NOTE: End date changed to 9/30/2021 per PI (Ed., 5/4/2020)

NOTE: End date changed to 8/31/2020 per PI (Ed., 8/17/18)

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight can potentially provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration exploration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The Veggie vegetable-production system on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad crops in the Veggie unit during spaceflight and assess the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional value, and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be to develop growth protocols for these crops in a spaceflight vegetable-production system. This will reduce the risk and close the gap of inadequate nutrition by helping us advance bioregenerative food production to supplement the packaged diet for future space exploration.

B. VEG-04A and B VEG-04A was conducted during Increment 57-58 and ran from June 4, 2019-July 9, 2019. VEG-04B was conducted during Increment 61-62 and ran from October 1, 2019-November 28, 2019, and both had ground controls run ~48 hours later. Science samples were returned from the ISS and were processed for microbiological food safety and chemical analyses.

An article, Pick-and-eat space crop production flight testing on the International Space Station, was published in Feb. 2024 in a special edition of The Journal of Plant Interactions, Article Collection on “Plant Astrobiology.” The citation is:

Jess M. Bunchek, Mary E. Hummerick, LaShelle E. Spencer, Matthew W. Romeyn, Millennia Young, Robert C. Morrow, Cary A. Mitchell, Grace L. Douglas, Raymond M. Wheeler & Gioia D. Massa (2024) Pick-and-eat space crop production flight testing on the International Space Station, Journal of Plant Interactions, 19:1, 2292220, DOI: 10.1080/17429145.2023.2292220 https://doi.org/10.1080/17429145.2023.2292220 and as of 6/25/2024 it has been viewed 1,234 times. This publication includes data on mizuna crop yield, chemistry, nutrient composition and microbial food safety, as well as human sensory data. The behavioral health data from VEG-04 A and B have been analyzed but we are still compiling these data with data in HRF-VEG and VEG-05 studies, and preliminary analyses are provided below.

C. HRF VEG All planned HRF VEG studies have been completed, and no additional tests have been added. Data are being compiled for publication as part of the BHP data set for this study.

D. VEG-05 Experiment Summary The Veggie (Vegetable Production System) on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component for the ISS food system as the first step towards bioregenerative supplemental food production. In the Veggie unit, during spaceflight, salad plants will be grown, focusing on the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional content, and behavioral health benefits of the fresh produce for the Solanum lycopersicum "Red Robin" dwarf Tomato cultivar. The VEG-05 experiment will test different red-to-blue light ratios using the Veggie units on the ISS. Tomato plants will be grown in Veggie for ~100 days and astronaut crewmembers will provide plant care and pollination, and harvest fruit 3 times during and at the end of this growth period. Crewmembers will be asked to complete self-report surveys, including the Profile of Mood States – Short Form (POMS-SF) and a Veggie-specific questionnaire pre-flight, in-flight, and post-flight. They will also be asked to perform an organoleptic evaluation of a portion of the fresh produce they consume at harvest.

Consumed tomato samples will have their mass measured on ISS prior to crew consumption, and half of the produce will be frozen and returned to Earth for post-flight microbiological food safety and nutritional analysis. The goal of this experiment is to help define light colors and horticultural best practices to achieve high yields of safe, nutritious appealing tomato fruit to supplement a space diet of prepackaged food.

VEG-05 Flight Operations SpX-CRS-26 launched the VEG-05 payload on Nov. 26, 2022, and it docked on Nov. 27. Initiation of VEG-05 was started on 12/9/2022, but challenges led to subsequent initiation activities on 12/12/2022 and plant growth initiation on 12/14/2022, with the first ground control started 12/16/2022. This was later terminated due to poor germination and a second ground control was run 2/1/2023-05/12/2023. Both flight and ground control ran 100 days, with harvests of fruit at day 83, day 90, and day 100. In total, from the five surviving red-rich lighted plants in flight, only 5 ripe fruits were produced, and from the four surviving blue-rich lighted flight plants, 10 fruits were produced with only 6 of these ripe by day 100. A large number of photos and some video tours are part of the plant data collected. Due to the challenges with growth enumerated in the previous report, all planned activities were not accomplished. Human subject surveys were conducted but there were insufficient fruit samples for sensory evaluation. All fruit and plant samples were returned frozen at or below -80ºC. Plant samples included leaves and adventitious roots of surviving plants. Water samples were returned from root mat reservoirs, with the post-growth samples collected the day after harvest. Only a few swabs were taken from the flight Veggies from bungees only, due to harvest activities running over time. The planned samples from bellows and the Veggie fans were not collected. Pillows 1, 3, 7, and 10 were also collected and returned for analysis of roots, wicks, and substrates.

Because of the small sample sizes and factors affecting growth on the ISS, the objectives of assessing the light quality effect (red: blue light treatments) will not be fully achieved. Revised objectives of this study include comparing stressed flight plants with normal ground plants to determine the impact of plant overwatering stress in space on food safety and the plant microbial community, to determine nutrient content changes in fruit and leaves from stressed plants, and to evaluate stress metabolism changes in returned tissue by transcriptomic analysis. Postflight analysis was conducted with the following analyses being prioritized: A. culturable microbiology and food safety as well as molecular microbial community analysis of 1. ripe fruit; 2. leaves, stems, and adventitious roots; 3. pillow components (roots, wicks, and substrates); 4. swabs; and 5. water samples from root mats before and after growth. B. transcriptomics of leaf tissue and adventitious roots, and C. elemental analysis of leaf tissue. Unfortunately, there was insufficient tissue to conduct the analyses of leaves; however, elemental analyses were performed on a few of the harvested fruit.

Microbial Analyses Total Culturable Microorganisms were assessed with determinations of Aerobic Plate Counts (APC)/Bacteria and Total Yeast and Mold Count. Specific pathogens were screened for including Generic E. coli/Coliforms using Petrifilm, Salmonella sp. using enrichment/selective media, and Staphylococcus aureus using Petrifilm. Any colonies that grew in these methods were isolated and identified using the Biolog MicroID system and MicroSEQ fungal and bacterial gene sequencing ID. Screening for selected pathogens yielded negative results and this was confirmed by community 16S and ITS sequencing.

We observed no difference between red-rich and blue-rich treatments in bacterial and fungal CFUs in-flight samples. The red-rich ground control surface swabs and adventitious roots had significantly lower bacterial counts than the blue-rich treatment, while the fungal counts were lower in the red-rich wicks, leaves, and adventitious roots. Blue-rich fruit samples, surface swabs and leaves were lower in the ground controls than in the flight samples for both bacteria and fungi.

Molecular Analyses - Microbiome Community sequencing was performed using the 16S rRNA gene for bacterial taxonomic identification with the QIIME 2.0 with Silva approach. ITS was used for fungal taxonomic identification using KRAKEN2 with the UNITE database.

If we look at a heat map of the total microbiome, we see differences between flight and ground, and we see that true roots (from plant pillows) and adventitious roots had higher community diversity than leaf or fruit samples. When we look at 16S details by sample we see Pseudomonas dominating many of the samples, and if we remove that we start to see different clustering appear.

Molecular Analyses - Transcriptome Transcriptomics of leaf and adventitious roots sampled was performed with the GeneLab RNA-Seq Consensus Pipeline-RCP. We looked at the percentage distribution of differentially expressed genes (DEGs) in adventitious roots and leaves of ground and flight plants grown with either the red-rich or blue-rich lighting treatments and saw that leaf and root tissue in blue-rich lighting showed a higher percent of DEGs.

When Principal Component Analyses (PCA) are performed on these data, we start to see leaf and root DEGs clustered based upon condition (flight versus ground) and gene expression completely separated based on tissue type, but there is overlap in both tissue types between flight and ground. If PCA plots are coded to differentially represent red-rich and blue-rich treatments, there is considerable overlap between ground and flight samples in red-rich treatments, but blue-rich samples segregate between these locations.

Work is underway to interpret the differential gene expression observed in VEG-05. In leaves, there is strong differential regulation of genes in blue-rich grown samples in flight. Biological processes are identified, such as light-harvesting photosystem I, cell division, and protein regulation. There is a different group of upregulated genes in blue-rich grown ground control leaf samples with functions such as cell cycle regulation and protein biosynthesis being modified. In adventitious roots, we see some similar responses as in the leaf tissue including changes in photosynthesis, ATP energy production, and protein biosynthesis. This is not unexpected because the adventitious roots did contain some chlorophyll.

In the red-rich grown plant samples, there were fewer DEGs. For leaves, these involve respiration, mitochondria and energy production in the form of electron transport. In adventitious roots, DEGs are showing that these are responding to red-light differential regulation in photosynthesis and energy production, protein biosynthesis. When gene ontology is looked at, we start to see a broad categorization of the genes involved. For adventitious roots under the same conditions, we see similar responses to the leaves but also responses to stress, hydrogen peroxides catabolic processes or redox stress.

Overall, there appear to be more DEGs in leaf and adventitious root samples in blue-rich light compared to red-rich light. A functional enrichment analysis of DEGs showed similarities between leaf and adventitious root samples, as expected. Some of the hallmarks of spaceflight were detected in flight samples including stress response, lignin, radiation stress, and hydrogen peroxide catabolic processes. Further analyses are underway, and two manuscripts on the VEG-05 plant data are being prepared. While not generating the desired information on spaceflight growth responses of healthy crops, our team is hopeful that these analyses will shed light on tomato responses to stress in this environment as plant overwatering stress is a mission-relevant condition that could occur in future space crop growth systems.

Behavioral Health data from VEG-04, HRF VEG, and VEG-05 Plants as a Potential Spaceflight Behavioral Health and Performance (BHP) Countermeasure Plants are a potential countermeasure for the stresses of living in space. Caring for plants and eating fresh food have been shown to serve as a psychological benefit for previous astronauts and for others in analogous environments such as Antarctica (Schlacht et al., 2019, Vessel & Russo, 2015). Gardening can be therapeutic and has been used as such in terrestrial settings (Odeh & Guy, 2017). These effects may carry over or even be more pronounced in austere resource-limited environments such as spaceflight. It can reduce stress and increase sensory stimulation and enjoyment (Vessel & Russo, 2015). Growing crops for consumption may also offer astronauts a form of meaningful and engaging work as they care for living things in an austere environment away from Earth’s nature and as they provide sustenance for their crew. Additionally, fresh produce may enhance the diet and encourage consistent consumption by avoiding menu fatigue, which may further support psychological health and well-being by supporting a healthy brain and body (Douglas et al., 2022). Despite these potential benefits, difficulty in crop production and struggling or dying plants may reduce positive effects. The data on the psychological effects of growing crops in spaceflight is limited.

