The application of a HACCP-informed risk assessment to mizuna and tomato crops grown on ISS in Veggie to develop a baseline food safety plan within the constraints of space mission food production

Introduction

With the rapid growth of Controlled Environment Agriculture (CEA) in the fresh food production market, new obstacles are being encountered with regard to food safety and plant disease control. Lessons learned from terrestrial CEA can be applied to space crop production (Hamilton et al., 2023; Topalcengiz et al., 2024). However, the scale of production from the small plant growth units installed on the ISS, such as NASA’s Veggie and Advanced Plant Habitat (APH), the end consumer (astronaut), and the unique environmental conditions crops are subjected to in space make it challenging to directly compare to terrestrial agriculture systems’ challenges. Furthermore, terrestrial CEA operations depend on customer requirements often verified by 3^rd party audits with standards developed by blending elements of conventional farming and processing facilities (CEA Food Safety Coalition, 2021; CEA Alliance, 2023), whereas space-based CEA currently has no developed standards. NASA has a long history of growing crops in space for basic biological research and, with the deployment of the Veggie plant growth unit in 2014, for consumption by the astronauts (Paul et al., 2013; Massa et al., 2017a; Kordyum and Hasenstein, 2021; Hughes and Kiss, 2022; De Micco et al., 2023; Maffei et al., 2024). A variety of leafy greens and, most recently in the VEG-05 experiment, tomatoes, have been grown in the Veggie hardware, and pre- and post-flight samples have been analyzed for microbial content (Khodadad et al., 2020; Hummerick et al., 2021; Bunchek et al., 2024; Spern et al., 2026). As a ready-to-eat food source for the crew, the crops grown on the ISS must be safe and free of potential foodborne pathogens. Hazard Analysis Critical Control Point (HACCP) plans are utilized in food production and agricultural operations to prevent food borne illness and reduce hazards to a level that is not reasonably likely to occur (Awuchi, 2023; Stenzel et al., 2023; FDA, 2024). This approach originated with the beginning of human space flight by NASA to ensure the quality of food prepared for astronauts, and has since been implemented globally to meet international food safety standards (Awuchi, 2023). Inherent limitations of crop systems in spaceflight may not support a HACCP plan in the classical sense; however, the framework can be applied where possible. HACCP is based on the following seven principles to control and prevent food safety hazards (https://www.fda.gov/food/hazard-analysis-critical-control-point-haccp/haccp-principles-application-guidelines)

1. Conduct a hazard analysis

2. Determine critical control points

3. Establish critical limits

4. Establish monitoring procedures

5. Establish corrective actions

6. Establish verification processes

7. Establish record keeping

The US Food and Drug Administration’s Food Safety Modernization Act (FSMA) addresses requirements for agricultural operations and producers and establishes minimum standards for safe growing and harvesting fresh produce. These standards as well as those set by other regulatory agencies, are good guidelines to define best practices in the production of fresh crops in space-based systems like Veggie, APH, or future exploration crop growth systems. While applying the classic HACCP approach to space crop production presents challenges and exposes gaps in its implementation, applying the framework of HACCP to vegetable production on ISS will lower the risk of foodborne illness from space-grown crops and inform development of standards and technologies that close these gaps. The FDA provides guidance (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industry-guide-minimize-microbial-food-safety-hazards-fresh-fruits-and-vegetables) specifically for the implementation of Good Agricultural Practices (GAP), outlining the potential sources of microbial contamination and stressing the importance of preventive measures to minimize contamination in the first place. In terrestrial agriculture, human and animal feces are the major source of foodborne pathogens often introduced in field irrigation water via contaminated irrigation water (Holvoet et al., 2015; Mogren et al., 2018; Pachepsky et al., 2018; Alegbeleye et al., 2018; Kim et al., 2023), while nutrient solutions deployed in indoor agriculture operations can carry various microorganisms, including bacteria, protozoa, and chemical contaminants (CEA Alliance, 2023). The advantage of crop systems designed for space missions is the precise control and the ability to prevent certain types of contamination that may be difficult in terrestrial agricultural practices.

Prevention of foodborne illness and crop disease is especially critical for controlled environment space farming. If the introduction of human pathogens were to occur, the risks to the crew of exposure to foodborne pathogens could be increased over terrestrial risks, considering the association of immune dysregulation with spaceflight (Borchers et al., 2002; Rizzo et al., 2012; Crucian et al., 2015; Winer et al., 2026).

