One of the main tasks of NASA's planetary protection program is to prevent the forward contamination of extraterrestrial environments with Earth life, and in turn preserve other planets and the integrity of future life detection missions. Despite information regarding bacterial diversity in NASA's clean rooms, little is known about the presence of Archaea. Archaeal community analysis of spacecraft-associated surfaces is important, as they are considered by some to represent terrestrial life most capable of surviving on Mars. The first insights into the archaeal diversity of clean rooms where spacecraft assembled are attempted. Nucleic acid sequences clustering with uncultivable Archaea within the Eury-and Crenarchaeota were retrieved from 8 of 26 samples collected from several spacecraft assembly clean rooms. Due to their potential capability to survive and proliferate in Martian conditions, screening for Archaea on spacecraft surfaces and instruments that are associated with future life detection missions may be necessary.

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The Regenerative Enclosed Life Support Module Simulator (REMS) was designed to simulate the conditions aboard the International Space Station (ISS). This unique terrestrial, encapsulated environment for humans and their associated organisms allowed investigations into the microbial communities within an enclosed habitat system, primarily with respect to diversity, phylogeny and the possible impact on human health. To assess time-and/or condition-dependent changes in microbial diversity within REMS, a total of 27 air samples were collected during three consecutive months. The microbial burden and diversity were elucidated using culture-dependent and culture-independent molecular methods. The results indicate that during controlled conditions the total microbial burden detected by culture-dependent techniques (below a detectable level to 10 2 cells m x3 of air) and intracellular ATP assay was significantly low (10 2 -10 3 cells m x3 of air), but increased during the uncontrolled post-operation phase (y10 4 cells m x3 of air). Only Gram-positive and a-proteobacteria grew under tested culture conditions, with a predominant occurrence of Methylobacterium radiotolerans, and Sphingomonas yanoikuyae. Direct DNA extraction and 16S rDNA sequencing methodology revealed a broader diversity of microbes present in the REMS air (51 species). Unlike culture-dependent analysis, both Gram-positive and proteobacteria were equally represented, while members of a few proteobaterial groups dominated (Rhodopseudomonas, Sphingomonas, Acidovorax, Ralstonia, Acinetobacter, Pseudomonas, and Psychrobacter). Although the presence of several opportunistic pathogens warrants further investigation, the results demonstrated that routine maintenance such as controlling the humidity, crew's daily cleaning, and air filtration were effective in reducing the microbial burden in the REMS. Sample collectionSamples were collected from the REMS during three different sampling trips in three consecutive months using a C. Moissl et al. 132

Sublacustrine hydrothermal vents, geysers, and fumaroles impart regions of Yellowstone Lake with distinctive chemical compositions that generate unique freshwater habitats and support diverse microbial life. Some microbial communities within Sedge Bay manifest themselves as accumulations of white-colored films on the surfaces of aquatic macrophytes located within the hydrothermal flow of vents. It was hypothesized that the white films were the product of microbial growth, particularly sulfur-oxidizing bacteria. An investigation of the relevant biological compounds in the vent waters was conducted. Microscopy, non-culture molecular techniques, and phylogenetic analysis were used to assay the bacterial diversity associated with the films. Microscopic analysis of the white films revealed the presence of long filaments (>200 μm) that contained sulfur granules. Filaments with these characteristics were not detected on the normal macrophyte samples. Nucleic acids were extracted from the surface of macrophyte coated with the white film (SB1, SB2) and from the surface of an uncoated macrophyte (SC). 16S ribosomal (rRNA) genes were amplified with the polymerase chain reaction (PCR) and cloned. Amplified ribosomal DNA restriction analysis (ARDRA) was used to examine 100 clones from each library and identify unique phylotypes. S(Chao1) and the Shannon Index, mathematical measures of richness and heterogeneity, were employed to assess the ARDRA pattern diversity of each sample. The SC community contained 50 unique phylotypes, predominantly cyanobacteria and proteobacteria, and was the most heterogeneous. SB1 and SB2 communities were less heterogeneous and dominated by Thiothrix. Dilution to extinction PCR conducted with specific primers indicated that the relative abundance of Thiothrix 16S rRNA gene copies in all three samples were similar. However, reduced sulfur compounds from the vent resulted in a more narrow habitat that supported the sulfur-oxidizing Thiothrix in the white film to the exclusion of cyanobacteria and other proteobacteria found on the normal macrophyte. The majority of 16S rRNA gene sequences obtained in this study displayed similarities ≤98% to any known sequence in public data bases which suggests an abundance of new bacterial species in Sedge Bay.