As such, we examined behavioral outcomes associated with tending to plants and consuming crops grown during long-duration missions. Here, we further characterized which aspects of horticulture are most salient and psychologically beneficial to astronaut health and well-being. We hypothesized that engaging with plants during a mission would have a beneficial effect as evidenced by reported positive mood and enhanced well-being, meaningfulness, connection to Earth, relationships, and performance. We also hypothesized that interacting with the plants would be a source of positive sensory stimulation for the crew. Preliminary in-flight data analysis results are described below. More comprehensive analyses are underway in preparation for the final report.

Methods for the In-flight Phase for BHP Aims Participants Participants were 27 astronauts who interacted with the plant systems VEG-04, VEG-05, and HRF-VEG (Human Research Facility – Veggie). HRF-VEG data were collected from other space biology investigations of plants that were being conducted on the ISS between the VEG-04 and VEG-05 tests. HRF-VEG data included VEG-03 I, J, K, and L and PH-04. Total N sizes noted below vary by different plant activities, depending on what each astronaut may or may not have experienced during their mission. There was a total of 106 in-flight observations. Measures POMS-SF. The Profile of Mood States Short Form (Curran et al., 1995) was given to participants every three weeks around plant growth. Results forthcoming. BHP Veggie Questionnaire. The Behavioral Health and Performance Veggie Questionnaire was given to participants monthly, beginning with plant growth initiation. Plant and crop growth experience items examined enjoyment and time spent performing specific activities with the plants; behavioral health outcomes such as engagement, demanding, meaningful, and effects on well-being, mood, performance, and relationships with others; connections to the Earth; desire to work with and eat plants; sensory stimulation for sight, smell, touch, and taste; human factor aspects of time, training, and supplies; and experiences with struggling or dying plants. Items were initially offered on a 201 or 101-point visual analog scale (VAS), with higher ends of the scale indicating more positive outcomes, e.g., enhanced enjoyment, meaning, and engagement. The lower ends of the scale were anchored with more negative outcomes, e.g., diminished enjoyment, etc. The scale midpoints were neutral in that they neither enhanced nor diminished. Data was binned to a 7-point Likert scale at the direction of the study’s biostatistician for use in our analyses. Participants selected a specific survey version based on whether they interacted with the plant system(s) or not, and we included only the scores from the crewmembers who participated in any given activity.

Preliminary Results and Discussion for BHP Aims Generally, engaging in tasks related to the plants and crop growth (a.k.a., Veggie tasks), was enjoyable. The highest ratings were related to consuming the harvested plants and voluntary viewing, suggesting that some crewmembers found time to experience the plants and nature as a source of enjoyment. Tending to the plants was also moderately enjoyable (e.g., plant thinning, pollinating, wick opening, and harvesting), indicating that there are BHP benefits from this work task.

The BHP impacts of whether the task was engaging, meaningful, supported well-being, or was demanding for a crewmember also indicates generally positive trends. Engaging, meaningful, and supporting well-being were strongly, and positively correlated with each other, which is consistent with behavioral health and workplace research that suggests that engaging and meaningful work can support well-being. Scores for these three positively-oriented variables increase over time, suggesting that the benefits of working with plants – that is, seeing them grow, tending to them, continuing to enjoy them as nature, and ultimately, consuming the fresh produce – becomes a greater source of behavioral health benefits as the long-duration mission continues.

For plant activities, not only are they generally enjoyable for those crewmembers engaging with the plants, but the data show voluntary viewing, watering, harvesting, and consuming as consistently, positively related to the task being engaging and meaningful, and enhancing well-being. Preliminary analyses suggest that working with plants in space as well as consuming them, can be a behavioral health countermeasure for long-duration spaceflight. Additional analyses will be completed for the final report and a publication is in the works.

References Curran, S. L., Andrykowski, M. A., & Studts, J. L. (1995). Short form of the profile of mood states (POMS-SF): psychometric information. Psychological assessment, 7(1), 80. Douglas, G. L., DeKerlegand, D., Dlouhy, H., Dumont-Leblond, N., Fields, E., Heer, M., ... & Zwart, S. R. (2022). Impact of diet on human nutrition, immune response, gut microbiome, and cognition in an isolated and confined mission environment. Scientific Reports, 12(1), 20847. Odeh, R., & Guy, C. L. (2017). Gardening for therapeutic people-plant interactions during long-duration space missions. Open Agriculture, 2(1), 1-13. Schlacht, I. L., Kolrep, H., Daniel, S., & Musso, G. (2020). Impact of plants in isolation: The EDEN-ISS human factors investigation in Antarctica. In Advances in Human Factors of Transportation: Proceedings of the AHFE 2019 International Conference on Human Factors in Transportation, July 24-28, 2019, Washington DC, USA 10 (pp. 794-806). Springer International Publishing. Vessel, E. A., & Russo, S. (2015). Effects of Reduced Sensory Stimulation and Assessment of Countermeasures for Sensory Stimulation Augmentation. A Report for NASA Behavioral Health and Performance Research: Sensory Stimulation Augmentation Tools for Long Duration Spaceflight (NASA/TM-2015-218576). NASA Johnson Space Center, Houston, TX.

NOTE: End date changed to 9/30/2021 per PI (Ed., 5/4/2020)

NOTE: End date changed to 8/31/2020 per PI (Ed., 8/17/18)

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight can potentially provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration exploration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The Veggie vegetable-production system on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad crops in the Veggie unit during spaceflight and assess the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional value, and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be to develop growth protocols for these crops in a spaceflight vegetable-production system. This will reduce the risk and close the gap of inadequate nutrition by helping us advance bioregenerative food production to supplement the packaged diet for future space exploration.

Some chemical analyses were delayed due to COVID-19 and equipment failures, but these now have been completed. These data have all been analyzed, and the team is writing up the VEG-04 plant, chemistry, microbial, and organoleptic results for publication in a special edition of The Journal of Plant Interactions, Article Collection on “Plant Astrobiology”, with a submission deadline of August 31, 2023. The behavioral health data from VEG-04 A and B have been analyzed, but we are still compiling these data with data in HRF-VEG studies.

HRF VEG Plans for HRF VEG, essentially the collection of human data-of-opportunity on plant growth tests – including VEG-03 I, J, K, and L, and PH-04 – were approved by the Human Research Program Control Board on Sept. 17, 2020. The Institutional Review Board (IRB) and crew informed consent briefings were modified to include these additional experiments; all crew members for the possible missions received Informed Consent Briefings; and organoleptic and veggie questionnaires were modified to allow the additional crops (‘Outredgeous’ lettuce, ‘Dragoon’ lettuce, ‘Wasabi’ mustard, ‘Red Russian’ Kale, ‘Extra Dwarf’ Pak Choi, ‘Amara’ mustard, and ‘Española improved’ Hatch Chile peppers) and additional hardware (Advanced Plant Habitat) to be evaluated by participating subjects. Collection of plant data was not a part of the HRF VEG studies.

All planned HRF VEG studies have been completed, and no additional tests have been added. Data are being compiled.

VEG-05 Experiment Summary The Veggie (Vegetable Production System) on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component for the ISS food system as the first step towards bioregenerative supplemental food production. In the Veggie unit, during spaceflight, salad plants will be grown, focusing on the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional content, and behavioral health benefits of the fresh produce for the Solanum lycopersicum ‘Red Robin’ dwarf Tomato cultivar. The VEG-05 experiment will test different red to blue light ratios using the Veggie units on the ISS. Tomato plants will be grown in Veggie for ~100 days and astronaut crewmembers will provide plant care and pollination, and harvest fruit 3 times during and at the end of this growth period. Crewmembers will be asked to complete self-report surveys, including the Profile of Mood States - Short Form (POMS-SF) and a Veggie-specific questionnaire pre-flight, in-flight, and post-flight. And they will also be asked to perform an organoleptic evaluation of a portion of the fresh produce they consume at harvest.

Consumed tomato samples will have their mass measured on the ISS prior to crew consumption, and half of the produce will be frozen and returned to Earth for post-flight microbiological food safety and nutritional analysis. The goal of this experiment is to help define light colors and horticultural best practices to achieve high yields of safe, nutritious appealing tomato fruit to supplement a space diet of prepackaged food.

Experiment Verification Test (EVT) EVT began April 27, 2022, and ran for 99 days, with completion on August 4th, 2022. EVT consisted of 12 plant pillows with pillows 1-6 in the blue-rich light treatment ((330 micromoles, Red setting: 150 micromoles, Blue setting: 150 micromoles, Green setting: ON (30 micromoles)), and pillows 7-12 in the red-rich light treatment ((330 micromoles, Red setting: 270 micromoles, Blue setting: 30 micromoles, Green setting: ON (30 micromoles)). We had initially planned to add water to the root mats at day 45, and to water plant pillows every other day at this point. Six days after water was added to the root mats, we observed wilting of all plants and the root mats were dry (Days after Initiation or "DAI" 51). Some leaves were lost during this wilting event and plants had some recovery after refilling the root mat. To mitigate wilting, water was added to the pillows in excess of the planned volume, and the root mats were filled for a second time. This seemed to remedy the issue, but the plants again used up all the water within 6 days (though not to the point of wilting). The team monitored and added water to the root mats every 6 days, with watering to pillows every other day, and root mats continued to wick, and plants generally grew well. Leaking was observed after the first wilting event, and refill of the root mats and periodically after this when water was added to pillows, indicating that the plant pillows were back at capacity. A second wilting event occurred at 75 days after initiation, when plants again used all the water and emptied the root mat.

The first flower was observed in the red-rich treatment on DAI 32 with flowers open on DAI 34. The first flowers emerged in the blue-rich treatment on DAI 36 and opened on DAI 37. All plants had flowers by DAI 41. The first fruit was noted on DAI 47, and all plants had fruit visible by DAI 49. There were signs of intumescence on many leaves; intumescence is a physiological growth disorder often seen in tomatoes grown under narrow spectrum light sources, and this does not present a cause for concern. We did see slightly different rates of flowering, fruit formation, and fruit ripening between the two light treatments, with the red-rich light treatment generally being in advance of the blue-rich light treatment. We had ripe fruit detach prior to the first harvest, one each from plants 7 and 11 at 80 DAI, and one from plant 2 at 81 DAI, as well as an unripe fruit from plant 9 the same day. Three harvests were conducted over the course of the EVT, with the first harvest at 83 DAI, the second harvest at 90 DAI, and the final harvest of flowers and plant samples at 99 DAI. The mass measuring device that was planned to be used in the flight experiment was tested for the EVT harvests. EVT success criteria were analyzed.

