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Microbial Monitoring of Spacecraft Environments: Automated Sample Preparation for Sequencing-Based Monitoring


Scope Title:

Microbial Monitoring of Spacecraft Environments: Automated Sample Preparation for Sequencing-Based Monitoring

Scope Description:

Microbial monitoring of the spaceflight environment, including surfaces, water, and air, is required by the medical operations community and enables crew health risk assessments. To date, this monitoring has relied on culture-based analysis in which the samples must be returned to Earth for identification. This data is used to assess risk to the vehicle and crew health, as well as to evaluate the effectiveness of the engineering controls in place and define any required remediation activities. While this method has served the International Space Station (ISS) well, it results in a bias towards the detection of culturable organisms, an inherent delay between sample collection and ground-based analysis, and an increase in the number of potential pathogens on the spacecraft. Moreover, sample return will not be possible on exploration missions. As such, a near real-time monitoring system capable of in situ analysis is absolutely critical. Significant strides toward in situ microbial monitoring have been made through the implementation of nanopore sequencing onboard the ISS. Sequencing was first demonstrated onboard the ISS in 2016 using Oxford Nanopore Technologies MinION. Following this demonstration, miniPCR (polymerase chain reaction), a small thermal cycler, was paired with the MinION, and a complete sample-to-answer method was validated. Following validation in subsequent payload experiments, this analysis method was transitioned to medical operations hardware and is currently being evaluated by Crew Health Care Systems to replace culture-based monitoring methods that require sample return; the current NASA microbial monitoring requirements have evolved to allow for the inclusion of this technology. Moreover, these updated requirements are in place for future programs, and this current manual sequencing-based method has been baselined for Gateway. The current sequencing-based method is manual, involves substantial crew time, and involves numerous consumables and piece parts. NASA is soliciting an automated system yielding a sequence-ready sample.

With the movement of the field toward metagenomic assessments, the increase in portability of these platforms, NASA’s acceptance of this molecular-based analysis and the evolution of requirements to include this technology, and the baselining of this molecular method as the monitor for future programs, this is a key time to seek an automated solution for sample preparation to enable sequencing. Innovations needed are:

DNA Extraction

Based on data from the ISS, spacecraft surfaces are relatively clean microbially, and swabs from these surfaces are considered to be low biomass. Optimal and efficient extraction from varied sample sources is needed, including swabs, wipes, and filters.

DNA Purification

Removal of cellular and source sample debris. This is critical for downstream processing.

DNA Amplification

While nontargeted metagenomics is the ultimate goal, the ability to amplify DNA as needed is ideal. For example, fungal identification is required, and it can be problematic to obtain sequencing reads from low levels of spores. The proposer does not need to define PCR targets but, rather, to describe the capability of the platform to perform this reaction. Another purification will likely be required following amplification.

Library Preparation

All sequencers require that DNA be put into a format that can be detected. As nanopore sequencing has been selected for future programs, providing libraries compatible with nanopore sequencing chemistry is required.


  • Provide DNA from low biomass environmental samples from surface swabs, surface wipes, and water/air filters.
  • Provide purified, nanopore sequence-ready DNA.
  • At Phase I, describe the ability to include an amplification reaction.
  • Overall platform should need the minimal mass, volume, and power as required.
  • Provide complete description of consumables required for toxicology assessment.
    • Provide the needed containment for the consumables per NASA Safety.
  • All consumables should be able to be produced in a temperature-stable format with a shelf life minimum of at least 3 years.
  • The overall platform should be stable at ambient conditions for at least 3 years with consumables replaced as required.
  • The overall platform should be able to process multiple samples at once.
    • A minimum of 16 samples should either be processed simultaneously or sorted and processed sequentially.
  • The overall platform should have a high ease of use and require minimal hands-on crew time (less than 1 hr, not including sample collection time).
    • Current manual prep and sequencing require ~5 hr of hands-on crew time, a 50% reduction to no more than 2.5 hr is desired. 

Expected TRL or TRL Range at completion of the Project: 2 to 6

Primary Technology Taxonomy:

  • Level 1 06 Human Health, Life Support, and Habitation Systems
  • Level 2 06.3 Human Health and Performance

Desired Deliverables of Phase I and Phase II:

  • Research
  • Analysis
  • Prototype
  • Hardware
  • Software

Desired Deliverables Description:

At the completion of Phase I, it is anticipated that a report detailing the proof of concept of a fully automated system will be provided. While a full prototype is not expected, laboratory test data detailing DNA extraction, purification, and library preparation should be delivered. Reporting should also discuss the ability to multiplex samples and reuse of the system. Additional data provided at the end of Phase I includes, but is not limited to, detailed schematics of the platform, test data from designs, test data of individual components (if included), and a plan for movement to Phase II. During development, attention should be directed toward mass, volume, power requirements, stowage conditions, ease of use, required consumables, toxicity of reagents, shelf stability of reagents, and level of biomass required for successful results (with low biomass samples expected from spacecraft environments).


