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Preventing the Forward Contamination of Europa A Calculating the Probability of Contamination, Pc, In the main text of this report the task group concluded that for each mission to Europa the probability of contaminating a europan ocean with a viable terrestrial organism at any time in the future should be less than 10-4 per mission. The task group offers the following calculation as an example of a methodology NASA can use to certify that a particular mission meets the 10-4standard. The calculation is based on several important assumptions and caveats, including the following: The values assigned to individual parameters are not definitive; Parameter values are plausible, but err on the side of conservatism; No attempt was made to account for uncertainties in the parameters; All parameters are assumed to be independent and uncorrelated; and The values of particular parameters will change as new information is gathered. NASA may decide that more detailed calculations and considerations are necessary or that the calculations for a particular mission show that the probability threshold of 10-4is exceeded at some high level of confidence. Whichever the case may be, the onus is on NASA to determine values of the various parameters in the calculation and to certify them as part of the planetary protection plan for each Europa mission. Expressing the 10-4 standard in terms of the delivery of viable organisms to an ocean allows spacecraft designers and mission planners to take advantage of the bioload reduction attributable to the high radiation environment that occurs naturally in transit to Jupiter, in orbit around Jupiter while the spacecraft maneuvers to rendezvous with Europa, in orbit around Europa, and at and near Europa’s surface following a controlled or uncontrolled landing. In this context, it is expected that the requirement will be satisfied by meeting a level of bioload reduction prior to launch that depends on the mission plan, with landers or short-lived orbiters requiring a more stringent level of cleanliness than long-lived orbiters. A SAMPLE METHODOLOGY The task group suggests that this analysis consider four main types of organisms: Type A—Typical, common microorganisms of all types (bacteria, fungi, etc.); Type B—Spores of microorganisms that are known to be resistant to environmental insults (such as desiccation, heat, and radiation); Type C—Spores that are especially radiation-resistant; and Type D—Rare but highly radiation-resistant nonspore microorganisms (e.g., Deinococcus radiodurans). The basis for the above categorization is radiation sensitivity. Although each species will have a somewhat different survival response to ionizing radiation, these are the four general categories that can be readily distinguished by straightforward assay protocols. Calculation of NXs, the number of organisms of type X that survive to grow in the europan ocean environment, functionally depends on the initial assayed contamination level (bioload) of the spacecraft, expressed as NX0 organisms of type X as follows: NXs = NX0 F1 F2 F3 F4 F5 F6 F7. In this equation, the variables F1 through F7 reflect the various factors that affect the bioload and the survival and growth of organisms for the Europa mission. These factors are explained in detail below.
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Preventing the Forward Contamination of Europa If the sum of all NXs is found to be much less than one, it is known by the Poisson statistic that this sum is equal to the probability of any organism being successful. Hence, Pc = Sum (NXs) in the limit of a small value (e.g., 10-4). Microbial Populations On Spacecraft To begin the calculation, it is important to recognize that different classes of organisms are critical to these calculations, and methods must be devised to estimate their abundances. It is assumed that the spacecraft will be cleaned and/or treated to reduce its bioburden of organisms. As a starting point, the current procedures for cleaning and validating a Mars lander that does not carry life-detection experiments will be assumed. Under these procedures, the lander spacecraft is certified to be carrying a total available bioload of not more than 300,000 culturable “spores,” where the “spores” are defined to be heat-shock-resistant organisms. However, some portions of a Mars spacecraft are solid materials, encapsulated components, and occluded surfaces, and they are not included in the above levels of bioload because they are not “available” for release to the martian environment. For the sake of argument, however, the task group considered a worst-case scenario, in which the long-term corrosive action of ocean water ultimately liberates all