The Future of in-Situ Sequencing-Based Microbial Monitoring: Development of a Shelf-Stable Method for Artemis and Beyond

Microbial monitoring onboard the International Space Station (ISS) is essential for assessing the efficiency of the Environmental Control and Life Support Systems (ECLSS) and providing insight into potential risk to both crew and spacecraft. Historically, this monitoring required the need to culture organisms onboard, return these cultures to Earth, and then complete the identifications, a process that would take months. Over the past decade, and through numerous payloads, advances in molecular biology have enabled in-flight microbial identifications using nanopore sequencing. The swab-to-sequencer method resulting from these efforts was transitioned from research to operations for microbial monitoring under the Crew Health Care Systems (CHeCS) BioMole. Collectively, these accomplishments have propelled the swab-to-sequencer method to be selected as the Microbial Surface Monitor (MSM) for Gateway, as well as a payload on Artemis IV. However, the lack of cold stowage availability for Artemis requires modifications to the entire method due to the thermal instability of the reagents required for sample preparation. To achieve this, new development, optimization, and validations were undertaken. Key considerations included enzyme concentration, buffer compatibility, and equal or enhanced sensitivity and specificity. At each step, thorough side-by-side comparisons with the current ISS method were performed. The development of a robust shelf-stable method will ensure continued sequencing-based microbial monitoring for Artemis and beyond, providing data in near real-time, enhancing risk response time, and yielding clear insight into the microbiome of spacecraft.