Polyextremophile engineering: a review of organisms that push the limits of life.

Nature exhibits an enormous diversity of organisms that thrive in extreme environments. From snow algae that reproduce at sub-zero temperatures to radiotrophic fungi that thrive in nuclear radiation at Chernobyl, extreme organisms raise many questions about the limits of life. Is there any environment where life could not "find a way"? Although many individual extremophilic organisms have been identified and studied, there remain outstanding questions about the limits of life and the extent to which extreme properties can be enhanced, combined or transferred to new organisms. In this review, we compile the current knowledge on the bioengineering of extremophile microbes. We summarize what is known about the basic mechanisms of extreme adaptations, compile synthetic biology's efforts to engineer extremophile organisms beyond what is found in nature, and highlight which adaptations can be combined. The basic science of extremophiles can be applied to engineered organisms tailored to specific biomanufacturing needs, such as growth in high temperatures or in the presence of unusual solvents.

Use of NanoSIMS to Identify the Lower Limits of Metabolic Activity and Growth by Serratia liquefaciens Exposed to Sub-Zero Temperatures.

Serratia liquefaciens is a cold-adapted facultative anaerobic astrobiology model organism with the ability to grow at a Martian atmospheric pressure of 7 hPa. Currently there is a lack of data on its limits of growth and metabolic activity at sub-zero temperatures found in potential habitable regions on Mars. Growth curves and nano-scale secondary ion mass spectrometry (NanoSIMS) were used to characterize the growth and metabolic threshold for S. liquefaciens ATCC 27,592 grown at and below 0 °C. Cells were incubated in Spizizen medium containing three stable isotopes substituting their unlabeled counterparts; i.e., 13C-glucose, (15NH4)2SO4, and H218O; at 0, -1.5, -3, -5, -10, or -15 °C. The isotopic ratios of 13C/12C, 15N/14N, and 18O/16O and their corresponding fractions were determined for 240 cells. NanoSIMS results revealed that with decreasing temperature the cellular amounts of labeled ions decreased indicating slower metabolic rates for isotope uptake and incorporation. Metabolism was significantly reduced at -1.5 and -3 °C, almost halted at -5 °C, and shut-down completely at or below -10 °C. While growth was observed at 0 °C after 5 days, samples incubated at -1.5 and -3 °C exhibited significantly slower growth rates until growth was detected at 70 days. In contrast, cell densities decreased by at least half an order of magnitude over 70 days in cultures incubated at ≤ -5 °C. Results suggest that S. liquefaciens, if transported to Mars, might be able to metabolize and grow in shallow sub-surface niches at temperatures above -5 °C and might survive-but not grow-at temperatures below -5 °C.

Taxonomic and functional analyses of intact microbial communities thriving in extreme, astrobiology-relevant, anoxic sites.

BACKGROUND: Extreme terrestrial, analogue environments are widely used models to study the limits of life and to infer habitability of extraterrestrial settings. In contrast to Earth's ecosystems, potential extraterrestrial biotopes are usually characterized by a lack of oxygen. METHODS: In the MASE project (Mars Analogues for Space Exploration), we selected representative anoxic analogue environments (permafrost, salt-mine, acidic lake and river, sulfur springs) for the comprehensive analysis of their microbial communities. We assessed the microbiome profile of intact cells by propidium monoazide-based amplicon and shotgun metagenome sequencing, supplemented with an extensive cultivation effort. RESULTS: The information retrieved from microbiome analyses on the intact microbial community thriving in the MASE sites, together with the isolation of 31 model microorganisms and successful binning of 15 high-quality genomes allowed us to observe principle pathways, which pinpoint specific microbial functions in the MASE sites compared to moderate environments. The microorganisms were characterized by an impressive machinery to withstand physical and chemical pressures. All levels of our analyses revealed the strong and omnipresent dependency of the microbial communities on complex organic matter. Moreover, we identified an extremotolerant cosmopolitan group of 34 poly-extremophiles thriving in all sites. CONCLUSIONS: Our results reveal the presence of a core microbiome and microbial taxonomic similarities between saline and acidic anoxic environments. Our work further emphasizes the importance of the environmental, terrestrial parameters for the functionality of a microbial community, but also reveals a high proportion of living microorganisms in extreme environments with a high adaptation potential within habitability borders. Video abstract.