organisms, wherever they reside. To adjust the estimate of bioload, typical values for surface and buried microbial density on items manufactured inside and outside of controlled environments (e.g., clean rooms) must be taken into account. These values are given in current planetary protection guidelines.1 From a series of many thousands of samplings of the Viking landers and subsequent culture studies, information is available on the types of organisms present under clean room assembly conditions. Chemolithoautotrophic organisms were not specifically tested for because at the time, it was widely expected that organic compounds would be present in the martian soil. The Viking planetary protection studies characterized only the aerobic, mesophilic organisms that grow on trypticase soy agar (TSA) plates. The ratio of total culturable cells to “spores” was found to be quite variable but typically ranged from 3:1 to 60:1.2 Under present protocols the spacecraft assay is for heat-shock-resistant organisms, presumed to be spores. To be safe, the task group assumed a value of 50 (see the next paragraph) as the factor by which to multiply the number of heat-shock-resistant organisms measured for a given spacecraft to estimate the actual number of organisms that could grow in culture. This value is significantly higher than the average observed. More than 55 percent of the organisms were gram-positive cocci (Staphylococcus and Micrococcus), characteristic of organisms found in the human body. Bacillus organisms accounted for about 24 percent, the Corynebacterium-Brevibacterium group accounted for about 15 percent, and Actinomycetes and yeasts constituted the remaining few percent.3 In studies done on the Surveyor spacecraft before they were sent to the Moon, aerobes outnumbered anaerobes by 5 to 1, both overall and for just the “spore” fraction.4 Even though autotrophy was not assayed, a search was made for psychrophilic microorganisms, but none were detected on spacecraft surfaces.5 The factor-of-50 multiplier (see preceding paragraph) takes into account these additional anaerobic microorganisms, which were not assayed in the Viking studies. In summary, the number of chemolithotrophic organisms on spacecraft is small compared to the number of more common heterotrophs. Since organic compounds may be present in the putative europan ocean, the task group accepts the nominal spacecraft assays that use TSA as the growth medium and assumes them to be indicative of the overall population of organisms that pose a contamination threat. Type A organisms are all those organisms that are culturable using the standard TSA plating technique. Type B are those known as “spores ” in the standard protocol, as determined by their resistance to heat shock. Type C are a subset of Type B and are resistant to higher radiation doses. Type D are a subset of Type A and are also resistant to high radiation doses. To avoid unnecessarily elaborate testing and analysis procedures, the task group suggests that Types C and D be determined by a simple screening test using exposure to 60Co or by some other well-established procedure for dosing with ionizing radiation. The classification criteria suggested at this time are as follows: Type C—Organisms with 10 percent or greater survival above 0.8 Mrad; and Type D—Organisms with 10 percent or greater survival above 4.0 Mrad. For this example, the task group took a level of 10 times the Mars available “spore” bioburden, or 3 × 106 culturable heat-shock-resistant organisms for the total spacecraft bioload, based on typical spacecraft sizes and
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Preventing the Forward Contamination of Europa proportions of high-technology components for which cleanliness precautions are taken during manufacture. For an actual spacecraft, this level will be determined by both assays and careful inventory of nonassayable parts and their mode of manufacture. In this calculation, starting populations for each type were estimated as follows: Type B was 3 × 106 “spores.” Type A was estimated as 50 times that of Type B, based on previous experience in spacecraft assembly areas (see discussion above). Since data on the radiation resistance of spacecraft microbes are not available at this time, Type C was arbitrarily taken as 0.1 percent of Type B and Type D as 0.1 percent of Type A. All of these population values should be determined by actual measurements of the spacecraft microorganisms. As will be seen in this and most examples, the Type D organisms are the largest problem, although all four types must be analyzed. F1—Total Number of Cells Relative to Cultured Cells It is now recognized, through the use of molecular probes, that of the organisms in any given ecological environment, laboratory cultivation is successful for only a very small fraction of those present. Thus, unsuccessful laboratory cultivation does not imply that the organisms are not viable. Indeed, only 0.2 percent to 0.3 percent of the organisms found in sediments and soils can be cultured using current techniques. 