Microbial food safety in space production systems

Full text

Microbial food safety
in space production systems
A white paper submitted to the Decadal Survey on Biological and Physical Sciences Research in Space
2023-2032
Jessica Audrey Lee
Space Biosciences Research Branch, NASA Ames Research Center - jessica.a.lee@nasa.gov
Jeffrey K. Brecht, Horticultural Sciences Department, University of Florida
Sarah Castro-Wallace, NASA Johnson Space Center
Frances M. Donovan, Bioengineering Branch, NASA Ames Research Center
John A. Hogan, Bioengineering Branch, NASA Ames Research Center
Tie Liu, Horticultural Sciences Department, University of Florida
Gioia D. Massa, Exploration Research and Technology Programs, NASA Kennedy Space Center
Macarena Parra, Engineering Systems Division, NASA Ames Research Center
Steven A. Sargent, Horticultural Sciences Department, University of Florida
A. Mark Settles, Bioengineering Branch, NASA Ames Research Center
Nitin Kumar Singh, Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory
Yo-Ann Velez Justiniano, NASA Marshall Space Flight Center
Cover design: Miki Huynh

1
While traveling to deep space is difficult for many reasons, food is a crucial one. Round-trip Mars
mission scenarios last 3 years, demanding food with a shelf-life of 5 years [1]. This means that feeding
human crew sustainably for long-duration missions beyond low Earth orbit (LEO) will ultimately lead
to a paradigm shift away from the current Earth-based food production system, which depends upon
storing and transporting prepackaged foods, and toward bio-regenerative production of food in space
[1,2]. Pharmaceuticals and nutritional supplements face similar shelf-life challenges [2,3]. Moreover,
the methods we currently use to detect dangerous microbes in food require sample return to Earth, a
situation not viable for deep-space missions. While the science behind generating foods and
bioproducts is covered by other white papers, in this paper we discuss a crucial gap uniting all of them:
how to ensure that such products are free of unwanted microbial contamination and safe for crew to
consume. Because Earth-based food safety systems cannot be directly applied in space, safety
assurance is currently a critical bottleneck in the space production of food and other bioproducts.
Future sustainable deep-space missions will require NASA to devote more resources in the
coming decade to understanding the biological and physical science principles underlying
microbial food safety in space, and to developing efficient, reliable methods in this area.
BACKGROUND
NASA's current space food system is Earth-dependent and mostly sterile. The food system
currently feeding the International Space Station (ISS) crew consists of a rotating menu with ~200
items prepared on Earth, described in depth elsewhere [1,4,5]. All foods must be stable at room
temperature, as the ISS has no dedicated cold storage for food. Pre-packaged food types include
thermostabilized (retort process) dishes, irradiated meat items, dried and freeze-dried foods, extended
shelf-life bread products, dry beverage mixes, and natural-form foods, supplemented by a few fresh
foods as preference items and, only recently, some space-grown crops. Food safety in processed foods
relies on safe production methods: the Hazard Analysis and Critical Control Point (HACCP) system
and Good Manufacturing Practices (GMPs). Foods that are not commercially sterile must meet
standards for microbial tolerances (2x104 CFU/g total aerobic count; 103 CFU/g yeasts and molds;
102 CFU/g coliform or coagulase-positive Staphylococci; no Salmonella) [6]. Crew are trained to discard
uneaten food within 2 hours of preparation, and are not permitted to consume fermented foods,
probiotics, or other products with live microorganisms. No cases of foodborne illness have ever been
reported [5]. Thus, from a food safety perspective, the current system has been considered sufficiently
reliable for a future mission to Mars. But where the system is not sufficient is in the areas of nutritional
value and acceptability. And because solving the nutrition and acceptability problems will likely entail
the development of novel production methods, a novel safety assurance system will also be required.
Sustainable exploration beyond LEO will ultimately require bio-regenerative food production
in space. Initial missions to Mars would require foods with a minimum shelf life of 5 years at room
temperature, to allow for transit time, potential prepositioning of food, and contingencies [1].