Microbiome dynamics during the HI-SEAS IV mission, and implications for future crewed missions beyond Earth.

BACKGROUND: Human health is closely interconnected with its microbiome. Resilient microbiomes in, on, and around the human body will be key for safe and successful long-term space travel. However, longitudinal dynamics of microbiomes inside confined built environments are still poorly understood. Herein, we used the Hawaii Space Exploration Analog and Simulation IV (HI-SEAS IV) mission, a 1 year-long isolation study, to investigate microbial transfer between crew and habitat, in order to understand adverse developments which may occur in a future outpost on the Moon or Mars. RESULTS: Longitudinal 16S rRNA gene profiles, as well as quantitative observations, revealed significant differences in microbial diversity, abundance, and composition between samples of the built environment and its crew. The microbiome composition and diversity associated with abiotic surfaces was found to be rather stable, whereas the microbial skin profiles of individual crew members were highly dynamic, resulting in an increased microbiome diversity at the end of the isolation period. The skin microbiome dynamics were especially pronounced by a regular transfer of the indicator species Methanobrevibacter between crew members within the first 200 days. Quantitative information was used to track the propagation of antimicrobial resistance in the habitat. Together with functional and phenotypic predictions, quantitative and qualitative data supported the observation of a delayed longitudinal microbial homogenization between crew and habitat surfaces which was mainly caused by a malfunctioning sanitary facility. CONCLUSIONS: This study highlights main routes of microbial transfer, interaction of the crew, and origins of microbial dynamics in an isolated environment. We identify key targets of microbial monitoring, and emphasize the need for defined baselines of microbiome diversity and abundance on surfaces and crew skin. Targeted manipulation to counteract adverse developments of the microbiome could be a highly important strategy to ensure safety during future space endeavors. Video abstract.

Addition of anaerobic electron acceptors to solid media did not enhance growth of 125 spacecraft bacteria under simulated low-pressure Martian conditions.

To protect Mars from microbial contamination, research on growth of microorganisms found in spacecraft assembly clean rooms under simulated Martian conditions is required. This study investigated the effects of low atmospheric pressure on the growth of chemoorganotrophic spacecraft bacteria and whether the addition of Mars relevant anaerobic electron acceptors might enhance growth. The 125 bacteria screened here were recovered from actual Mars spacecraft. Growth at 7 hPa, 0 °C, and a CO2-enriched anoxic atmosphere (called low-PTA conditions) was tested on five TSA-based media supplemented with anaerobic electron acceptors. None of the 125 spacecraft bacteria showed active growth under the tested low-PTA conditions and amended media. In contrast, a decrease in viability was observed in most cases. Growth curves of two hypopiezotolerant strains, Serratia liquefaciens and Trichococcus pasteurii, were performed to quantify the effects of the added anaerobic electron acceptors. Slight variations in growth rates were determined for both bacteria. However, the final cell densities were similar for all media tested, indicating no general preference for any specific anaerobic electron acceptor. By demonstrating that a broad diversity of chemoorganotrophic and culturable spacecraft bacteria do not grow under the tested conditions, we conclude that there may be low risk of growth of chemoorganotrophic bacteria typically recovered from Mars spacecraft during planetary protection bioburden screenings.

The Hypopiezotolerant Bacterium, Serratia liquefaciens, Failed to Grow in Mars Analog Soils under Simulated Martian Conditions at 7 hPa.