Microbiological analysis of tomato fruit from VEG-05 Experiment Verification Test (EVT)

The VEG-05 EVT consisted of an initial fruit harvest completed on 7/19/22, an interim harvest completed on 7/26/22, and a final harvest completed on 8/4/22. For the first two harvests, tomatoes were obtained from twelve plants, with the final harvest consisting of fruit from eleven plants. The team performed standard food safety testing on all samples. All samples were processed for aerobic plate counts (APC), yeast and mold counts, coliform/E. coli, S. aureus, and Salmonella sp. All three harvests yielded fungal counts that fell below the detection limit. Detection limits vary due to individual sample weights but are all below 70 (the highest detection limit) colony-forming units per gram fresh weight (CFU/gfw) for yeast and mold and 7 for Coliform/E. coli and S. aureus. No Salmonella were detected. The highest counts were on the fruit from the 2nd harvest and the colony type indicated one species of Bacillus. This identification has not been confirmed.

VEG-05 Flight Operations Flight Preparation VEG-05 plant pillows were prepared at the end of October 2023. All pillows were packed and sewn with all materials sanitized appropriately (pillows and wicks were treated with ethylene oxide and allowed to offgas, substrates were sieved, washed with deionized (DI) water to remove dust, autoclaved, and oven dried. Seeds were planted in a laminar flow hood. After allowing seeds to dry, pillows were packed in bags and sealed for flight. They were then turned over for flight packing and placement on the rocket.

Veggie Light Mapping SpX-CRS-26 launched the VEG-05 payload on Nov. 26, 2022, and it docked on Nov. 27. The first VEG-05-related activity was the light meter activity. Here an astronaut used the light meter with a Photosynthetically Active Radiation (PAR) sensor to measure both the external ambient light in the Veggie units when the Veggie lights were off, as well as the light output for red, blue, and green lights at different settings at a 10 cm height and 5 positions on the baseplate in both Veggie units. From the data, steps were conducted to determine the appropriate light setpoints for the flight Veggie lights to get the same PAR output with the different light ratios of 90: 10 Red: Blue and 50: 50 Red: Blue. Using similar calculations and confirming measurements, ground settings for Veggie units were also determined. Using this approach, we were able to get an average PAR per unit of approximately 277-281 µmol/m2/s, which is lower than the planned intensity of 330 µmol/m2/s, but it was the highest level that could be obtained given the limitations of the flight hardware.

Flight and Ground Control Initiation Initiation of VEG-05 was started on 12/9/2022 but there were challenges. On 12/14/2022 all pillows were watered, and photos were taken, and the height was set, and the fans were turned on low. The lights were turned on at setpoints later that day. Lights were started later to have lights on during the night cycle running from 16:00-8:00 to keep the Veggie lights from interfering with other activities in Columbus. The ground control experiment was initiated on 12/16/2022. Plant pillows dried out much earlier than expected, and this drying was at a critical period during seed germination, when plants are most vulnerable. Emergency water was provided to try and rescue germination. This was partially successful in flight due to the behavior of water, and ultimately all but 3 flight pillows (9 of 12) had plants in them, though germination was delayed for most and few had more than 1 plant. Even though water was added more quickly on the ground, only 3 of 12 ground pillows ended up with plants and all were in the blue-rich treatment. The root cause of this premature drying was a low relative humidity event on the ISS starting at the end of DAI 2, which caused increased evaporation from the pillows and drying much earlier than anticipated. Due to the poor germination on the ground, after it was obvious that no additional ground plants would germinate, a decision was made to terminate the original ground control and re-run this asynchronously. Because in flight an extra 100 mL of water was added on day 9 to try and rescue the dry pillows, and due to the fluid physics differences between ground and flight, the restarted ground control was run at the same environmental conditions, but the additional water was split into multiple additions and dosed daily from DAI 3 until DAI 10 to get the pillows through the drying events. Following this action, we had plants germinate successfully in all plant pillows. The asynchronous ground control was restarted at the beginning of Feb. 2023 and ran through May, with successful growth of all 12 plants.

Both flight and ground control ran 100 days, with harvests of fruit at day 83, day 90, and day 100. Flight plants had uneven growth, and following the early drying events, excess water was frequently observed, which led to a variety of plant stress responses, including uneven plant growth, excess adventitious root formation, flower and fruit abortion, and visible microbial growth. This was likely due to overperformance and uncontrolled wicking from the root mat reservoir. While flight plants received approximately half the water that ground plants received, the uneven growth of these and conditions of the flight environment caused significant overwater stress to occur with flight plants. While ground control plants required water to both the root mat and plant pillows throughout their life after the initial root mat fill at DAI 45, flight plants required very little water added to pillows and primarily the root mat was used after DAI 45.

The root mat appeared to wick water to excess. Judging wetness was often difficult; and due to the length of the experiment, adding water daily to plant pillows was not practical, so the root mats continued to be used. In total, from the five-surviving red-rich lighted plants, only 5 ripe fruit were produced, and from the four-surviving blue-rich lighted plants, 10 fruit were produced, with only 6 of these ripe by day 100. Pillows were also collected and returned for analysis of roots, wicks, and substrates. Swab samples and pre- and post-growth water samples from the root mat were collected.

Because of the small fruit number and the unsatisfactory growth, crewmembers were not allowed to consume the tomatoes, and all fruit, as well as large branches with leaves, samples of the adventitious roots, two plant rooting pillows from each treatment, microbial sampling swabs, and some water samples were returned for analysis.

The restarted ground control had significantly better plant growth, primarily due to the ability of excess water to drain out of the plant pillows. Plants showed normal growth and fewer adventitious roots. All harvest dates had excellent fruit production. Interestingly, when compared to the yields from EVT, blue-rich plants produced a similar average fruit mass, but red-rich plants produced a much lower mass of ripe fruit.

Because of the small sample sizes and factors affecting growth on the ISS, objectives of assessing light quality effect (red: blue light treatments) will not be achieved. Revised objectives of this study include to compare stressed flight plants with normal ground plants to determine the impact of plant overwatering stress in space on food safety and the plant microbial community, to determine nutrient content changes in fruit and leaves from stressed plants, and to evaluate stress metabolism changes in returned tissue by transcriptomic analysis. Postflight analysis is underway. While not generating the desired information on spaceflight growth responses of healthy crops, our team is hopeful that these analyses will shed light on tomato responses to stress in this environment, as plant overwatering stress is a mission-relevant condition that could occur in future space crop growth systems.

NOTE: End date changed to 9/30/2021 per PI (Ed., 5/4/2020)

NOTE: End date changed to 8/31/2020 per PI (Ed., 8/17/18)

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight can potentially provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration exploration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The Veggie vegetable-production system on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad crops in the Veggie unit during spaceflight and assess the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional value, and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be to develop growth protocols for these crops in a spaceflight vegetable-production system. This will reduce the risk and close the gap of inadequate nutrition by helping us advance bioregenerative food production to supplement the packaged diet for future space exploration.

Some chemical analyses were delayed due to COVID-19 and equipment failures, but these now have been completed. These final data are being analyzed and the team is writing up the VEG-04 plant, chemistry, microbial, and organoleptic results for publication in a special edition of Frontiers in Plant Science, “Higher Plants, Algae and Cyanobacteria in Space Environments, Volume II” for fall of 2022. The behavioral health data from VEG-04 A and B have been analyzed but we are considering if we want to publish these with the data from other studies or as a stand-alone component at this point.

HRF VEG Plans for HRF VEG, essentially the collection of human data-of-opportunity on plant growth tests including VEG-03 I, J, K, and L and PH-04, were approved by the NASA Human Research Program Control Board on Sept. 17, 2020. The Institutional Revew Board (IRB) and crew informed consent briefings were modified to include these additional experiments; all crew members for the possible missions received Informed Consent Briefings; and organoleptic and veggie questionnaires were modified to allow the additional crops (‘Outredgeous’ lettuce, ‘Dragoon’ lettuce, ‘Wasabi’ mustard. ‘Red Russian’ Kale, ‘Extra Dwarf’ Pak Choi, ‘Amara’ mustard, and ‘Española improved’ Hatch Chile peppers) and additional hardware (Advanced Plant Habitat) to be evaluated by participating subjects. Collection of plant data was not a part of the HRF VEG studies.

At this point all planned HRF VEG studies have been completed, and no additional tests have been added. The final HRF VEG test was the PH-04 test growing ‘Española improved’ Hatch Chile peppers in the Advanced Plant Habitat which grew July 12th to Nov. 26th, 2021 (137 days). Behavioral health and organoleptic data were collected.

VEG-05 Work to prepare VEG-05 for flight was completed in parallel with testing on the ISS of the Passive Orbital Nutrient Delivery System (PONDS) hardware. Because the PONDS hardware had yet to be successfully tested in flight, it was decided to test tomato plant growth both in PONDS units and in Veggie plant pillows.

Prior to this, in late 2018-early 2019 we grew two ‘Red Robin’ dwarf tomato plants in #6 plant pillows and 2 plants in #3 plant pillows – the only time tomato growth had been tested in actual flight pillows. Of these, only 1 plant in each pillow type survived, which we believe was caused by early overwatering and excessive fertilizer salt buildup. Additionally, both plants were severely epinastic (leaves curled or bent), though the reasons for this are unknown. ‘Red Robin’ is prone to epinasty when stressed. In this early pretest, both surviving plants produced roughly the same amount of fruit (21 in the #3 pillow and 24 in the #6 pillow). Other harvest data were lost due to the government shutdown in December through January 2019.

Pre-SVT Fertilizer Testing: Given the challenges with salt stress and the limited knowledge of tomato growth in both PONDS and pillows, the team was given an opportunity to do a pre-Science Verification Test (pre-SVT) definition test under flight-like conditions using Veggie hardware. For the pillows, a method to lengthen the wicks and pin them back away from the plants was tested to keep salt from burning the plant stems. Also, the Calcium Nitrate used in previous tests had been shown to release very quickly and contribute to burning. An alternate form of calcium, Calcium Carbonate, was tested in this pre-SVT test. After considerable assessment of the results of previous tests with tomatoes, and team discussions, two different low fertilizer formulations totaling not more than 10 g/L were selected for testing. There were 3 pillows and 3 PONDS of each of two treatments.

The Pre-SVT fertilizer test was initially designed to run 45 days to the onset of flowering; however, the opportunity arose to continue this test longer to start to assess fruit formation, so the test was run for 53 days. Two Veggie facilities were used, both with the Red-rich light treatment (330 micromoles, Red setting: 270 micromoles, Blue setting: 30 micromoles, Green setting: ON (30 micromoles)). The photoperiod was 16 h on/ 8 h off and the fan setting was supposed to be low, but fans were initially accidently set to high and then reset after starting. For pillows, a new way to pin the wicks back was devised as “beltloops” sewn through the pillows. The wicks extended over the gasket and thus were considerably longer than previous Veggie wicks. Also, the pillow shades had to be trimmed to account for the wicks, and the combination of longer wicks plus wicks touching shades led to an unforeseen issue where the pillows evaporated more water than anticipated. The pillow shades also absorbed and wicked water, providing a large evaporative surface, and because of this (and possibly the high fan setting early on) the pillows dried out from their initial priming in 4 days, prior to additional water being added or any anticipation that water would be needed. The team decided to replant the pillows and to shorten the wicks and pin them to the gasket instead of the pillow surface to reduce excess evaporation. The pillows were restarted 1 week later and operations for pillows were offset from the PONDS plants by one week.