Currently, NASA's pre-packaged foods have a stated shelf life of 2 years [5], due not to safety concerns
but to declines in acceptability and nutrient content. According to one study, Vitamins C and B1
degrade sufficiently quickly that within three years, the standard ISS diet would not provide adequate

2
levels for crew health; vitamins A, B6, and B12 also showed some degradation [7]. Deep-space
radiation may accelerate food degradation further, though data are lacking [5]. Space explorers thus
face one of the same challenges that faced explorers of Earth centuries ago, when vitamin C deficiency
(scurvy) incurred disastrous losses on historic voyages such as Anson's global circumnavigation [8].
In addition to maintaining essential nutrition, numerous other reasons exist for deep-space
explorers to consume fresh food. Several compounds typically found in plants could serve as dietary
radioprotective agents [9]. Including probiotics in the diet may potentially counteract the microbiome
shifts observed in astronauts [10,11]. In situ resource utilization (ISRU) through bio-regenerative food
production could ultimately save space and mass in food transport; for instance, through fixation of
waste CO2 and by minimizing food packaging, which comprises 15-17% of the current food system
[5]. Finally, the psychological benefits of caring for plants, eating diverse foods, and preparing food
may sustain crew mental health on long-duration missions [8,12,13]. Thus, while initial missions to
Mars will likely depend primarily on the current system of pre-packaged foods, a sustainable human
presence in deep space will ultimately require bio-regenerative food production [1,2].
The landscape of microbial food safety risks is fundamentally different in space. Microbial
food safety assurance entails 1) assessing the risk of foodborne illness to consumers and quantifying
acceptable levels of risk; 2) establishing a method of production that minimizes risk; and 3) testing to
ensure that food products meet standards. Risk assessments are context-dependent;
crucially, many
of the factors that play into food safety risk assessments are different in the space
environment, but we do not have sufficient data to recalculate risks accordingly.
These include:
● Constrained resources and dramatically different supply chains. Unique environments and
novel production systems require new solutions for monitoring, processing, and storage.
● Reduced medical care availability. On Earth, we accept that 7-20% of Americans will suffer
from foodborne illness and mitigate the consequences with medical treatment [14], but in space,
the scope of medical care is limited, which strongly encourages prevention rather than treatment.
● Compromised immune response in human crew. Spaceflight-induced immune system [15]
and gut microbiome stress [10,11] may require stricter microbial contamination control [16].
● Potential for increased microbial virulence. Safety methods must account for unpredictable
changes in virulence and biofilm formation of both pathogens and non-pathogens [17,18].
● Microorganisms will likely feature prominently in space-produced foods and
biopharmaceuticals. Without animal husbandry, a sustainable food system will require
engineered microorganisms to produce essential micronutrients for the near future, and microbial
bioreactors will likely precede agriculture as the first food production systems on Mars [2].
Space-based foods currently in development have limited microbial management processes.
Here is a summary of the status of vegetable crops, microbial foods, and recycled water on the ISS.
A) Horticultural crops
. Fresh produce (leafy greens, radishes, peppers, and soon tomatoes) has been
grown periodically on the ISS for crew consumption since 2015 [19,20], but production is still limited
and far from standardized. In contrast with pre-packaged foods, microbial safety standards for on-
orbit produce are assessed on a case-by-case basis. Initial approval for crew consumption of crops by
flight surgeons and safety boards was based primarily on data from ground studies. HACCP plans
have now been developed for space-grown produce; in addition to considerable preflight precautions,
crew procedures include wearing gloves when handling plants, and precautionary sanitizing. Crops are
grown from sterilized seeds and have been found, by microbial analyses of frozen samples returned
to Earth (e.g., [21]), to be colonized during growth by microbes from the surroundings. Microbial
levels on fresh produce grown on ISS tend to be higher than terrestrial ground controls, and

3
microorganisms that may be associated with health risks have occasionally been found on produce,
but only at levels well below levels of possible concern for human health. Sanitization of crops to
enable consumption currently entails gently pressing leaves of leafy produce between layers of Pro-
San® sanitizing wipes, or wiping non-leafy produce, for 30 seconds [19]. This approach is labor-
intensive and has the potential to miss microbial contaminants or damage produce.
B) Microbially produced foods and supplements
. The BioNutrients project is a series of ISS
experiments using microbes to produce micronutrients with short shelf-life in pre-packaged foods.
