Molecular Approaches for Quantifying and Characterizing the Microbiome of Low-Biomass Spacecraft Environments

Microbial contamination poses a critical risk to the scientific integrity of astrobiology missions, particularly those aimed at detecting life or returning samples from potentially habitable extraterrestrial environments. To comply with NASA Planetary Protection (PP) standards, spacecraft surfaces are currently evaluated using the NASA Standard Assay (NSA) - a cultivation-based method that detects only heat-shock-resistant, aerobic, heterotrophic, spore-forming bacteria. The overarching goal of this study is to establish and validate a next-generation molecular framework for quantifying (qPCR) and characterizing microbial burden (shotgun metagenome sequencing) on spacecraft-associated environmental surfaces. We employ an integrated approach combining optimized sample collection, metagenomic sequencing, and dPCR-based enumeration to enable high-resolution, contamination-resilient microbial surveillance in support of NASA's evolving PP needs. This study addresses a critical knowledge and capability gap by directly comparing molecular tools for determining cleanliness of spacecraft associated surfaces and evaluating their sensitivity, specificity, and reproducibility across a range of surface types and bioburden levels.