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Hang N Nguyen

Publications and source records attributed to Hang N Nguyen.

8 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 NTRS2024

Overview of the International Space Station’s Water and Cabin Air Quality: A Five-Year Status

Since the beginning of the International Space Station (ISS), water and air quality have been monitored to ensure crew health and verify the performance of the regenerative Environmental Control and Life Support (ECLS) systems. Over the last 25 years, the ISS has evolved greatly with significant changes to operations, crew complement sizes, visiting vehicles, payloads, and upgrades within the regenerative hardware, seen through Technology Demonstration integrations. In particular, better assessment and prevention of volatile organic releases from payloads and crew hygiene products, and implementation of advanced sorbents both on the air and water strings have been successful in reducing contaminant loads. Data on air and water quality for the last five years on ISS will be presented (nominal and contingency air grab samples, in-flight monitoring for air and water quality, and water samples from all segments of the ISS water system), including some notable events. The available data demonstrate the performance of existing ECLS systems and overall status of how the approach to air and water quality have evolved through the new ISS architecture baseline operations.

air quality↗
NASA NTRSDate not supplied

Method Development for In-situ Detection of Latent Herpesvirus DNA from Saliva using Nanopore Sequencing

Research toward latent herpesvirus reactivation has been intensively addressed through Space Shuttle and International Space Station (ISS) investigations. This work has provided the understanding that persistent reactivation of herpesviruses from asymptomatic crew can be detected through viral shedding in saliva, urine, and blood. Occasionally, viral reactivation from the latency stage can pose a threat to crew health (clinical manifestation) before, during, and after flight missions. Furthermore, previous work detailing correlations with immunity indicate that monitoring viral reactivation could be implemented to assess potential immune dysfunction. While in-flight monitoring is desirable, there is no well-established procedure or method for real-time evaluations, and research to date has relied on postflight, ground-based analysis. The development of portable molecular technologies like the miniPCR™ (miniPCR Bio) thermal cycler and the MinION™ sequencer (Oxford Nanopore Technologies) have confirmed that real-time monitoring is possible in extreme and low resource environments. These devices, combined with simple sample preparation methods, have been used to demonstrate bacterial identification onboard the ISS, as well as rapid viral detection in remote locations on Earth. The work here builds upon previous molecular advancements onboard the ISS toward the development and validation of a spaceflight-compatible method for viral detection from crew samples. Several herpesviruses can be detected in saliva, which provides a non-invasive means to collect samples for monitoring. While the basis for this method lies in previous spaceflight investigations, key points for method optimization include DNA extraction from saliva, viral primer selection, and bioinformatic processes for data analysis. To increase viral yield, numerous DNA extraction methods have been evaluated and will be discussed in detail. For initial development and testing, varicella-zoster virus (VZV) is being targeted though open reading frame 51 and 63 (ORF51, ORF63), as the replication origin-binding protein is highly expressed during latency. Optimization of the thermal cycling parameters has resulted in the ability to test the entire process. The full method has been tested with both viral VZV DNA standards and saliva spiked with varying concentrations of VZV. Viral sequence data were mapped to the reference sequence using minimap2. Prior to mapping, DNA sequencing reads were filtered for length and quality, barcodes were removed, and alignment identity calculated. Following further assessments, statistics were compared across multiple sequencing experiments and are being used to determine the success of the protocols. Forward work will include the incorporation of herpes simplex virus 1 (HSV-1) and Epstein-Barr virus (EBV) primers as well as the validation of results to the terrestrial qPCR standard assay. Upon full validation of the developed method, saliva will be collected from 20 healthy subjects and spiked with viral DNA. These samples will be split and assayed with the MinION and standard qPCR assay.

Hang N Nguyen↗
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

In-Situ Electrochemical Generation and Utilization of Hydrogen Peroxide for Disinfection

Disinfection needs to meet personal hygiene requirements at the International Space Station (ISS) is currently accomplished through the use of pre-packaged, disposable, wetted wipes, which represent an appreciable carry-along mass and disposal burden. However, as human missions travel further into the solar system the availability of resources to resupply will be diminished. Therefore, next-generation system should use onboard utilities to create on demand disinfectants, thereby reducing the dependence on earth-based supplies and further eliminating storage and disposal problems. Accordingly, we are developing an in-situ approach to electrochemically generate hydrogen peroxide disinfecting solution utilizing onboard life support supplies (Air/Water) to neutralize surface microorganisms present in closed living systems. As discussed within our 2019 and 2021 ICES papers (ICES-2019-38 and ICES-2021-273), we have continued to improve our TRL by scaling the electrochemical generation production process and validating the system in a zero-gravity parabolic loop flight test. In this paper we will demonstrate a system that can achieve over 1 L of >2 w/w% peroxide per day with DI water and air feeds. The hydrogen peroxide solutions generated were sent to NASA for microbial control property characterization. Overall, the electrochemical peroxide generation system offers a more economical and practical alternative, with the disinfectant being generated on demand and in-situ (using available life support materials of Air/Water); and applied to reusable cloths. The specific application of interest to this program is crew contact surfaces in space vehicles, but this technology could be utilized for waste water disinfection, heat exchanger biofouling remediation, and laundry applications. The peroxide generation system will also be able to address Earth-based needs in various settings such as field hospitals, restaurants, military, warehouses, movie theatres, among many others.

hydrogen peroxide↗
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↗