6,7 In eutrophic samples of activated sludge, the fraction is not so small,8,9 but in seawater the fraction of successfully cultivated microorganisms is very small.10,11 Because a significant component of spacecraft contamination is known to come from soil (the other major component comes from organisms associated with the human body), the task group assumed conservatively that laboratory cultivation underestimates actual microbial abundance by a factor of 1,000, for each type of microbial subpopulation. F2—Bioburden Reduction Treatment This factor accounts for any treatment of the entire spacecraft, after assembly, that reduces bioload. For example, the Viking lander spacecraft were heat treated and the value of bioload reduction was specified to be 10-4. Other approaches would be to expose the spacecraft to penetrating ionizing irradiation to destroy all microorganisms, or to chemicals such as hydrogen peroxide or ethylene oxide gas to kill many surface organisms. For this sample calculation, no special treatments are assumed, so no credit for bioload reduction can be taken and this factor must be set to 1.0. F3—Cruise Survival Fraction During the cruise phase of the journey from Earth to Europa, the spacecraft is exposed to the ultrahigh vacuum and ultraviolet irradiation environment of deep space. However, since spacecraft are generally wrapped in opaque thermal protection blankets, it is only these outermost surfaces that are exposed to ultraviolet irradiation. Bacterial spores are known to be generally resistant to high vacuum, even for long periods of time. For this reason, no credit is taken for this remediating factor for organisms of Type B and C (i.e., F3 = 1.0). Ordinary vegetative cells, Type A, are often susceptible to inactivation by extreme vacuum, so the task group took a value of 0.1 for the survival of these cells. Type D cells are radiation-resistant vegetative microorganisms, with some species being highly resistant to desiccation and others not. Hence, for Type D cells the task group assumed a survival fraction of 0.5. F4—Radiation Survival A mission to Europa might place a spacecraft in Europa orbit for several weeks before its orbit decayed and the spacecraft impacted Europa’s surface. During orbit, and later, on the surface, the spacecraft components and any microorganisms on board could be exposed to up to several megarads of radiation from Jupiter’s radiation belts. The longer the spacecraft stayed in orbit, the higher the radiation dose it would receive. Once impact occurred, spacecraft debris would be exposed to radiation on the surface at a dose rate of 10 to 100 Mrad per month (see Figure 2.3 in Chapter 2). Notwithstanding the possibility of some radiation shielding on board the spacecraft or of burial in the Europa surface, it is highly probable that many contaminant microorganisms transported to the surface would ultimately accumulate several megarads of radiation damage. Microorganisms exhibit exponential declines in survival at high doses of ionizing radiation according to the following relationship:
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Preventing the Forward Contamination of Europa N = N0exp(-D/D0) where N0 = the initial cell number, N = the number of survivors that form colonies, D = the radiation dose, and D0= the D37 dose (at which 37 percent of the population survives). This equation indicates that increasing the dose by a factor of 10 should reduce the survival of microorganisms by a factor of 22,000. The survival of the bacterium Escherichia coli falls to 0.1 percent at 1 × l05 rad;12for B. subtilis, it falls to 0.1 percent at 6 × 104 to 3 × l05 rad;13,14for Bacillus pumilus, at 9 × 105 rad;15 and for D. radiodurans, at 1.8 × 106 rad.16 The physiologic state of the cells, determined by the nutrient conditions at the time of irradiation, is an important factor in determining radiation resistance. Similarly, the presence of oxygen and water greatly potentiates the effect of ionizing radiation on living systems. In general, dry and/or anoxic cells are much more difficult to inactivate than is a fully hydrated cell growing aerobically. Thus, it should be noted that the nutrient conditions of the test subjects in the studies mentioned above were not equivalent.17,18,19,20The reported numbers, therefore, are not absolute—they only reflect a trend of resistance. The radiation sensitivity of all cell types is increased during rapid, exponential growth. In