The search for life on Mars is predicated on the idea that Earth and Mars life (if present) should be both carbon- and water-based with similar forms of evolution. However, the astrobiology community can currently only investigate plausible Martian microbial ecosystems by using Terran life-forms as proxies. In order to examine how life might persist on Mars, we used a hypopiezotolerant bacterium (def., able to grow at 7-10 hPa)-Serratia liquefaciens-in growth assays with four Mars analog soils conducted under a subset of simulated Martian conditions including 7 hPa, 0 °C, and a CO2-enriched anoxic atmosphere (called low-PTA conditions). The four Mars analog soils included an Aeolian dust analog, the Mars JSC-1 analog, a Phoenix lander-site simulant, and a high-Salts analog. Serratia liquefaciens cells were able to grow at 30 °C in a liquid minimal basal medium (MBM) supplemented with 10- or 20-mM sucrose, Spizizen salts, and micronutrients. When the four analog soils were doped with both MBM and cells of S. liquefaciens, and subsequently incubated at 30 °C for 72 h, cell densities increased between 2-logs (Phoenix analog) and 4-logs (Aeolian and JSC-1 analogs); the Salts analog led to complete inactivation of S. liquefaciens within 24 h. In contrast, when the experiment was repeated, but incubated under low-PTA conditions, S. liquefaciens cells were either killed immediately by the Salts analog, or decreased by > 5 logs over 28 d by the Aeolian, JSC-1, and Phoenix analogs. The failure of S. liquefaciens to grow in the analog soils under low-PTA conditions was attributed to the synergistic interactions among six factors (i.e., low pressure, low temperature, anoxic atmosphere (i.e., the low-PTA conditions), low-pH in the Salts soil, dissolved salts in all analogs, and oligotrophic conditions) that increased the biocidal or inhibitory conditions within the analog soils. Results suggest that even if a hypopiezotolerant Terran microbe is displaced from a spacecraft surface on Mars, and lands in a hydrated and nutrient-rich niche, growth in the Martian regolith is not automatically assured.

Exploring Microbial Activity in Low-pressure Environments.

The importance of hypopiezophilic and hypopiezotolerant microorganisms (i.e., life that grows at low atmospheric pressures; see section 2) in the field of astrobiology cannot be overstated. The ability to reproduce and thrive at Martian atmospheric pressure (0.2 to 1.2 kPa) is of high importance to both modeling the forward contamination of its planetary surface and predicting the habitability of Mars. On Earth, microbial growth at low pressure also has implications for the dissemination of microorganisms within clouds or the bulk atmosphere. Yet our ability to understand the effect of low pressure on microbial metabolism, growth, cellular structure and integrity, and adaptation is still limited. We present current knowledge on hypopiezophilic and hypopiezotolerant microorganisms, methods for isolation and cultivation, justify why there should be more focus for future research, and discuss their importance for astrobiology.

Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

Space Station conditions are selective but do not alter microbial characteristics relevant to human health.

The International Space Station (ISS) is a unique habitat for humans and microorganisms. Here, we report the results of the ISS experiment EXTREMOPHILES, including the analysis of microbial communities from several areas aboard at three time points. We assess microbial diversity, distribution, functional capacity and resistance profile using a combination of cultivation-independent analyses (amplicon and shot-gun sequencing) and cultivation-dependent analyses (physiological and genetic characterization of microbial isolates, antibiotic resistance tests, co-incubation experiments). We show that the ISS microbial communities are highly similar to those present in ground-based confined indoor environments and are subject to fluctuations, although a core microbiome persists over time and locations. The genomic and physiological features selected by ISS conditions do not appear to be directly relevant to human health, although adaptations towards biofilm formation and surface interactions were observed. Our results do not raise direct reason for concern with respect to crew health, but indicate a potential threat towards material integrity in moist areas.

A Low-Diversity Microbiota Inhabits Extreme Terrestrial Basaltic Terrains and Their Fumaroles: Implications for the Exploration of Mars.

A major objective in the exploration of Mars is to test the hypothesis that the planet hosted life. Even in the absence of life, the mapping of habitable and uninhabitable environments is an essential task in developing a complete understanding of the geological and aqueous history of Mars and, as a consequence, understanding what factors caused Earth to take a different trajectory of biological potential. We carried out the aseptic collection of samples and comparison of the bacterial and archaeal communities associated with basaltic fumaroles and rocks of varying weathering states in Hawai'i to test four hypotheses concerning the diversity of life in these environments. Using high-throughput sequencing, we found that all these materials are inhabited by a low-diversity biota. Multivariate analyses of bacterial community data showed a clear separation between sites that have active fumaroles and other sites that comprised relict fumaroles, unaltered, and syn-emplacement basalts. Contrary to our hypothesis that high water flow environments, such as fumaroles with active mineral leaching, would be sites of high biological diversity, alpha diversity was lower in active fumaroles compared to relict or nonfumarolic sites, potentially due to high-temperature constraints on microbial diversity in fumarolic sites. A comparison of these data with communities inhabiting unaltered and weathered basaltic rocks in Idaho suggests that bacterial taxon composition of basaltic materials varies between sites, although the archaeal communities were similar in Hawai'i and Idaho. The taxa present in both sites suggest that most of them obtain organic carbon compounds from the atmosphere and from phototrophs and that some of them, including archaeal taxa, cycle fixed nitrogen. The low diversity shows that, on Earth, extreme basaltic terrains are environments on the edge of sustaining life with implications for the biological potential of similar environments on Mars and their exploration by robots and humans.

