The research, published in the journal Microbiome, describes a 21-day continuous-occupation trial in which a student crew lived inside the ILMAH while researchers sampled surfaces across eight modules. Rather than simply cataloging all the DNA left behind on surfaces, the team, led by Haley O. Boles of the University of North Dakota and Kasthuri Venkateswaran of NASA’s Jet Propulsion Laboratory, used viability-targeted methods designed to distinguish living microorganisms from dead biomass, a distinction that matters enormously for assessing actual risk to crews on long-duration missions.

The technique at the heart of the study is propidium monoazide treatment combined with digital PCR, known as PMA-dPCR. Propidium monoazide is a DNA-binding dye that cannot penetrate intact cell membranes; it infiltrates only dead cells, where it binds DNA and prevents amplification. By applying the dye before quantifying 16S rRNA gene copies, the researchers effectively filtered their measurements down to organisms with intact, presumably viable, cells.

The results revealed dramatic spatial heterogeneity in viable bacterial loads, which ranged from 10^8 to 10^11 16S rRNA gene copies per square meter of surface. High-traffic modules, including the Entrance, Kitchen, Bathroom, and Crew Quarters, carried consistently higher burdens than low-traffic areas, with a difference of roughly 4.11 log units, a gap that was highly statistically significant. Fungal loads, by contrast, were far more uniform across the habitat and did not differ meaningfully by location, suggesting that fungi distribute themselves more evenly regardless of where crew members spend their time.

To corroborate the PMA-dPCR measurements, the team measured intracellular ATP, the energy-carrying molecule found in all living cells, which tracked closely with the viable cell counts across modules. Culture-based counts using ready-to-use dry-sheet culture media told a different story: they were two to three orders of magnitude lower than the viability-adjusted molecular estimates. That gap points to a substantial viable-but-nonculturable fraction, a well-known phenomenon in microbiology in which cells are alive and metabolically active but will not grow under standard laboratory conditions. In a habitat context, it means routine culturing alone would dramatically understate the living microbial burden surrounding a crew.

From the habitat’s surfaces, the researchers isolated and characterized 240 strains, 140 bacteria and 100 fungi, encompassing 42 bacterial and 26 fungal species. Among these were three putative novel fungal taxa, a reminder that even analog habitats on Earth can harbor microbes new to science. Amplicon sequencing of the 16S rRNA gene from PMA-treated samples showed that community structure was driven by location, dominated by the genus Pseudomonas, with episodic enrichment of Acinetobacter in the quieter, less-visited modules.

Shotgun metagenomic sequencing, which reads all genetic material in the samples rather than just a single marker gene, added functional depth. It confirmed strong spatial structuring of the viable microbiome: high-traffic modules were enriched in Pseudomonas, while low-traffic modules hosted greater fungal prevalence and higher taxonomic diversity. The functional potential of these communities followed the same geographic logic, with the busiest areas concentrating metabolic and survival machinery adapted to frequent disturbance and resource scarcity.

Perhaps the most consequential findings concern biosafety. Metagenomics detected low-abundance viable biosafety level-2 taxa, including Salmonella enterica and Serratia species, and revealed that antimicrobial resistance genes and virulence factor signatures were concentrated in the high-traffic modules, largely carried by Pseudomonads. The picture that emerges is not a random smear of contamination but a selectively filtered ecosystem in which the most resilient, potentially problematic organisms accumulate precisely where humans touch most often.

The authors conclude that the ILMAH supports a spatially differentiated, stress-tolerant core microbiome maintained by human occupancy and selective survival. Interestingly, the viability-based approach cuts both ways: while total DNA measurements can overestimate the diversity of living organisms by including fragments from dead cells, the community that does survive is low in richness yet high in resilience. In other words, sealed habitats select for a small cast of tough generalists rather than a wide array of species.

The practical implications reach well beyond spaceflight. The integrative workflow established in this study, combining PMA-dPCR, ATP measurement, culture, amplicon sequencing, and shotgun metagenomics, offers a template for biosurveillance in any sealed ecological system, from submarines and hospital isolation wards to deep-space transit vehicles. As agencies plan multi-year missions to Mars, knowing which microbes are alive, where they cluster, and what they can do may prove as important to crew health as monitoring radiation or air quality. This study provides the first rigorous, viability-aware baseline for that task.

Subject of Research: The viable microbial ecology and functional potential of the Integrated Lunar-Mars Analog Habitat, characterized using viability-targeted multi-omics analyses during a 21-day human occupation trial.

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

Article References: Boles, H. O., Hernandez, E., Kahsen, J., Suzuki, T., Okochi, N., Jeon, T., Naik, S., Joshi, N., Tumeo, A., Lin, W.-J., Miliotis, G., de León, P., Green, S. J., & Venkateswaran, K. (2026). Viable microbial ecology and functional potential of the Integrated Lunar-Mars Analog Habitat revealed by multi-omics analyses. Microbiome. https://doi.org/10.1186/s40168-026-02516-4

Image Credits: AI Generated

DOI: 10.1186/s40168-026-02516-4

Keywords: ILMAH, space microbiome, PMA-dPCR, metagenomics, Pseudomonas, antimicrobial resistance, viable but nonculturable, analog habitat, biosurveillance, spaceflight, 16S rRNA, ATP