Plants in both pillows and PONDS units grew well and appeared healthy. At harvest we assessed the height of each plant; the diameter in two dimensions; the number of flowers, leaves, and fruit; the leaf area and chlorophyll content (Soil Plant Analysis Development/SPAD); the leaf fresh mass; and the fruit fresh mass. Although both sets of plants had fruit, none were ripe.

In addition to the plant data, water from the PONDS cylinders was analyzed at the completion of the growth cycle. Data indicated few nutrients present in the water, with Electrical Conductivity (EC) between 77-228 µS/cm (for reference tap water often exceeds 600 µS/cm). In both PONDS and pillows, the higher fertilizer (Treatment 2) showed the best growth (fruit) or growth potential (flowers) and leaves, as well as in pillows the highest chlorophyll concentration. Since the amount of fertilizer in the PONDS water at the completion of the pre-SVT test was so low (at only 53 days when the planned growth period is 104 days), there was concern that even the higher level of treatment 2 was insufficient to maintain flowering and fruiting. Because of these factors, the team decided to select the highest treatment to move forward, but also to add an additional even higher treatment for comparison in SVT and again to split the plants with 3 pillows or PONDS per treatment.

Science Verification Test (SVT): SVT was initiated on January 4, 2022, with the two fertilizer treatments in both plant pillows and PONDS units. Each pillow and PONDS unit contained three Red Robin Tomato seeds. Two Veggie facilities were used, both with the Blue-rich light treatment (330 micromoles, Red setting: 150 micromoles, Blue setting: 150 micromoles, Green setting: ON (30 micromoles)). The photoperiod was 16 h on/ 8 h off and the fan setting was low. The primary difference between the settings for this test and the pre-SVT test was that the Red-rich light treatment was used in Pre-SVT, while the Blue-rich treatment was used in SVT.

All plants showed excellent flowering and fruit production except the plant in pillow 5 which had abnormal growth. Although this plant flowered, flowering was very delayed, and all flowers aborted, leading to no fruit production. This abnormality was never previously seen with Red Robin tomato.

In general from SVT, fruit ripened later than anticipated (original harvest plans for DAI 80, 90, 104 were modified to DAI 90, 97, 104). The number of fruit produced and the size of the fruit was greatest in the first harvest and decreased over time. Fertilizer Treatment 2 (a slightly higher concentration) produced a slightly greater fruit mass on average than Treatment 1. Some unripe fruit and flowers remained at the final harvest. While growth could have continued longer, it is anticipated that fruit yield would continue to decline. Photos taken when Veggie lights are off are easier to use to detect flowering and fruiting, judge plant health, and determine ripeness level of fruit. Plant data will not be taken inflight, so these were not collected during SVT. Three mature leaves from each plant will be collected in flight and returned for analyses, similar to plant sampling that was performed in PH-04. Some fungus was observed on several stems and one leaf, likely due to excess water. After this was observed, water volumes were reduced, the leaf with fungus was removed, and stems were cleaned with ProSan® wipes. Fungal growth did not reoccur.

Microbiological analysis of tomato fruit from VEG-05 SVT: The VEG-05 SVT consisted of an initial fruit harvest completed on 4/4/22, an interim harvest completed on 4/11/22, and a final harvest completed on 4/18/22. At each harvest, tomatoes were obtained from five plants and standard food safety testing was performed on both sanitized and non-sanitized samples. Fruit samples were sanitized by wiping with wipes containing 1% ProSan solution for 30 seconds. All samples were processed for analysis of aerobic plate counts (APC), total yeasts and molds (Y & M), coliform/E. coli, S. aureus, and Salmonella sp. All three harvests yielded microbial and fungal counts that fell below the detection limits. Detection limits vary due to individual sample weights but are all below 70 (the highest detection limit) CFU/gfw for APC and Y & M and 7 for Coliform/E. coli, and S. aureus. No Salmonella was detected.

Experiment Verification Test (EVT): After completion of SVT, results were presented to NASA Biological and Physical Sciences (PBS) management in an EVT Readiness Review on April 25, 2022, and the team received approval to proceed with EVT. EVT began April 27, 2022, and is scheduled to run for 104 days with completion on August 9, 2022. EVT consists of 12 plant pillows with pillows 1-6 in the blue-rich light treatment ((330 micromoles, Red setting: 150 micromoles, Blue setting: 150 micromoles, Green setting: ON (30 micromoles)), and pillows 7-12 in the red-rich light treatment ((330 micromoles, Red setting: 270150 micromoles, Blue setting: 30 micromoles, Green setting: ON (30 micromoles)). SVT fertilizer treatment 2 will be used for all pillows. We had initially planned to add water to the root mats at day 45, and to water plant pillows every other day. This was successful in EVT, but 6 days after water was added to the root mats, we observed wilting of all plants and the root mats were dry. Some leaves were lost during this wilting event. To mitigate wilting, water was added to the pillows in excess of the planned volume, and the root mats were filled for a second time. This seemed to remedy the issue, but the plants again used up all the water within 6 days (though not to the point of wilting). The team has been monitoring and adding water every 6 days, and root mats continue to wick, and plants are growing well. Leaking was observed after the first wilting event and refill of the root mats, indicating that the plant pillows were back at capacity at that time.

EVT continues to progress. At this point, all plants appear to be flowering and setting fruit. The method of pollination was changed from tapping individual plants to instead knocking on the Veggie baseplate. This approach was successfully performed in analog testing, and it minimized damage to plants and potential loss of leaves, flowers, and fruit. It is working very well for EVT, and it also significantly reduces the time needed to pollinate all plants. Plans are for fruit to be harvested at day 90, 97, and 104. The mass measuring device will be used in the flight experiment and the ground unit will be tested for the EVT harvests.

NOTE: End date changed to 9/30/2021 per PI (Ed., 5/4/2020)

NOTE: End date changed to 8/31/2020 per PI (Ed., 8/17/18)

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight can potentially provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration exploration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The Veggie vegetable-production system on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad crops in the Veggie unit during spaceflight and assess the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional value, and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be to develop growth protocols for these crops in a spaceflight vegetable-production system. This will reduce the risk and close the gap of inadequate nutrition by helping us advance bioregenerative food production to supplement the packaged diet for future space exploration.

VEG-04A was conducted during Increment 57-58 and ran from June 4, 2019-July 9, 2019. VEG-04B was conducted during Increment 61-62, and ran from October 1, 2019-November 28, 2019, and both had ground controls run ~48 hours later. Science samples were returned from ISS and were processed for microbiological food safety and chemical analyses. All plant samples have undergone chemical and microbiological analyses. Microbiological data analysis was finished, and results were presented in November of 2020 at the American Society for Gravitational and Space Research Annual meeting. In summary, inconsistent differences were seen in bacterial counts between the red and blue light treatments and the 2 experiments 04A and 04B. Microbial counts were higher on flight leaves than ground control leaves and counts increased with the repeated harvest method used in the VEG-04B experiment. Seven critical control points to prevent microbiological contamination have been identified for Veggie grown leafy greens from ground processing through harvest. All cultured bacterial and fungal isolates were identified and archived.

Some chemical analyses remain in work after being postponed due to COVID-19. This work was not able to be added to the mission essential list of onsite work until late spring 2021. The completion of the chemical analysis of the leaf tissue from VEG-04B was resumed after COVID restrictions were lifted at Kennedy Space Center for this project. This has been delayed again due to equipment failure. The equipment has been replaced and will be installed in the next few weeks enabling the extraction of the leaf tissue to analyze for phenolic compounds as well as oxygen radical absorbance capacity (ORAC) completing the elemental and nutritional chemistry data set for these experiments. The new equipment EDGE Automated Solvent Extraction System (CEM) can extract a wide range of samples 3 times faster than the previous extraction system. It has the capability to filter, cool, and wash extracted samples. Additionally, it is equipped with a Q-Dry Solvent Evaporator.

The VEG-04 plant, microbiological, chemical, organoleptic, and behavioral health data to date were presented at the Human Research Program Investigator Workshop held virtually in Feb. 2021.

HRF VEG

Plans for HRF VEG, essentially the collection of human data of opportunity on plant growth tests including VEG-03 I, J, K, and L and PH-04, were approved by the Human Research Program Control Board on Sept. 17, 2020. The Institutional Review Board (IRB) and crew informed consent briefings were modified to include these additional experiments, all crew members for the possible missions received Informed Consent Briefings, and organoleptic and veggie questionnaires were modified to allow the additional crops (‘Outredgeous’ lettuce, ‘Dragoon’ lettuce, ‘Wasabi’ mustard, ‘Red Russian’ Kale, ‘Extra Dwarf’ Pak Choi, ‘Amara’ mustard, and ‘Española improved’ Hatch Chile peppers) and additional hardware (Advanced Plant Habitat) to be evaluated by participating subjects. Collection of plant data is not a part of the HRF VEG studies.

VEG-03 I and J were grown Jan. 4 through Feb. 2, 2021. VEG-03 I consisted of a mixture of crops and VEG-03 J consisted of tests of a new seed film technology with ‘Outredgeous’ red romaine lettuce. Crew were able to eat only crops grown in VEG-03 I. Immediately following these growth tests, VEG-03 K and VEG-03 L were conducted from Feb. 8 through April 13th with ‘Amara’ mustard and ‘Extra Dwarf’ Pak Choi, respectively, and both crops could be eaten. Surveys were collected from participating crew aboard during these tests and analysis is in work.

The PH-04 test growing ‘Española improved’ Hatch Chile peppers in the Advanced Plant Habitat was initiated July 12th. Plants will grow for 120 days and behavioral health and organoleptic data will be collected. VEG-05 is currently planned for no earlier than spring of 2022.

Purdue University Research. Purdue University MS student Asmaa Morsi, who successfully completed and defended her master’s Thesis at the end of 2019 is completing work on two manuscripts related to fertilizer use in Veggie-grown crops.

