Integrated Microbial Surveillance in Confined Habitats Using a Multi-Omics Approach to Assess Bioburden, Microbial Diversity, and Functional Characteristics

Space habitats are isolated systems where microbial populations are primarily influenced by human occupants, cargo, and life support infrastructure. In such environments, microbial succession may lead to the dominance of opportunistic pathogens, persistent biofilms, and material degradation- all of which can compromise astronaut health and spacecraft integrity. The primary objective of this study was to evaluate microbial dynamics within the Integrated Lunar Mars Analog Habitat (ILMAH) - a human-occupied closed system here on Earth designed as an analog for future space habitats. A 21-day continuous occupation scenario was simulated to monitor and characterize how microbial communities responded to human activity in a sealed, resource-limited environment. To achieve this, a multi-faceted and integrated microbial diversity pipeline was employed, combining culture-based, biochemical, molecular, and shotgun metagenomic techniques to provide a robust, layered understanding of microbial presence, viability, and functional potential. The Kikkoman Easy Plate assay was used in place of traditional agar-based cultivation to enumerate viable bacterial and fungal populations. ATP bioluminescence assays were conducted to estimate total biological activity rapidly. Digital PCR (dPCR) was performed to detect microbial gene targets, including universal 16S rRNA gene (bacteria) and ITS (fungi) targets. Shotgun metagenomic sequencing was carried out to obtain a comprehensive taxonomy of viable microbes after PMA treatment and their functional profiles of the microbial communities. Through this approach, a validated microbial surveillance workflow was established, offering foundational insights for application in future NASA missions.