Browsing by Author "Castro, Christian"
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Item Culture-Independent Microbial Air Profiling using a Spaceflight-Compatible Nanopore Sequencing Method(51st International Conference on Environmental Systems, 7/10/2022) Dunbar, Brandon; Nguyen, Hang; Stahl-Rommel, Sarah; Sharp, G. Marie; Castro, Christian; Castro-Wallace, SarahMicrobial 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 on a quarterly basis using an impaction air sampler. With this method, microbial cells and spores are pulled onto plates containing 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, eliminates the need for sample return, and reduces 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.Item Redefining Spaceflight Microbiology: The Evolution of In Situ Nanopore Sequencing for Microbial Monitoring of Crewed Spacecraft(2024 International Conference on Environmnetal Systems, 2024-07-21) Mena, Christian; Stahl-Rommel, Sarah; Nguyen, Hang; Castro, Christian; Dunbar, Brandon; Rydzak, Patrick; Castro-Wallace, SarahMicrobial monitoring onboard the International Space Station (ISS) is essential to assess risks to both spacecraft and crew. Since human occupation began, onboard microbial culture followed by ground-based analysis has provided data descriptive of a human-occupied environment with fluctuations associated with crew turnover and process escapes from the Environmental Control and Life Support Systems (ECLSS). While this culture-based process has served as the gold standard to alert NASA to anomalies and to provide confidence in the operational controls, the data are biased towards organisms that can be cultured. In 2017, the paradigm that samples had to be returned to Earth for analysis was shifted when unknown microbes, collected and cultured from ISS surfaces, were identified onboard through the use of nanopore sequencing. The following year, culture was removed from the process, and a direct swab-to-sequencer, culture-independent method was validated onboard the ISS. Based on the success of these payloads, the Crew Health Care Systems (CHeCS) BioMole Facility was established. BioMole consists of the hardware, consumables, and procedures needed to support sample preparation and nanopore sequencing. Under the BioMole umbrella, the swab-to-sequencer method and a parallel approach for water analysis has been validated, and a full sample-to-answer process has been demonstrated with the inclusion of onboard data analytics. On the ground, fungal identification and air analysis have also been validated. Just as nanopore sequencing has resulted in the evolution of space-based microbial monitoring, improvements in the technology continue to provide opportunities for further expansion of sequencing capabilities. While previous work (since 2017) has focused on targeted sequencing, metagenomics has emerged as the next phase in environmental sample monitoring using nanopore sequencing. A review of nanopore sequencing for microbial monitoring, as well as the benefits and limitations of metagenomic sequencing, will be discussed here.