Microbial Diversity and Its Relationship to Planetary Protection
Ronald L. Crawford *
Environmental Biotechnology Institute, University of Idaho, P.O. Box 441052, Moscow, Idaho 83844-1052
As summarized by Rummel (56) and Rummel and Meyer (57), while exploring our solar system and the universe at large, spacefaring nations must be committed to avoiding biological contamination of other planetary systems while also protecting the Earth from potential harm caused by materials returned from space. Most scientists accept this, and there are international treaties and regulations addressing these issues (6, 62). Thus, planetary protection is now a part of planning for all extraterrestrial missions (64), and the rules regarding these activities are prepared by an international group known as the Committee on Space Research (COSPAR) (Paris, France). Spacefaring nations generally adhere to the scientific and technical standards developed by COSPAR.
COSPAR describes five categories for interplanetary missions, and there are suggested ranges of planetary protection requirements for each category. The following descriptions are set forth in the COSPAR regulations (6). Category I includes any mission to a target body that is not of direct interest for understanding the process of chemical evolution or the origin of life; no protection of such bodies is warranted, and no planetary protection requirements are imposed by COSPAR policy. Category II missions are missions whose target bodies are of significant interest relative to the process of chemical evolution and the origin of life but in which there is only a remote chance that contamination carried by a spacecraft could jeopardize future exploration. COSPAR requires only simple documentation that includes preparation of a short planetary protection plan in the form of an outline of intended or potential impact targets, brief pre- and postlaunch analyses detailing impact strategies, and a postencounter and end-of-mission report providing the location of impact, if such an event occurs. Category III missions (mostly flyby and orbiter missions) are missions to a target body of chemical evolution and/or origin-of-life interest or for which scientific opinion indicates that there is a significant chance of contamination that could jeopardize a future biological experiment. COSPAR requires documentation of planetary protection issues and some implementation of protection procedures that include at a minimum trajectory biasing, the use of cleanrooms during spacecraft assembly and testing, and possibly spacecraft bioburden reduction. An inventory of bulk constituent organics is required if the probability of impact is significant. Category IV missions (mostly probe and lander missions) target a body of chemical evolution and/or origin-of life-interest or for which scientific opinion indicates that there is a significant chance of contamination that could jeopardize future biological experiments. COSPAR requires detailed documentation of planetary protection issues, including a bioassay to enumerate spacecraft bioburden, an analysis of the probability of contamination that may include trajectory biasing, use of cleanrooms during spacecraft assembly, bioload reduction, partial sterilization of any direct contact hardware, and a bioshield for that hardware. The requirements and compliance are similar to those imposed for the Viking missions, with the exception of complete lander or probe sterilization. Category V comprises all return-to-Earth missions, where the concern is the protection of the terrestrial system comprising the Earth and the Moon. The Moon must be protected from back-contamination to retain freedom from planetary protection requirements for Earth-Moon travel. For solar system bodies deemed by scientific opinion to have no indigenous life forms, an "unrestricted Earth return" subcategory is defined; missions in this subcategory have planetary protection requirements on the outbound phase only that correspond to the category of that phase (typically category I or II). For all other category V missions, in a subcategory defined as "restricted Earth return," the highest degree of concern is expressed by the absolute prohibition of destructive impact upon return, the need for containment throughout the return phase of all returned hardware which directly contacted the target body or unsterilized material from the body, and the need for containment of any unsterilized sample collected and returned to Earth. Postmission, timely analysis of the unsterilized, returned sample(s) is required under strict containment, using the most sensitive techniques. If any sign of a nonterrestrial replicating entity is found, the returned sample must remain contained unless it is treated by an effective sterilizing procedure.
None of the suggested disinfection procedures for categories I to IV actually requires sterilization of the entire spacecraft; however, the COSPAR regulations are often more specific for certain locations and types of missions. For example, if a "special region" of Mars is to be accessed though horizontal or vertical mobility, either the entire landed system must be sterilized to the Viking poststerilization biological burden levels or the subsystems which directly contact the special region must be sterilized to these levels, and a method of preventing their recontamination prior to accessing the special region must be provided. If an unplanned condition (e.g., a hard landing) could result in a high probability of inadvertent biological contamination of the special region by the spacecraft, the entire landed system must be sterilized to Viking poststerilization biological burden levels. COSPAR defines a special region as an area within which terrestrial organisms are likely to propagate or a region which is thought to have a high potential for the existence of extant life forms. Readers who are interested in such details are referred to the COSPAR document (6) and other relevant NASA and National Research Council documents (3-5).
Since scientific investigations using Earth-launched spacecraft frequently target Mars, there are particular and immediate needs for planetary protection planning related to study of this planet. The concern that forward contamination of Mars might complicate the search for extraterrestrial life has been increased by data indicating that there is frequent meteorite exchange between Mars and Earth (25) and by the strong probability that living bacterial spores can survive interplanetary transfer (42, 46). This raises the distinct possibility that Martian life could resemble life on Earth (37). Irrespective of scientific issues, it is also necessary to address public concerns about potential back-contamination of the Earth (5, 19) and ecological contamination of Mars with Earth organisms, as well as to fulfill formal legal requirements of various laws, such as the National Environmental Policy Act that are already in place (50). Unfortunately, there is no universal appreciation of the difficulty of accomplishing these goals given our limited knowledge of microbial ecology and diversity even here on Earth. The fact is that we do not even know the identities of most of the microorganisms that are actually on the surfaces or within the interiors of our spacecraft.
