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Measurement techniques for determining microbial contamination from occupied space suits
Microbial mats are living examples of the most ancient biological communities on Earth. As Earth's earliest ecosystems, they are centrally important to understanding the history of life on our planet and are useful models for the search for life elsewhere. As relatively compact (but complete) ecosystems, microbial mats are also extremely useful for educational activities. Mats may be used to demonstrate a wide variety of concepts in general and microbial ecology, including the biogeochemical cycling of elements, photosynthesis and respiration, and the origin of the Earth's present oxygen containing atmosphere. Microbial mats can be found in a number of common environments accessible to teachers, and laboratory microbial mats can be constructed using materials purchased from biological supply houses. With funding from NASA's Exobiology program, we have developed curriculum and web-based activities centered on the use of microbial mats as tools for demonstrating general principles in ecology, and the scientific process. Our web site (http://microbes.arc.nasa.gov) includes reference materials, lesson plans, and a "Web Lab", featuring living mats maintained in a mini-aquarium. The site also provides information as to how research on microbial mats supports NASA's goals, and various NASA missions. A photo gallery contains images of mats, microscopic views of the organisms that form them, and our own research activities. An animated educational video on the web site uses computer graphic and video microscopy to take students on a journey into a microbial mat. These activities are targeted to a middle school audience and are aligned with the National Science Standards.
Microbial induced sedimentary structures (MISS) are frequently discussed as potential astrobiological markers on Mars and other planetary bodies[1,2,3]. The presence of fossilized microbial mat structures from the Archaean demonstrates the ability of microbial mats to remain preserved for billions of years[3,4,5].To be preserved microbial mats must survive erosion to be buried in their depositional environment[5]. Erosion of mats is typically due to water, but mats also exist in aeolian environments where wind is a dominant a gent of erosion. The susceptibility of microbial mats to erosion due to aeolian a nd subaqueous sediment transport has important implications for understanding the distribution a nd preservation of potential biological materials on the surface of Mars, Titan, a nd the early Earth. Here we measure the erodibility of microbial m a t s by water a nd wind using field experiments on hyper-saline tidal flats at Padre Island, Texas. The sandy sedimentary environment of Padre Island is a promising astrobiological analogue for Mars and Titan.
Extension of human habitation into space requires that humans carry with them many of the microorganisms with which they coexist on Earth. The ubiquity of microorganisms in close association with all living things and biogeochemical processes on Earth predicates that they must also play a critical role in maintaining the viability of human life in space. Even though bacterial populations exist as locally adapted ecotypes, the abundance of individuals in microbial species is so large that dispersal is unlikely to be limited by geographical barriers on Earth (i.e., for most environments "everything is everywhere" given enough time). This will not be true for microbial communities in space where local species richness will be relatively low because of sterilization protocols prior to launch and physical barriers between Earth and spacecraft after launch. Although community diversity will be sufficient to sustain ecosystem function at the onset, richness and evenness may decline over time such that biological systems either lose functional potential (e.g., bioreactors may fail to reduce BOD or nitrogen load) or become susceptible to invasion by human-associated microorganisms (pathogens) over time. Research at the John F. Kennedy Space Center has evaluated fundamental properties of microbial diversity and community assembly in prototype bioregenerative systems for NASA Advanced Life Support. Successional trends related to increased niche specialization, including an apparent increase in the proportion of nonculturable types of organisms, have been consistently observed. In addition, the stability of the microbial communities, as defined by their resistance to invasion by human-associated microorganisms, has been correlated to their diversity. Overall, these results reflect the significant challenges ahead for the assembly of stable, functional communities using gnotobiotic approaches, and the need to better define the basic biological principles that define ecosystem processes in the space environment. Copyright 2004 Springer-Verlag.