BioNutrients-1 demonstrates the growth of yeast engineered to produce zeaxanthin and beta-carotene,
two carotenoid compounds important for photoprotection, vision, and oxidative stress mitigation,
which are also sensitive to ionizing radiation and heat. The cultures were flown to the ISS in
dehydrated form in April 2019 and so far have demonstrated three on-orbit operations spanning two
years of storage in space [22]. BioNutrients-2, to fly in 2022, improves the bioreactor to a lower-mass
FEP bag, and expands the microbial species and products: included are the fermented foods yogurt
and kefir, and microbes engineered to produce follistatin, a muscle-enhancing protein and potential
reduced gravity countermeasure for astronauts [23]. As demonstrated by BioNutrients, microbial
cultures may be used to produce not only familiar fermented foods but also essential nutritional
supplements and pharmaceuticals for crew health. Other related future applications of cell culture
(microbial and eukaryotic) may include probiotic supplements and lab-grown meat, all of which will
require novel food safety protocols. Although the BioNutrients products use microbial strains
commonly used in food production, they are returned to Earth without being consumed, as there is
currently no method for inactivating the microbes, and ISS food systems policy does not allow crew
to consume high abundances of any live microorganisms.
C) Water recycling
. One of the longest-lived examples of microbial management in the space food
system is the Water Recovery System (WRS), launched in 2008 to generate potable water from urine
distillate, humidity condensate, and Sabatier product water, as well as the occasional off-loading of
ground-supplied water [24]. This system recycles >90% of ISS water, and its products are used for
drinking, washing, and rehydrating food. The WRS consists of the Urine Processor Assembly (UPA)
and the Water Processor Assembly (WPA). The reservoir for these products, the wastewater tank,
does not have a means of microbial control and, a year into operation, saw decreased outflow due to
an obstructing biofilm [24]. The issue has been remedied: while the wastewater tank likely contains a
thriving microbial community, catalytic oxidation, filtration, and the addition of a biocide (iodine, I2)
during processing serve to reduce microbial contamination in the potable water substantially. The ISS
water is held to stringent acceptability limits (50 CFU/mL total bacteria, assessed monthly; no
detectable coliforms per 100 mL, quarterly [25]). There has never been a true positive coliform
detected, and, apart from the early checkout process [26], the levels of culturable bacteria are well
below the requirement. This system has proven a reliable source of safe water and will likely serve as
an example for crewed missions and deep space exploration. However, microbial monitoring for the
WRS entails crew culturing bacteria and subsequent return to Earth for DNA sequencing. The present
methods have significant limitations, including the length of time between sample collection on-orbit
and subsequent analysis, and possible degradation during transport from ISS back to the JSC
Microbiology Laboratory on Earth.
Space-based monitoring and detection of microbial communities are in the early stages of
development (Table 1). Nucleic acid technologies have seen the most progress, including spaceflight
technology demonstrations, due to their diverse applications. However, none have yet been tested on
food samples. Table 1 summarizes these, as well as selected other methods used by the food industry
that have potential for space food applications but need development.

4
Table 1. Current and future technologies that could support in-flight pathogen detection.
