Supplementary material from "An Ensemble Binning Approach to Identify Functional Diversity in Cleanroom Environments"

Cleanroom environments, crucial for spacecraft assembly, are subject to stringent sterilisation protocols to minimise 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. This approach generated 26 medium and high quality, non-redundant metagenome-assembled genomes (MAGs) spanning six bacterial phyla. Functional analysis of these MAGs identified potential metabolic pathways for the degradation of commonly used cleaning agents, suggesting that these compounds serve as carbon sources. Furthermore, genomic analyses identified diverse physiological tolerances, with many MAGs identified as possessing polyextremophilic traits, including resistance to high salinity, alkalinity, and temperature. Growth Rate Index 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 provide critical insights for refining planetary protection protocols.