The ability to grow safe, fresh food to supplement packaged foods of astronauts in space has been an important goal for NASA. Food crops grown in space experience different environmental conditions than plants grown on Earth (e.g., reduced gravity, elevated radiation levels). To study the effects of space conditions, red romaine lettuce, Lactuca sativa cv ‘Outredgeous,’ plants were grown in Veggie plant growth chambers on the International Space Station (ISS) and compared with ground-grown plants. Multiple plantings were grown on ISS and harvested using either a single, final harvest, or sequential harvests in which several mature leaves were removed from the plants at weekly intervals. Ground controls were grown simultaneously with a 24–72 h delay using ISS environmental data. Food safety of the plants was determined by heterotrophic plate counts for bacteria and fungi, as well as isolate identification using samples taken from the leaves and roots. Molecular characterization was conducted using Next Generation Sequencing (NGS) to provide taxonomic composition and phylogenetic structure of the community. Leaves were also analyzed for elemental composition, as well as levels of phenolics, anthocyanins, and Oxygen Radical Absorbance Capacity (ORAC). Comparison of flight and ground tissues showed some differences in total counts for bacteria and yeast/molds (2.14 – 4.86 log10 CFU/g), while screening for select human pathogens yielded negative results. Bacterial and fungal isola 1787 frontplantsci Frontiers in Plant Science Front Plant Sci Frontiers Media SA PMC7067979 7067979 7067979 32210992 10.3389/fpls.2020.00199 Microbiological and Nutritional Analysis of Lettuce Crops Grown on the International Space Station Khodadad Christina L M 1 Hummerick Mary E 1 Spencer LaShelle E 1 Dixit Anirudha R 1 Richards Jeffrey T 1 Romeyn Matthew W 2 Smith Trent M 2 Wheeler Raymond M 2 Massa Gioia D 2 * 1 AECOM Management Services, Inc., LASSO, Kennedy Space Center, Merritt Island, FL, United States 2 NASA UB, Kennedy Space Center, Merritt Island, FL, United States Edited by: Roberta Paradiso, University of Naples Federico II, Italy Reviewed by: Francesco Di Gioia, Pennsylvania State University (PSU), United States; Walter Chitarra, Council for Agricultural Research and Economics, Italy ✉ *Correspondence: Gioia D. No use, distribution or reproduction is permitted which does not comply with these terms. Abstract The ability to grow safe, fresh food to supplement packaged foods of astronauts in space has been an important goal for NASA. Food crops grown in space experience different environmental conditions than plants grown on Earth (e.g., reduced gravity, elevated radiation levels). To study the effects of space conditions, red romaine lettuce, Lactuca sativa cv ‘Outredgeous,’ plants were grown in Veggie plant growth chambers on the International Space Station (ISS) and compared with ground-grown plants. Veggie is a small plant growth chamber designed and built by Orbital Technologies Corporation (Now Sierra Nevada Corp., Madison, WI, United States) to grow vegetable crops in space ( Morrow et al., 2005 ; Morrow and Remiker, 2009 ). The first Veggie plant growth chamber was launched to the International Space Station (ISS) in April, 2014 along with eighteen plant (rooting) pillows for the VEG-01 experiment. Veggie is a simple plant growth facility that uses LED lights and fans to circulate ISS air through the plant growth volume. Previous studies with salad crops have focused on combinations of environmental conditions (e.g., light, temperature, CO 2 concentration) needed to optimize plant growth for space settings ( Richards et al., 2004 , 2006 ). While the macro nutritional value of crops was often evaluated in productivity studies (calories, protein, fats, and carbohydrates), micro nutritional data are limited ( McKeehen et al., 1996 ; Wheeler et al., 1996 , 1997 ). In order to utilize “pick-and-eat” produce such as the lettuce crops grown in VEG-01 and 03 as a supplemental food for space, nutritional assessment is essential. Increased temperatures have been shown to decrease macronutrient content in red leaf lettuce while increasing flavonoids and phenolics ( Sublett et al., 2018 ). Studies have shown that increased humidity initiates a positive response in biomass yield in many agricultural crops while concomitantly decreasing transpiration rate altering the uptake of water and some nutrients ( Tibbitts, Mizuna, barley and radish from Lada were stored at −80°C after harvest until analysis was performed