Crop loss from the introduction of plant pathogens could impact astronaut nutrition if crops are a key part of future space diets. Also, cleaning hardware to remove contaminants may be more challenging in flight with resource and materials limitations. One of the components in the development of a disease prevention plan is to identify microbial risks associated with individual crops, the systems in which they are grown, and the associated horticultural practices. The investigation of microbial load on space-grown crops is ongoing, especially since the deployment of the Veggie facilities on ISS (Khodadad et al., 2020; Hummerick et al., 2021; Bunchek et al., 2024; Spern et al., 2026). Unlike processed food products in the astronaut diet, there are not yet enough data from space crop systems for NASA to develop microbiological standards for fresh grown produce harvested and consumed by the astronauts. The only close comparison is CEA-grown fresh produce, which must be free from pathogens but does not have limits for general microbiological levels. Instead, these systems rely on safe handling, high levels of cleaning and sanitation, and pathogen testing of inputs, food contact surfaces, and final product. The same levels of cleaning are not possible in spaceflight due to limitations on water and chemical cleansers, and microbiological testing capabilities prior to consumption are not available.

Crops, mostly leafy greens, have been grown in the Veggie facilities in a series of technological demonstrations and experiments, and for each of these crops, microbiological analysis was performed from Veggie ground test samples before they were approved for crew consumption on ISS. In addition, harvested produce grown on ISS has been returned to Earth for analysis. Results indicate that ISS-grown crops carry higher numbers of bacteria and fungi than corresponding ground controls (Khodadad et al., 2020; Hummerick et al., 2021; Bunchek et al., 2024; Spern et al., 2026).

Prior to the establishment of the food safety plan, prerequisite programs were developed to reduce the likelihood of introducing potential hazards (USDA, FSIS Guidelines | Food Safety and Inspection Service, usda.gov). As applied to crops grown in Veggie, this prerequisite program included standard operating procedures for sanitization of plant growth materials and seeds and other safe handling techniques.

Here we have applied the HACCP planning principles as a risk assessment to the cultivation of crops on the ISS in the Veggie hardware. This work focuses on the VEG-04 and VEG-05 experiments in which mizuna and tomato were grown, respectively, under different lighting and harvest regimes in the unique environment of the ISS (Fig. 1). Baseline microbiological assessments of mizuna and tomatoes from experiment verification testing, as well as ground and flight experiments, are presented here. The approach described in this work is a modification of traditional HACCP plans given that there is currently limited microbiological information available for crop growth in spaceflight. The resulting data and baseline food safety plan will be used to inform development of standards and technologies that close the food safety gaps.

Figure 1.

Methods

Results

Hazard Analysis: Microbial assessment

VEG-04A and B: EVT, ground and flight mizuna

By performing microbial food safety screening and analysis on crops grown in the Veggie system during pre-flight testing, we determined the microbial load typical of the crops grown, specifically leafy greens and tomatoes. Table 3 shows the microbial counts from the EVT and the VEG-04A experiment ground controls, and flight samples reported previously by Bunchek et al., 2024. EVT testing provided an opportunity to determine baseline microbial loads and the efficacy of produce sanitization on plants grown using the same materials under the same conditions as the flight experiments. Johnson Space Center (JSC) food technology and environmental health reviewed EVT data, and flight medicine provided final approval before the crew was allowed to consume the produce during the subsequent flight experiment. The mean aerobic plate count (APC) and fungal counts per gram of unsanitized leaf tissue from VEG-04A EVT were low, with the exception of one plant out of the five sampled from the EVT grown under red-rich light at 1.2 × 10⁶ CFU/g. The other four plants from the set of five did not exceed 1.4 × 10⁴ CFU/g (Table 3).

Table 3.

Mean bacterial (APC) and fungal counts on mizuna leaves (CFU/g) from the VEG-04A experiments, including the EVT, Ground control, and Flight.

	Mizuna VEG-04A-Red-rich					Mizuna VEG-04A-Blue-rich
	EVT		Ground Control		Flight	EVT		Ground Control		Flight
	BS	AS	BS	AS	BS	BS	AS	BS	AS	BS
APC	1.2×106 (1/5)	61	184	311	1.7×106 (5/5)	1,100	95	1,300	295	5.2×104 (2/3)
Fungi	268	61	124	74	3.4×104	106	95	555	94	3.9×103
Pathogen screening	NEG	NEG	NEG	NEG	NEG	NEG	NEG	NEG	NEG	NEG

[i] BS=before sanitizing; AS = after sanitizing. EVT (n=6 per light treatment). Ground control (n=6 per light treatment) and Flight samples (n=5 in red-rich and n=3 in the blue-rich treatment) as described in Buncheck et al, 2024. The number of plants of total evaluated, in which counts exceed the NASA thermostabilized standard for APC, is shown in parentheses. <DL=below detection limit. Pathogen screening included Salmonella spp, Staphylococcus aureus, coliforms, and E. coli.