the exponential growth phase, D. radiodurans’s viability falls to less than 0.1 percent after a dose of 1 Mrad. By contrast, during the stationary phase (after the growth nutrients have been depleted) this vegetative bacterium is very much more resistant to radiation. Numerous reports show that in the stationary physiologic state, its D37 is 1.75 Mrad (at 5 to 22 °C). At lower temperatures (-70 °C), the D37 of D. radiodurans is even more dramatic: 3.0 Mrad.21 And, remarkably, it was shown that the bacterium can grow at >6,000 rad per hour without any effect on viability.22 It should be noted that a combination of radiation-resistant and mildly thermophilic characteristics has been identified in two members of the Deinococcal family, D. geothermalis and D. murrayi.23 Table A.1 presents the current best estimates of the radiation sensitivities that should be assumed for the four types of organism when assessing the effect of exposure to radiation in space. When the predicted survival fraction is below 10-10, no lower value is assumed because of the difficulty of verification in practical laboratory experiments. For Type A and Type B organisms, the survival fractions are purely exponential with dose. For Type C and Type D organisms, there is a significant “shoulder” of high survivability until a pure exponential curve comes into play at higher doses. TABLE A.1 Radiation Sensitivity of Microorganisms Dose (Mrad) Type D Type C Type B Type A 0.1 9.90 × 10-1 9.00 × 10-1 3.53 × 10-1 1.15 × 10-2 0.3 9.50 × 10-1 8.00 × 10-1 4.39 × 10-2 1.53 × 10-6 1.0 8.00 × 10-1 3.63 × 10-2 3.00 × 10-5 1.00 × 10-10 4.0 1.00 × 10-1 2.30 × 10-9 1.00 × 10-10 1.00 × 10-10 6.0 1.00 × 10-3 1.00 × 10-10 1.00 × 10-10 1.00 × 10-10 7.0 1.00 × 10-5 1.00 × 10-10 1.00 × 10-10 1.00 × 10-10 8.0 1.00 × 10-8 1.00 × 10-10 1.00 × 10-10 1.00 × 10-10 F5—Probability of Landing at an Active Site Much of the europan surface is considered geologically young, with some parts showing evidence of relatively recent activity. The factor F5 represents the likelihood of landing at a geologically active site on the europan surface. Landing at such a site could allow geologic activity to transport some or all of the spacecraft to a depth sufficient to shield it from the sterilizing effect of the surface radiation environments and eventually allow it to reach a europan ocean. Since a lethal radiation dose at a depth of 1 meter below the surface is accumulated in 7,000 years (see factor F6), F5 is the probability that the spacecraft will land at a site where burial to a depth of significantly more than 1 meter will occur in less than 7,000 years. Extrusive volcanic activity could bury the spacecraft and protect it against the radiation environment, and the spacecraft, or some of its parts, eventually could be carried to a depth where it could interact with a global ocean Assuming an average age of 50 million years, resurfacing models give the probability of activity at any location as on the order of 10 -3 in 7,000 years.24Nonetheless, highly active areas are of particular concern and may
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Preventing the Forward Contamination of Europa cover as much as 10 percent of Europa’s surface. Thus, the task group assigned the very conservative value of 0.1 to factor F5, the probability of landing at a site where activity might bury the spacecraft or a significant part of it within 7,000 years, allowing eventual access to a global ocean. F6—Burial Fraction Once a spacecraft is on the europan surface, the probability of an organism reaching the ocean would depend on the lag time until the spacecraft was buried at depths greater than about 10 meters to totally protect it from radiation. It would also depend on the intrinsic shielding within the spacecraft and the extent to which a portion of the spacecraft was buried into the near surface upon coming into contact with it (e.g., in the case of a crash of an orbiter, or a purposeful penetration by a hard lander). The dose profile within the surface ice is a strong function of depth (approximately, inverse square), as shown in Figure 2.3 in Chapter 2. For example, if a portion of the spacecraft is buried to 10 cm, it will take only 90 years to accumulate 7 Mrad of dose, but if it is buried to 1 meter the time to 7 Mrad will be 7,000 years. For this illustrative calculation, 50 percent of the spacecraft was assumed to be protected by being buried in ice to a depth of 1 meter or more. F7—Probability That an Organism Survives and Proliferates It is the consensus of all members of the task group that the likelihood that some terrestrial organism can survive and proliferate in an arbitrary environment on some other body is intrinsically small. However, because such extreme diversity