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Appl. Environ. Microbiol. doi:10.1128/AEM.03007-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean-room Environments

Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA

^* To whom correspondence should be addressed. Email: kjvenkat{at}jpl.nasa.gov.

	Abstract

In assessing the bacterial populations present in spacecraft assembly, test, and launch-preparation facilities, extremophilic (requiring severe conditions for growth) and extremo-tolerant (tolerant to extreme conditions) bacteria were isolated. Several cultivation approaches were employed to select for, and identify, bacteria that not only survive the nutrient-limiting conditions of clean-room environments, but can also withstand even more inhospitable environmental stresses. Due to their proximity to space-faring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct NASA clean-rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4° C to 65° C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremo-tolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (Q-PCR-based) burdens. Intracellular-ATP concentrations suggested a viable microbial presence ranging from below detection limits to 10⁶ cells/m². However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (- and -proteobacteria, actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multi-assay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean-rooms, and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.

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