VEG-04B plants were harvested using a “cut-and-come-again” method with three consecutive harvests during the EVT (Table 4) as well as during the flight growth test on the ISS and the corresponding ground control (Bunchek et al., 2024).

Table 4.

Mean bacteria (APC) and fungal counts on mizuna leaves (CFU/g) from the VEG-04B EVT (n=6 per light treatment).

	EVT Mizuna VEG-04B-Red-rich			EVT Mizuna VEG-04B-Blue-rich
	Harvest 1	Harvest 2	Harvest 3	Harvest 1	Harvest 2	Harvest 3
APC BS	2.1×106 (4/6)	8.2 × 103	3.1 × 104 (2/5)	8.2 × 105 (2/5)	3.3 × 105 (4/5)	6.7 × 106 (5/5)
APC AS	1.6 × 103	347	6.4 × 103	3.1 × 103	4.1 × 103	2.8 × 103
Fungi BS	1.2 × 103	400	9.2 × 103	260	31	2.2 × 105
Fungi AS	22	2.1 × 103	899	<DL	573	498
Pathogen screening	NEG	NEG	NEG	NEG	NEG	NEG

[i] BS=before sanitizing, AS=After sanitizing. Ground control (n=6 per light treatment) and flight samples (n=6 until harvest 1, then n=5 in red-rich and n=3 in the blue rich treatment) as described in Bunchek et al, 2024. Number of plants of total evaluated in which counts exceed NASA thermostabilized standard for APC are shown in parenthesis. <DL=below detection limit. Pathogen screening included Salmonella spp, Staphylococcus aureus, coliforms, and E. coli.

Sanitizing produce with wipes (Bunchek et al., 2024) appeared to be effective in lowering the average microbial load on the mizuna leaves as demonstrated in the EVT and ground controls (Table 4).

As a comparison to NASA standards for non-thermostabilized food, which state “no more than two samples out of five exceed 1 × 10⁴ CFU/g APC or 1 sample out of 5 exceed 2 × 10⁴ CFU/g”, VEG-04A and B mizuna after sanitizing met this standard. Perhaps more relevant to fresh produce, since APC is not necessarily a safety indicator, is the comparison to similar greens from market or field reported to be in the range of 1 × 10⁴ to 1 × 10⁸ CFU/g (International Fresh Produce Association, 2010; Valentin-Bon and Jacobson, 2008; Soendjojo, 2013; Jackson et al., 2013; Korir et al., 2016; Bohaychuk et al., 2016; Zhang et al., 2018; Zoellner et al., 2018; Maffei et al., 2024).

Comparisons of APC counts from VEG-04A and B mizuna (Fig. 2) between other Veggie tests with mizuna on ISS, as well as produce grown in controlled environment chambers at KSC, and similar market-acquired leafy produce (unpublished data) illustrate that Veggie-grown mizuna is in the range of the Market mustard greens. Additionally, the VEG-04A greens grown in flight were orders of magnitude lower than market greens, as are controlled environment-grown mizuna samples. VEG-04B mizuna showed a much wider range in counts when all three harvests were combined. Our analysis indicates no potential pathogens were identified from these crops, with the exception of a low number (88 CFU/g) of S. aureus in the 2nd and 3rd harvests on the same plant in VEG-04B, indicating a safe product and adequate control procedures.

Figure 2.

VEG-05 tomatoes

Tomato samples from the EVT harvests yielded generally low APC and Fungi CFU/g. Fungal counts were all below detection limits, and tomatoes from the second harvest from the blue-rich treatment yielded the highest counts (Table 5). APCs from the VEG-05 ground control samples (16 fruits) from the red-rich treatment ranged from 12 to 1.3 × 10³ CFU/g, and from the blue-rich treatment (14 fruits), 8 to 136 CFU/g. There was a lower harvest yield in the flight experiments due to plant stress leading to flower and fruit abortion, as well as visible microbial growth. Only one ripe fruit was evaluated microbiologically from the red-rich light treatment, and four were evaluated from the blue-rich treatment, and APCs ranged from 13 CFU/g in the red-rich tomato sample to 1.8 × 10³ from the blue-rich samples, in spite of the microorganisms observed growing on the plants. Fungal counts were low in the ground controls in both lighting regimes, while flight samples were higher, ranging from 2.5 × 10³ to 1.3 × 10⁴. All samples were negative for E. coli, S. aureus, and Salmonella sp. Tomatoes were not consumed in flight due to plant stress and low yield.

Table 5.