Chemical analyses of Mars soils and sediments from previous landed missions have demonstrated that Mars surface materials possessed major (e.g., P, K, Ca, Mg, S) and minor (e.g., Fe, Mn, Zn, Ni, Cl) elements essential to support microbial life. However, the detection of microbial essential organic-carbon (C) and nitrate have been more elusive until the Mars Science Laboratory (MSL) rover mission. Nitrate and organic-C in Gale Crater, Mars have been detected by the Sample Analysis at Mars (SAM) instrument onboard the MSL Curiosity rover. Eolian fines and drilled sedimentary rock samples were heated in the SAM oven from approximately 30 to 860 degrees Centigrade where evolved gases (e.g., nitrous oxide (NO) and CO2) were released and analyzed by SAM’s quadrupole mass spectrometer (MS). The temperatures of evolved NO was assigned to nitrate while evolved CO2 was assigned to organic-C and carbonate. The CO2 releases in several samples occurred below 450 degrees Centigrade suggesting organic-C dominated in those samples. As much as 7 micromoles NO3-N per gram and 200 micromoles CO2-C per gram have been detected in the Gale Crater materials. These N and C levels coupled with assumed microbial biomass (9 x 10 (sup -7) micrograms per cell) C (0.5 micrograms C per micrograms cell) and N (0.14 micrograms N per micrograms cell) requirements, suggests that less than 1 percent and less than 10 percent of Gale Crater C and N, respectively, would be required if available, to accommodate biomass requirements of 1 by 10 (sup 5) cells per gram sediment. While nitrogen is the limiting nutrient, the potential exists that sufficient N and organic-C were present to support limited heterotrophic microbial populations that may have existed on ancient Mars.
In this study, using NextGen sequencing of the collective 16S rRNA genes obtained from two sets of samples collected from Lake Obersee, Antarctica, we compared and contrasted two bioinformatics tools, PICRUSt and Tax4Fun. We then developed an R script to assess the taxonomic and predictive functional profiles of the microbial communities within the samples. Taxa such as Pseudoxanthomonas, Planctomycetaceae, Cyanobacteria Subsection III, Nitrosomonadaceae, Leptothrix, and Rhodobacter were exclusively identified by Tax4Fun that uses SILVA database; whereas PICRUSt that uses Greengenes database uniquely identified Pirellulaceae, Gemmatimonadetes A1-B1, Pseudanabaena, Salinibacterium and Sinibacteraceae. Predictive functional profiling of the microbial communities using Tax4Fun and PICRUSt separately revealed common metabolic capabilities, while also showing specific functional IDs not shared between the two approaches. Combining these functional predictions using a customized R script revealed a more inclusive metabolic profile, such as hydrolases, oxidoreductases, transferases; enzymes involved in carbohydrate and amino acid metabolisms; and membrane transport proteins known for nutrient uptake from the surrounding environment. Our results present the first molecular-phylogenetic characterization and predictive functional profiles of the microbial mat communities in Lake Obersee, while demonstrating the efficacy of combining both the taxonomic assignment information and functional IDs using the R script created in this study for a more streamlined evaluation of predictive functional profiles of microbial communities.
In accordance with NASA Planetary Protection (PP) policy requirements, flight project hardware may be required to undergo microbial reduction processes to prevent the forward contamination of target planetary bodies with Earth organisms. Heat microbial reduction (HMR) is the most commonly employed modality used at JPL for reducing the microbial bioburden on flight hardware. In 2013, longstanding HMR specifications were abandoned, and revised specifications were developed which integrated the latest findings on bacterial spore heat resistivity. Revised decimal reduction values (D-values) for time-temperature lethality curves (110 °C to 200 °C) were developed to account for “hardy” bacterial spores that exhibit greater heat resistance than previously understood. Presented here is a comparative analysis of the revised NASA HMR specifications against empirical data compiled from recent JPL studies, and peer-reviewed, published literature. Bacillus sp. strain ATCC 29669 displayed high heat resistance, and this strain’s 4-log heat lethality curve was comparable to the revised 4-log specification. Spores of Bacillus atrophaeus ATCC 9372 displayed less heat resistance, and exhibited D-values which were less than the revised 3-log microbial reduction specifications. Extrapolations indicate that the current 6-log reduction credit applied to 350 °C for 1 hr. and 500 °C for 0.5 sec. is highly conservative. Projections indicate that a 10- to 18-log reduction of both hardy and non-hardy spores may be achievable at bakeouts of 350 °C for 1 hr. The findings reported here indicate the revised NASA HMR specifications from 110 °C to 200 °C are appropriate for achieving 4-log and 6-log reductions with hardy spore populations; however, for non-hardy spore populations, or for temperatures above 200 °C, the specifications are exceedingly conservative.
