Microbiome and Metagenome Analyses of a Closed Habitat During Human Occupation
Authors: Mohan Ganesh Babu Malli , Parker Ceth , Urbaniak Camilla , Singh Nitin , Hood Anthony , Minich Jeremiah , Knight Rob , Rucker Michelle A , Venkateswaran Kasthuri 
Published: April 2020

Abstract
Background: Microbial contamination during long-term confinements of space exploration present potential risks for both crew members and spacecraft life support systems. As NASA prepares for manned missions beyond low Earth orbit, deeper 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 recently developed a microbial swab kit, designed specifically to be used during astronaut Extravehicular Activity (EVA). The EVA swab kit was designed in such a way that it could be held easily within an astronaut’s bulky glove and or by a robot’s manipulator, making it suitable for microbial sample collection in remote and extreme environments. The previously tested (in laboratory and controlled settings) EVA swab kit was used in this study to sample various surfaces from a submerged, closed, analog habitat in order to characterize the microbial populations in this unique human habitat. Results: Samples were collected from various locations across the habitat of which were constructed from various surface materials (linoleum, dry wall, particle board, glass, and metal) and microbial populations examined by culture, qPCR, microbiome 16S rRNA gene sequencing and shot gun metagenomics. Propidium monoazide treated samples identified the viable/intact microbial population of the habitat. The cultivable microbial population ranged from below the detection limit (BDL) to 10 6 CFU/sample and their identity was characterized using Sanger sequencing. Next-generation sequencing (NGS; both 16S rRNA amplicon and shotgun) were used to characterize the microbial dynamics, community profiles and functional attributes (metabolism, 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 linoleum, dry wall, and particle board (LDP) surfaces, while members of Firmicutes ( Leuconostocaceae ) and Proteobacteria ( Enterobacteriaceae ) were high on the glass/metal surfaces. Non-metric multidimensional scaling (NMDS) determined from both 16S rRNA and metagenomic analyses revealed differential microbial speciation between LDP surfaces and glass/metal surfaces. The shotgun metagenomics sequencing showed bacterial predominance of Brevibacterium (53.6%), Brachybacterium (7.8%), Pseudonocardia (9.9%), Mycobacterium (3.7%), and Staphylococcus (2.1%); while fungal analyses revealed Aspergillus and Penicillium dominance. Conclusion: This study provides the first assessment of monitoring cultivable and viable microorganisms on surfaces within a submerged, closed, analog habitat. The analyses presented herein suggests that the surface material plays a role in microbial community structure as the microbial populations differed between LDP and metal/glass surfaces. 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. Finally, while a few species were associated with previously cultivated isolates from the International Space Station and MIR spacecraft, the majority of the microbial ecology of the submerged Analog habitat differs greatly from that of previously studied analog habitats.