at Kennedy Space Center, Merritt Island, FL, United States. A range of microbial densities as indicated by aerobic bacterial and fungal plate counts was found depending on plant type and location. The four samples of mizuna, the only leafy green tested, were grown in different Lada root modules at different times and counts ranged from 3.1 × 10 3 – 8.7 × 10 5 . Materials and Methods Plant Growth and Harvest The Veggie production system on ISS is a small plant growth chamber designed and built by ORBITEC (Madison, WI, United States) to grow crops in space ( Morrow et al., 2005 ; Morrow and Remiker, 2009 ). The Veggie production system, equipped with LED lighting and a passive watering system, launched to the ISS in 2014 aboard Space X’s third Commercial Resupply (CRS-3) mission ( Figure 1 ). Red romaine lettuce Lactuca sativa cv. ‘Outredgeous’ was grown in Veggie rooting pillows as described by Massa et al. (2017a) . These are persistent and common in station potable water as well as terrestrial water sources and have been isolated and identified from Space Shuttle potable water ( Koenig and Pierson, 1997 ; Ryan et al., 2007 ) so it is not unexpected that these were also isolated in the roots and leaves of both flight and ground control plants. Plants, regardless of growth conditions, harbor an indigenous population that may affect plant health and, if utilized as a food source, human health.

Plants in space refer to plants that are grown in the physical universe known as outer space or outer Earth’s atmosphere, which is the typical orbit range of the Space Shuttle missions and of the International Space Station (ISS), where most of human spaceflight and research has taken place. It was focused on improving human habitability through the supply of a continuous source of fresh food for the crews. The International Space Station continues to be a platform for researchers to investigate how various spaceflight conditions affect plant growth and development in different environmental conditions than plants grown on earth. The ability to grow safe and fresh vegetables to supplement the packaged foods of astronauts in space has been an important goal for the National Aeronautics and Space Administration (NASA). Among crop plants, vegetables rich in nutrients and minerals, short duration and more responsive to environmental parameters are the best to grow in ISS. The primary goals of NASA in using these modular plant growth systems are to investigate both the nutritional impact of supplementing astronaut diets with fresh produce as well as the psychosocial benefits of crew tending plants during extended duration spaceflight missions. 3Department of Agricultural Entomology, University of Agricultural Sciences, Bangalore-560065, India. *Corresponding author E-mail: suneethavsn@gmail.com (Date of Receiving : 25-08-2025; Date of Acceptance : 01-11-2025) ABSTRACT Plants in space refer to plants that are grown in t he physical universe known as outer space or outer Earth’s atmosphere, which is the typical orbit rang e of the Space Shuttle missions and of the International Space Station (ISS), where most of hu man spaceflight and research has taken place. It wa s focused on improving human habitability through the supply of a continuous source of fresh food for th e crews. The International Space Station continues to be a platform for researchers to investigate how various spaceflight conditions affect plant growth and development in different environmental conditions than plants grown on earth. The ability to grow saf e and fresh vegetables to supplement the packaged foods of astronauts in space has been an important goal for the National Aeronautics and Space Administration (NASA). Among crop plants, vegetable s rich in nutrients and minerals, short duration and more responsive to environmental parameters are the best to grow in ISS. The challenges of watering plants in microgravity (Stankovic, 2018). As well, to grow plants in space requires cultivars th at are well suited to growing and sustaining in the space environment. 