Human presence impacts fungal diversity of inflated lunar/Mars analog habitat

Abstract

Background: An inflatable lunar/Mars analog habitat (ILMAH), simulated closed system isolated by HEPA filtration, mimics International Space Station (ISS) conditions and future human habitation on other planets except for the exchange of air between outdoor and indoor environments. The ILMAH was primarily commissioned to measure physiological, psychological, and immunological characteristics of human inhabiting in isolation, but it was also available for other studies such as examining its microbiological aspects. Characterizing and understanding possible changes and succession of fungal species is of high importance since fungi are not only hazardous to inhabitants but also deteriorate the habitats. Observing the mycobiome changes in the presence of human will enable developing appropriate countermeasures with reference to crew health in a future closed habitat.

Results: Succession of fungi was characterized utilizing both traditional and state-of-the-art molecular techniques during the 30-day human occupation of the ILMAH. Surface samples were collected at various time points and locations to observe both the total and viable fungal populations of common environmental and opportunistic pathogenic species. To estimate the cultivable fungal population, potato dextrose agar plate counts method was utilized. The internal transcribed spacer region-based iTag Illumina sequencing was employed to measure the community structure and fluctuation of the mycobiome over time in various locations. Treatment of samples with propidium monoazide (PMA; a DNA intercalating dye for selective detection of viable microbial populations) had a significant effect on the microbial diversity compared to non-PMA-treated samples. Statistical analysis confirmed that viable fungal community structure changed (increase in diversity and decrease in fungal burden) over the occupation time. Samples collected at day 20 showed distinct fungal profiles from samples collected at any other time point (before or after). Viable fungal families like Davidiellaceae, Teratosphaeriaceae, Pleosporales, and Pleosporaceae were shown to increase during the occupation time.

Conclusions: The results of this study revealed that the overall fungal diversity in the closed habitat changed during human presence; therefore, it is crucial to properly maintain a closed habitat to preserve it from deteriorating and keep it safe for its inhabitants. Differences in community profiles were observed when statistically treated, especially of the mycobiome of samples collected at day 20. On a genus level Epiccocum, Alternaria, Pleosporales, Davidiella, and Cryptococcus showed increased abundance over the occupation time.

Keywords: Closed habitat; Mycobiome; Succession; Surface.

Fig. 1

Picture of the closed habitat from outside

Fig. 2

Statistical analysis of cultivable fungal diversity detected through the 30-day habitation period at all the locations based on colony-forming unit (CFU) counts. To assess the difference between fungal abundances in cultivable sample categories (based on time—A and location—B), we applied the following univariate statistics. The normal distribution of the populations were tested using Shapiro-Wilk normality test, and as most of them were not normally distributed (p value <0.05), we used a Kruskal-Wallis test coupled to a Dunn’s test to investigate differences in the tested populations. Resulting p values were corrected using the Benjamini-Hochberg correction. A CFU counts before crew occupation T₀—a were statistically different from CFU counts at T₂₀ and T₃₀—b, but no statistical difference was observed between T₀ and T₁₃ counts—ab. Additionally, no statistical differences were observed between any other time points. B CFU counts in the bedroom—a, differed significantly from the CFU counts in lab—b, but no statistical differences were observed between bedroom and kitchen or toilet—ab. No statistical differences were observed between any other locations

Fig. 3

Cultivable fungal diversity detected through the 30-day habitation period at all the locations based on internal transcribed spacer (ITS) sequences. The phylogenetic tree was constructed using neighbor-joining method (bootstrap 1000). In total, 117 isolates were collected 113 of which were successfully sequenced (4 strains either did not show growth or did not respond to the sequencing methods attempted). The numbering of the isolates is explained as follows: F = fungi, first number (0–4) will be the sample collection day (0 = T₀, 2 = T₁₃, 3 = T₂₀, 4 = T₃₀), second number (1–8) will be sampling location, and the third number (1–5) is the replicate number of the isolate. For example, F23-02 will be a fungal strain, isolated from T₂₀, at location number 3 and a second isolate. Frequency of isolates is given as a frequency bar after the name of fungus. Colors of the bars correspond to the collection time (single or multiple)

Fig. 4

NMDS ordinations based on Bray-Curtis distances between all samples. a Ordination displaying the distance between non-PMA-treated samples taken at the different time points. b The distance between PMA-treated samples taken at the different time points. c The distance between non-PMA-treated samples taken at the different locations. d NMDS ordination displaying the distance between PMA-treated samples taken at the different locations. A “P” after the respective variable indicates that these are the samples treated with PMA. Plots a, c, and b, d represent the same data but differ in colors to underscore the focus on distribution over time and location, respectively

Fig. 5

Linear representation of alpha diversity averages change over time for cultivable, viable and total mycobiome

Fig. 6

Dominant fungal population and succession patterns observed in 30-day occupation period of the ILMAH system. The OTUs presented in the bar graph are the most abundant. T₂₀ surface samples show different fungal profile when compared to other time points. T₃₀ samples show increase in fungal diversity when compared to other time point

Fig. 7

Box plots of viable dominant fungal families and their succession patterns observed in 30-day occupation period of the ILMAH system. The OTU counts presented in the boxplots are the most abundant. Each time point is represented in a different color: T₀—green, T₁₃—orange, T2₀—red, T3₀—purple

Fig. 8

Heat map of the taxa that showed a significant correlation (p value 0.01) with the factor time in the PMA-treated sample set. The color blue indicates a low abundance of the single OTU in the respective sample, and orange indicates a high abundance of the single OTU in the respective sample. Each column represents one sample collected throughout the study. The numbering pattern is explained as follows 30 means 30-day study. The first number (0–4) will be the sample collection day (0 = T₀, 2 = T₁₃, 3 = T₂₀, 4 = T₃₀), second number (1–8) will be sampling location, and P stands for PMA-treated samples. For example, 30.06P will be a sample collected during the 30-day study from T₀ at location 6