Spacecraft assembly facilities such as those used by NASA at the California Institute of Technology's Jet Propulsion Laboratory (JPL), the Johnson Space Center, and the Kennedy Space Center are unique microbiological environments. They are extremely oligotrophic (nutrient poor and high stress) because they are rigorously and repeatedly cleaned with antimicrobial agents, particulates are continuously filtered from the air circulating through the facilities, the atmospheres within the facilities are maintained at low humidity, and most surfaces are comprised of man-made materials, such as polished metals. Thus, these facilities are highly selective for indigenous communities of microorganisms that resist desiccation, chemical sterilization agents, and high-energy radiation (36, 68). Interestingly, radiation resistance is observed in these communities even though the facilities themselves are not exposed to unusual radiation other than normal lighting. The local sources of microbes, however, are sometimes subject to high solar light intensities, and these sources provide the microbial forms that eventually are subject to the selective pressure of the cleanroom environment (49).
Since we will be searching Mars for extraterrestrial life, this planet is of special concern regarding issues of planetary protection. Therefore, we need to protect Mars from contamination and thereby protect the integrity of future science missions to the planet. As discussed by Barengoltz (8) and others (30, 39), future sample acquisition flight missions to Mars pose a number of specific protection issues. There is concern for contamination of Earth by possible Mars organisms (19). Also, there is a need for robust anticontamination procedures for the forward protection of Mars and for the sake of future missions. We clearly need to ensure that terrestrial microbes from acquisition missions do not contaminate samples analyzed in situ or after return to Earth. The microbial forms that survive within spacecraft assembly facilities can potentially contaminate spacecraft assembled in them and thus ultimately their destinations.
It has been known since the Viking missions, in which dry-heat sterilization of the spacecraft was employed (48, 49), and from research done since then in various world laboratories (66) that Earth microorganisms may significantly contaminate space-qualified materials. It also is recognized that endospores are of special concern. The moderate levels of dry heat or chemical disinfectants that do not harm the spacecraft or its instruments often are insufficient to kill endospores (67). To complicate matters, recent work at JPL characterizing numerous cultivable bacteria from spacecraft assembly facility environments showed that these bacteria have unusual resistance to both physical and chemical antimicrobial agents (36, 68-70). For example, Link et al. (37) identified spores of Bacillus pumilus as major culturable bacterial contaminants found on and around spacecraft within the spacecraft assembly facility at the NASA Jet Propulsion Laboratory. One strain, B. pumilus SAFR-032, exhibited the highest degree of spore UV resistance observed for any Bacillus spp. encountered to date. The observed cultivable strains were mostly gram-positive strains dominated by Bacillus species; however, preliminary work using molecular tools (e.g., characterization of numerous 16S rRNA genes amplified by PCR from JPL assembly facility community DNA; analyses of ATP, lipopolysaccharide, and ribosomal or spore-specific DNA) indicated that many uncultivable and as-yet-unstudied bacteria also are present in spacecraft assembly environments (35, 68). These microorganisms, as demonstrated by their presence in these harsh environments, must have characteristics similar to those of cultivated, environmentally robust microorganisms. The uncultivated forms include numerous uncultivated gram-negative strains. Thus, the true diversity of these uncultivable communities has not been assessed. This is a distinct limitation in our present knowledge base that must be surmounted in order to achieve a scientifically valid program for planetary protection.
The particular concern about the potential for survival of Bacillus spores on Mars was examined by Schuerger et al. (58). These authors conducted experiments in a Mars simulation chamber to characterize the survival of endospores of Bacillus subtilis deposited on aluminum coupons and exposed to high UV irradiation and simulated Martian conditions. The variables examined were pressure, gas composition, and temperature alone or in combination with Mars-normal UV-visible-near-infrared light environments. The authors' data indicated that more than 99.9% of the bacterial populations on sun-exposed surfaces of a spacecraft are likely to be inactivated within a few minutes on the surface of Mars and that within one Mars day, the bacterial populations on sun-exposed surfaces of a spacecraft would be sterilized, thereby minimizing the prospect of forward contamination of Mars by contaminated spacecraft. These types of data are very encouraging regarding the potential for minimizing possible forward contamination of extreme extraterrestrial locations, such as Mars; however, some spores are much more resistant to Mars-like conditions that those of B. subtilis, and many areas of a lander may not be fully exposed to sunlight while on Mars. Thus, more research similar to that of Schuerger et al. (58) but employing more resistant spores (37) is needed.
Source: Applied and Environmental Microbiology, August 2005, p. 4163-4168, Vol. 71, No. 8