Given our present lack of knowledge of the microbial diversity within spacecraft assembly facilities, what should be the scientific community's research objectives to fill in this missing information? The overall objective of a research program, at a minimum, should be to determine and compare the phylogenetic diversities of microbial communities within the extreme oligotrophic environments of several spacecraft assembly facilities. This research should primarily employ modern molecular and genomics-based tools, since these methods represent the present state of the art for characterizing both the cultivable and uncultivable microbes in a community. At least one spacecraft facility should be representative of a facility located in an arid geographic region. Another should be representative of a spacecraft assembly facility located in a subtropical marine coastal environment. These environments are representative of locations where NASA assembles spacecraft immediately prior to launch. It should be expected that the microbial communities of these types of facilities would be different, reflecting the sources of microbes from the local environments (49).
Although present molecular biology-based tools provide powerful techniques for characterizing microbial community structure and diversity, it must be recognized that even the best of these tools have limitations. There are challenges in extraction of truly representative DNA pools from many environments, and the extracted DNA must be free of contaminants that inhibit enzymes (e.g., Taq DNA polymerase) that are used in subsequent operations, such as PCR and cloning (65). Thus, DNA extraction protocols must be carefully examined for individual environments to maximize DNA yields. Sample size must be considered, particularly when microbial populations are small and/or dispersed (52). PCR techniques can have biases that must be recognized, and choices of PCR primers must be made carefully to minimize preferential amplification of some templates (2, 7). It is also sometimes difficult to determine if DNA isolated from a specific environment is derived from dead or living cells. Although it is becoming possible to overcome or minimize some of these known limitations of molecular biology-based metagenomic techniques (35), we must recognize that there likely will be unknown members of many microbial communities that will resist detection by the characterization methods developed to date. Undoubtedly, this will also be true for our spacecraft. However, in characterizing the microbial communities that we are sending into space, it will behoove us to use state-of-the-art methods. Improved methods should be adopted quickly as they are proven in the field.
Preliminary data from the study of microorganisms isolated from the JPL spacecraft assembly facility indicate that microbial strains that survive in this environment are unusually resistant to desiccation, H2O2, UV light, and gamma radiation (36, 68-70). If we can understand the mechanisms of these types of resistance at the fundamental level of genes and proteins, space scientists will be able to use this information to make appropriate changes in cleaning technologies to defeat microbial survival. Therefore, some emphasis should be placed on fundamental research on the genetics and structural biology of the unique microbes found in spacecraft assembly areas.
Since strains of bacteria that survive in the spacecraft assembly facility environment almost certainly reflect robust members of the surrounding environment, more research on the microbial diversity of areas near assembly facilities is needed. For example, the environment near JPL is dominated by arid land. Cultivable bacteria found in JPL spacecraft assembly facilities are dominated by Bacillus species, and not surprisingly, strains of Bacillus appear to be common in desert soils (1, 34, 45, 53, 54). The species that have been observed include B. subtilis, Bacillus stearothermophilus, and a new species, Bacillus mojavensis. However, actinomycetes also are frequently isolated from desert environments, and Streptomyces species are observed most often (16, 18, 21, 29, 41, 51). Actinomycetes, however, have yet to be well described for spacecraft assembly facilities. They are almost certainly present, as indicated by culture-independent techniques that have revealed the presence of many gram-positive and gram-negative microorganisms, as well as actinomycetes and fungi (35). Cyanobacteria have received considerable attention as inhabitants of desert environments, where they are found in sun-impacted soil crusts and endolithically in rocks (9, 10, 12, 23, 47). These photosynthetic, chemoautotrophic microbial forms might also find their way into the lighted, oligotrophic environments of spacecraft assembly facilities, where they might be well adapted for survival; these microbial forms await investigation as part of the community of spacecraft assembly facilities and/or on spacecraft surfaces.
Since many microbial forms appear to be resistant to present sterilization technologies, novel sterilization procedures should be examined. This area has been the focus of some research, but modern instrument packages are not sufficiently robust to withstand some of the previous sterilization treatments, such as use of strong chemicals, penetrating radiation, and heat treatment of spacecraft parts and components carried out before the final assembly of the spacecraft. Likewise, the use of plasmas or gaseous radiation sterilization of the whole spacecraft, as sometimes employed presently (17), needs improvement or replacement. The ultimate objective of such a research program would be to allow sterilization (not just to clean to a specified level) of a complete spacecraft without damage to its structure or instrument packages.
In summary, as discussed by scientists such as Rummel (56), Mancinelli (39), and Horneck et al. (30), planetary protection issues of great importance include minimization of the inevitable deposition of Earth microbes by humans on the surface of Mars or other potentially life-bearing locations in our solar system (59) and prevention of Martian subsurface contamination by Earth microbes and organic material. The natural environments of places in our solar system that may harbor life or complex forms of organic chemicals should be protected so that they retain their value for scientific purposes as humans design planetary missions to search for organic material (60) on and beneath the surface of other planets or to study the chemistry and mineralogy (61) of extraterrestrial landing sites.
ACKNOWLEDGMENTS
I thank NASA for providing a Summer Faculty Fellowship in Residence at the California Institute of Technology's Jet Propulsion Laboratory in Pasadena, Calif., in June to August 2002, where I first became acquainted with the topic of planetary protection.
Cornelia Sawatzky provided valuable editorial assistance.
FOOTNOTES
* Mailing address: Environmental Biotechnology Institute, University of Idaho, Food Research Center 204, P.O. Box 441052, Moscow, ID 83844-1052. Phone: (208) 885-6580. Fax: (208) 885-5741. E-mail: crawford@uidaho.edu .
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