Metabolic fingerprints of Serratia liquefaciens under simulated Martian conditions using Biolog GN2 microarrays.

Microorganisms growing at atmospheric pressures of 0.7 kPa may have a significant impact on the search for life on Mars. Data on their nutrient requirements in a simulated Martian environment are required to ascertain both the potential risk of forward contamination and the potential of past or present habitability of Mars. Serratia liquefaciens can grow at concomitant conditions of low pressure, low temperature, and anoxic atmosphere. Changes in the metabolic fingerprint of S. liquefaciens grown under varying physical conditions including diverse atmospheric pressures (0.7 kPa to 101.3 kPa), temperatures (30 °C or 0 °C), and atmospheric gas compositions (Earth or CO2) were investigated using Biolog GN2 assays. Distinct patterns for each condition were observed. Above 10 kPa S. liquefaciens performed similar to Earth-normal pressure conditions (101.3 kPa) whereas below 10 kPa shifts in metabolic patterns were observed. The differences indicated a physiological alteration in which S. liquefaciens lost its ability to metabolize the majority of the provided carbon sources at 0.7 kPa with a significant decrease in the oxidation of amino acids. By measuring the physiological responses to different carbon sources we were able to identify nutritional constraints that support cellular replication under simulated shallow Mars subsurface conditions.

Beyond Chloride Brines: Variable Metabolomic Responses in the Anaerobic Organism Yersinia intermedia MASE-LG-1 to NaCl and MgSO4 at Identical Water Activity.

Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure. Microorganisms have evolved a number of adaptations, both structural and metabolic, to counteract osmotic stress. These strategies are well-understood for organisms in NaCl-rich brines such as the accumulation of certain organic solutes (known as either compatible solutes or osmolytes). Less well studied are responses to ionic environments such as sulfate-rich brines which are prevalent on Earth but can also be found on Mars. In this paper, we investigated the global metabolic response of the anaerobic bacterium Yersinia intermedia MASE-LG-1 to osmotic salt stress induced by either magnesium sulfate (MgSO4) or NaCl at the same water activity (0.975). Using a non-targeted mass spectrometry approach, the intensity of hundreds of metabolites was measured. The compatible solutes L-asparagine and sucrose were found to be increased in both MgSO4 and NaCl compared to the control sample, suggesting a similar osmotic response to different ionic environments. We were able to demonstrate that Yersinia intermedia MASE-LG-1 accumulated a range of other compatible solutes. However, we also found the global metabolic responses, especially with regard to amino acid metabolism and carbohydrate metabolism, to be salt-specific, thus, suggesting ion-specific regulation of specific metabolic pathways.

Lack of correlation of desiccation and radiation tolerance in microorganisms from diverse extreme environments tested under anoxic conditions.

Four facultative anaerobic and two obligate anaerobic bacteria were isolated from extreme environments (deep subsurface halite mine, sulfidic anoxic spring, mineral-rich river) in the frame MASE (Mars Analogues for Space Exploration) project. The isolates were investigated under anoxic conditions for their survivability after desiccation up to 6 months and their tolerance to ionizing radiation up to 3000 Gy. The results indicated that tolerances to both stresses are strain-specific features. Yersinia intermedia MASE-LG-1 showed a high desiccation tolerance but its radiation tolerance was very low. The most radiation-tolerant strains were Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1. In both cases, cultivable cells were detectable after an exposure to 3 kGy of ionizing radiation, but cells only survived desiccation for 90 and 30 days, respectively. Although a correlation between desiccation and ionizing radiation resistance has been hypothesized for some aerobic microorganisms, our data showed that there was no correlation between tolerance to desiccation and ionizing radiation, suggesting that the physiological basis of both forms of tolerances is not necessarily linked. In addition, these results indicated that facultative and obligate anaerobic organisms living in extreme environments possess varied species-specific tolerances to extremes.

The UK Centre for Astrobiology: A Virtual Astrobiology Centre. Accomplishments and Lessons Learned, 2011-2016.