Method
Ground-based
technology
Platforms tested in flight
Limitations of method
Nucleic acid
amplification
and
sequencing
• Real-time PCR
• Genomic
sequencing
• LAMP-BART
[27]
SHERLOCK
(CRISPR-Cas)
[28]
• CRISPR-chip
[29]
• WetLab-2: sample extraction (custom
platform; SimplePrep X8 validated on
the ground) & qPCR / RT-qPCR
(Cepheid SmartCycler) [30,31]
• BEST: Biomolecule Extraction and
Sequencing Technology (miniPCR
extraction, MinION sequencing). First
space DNA sequencing; on-orbit data
analysis demonstrated [32–34]
• Razor EX: qPCR platform [35]
• µTitan: custom automated nucleic acid
extraction for diverse low-biomass
samples (tested in parabolic flight) [36]
• Time-intensive
• Requires sample prep
• Reagents have short shelf-life
(lyophilization is potential fix)
• Data usually downlinked to
ground for analysis (in-flight
analysis in development)
• Does not determine live/dead
(viability PCR is potential fix)
• PCR detects only specified
pathogens
Chemical
sensors
• E-nose [37,38]
• E-tongue [39]
• surface-
enhanced
Raman
spectroscopy
[40,41]
• E-nose
• Detection limits for VOCs
• Sensor drift
• May require culturing of food
item to detect
• Raman requires sample
adherence to metal nanoparticles;
error-prone due to food particles
Antigen or
protein
• RAPID testing
[42,43]
• lateral flow
assays [44]
n/a
• Detects only specified pathogen
• Requires sample prep
• Reagent storage
Culture-
based
• PetriFilm plates
[45]
n/a
• Plates have ~1 yr shelf life
• Plating in lowered gravity
unknown
• Amplifying potential pathogens
GAPS AND RECOMMENDATIONS
A. Risk assessment and policy revision. The first step in establishing a coherent food safety
assurance plan is to quantify the risks of microbial contamination and establish standards for control
and monitoring, as the standards for pre-packaged foods are not appropriate for space-produced bio-
regenerative foods. Recent reviews on space-produced food conspicuously lack safety discussions, and
one recent review dedicated to safety acknowledged the dearth of data [4]. There has been one report
of a Quantitative Microbial Risk Assessment for exposure of ISS crew to Salmonella spp., Staphylococcus
aureus, and Pseudomonas spp. through consumption of lettuce and radishes grown in space [46], which
found the annual risk of infection to be as high as 10-1 for some scenarios. However, due to the paucity
of data, it used numerous assumptions and presented a worst-case scenario for a conservative estimate.
More data are needed to constrain risk estimates and generate targets and guidelines for safe food.
Given the potential future role of microbial foods in space, developing sustainable safety practices for
bio-regenerative space foods may entail a paradigm shift away from a philosophy of "the only good
bugs are dead bugs," toward a focus on maintaining healthy crew / station microbiomes.
B. Developing space-specific food production pipelines with safety in mind.
The development
of microbial space bioproducts should consider the downstream steps required for food safety
assurance: for instance, design of bioreactors that facilitate microbial monitoring and product
extraction, and methods for maintaining quality in recycled water (i.e., biocides) that do not interfere
with the growth of crops or microbial cultures for which the water is used. Horticultural crops can

5
themselves be improved through the identification of new cultivars with rapid flowering, high yield,
and extended shelf life and nutrient retention [47], or by engineering such traits using CRISPR/Cas
[48]. Hydroponic farming and machine learning technologies for growth monitoring can also aid in
producing crops with delayed senescence [49,50]. While all ISS crops are currently grown from sterile
seeds, inoculation of crops with probiotic or beneficial microbiota might improve crop health and
lower the risk from pathogens [51]; however, extensive research is required. Finally, HACCP plans are
the current standard for minimizing safety risk through the entire food production pipeline [52], and
will need to be developed for diverse production platforms, both microbial and agricultural. Process-
driven protection practices should be developed with input from flight crew.
C. Food storage and quality monitoring.
Improved plans for postharvest handling, processing, and
storage, such as refrigeration, freezing, and dehydration, can help to prevent spoilage and food waste
and increase food supply flexibility. Methods for processing and storage at microgravity, and
packaging with necessary barrier and sustainability properties, require development. Passive
refrigeration/freezing utilizing the low temperatures and vaccum of space should be explored.
Furthermore, the development of non-destructive and rapid methods for monitoring senescence and
deterioration in produce, such as state-of-the-art food shelf-life modeling, imaging-based sensors, and
machine learning approaches, will aid in enabling the storage of produce to meet shelf-life goals [53].
D. Microbial control.
Food system process controls are standardized methods to reduce the chance
of contamination. These procedures reduce the microbe population by inhibiting growth or killing
live microbes. The only process control developed for space-produced foods is the use of sanitizing
wipes on space-grown vegetables. Other methods need to be developed, such as:
● Heat-based microbial inactivation, such as cooking or pasteurization. As evidenced by the
Zero G Space Oven [54], reduced heat transfer due to the altered convective flows in reduced
gravity [55] can make the simplest of Earth food processing methods complicated in space. Both
basic research and development of heating techniques are required in this area.