Mean bacteria (APC) and fungal counts (CFU/g) on tomatoes per gram from the VEG-05 EVT (n=6 per light treatment).

	EVT Tomato VEG-05-Red-rich			EVT Tomato VEG-05-Blue-rich
	Harvest 1	Harvest 2	Harvest 3	Harvest 1	Harvest 2	Harvest 3
APC	54	504	21	1000	74,300 (2/6)	70
Fungi	<DL	<DL	<DL	<DL	<DL	<DL
Pathogen screening	NEG	NEG	NEG	NEG	NEG	NEG

[i] Number of plants out of the total analyzed in which counts exceed the NASA thermostabilized standard for APC are shown in parentheses. <DL=below detection limit. Pathogen screening included Salmonella spp, Staphylococcus aureus, coliforms, and E. coli.

A comparison was made between the VEG-05 grown tomatoes and KSC chamber-grown and market-acquired tomatoes (unpublished data) (Fig 3), and results show that the APC from Ground control and red-rich flight tomatoes were lower than Market tomatoes on average. Blue-rich flight tomatoes were in line with the market-grown tomatoes regarding bacterial counts.

Figure 3.

Discussion

The microbial load of mizuna grown in Veggie in earlier technology demonstration tests VEG-03D and VEG-03E (mixed crops with mizuna growing together in Veggie with various lettuce varieties), and tests of mizuna grown in the Russian Lada plant growth hardware on the ISS (Hummerick et al., 2010, 2011) ranged from 1.4 × 10³ to 2.6 × 10⁷ CFU/g bacteria and 2.4 × 10⁴ to 1.3 × 10⁵ CFU/g fungi (Hummerick et al., 2011, 2021). The repeated harvest method used in the VEG-04B experiments was also used in the LADA experiments with mizuna and, as in VEG-04B, bacterial and fungal counts increased with each subsequent harvest in the flight experiments (Hummerick et al., 2012, 2011; Bunchek et al., 2024). This trend was evident in the EVT harvests reported here only under blue-rich light treatment (Table 4). Microbial counts did not increase in the ground controls of VEG-04B (Bunchek et al., 2024).

The ISS microgravity environment, as well as other environmental factors such as high localized humidity in the Veggie flight chambers, may account for the differences between flight and ground control microbial growth dynamics (Bunchek et al., 2024). In addition, increased human handling, longer growth durations, and increased availability of microbial nutrients released due to leaf damage during harvest may increase microbial load, which may be a factor aside from harvest yield in determining the optimal harvest method for future space crops.

Since S. aureus is a commensal bacterium associated with the human microbiome, its presence on the leaves of one plant from the VEG-04B flight experiment emphasizes the necessity of safe handling procedures such as the correct use of gloves during and handwashing before harvest. Sequencing data collected from a variety of leafy greens grown in Veggie tech demos, VEG-01A and B, and VEG-03 C, D, E, and F, demonstrated the absence of human-associated pathogens on leaves (Khodadad et al., 2020; Hummerick et al., 2021). These additional data support the effectiveness of Veggie pre-flight and in-flight procedures to minimize the introduction of human pathogens to the crops grown in Veggie.

One of the goals of the VEG-04 and 05 experiments was to compare impacts of the different lighting regimes on crop yields, nutritional content, and microbial food safety. The different lighting treatments had no significant impact on microbial counts. The greatest differences in bacterial and fungal counts can be attributed to ground vs flight conditions. While CO₂, humidity, and temperature exterior to the plant growth compartments are the same between the ISS and the ground control chambers, there are variations in humidity and temperature inside the Veggie facilities. In the VEG-04 and 05 experiments, high humidity and water accumulation may have contributed to higher counts in ISS-grown plants than in ground controls (Bunchek et al., 2024; Spern et al., 2026). Humidity is a factor in increased bacterial and fungal counts on plant surfaces and seems to be the trend when comparing ISS-grown plants and the corresponding ground controls (Hummerick et al., 2021; Khodadad et al., 2020; Bunchek et al., 2024; Spern et al., 2026). Elevated humidity levels create a moist environment that promotes the growth and proliferation of microorganisms, including bacteria and fungi. This can lead to a greater accumulation of microbial populations on leaves and stems, increasing the risk of infection and disease development. Additionally, prolonged exposure to high humidity suppresses transpiration rates and can weaken plant defenses, increasing susceptibility to pathogens and ultimately impacting crop health and yield (Talley et al., 2002; Li et al., 2014; Schuerger et al., 2021; Yao et al., 2023; Yasuda et al., 2025).

Ideally, hardware created for sustainable crop production in space should regulate environmental factors that influence the microbiological quality of the crops.

