Digging deep for viable microbial diversity of a cleanroom facility. JPL viable microbiome

Cleanrooms are assumed to be microbial-free environments protecting human health or industrial product assembly. The recent molecular microbial community analyses of cleanrooms has indicated the presence of large amounts of microbial diversity despite the harsh environmental conditions for microbial life. This study involved the examination of the microbial input from the surrounding built environment into a spacecraft assembly cleanroom in which only 1% of the microorganisms were viable. These viable yet low biomass microbiomes were examined using adenosine tri-phosphate (ATP) assay, propidium monoazide (PMA) treatment (which masks compromised cells for DNA-based analyses) followed by 16S rRNA qPCR and amplicon sequencing. In general, PMA treatment prior to DNA extraction enabled the detection of underrepresented genera. Although the remaining viable microbial diversity was lower in the cleanroom (Shannon-Wiener index H’: 5.8) compared to the adjoining facility (Shannon-Wiener index H’: 6.1), the diversity was unexpectedly high despite the extremely low biomass (~6.2*103 16S rRNA gene copies per m2 floor surface) of the cleanroom. In addition to microbial exchange, survival specialists like bacterial spore formers and archaeal halophiles and mesophiles were found to be enriched in the cleanroom. The findings included the exclusive presence of Haloferax and Sporosarcina in the cleanroom, which are considered to be novel indicator genera of clean built environments. Our results provide evidence that Archaea are alive in cleanrooms and that despite the implementation of strict cleaning and rigorous control measures, these procedures were strong drivers but did not reduce the diversity of the indoor microbiome.