NOTE: End date changed to 8/31/2020 per PI (Ed., 8/17/18)

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight can potentially provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration exploration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The Veggie vegetable-production system on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad crops in the Veggie unit during spaceflight and assess the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional value, and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be to develop growth protocols for these crops in a spaceflight vegetable-production system. This will reduce the risk and close the gap of inadequate nutrition by helping us advance bioregenerative food production to supplement the packaged diet for future space exploration.

Flight Experimentation

Pick-and-eat Salad-crop Productivity, Nutritional Value, and Acceptability to Supplement the ISS Food System (VEG-04A, VEG-04B, and VEG-05) is a set of hybrid experiments of plant research with human organoleptic and behavioral research. These experiments are sponsored by the Human Research Program but are implemented in partnership with the Space Biology Program.

VEG-04A was conducted during Increment 57-58, and ran from June 4, 2019-July 9, 2019. VEG-04A grew Mizuna mustard, a leafy green crop, for approximately 28 days under 2 different light quality treatments in space. VEG-04B was conducted during Increment 61-62, and ran from October 1, 2019-November 28, 2019, with a ground control run 48 hours later. VEG-04B also grew Mizuna mustard, but this time for approximately 2 months under the same 2 different light quality treatments in space, with multiple harvests of the leafy produce. For both VEG-04 A and VEG-04B, the impact on crop growth was analyzed in terms of the differences observed in plant yield, nutritional composition, and microbial levels. Ground studies were conducted in parallel to determine the effects of spaceflight on plant growth. Consenting crew members, after eating the vegetables, were asked to rate the flavor, texture, tenderness, etc. of the produce grown under the different light treatments. Crew members also participated in surveys to evaluate their moods and assess any psychological benefits from interacting with plants in the spaceflight environment.

For VEG-04A, 6 pillows were initiated in each light treatment. Mizuna germination was noted in all 12 pillows. All objectives were conducted as expected, despite a plant establishment anomaly. Five pillows grew successfully to harvest in the red-rich light treatment, while 3 pillows grew successfully in the blue-rich light treatment. Plant mortality was attributed to too much water added early in the experiment. For VEG-04B, 6 pillows were initiated in each light treatment. Mizuna germination was noted in all 12 pillows. All objectives were conducted as expected, despite a plant establishment anomaly and a fungal anomaly. At initiation, the crew noticed loose plant growth media in the bag containing pillow 3. This pillow was successfully installed 1 day after initiation of the others after the ground team had determined the risk to the crew and ISS environment, the cause of the leaked media, and cleanup requirements. Plants in five pillows grew successfully in the blue-rich light treatment; however, the seedlings in plant 7 dried out before the first watering procedure and did not survive. Plants in six pillows grew successfully to the first harvest in the red-rich light treatment; however, pillow 2 was removed following this activity after fungal growth was detected on the base of the plant.

Data from flight include photos, videos, and downlinked water volumes added. Samples from each harvest were collected for science (1 harvest in VEG-04A and 3 in VEG-04B). The remaining produce was subjected to organoleptic analysis or retained for consumption. Science samples were returned and have been processed for microbiological food safety and chemical analyses. Plant sample elements have been assessed, and fungi and bacteria have been identified via traditional microbiological techniques and MicroSeq. Plant pillows and swab samples have been analyzed for microbial constituents, with a focus on bacteria and fungi isolated for identification. Survey data have all been collected.

Within each study, plant growth did not differ across light treatment or location (flight versus ground). In flight plants, Mizuna grown in the blue-rich treatment produced more biomass when grown longer, and on the ground, mizuna produced more biomass in both light treatments when grown longer, but growth of this crop declined over time with the repeated harvests. On average, bacterial and fungal counts were significantly lower on ground control samples than flight samples, and microbial counts increased with repeated harvests. Light treatment did not influence any elements in tissues tested; however, the growth duration did impact levels of several elements. Antioxidant and phenolic analyses remain incomplete due to COVID-19. Organoleptic scores were generally higher in flight, and ground tasters considered samples more bitter. Amount of interaction and responses to Veggie varied widely by individual. Half of all crew time spent interacting with Veggie (47%) was Setup and Watering. Consumption and Voluntary Viewing accounted for 13% of crew time. Enjoyable tasks had higher impact than non-enjoyable tasks and interacting with Veggie was generally viewed as positive. These tests on ISS are helping to mitigate the risk of an inadequate food supply for long-duration missions by adding fresh vegetables and key nutrients to the crew diet and indicating which plant care activities are providing behavioral health benefits for the crew.

Purdue University Research - Optimizing Controlled-Release Fertilizer for Lettuce and Mizuna Grown on the International Space Station

Astronaut diets on the ISS depend on resupplied packaged food. However, missions to Mars of 3-5 years will not accommodate re-supply. In addition, many human macro- and micro-nutrients degrade during long-term storage. Thus, growing nutritional plants aboard ISS is essential for providing astronauts with fresh, healthy produce. NASA is using Veggie to grow fresh salad crops aboard ISS to provide astronauts with healthy diets. In Veggie plants are grown with roots in a baked-ceramic substrate (arcillite) incorporating controlled-release fertilizer (Nutricote prills) and wicks delivering water by capillary action from a reservoir.

The fertilizer prills release nutrients into arcillite slowly over time. Different controlled-release types have the same amount of fertilizer but release it over different time periods. The Purdue Mitchell lab in collaboration with NASA is testing growth of salad crops within Veggie analogs under ISS-like environments in a growth chamber. Specifically, we are evaluating effects of different controlled-release fertilizer treatments as well as different substrate particle sizes on “cut-and-come-again” harvest scenarios, comparing productivity and quality of Red Romaine lettuce as well as Mizuna mustard.

ISS environments being mimicked include temperature, CO2, relative humidity, and photoperiod. Arcillite medium contained one of two different fertilizer mixes: 7.5g 18-6-8 T 70 + 7.5g 18-6-8 T100, or 7.5g18-6-8 T70 +7.5g 18-6-8 T180 fertilizer/liter medium. LED Light treatment was the red-rich light treatment tested in VEG-04 A and B. Plants are grown under those conditions for 8 weeks, and harvested three times at 28, 42, and 56 days from planting. At each harvest, yield parameters as well as tissue mineral content have been measured for optimum fertilizer treatment selection.

Lettuce and Mizuna plants grown in a mix of 100% fine substrate particles (Profile) and fertilizer treatment of 50% T100:50%T70 had the higher yield as well as nitrogen content compared to those grown in 50%T180:50%T70. Growing mizuna plants in 100% profile resulted in higher shoot fresh weight; although no significant differences occurred for shoot dry weight. In addition, there was no significant interaction between substrate and fertilizer, which is reported by other research as one of the advantages of using controlled-release fertilizer.

M.S. Student Asmaa Morsi successfully presented and defended her thesis work in November 2019.

NOTE: End date changed to 8/31/2020 per PI (Ed., 8/17/18)

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight has the potential to provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration exploration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The Veggie vegetable-production system on the International Space Station (ISS) offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad plants in the Veggie unit during spaceflight, and assess the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional value and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be the development of growth protocols for these crops in a spaceflight vegetable-production system. This will help reduce the risk and close the gap of inadequate nutrition by helping us advance the development of bioregenerative food production to supplement the packaged diet for future space exploration.

Crop Testing

Mizuna: After the VEG-04 experiment with mizuna was transitioned to become a plant pillow experiment in Veggie, ground testing was required to optimize fertilizer formulation and growth duration for this different growing system. It was decided to split the VEG-04 mizuna testing for flight into two tests, VEG-04A, lasting 28 days, and VEG-04B, a 56 day test with repeated harvests. Three ground tests were conducted -- the first a 28 day test to quickly narrow down fertilizer for VEG-04A preflight verification testing. The second and third tests were 56 day tests which further narrowed down the fertilizer and also the timing of harvests. The VEG-04A verification testing for flight proceeded in parallel with these tests. VEG-04A testing revealed issues of differential thermal heating through absorbance of visible radiation and re-radiation of infrared energy. The two light treatments selected for flight – 90% red: 10% blue plus green compared to 50% red: 50% blue plus green resulted in differential heating and water use in the plant pillows. This was mitigated by the addition of reflective plant pillow shades. Following fertilizer testing a change was made in the levels of fertilizer for fight. VEG-04A launched in Dec. 2018, and plants for that test were grown on ISS (with ground controls on a 52-hour delay) in June-July of 2019.

Several preflight verification tests had to be conducted for VEG-04B, and it was found that previous ground tests in analog systems did not act as good analogs for Veggie and plants in Veggie showed much worse growth than they did in ground analogs under the same conditions. This required changes in levels of fertilizer, spacing from lights, horticultural procedures, and water applications when compared to planned treatments. Ground verification testing and a water use test helped to clarify methods changes and the VEG-04B plant pillows launched in July of 2019 for planned growth in September.

During all preflight testing crew procedures and surveys were finalized, crew were consented, and preparations were made for the human subject aspects of the research. Additionally, ground samples were grown from the VEG-04A selected fertilizer, and organoleptic analyses of plants grown under different light settings were conducted. Analyses indicated that produce was acceptable to tasters, and there were no important changes in overall appeal over time, or between light treatments.

Dwarf tomato: Tomato testing was generally put on hold as the PONDS (Passive Orbital Nutrient Delivery System) hardware was redesigned for better functionality in microgravity. One test was conducted to grow tomatoes in plant pillows of either 250 mL or 500 mL substrate capacity in Veggie. While tomato plants grew in both pillow sizes, only 50% of the tomato plants survived. Fruit were produced on remaining plants and roughly the same amounts of fruit were produced in both.

HACCP plan development: A hazard analysis critical control point (HACCP) plan has been developed, based on baseline microbiological data and a risk assessment for crops grown in the Veggie. The HACCP plan consists of clarification of process step control points, identification of food safety hazards at this point, and determination of methods to reduce the hazard. The following seven points have been identified:

1. Ground processing of pillows/PONDS, where introduction of microbes via handling and materials could occur and a plan to sterilize components and aseptic technique while assembling will help mitigate this hazard.

2. Ground processing of seeds, where introduction of microbes via handling and indigenous microbes present on seeds could present a hazard and this can be mitigated by disinfection, certification of pathogen free seed, and use of sanitary handling practices.

3. Integration with the Veggie hardware, where introduction of microbes via handling could occur and use of sanitary handling will help mitigate this risk.

4. Watering, where introduction of microbes via water supply or unsanitary handling is possible and can be mitigated by ensuring that water is potable quality and treated with biocide.

5. Growth of plants, where potential contamination from air and human presence, and an increase in indigenous flora due to availability of nutrients are possible risks, and use of sanitary handling and minimizing handling of plants before harvest will help mitigate this risk.

6. Harvest of crops, where introduction of microbes due to harvest procedures/human handling presents a risk, and sanitized instruments should be used, and gloves worn to mitigate it.

