Planning future space explorations, involving potential human missions to Mars, would require constructing a safe closed habitat [1, 2]. An inflatable lunar/Mars analog habitat (ILMAH) is a unique, simulated closed environment (isolated by HEPA filtration) that can be utilized to overcome challenges associated with both technical and scientific issues [3]. Because the ILMAH mimics International Space Station (ISS) conditions and is treated as a prototype habitat for future space explorations, microbiological characteristics of such a closed environment is of high interest to the National Aeronautics and Space Administration (NASA). The environmentally controlled ILMAH is an easily accessible system that enables samples to be collected and analyzed at multiple times at relatively low cost. Understanding the microbiome of a closed system and its association with human inhabitation will help to assess the correlation between human health and microbiome of the habitat as well as the influence of microorganism on the habitat deterioration [4,5,6].

The highly specialized structure of the simulated ILMAH keeps its inhabitants in isolation from the outside environment. Except for the exchange of the air between outdoor and indoor environments via an advanced environmental control system, the ILMAH mimics the ISS and other future habitats of human explorers on the other planets [3]. This unique feature of the ILMAH allows observing the changes in the microbiome during human occupation. The bacteriome of the ILMAH was recently reported [7], as in the case of most of the studies reporting on bacterial microbiomes [8]. The molecular fungal diversity of Japanese Experimental Module—Kibo, on the ISS, revealed abundance of fungi associated with astronauts, but succession of viable fungal population in their habitat was not addressed [9]. The skin fungal microbiota of 10 Japanese astronauts showed temporal changes before, during, and after their stay on the ISS. The molecular fungal diversity associated with various body parts was reduced during the spaceflight when compared to pre-flight data. However, the ratio of Malassezia genetic signatures to all fungal gene copies (including dead fungal cells) increased during their stay at the ISS—but the viability of these fungi was not confirmed [10]. This is the first report that thoroughly characterizes the mycobiome of a simulated habitat meant for the future human habitats on other planets.

Utilization of next generation sequencing (NGS) techniques enables more in-depth analysis of indoor microbiomes [11]. Many studies focus on the bacterial microbiome of intensive care units [8, 12,13,14], pharmaceutical clean rooms [15,16,17], or tissue banks [18] since their microbial composition has an impact on human health and life. Nosocomial infections acquired in hospitals and other health care facilities remain the sixth leading cause of death in the hospitals in USA [19, 20]. Nosocomial infections are mostly caused by various fungal species that belong to the Candida genus and filamentous fungus Aspergillus fumigatus [21,22,23]. Therefore, it remains important to screen future closed habitats for the presence of opportunistic pathogens that can affect health of immunocompromised astronauts. So far, majority of the indoor microbiome studies have focused on the bacterial microbiome without analyzing the mycobiome. In addition, those few studies that characterized fungi of indoor environments focused on culture-based populations [24,25,26,27]. In those cases, where new molecular techniques were implemented [28,29,30,31] viable fungi were not differentiated from the total population (viable and dead) [32]. The internal transcribed spacer region-based iTag Illumina sequencing coupled with the propidium monoazide (PMA) treatment used in this study can determine the viable mycobiome.

Fungi are extremophiles that can survive harsh conditions such as low nutrient [33], desiccation [34], high/low temperatures [35, 36], acidic/alkaline [37, 38], radiation [39, 40], and other environments [41, 42]. Fungal species not only have been isolated from all known environments on Earth, including barren lands like deserts, caves, or nuclear accident sites, but also are known to be difficult to eradicate from other types of environments including indoor and closed spaces [8, 36, 42, 43].

Characterizing and understanding possible changes to, and succession of, fungal species in the ILMAH is of high importance since some of the fungi are extremophiles that are not only potentially hazardous to inhabitants but also can deteriorate the habitat itself [25, 44, 45]. It was previously reported that people spending a significant amount of time indoors might suffer from so called “sick building syndrome” (SBS). SBS is characterized by health- and comfort-related syndromes (e.g., headache, tiredness) that ease after leaving a building. Fatigue and discomfort might be caused not only by physical characteristics of the closed system (humidity, temperature, lighting) but also by biological contamination from both bacteria and fungi [46, 47]. Fungal pathogens’ presence in indoor areas might pose health hazards for people exhibiting immunodeficiency [48]. Pathogenic fungi produce a range of secondary metabolites (SMs) that influence their virulence (e.g., melanins, siderophores, or species-specific toxins), induce allergies, and cause diseases (e.g., aspergillosis, candidiasis, or cryptococcosis) [48, 49]. Prolonged stays in closed habitats (e.g., ILMAH, ISS, etc.) might be stressful for inhabitants and lead to a decrease in immune response; therefore, assessing the presence of any opportunistic pathogens is vital [50].

Previous reports on the mycobiome in NASA clean rooms and on the ISS documented NGS results from samples collected from various locations, but none of the studies focused on the analysis of the microbial succession of systematically collected samples [28, 32]. The bacterial and archaeal microbiome succession of the ILMAH using NGS has been carried out [7]. This is the first report characterizing the succession of fungi in a simulated closed system meant for human habitation on other planets utilizing both traditional and state-of-the-art molecular techniques. In addition, attempts were made during this study to elucidate the temporal and spatial distribution of the fungal population and diversity in a closed human habitat.