NOTE: End date changed to 09/30/2024 per F. Hernandez/ARC (Ed., 10/10/23).

NOTE: Project dates for this investigation have changed; the period of performance (POP) is now 10/1/2021-09/30/2023 per F. Hernandez/ARC. Original POP was 09/1/2021 - 08/31/2024 (Ed. 1/19/22).

The media shelf-life storage methods reported in Fisher et al. (2024) were not optimal for implementation in flight. Lyophilized media is very stable but requires additional crew time to rehydrate the media. Liquid media stored in FEP bioreactor bags accumulated gas within the bag over time. Although the gas did not impact A. platenesis growth, it indicated that the media composition is altered and poses a risk of over pressurizing the FEP bioreactor bags. We completed a second long-term media storage test with liquid media in FEP bioreactor bags that are placed in a Mylar secondary containment bag. This storage experiment showed 9-month old media supported A. platensis growth at a nearly identical rate to freshly prepared media. No excess gas was observed during this test, indicating that 9-month ambient storage of liquid media is acceptable to meet our science requirements.

We also made significant progress in developing improved cryopreservation methods to store live A. platensis cells. Our new method has been validated for storage periods longer than 8 months and we adapted the concept of operations for flight to use cryopreserved cells for the initial inoculum of each long-term culture.

We engaged the Washington State University J.J.Murdock Metabolomics Laboratory Service Center to characterize untargeted metabolomics sample analysis for A. platensis. Our initial experiment compared flask grown cultures to FEP Bioreactor bag cultures. Untargeted metabolomics of the biomass from these cultures showed eight outlier metabolites that are relatively increased in the FEP bioreactor.

NOTE: Project dates for this investigation have changed; the period of performance (POP) is now 10/1/2021-09/30/2023 per F. Hernandez/ARC. Original POP was 09/1/2021 - 08/31/2024 (Ed. 1/19/22).

1) Culture growth parameters: We determined growth parameters for the NIES-39 strain of A. platensis to result in a 13-to-15-day growth cycle. This growth cycle is slower than optimal growth in order to reduce experiment mass and volume and to limit crew time to eleven serial passages for 5-to-6-month exposure to spaceflight. We optimized the media composition, light intensity, growth temperature, inoculum quantity, and bioreactor bag material.

2) Live cell storage conditions: We developed a method for storing A. platensis inoculated cultures alive in bioreactor bags in dark, soft stowage conditions for up to 10 weeks prior to transfer to lighted growth conditions. This method allows the spaceflight experiment to be initiated at NASA Ames Research Center and integrated into a resupply flight without a late load requirement. Initial experiments compared additions of different carbon sources to the media, dry storage of cell pellets, and a variety of temperature conditions.

3) Media shelf life: We determined that Zarrouk’s media has at least a 6-month shelf life when stored in flurorinated ethylene propylene (FEP) bioreactor bags either as liquid media or in dry salt form.

4) Cell morphology imaging: We confirmed that A. platensis trichome structure can be imaged directly from cultures in FEP bioreactor bags.

5) Biomass harvest methods: We developed a filtration method to dewater cultures and concentrate biomass for storage in ultralow freezers. This method reduces the volume of frozen material that will need to be returned and simplifies biomass sample processing in the laboratory.

6) Cryopreservation of live cells: We tested a variety of methods to cryopreserve A. platensis cultures and confirmed that adding 10% dimethyl sulfoxide (DMSO) to cultures according to Shiraishi (2016) resulted in viable frozen cells for short duration storage at -80°C. However, long-term viability of the cryopreserved cells declined more rapidly than reported. We are currently testing a variety of alternative cryoprotectants to screen for more robust protocols to return live cells from each passage of the experiment.

7) Multi-omics extraction methods: We tested a variety of sample processing methods for DNA, RNA, and protein extractions. Input biomass processes tested included frozen cultures, frozen dewatered biomass, and lyophilized biomass. Cell disruption methods included heat lysis and bead beating for different time intervals. In addition, multiple extraction protocols were used for DNA and RNA. DNA and RNA extractions were characterized for total quantity extracted per unit dry biomass, purity based on UV/Qubit spectroscopy, and molecular integrity using a bioanalyzer.

8) Metabolomics & Nutritional Analysis: We engaged a metabolomics laboratory service center to characterize untargeted metabolomics sample analysis for A. platensis. Our initial experiment will compare flask-grown cultures to FEP bioreactor bag cultures. Samples were submitted in July 2023, and analyzed data is expected in October 2023. We also completed a literature survey of spirulina nutritional composition to rank nutrients for priority of analysis based on human recommended daily values.

First, we will examine inoculum volume and growth temperature to establish a matrix of culture growth duration that will allow eight passages within the growth window scheduled for the flight hardware. The target growth period is 5-6 months of serial passages.

Second, we will determine if frozen cultures can be used for multiomics, nutritional, and cell biology analysis. Specifically, we will determine the quantity and quality of DNA, RNA, protein, and metabolites that can be extracted from frozen cultures. We will compare lyophilization to multiple methods for thawing cultures. Culture volumes and biomass quantities needed for genome sequencing, transcriptomics, proteomics, and nutritional composition analysis will be determined. These data will be used to define the total culture volume required to complete all analyses.

Third, we will test conditions to allow storage of live A. platensis in dark, ambient conditions. These experiments will determine payload handover conditions.