is found among terrestrial microorganisms, this probability cannot be assumed to be zero. As difficult as it is to make such an estimate, the task group hazarded a guess by considering four factors that are pertinent to the survival and growth of any organism in any environment: It must survive the physicochemical properties of all environments to which it is exposed on the way to a final environment in which it can prosper; The final environment must provide key nutrients; A source of energy that the organism can exploit must be available; and The organism must be able to grow and reproduce in the final environment in which these nutrients and energy resources are available. As is indicated in the following considerations, this probability may well be as low as 10-6. Yet, because every spacecraft possesses a certain bioload, the extremely low probability is not per se sufficient to eliminate a need to maintain cleanliness and to control the bioburden. The task group analyzed this probability by breaking it down into four separate components (subfactors 7a, 7b, 7c, and 7d), each of which must be satisfied for an organism to survive and multiply once it reaches the europan ocean. F7a—Survivability of Exposure Environments Other than the correlation between radiation and desiccation resistance, very little is known about the survival of terrestrial organisms in combinations of environmental extremes. Given this lack of information, the task group assumes that the ability to survive in one set of physical and chemical conditions does not predispose an ability to survive in other conditions. The factors relevant to survival on Europa include pH, ionic strength, toxic ions, cold temperatures throughout the ocean, and the high pressures at depth. Organisms that do not lyse or become poisoned in this environment and are psychrotolerant and barotolerant could survive in a dormant state as currents move them to different regions of the hydrosphere. For this calculation, 20 percent of organisms are assumed to survive. This conservative value may need to be revised downward if recent suggestions that both hydrogen peroxide and sulfuric acid are relatively abundant in Europa’s surface ice are confirmed.25,26 F7b—Availability of Nutrients Elemental nutrients are needed by organisms to synthesize key biomolecules. Especially important are carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. Other elements, such as sodium, potassium, chlorine, magnesium, and calcium, are needed to maintain electrolyte balance. Virtually all of these are likely to be available in a salty ocean, in various forms of dissolved compounds or—in some cases—as gases. Trace ions, such as the transition elements needed as enzyme cofactors, are generally present in terrestrial seawater and should be present in a europan ocean. The
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Preventing the Forward Contamination of Europa balance of ions, or the potential nonavailability of some key resource such as phosphorus or nitrogen, could, however, be limiting. Certain molecular nutrients are required by many organisms. The task group took the probability that the entire suite of needed components are present as 50 percent. F7c—Suitability of Energy Sources If life already exists on Europa, primary production by chemoautotrophs might support a food web that produces organics and hence contains heterotrophic populations. The task group made a few a priori estimates of the likelihood that an Earth microbe, either heterotrophic or autotrophic, would grow in a novel chemosphere or the likelihood that it would displace indigenous microbiota in a novel biosphere. This lack of fundamental knowledge requires a conservative approach to europan exploration. Chemolithotrophic microorganisms include both aerobes and anaerobes. Energy sources for aerobic varieties include hydrogen, ammonia, reduced iron, and sulfur. These serve as electron donors for the respiration of oxygen. Anaerobic chemolithotrophs have the capacity to use these same energy sources when there is an appropriate electron-accepting species other than oxygen, such as nitrate, nitrite, or carbon dioxide. Both anaerobes and aerobes are widespread in soils and are therefore expected to be potential contaminants in assembly areas that have unfiltered conduits to outside air.27 Since anaerobic chemolithotrophs may be more suited to a europan ocean whether or not indigenous biota are present, the task group discussed only this group in more detail. Although their requirement for an oxygen-free environment for growth would seem to suggest that anaerobes would be less likely contaminants in an assembly area, it has been well documented in numerous studies that viable anaerobic bacteria are present in