Defining Core Cleanroom Microbiomes to Inform a Probabilistic Risk Assessment Framework for Planetary Protection

Kurzfassung

Planetary protection (PP) aims to minimise the forward contamination of extraterrestrial environments by terrestrial microorganisms. Initial guidelines for planetary protection were directed by a probabilistic approach (1 ´ 10-4). Post-Viking, bioburden limits/ spore counts were introduced to the policy for target bodies like Mars, as it was concluded that Mars was less hospitable than initially believed. Yet, the probabilistic approach is still applied to Category III and IV (e.g., Europa Clipper) and Category V (e.g. Mars sample return) missions. This approach uses mathematical models to calculate the probability of the initial microbial contamination from a spacecraft contaminating a target body. It could benefit more complex missions where there is a need for a more advanced approach to planetary protection. Existing approaches rely largely on measuring microbial spores as a proxy for contamination. However, to develop a probabilistic approach it is critical to identify the problematic microorganisms that exist in cleanrooms. These microorganisms have adapted to extreme conditions of cleanroom facilities, for example, desiccation, nutrient limitation, and exposure to cleaning agents. Here, we present a framework for characterising core microbial taxa across multiple spacecraft-associated cleanrooms using integrated culture-dependent and -independent approaches (e.g., amplicon sequencing, metagenomics, and viability assays). By analysing temporal and spatial datasets, we define core taxa as those consistently detected across environments and resilient to standard cleaning protocols. Functional profiling further reveals metabolic traits linked to stress tolerance, dormancy, and potential survivability under space-relevant conditions. We propose that these core organisms represent a disproportionately relevant subset for forward contamination risk, and should form the basis of a probabilistic approach. By incorporating organism-specific traits—such as resistance to radiation, desiccation, and low temperatures—alongside likelihood of transfer and survival during mission phases, models can move beyond total bioburden to a risk-weighted microbial inventory. This approach provides a step change in policy protection development by linking microbial ecology with mission-specific risk profiles. Ultimately, identifying and quantifying core cleanroom microbiomes enables more targeted mitigation strategies, supports evidence-based policy evolution, and enhances our ability to protect potentially habitable environments on Mars and icy worlds.