μTitan system performs equal to or greater than a similar commercially available, earth-based, automated nucleic acid extraction device. The μTitan system was also tested in Yellowstone National Park (YNP) with the WCMR, to mimic a remote setting, with limited resources. The performance of the device at YNP was comparable to that in a laboratory setting. Such a portable, field-deployable, nucleic extraction system will be valuable for environmental microbiology, as well as in health care diagnostics.Item Carbon-dependent chromate toxicity mechanism in an environmental Arthrobacter isolate(2018-08) Field, Erin K.; Blaskovich, John P.; Peyton, Brent M.; Gerlach, RobinArthrobacter spp. are widespread in soil systems and well-known for their Cr(VI) reduction capabilities making them attractive candidates for in situ bioremediation efforts. Cellulose drives carbon flow in soil systems; yet, most laboratory studies evaluate Arthrobacter-Cr(VI) interactions solely with nutrient-rich media or glucose. This study aims to determine how various cellulose degradation products and biostimulation substrates influence Cr(VI) toxicity, reduction, and microbial growth of an environmental Arthrobacter sp. isolate. Laboratory culture-based studies suggest there is a carbon-dependent Cr(VI) toxicity mechanism that affects subsequent Cr(VI) reduction by strain LLW01. Strain LLW01 could only grow in the presence of, and reduce, 50 μM Cr(VI) when glucose or lactate were provided. Compared to lactate, Cr(VI) was at least 30-fold and 10-fold more toxic when ethanol or butyrate was the sole carbon source, respectively. The addition of sulfate mitigated toxicity somewhat, but had no effect on the extent of Cr(VI) reduction. Cell viability studies indicated that a small fraction of cells were viable after 8 days suggesting cell growth and subsequent Cr(VI) reduction may resume. These results suggest when designing bioremediation strategies with Arthrobacter spp. such as strain LLW01, carbon sources such as glucose and lactate should be considered over ethanol and butyrate.Item Endophytic fungal production rates of volatile organic compounds are highest under microaerophilic conditions(2017-11) Schoen, Heidi R.; Knighton, W. Berk; Peyton, Brent M.Volatile organic compound (VOC) production from an endophytic fungus was quantified at four oxygen concentrations (0, 1, 13 and 21 %) throughout culture growth phases. The filamentous fungus, a Nodulisporium sp. (designated TI-13), was grown in a solid-state reactor with an agricultural byproduct, beet pulp, as the solid substrate. The VOCs, with potential applications as biofuels, natural flavour compounds and bioactive mixtures, were measured with a recently introduced platinum catalyst and proton transfer reaction mass spectrometry quantification system. The highest-specific production rates of carbon number four and higher VOCs were observed under microaerophilic conditions, which is the expected environment within the plant host. Specific production rates of two ester compounds increased by at least 19 times under microaerophilic conditions compared with those under any other oxygen concentration studied. Total VOC production, including small molecules such as ethanol and acetaldehyde, increased by 23 times when compared between aerobic and anoxic conditions, predominately due to increased production of ethanol. Additionally, total specific production for all 21 compounds quantified was highest under reduced oxygen conditions.Item Assessment of Nannochloropsis gaditana growth and lipid accumulation with increased inorganic carbon delivery(2018-04) Pedersen, Todd C.; Gardner, Robert D.; Peyton, Brent M.Algal biomass refineries for sustainable transportation fuels, in particular biodiesel, will benefit from algal strain enhancements to improve biomass and lipid productivity. Specifically, the supply of inorganic carbon to microalgal cultures represents an area of great interest due to the potential for improved growth of microalgae and the possibility for incorporation with CO2 mitigation processes. Combinations of bicarbonate (HCO3−) salt addition and application of CO2 to control pH have shown compelling increases in growth rate and lipid productivity of fresh water algae. Here, focus was placed on the marine organism, Nannochloropsis gaditana, to investigate growth and lipid accumulation under various strategies of enhanced inorganic carbon supply. Three gas application strategies were investigated: continuous sparging of atmospheric air, continuous sparging of 5% CO2 during light hours until nitrogen depletion, and continuous sparging of atmospheric air supplemented with 5% CO2 for pH control between 8.0 and 8.3. These gas sparging schemes were combined with addition of low concentrations (5 mM) of sodium bicarbonate at inoculation and high concentration (50 mM) of sodium bicarbonate amendments just prior to nitrogen depletion. The optimum scenario observed for growth of N. gaditana under these inorganic carbon conditions was controlling pH with 5% CO2 on demand, which increased both growth rate and lipid accumulation. Fatty acid methyl esters were primarily comprised of C16:0 (palmitic) and C16:1 (palmitoleic) aliphatic chains. Additionally, the use of high concentration (50 mM) of bicarbonate amendments further improved lipid content (up to 48.6%) under nitrogen deplete conditions when paired with pH-controlled strategies.