Terrestrial biotas from microbially dominated hypersaline environments will help us understand microbial fossilization processes. Hypersaline tolerant biota from Storr's Lake, San Salvador Island (Bahamas), Mono Lake (California), and the Dead Sea (Israel) represent marine and nonmarine sites for comparative studies of potential analogs for interpreting some Mars meteorites and Mars sample return rocks [1,2,3,4,5,6]. The purpose of this study is to compare microbial fossilization processes, the dominant associated minerals, and potential diagenic implications.
Microbial mats are living examples of the most ancient biological communities on earth, and may also be useful models for the search for life elsewhere. They are centrally important to Astrobiology. In this lecture, we will present an introduction to microbial mats, as well as an introduction to our web-based educational module on the subject of microbial ecology, featuring living mats maintained in a mini "Web Lab" complete with remotely-operable instrumentation. We have partnered with a number of outreach specialists in order to produce an informative and educational web-based presentation, aspects of which will be exported to museum exhibits reaching a wide audience. On our web site, we will conduct regularly scheduled experimental manipulations, linking the experiments to our research activities, and demonstrating fundamental principles of scientific research.
Photosynthetic microbial mats are remarkably complete self-sustaining ecosystems at the millimeter scale, yet they have substantially affected environmental processes on a planetary scale. These mats may be direct descendents of the most ancient biological communities in which even oxygenic photosynthesis might have developed. Photosynthetic mats are excellent natural laboratories to help us to learn how microbial populations associate to control dynamic biogeochemical gradients.
Between April 2009 and July 2011, the NASA Haughton-Mars Project (HMP) led the Northwest Passage Drive Expedition (NWPDX), a multi-staged long-distance crewed rover traverse along the Northwest Passage in the Arctic. In April 2009, the HMP Okarian rover was driven 496 km over sea ice along the Northwest Passage, from Kugluktuk to Cambridge Bay, Nunavut, Canada. During the traverse, crew members collected samples from within the rover and from undisturbed snow-covered surfaces around the rover at three locations. The rover samples and snow samples were stored at subzero conditions (-20C to -1C) until processed for microbial diversity in labs at the NASA Kennedy Space Center, Florida. The objective was to determine the extent of microbial dispersal away from the rover and onto undisturbed snow. Interior surfaces of the rover were found to be associated with a wide range of bacteria (69 unique taxa) and fungi (16 unique taxa). In contrast, snow samples from the upwind, downwind, uptrack, and downtrack sample sites exterior to the rover were negative for both bacteria and fungi except for two colony-forming units (cfus) recovered from one downwind (1 cfu; site A4) and one uptrack (1 cfu; site B6) sample location. The fungus, Aspergillus fumigatus (GenBank JX517279), and closely related bacteria in the genus Brevibacillus were recovered from both snow (B. agri, GenBank JX517278) and interior rover surfaces. However, it is unknown whether the microorganisms were deposited onto snow surfaces at the time of sample collection (i.e., from the clothing or skin of the human operator) or via airborne dispersal from the rover during the 12-18 h layovers at the sites prior to collection. Results support the conclusion that a crewed rover traveling over previously undisturbed terrain may not significantly contaminate the local terrain via airborne dispersal of propagules from the vehicle. Key Words: Planetary protection-Contamination-Habitability-Haughton Crater-Mars. Astrobiology 15, 478-491.
Advantages of new approach for differentiating live cells/ spores from dead cells/spores. Four examples of Salmonella outbreaks leading to costly destruction of dairy products. List of possible collaboration activities between JPL and other industries (for future discussion). Limitations of traditional microbial monitoring approaches. Introduction to new approach for rapid measurement of viable (live) bacterial cells/spores and its areas of application. Detailed example for determining live spores using new approach (similar procedure for determining live cells). JPL has developed a patented approach for measuring amount of live and dead cells/spores. This novel "molecular" method takes less than 5 to 7 hrs. compared to the seven days required using conventional techniques. Conventional "molecular" techniques can not discriminate live cells/spores among dead cells/spores. The JPL-developed novel method eliminates false positive results obtained from conventional "molecular" techniques that lead to unnecessary delay in the processing and to unnecessary destruction of food products.
Detection and study of microbial population in upper atmosphere - simulated high altitude sampling with electrostatic precipitator
Mathematical models and computer program for microbial burden prediction - users manual
Computer program improvements of mathematical models for microbial burden prediction
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