The UK Centre for Astrobiology (UKCA) was set up in 2011 as a virtual center to contribute to astrobiology research, education, and outreach. After 5 years, we describe this center and its work in each of these areas. Its research has focused on studying life in extreme environments, the limits of life on Earth, and implications for habitability elsewhere. Among its research infrastructure projects, UKCA has assembled an underground astrobiology laboratory that has hosted a deep subsurface planetary analog program, and it has developed new flow-through systems to study extraterrestrial aqueous environments. UKCA has used this research backdrop to develop education programs in astrobiology, including a massive open online course in astrobiology that has attracted over 120,000 students, a teacher training program, and an initiative to take astrobiology into prisons. In this paper, we review these activities and others with a particular focus on providing lessons to others who may consider setting up an astrobiology center, institute, or science facility. We discuss experience in integrating astrobiology research into teaching and education activities. Key Words: Astrobiology-Centre-Education-Subsurface-Analog research. Astrobiology 18, 224-243.

Preparing for the crewed Mars journey: microbiota dynamics in the confined Mars500 habitat during simulated Mars flight and landing.

BACKGROUND: The Mars500 project was conceived as the first full duration simulation of a crewed return flight to Mars. For 520 days, six crew members lived confined in a specifically designed spacecraft mock-up. The herein described "MIcrobial ecology of Confined Habitats and humAn health" (MICHA) experiment was implemented to acquire comprehensive microbiota data from this unique, confined manned habitat, to retrieve important information on the occurring microbiota dynamics, the microbial load and diversity in the air and on various surfaces. In total, 360 samples from 20 (9 air, 11 surface) locations were taken at 18 time-points and processed by extensive cultivation, PhyloChip and next generation sequencing (NGS) of 16S rRNA gene amplicons. RESULTS: Cultivation assays revealed a Staphylococcus and Bacillus-dominated microbial community on various surfaces, with an average microbial load that did not exceed the allowed limits for ISS in-flight requirements indicating adequate maintenance of the facility. Areas with high human activity were identified as hotspots for microbial accumulation. Despite substantial fluctuation with respect to microbial diversity and abundance throughout the experiment, the location within the facility and the confinement duration were identified as factors significantly shaping the microbial diversity and composition, with the crew representing the main source for microbial dispersal. Opportunistic pathogens, stress-tolerant or potentially mobile element-bearing microorganisms were predicted to be prevalent throughout the confinement, while the overall microbial diversity dropped significantly over time. CONCLUSIONS: Our findings clearly indicate that under confined conditions, the community structure remains a highly dynamic system which adapts to the prevailing habitat and micro-conditions. Since a sterile environment is not achievable, these dynamics need to be monitored to avoid spreading of highly resistant or potentially pathogenic microorganisms and a potentially harmful decrease of microbial diversity. If necessary, countermeasures are required, to maintain a healthy, diverse balance of beneficial, neutral and opportunistic pathogenic microorganisms. Our results serve as an important data collection for (i) future risk estimations of crewed space flight, (ii) an optimized design and planning of a spacecraft mission and (iii) for the selection of appropriate microbial monitoring approaches and potential countermeasures, to ensure a microbiologically safe space-flight environment.

The responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses.

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today.

Deinococcus phoenicis sp. nov., an extreme ionizing-radiation-resistant bacterium isolated from the Phoenix Lander assembly facility.

A bacterial strain, designated 1P10ME(T), which was resistant to extreme doses of ionizing radiation, pale-pink, non-motile, and a tetrad-forming coccoid was isolated from a cleanroom at the Kennedy Space Center, where the Phoenix spacecraft was assembled. Strain 1P10ME(T) showed optimum growth at 30 °C, with a pH range for growth of 6.5-9.0 and was highly sensitive to sodium chloride, growing only in medium with no added NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 1P10ME(T) represents a novel member of the genus Deinococcus, with low sequence similarities (<93.5%) to recognized species of the genus Deinococcus. The predominant cellular fatty acid was C15:1ω6c. This novel strain exhibits extreme resistance to gamma radiation (D10 >8 kGy) and UV (D10 >1000 Jm(-2)). The results of our polyphasic taxonomic analyses suggest that strain 1P10ME(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus phoenicis sp. nov. is proposed. The type strain is 1P10ME(T) ( = NRRL B-59546(T) = DSM 27173(T)).

