Clean room microbiome complexity impacts planetary protection bioburden

Area of Science:

• Microbiology
• Space Science
• Environmental Monitoring

Background:

• The Spacecraft Assembly Facility (SAF) at NASA's Jet Propulsion Laboratory is critical for constructing planetary protection (PP)-sensitive missions.
• The Mars 2020 Perseverance Rover construction occurred in the SAF, necessitating a thorough understanding of its microbial environment.

Purpose of the Study:

• To investigate the temporal and spatial distribution of bacterial populations (total, viable, cultivable, spore) within the SAF.
• To compare traditional culture-based methods with molecular techniques for cleanroom bioburden assessment.

Main Methods:

• Collected 98 floor samples from the SAF over six months prior to Mars rover construction.
• Utilized traditional NASA Standard Assay (NSA) for spore isolation and Sanger sequencing.
• Employed 16S rRNA gene-targeted amplicon sequencing and propidium monoazide (PMA)-treated amplicon sequencing.
• Applied single-cell genomics for microbial analysis.

Main Results:

• Traditional NSA predominantly isolated spore-forming bacteria (97% of 130 isolates), identifying Bacillus subtilis and Virgibacillus panthothenticus as key genera.
• 16S rRNA sequencing detected 51 additional genera missed by NSA.
• PMA-treated amplicon sequencing revealed 54 genera, with ~86% non-spore-formers and ~14% spore-formers.
• Common genera identified included Sphingobium, Geobacillus, Bacillus, and Acinetobacter, with variations in spatial and temporal abundance.

Conclusions:

• Traditional spore assays are insufficient for comprehensive cleanroom bioburden assessment.
• Molecular methods are essential to detect planetary protection-relevant genera missed by culture-based assays.
• A paradigm shift towards molecular sequencing is recommended for monitoring bioburden in aerospace, pharmaceutical, and medical industries.