oxic, well-drained soils.28,29Recoverable anaerobes include non-spore-forming autotrophic types such as methanogens as well as many spore-forming varieties, most notably the acetogens. Both methanogens and acetogens have the capacity to grow on molecular hydrogen and carbon dioxide, using these substrates for carbon and energy, yielding either methane or acetate as the main end product of metabolism. In addition, many acetogens also have the capacity to grow heterotrophically. Sulfate reducers combine sulfate salts and reducing species such as molecular hydrogen as an energy source and produce reduced compounds such as elemental sulfur or hydrogen sulfide. The relative and absolute abundance of anaerobic species has been estimated for a variety of soils using culture-based methods. The task group emphasizes, as discussed above, that these methods have probably greatly underestimated the abundance of most chemolithotrophic populations.30 For example, a study of autotrophic H2/CO2- consuming methanogens showed that the numbers determined by culturing were about an order of magnitude lower than the numbers estimated by direct microscopic examination.31Facultative anaerobes generally constitute about 10 percent of the total aerobic population and are several orders of magnitude more common than the obligate anaerobes. In one study of the soil in a beech forest, the concentration of H2/CO2-utilizing anaerobes ranged from approximately 10 to 1,000 cells/g. Since these results are culture-based, they are almost certainly significant underestimates.32 The task group also noted that people can harbor significant numbers of autotrophs, including methanogens and acetogens, in their gut. 33 Thus, autotrophic anaerobes are anticipated to be common contaminants in most spacecraft assembly environments. The probability that for any given assembly of organisms found on a spacecraft there will be a species that is capable of utilizing the exact energy couples available in the europan ocean is, of course, small because of the natural diversity of these populations; this factor is taken as 0.001. F7d—Suitability for Active Growth Here the task group considered the two most likely growth environments: the rock-water interface at the bottom of an ocean and the water-ice interface at the top of the ocean. At the rocky boundary, rock-water hydrothermal conditions analogous to Earth’s deep-sea hydrothermal vents may occur and produce energy couples that can be exploited. However, because of the extreme depth of Europa’s ocean (~80 to 170 km) compared to Earth’s, which is only a few kilometers deep, the environment will be at a much higher pressure, even given the lower gravitational attraction of this smaller body. To prosper here, organisms would have to be highly barophilic. Alternatively, psychrophilic organisms might inhabit the water-ice interface just below the surface ice layer, exploiting the reaction of chemically reduced components in the ocean water with oxidized species from the ice surface or utilizing organic or other compounds in the water itself. Such organisms would have to be highly psychrophilic, however, because of the near-freezing temperatures that would prevail. For this sample calculation, the task group took the likelihood of a suitable organism to be no more than 1 percent of the organisms that are preadapted to the other environmental factors given previously.
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Preventing the Forward Contamination of Europa Joint Probability for F7 The four subfactors that make up F7are summarized in Table A.2, along with the calculated joint probability. TABLE A.2 Basis of F7 Subfactor Probability F7a – Survivability of exposure environment 2.0 × 10-1 F7b – Availability of nutrients 5.0 × 10-1 F7c – Suitability of energy sources 1.0 × 10-3 F7d – Suitability for active growth 1.0 × 10-2 F7 1.0 × 10-6 Conclusions from the Illustrative Calculation The conclusion from this admittedly highly approximate type of calculation are given in Table A.3. They indicate that sterilization of a relatively clean spacecraft by the natural radiation environment can be sufficient to protect the europan ocean environment. The task group notes that the 10-4 standard is met if all portions of the spacecraft receive a radiation dose of 7 Mrad. But a 6-Mrad dose would fall far short of being sufficient to achieve the 10-4 standard. On the other hand, an 8-Mrad exposure would appear to give extremely favorable results regardless of most other assumptions. TABLE A.3 Probability of Contamination Type D Type C Type B Type A Number of Culturable Organisms on Spacecraft 1.5 × 105 3.0 × 103 3.0 × 106 1.5 × 108 F1—Total cells/CFUs 1.0 × 103 1.0 × 103 1.0 × 103 1.0 × 103 F2—Bioburden reduction treatment 