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Christian L Castro

Publications and source records attributed to Christian L Castro.

5 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS2026

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.

Christian G Mena↗
NASA NTRSDate not supplied

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 both the crew and the vehicle environment. Additionally, understanding of the atmospheric microbial profiles will be essential toward Mars-forward planetary protection planning. Currently, onboard the International Space Station (ISS), the air is monitored on a quarterly basis using an impaction air sampler. With this method, bacterial and fungal cells and spores are pulled onto plates containing growth medium. Following five days of ambient incubation onboard the ISS, the crew compares the growth to density charts and provides the approximations to the ground. Upon return of the culture plates to the Johnson Space Center’s Microbiology Laboratory, the isolates present are identified for crew health risk assessments. As NASA moves beyond the low-Earth orbit of ISS, sample return will be impractical. As such, a near real-time monitoring capability for the assessment of the spacecraft atmosphere is necessary. Significant strides have been made in recent years to utilize a molecular-based method for microbial profiling of ISS surfaces. The developed method is not dependent on microbial culture, thus removing the associated risk to the crew from high microbial levels, the bias toward detecting only the culturable organisms, and eliminates the need for sample return. The work described here details the evaluation of three different air sampling platforms whose product is amenable to downstream molecular processing and nanopore sequencing. 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 could not be identified through the culture-based method. The developed method will enable the generation of near real-time microbial profiles of the spacecraft atmosphere.

Brandon J Dunbar↗
NASA NTRSDate not supplied

Development of Computational Environmental Microbiome Workflows for the Laboratory and the International Space Station

Identification of microorganisms in the spaceflight environment is critical for crew health risk assessment on the International Space Station (ISS). Since 2017, nanopore sequencing technology has been used to support thein situ identification of microbial species during spaceflight. Beginning in 2018, a culture-independent, swab-to-sequencer method was implemented onboard the ISS to provide a more thorough insight of the ISS microbiome. Eliminating microbial culture enables identification of difficult-to-culture organisms, reduces risks associated with potentially pathogenic cultures, and could significantly reduce the time from sample-to-answer. However, this molecular-based approach generates large metagenomic datasets that require substantial computational resources for analysis. To process nanopore-generated sequencing data, the JSC Microbiology Laboratory established a bioinformatics workflow on Amazon EC2 under the security guidance of the NASA Science Managed Cloud Environment (SMCE).This resource allows for the development, testing, and accessing of computational tools for processing large and complex datasets. The work described here will address the downlinking of data from the ISS, the automated pipeline developed to identify targeted bacterial and fungal organisms, and the time from sampling onboard to microbial identification. The pipelines have been enhanced to address high and low biomass samples using optimization based on sample source (air, water, or surface) and type of collection (filter, colony, or swab).The resulting microbiome data can be assessed beyond microbial identifications to gain understanding toward population changes over time, potential selective environmental pressures, and evaluating correlations with a wide range of additional data sets. Metagenome analysis pipelines in development could allow for simultaneous identification of microbial species, gene function, and gene pathways present in the environment. Beyond the ground processing, the developed analysis pipeline is currently deployed onboard the ISS to allow for near real-time assessments of the ISS microbiome. This study serves as a critical foundation for exploration missions, where rapid microbiome analyses will be required.

G. Marie Sharp↗
NASA NTRSDate not supplied

Method Development for In Situ Microbiome Profiling of the Water Recovery System’s Wastewater Tank Onboard the International Space Station

A distinctive microbial community has inhabited the wastewater tank within the International Space Station Water Recovery System (WRS) for over 14 years and experienced the stressors associated with the microgravity environment. The WRS generates potable water for the crew from urine distillate, humidity condensate, Sabatier product water, and the occasional off-loading of ground-supplied water. The reservoir for these products, the wastewater tank, does not have a means of microbial control. While samples are occasionally collected for analysis, the time between sample collection and the return to Earth, as well as the lack of preservation, results in a skewed depiction of the microbiome. Routinely observed from these returned samples are high counts (105 – 106 colony forming units per mL) and two prevailing genera, Burkholderia and Ralstonia. The wastewater tank likely contains a more diverse microbiome, as a higher diversity of bacteria and fungus has been noted upstream and downstream of the tank. To characterize the microbial profile of the tank, analysis needs to occur at the time of sample collection. Toward this goal, a method for in situ analysis based on nanopore sequencing was developed. The filter-to-sequencer method evolved from previous work that has been validated onboard the ISS (BEST payload and the BioMole Crew Health Care Systems Facility). The method, including filtration, DNA extraction, purification, amplification, library preparation, and nanopore sequencing will be described. Additionally, data collected with this method from both ISS and terrestrial samples will be detailed. The consumables needed to support in situ analysis of the tank are set to the launch to the ISS in the spring of 2023. This investigation will allow for the first accurate characterization of the microbiome of the tank providing insight for crew health, planetary protection, and has the potential to enable engineering controls for future space station water systems.

Sarah Stahl-Rommel↗