Culture-Independent Microbial Air Profiling using a Spaceflight-Compatible Nanopore Sequencing Method

Microbial monitoring of spacecraft air is critical toward assessing the efficacy of microbial controls within the environmental control and life support systems to protect the crew and vehicle environment. Currently, onboard the International Space Station (ISS), the air is monitored every quarter using an impaction air sampler. With this method, microbial cells and spores are pulled onto plates containing a growth medium. Following onboard incubation, the crew reports approximate microbial levels to the ground, but sample return is required for identification. Upon return of the plates, the isolates present are identified for crew health risk assessments. As NASA moves beyond low-Earth orbit, sample return will be impractical, and a near real-time monitoring capability is essential. Significant strides have been made in recent years to utilize a molecular-based method for microbial profiling of ISS surfaces. The developed method is independent of microbial culture, thus removing the bias toward detecting only culturable organisms, eliminating the need for sample return, and reducing the risk to crew health from exposure to high microbial levels. The work described here details the evaluation of three different air sampling platforms whose product is amenable to downstream molecular processing. The three samplers were compared in terms of mass and power requirements, ease of use, and the resulting data. For the two highest-ranking samplers, a basic concept of operations was developed to transfer the sample into the already established preparation and sequencing process. Using these concepts of operations, an in-depth comparison of the molecular data generated was compared to the historical culture-based method. Data from both methods detailed similar microbial profiles, while the molecular method detailed microbial identifications that were lacking from the culture data. The developed method will enable the generation of near real-time microbial profiles of the spacecraft atmosphere.