1.0 1.0 1.0 1.0 F3—Cruise survival fraction 0.50 1.0 1.0 0.10 F4—Radiation survival fraction* 1.0 × 10-5 1.0 × 10-10 1.0 × 10-10 1.0 × 10-1 F5—Probability of landing at an active site 0.10 0.10 0.10 0.10 F6—Fraction buried under more than 1 m of ice 0.50 0.50 0.50 0.50 F7—Probability of survival and proliferation 1.0 × 10-6 1.0 × 10-6 1.0 × 10-6 1.0 × 10-6 Product of Factors and Organisms 3.8 × 10-5 1.5 × 10-11 1.5 × 10-8 7.5 × 10-8 Sum 3.8 × 10-5 * Values for 7-Mrad dose Another conclusion reached from this particular sample analysis is that to meet the requirement that Pc ≤ 10-4, it will be necessary to do at least one of the following: Demonstrate that no Type C or Type D organisms are on the spacecraft; or Demonstrate that the probability of impacting the surface is less than 10-4 for the entire time the spacecraft is in the vicinity of Europa (regardless of whether the spacecraft is operational or not); or Show by probabilistic calculations that the 10-4 standard can be met through a combination of spacecraft cleaning, selective and/or whole-spacecraft sterilization, and exposure of the spacecraft to the radiation environment at Europa for a long enough period of time to reduce the bioload to the required level (“near sterilization”). Option 1 is not practical because only a small fraction of microorganisms are culturable. Option 2 may not be possible because of the chaotic perturbations on spacecraft orbits near Europa. Option 3 is achievable using current practices.
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Preventing the Forward Contamination of Europa Any given mission will need to be individually analyzed. The illustrative calculation presented here indicates that the administration of a dose of at least 7 Mrad to all portions of the spacecraft that have not been previously sterilized and protected from in-flight recontamination is needed to complete sterilization of the most radiation-resistant organisms (Type D). If Type D organisms are present at lower levels than assumed in the example, a lower exposure will be adequate. To achieve the desired final value, additional approaches could be taken, including the following: Bioburden reduction—The task group’s calculation assumed that no pre-launch sterilization procedures are administered to reduce the bioburden of the spacecraft (i.e., F2 was assumed to be 1.0). Many technologies could be applied to reduce the bioburden, including heat treatment of the entire spacecraft after cleaning (as was done for Viking), exposure to gaseous chemicals, ultraviolet irradiation of various surfaces, and so forth. Clean-room ecology—Research to elucidate the populations of organisms in the four categories may show that the assumed number is higher than the number of organisms present in actual assembly conditions for Europa spacecraft. Impact circumstances—Another factor that could significantly affect the outcome of such calculations are the analyses of how an orbiter spacecraft might impact the surface and to what depths it would be buried. Resurfacing rates at various locations on Europa, and the fractional areas covered, are also highly relevant to this problem. REFERENCES 1 National Aeronautics and Space Administration, Office of Space Science, Planetary Protection Provisions for Robotic Extraterrestrial Missions ,NPG 8020.12B, Washington, D.C., 1999, Appendix A. 2 J.R. Puleo et al., “Microbiological Profiles of the Viking Spacecraft,” Applied Environmental Microbiology 33: 379, 1977. 3 J.R. Puleo et al., “Microbiological Profiles of the Viking Spacecraft,” Applied Environmental Microbiology 33: 379, 1977. 4 M.S. Favero, “Microbiologic Assay of Space Hardware,” Environmental Biology and Medicine 1: 27, 1971. 5 J.R. Puleo ey al., “Microbiological Profiles of the Viking Spacecraft,”Applied Environmental Microbiology 33: 379, 1977. 6 V. Torsvik, J. Gokosyr, and F.L. Daae, “High Diversity of DNA Soil Bacteria,” Applied Environmental Microbiology 56: 782, 1990. 7 J.G. Jones, “The Effect of Environmental Factors on Estimated Viable and Total Populations of Planktonic Bacteria in Lakes and Experimental Enclosures, ” Freshwater Biology 7: 67, 1977. 8 M. Wagner et al., “Probing Activated Sludge with Proteobacteria-Specific Oligonucleotides: Inadequacy of Culture-Dependent Methods for Describing Microbial Community Structure,” Applied Environmental Microbiology 59: 1520, 1993. 9 M. Wagner et al., “Development of rRNA-Targeted Oligonucleotide Probe Specific for the Genus Acinetobacter and Its Application for In Situ Monitoring in Activated Sludge,” Applied Environmental Microbiology 60: 792, 1994. 10 K. Kogure, U. Shimidu, and N. Taga, “Distribution of Viable Marine Bacteria in Neritic Seawater Around Japan,” Canadian Journal of Microbiology 26: 318, 1980.
