NOTE: Extended to 09/30/2023 per F. Hernandez/ARC (Ed., 9/6/22)

NOTE: Extended to 12/31/2022 per F. Hernandez/ARC (Ed., 9/23/21)

NOTE: Extended to 9/30/2021 per F. Hernandez/ARC (Ed., 9/11/20)

NOTE: Extended to 9/30/2020 per F. Hernandez/ARC (Ed., 7/23/19)

NOTE: Extended to 9/30/2019 per F. Hernandez/ARC (Ed., 4/2/19)

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

An oral presentation titled, “Microbial Evolution in the Spaceflight Environment” was given at the 38th ASGSR annual meeting in Houston, TX, in November 2022. See the Bibliography for full citation (Ed., 6/1/23).

NOTE: Extended to 12/31/2022 per F. Hernandez/ARC (Ed., 9/23/21)

NOTE: Extended to 9/30/2021 per F. Hernandez/ARC (Ed., 9/11/20)

NOTE: Extended to 9/30/2020 per F. Hernandez/ARC (Ed., 7/23/19)

NOTE: Extended to 9/30/2019 per F. Hernandez/ARC (Ed., 4/2/19)

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Due to the COVID-19 pandemic, laboratory access and research capabilities were severely limited for a part of the reporting period. Research activities have focused on redesign of the cell cassettes, with iterative modifications to the design. Additional testing of fully assembled high-fidelity hardware was possible; also possible was needed biocompatibility testing on new materials used in the updated design. Preliminary growth tests with new spore and media preps were performed. A new racetrack insert design was finalized and printing of new tracks is ongoing for both Science Verification Tests (SVT) and Experiment Verification Tests (EVT). The SVT is currently underway.

The Experimental Requirements Document (ERD) was updated (Revision A), signed, and released on January 31, 2022, with success criteria refined to incorporate lessons learned from the original flight and hardware. A draft Return to Work Plan was originally submitted to Exobiology Branch line management on April 6, 2021, as previously reported. Several iterations and revisions were made prior to formal submission to the NASA Ames Reserch Center (ARC) Space Science and Astrobiology Division on June 1, 2021, and submission to the ARC Science Directorate for approval on June 12, 2021. The work plan was approved on August 24, 2021, which allowed for laboratory access for the project.

In June 2021, an ARC Space Biology Space Life Sciences Training Program (SLSTP) Research Associate joined the project and began a remote internship working to improve the flight growth media based on data collected from the original flight. The project used a genomics approach to improve the selected genomic DNA used for the DNA-amended growth media, in order to make the overall experiment more reproducible and more relevant to the Space Biology and Astrobiology community. The results were presented at the American Society for Gravitational and Space Research (ASGSR) annual meeting in November 2021.

NOTE: Extended to 9/30/2021 per F. Hernandez/ARC (Ed., 9/11/20)

NOTE: Extended to 9/30/2020 per F. Hernandez/ARC (Ed., 7/23/19)

NOTE: Extended to 9/30/2019 per F. Hernandez/ARC (Ed., 4/2/19)

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Due to the COVID-19 pandemic, laboratory access and research capabilities were severely limited. The ground control samples were successfully shipped to Ames Research Center (ARC) on September 21, 2020. Similarly, the flight samples were successfully shipped to ARC from Johnson Space Center on October 6, 2020. All experimental samples (flight and ground) are accounted for, safely stowed at -80°C at ARC, and were maintained in a frozen state during all shipments and transfers and are in good condition. Unfortunately, it is not currently possible to analyze these samples.

Formal approval for a re-flight was completed February 10, 2021. With no lab access, research activities have focused on redesign of the cell cassettes, and limited testing of hardware (volumes, camera angles, valve tests). Additionally, the Experimental Requirements Document (ERD) was updated, and success criteria refined to incorporate lessons learned from the original flight and hardware. This process was ongoing at the end of this reporting period. A draft Return to Work Plan was originally submitted to ARC management on April 6, 2021, with several iterations and revisions made prior to formal submission for approval on June 12, 2021.

In June 2021, an ARC Space Biology Space Life Sciences Training Program (SLSTP) Research Associate joined the project and began a remote internship working to improve the flight growth media based on data collected from the original flight. This project has just begun.

The overall experimental framework and preliminary results from our flight and ground experiments were presented as an invited virtual oral presentation to the Department of Biological Sciences, Tokyo Metropolitan University, Tokyo Japan, in June, 2020 (Note: Talk was given before the current reporting period but after submission of the previous year’s report so is included here).