446 Plants in space : Growing vegetables in space- A review Importance of growing plants in space One astronaut on the International Space Station requires approximately 1.8 kilograms of food and packaging per day. For a long-term mission, such as a four-men crew, three-year mission, this number can grow to as much as 10,886 kg (Cooper et al., 2011). Tokyo Bekana) to continuously elevated carbon dioxide in a simulated Space Station "Veggie " crop-production environment. Studies reported that ‘Tokyo Bekana’ was very sensitive to continuously elevated CO 2 in such a growth environment and indicated the need for improved environmental contr ol of CO 2 and possibly root-zone factors for successful crop production in the ISS spaceflight environment. Khodadad et al . (2020) conducted an experiment on the microbiological and nutritional analysis of let tuce crops grown on the International Space Station. Thi s study indicated that leafy vegetables could produce safe, edible, fresh food for astronauts’ diets and provide baseline data for continual operation of th e veggie plant growth units in the ISS. Advanced Plant Habitat (APH) The Advanced Plant Habitat (APH) is a fully automated plant growth facility that will be used t o grow plants on the International Space Station. The Radish plants grown in the Advanced Plant Habitat were harvested on Nov. 30th, 2020 on the International Space Station. Harvested produce is brought back to earth for microbiological and nutri ent analysis in laboratories. Chiili is grown in 2021 f or a period of 137 days. It is the longest and spiciest vegetable in ISS. John et al . (2021) conducted an experiment on space flight cultivation for radish (Raphanus sativus ) in the Advanced Plant Habitat and reported that the strategies to optimise the growth substrate, watering regimen, light settings, and planting design that produced good-sized radishes, minimised competition, and allowed for easy harvesting. International Journal of Chemical Studies , 8(4), 2936–2938. John, S., Abou-Issa, F., and Hasenstein, K. H. (202 1). Space flight cultivation for radish ( Raphanus sativus ) in the advanced plant habitat. Gravitational and Space Research , 9(1), 121–132. Khodadad, C. L., Hummerick, M. E., Spencer, L. E., Dixit, A. R., Richards, J. T., Romeyn, M. W., and Massa, G. D . (2020). Microbiological and nutritional analysis of lettuce crops grown on the International Space Station. Frontiers in Plant Science , 11 , 1–15. Maggi, F., and Pallud, C. (2010). Martian base agri culture, The effect of low gravity on water flow, nutrient cycle s, and microbial biomass dynamic. Advances in Space Research , 46 , 1257–1265. Massa, G. D., Wheeler, R. M., Morrow, R. C., and Le vine, H. (2016). Growth chambers on the International Space Station for large plants. Acta Horticulturae , 1134 , 215– 222. Musgrave, M. E., Kuang, A., Tuominen, L. K., Levine , L. H., and Morrow, R. C. (2005). Seed storage reserves and glucosinolates in Brassica rapa L. grown on the International Space Station. Journal of the American Society for Horticultural Science , 130 (6), 848–856. Paul, A. L., Elardo, S. M., and Ferl, R. J. (2022). Plants grown in Apollo lunar regolith present stress-associated 449 S.N. Darshan et al . transcriptomes that inform prospects for lunar expl oration.

ral sensing to monitor crop stress. In a plant growth chamber in the KSC Space Life Sciences Lab, plant physiologist Ray Wheeler checks radishes being grown using hydroponic techniques. Wheeler and other colleagues are researching plant growth under different types of light, different CO2 concentrations and temperatures. The Lab is exploring various aspects of a bioregenerative life support system. Such research and technology development will be crucial to long-term habitation of space by humans. (Photo by NASA) UM – Okay, let's talk food and space. Do you see future colleagues growing their own food in space, essentially eating fresh foods? RW – I definitely do. In fact, it's already happening with U.S. and other astronauts on the International Space Station. I think this will continue to expand as mission durations and distances increase. I would hope that some of these future astronauts would be some of my current colleagues, but we are all collegial in the space exploration community. UM – For space exploration, say over a year, how much space is available to grow/store food? RW – As a community of crop researchers, I think we would all say the more space available the better, but we realize there will be constraints. We know with the two veggie units on the space station, we have about 0.3 m2 of growing area. I am personally hopeful that we can at least get this to about 0.5 m2 with a plant vegetable production system called OHALO that we plan to test on station. I think further increases may be possible in the future for a Mars mission. When we get to surface settings on the Moon and Mars, then we might be able to evolve to larger crop production systems as the mission infrastructure expands. We know that with high light intensity and optimized crop growth environments, 20-25 m2 of crops can produce enough oxygen for one person and perhaps about half of a person's dietary calories; having enough light and optimizing growth are key to achieving those goals, th

