An ensemble binning approach to identify functional diversity in cleanroom environments

Abstract Cleanroom environments, crucial for spacecraft assembly, are subject to stringent sterilization protocols to minimize microbial contamination. However, tolerant microbes can persist and pose a potential risk for planetary protection. This study employs an ensemble binning approach, integrating multiple metagenome binning programs, to analyse published metagenomic datasets generated from NASA cleanrooms to investigate functional diversity within cleanrooms. Twenty-six medium and high-quality, non-redundant metagenome-assembled genomes (MAGs) spanning six bacterial phyla were generated. Functional analysis of these MAGs identified potential metabolic pathways for the degradation of commonly used cleaning agents, suggesting that these compounds could serve as carbon sources. Furthermore, genomic analyses identified diverse physiological tolerances, with many MAGs possessing polyextremophilic traits, including resistance to high salinity, temperature and alkalinity. Growth rate index (GRiD) analysis also suggested some MAGs were actively replicating within the cleanroom environments. This study demonstrates the power of ensemble binning in revealing the functional diversity and adaptive strategies of cleanroom microbiomes and provides critical insights for refining planetary protection protocols. This article is part of the theme issue ‘Planetary Protection for sustainable space exploration’.