NOTE: Extended to 9/30/2020 per F. Hernandez/ARC (Ed., 7/23/19)

NOTE: Extended to 9/30/2019 per F. Hernandez/ARC (Ed., 4/2/19)

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

As previously reported, the Experimental Verification Test (EVT) Readiness Review was successfully completed on May 9, 2019. EVT began on May 15, 2019 and was completed on June 12, 2019, with all acceptable success criteria being met or exceeded. The Flight Readiness Review (FRR) was successfully presented and approved at NASA Ames Research Center (ARC) on June 24, 2019. Members of the Science team first arrived at Kennedy Space Center (KSC), Merritt Island, FL, on July 12, 2019 to assemble the flight and ground control experiments. All of the work was performed in the Space Station Processing Facility (SSPF) at KSC. The first 3 days involved laboratory set-up, safety training, inventory and assembly of the 84 flight cassettes (2 strains, 3 media types, 7 replicates per treatment, 2 gravity treatments (microgravity, 1-g)) for the flight experiment. On day 4 the different media types were prepared with the associated foreign DNA (high complexity, low complexity, DNA-free), loaded into syringes, and integrated into the 84 flight cassettes (Figure 1). On day 5, 42 cassettes for the ground control experiment were assembled and the different media types were loaded into syringes and integrated to the cassettes, as per the flight experiment set-up. QA tests were performed on all of the cassettes, before and after integration of the media. Serial numbers and other metadata associated with cassette assembly and media loading operations were recorded. Loaded cassettes were placed in the 25°C incubator and checked at 24, 48, and 62 hours to assess potential contamination of the media. No contamination was observed on any of the flight or ground control media. Flight and control cassettes were delivered to payload developer (PD) for integration on July 17 and 20, respectively. Handover of the integrated flight hardware for flight occurred on July 18, 2019.

The flight experiment launched aboard SpaceX CRS-18 on July 25, 2019 and was activated onboard the ISS on August 27, 2019. The experiment ended on September 15 after 19 days of growth, and the samples were moved into cold stowage at 4°C. ISS crew imaged each cassette as they were transferred to cold stowage. Samples were subsequently transferred to -80°C on September 24, 2019. The ground control experiment was activated by the PD on August 30, 2019 and the ground control experiment ended on September 24, 2019.

No contamination occurred in the flight or ground control samples, and all but 8 of the 126 replicates germinated (94%). Some challenges occurred during the experiment, causing loss of some replicates as well as imaging data. However, a sample size of at least n=3 was preserved for all treatments. Flight samples returned to Earth on SpaceX CRS-20 on April 7, 2020. Ground control samples are currently stored at -80°C by the PD, and flight samples are currently stored at -80°C at NASA Johnson Space Center (JSC), awaiting return to Ames Research Center (ARC) for isolation and genomics work to proceed. Preliminary imaging results from the ground control experiment indicate by-strain differences in growth, as well as a treatment effect from the different DNA types in the growth media.

The overall experimental framework and preliminary results from our flight and ground experiments were presented as an oral presentation at the American Society for Gravitational and Space Research 35th Annual meeting, Denver, CO, USA, Nov. 20-23, 2019.

NOTE: Extended to 9/30/2019 per F. Hernandez/ARC (Ed., 4/2/19)

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Subsequent to the conclusion of the SVT tasks May 1, 2019, the Experimental Verification Test (EVT) Readiness Review was successfully completed on May 9, and EVT began on May 15, 2019. For EVT, a full mock-up in 6 flight-like MVP modules (akin to the ground control) was performed. 42 cell cassettes were assembled and loaded with spores and media (7 per module) in a semi-randomized nature, with the full experimental matrix (2 mutants, three media types, 7 replicates per treatment). A 27-day growth experiment was initiated with each camera (2 per module) taking images every two hours, at three light intensities for the duration. In all, almost 12,000 high-resolution images of the bacterial tracks were taken as they propagated. At the completion of the EVT growth experiment, all 42 cassettes were frozen at -80°C to replicate on-station storage. After two days, 18 cassettes (n=3 for each treatment) were thawed, and used for bacterial isolation efforts and DNA extractions. All isolations were successful, and all DNA extractions produced sufficient mass and quality of DNA for downstream sequencing analysis. EVT concluded on June 12, 2019, with all acceptable success criteria, as outlined in the ERD, being met or exceeded. The Flight Readiness Review is currently scheduled for June 24, 2019. If the review is successful, this experiment is anticipated to launch aboard SpaceX-18 (no earlier than July 21, 2019) with sample return to occur no earlier than SpaceX-19 reentry (January 2020).