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Preventing the Forward Contamination of Europa 11 R.L. Ferguson, E.N. Buckley, and A.V. Palumbo, “Response of Marine Bacterioplankton to Differential Filtration and Confinement,” Applied Environmental Microbiology 47: 49, 1984. 12 J.R. Battista, “Against All Odds: The Survival Strategies of Deinococcus radiodurans,” Annual Reviews of Microbiology 51: 203, 1997. 13 G.D. Ledney et al., “Neutron and Gamma Radiation Killing of Bacillus Species Spores: Dosimetry, Quantitation, and Validation Techniques,” Technical Report 96-1, Armed Forces Radiobiology Research Institute, Bethesda, Md., 1996. 14 G.J. Silverman, “Sterilization and Preservation by Ionizing Radiation,” Disinfection Sterilization and Preservation, 4th edition, S.S. Block (ed.), Lea and Febiger, Philadelphia, Pa., 1991, page 556. 15 G.D. Ledney et al., “Neutron and Gamma Radiation Killing of Bacillus Species Spores: Dosimetry, Quantitation, and Validation Techniques,” Technical Report 96-1, Armed Forces Radiobiology Research Institute, Bethesda, Md., 1996. 16 M.J. Daly et al., “In Vivo Damage and recA-Dependent Repair of Plasmid and Chromosomal DNA in the Radioresistant Bacterium Deinococcus radiodurans,” Journal of Bacteriology 176: 3508, 1994. 17 G.D. Ledney et al., “Neutron and Gamma Radiation Killing of Bacillus Species Spores: Dosimetry, Quantitation, and Validation Techniques,” Technical Report 96-1, Armed Forces Radiobiology Research Institute, Bethesda, Md., 1996. 18 J.R. Battista, “Against All Odds: The Survival Strategies of Deinococcus radiodurans,” Annual Reviews of Microbiology 51: 203, 1997. 19 M.J. Daly et al., “In Vivo Damage and RecA-Dependent Repair of Plasmid and Chromosomal DNA in the Radioresistant Bacterium Deinococcus radiodurans,”Journal of Bacteriology 176: 3508, 1994. 20 G.J. Silverman, “Sterilization and Preservation by Ionizing Radiation,” inDisinfection terilization and Preservation, 4th edition, S.S. Block (ed.), Lea and Febiger, Philadelphia, Pa., 1991, page 556. 20 G.J. Silverman, “Sterilization and Preservation by Ionizing Radiation,” inDisinfection terilization and Preservation, 4th edition, S.S. Block (ed.), Lea and Febiger, Philadelphia, Pa., 1991, page 556. 22 C. Lange et al., “Construction and Characterization of Recombinant Deinococcus radiodurans for Organopollutant Degradation in Radioactive Mixed Waste Environments, ” Nature Biotechnology 16: 929, 1998. 23 A.C. Ferreira et al., “Deinococcus geothermalis and Deinococcus murrayi, Two Extremely Radiation-Resistant and Slightly Thermophilic Species from Hot Springs,” International Journal of Systematic Bacteriology 47: 939, 1997. 24 R.T. Pappalardo and R.J. Sullivan, “Evidence for Separation Across a Gray Band on Europa,” Icarus 123: 557, 1996. 25 R.W. Carlson et al., “Hydrogen Peroxide on the Surface of Europa,” Science 283: 2062, 1999. 26 R.W. Carlson, R.E. Johnson, and M.S. Anderson, “Sulfuric Acid on Europa and the Radiolytic Sulfur Cycle,” Science 286: 97, 1999.
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Representative terms from entire chapter: