Comprehensive Measurement of Microbial Burden in Nutrient-Deprived Cleanrooms

Spacecraft surfaces that are destined to land on potential life harboring celestial bodies are rigorously cleaned and monitored using the traditional NASA standard spore assay (NSA). This method specifically measures NSA spore formers, which are cultivable and heat shock resistant. Since the vast majority of microbial cells cannot be characterized via the NSA, it is necessary to utilize state-of-the art molecular techniques to better understand the total, viable, cultivable, and spore populations present in nutrient-deprived low biomass cleanrooms where spacecraft are assembled.

Approximately 100 surface wipe samples were collected in the spacecraft assembly facility (SAF) environment over a 6-month period. The samples were processed and the microbial bioburden was analyzed using traditional and molecular assays. Traditional plating methods were used to determine the cultivable and NSA spore former populations. Molecular assays were used to determine the total organism (TO) and the viable organism (VO) populations. The total population was measured using adenine triphosphate (ATP) and qPCR. The viable population was measured using internal ATP, propidium monoazide (PMA)-qPCR, and flow cytometry assays. Additionally, iTag-based microbiome, multi-gene metagenome, and single cell genome analyses were performed to comprehensively measure the microbial burden and diversity of the SAF.

Based on the results, it was possible to establish a ratio between NSA spore counts and VO. The spore to VO ratio will be used to estimate the viable microbial population on spacecraft surfaces via the traditional NSA spore measurements. The Mars 2020 mission will carry a sample caching system that will collect samples of Mars rock and regolith. These samples may potentially be brought back to Earth in the future for analysis. Hence, developing statistically sound genetic inventory and microbial burden analyses are essential.