7. Post-harvest handling, where microbial presence established during plant growth may be introduced via handling, and crops should be sanitized before consumption following procedures to mitigate this. As well the Veggie facility should be thoroughly sanitized.

Packing and transport of plant pillows and PONDS are not considered control points and no additional mitigation is needed for these steps. Data from verification and flight tests continue to be taken to validate these HACCP points and mitigation steps.

Purdue University Research: During the present reporting period, our team members from Purdue University worked with Mizuna and ‘Outredgeous’ lettuce, two candidate salad-crop species. A ground-based Mizuna study was conducted with two main objectives: to investigate the effect of a cut-and-come-again procedure effect on biomass yield and mineral content of Mizuna over time, and to evaluate controlled-release fertilizer treatments for growing Mizuna under ISS conditions.

During the study, two Nutricote fertilizer treatments were evaluated. Mizuna plants grown under the mix with T180, the slower release fertilizer, had higher yield during the first harvest. However, plants grown under the mix with T100 had a higher increase in yield during the second harvest and less decrease for the third harvest. So, the T100 mix ended up with higher total yield for the three harvests. The T100 mix gave an increase in micro-nutrients but decrease in macro-nutrients from harvest to harvest. The T180 mix gave an increase in P and Mg content, but a decrease in N, K, Na, Ca, and S, and an increase in micro-nutrients from harvest to harvest. Both treatment mixes led to a higher percentage of Al, Cu, and Fe in root tissues, and the T180 mix-grown plants showed a higher level of Mn.

A similar test was performed with ‘Outredgeous’ lettuce. Lettuce plants grown with the T100 mix had higher fresh weight than plants grown under the T180 treatment. We concluded that both Mizuna and lettuce grew better with a fertilizer mix with T100. Further studies are needed to understand the response of Mizuna to T180 fertilizer mix. At the end of each experiment, leachate samples were collected from each substrate for electrical conductivity (EC) measurements. Despite the higher yield for T100 fertilizer treatment, leachate samples indicated high EC for both T100 and T180. This led to an examination of the root environment to determine if it may limit fertilizer uptake. Substrate physical-properties including container capacity, air space, total porosity, and bulk density for different substrate combinations were measured. Analysis indicated that standard substrate physical properties can be met with the mix of 60%Turface: 40% Profile.

SNC ORBITEC Research: The SNC ORBITEC testing assessed a range of wick materials, wick configurations, wick processing steps, and seed-placement position to determine effectiveness for germinating seeds in Veggie plant pillows. Since a large number of plant pillows were not available for this assessment, a cup system mimicking a plant pillow was developed. The substrate formulation used inside the cups was the same as that used for the VEG-04B flight experiment.

Tested wicks were cut from a variety of materials, passed through a foam gasket and lid, and positioned in the cup while the substrate is filled and packed around each of the paired wicks (depending on the configuration used). ‘Outredgeous’ lettuce was used as a test species. Each cup was bottom watered in a controlled environment room under similar temperature and light to the ISS with slightly higher humidity and Earth-ambient CO2.

Wick material, wick configuration, and seed placement were evaluated. Five wick materials that should be safe for ISS were tested: Shamtastic (85% rayon and 15% olefin), Crew wipe (polypropylene), Synthetic gauze (polyester-rayon), Nomex (aramid polymer), and Capmat 2 (non-woven polyester). Most of these wicks, aside from the crew wipes, have an open fibrous structure. They are also thicker than the crew wipe wicks currently used in Veggie pillows. Wick configurations tested included 1) wicks cut flush to the foam gasket, 2) wicks cut so they protrude 2 cm above wick gasket, and with the two wick pieces spread at the base, and 3) wicks cut so they protrude 2 cm above the foam gasket, with the two wick pieces together at base. Seed position treatments included 1) placing the seeds at the midpoint of the gasket thickness, and 2) placing the seeds just below the gasket. Seedlings were thinned, and the germination rate was recorded. At 21 days, plants were assessed for any damage, measured to obtain height, and then harvested. Fresh and dry weights were collected from harvested plants. Wicks were assessed for salt accumulation and contamination.

In general, the crew wipe and synthetic gauze materials had the highest germination rates. Germination did not appear to be impacted by wick configuration or by seed location. Plants grew best in the Crew Wipe wicks, closely followed by Synthetic gauze wicks, and then by the Shamtastic wicks. The Nomex and CapMat 2 wicks showed consistently poor growth. Plant growth did not appear to significantly differ due to wick configuration or seed placement. So far, the crew wipe and synthetic gauze materials appear to perform better than the other wick materials tested. A long wick opening away from the plant stem may be slightly better than a wick cut flush to the gasket. Salt deposits seemed to be more apparent on the crew wipes, though this might be due to either the tight fiber configuration causing more salt deposition, or this configuration just making salt deposition more visible. The Shamtastic material appeared particularly susceptible to mold development and degradation than the other wick materials. For this reason we will not evaluate this wick material in more detail.

Upcoming assessments will include testing of additional wick materials. In addition, some seeds will be grown without wicks for comparison. Other wick treatments will include additional wick lengths and autoclaved wicks vs non autoclaved wicks to determine if this step helps reduce mold and algae growth.

NOTE: Element change to Human Health Countermeasures; previously Space Human Factors & Habitability (Ed., 1/18/17)

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight has the potential to provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration exploration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The Veggie vegetable-production system on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad plants in the Veggie unit during spaceflight, and assess the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, organoleptic acceptability, nutritional value and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be the development of growth protocols for these crops in a spaceflight vegetable-production system. This will help reduce the risk and close the gap of inadequate nutrition by helping us advance the development of bioregenerative food production to supplement the packaged diet for future space exploration.

Mizuna: A number of tests were conducted with mizuna in both analog PONDS and in flight-like PONDS (water and plant containment) systems in preparation for the preflight science verification test. Each flight PONDS unit would hold one plant in a cylinder of granular rooting medium of arcillite, with the cylinder then supported in a plastic container that holds water. Two experiments were conducted in analog PONDS hardware focusing on the four different red (R): blue (B) light ratios of interest, and data on crop parameters including assessments of plant growth (fresh mass, area, volume, relative chlorophyll, leaf number, leaf area, dry mass) and chemistry/nutrient assessments (Ca, Fe, K, Mg, Lutein, Zeaxanthin) were conducted. 90% R :10% B light treatments and 50% R : 50% B treatments showed better fresh mass and lutein than in other treatments and because of these responses and similar levels of other nutrients to other treatments, these were the two light levels selected for follow on testing and flight experimentation. Additional ground tests were conducted comparing analog PONDS and flight-like PONDS systems in preparation for the preflight science verification test. At Kennedy Space Center (KSC), different methods of the cut-and-come-again, i.e., repetitive harvest techniques were tested in both types of hardware, with the “standard” removal of the outer oldest leaves compared to “major” removal, which removed large and medium leaves. Interestingly the type of PONDS analog had an impact on the most successful harvest strategy. Also there was a large impact on growth, with plants grown in the flight-like analog achieving a much greater mass than those in the original analog, likely due to a greater volume and oxygen exchange capacity. Although both techniques were similarly effective it was decided to proceed with the major cut-and-come-again approach for flight testing due to the ease of this technique and an ability to reduce chances of broken leaves in the Veggie hardware.

At Purdue University, mizuna plants were grown in a growth chamber mimicking the same environmental and cultural conditions as for the Veggie plant-growth system on ISS in original Plants grew for 56 days under one of three light treatments: 90% Red : 10% Blue; 70% Red : 30% Blue; or 50% Red : 50% Blue. Large and medium-sized leaves from the three light treatments were harvested two times during the experiment: The first harvest occurred 28 days from planting, and the second harvest at 38 days from planting. In addition, whole plants were harvested at 56 days to end the experiment. Data from all harvests are consistent with the fact that red light enhances leaf expansion and blue light increases leaf number. Overall, a decrease in yield was noted over time and it is postulated that this decrease relates to mineral deficiency. Current studies are testing optimum fertilizer ratios for growing mizuna plants with cut-and-come-again harvesting practices. Similar work is also being conducted at KSC in the plant pillows. Although the PONDS hardware and analogs worked effectively on Earth, preliminary flight tests of this hardware indicated that it was not as effective in microgravity. Thus the VEG-04 experiment with mizuna has been transitioned to become a plant pillow experiment in Veggie and ground testing is required to optimize fertilizer formulation and growth duration for this different growing system.

Mizuna plants were grown at KSC in Veggie analog conditions and PONDS analog hardware under the two red: blue light treatments selected for flight evaluation and shipped overnight to the Johnson Space Center Space Food Systems Laboratory for evaluation. Produce was evaluated on a 9-point hedonic scale (1=dislike extremely – 9=like extremely) for Overall Acceptability, Appearance, Color, Aroma, Flavor, and Texture, and a 5 point Just-about-right scale (3= just about right, <3=too little, >3=too much) for Crispness, Tenderness, and Bitterness. The mizuna samples were not statistically significantly different in overall acceptability and fell within the range of most ISS products, which generally score from ~6.5-7.5.

Dwarf tomato: In preparation for flight tomato seed orientation was determined to be able to have the root emerge in the substrate. Tomato seed surface sterilization techniques were also established so ensure that plants would start germination without a microbial load on their seed surfaces. Preliminary fertilizer testing of ‘Red Robin’ tomato plants growing in original PONDS analog hardware was completed at KSC in the summer of 2017, and results indicated that plants grown using the T90 nano 14-4-14 fertilizer produced a slightly greater fresh mass of ripe fruit than plants grown with T100 14-4-14 nutricote fertilizer of standard prill size. Further testing was conducted in the fall of 2017 with tomato plants grown in these analog PONDS units using T90 fertilizer with the 4 light treatments discussed above for mizuna: 90 R : 10 B, 70 R : 30 B, 50 R : 50 B, and split. On average the 50 R : 50 B and 90 R : 10 B treatments produced the greatest amount of fruit and contained some of the best nutritional properties of tested light treatments. Similar tomato light and fertilizer experiments were conducted at ORBITEC/SNC; however, these tests were conducted with a coarser arcillite substrate and a slightly different PONDS analog configuration. Plants seemed less healthy, and in many cases plants grown with the T90 fertilizer did not survive. The analog PONDS growth system appears to have great sensitivity to arcillite particle size and fertilizer prill size. Smaller fertilizer prills like the T90 nano may sift down in this system and not provide sufficient nutrients in the growing zone in a coarser rooting substrate.