The overall experimental framework and results from our science validation tests were presented as an oral presentation at the Joint CSA/ESA/JAXA/NASA (Canadian Space Agency/European Space Agency/Japan Aerospace Exploration Agency/NASA) Increments 59 and 60 Science Symposium in February, 2019.

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

The overall experimental framework and results from our science validation tests were presented as an oral presentation at the 33rd American Society for Gravitational and Space Research (ASGSR) meeting held in Renton, Washington in October, 2017.

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

For the current reporting period, our primary objectives have been to finalize appropriate experimental procedures with respect to conditions onboard ISS, including growth under elevated CO2 , testing for background DNA contamination, maintaining sterility in the context of EMCS assembly, growth under EMCS-like conditions including long-term growth of 20-days or more, and optimization of media and track design. Experiments underway include final track design tests, and DNA-uptake tests.

Printing and Sterilization –We have advanced our printing protocols to extend bacterial growth, based on the ‘dumbbell pattern’ previously reported, by narrowing the path for growth, removing the starting circle for growth, and adding a grid pattern to the wax to assist with imaging analysis. Some final experiments are underway to optimize growth duration and propagation speed.

One of the challenges of adapting the seed cassettes to heterotrophic bacteria and complex media is the risk of contamination and a need for sterilization of the hardware pre-flight (excepting the bacterial spores). We have solved this challenge with an ultraviolet light (UV) protocol, with multiple, extended exposures, which has resulted in minimal to no contamination. In combination with autoclave-sterilization of the seed cassette base and inserts, we developed reliable assembly procedures with sterilized hardware (excluding the seed cassette cover, which must be rendered aseptic-only).

Growth and Propagation –We have subsequently determined growth under elevated ambient CO2 conditions, as found onboard ISS. CO2 appeared to have no effect on bacterial growth. We have also solved the challenge of extensive growth on very small volumes of medium by designing a highly rich, buffered medium with added nutrients to extend growth and induce catabolite repression.

Stasis – We have demonstrated growth from spores after long-term storage (dried 10 months), using SBM medium dried for six months, under EMCS-like conditions. Dried medium/spore combinations, when rewetted with sterile, distilled water, after extensive periods of time in an inert/dry state, began growth in less than 24-hours post-wetting. We are now working to optimize the speed and distances capable for a given volume of medium, with growth currently continuing along the tracks for 20+ days.

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Our primary objectives thus far in this science flight definition phase have been to select appropriate experimental mutants of Bacillus subtilis strain 168, determine biocompatibility and types of growth media, growth surfaces, and materials/patterns to be used for track printing. Efforts are underway towards developing suitable sterilization protocols of growth surfaces post-printing. We have begun work to determine growth rates, and establish reliable ‘wake-up’ of Bacillus spores, and propagation along printed paths. We have also worked to develop methods for the image analysis under EMCS-like conditions.

Printing and Sterilization – Preliminary printing trials revealed no problems with either chromatography paper, or the polyethersulfone (PES) membranes similar to the materials used in the EMCS seed cassette containers. We have tested several track patterns, and are currently optimizing track width. Post printing sterilization has also been demonstrated successfully with ethanol washes of the printed filters. Ethanol causes minimal distortion to the wax, or membrane materials, and currently is superior to other approached such as autoclaving, which deform/melt the materials.

Growth and Propagation – Biocompatibility tests have shown that Bacillus appears to grow well on the PES membranes, as well as on chromatography paper, and glass fiber filter paper, although the rates do vary between materials. There does not seem to be any inhibition of growth due to the wax, though tests continue to determine this. Initial media tests are underway, with as are procedures for sporulation / wake-up protocols for preparing inert cells for spaceflight. Growth rate experiments are underway for our preliminary mutant lines to determine optimal growth temperature, as well as effects of materials on overall growth rate. We are also working to optimize track widths, media concentrations and experimental conditions for consistent propagation at reliable speeds.

Imaging – We have developed methods for correlating biomass with image data as can be collected with the EMCS hardware. Using open source software for pixel analysis, we have successfully demonstrated that correlations between image pixel value, and total biomass, can be made. It is our hope that such data will be valuable, in conjunction with growth speed along the printed paths, for determining number of generations and growth rate.