Abstract One of NASA’s goals is growing edible crops in spaceflight to supplement the astronauts’ diet with fresh, safe-to-eat vegetables. Growing plants in spaceflight presents challenges to achieve optimal plant growth and productivity. It is important to understand the optimal conditions for crops grown in spaceflight. The Advanced Plant Habitat (APH), an enclosed, environmentally controlled plant growth facility on the International Space Station (ISS) has been used to grow plants and monitors the environment. A technology demonstration mission on the ISS used the APH to grow chile peppers, Capsicum annuum, cv. NuMex Española Improved to maturity. Over 25 peppers were successfully harvested as a mix of red and green peppers at 109 and 137 days after initiation. Half of the fruit were consumed by the crew, and half were frozen at − 80 °C and returned with the science carrier (SC) to Kennedy Space Center, FL to determine microbial load and food safety. Bacterial and fungal counts, ranged from below detection limit to 111 CFU/gram fresh weight found on a single pepper only. Foodborne pathogens were not detected. Investigation of the communities using the V4 region of the 16S rRNA gene revealed taxonomic variations between the SC quadrants as well as between hardware components and plant tissues, indicating possible vertical or horizontal transfer of some bacteria. An investigation into the bacterial communities indicated 13 genera and 1 unidentified microbe as possible core 1579 scirep Scientific Reports Sci Rep Nature Publishing Group PMC13096386 13096386 13096386 41577717 10.1038/s41598-025-20440-9 Evaluating microbial community profiles of Chile peppers grown on the International Space Station provides implications for fruiting crops Khodadad Christina L M 1 Dixit Anirudha R 2 ✉ Hummerick Mary E 1 Spencer LaShelle E 1 Spern Cory J 1 Torres Jacob 3 Monje Oscar 2 Richards Jeffrey T 2 Gooden Jennifer 2 Curry Aaron B 3 Massa Gioia D 4 DuFour Nicole 4 Poulet Lucie 5 Romeyn Matthew W 4 Wheeler Raymond M 4 1 Noetic Strategies, Inc., Lincoln Mills Building #1, 1300 Meridian St N Suite 3000-b, Huntsville, AL 35801 USA 2 AETOS Systems Inc., 1525 Perimeter Parkway, Suite 245, Huntsville, AL 35806 USA 3 Engineering Research and Consulting LLC, 308 Voyager Way NW, Suite 200, Huntsville, AL 35806-3200 USA 4 NASA, Exploration Research & Technology (ER&T) Programs, Kennedy Space Center, FL, 32899 USA 5 Clermont Auvergne INP, CNRS, Institut Pascal, Université Clermont Auvergne, 63000 Clermont-Ferrand, France ✉ Corresponding author. Abstract One of NASA’s goals is growing edible crops in spaceflight to supplement the astronauts’ diet with fresh, safe-to-eat vegetables. Growing plants in spaceflight presents challenges to achieve optimal plant growth and productivity. It is important to understand the optimal conditions for crops grown in spaceflight. The Advanced Plant Habitat (APH), an enclosed, environmentally controlled plant growth facility on the International Space Station (ISS) has been used to grow plants and monitors the environment. A technology demonstration mission on the ISS used the APH to grow chile peppers, Capsicum annuum, cv. NuMex Española Improved to maturity. Since 2000, the International Space Station (ISS), a semi-closed, man-made environment built for space exploration and human habitation has become the largest space laboratory and being permanently crewed has provided support to spaceflight experiments including plant studies and validation of plant habitats on station. These studies included the Advanced Astroculture (ADVASC), in 2000, with Arabidopsis thaliana and soybean 1 – 3 ( Glycine max ) followed by the Biomass Production System (2000, Triticum aestivum L. cv. USU Apogee) 4 – 6 and then Lada 7 , 8 . The Lada greenhouse was developed and flown in 2002 using Mizuna ( Brassica rapa var. 1 An Advanced Plant Habitat (APH) science carrier/root tray being prepared for flight showing 4 independent quadrants in preparation for the chile pepper seed planting. Each quadrant shows a different stage of planting with the front left quadrant ready for seed insertion ( A ). Pepper plants in the APH on ISS at flowering stage ( B ), Pepper plants in APH in fruiting stage, each plant producing peppers ( C ), and science