A 4-person, 180-day controlled ecological life support system (CELSS) confinement experiment ('Green Star 180') showed that the salivary and faecal microbiomes and the plasma, urinary and faecal metabolomes of confined crew members remain broadly stable while undergoing discrete, phase-structured shifts: faecal taxa labelled anti-inflammatory trended upward and proinflammatory taxa downward, differential faecal species correlated (|r|>0.6) with the plasma phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratio, entry into confinement enriched salivary periodontitis-associated taxa together with metabolites linked to biofilm formation and alveolar bone loss, and Streptococcus salivarius subsp. salivarius and S. dentisani were differentially abundant at both oral and gut sites. All of these observations are correlational in a cohort of four. The unresolved problem is whether these confinement-associated microbial and metabolomic shifts causally affect host inflammation and clinical disease risk, whether they generalize to larger cohorts, other CELSS implementations, and real spaceflight, whether the oral-gut co-occurring taxa reflect transmission rather than shared environmental selection, and whether the associated metabolites and taxa predict clinical endpoints during long missions. Follow-up work through 2026 has replicated parts of the descriptive picture in other closed habitats but has not established causality, strain-level transmission, or prospective clinical prediction, so the problem remains open.

Background

Controlled ecological life support systems (CELSS, also called bioregenerative life support systems) are closed habitats in which food, water and atmosphere are regenerated largely by plants and microbes; they are ground analogues for spacecraft and planetary bases on long-duration missions. Because crews live for months in a sealed, diet-controlled, shared microbial environment, CELSS experiments are used to ask how long-term confinement itself — as distinct from microgravity or radiation — reshapes the human microbiome (the microbial communities at body sites such as the gut and the mouth) and the metabolome (the small-molecule profiles of plasma, urine and faeces), and whether such reshaping matters for health.

The largest such study of its time with multi-omic sampling was the 'Green Star 180' experiment: four healthy volunteers ('simulated astronauts') lived 180 days in a CELSS in Shenzhen, China (2016), through a simulated Mars-mission profile with distinct phases (space travel, 'Mars solar day', and post-confinement). Feng and colleagues reported in Gut a time-series multi-omics analysis of this cohort: salivary and faecal microbiome profiling by 16S rRNA gene sequencing (which identifies and quantifies bacterial taxa through a standard phylogenetic marker gene) at 18 time points before, during and after confinement, paired with plasma, urinary and faecal metabolomics. Their findings included: (i) overall the oral and gut microbiota stayed generally stable, with discrete phase-structured changes; (ii) directional trends in alpha diversity (within-sample diversity), measured by the phylogenetic diversity whole-tree index (the total branch length of the evolutionary tree spanned by a sample's species), and in position along the first principal coordinate axis (the leading axis of a between-sample ordination summarizing the greatest variation in community composition) moved in opposite directions in faeces versus saliva but did not reach statistical significance (p≥0.05); (iii) the Firmicutes:Bacteroidetes phylum balance (the ratio between the two dominant gut bacterial phyla) shifted concomitantly at both sites between mission phases; (iv) faecal taxa characterized in the literature as anti-inflammatory increased over confinement while putatively proinflammatory taxa decreased; (v) correlation-network analysis linked differential faecal species to the plasma ratio of phosphatidylcholine to phosphatidylethanolamine (PC:PE), a membrane-lipid ratio previously implicated in inflammatory and metabolic liver disease; (vi) salivary entry-phase enrichments concerned taxa associated with periodontitis (gum disease driven by dental-plaque biofilms), and the co-enriched salivary metabolites are associated with biofilm formation, alveolar bone loss (jawbone resorption around teeth, a hallmark of periodontal disease) and systemic disease; and (vii) two Streptococcus species (S. salivarius subsp. salivarius and S. dentisani) were differentially abundant and co-present in both saliva and faeces, raising — but not resolving — the question of oral-to-gut transmission versus independent site-specific dynamics under a shared, closed environment.