NASA’s Ohalo III crop production system is the newest technology being tested at Kennedy Space Center (https://techport.nasa.gov/projects/97036). Ohalo III features active sensor-based water management, along with precise nutrient delivery and humidity, temperature, and CO₂ control, and while it recycles water, it also utilizes the ISS atmosphere to regulate CO₂ and O₂. Ohalo III was designed to validate diverse hosted hardware concepts, ranging from media-based substrates to hydroponics, to enable continuous space-based crop production. The Utah Reusable Root Module (URRM) (https://techport.nasa.gov/projects/183165) is a hosted hardware component for Ohalo III, engineered to support repetitive crop cycles of pick-and-eat crops with conditions that will foster balanced microbial communities. This approach minimizes microbial contamination and disease, thereby enhancing both crop safety and productivity (Garrido-Ruiz et al, 2025).

While sustainable crop production is a goal, Veggie technological demos and experiments served as proof-of-concept research for crop productivity in space utilizing a granular substrate and rooting pillows that were not designed for in situ reuse. Ohalo III is moving toward sustainable repetitive crop production. Fully hydroponic approaches are also being investigated as additional sustainable crop production methods. Pre-flight and in-flight control point processes are described in this paper in Tables 1 and 2. Following pre-flight procedures is critical to prevent contamination of the Veggie facilities and plant growth pillows. With these controls in place, in-flight sources of contamination are limited to ISS air, water, and human contact with the plants. Massa et al. (2017b) describe the procedures for pre-flight preparation of pillows and seed sanitization.

Crop failure is not generally recognized as a hazard in the traditional sense in HACCP; however, it represents a critical risk factor with direct implications for crew sustenance and, consequently, space exploration mission success. Poor yields and inedible crop material can foster microbial contamination in an agricultural system, including those planned for space flight.

Although this is not a formal HACCP plan since controls such as safe handling are GAPs rather than Critical Control Points, and validation steps such as microbiological testing are not possible prior to consumption in spaceflight, the testing done here will be used to develop standards for different crops in spaceflight and reduce risk.

Conclusion and future work

Crops have been grown successfully in the Veggie facility on ISS since 2014 and have provided fresh produce for the crew to consume. Microbiological monitoring performed at KSC laboratories indicates that, under current operational procedures, crops are microbiologically safe; however, a gap remains in the real-time quality assurance testing of the crops on ISS.

Nucleic acid amplification and sequencing technologies have been tested for real-time monitoring of air, water, and surfaces on the space station with some success (Castro Wallace et al., 2017; Khodadad et al, 2021). While not yet tested with samples of space-grown produce to screen for microbiological food safety, there is potential for this use; however, more development is needed. The Biomolecule Extraction and Sequencing technology (BEST), a swab-to-sequencer method employing a miniPCR and MinION sequencing, was tested and compared to results from traditional culture-based methods, and a close similarity was found between data sets (Castro Wallace et al, 2017). The development of a rapid method to diagnose plant disease, also a nucleic acid-based method, was developed by Haveman et. al. (2023) and could potentially be adapted for detection of food-borne pathogens. A single HACCP plan cannot be applied to “space crop production” in general, as each plant growth system is different in operation and microbial exposure risks. Veggie is an open system sharing the same airborne microbes as the surrounding space station environment, while the Advance Plant Habitat (APH) is a closed system with internal atmospheric controls and a closed water delivery system (Morrow et al., 2016; Monje et al., 2020), thus only exposing crops to the ISS environment during infrequent door-open crew operations. Each plant growth system has hosted a wide variety of plant experiments and technological demonstrations, some for fundamental plant research and others for crop production for consumption by astronauts. Ideally, implementing a HACCP plan requires dedicating the plant growth system exclusively to food crops and clearly defining all food products and potential hazards. NASA’s Ohalo III prototype crop production system is currently being built and tested at the Kennedy Space Center, with launch scheduled no earlier than December 2026. Operational validation of water delivery and crop production operations with Ohalo III is intended to inform the future development of next-generation crop production systems for long-duration space missions. Ohalo III can accommodate various hardware configurations to test different growth approaches, produce fresh crops to improve whole-food nutrition, and diversify astronaut crew diets. To ensure system operations and food safety, the Ohalo project integrates microbial risk mitigation directly into the hardware design and is implementing a more comprehensive HACCP framework for each crop type and hosted hardware configuration before deployment. Ultimately, the inherent resource constraints of spaceflight, specifically regarding water availability, chemical sanitation, and in-flight testing, are expected to serve as a catalyst for developing next-generation, high-efficiency food safety technologies necessary to realize deep-space crop production.