HACCP plan development: A hazard analysis critical control point (HACCP) plan is being developed based on baseline microbiological data and a risk assessment for crops grown in the VEGGIE in order to provide fresh food for the crew. The goal of this work is to develop a plan based on an evaluation of potential microbial risks associated with crops grown in Veggie, harvest methods, and pre and post-harvest procedures for reducing and/or preventing microbial contamination, the primary goal of a HACCP plan being prevention. Understanding the normal microbial populations on the variety of produce grown in Veggie and contamination prevention is necessary to provide safe palatable fresh food especially since specific and real time microbial monitoring methods are currently lacking for this purpose on ISS.

The hazard analysis includes verification of the procedures, and baseline microbiological data collected from ground and flight studies to ascertain normal microbial populations on the crops and Veggie components. As part of this work both ‘Tokyo Bekana’ Chinese cabbage and mizuna have been assessed for baseline microbial populations and specific pathogens when grown under the different test configurations. After the first harvest, microbial numbers generally increased in mizuna plants over repetitive harvest times but these numbers were similar between the major and standard cut-and-come-again harvest methods. Produce sanitizing wipes were very effective at removing both aerobic bacterial and fungal contaminants from leaves. ‘Red Robin’ tomato baseline assessment is underway.

Preflight testing and flight preparation: A series of preflight tests must be conducted before candidate payloads proceed to flight. Initially an Experiment Requirements Document (ERD) must be developed. This involves defining success criteria for flight. Following approval of the ERD, a preliminary Science Verification Test (SVT) is conducted in the flight hardware under ISS environmental conditions. The goal of the SVT is to test an investigation under flight-like conditions and to answer key questions within the investigation. For the VEG-04 and VEG-05 key open questions were on the watering frequency for the crops. Therefore in both the VEG-04 and VEG-05 SVT plant water use was tracked on a daily basis. Following SVT, if success criteria were met, an investigation can gain approval to move on to the Experiment Verification Test (EVT). All conditions, as nearly as possible, should replicate those that are planned for use in flight. If an EVT is successful, a payload is approved for flight. If either an SVT or an EVT fails to meet sufficient success criteria, a delta verification test will be conducted. In the period of performance for this grant an SVT and EVT were conducted for VEG-04, and an SVT was conducted for VEG-05.

The VEG-04 and VEG-05 SVT tests were conducted using the PONDS growth hardware. These tests were run at ISS conditions, and plant water use was monitored. For VEG-04 SVT, plants were harvested four times over a 55 day growth period. Regrowth was not exceptional, and thus a decision was made to modify the fertilizer formulation for the EVT. While the modification appeared successful, EVT had additional challenges, including faster growth than was observed for SVT, leading to increased water use and wilting of plants. At the same time the PONDS tech demo on the ISS did not work as expected in microgravity. These circumstances led to a change in plans and VEG-04 will now use plant pillows instead of the PONDS hardware. A delta EVT is being planned for VEG-04 in late August 2018. The VEG-05 SVT of tomatoes in PONDS was successful, and currently it is planned that tomatoes will continue to be grown in PONDS with modification of PONDS components and operations. Modifications of this system are being developed under and independent contract and a tech demo will be tested in ISS as early as December of 2018, with an EVT possible for VEG-05 in January 2019.

Questionnaires to survey astronaut mood in response to plant growth, as well as organoleptic analysis ratings for on-orbit produce consumption have been approved through the Johnson Space Center (JSC) eIRB process. Questions will be asked of all enrolled US Orbital Segment (USOS) crew members that will fly during the time that plants will be grown on ISS. Data will be collected pre-flight, in flight, and post flight. Organoleptic evaluations will be conducted by crew who are enrolled and available to taste produce during harvest events. Evaluation criteria for both VEG-04 and VEG-05 have been developed and entry of these evaluations has been made into a data collection platform. Informed consent briefings and crew participant enrollment in the studies are ongoing. Four participants have consented to date.

In addition to preparing science for the flight, the grant team has worked closely with payload developers to develop the contents for science and support kits which are being used for the preflight testing. Kits are being developed for the flight payloads and will be launched as accompanying hardware. A quantum meter was selected, manifested, and launched to the ISS in June of 2018. This meter will be used to measure the incident stray light coming into the Veggie hardware from overhead ISS lighting when Veggie lights are off, and allow us to set up similar ambient lighting conditions for ground control experiments. A divider has been built to separate the Veggie units to keep the light environments separate on the ground, and on the ISS, if needed (depending on their locations). Additionally it will provide a white surface which will allow good reflection of escaping light back into the chamber, thus helping to increase light uniformity within the Veggie chamber. ORBITEC/SNC has tested a number of materials to act as a reflective bellows, providing an essentially one-way mirror effect, reflective when the Veggie interior lighting is off, but translucent when the Veggie lighting is on. A good candidate material has been identified and may be used in future Veggie hardware upgrades if the science drives a need to reduce stray light into Veggie.

Preflight preparation continues at all locations with the first flight operations scheduled to start in fall 2018 and subsequent flight tests in 2019.

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

The capability to grow nutritious, palatable food for crew consumption during spaceflight has the potential to provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce launch mass as we move toward longer-duration missions. However, studies of edible produce during spaceflight have been limited, leaving a significant knowledge gap in the methods required to grow safe, acceptable, nutritious crops for consumption in microgravity. The “Veggie” vegetable-production system on the ISS offers an opportunity to develop a “pick-and-eat” fresh vegetable component to the ISS food system as a first step to bioregenerative supplemental food production. Our goal is to grow salad plants in the Veggie unit during spaceflight, and assess on the impact of light quality and fertilizer formulation on crop morphology, edible biomass yield, microbial food safety, acceptability, nutritional value, and behavioral health benefits. Our work will help define light color ratios, fertilizer composition, and horticultural best practices to achieve high yields of safe, nutritious leafy greens and tomatoes to supplement a space diet of prepackaged food. Our final deliverable will be the development of growth protocols for these crops in a spaceflight vegetable-production system. This will help reduce the risk and close the gap of inadequate nutrition by helping us advance the development of bioregenerative food production to supplement the packaged diet for future space exploration.

Chinese cabbage: Following down selection to a top leafy green (‘Tokyo bekana’ Chinese cabbage) and tomato (‘Red Robin’ dwarf tomato) varieties, testing was conducted using analog Veggie growth systems using the four light treatments and three fertilizer treatments selected. Plants were grown in analog Veggie pillows using a mixture of arcillite and vermiculite substrate. Although plants were grown under identical conditions in both locations, differences in growth between crops grown at the different locations were large and these tended to confound the light and fertilizer variables. Under ISS and Veggie-relevant conditions, however, the ‘Tokyo bekana’ Chinese cabbage exhibited moderate to severe stress symptoms. Symptoms included yellowing (chlorosis) and necrotic lesions on leaves. This symptomology led Purdue team members to investigate the underlying causes of these stress responses, while Kennedy Space Center (KSC) team members focused on trying to mitigate the response via fertilization.

A series of tests at Purdue attempted to determine the sources of this stress. Chemistry of plant samples from the first two pillow tests indicated high levels of Manganese in plant tissues. Tests comparing rinsed and un-rinsed arcillite attempted to determine if these excessive levels were due to minerals leaching from the substrate. Additional testing was performed with different particle sizes of arcillite. Data from these tests showed no significant differences in plant growth in response to particle size or substrate rinsing. Additional testing conducted at Purdue substituted green lighting for red lighting and canopy separation distance from the light cap. Neither approach positively impacted plant growth, nor reduced observed stress responses.

While the Purdue team worked to track down the source of Chinese cabbage stress, the KSC team attempted to mitigate that stress via fertilizer supplementation. For this reason, the most stress-inducing lighting treatment of 90% Red: 10% Blue was used. Regardless of treatment Chinese cabbage still showed signs of stress.

The Purdue team then conducted studies growing plants under either 600 ppm CO2 or elevated CO2 at 2800 ppm, the conditions found on the ISS. In addition to growing plants for 28 days under these conditions, they also swapped trays of plants between conditions at 14 days and 21 days. They found that not only was plant growth better when plants were grown for longer under 600 ppm but damage to leaves was also directly dependent on receiving lower CO2, especially during the last two critical weeks of growth. These results led to the examination of photosynthetic responses of this crop to different CO2 levels. When grown under three different CO2 levels of 450, 900, and 1350 ppm, Chinese cabbage showed large differences in many growth parameters, including fresh and dry masses of tissues, with the best growth seen at the lowest CO2 level.

This response is quite different than many other plants studied and makes this crop interesting physiologically. Note, most so-called C3 photosynthesis plants typically show increased photosynthetic rates when CO2 is elevated from ambient (Drake et al., 1997). This feature, however, likely indicating the stress observed under the elevated CO2 levels of ISS, makes this plant unsuitable to grow in the Veggie chamber. For this reason, Mizuna was selected as the crop for subsequent spaceflight experimentation.

Mizuna: Mizuna showed similar yield to Chinese cabbage in PONDS (Passive Orbital Nutrient Delivery System) tests but had very few indications of stress. Earlier crop selection testing also indicated that Mizuna was an excellent candidate for spaceflight from a nutritional and organoleptic perspective (Massa et al., 2015). In addition, mizuna has been grown successfully in space before in the Russian Lada chamber on ISS, giving us more confidence in moving from Chinese cabbage to mizuna (Sugimoto et al., 2014). Initial tests with Mizuna used older seeds. The crop grew very robustly in the PONDS analog watering system with Veggie-analog lighting and testing on this crop with different lighting treatments is underway. Moving forward testing will continue with Mizuna which will be the crop grown in VEG-04 and ‘Red Robin’ which will be grown in the VEG-05 tests.

Dwarf tomato: KSC and Purdue conducted one full trial of tomato plants in rooting pillows. Results indicated good fruit production but with large amounts of variability in response due to location of testing. Also, plants demonstrated stress responses including stunting, nutrient defects, leaf curling, purpling of leaf and stem, and leaf senescence and abscission. Some of these impacts may have been due to the crop variety, some to the cultivation conditions, and some to the environment. Tomatoes are currently being grown in analog PONDS systems with an augmented fertilizer composition at KSC, and similar testing is starting in the larger growth rooms at SNC/ORBITEC. This experiment will be harvested in August, 2017. In addition to the fertilizer comparison, pollination methods are being tested, with a small soft brush being compared to manual plant shaking. Watering System: During the past year, the Veggie team has modified the Veggie watering system. The original system of plant pillows with a wicking reservoir was found to provide insufficient water to the plants (Massa et al., 2017) in earlier flight testing. Because of this the Veggie team has selected the PONDS (Passive Orbital Nutrient Delivery System) as the next watering system which will be used for the upcoming VEG-04 and VEG-05 flights for this project. The contract for the development of this hardware required for pre-flight testing and flight experimentation has been awarded to TechShot, and they have subcontracted out to Tupperware for these units. Our project team has had input into the design requirements for this hardware. Preliminary units are scheduled to arrive in Nov. 2017 for the initial science verifications tests with the Mizuna crop.