The authors were explicit that these results are correlative in a four-person cohort: associations such as the faecal-taxon–plasma PC:PE coupling suggest plausible links to host inflammation but do not demonstrate causation. In the years since, related closed-habitat studies (notably the 370-day, multicrew 'Lunar Palace 365' experiment in the Lunar Palace 1 facility) have mapped salivary and gut microbial dynamics, in one case paired with salivary cytokines (immune-signalling proteins such as IL-6, IL-10 and TNF-α), and short 28-day confinement operations have paired gut microbiota with serum metabolomics and inflammatory proteins. Real-spaceflight multi-omics (the NASA Twins Study and the Inspiration4/SOMA programme) have sampled astronauts' microbiomes but at tiny sample sizes and without causal or prospective-clinical designs. Authoritative reviews of the space microbiome (Clinical Microbiology Reviews, 2024) still describe the long-term health implications of spaceflight-associated microbial changes as not yet known and the expected chronic outcomes as not yet observed in space. Strain-level metagenomic work in general (non-confinement) populations has since shown that oral microbes transmit horizontally between cohabiting individuals and can seed the gut, making the transmission hypothesis testable but leaving it untested in confinement cohorts.

Problem Statement

Determine the host-health significance of the microbiome and metabolome shifts observed during long-term isolation confinement in the Green Star 180 CELSS study — that is, convert the reported correlative observations into validated knowledge by establishing: (1) whether the confinement-associated shifts (rise of putatively anti-inflammatory and decline of putatively proinflammatory faecal taxa; coupling of differential faecal taxa to the plasma phosphatidylcholine:phosphatidylethanolamine ratio; enrichment of salivary periodontitis-associated taxa and biofilm- and alveolar-bone-loss-associated metabolites at habitat entry) causally affect host inflammation or clinical disease risk, as opposed to being benign correlates of the confined regimen; (2) whether the reported compositional and metabolomic trends, several of which were directional but not statistically significant in the four-person cohort, replicate in larger cohorts, in modified CELSS implementations, and during real spaceflight; (3) whether the oral-gut co-occurring Streptococcus taxa (S. salivarius subsp. salivarius and S. dentisani) reflect person-to-person or oral-to-gut transmission under the closed habitat, shared environmental selection, or independent site-specific dynamics; and (4) whether the metabolites and taxon shifts associated with biofilm formation, alveolar bone loss, and systemic-disease signatures predict clinical endpoints during long missions. These four components form one translational objective: to decide whether confinement-associated microbial and metabolomic shifts are validated indicators and modifiable mediators of crew health risk during long-duration spaceflight, or merely correlates. The four components are independently resolvable: establishing or refuting any one of them leaves the others open.