Insights into the microbial diversity and bioburden in a South American spacecraft assembly clean room.

In this study, samples from the spacecraft assembly clean room BAF (final assembly building), located at Centre Spatial Guyanais in Kourou, French Guiana, were characterized by qualitative and quantitative methods to determine the bioburden and biodiversity. The cultivation assays mainly focused on extremotolerant microorganisms that have special metabolic skills, such as the ability to grow without oxygen, fix nitrogen, grow autotrophically, or reduce sulfate. A broad range of media and growth conditions were used to simulate possible extraterrestrial environments and clean room buildings. In addition to these alternative cultivation assays, the ESA standard protocol for bioburden estimation was also applied. The phylogenetic analysis of the isolates (mainly facultative anaerobes) showed an extraordinarily broad cultivable biodiversity. Overall, 49 species were isolated and identified as members of the bacterial phyla Actinobacteria, Firmicutes, α-, ß-, γ-Proteobacteria, and Bacteroidetes/Chlorobi. In addition to cultivation-based analyses, molecular techniques were also applied, including construction of a 16S rRNA gene clone library. The results indicate a wide-ranging microbial diversity (12 bacterial phyla, 34 families) that not only confirms the results of the cultivation efforts but also deepens our understanding of the noncultivable variety. Our investigations hint at a very broad, mainly uncultivated microbial diversity.

Lessons learned from the microbial analysis of the Herschel spacecraft during assembly, integration, and test operations.

Understanding microbial diversity in spacecraft assembly clean rooms is of major interest with respect to planetary protection considerations. A coordinated screening of different clean rooms in Europe and South America by three German institutes [Deutsches Zentrum für Luft- und Raumfahrt (DLR), Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), and the Institute of Microbiology and Archaea Center, University of Regensburg] took place during the assembly, test, and launch operations of the Herschel spacecraft in 2006-2009. Through this campaign, we retrieved critical information regarding the microbiome within these clean rooms and on the Herschel spacecraft, which served as a model for upcoming ESA mission preparations. This "lessons learned" document summarizes and discusses the data we obtained during this sampling campaign. Additionally, we have taken the opportunity to create a database that includes all 16S rRNA gene sequences ever retrieved from molecular and cultivable diversity studies of spacecraft assembly clean rooms to compare the microbiomes of US, European, and South American facilities.

Paenibacillus phoenicis sp. nov., isolated from the Phoenix Lander assembly facility and a subsurface molybdenum mine.

A novel Gram-positive, motile, endospore-forming, aerobic bacterium was isolated from the NASA Phoenix Lander assembly clean room that exhibits 100 % 16S rRNA gene sequence similarity to two strains isolated from a deep subsurface environment. All strains are rod-shaped, endospore-forming bacteria, whose endospores are resistant to UV radiation up to 500 J m(-2). A polyphasic taxonomic study including traditional phenotypic tests, fatty acid analysis, 16S rRNA gene sequencing and DNA-DNA hybridization analysis was performed to characterize these novel strains. The 16S rRNA gene sequencing convincingly grouped these novel strains within the genus Paenibacillus as a separate cluster from previously described species. The similarity of 16S rRNA gene sequences among the novel strains was identical but only 98.1 to 98.5 % with their nearest neighbours Paenibacillus barengoltzii ATCC BAA-1209(T) and Paenibacillus timonensis CIP 108005(T). The menaquinone MK-7 was dominant in these novel strains as shown in other species of the genus Paenibacillus. The DNA-DNA hybridization dissociation value was <45 % with the closest related species. The novel strains had DNA G+C contents of 51.9 to 52.8 mol%. Phenotypically, the novel strains can be readily differentiated from closely related species by the absence of urease and gelatinase and the production of acids from a variety of sugars including l-arabinose. The major fatty acid was anteiso-C(15 : 0) as seen in P. barengoltzii and P. timonensis whereas the proportion of C(16 : 0) was significantly different from the closely related species. Based on phylogenetic and phenotypic results, it was concluded that these strains represent a novel species of the genus Paenibacillus, for which the name Paenibacillus phoenicis sp. nov. is proposed. The type strain is 3PO2SA(T) ( = NRRL B-59348(T)  = NBRC 106274(T)).