and radiation during the cruise phase will correspond to a subset of those that will succumb during orbit in a high-radiation flux around Europa or other icy moons. The factor F4 (radiation survival fraction) is part of F3 (cruise survival fraction), and F3, F4, and F6 (burial fraction) reflect non-independent measures of bio-reduction factor due to radiation flux. In this example, burial fraction dictates the radiation dose profile as a function of depth. The level of protection offered by burial over unit time correlates with estimates of radiation sensitivity as reflected by F4.

The environmental factors F7a through F7d constrain the survivability of organisms on or in the spacecraft and their ability to proliferate for a combined bio-load reduction of 10–6, but these factors either lack independence or use “survivability” as a substitute for the probability of growth, Pg, which is impossible to estimate. With respect to the independence of these factors, F7a will include radiation sensitivity as measured by F4. The factors F7b through F7d reflect non-independent environmental resources required for growth. The combination of the factors F7a through F7d substitutes for Pg, which most planetary protection studies assume to be unitary because of the complexity of predicting whether a microbe can or cannot grow under a given set of environmental conditions. By assigning probabilities less than 1 for the non-independent bio-reduction factors and the Pg-like estimates for “organism survivability and proliferation,” the Coleman-Sagan calculation can reduce the value of NXs by several orders of magnitude. Yet, with the exception of the geologically influenced parameter F5, all of these factors have dependencies on other factors.

Because the overall uncertainty factor in the final result from the Coleman-Sagan equation is greater than the uncertainty factor for the least-constrained variable, a three or four order-of-magnitude uncertainty in estimates of the number of organisms on spacecraft would lead to approximately a three or four order-of-magnitude uncertainty in the overall probability of contamination.

Even greater uncertainty arises from the inability to confidently assign values to many of these factors, including estimates of the number of viable microbes NX0, on the spacecraft prior to launch. As described in Chapter 1 of this report, the standard NASA assay of heat-resistant microbes serves as an indicator of the number of spores on the sampled spacecraft surfaces. These measurements provide no information about the number of heat-sensitive but radiation- and vacuum-resistant microbes on a spacecraft, nor do these surveys provide accurate estimates of heat-resistant spores that are refractory to cultivation. Over the past two decades culture-independent microbial diversity investigations based on comparisons of highly conserved sequences (ribosomal RNA genes) in Bacteria and Archaea demonstrate that microbiologists have successfully cultivated only a small fraction (<1 percent) of the different kinds of single-cell organisms that occur in nature.3 Deep-sequencing surveys suggest that microbial diversity may be 1,000 to 10,000 times greater than estimates from cultivation-based studies and that most of this novelty corresponds to low-abundance taxa described as the “rare biosphere.”4,5 Similar analyses of simple mock communities containing one or a few taxa suggested that sequencing errors can lead to inflated estimates of microbial diversity.6 More recent studies show that a 2 percent single-linkage preclustering methodology followed by an average-linkage clustering based on pair-wise sequence alignments more accurately predicts expected complexity of mock communities of known taxonomic composition. However, this analytical paradigm does not reduce the fraction of novel taxa in the long-tailed rank abundance curves that define the rare biosphere for complex, naturally occurring microbial communities. This implies that the standard spore assay likely underdetermines the number of heat-resistant organisms on a spacecraft. If many spore-forming organisms cannot grow under laboratory conditions, then growth-based assays of survival will not accurately report the size of the surviving populations.

Given current technology, non-rigorous estimates of NX0 can lead to significant underestimates of the number of organisms delivered to the target body. Estimates for other bioload reduction factors suffer similar uncertainties. The current inability to cultivate most of the different microbes that comprised by a community makes it impossible to estimate what fraction of a community succumbs to radiation and ultralow vacuum during cruise or orbit in a high-radiation environment. Because the overall uncertainty factor in the final result from the Coleman-Sagan equation is greater than the uncertainty factor for the least-constrained variable, a three or four order-of-magnitude

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