References

  1. Feng Q, Lan X, Ji X, Li M, Liu S, Xiong J, Yu Y, Liu Z, Xu Z, He L, Chen Y, Dong H, Chen P, Chen B, He K, Li Y. Time series analysis of microbiome and metabolome at multiple body sites in steady long-term isolation confinement. Gut. 2021;70(7):1409.1-1412. doi:10.1136/gutjnl-2020-320666. https://doi.org/10.1136/gutjnl-2020-320666 (PMID 32912857)
  2. Dong HS, Chen P, Yu YB, Zang P, Wei Z. Simulated manned Mars exploration: effects of dietary and diurnal cycle variations on the gut microbiome of crew members in a controlled ecological life support system. PeerJ. 2019;7:e7762. doi:10.7717/peerj.7762. https://doi.org/10.7717/peerj.7762
  3. Chen Y, Xu C, Zhong C, Lyu Z, Liu J, Chen Z, Dun H, Xin B, Xie Q. Temporal characteristics of the oropharyngeal and nasal microbiota structure in crewmembers stayed 180 days in the controlled ecological life support system. Front Microbiol. 2020;11:617696. doi:10.3389/fmicb.2020.617696. https://doi.org/10.3389/fmicb.2020.617696
  4. Yuan M, Custaud MA, Xu Z, Wang J, Yuan M, Tafforin C, Treffel L, Arbeille P, Nicolas M, Gharib C, Gauquelin-Koch G, Arnaud L, Lloret JC, Li Y, Navasiolava N. Multi-system adaptation to confinement during the 180-day controlled ecological life support system (CELSS) experiment. Front Physiol. 2019;10:575. doi:10.3389/fphys.2019.00575. https://doi.org/10.3389/fphys.2019.00575
  5. Hao Z, Zhu Y, Fu Y, Yang J, Meng C, Dong C, Liu H. Effects of long-term enclosed environment on human health based on the analysis of salivary microbiota and cytokines. Microbiol Spectr. 2022;10(2):e00254-22. doi:10.1128/spectrum.00254-22. https://doi.org/10.1128/spectrum.00254-22
  6. Hao Z, Meng C, Li L, Feng S, Zhu Y, Yang J, Han L, Sun L, Lv W, Figeys D, Liu H. Positive mood-related gut microbiota in a long-term closed environment: a multiomics study based on the 'Lunar Palace 365' experiment. Microbiome. 2023;11:88. doi:10.1186/s40168-023-01506-0. https://doi.org/10.1186/s40168-023-01506-0
  7. Wang W, Hao Z, Guo Y, Ma X, Cui J, Liu H. Temporal dynamics of human gut microbiota revealed by the 'Lunar Palace 365' experiment. Acta Astronaut. 2026;245:126-136. doi:10.1016/j.actaastro.2026.02.048. https://doi.org/10.1016/j.actaastro.2026.02.048
  8. Sun F, Qin G, Zeng H, Guan M, Liu H, Qiu H, Wei T, Hu H. Effects of prolonged confined space operations on human gut microbiota and serum metabolome. Hum Nutr Metab. 2025;42:200347. doi:10.1016/j.hnm.2025.200347. https://doi.org/10.1016/j.hnm.2025.200347
  9. Etlin S, Rose J, Bielski L, Walter C, Kleinman AS, Mason CE. The human microbiome in space: parallels between Earth-based dysbiosis, implications for long-duration spaceflight, and possible mitigation strategies. Clin Microbiol Rev. 2024;37(3):e00163-22. doi:10.1128/cmr.00163-22. https://doi.org/10.1128/cmr.00163-22
  10. Garrett-Bakelman FE, Darshi M, Green SJ, Gur RC, Lin L, Macias BR, McKenna MJ, Meydan C, Mishra T, Nasrini J, Piening BD, Rizzardi LF, Sharma K, Siamwala J, Taylor L, Vitaterna MH, Afkarian M, Afshinnekoo E, Ahadi S, Ambati A, et al. The NASA Twins Study: a multidimensional analysis of a year-long human spaceflight. Science. 2019;364(6436):eaau8650. doi:10.1126/science.aau8650. https://doi.org/10.1126/science.aau8650
  11. Valles-Colomer M, Blanco-Míguez A, Manghi P, Asnicar F, Dubois L, Golzato D, Armanini F, Cumbo F, Huang KD, Manara S, Masetti G, Pinto F, Piperni E, Punčochář M, Ricci L, Zolfo M, Farrant O, Goncalves A, Selma-Royo M, Binetti AG, Becerra JE, et al. The person-to-person transmission landscape of the gut and oral microbiomes. Nature. 2023;614(7946):125-135. doi:10.1038/s41586-022-05620-1. https://doi.org/10.1038/s41586-022-05620-1
  12. Overbey EG, Ryon K, Kim J, Tierney BT, Klotz R, Ortiz V, Mullane S, Schmidt JC, MacKay M, Damle N, et al. Collection of biospecimens from the Inspiration4 mission establishes the standards for the Space Omics and Medical Atlas (SOMA). Nat Commun. 2024;15:4964. doi:10.1038/s41467-024-48806-z. https://doi.org/10.1038/s41467-024-48806-z
  13. Yang J, Kim HD, Barrila J, Lee SH, Nickerson CA, Ott CM, Israel SA, Choukér A, Yang JY. Navigating mental health in space: gut-brain axis and microbiome dynamics. Exp Mol Med. 2025;57(6):1152-1163. doi:10.1038/s12276-025-01488-z. https://doi.org/10.1038/s12276-025-01488-z