Microbiome and Metagenome Analyses of a Closed Habitat During Human Occupation
Authors: Mohan Ganesh Babu Malli , Parker Ceth W , Urbaniak Camilla , Singh Nitin K , Hood Anthony Drew , Minich Jeremiah J , Knight Rob , Rucker Michelle A , Venkateswaran Kasthuri 
Published: November 2019

Abstract
Background: Microbial contamination during the long-term confinements of space exploration present a potential risk for both crew members and spacecraft life support systems. As NASA moves from low Earth orbit further into the solar system, the monitoring of microbial populations within closed human habitation will be necessary to ensure the safety of both the crew and the spacecraft. NASA’s Johnson Space Center has recently developed a microbial swab kit specifically for use during astronaut Extravehicular Activity (EVA). The EVA swab kit is designed to be held in an astronauts’ bulky gloves and or by a robot’s manipulator, and is thus suitable for microbial sample collection in remote and extreme locations. The ability of crew members to successfully use the EVA swab kit to sample the microbial communities of an Analog habitat was tested, resulting in the successful characterization of the microbial communities within this unique habitat. Results: Several samples (floor, dry wall, glass, and metal surfaces) were collected for estimating cultivable, viable, and metabolically active microbial population using the EVA swab kit. The cultivable microbial population ranged from below the detection limit (BDL) to 106 CFU/sample and their identity was characterized using molecular methods. Next-generation sequencing (NGS; both 16S rRNA amplicon and shotgun) were used to characterize the microbial dynamics, community profiles and functional analysis (metabolic, virulence, and antimicrobial resistance). The 16S rRNA amplicon sequencing revealed abundance of viable Actinobacteria (Brevibacterium, Nesternkonia, Mycobacterium, Pseudonocardia and Corynebacterium), Firmicutes (Virgibacillus, Staphylococcus and Oceanobacillus) and Proteobacteria (esp. Acinetobacter) on floor/wall surfaces, while members of Firmicutes (Leuconostocaceae) and Proteobacteria (Enterobacteriaceae) were high on the glass/metal surfaces. Through non-metric multidimensional scaling (NMDS) determined from both 16S rRNA and metagenomic analyses revealed differential microbial speciation between floor/wall surfaces and glass/metal surfaces. Conclusion: This study provides the first assessment of monitoring cultivable and viable microorganisms from a closed spacecraft Analog submerged habitat surfaces. Several statistical treatments suggested that the largest selective pressure on the microbial community structure was the surface type since different kinds of microorganisms were observed in the floor/dry wall surfaces when compared to the metal/glass surfaces, these samples also consistently grouped separately. The metal/glass surfaces had less complex community, lower bio-burden, and more closely resembled the controls. These results indicated that material choice is crucial when building closed habitats, even if they are simply analogs. Despite our results indicating the strong role that surfaces play in selecting for the live/viable microbial communities, our study also shows that there is a shared background community of non-viable microorganisms throughout the Analog habitat. Finally, the microbial ecology of the submerged Analog habitat differs greatly from that of previously studied Analog habitats, while a few species were associated with previously cultivated isolates from the International Space Station and MIR spacecraft.