Crew assessments: Questionnaires to survey astronaut mood in response to plant growth, as well as organoleptic analysis ratings for on-orbit produce consumption have been developed and were submitted to the Johnson Space Center (JSC) eIRB process. Questions will be asked of all USOS crew members that will fly during the time that plants will be grown on ISS. Organoleptic evaluations will be conducted by crew who are available to taste produce during harvest events.

Flight software development: Firmware modifications for the Veggie system controller were completed during the first project period. During the project period covered by this report the new firmware was tested on flight-like Veggie units and will be loaded on four ground Veggie units sent to KSC and one unit maintained in house at SNC/ORBITEC. The new firmware will be loaded on the two Veggies on the ISS when crew time is available. The firmware modifications changed the red and blue LED light intensity control method. There was interest expressed by KSC to make this modification for green LEDs also. This was determined by SNC/ORBITEC to be feasible but it has not been determined if it will be done at this time. It may be implemented at a later date. Once the new firmware is fully functional in all ISS Veggie units this task will be complete.

Preparation for Flight Experimentation: A second Veggie unit (Veggie SN 001) launched to the ISS aboard the Orbital ATK space station resupply mission (OA-7) on April 18th, 2017 from Kennedy Space Center. This unit is planned to be installed in the Columbus module EXPRESS (EXpedite the PRocessing of Experiments for Space Station) rack containing the original ISS Veggie (SN 002) in late summer or early fall of 2017. The two units will be used simultaneously for the VEG-04 and VEG-05 experiments to perform side-by-side testing with different light spectra for growth of Mizuna and Dwarf tomato, respectively.

Works Cited

Drake BG, Gonzalez-Meler MA, Long SP. 1997. More efficient plants: A consequence of rising atmospheric CO2? Annual Reviews of Plant Physiology and Plant Molecular Biology 48: 609-639.

Massa, GD, Dufour NF, Carver JA, Hummerick ME, Wheeler RM, Morrow RC, Smith TM (2017) VEG-01: Veggie hardware validation testing on the International Space Station. Open Agriculture 2:33-41.

Massa GD, Wheeler RM, Stutte GW, Richards JT, Spencer LE, Hummerick ME, Douglas GL, Sirmons T (2015) Selection of Leafy Green Vegetable Varieties for a Pick-and-Eat Diet Supplement on ISS. International Conference on Environmental Systems Technical Paper, ICES-2015-252, 16 pp.

Sugimoto, M. Y. Oono, O. Gusev, T. Matsumoto, T. Yazawa, M. A. Levinshkikh, V.N. Sychev, G.E. Bingham, R. Wheeler and M. Hummerick. 2014. Genome-wide expression analysis of reactive oxygen species gene network in mizuna plants grown in long-term spaceflight. BMC Plant Biology 2014 14:4.

NOTE: Period of performance changed to 9/01/2015-8/31/2018 (previously 7/1/15-6/30/18) per G. Douglas/HRP (Ed., 4/3/16)

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.

Numerous graduate student candidates were interviewed at Purdue University and a Masters Student, Sam Burgner, was selected to work on this project with his tenure beginning in August, 2015. Sam travelled to KSC in October, 2015, and learned the construction and operation of the Veggie analog systems. Planning and consultation among KSC, Purdue, and ORBITEC personnel along with Florikan fertilizer partners was carried out in September-November, 2015 to determine the optimum fertilizer formulations to test with selected crops. Based on the expertise of the team it was decided that Chinese cabbage would be tested with three different formulations of Nurtricote 18-6-8 controlled release fertilizer. Testing would examine the release rate of this fertilizer, with the three test scenarios being A) 180-day release, B) a 2:1 ratio of 180-day release to 100-day release, and C) a 1:1 ratio of 180-day release to 100-day release. For tomato the same release rates would be tested but instead of testing the 18-6-8 (N-P-K) fertilizer, the Nutricote 14-4-14 will be tested. Our fertilizer consultants recommended that equal levels of nitrogen and potassium would be best for a fruiting crop like tomato, while a leafy crop like Chinese cabbage, needs more nitrogen. In addition, tomatoes require extra calcium to prevent physiological disorders in the fruit, so the team decided to supplement the N-P-K fertilizer with calcium carbonate and calcium nitrate.

Light treatments were also selected, which included: A) 90% Red, 10% Blue, B) 70% Red, 30% Blue, C) 50% Red, 50% Blue, and D) a split treatment of 90% Red, 10% Blue followed by a change to 50% Red, 50% blue. In all cases, the Red and Blue light would be provided with LEDs (light emitting diodes), similar to the lighting system in Veggie. For the split treatment, the team decided to switch from high red to equal red: blue light after the plants had completed ¾ of their growth. The goal of the split treatment is to enhance nutrients prior to harvest. Chinese cabbage is scheduled to grow for 28 days and tomatoes are scheduled for 3 months.

ORBITEC outfitted six Biomass Production System for Education (BPSe) Veggie analog systems with LED lights and shipped them to Purdue University where they were set up in controlled environment growth chambers. Similarly, six BPSe systems with fluorescent lights belonging to Kennedy Space Center were retrofitted with LED lights and returned to KSC. These were long lead items and arrived at KSC in late December 2015 and at Purdue in January 2016. Systems are larger than the veggie on ISS but are similarly adjustable in height, so lights can be maintained at a set distance above plants and this height can be adjusted upwards as plants grow. For preliminary trials lights were maintained at 10 cm above plants. These BPSe systems were installed and calibrated at both locations in January and February of 2016. Calibration involved extensive light mapping at defined points beneath the light caps at the proposed light settings. Because BPSe units have slider controls for the light intensity rather than digital controls, these set points needed to be established. Quantum light meters and spectroradiometers were used for light mapping and for precise calibration of each light system. The units were also mapped out to ensure that as many plant rooting pillows as possible could be placed in each. The systems allowed 12 plant pillows for Chinese cabbage containing 180 mL of substrate and 6 of the larger pillows for tomato containing 360 mL of substrate. The first growth trial with Chinese cabbage was initiated at the week of 2/29-3/4/16 at both KSC and Purdue. This followed several weeks of preparation of the Veggie analog growth system and analog plant pillow manufacturing.

Trial 1 was conducted with the four light treatments and three fertilizer treatments with 144 total plants grown between KSC and Purdue. Plants were grown for 28 days and photographed, then harvested. Harvested plant material was measured, weighed, assessed for chlorophyll content and leaf area, and then either frozen for chemical analysis or processed for microbiological assessment. Frozen plants were freeze dried and tissue was ground and extracted. Plant pillows were also oven dried to obtain pH and conductivity readings of the substrate. After a short period for cleaning a second trial was conducted with re-randomization of the light and fertilizer treatments within each BPSe unit. The light treatments that had previously been replicated at Purdue were replicated at KSC in trial 2. Plants and pillows were treated similarly. Chemical analysis of these trials is still underway. KSC is analyzing specific elements of interest to astronaut health, and measurement of antioxidants, phenolics, and anthocyanins. Purdue University is conducting analysis of nitrates and nitrites. KSC also conducted microbiology assays for aerobic plate counts and total yeasts and molds from a subset of plants. Microbiological results from the first two trials indicated larger microbial loads than expected. Expectations were based on microbial levels previously observed with this species in testing for the Veg-03 demonstration flight in Veggie.

Data are being compiled for statistical analysis at this time. Results will allow us to down select from the 12 possible combinations of fertilizer and light to a goal of the top four or five options for future assessment. The next sets of assessments will be organoleptic evaluation at JSC and the costly Vitamin K analysis at an outside lab. During later growth and at final harvest, some symptoms of stress were noted in the Chinese cabbage plants grown in the BPSe units. This stress had not been observed in prior growth studies, and our hypothesis is that this cultivar of Chinese cabbage suffers in response to narrow spectrum radiation. Symptoms observed included chlorosis and speckled bleaching of leaves. The severity of symptoms appeared to vary with light treatment but not with fertilizer treatment. A scoring guide was developed to allow quantification of stress responses at the different locations. Treatments with higher levels of red light (90% Red, 10% blue and the split treatment) appeared to have the highest proportion of stressed leaves. Additionally stress responses increased dramatically in all treatments between day 22 and day 28 of growth.

It was decided to hold further trials of Chinese cabbage pending chemistry, statistical analysis, and further investigation into plant stress, and to instead conduct a trial of tomato in four systems both at Purdue and KSC. In the remaining two systems at Purdue troubleshooting efforts will attempt to diagnose the causes of plant stress. These tests are ongoing, focusing on light intensity levels and looking at substituting other wavelengths for red light. One hypothesis is that light of a lower intensity but spread out over a longer duration to provide the same daily light integral (DLI) might prevent the observed stress. In the remaining two systems at KSC, the focus will be to conduct microbiological testing of Chinese cabbage. These tests are also on-going and our team is looking at seed surface sterilization and media sterilization as methods to reduce overall microbial loads. These sterilization techniques are used for spaceflight and will be used during future flight experimentation, so isolating the critical control points at this time is the goal of this testing. Meanwhile, ORBITEC has prepared their plant growth rooms for subsequent larger growth trials. These rooms will be used to conduct large capacity growth studies of selected light and fertilizer conditions, where harvested produce will be shipped to JSC for organoleptic evaluation or freeze dried and ground for Vitamin K evaluation.

Initial preparations are underway for flight tests of these crops following ground, down-selection of fertilizer and light. JSC behavioral health and performance Co-I Tom Williams is assessing appropriate crew surveys and IRB (Institutional Review Board) requirements. The KSC Veggie team has begun coordinating with the Human Research Program (HRP) to start planning the on-orbit testing. A second Veggie unit will be launched to the ISS and co-located near the current unit, which will allow side by side testing of two independent light treatments in ISS. This is a significant improvement on the initially proposed subsequent testing because environmental conditions for the two treatments will be identical. The Chinese cabbage test has been preliminarily planned as Veg-04, with the tomato test book kept as Veg-05. Due to issues with the current Veggie watering system providing insufficient water for longer duration crop studies it has been decided that Veg-04 and Veg-05 are on hold until a new Veggie watering system capable of sustaining the plants can be developed.

Specific aim 1: Evaluate the effects of four light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of leafy greens during flight-definition and flight testing.

Specific aim 2: Perform cultivar selection and evaluate the effects of four different red: blue light treatments and two different fertilizer compositions on the yield, morphology, organoleptic acceptability, and nutritional attributes of dwarf tomato during ground and flight tests.

Specific aim 3: Perform hazard analysis, develop plans for minimizing microbial hazards, and screen flight-grown produce for potential pathogens.