● Irradiation methods, such as gamma or UV irradiation [56]
● Novel approaches such as exposing food to cold plasma or other sterilizing technologies [5,57]
E. Monitoring microbes and detecting pathogens.
Standard terrestrial methods for pathogen
detection in food systems by culturing pathogens from samples are slow, require significant material
resources, and risk contaminating the crew habitat with pathogens. As shown in Table 1, an array of
diverse microbial monitoring technologies awaits development for application in space food safety. In
addition, non-invasive technologies, such as hyperspectral imaging [53,58], could be employed to
detect biotic stress in horticultural crops during growth as a means of assessing contamination risk.
CONCLUSION
The 2011 Decadal Survey listed "Food, nutrition, and energy balance in astronauts" as a cross-cutting
issue of highest priority, stating, "It is critical that NASA: Address nutrient stability over time as part
of planning for long-duration exploratory missions, and; Develop a food system that can support such
a mission" [59]. Many specific goals are described in NASA's Human Research Roadmap Risk of
Performance Decrement and Crew Illness Due to Inadequate Food and Nutrition [60]. While
progress has been made in the past decade toward
producing
plant and microbial foods, the
development of
food safety assurance
lags substantially behind. Closing the gaps described
above in the coming decade will require not only technology development but also fundamental
research in a wide range of disciplines in the biological and physical sciences, such as microbial
physiology and ecology, host-microbe interactions, human immunology, microbial detection, synthetic
biology, nutritional biochemistry, and heat and fluid dynamics in reduced gravity.

6
REFERENCES
1. Douglas GL, Wheeler RM, Fritsche RF. Sustaining astronauts: Resource limitations, technology needs,
and parallels between spaceflight food systems and those on Earth. Sustainability. 2021;13: 9424.
doi:10.3390/su13169424
2. Nangle SN, Wolfson MY, Hartsough L, Ma NJ, Mason CE, Merighi M, et al. The case for biotech on
Mars. Nat Biotechnol. 2020;38: 401–407. doi:10.1038/s41587-020-0485-4
3. McNulty MJ, Xiong Y (Mary), Yates K, Karuppanan K, Hilzinger JM, Berliner AJ, et al. Molecular
pharming to support human life on the Moon, Mars, and beyond. Critical Reviews in Biotechnology.
2021;41: 849–864. doi:10.1080/07388551.2021.1888070
4. Kim HW, Rhee MS. Space food and bacterial infections: Realities of the risk and role of science. Trends
in Food Science & Technology. 2020;106: 275–287. doi:10.1016/j.tifs.2020.10.023
5. Douglas GL, Cooper M, Bermudez-Aguirre D, Sirmons T. Evidence Report: Risk of performance
decrement and crew illness due to an inadequate food system. NASA Human Research Program
Human Health Countermeasures Element; 2016. Available:
https://humanresearchroadmap.nasa.gov/Risks/risk.aspx?i=176
6. NASA (National Aeronautics and Space Administration). NASA Spaceflight Human-System Standard
3001, Vol 2. Human Factors Habitability and Environmental Health. NASA-STD-3001 VOL 2. 2011.
Available: https://standards.nasa.gov/standard/nasa/nasa-std-3001-vol-2
7. Cooper M, Perchonok M, Douglas GL. Initial assessment of the nutritional quality of the space food
system over three years of ambient storage. npj Microgravity. 2017;3: 1–4. doi:10.1038/s41526-017-
0022-z
8. Preston E. How NASA is solving the space food problem. In: Eater [Internet]. 17 Sep 2015 [cited 19
Sep 2021]. Available: https://www.eater.com/2015/9/17/9338665/space-food-nasa-astronauts-mars
9. Montesinos CA, Khalid R, Cristea O, Greenberger JS, Epperly MW, Lemon JA, et al. Space radiation
protection countermeasures in microgravity and planetary exploration. Life. 2021;11: 829.
doi:10.3390/life11080829
10. Morrison MD, Thissen JB, Karouia F, Mehta S, Urbaniak C, Venkateswaran K, et al. Investigation of
spaceflight induced changes to astronaut microbiomes. Front Microbiol. 2021;12: 659179.
