Viable microbial ecology and functional potential of the Integrated Lunar-Mars Analog Habitat revealed by multi-omics analyses

Long-duration human missions to the Moon and Mars will rely on closed, self-sustaining habitats where microorganisms inevitably interact with crew, materials, and life-support systems. While routine cleaning and environmental control can limit microbial growth, viable populations may persist and adapt to resource limitation, environmental parameter fluctuations, and disinfection stress. Distinguishing living from dead biomass is therefore essential for risk assessment. However, few studies have examined the viable microbiome of confined, human-occupied analogs using multi-omics, viability-targeted approaches. Across a 21-day continuous-occupation trial in the Integrated Lunar-Mars Analog Habitat (ILMAH), propidium monoazide-treated digital PCR (PMA-dPCR) assay results revealed strong spatial heterogeneity in viable bacterial loads (10 8 to 10 11 16S rRNA gene copies m − 2 ), with consistently higher burdens in high-traffic modules (Entrance, Kitchen, Bathroom, Crew Quarters) than low-traffic modules (Welch’s Δ = 4.11 log₁₀; p < 0.001; ANOVA F₇,₃₂ = 6.7, p < 0.001). Viable fungal loads were more uniform and did not differ by location ( p = 0.38). Intracellular ATP tracked viable microorganisms and correlated with PMA-dPCR across modules, whereas culture counts determined using “ready-to-use dry-sheet culture media” were 2–3 logs lower, consistent with a substantial viable but nonculturable fraction. From 240 isolates, 140 bacteria and 100 fungi were characterized; 42 bacterial and 26 fungal species were identified, including three putative novel fungal taxa. Microbiome characterization using 16S rRNA gene amplicon sequencing from PMA-treated samples showed location-driven community structure (PERMANOVA p = 0.021) dominated by Pseudomonas , with episodic Acinetobacter enrichment in low-traffic areas. Shotgun metagenomic sequencing revealed strong spatial structuring of viable microbiomes, with high-traffic modules enriched in Pseudomonas , while low-traffic modules showed increased fungal prevalence and greater taxonomic diversity. Metagenomics further detected low-abundance viable biosafety level-2 taxa (e.g., Salmonella enterica , Serratia species) and concentrated antimicrobial resistance genes and virulence factor signatures in high-traffic modules, largely carried by Pseudomonas . The ILMAH habitat supports a spatially differentiated, stress-tolerant core microbiome maintained by human occupancy and selective survival. Viability-based assays reveal that while total DNA may overestimate viable microbial diversity, the living community is low in richness yet high in resilience. This integrative workflow establishes a foundation for biosurveillance and microbial-risk management in future long-duration spaceflight and other sealed ecological systems. Video Abstract