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Abstract EANA2025-71 |
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An Ensemble Binning Approach to Identify Functional Diversity in Cleanroom Environments
As planetary exploration intensifies, robust strategies to prevent forward contamination are critical to safeguarding life-detection experiments and preserving extraterrestrial environments. Cleanroom environments are therefore subject to stringent sterilisation protocols to minimise microbial contamination. However, tolerant microbes can persist and pose a potential risk for planetary protection. To investigate the functional diversity of these persistent microbes, published metagenomic datasets generated from NASA cleanrooms were analysed with an ensemble binning approach.
Metagenome-assembled genomes (MAGs) were assembled from 54 published metagenomes generated from cleanrooms at the NASA Jet Propulsion Lab, Kennedy Space Center's Payload Hazardous Servicing Facility and Lockheed Martin Aeronautics’ Multiple Testing facility (1–3) using three direct binning algorithms (Metabat2, MaxBin2, CONCOCT) (4–6). The resultant MAGs were dereplicated and quality filtered with DASTool and dRep (7,8), and annotated for functional potential using Prokka, DRAM, and BlastKOALA. Physiological tolerance and growth predictions were generated with GenomeSPOT and the GRiD pipeline respectively (9,10).
Following dereplication, 26 non-redundant MAGs were generated across six bacterial phyla. Functional profiling revealed a widespread presence of alcohol and aldehyde dehydrogenases, with some MAGs also encoding for enzymes relating to putative degradation pathways for cleaning agents, such as dodecylbenzene sulfonate and nonylphenol ethoxylate. Physiological predictions indicated that 13 MAGs were polyextremophiles, with proposed tolerances to ≥ 4 M NaCl, ≥ pH 9, and ≥ 40 °C. GRiD analysis identified ten MAGs exhibiting replication signatures, with some taxa persisting across multiple years and sampling sites.
These findings exemplify how ensemble binning can enhance genome recovery and functional insight in low-biomass environments. The detection of replicating, metabolically versatile, and extreme-tolerant microbes has implications for planetary protection and can be used to inform future bioburden mitigation strategies.
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