carrier after harvesting all 4 quadrants ( D ). Planting occurred in April 2021 at Kennedy Space Center, FL and launched to the International Space Station (ISS) in June 2021. Table 1 Nutricote fertilizer formulation for the PH-04 science carrier. Fruit and leaves were harvested on station by the crew members and immediately frozen at − 80 °C in the station’s Minus Eighty Laboratory Freezer on ISS (MELFI) and maintained at that temperature when received at Kennedy Space Center, FL until processing. Table 2 Samples taken from the science carrier/root tray returned from the International Space Station. It is crucial to understand how these environmental conditions affect plants for the success of long-duration missions, where freshly grown vegetables and fruit, like peppers, will serve as a primary food source for crew members on lunar or Mars missions, especially when resupply from Earth is limited or impossible. Prior technical demonstrations on the International Space Station (ISS) have shown that growing leafy green vegetables in space can be accomplished successfully and have provided the astronauts with a fresh vegetable supplement 38 , 39 . Plants grown in space experience different environmental conditions than on Earth and understanding these effects on plants and plant microbiota is necessary to accomplish successful crop cultivation. Studies on Earth have revealed that the microbiomes of plants can be responsible for plant growth and development as well as plant defense mechanisms providing purpose to the microbiomes of plants from the seeds 41 to the mature plants 42 – 46 .

Virtually all scenarios for the long-term habitation of spacecraft and other extraterrestrial structures involve plants as important parts of the contained environment that would support humans. Recent experiments have identified several effects of spaceflight on plants that will need to be more fully understood before plant-based life support can become a reality. The International Space Station (ISS) is the focus for the newest phase of space-based research, which should solve some of the mysteries of how spaceflight affects plant growth. Research carried out on the ISS and in the proposed terrestrial facility for Advanced Life Support testing will bring the requirements for establishing extraterrestrial plant-based life support systems into clearer focus.

The original finding aid described this as: Description: Close-up view of the Plant Generic Bioprocessing Apparatus (PGBA) /BioServe experiment in Rack 4 of the International Space Station (ISS) Destiny laboratory module. Subject Terms: Plants (Botany), Racks (Frames), Spaceborne Experiments, STS-112, U.S. Laboratory Date Taken: 10/13/2002 Categories: Experiment (Non-medical) Interior_Exterior: Interior Ground_Orbit: On-orbit Original: Digital Still Preservation File Format: TIFF

The original finding aid described this as: Description: Close-up view of the Plant Generic Bioprocessing Apparatus (PGBA) /BioServe experiment in Rack 4 of the International Space Station (ISS) Destiny laboratory module. Subject Terms: Plants (Botany), Racks (Frames), Spaceborne Experiments, STS-112, U.S. Laboratory Date Taken: 10/13/2002 Categories: Experiment (Non-medical) Interior_Exterior: Interior Ground_Orbit: On-orbit Original: Digital Still Preservation File Format: TIFF

Abstract In the period from March 2003 to April 2005 we fulfilled five experimental cultivations of genetically marked dwarf pea species in greenhouse Lada installed in the Russian segment (RS) of the International Space Station (ISS). The purpose of this series of experiments was to make morphologic and genetic analysis of pea plants grown in successive generations. According to our results, pea growth and development over the full cycle of ontogenesis (from seed to seed) taking place in space greenhouse Lada were not different as compared with the ground control plants. In addition, four successive pea crops gathered in space flight did not loose their reproductive functions and formed viable seeds. Genetic analysis of the plants grown from the “space” and “ground” seeds produced by the first to fourth successive crops was performed using the methods of chromosomal aberrations count and Random Amplified Polymorphic DNA (molecular method). No genetic polymorphism was found either in the experimental or control crops. This can serve as a sound argument for the supposition that the genetic apparatus of plants is not impacted by exposure of several successive generations to the conditions of space flight.