doi:10.3389/fmicb.2021.659179
11. Voorhies AA, Mark Ott C, Mehta S, Pierson DL, Crucian BE, Feiveson A, et al. Study of the impact of
long-duration space missions at the International Space Station on the astronaut microbiome. Scientific
Reports. 2019;9: 9911. doi:10.1038/s41598-019-46303-8
12. Magnus S. Sandra Magnus’ journal: Food and cooking in space. In: ISS Expedition 18 [Internet]. [cited
19 Sep 2021]. Available:
https://www.nasa.gov/mission_pages/station/expeditions/expedition18/journal_sandra_magnus_6.ht
ml
13. Odeh R, Guy CL. Gardening for therapeutic people-plant interactions during long-duration space
missions. Open Agriculture. 2017;2: 1–13. doi:10.1515/opag-2017-0001

7
14. Scallan E, Griffin PM, Angulo FJ, Tauxe RV, Hoekstra RM. Foodborne illness acquired in the United
States—Unspecified agents. Emerging Infectious Diseases. 2011;17. doi:10.3201/eid1701.p21101
15. Afshinnekoo E, Scott RT, MacKay MJ, Pariset E, Cekanaviciute E, Barker R, et al. Fundamental
biological features of spaceflight: Advancing the field to enable deep-space exploration. Cell. 2020;183:
1162–1184. doi:10.1016/j.cell.2020.10.050
16. US Department of Health and Human Services. People at risk of food poisoning. In: FoodSafety.gov
[Internet]. 26 Apr 2019 [cited 1 Oct 2021]. Available: https://www.foodsafety.gov/people-at-risk
17. Bijlani S, Stephens E, Singh NK, Venkateswaran K, Wang CCC. Advances in space microbiology.
iScience. 2021;24: 102395. doi:10.1016/j.isci.2021.102395
18. Singh NK, Wood JM, Karouia F, Venkateswaran K. Succession and persistence of microbial
communities and antimicrobial resistance genes associated with International Space Station
environmental surfaces. Microbiome. 2018;6: 204. doi:10.1186/s40168-018-0585-2
19. Massa GD, Dufour NF, Carver JA, Hummerick ME, Wheeler RM, Morrow RC, et al. VEG-01: Veggie
hardware validation testing on the International Space Station. Open Agriculture. 2017;2: 33–41.
doi:10.1515/opag-2017-0003
20. NASA (National Aeronautics and Space Administration). Veggie Fact Sheet. FS-2020-01-007-KSC. In:
NASA Facts [Internet]. Available:
https://www.nasa.gov/sites/default/files/atoms/files/veggie_fact_sheet_508.pdf
21. Khodadad CLM, Hummerick ME, Spencer LE, Dixit AR, Richards JT, Romeyn MW, et al.
Microbiological and nutritional analysis of lettuce crops grown on the International Space Station.
Front Plant Sci. 2020;11. doi:10.3389/fpls.2020.00199
22. Ball N, Kagawa H, Hindupur A, Sims K. BioNutrients-1: Development of an on-demand nutrient
production system for long-duration missions. 49th International Conference on Environmental
Systems ICES-2020-119. 2020. Available: https://ttu-ir.tdl.org/bitstream/handle/2346/86343/ICES-
2020-119.pdf
23. Ball N, Kagawa H, Hindupur A, Kostakis A, Hogan J, Villanueva A, et al. BioNutrients-2:
Improvements to the BioNutrients-1 nutrient production system. 50th International Conference on
Environmental Systems ICES-2021-331. 2021. Available: https://ttu-
ir.tdl.org/bitstream/handle/2346/87260/ICES-2021-331.pdf
24. Carter L. Status of the Regenerative ECLS Water Recovery System. 40th International Conference on
Environmental Systems AIAA 2010-6216. Barcelona, Spain; 2010. Available:
https://arc.aiaa.org/doi/pdf/10.2514/6.2010-6216
25. Yamaguchi N, Roberts M, Castro S, Oubre C, Makimura K, Leys N, et al. Microbial monitoring of
crewed habitats in space—Current status and future perspectives. Microbes and environments / JSME.
