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Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies
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Clean room microbiome complexity impacts planetary protection bioburden.

Ryan Hendrickson1, Camilla Urbaniak1, Jeremiah J Minich2

  • 1Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, USA.

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Molecular methods are crucial for accurately assessing cleanroom bioburden, as traditional spore assays miss many planetary protection-relevant bacteria. This highlights the need for advanced techniques to ensure spacecraft and other sensitive environments are truly clean.

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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.