At the completion of Phase I, expected deliverables will include:

  • Proof of concept for an automated sample-to-sequence-ready DNA system.
  • Laboratory demonstration of DNA extraction and purification from multiple sample sources.
  • Report describing the capability of an optional amplification reaction.
  • Laboratory demonstration of a library preparation compatible with nanopore sequencing.



Phase II


At the completion of Phase II, a full-scale prototype of the finished platform should be delivered to NASA. Documentation to accompany the prototype should include technical data sheets detailing the materials and consumables used, detailed instructions for operations, and design drawings. A final report should also be included that documents all development efforts, troubleshooting, and optimization that resulted in the final system. In addition, performance test data should be included detailing input source samples, DNA concentrations and purity achieved, and the total amount of prepared libraries generated. It is expected that some sequencing data will also be provided and discussed.


At the completion of Phase II, expected deliverables include:

  • A full-scale prototype of the finished platform.
  • A prototype demonstration of an automated solution for DNA extraction, purification, and library preparation from multiple sample sources.
  • A prototype demonstration of nanopore sequencing from the resulting prepped DNA.
  • Technical data sheets detailing materials and consumables.
    • Containment needed based on NASA Safety recommendations.  
    • Demonstrated temperature-stable consumables for at least 3 years.
  • Performance descriptions regarding storage at ambient temperature for up to 3 years (hardware, not consumables).
  • A 50% reduction in hands-on crew time compared to the current method (current method ~5 hr). 
    • Desired no more than 2.5 hr of hands-on time from sample collection to the initiation of sequencing. 


The proposers should clearly state the Technology Readiness Level (TRL) at which the research begins and what is expected at the end of Phase I and Phase II. Reference the TRL definitions here: Microsoft Word - TRL Definitions.doc (

State of the Art and Critical Gaps:

The state of the art in microbial identification, a NASA requirement needed for crew health assessments, is DNA sequencing, which is also the gold standard. NASA has been using DNA sequencing to identify returned cultures since 2006. The development of small, portable sequencers has provided the ability to place the monitor at the point of sample collection. NASA has achieved this, but the sample preparation required to support the generation of sequence-ready DNA involves multiple piece parts and consumables and requires a significant amount of hands-on time.


The gap is the lack of available commercial-off-the-shelf (COTS) automated solutions for DNA extraction from low biomass samples through library preparation in support of nanopore sequencing.


The development of a fully automated solution would extend far beyond microbial motioning and would be of great value to the planetary protection community and space biology researchers within the spaceflight industry. Beyond spaceflight, this technology would make substantial contributions to health care settings.

Relevance / Science Traceability:

This scope is included under the Space Operations Mission Directorate (SOMD) (previously the Human Exploration and Operations Mission Directorate (HEOMD)).

This work is needed to support Gateway and lunar microbial monitoring operations. While the focus of this work is directed toward the medical operations community within SOMD, including NASA’s international partners, it is extendable to all stakeholders with the goal of microbial analysis. These groups include, but are not limited to: OSMA, Planetary Protection Office, SMD-Planetary Sciences Division/Planetary Protection Research Program, the Human Research Program, AES Exploration Capabilities, space industry, academia, other government agencies, and SMD-Biological and Physical Sciences Division.


  1. Castro-Wallace, S. L., Chiu, C. Y., John, K. K., Stahl, S. E., Rubins, K. H., McIntyre, A. B. R., Dworkin, J. P., Lupisella, M. L., Smith, D.J., Botkin, D. J., Stephenson, T. A., Juul, S., Turner, D. J., Izquierdo, F., Federman, S., Stryke, D., Somasekar, S., Alexander, N., Yu, G., Mason, C. E., and Burton, A. S. (2017) Nanopore DNA Sequencing and Genome Assembly on the International Space Station. Scientific Reports. 7:18022. Nanopore DNA Sequencing and Genome Assembly on the International Space Station | Scientific Reports (
  2. Burton, A. S., Stahl, S. E., John, K. J., Jain, M., Juul, S., Turner, D. J., Harrington, E. D., Stoddart, D., Paten, B., Akeson, M., and Castro-Wallace, S. L. (2020) Off Earth Identification of Bacterial Populations Using 16S rDNA Nanopore Sequencing. Genes. 11(1), 76. Genes | Free Full-Text | Off Earth Identification of Bacterial Populations Using 16S rDNA Nanopore Sequencing (
  3. Stahl-Rommel, S., Jain, M., Nguyen H. N., Arnold, R.R., Aunon-Chancellor, S. M.,  Sharp, G. M., Castro, C., John, K. K., Juul, S., Turner, D. J., Stoddart, D., Paten, B., Akeson, M., Burton, A. S., and Castro-Wallace, S. L. (2021) Real-Time Culture-Independent Microbial Profiling Aboard the International Space Station Using Nanopore Sequencing. Genes. 12(1), 106. Genes | Free Full-Text | Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing (
  4. NASA Standard 3001 - Requirements:
    1. New requirements have not been made publicly available, but can be provided upon selection.

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