2014;29. doi:10.1264/jsme2.ME14031
26. Toon K, Lovell R. International Space Station United States on-orbit segment potable water dispenser
on-orbit functionality vs. design. 40th International Conference on Environmental Systems. Barcelona,
Spain: American Institute of Aeronautics and Astronautics; 2010. doi:10.2514/6.2010-6250

8
27. Yang Q, Domesle KJ, Wang F, Ge B. Rapid detection of Salmonell in food and feed by coupling loop-
mediated isothermal amplification with bioluminescent assay in real-time. BMC Microbiology. 2016;16:
112. doi:10.1186/s12866-016-0730-7
28. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, et al. Nucleic acid detection
with CRISPR-Cas13a/C2c2. Science. 2017;356: 438–442. doi:10.1126/science.aam9321
29. Hajian R, Balderston S, Tran T, deBoer T, Etienne J, Sandhu M, et al. Detection of unamplified target
genes via CRISPR–Cas9 immobilized on a graphene field-effect transistor. Nat Biomed Eng. 2019;3:
427–437. doi:10.1038/s41551-019-0371-x
30. Rapid nucleic acid isolation method and fluid handling devices (TOP2-276). Patent No. 10,975,425. In:
NASA Technology Transfer Program [Internet]. [cited 1 Oct 2021]. Available:
https://technology.nasa.gov/patent/TOP2-276
31. Parra M, Jung J, Boone TD, Tran L, Blaber EA, Brown M, et al. Microgravity validation of a novel
system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the
International Space Station. PLOS ONE. 2017;12: e0183480. doi:10.1371/journal.pone.0183480
32. Stahl-Rommel S, Jain M, Nguyen HN, Arnold RR, Aunon-Chancellor SM, Sharp GM, et al. Real-time
culture-independent microbial profiling onboard the International Space Station using Nanopore
sequencing. Genes (Basel). 2021;12. doi:10.3390/genes12010106
33. Boguraev A-S, Christensen HC, Bonneau AR, Pezza JA, Nichols NM, Giraldez AJ, et al. Successful
amplification of DNA aboard the International Space Station. npj Microgravity. 2017;3: 1–4.
doi:10.1038/s41526-017-0033-9
34. Castro-Wallace SL, Chiu CY, John KK, Stahl SE, Rubins KH, McIntyre ABR, et al. Nanopore DNA
sequencing and genome assembly on the International Space Station. Sci Rep. 2017;7: 18022.
doi:10.1038/s41598-017-18364-0
35. Khodadad CLM, Oubre CM, Castro VA, Flint SM, Roman MC, Ott CM, et al. A microbial monitoring
system demonstrated on the International Space Station provides a successful platform for detection of
targeted microorganisms. Life. 2021;11: 492. doi:10.3390/life11060492
36. Urbaniak C, Wong S, Tighe S, Arumugam A, Liu B, Parker CW, et al. Validating an automated nucleic
acid extraction device for omics in space using whole cell microbial reference standards. Frontiers in
Microbiology. 2020;11: 1909. doi:10.3389/fmicb.2020.01909
37. Tavares F. NASA’s E-Nose device advanced to “sniff” COVID-19 from human breath. 19 Mar 2021
[cited 1 Oct 2021]. Available: http://www.nasa.gov/feature/ames/e-nose
38. Marlaire RD. Ames Researcher Develops “New Nose” for NASA. 15 Jan 2009 [cited 1 Oct 2021].
Available: https://www.nasa.gov/centers/ames/research/2009/Jing_Li.html
39. E-Tongue (TOP2-285). In: NASA Technology Transfer Program [Internet]. [cited 1 Oct 2021].
Available: https://technology.nasa.gov/patent/TOP2-285
40. Langer J, Jimenez de Aberasturi D, Aizpurua J, Alvarez-Puebla RA, Auguié B, Baumberg JJ, et al.
Present and future of surface-enhanced Raman scattering. ACS Nano. 2020;14: 28–117.
doi:10.1021/acsnano.9b04224

9
41. Pérez-Jiménez AI, Lyu D, Lu Z, Liu G, Ren B. Surface-enhanced Raman spectroscopy: benefits, trade-
offs and future developments. Chem Sci. 2020;11: 4563–4577. doi:10.1039/D0SC00809E
42. Bicart-See A, Rottman M, Cartwright M, Seiler B, Gamini N, Rodas M, et al. Rapid isolation of
Staphylococcus aureus pathogens from infected clinical samples using magnetic beads coated with Fc-
mannose binding lectin. PLOS ONE. 2016;11: e0156287. doi:10.1371/journal.pone.0156287
43. RAPID: Testing for Food Contaminants. In: Wyss Institute [Internet]. 5 Jul 2018 [cited 1 Oct 2021].
Available: https://wyss.harvard.edu/technology/rapid/
44. Choi JR, Hu J, Tang R, Gong Y, Feng S, Ren H, et al. An integrated paper-based sample-to-answer
biosensor for nucleic acid testing at the point of care. Lab Chip. 2016;16: 611–621.
