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.

Microbial biodiversity assessment of the European Space Agency's ExoMars 2016 mission.

BACKGROUND: The ExoMars 2016 mission, consisting of the Trace Gas Orbiter and the Schiaparelli lander, was launched on March 14 2016 from Baikonur, Kazakhstan and reached its destination in October 2016. The Schiaparelli lander was subject to strict requirements for microbial cleanliness according to the obligatory planetary protection policy. To reach the required cleanliness, the ExoMars 2016 flight hardware was assembled in a newly built, biocontrolled cleanroom complex at Thales Alenia Space in Turin, Italy. In this study, we performed microbiological surveys of the cleanroom facilities and the spacecraft hardware before and during the assembly, integration and testing (AIT) activities. METHODS: Besides the European Space Agency (ESA) standard bioburden assay, that served as a proxy for the microbiological contamination in general, we performed various alternative cultivation assays and utilised molecular techniques, including quantitative PCR and next generation sequencing, to assess the absolute and relative abundance and broadest diversity of microorganisms and their signatures in the cleanroom and on the spacecraft hardware. RESULTS: Our results show that the bioburden, detected microbial contamination and microbial diversity decreased continuously after the cleanroom was decontaminated with more effective cleaning agents and during the ongoing AIT. The studied cleanrooms and change room were occupied by very distinct microbial communities: Overall, the change room harboured a higher number and diversity of microorganisms, including Propionibacterium, which was found to be significantly increased in the change room. In particular, the so called alternative cultivation assays proved important in detecting a broader cultivable diversity than covered by the standard bioburden assay and thus completed the picture on the cleanroom microbiota. CONCLUSION: During the whole project, the bioburden stayed at acceptable level and did not raise any concern for the ExoMars 2016 mission. The cleanroom complex at Thales Alenia Space in Turin is an excellent example of how efficient microbiological control is performed.

Quo vadis? Microbial profiling revealed strong effects of cleanroom maintenance and routes of contamination in indoor environments.

Space agencies maintain highly controlled cleanrooms to ensure the demands of planetary protection. To study potential effects of microbiome control, we analyzed microbial communities in two particulate-controlled cleanrooms (ISO 5 and ISO 8) and two vicinal uncontrolled areas (office, changing room) by cultivation and 16S rRNA gene amplicon analysis (cloning, pyrotagsequencing, and PhyloChip G3 analysis). Maintenance procedures affected the microbiome on total abundance and microbial community structure concerning richness, diversity and relative abundance of certain taxa. Cleanroom areas were found to be mainly predominated by potentially human-associated bacteria; archaeal signatures were detected in every area. Results indicate that microorganisms were mainly spread from the changing room (68%) into the cleanrooms, potentially carried along with human activity. The numbers of colony forming units were reduced by up to ~400 fold from the uncontrolled areas towards the ISO 5 cleanroom, accompanied with a reduction of the living portion of microorganisms from 45% (changing area) to 1% of total 16S rRNA gene signatures as revealed via propidium monoazide treatment of the samples. Our results demonstrate the strong effects of cleanroom maintenance on microbial communities in indoor environments and can be used to improve the design and operation of biologically controlled cleanrooms.

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.

Abundance and diversity of microbial inhabitants in European spacecraft-associated clean rooms.

The determination of the microbial load of a spacecraft en route to interesting extraterrestrial environments is mandatory and currently based on the culturable, heat-shock-surviving portion of microbial contaminants. Our study compared these classical bioburden measurements as required by NASA's and ESA's guidelines for the microbial examination of flight hardware, with molecular analysis methods (16S rRNA gene cloning and quantitative PCR) to further develop our understanding of the diversity and abundance of the microbial communities of spacecraft-associated clean rooms. Three samplings of the Herschel Space Observatory and its surrounding clean rooms were performed in two different European facilities. Molecular analyses detected a broad diversity of microbes typically found in the human microbiome with three bacterial genera (Staphylococcus, Propionibacterium, and Brevundimonas) common to all three locations. Bioburden measurements revealed a low, but heterogeneous, abundance of spore-forming and other heat-resistant microorganisms. Total cell numbers estimated by quantitative real-time PCR were typically 3 orders of magnitude greater than those determined by viable counts, which indicates a tendency for traditional methods to underestimate the extent of clean room bioburden. Furthermore, the molecular methods allowed the detection of a much broader diversity than traditional culture-based methods.

EXPOSE-E: an ESA astrobiology mission 1.5 years in space.

The multi-user facility EXPOSE-E was designed by the European Space Agency to enable astrobiology research in space (low-Earth orbit). On 7 February 2008, EXPOSE-E was carried to the International Space Station (ISS) on the European Technology Exposure Facility (EuTEF) platform in the cargo bay of Space Shuttle STS-122 Atlantis. The facility was installed at the starboard cone of the Columbus module by extravehicular activity, where it remained in space for 1.5 years. EXPOSE-E was returned to Earth with STS-128 Discovery on 12 September 2009 for subsequent sample analysis. EXPOSE-E provided accommodation in three exposure trays for a variety of astrobiological test samples that were exposed to selected space conditions: either to space vacuum, solar electromagnetic radiation at >110 nm and cosmic radiation (trays 1 and 3) or to simulated martian surface conditions (tray 2). Data on UV radiation, cosmic radiation, and temperature were measured every 10 s and downlinked by telemetry. A parallel mission ground reference (MGR) experiment was performed on ground with a parallel set of hardware and samples under simulated space conditions. EXPOSE-E performed a successful 1.5-year mission in space.