doi:10.1039/C5LC01388G
45. 3MTM Petrifilm Plates: Microbial Food Testing, Quality Indicator Test. [cited 1 Oct 2021]. Available:
https://www.3m.com/3M/en_US/food-safety-us/foodandbeveragetests/petrifilm-plates/
46. Mena KD. Risk reduction practices for growing produce in space: Promoting and protecting flight crew
health. Internal NASA Report. NASA Johnson Space Center; 2019.
47. Kwon C-T, Heo J, Lemmon ZH, Capua Y, Hutton SF, Van Eck J, et al. Rapid customization of
Solanaceae fruit crops for urban agriculture. Nat Biotechnol. 2020;38: 182–188. doi:10.1038/s41587-019-
0361-2
48. Gratz SJ, Wildonger J, Harrison MM, O’Connor-Giles KM. CRISPR/Cas9-mediated genome
engineering and the promise of designer flies on demand. Fly. 2013;7: 249–255. doi:10.4161/fly.26566
49. Wheeler RM. Plants for human life support in space: from Myers to Mars. Gravitational and Space
Research. 2010;23. Available:
http://gravitationalandspaceresearch.org/index.php/journal/article/view/490
50. Wheeler RM. Growing food for space and Earth: NASA’s contributions to vertical agriculture.
Presentation presented at: American Society of Horticultural Science; 2015 Aug 4; New Orleans, LA.
Available: https://simoc.space/wp-content/uploads/2018/09/NASA%e2%80%99s-Contributions-to-
Vertical-Agriculture-WHEELER-2015.pdf
51. Menendez E, Garcia-Fraile P. Plant probiotic bacteria: solutions to feed the world. AIMS Microbiol.
2017;3: 502–524. doi:10.3934/microbiol.2017.3.502
52. Ropkins K, Beck AJ. Evaluation of worldwide approaches to the use of HACCP to control food safety.
Trends in Food Science & Technology. 2000;11: 10–21. doi:10.1016/S0924-2244(00)00036-4
53. Guo X, Ahlawat Y, Zare A, Liu T. Evaluation of postharvest senescence in broccoli via hyperspectral
imaging. 2020 Dec p. 2020.12.16.423030. doi:10.1101/2020.12.16.423030
54. Space cookies: First food baked in space by astronauts. BBC News. 24 Jan 2020. Available:
https://www.bbc.com/news/world-51235555. Accessed 1 Oct 2021.
55. Lotto MA, Johnson KM, Nie CW, Klaus DM. The impact of reduced gravity on free convective heat
transfer from a finite, flat, vertical plate. Microgravity Sci Technol. 2017;29: 371–379.
doi:10.1007/s12217-017-9555-8

10
56. Gilbert SN, Almengor A, Harris J, Kelley C. Feasibility of UV LEDs in a spacecraft wastewater
application: Exploring biofilm control in the WPA wastewater tank. ICES-2021-372. 2021. Available:
https://ttu-ir.tdl.org/bitstream/handle/2346/87280/ICES-2021-372.pdf?sequence=1&isAllowed=n
57. Douglas GL, Massa GD, Hummerick ME, Hintze PE. Chapter 12 - Cold plasma to disinfect spaceflight
grown produce. In: Bermudez-Aguirre D, editor. Advances in Cold Plasma Applications for Food
Safety and Preservation. Academic Press; 2020. pp. 333–340. doi:10.1016/B978-0-12-814921-8.00012-8
58. Lu B, Dao PD, Liu J, He Y, Shang J. Recent advances of hyperspectral imaging technology and
applications in agriculture. Remote Sensing. 2020;12: 2659. doi:10.3390/rs12162659
59. NRC (National Research Council). Recapturing a future for space exploration: Life and physical
sciences research for a new era. Washington, DC: The National Academies Press; 2011.
doi:10.17226/13048
60. NASA (National Aeronautics and Space Administration). Human Research Roadmap. [cited 1 Oct
2021]. Available: https://humanresearchroadmap.nasa.gov/