Design of Spaceflight Hardware for Plant Growth in a Sealed Habitat for Experiments on the Moon

NASA is planning a return to the Moon, and long-duration human missions will benefit from the presence of plants (Duke et al., 1989; Blüm et al., 1994; Wolverton and Kiss, 2009; Shymanovich and Kiss, 2022). Most directly, plants provide a source of food. Plant systems can also be a key part of the recycling system for the air and solid wastes (e.g., Wolverton and Kiss, 2009; Vandenbrink and Kiss, 2016). Plants can also be used to produce useful pharmaceuticals and medicines (e.g., Menezes et al., 2015; Mortimer and Gilliham, 2022). For this reason, a key goal for near-term robotic lunar missions is to investigate the germination and growth of plants in the ionizing radiation environment and low gravity of the Moon.

Near-term robotic landers to the Moon (von Ehrenfried, 2020) have limited payload capacity and are solar powered, so they can only be fully operated during the 14 Earth-days maximum of the lunar daylight period. This capacity implies significant limitations on near-term plant experiments. For this reason, we consider if important plant science could be obtained from a minimal habitat, the LPX (Lunar Plant Experiment), with the following characteristics: a total volume of ~ 1 liter (1U Cubesat) hermetically sealed habitat with no gas handling or processing system, an overhead camera, a CO₂ sensor, no window and only interior LED lighting, and an internal water reservoir discharged after landing to initiate germination. In this paper, we report on the construction and operation of such a minimal habitat and show that it can successfully grow ~ 100 seeds of the model plant species Arabidopsis thaliana (as well as seeds of Brassica nigra) over a 10 Earth-day period and demonstrate that, with only a camera and a CO₂ sensor, fundamental plant science can be obtained on germination rate, initial plant growth, and cotyledon (seed leaf) development.

Figure 1 is a diagram of the minimal LPX habitat considered here showing the components internal to the habitat. These are: a plant growth platform, LED lights, a camera, a CO₂ sensor, and a water delivery system. Power and data handling are presumed to be provided by the lander, as well as an initiation command to activate the water delivery system. Knowing the temperature of the unit is important and this can be measured with probes attached to the outside of the metal habitat, although interior temperature sensors can also be considered.

Figure 1

The primary advantages of the LPX habitat for robotic lunar experiments are the small volume and mass and its suitability for conforming with 1U-shape requirements. LPX is self-contained, requiring only power in and data out connections to the main lander. Once started, it can operate automatically. Because the system is designed to operate in a lunar vacuum, it must be hermetically sealed to exacting standards. This aspect is most important in the design of the electrical pass-through for power and data. In addition, all components must be tested for long-term biocompatibility in a sealed environment—in particular no off-gassing from plastics or epoxy. This step is essential because components that may be adequate for plant growth in a ventilated Earth or ISS experiment can be toxic over time in a sealed environment (Kiss et al., 2009). Of course, a sealed volume also implies that plant metabolism will change the composition of the internal gas volume over time as seeds germinate and seedlings grow.

We have flown this basic design to ISS as part of the International Space University experiment, known as Hydra 1, which focused on the comparison of genetically modified and wild-type Arabidopsis thaliana grown together in the same sealed experiment (Kitto et al., 2021) over a 20-day experiment initiated after reaching the ISS by the addition of water to the seed platform. The camera and LED lights were interior to the plant chamber. The Hydra 1 habitat is shown in Figure 2.

Figure 2

In this paper, we report on a series of experiments that test the suitability of the LPX for plant growth. An overview of the LPX project is presented in Bowman et al. (2012). Here, our proposed experiments are designed to test the following specific questions: 1) Is there depletion of CO₂ in the sealed chamber? 2) Are there toxic effects on the plants from materials in the sealed chamber? 3) Is there alteration of the plants due to accumulation of plant byproducts (e.g., ethlyene) in the sealed chamber? 4) Overall, is there germination and plant growth for Arabidopsis and Brassica in the sealed chamber over a 10 Earth-day period?

We investigated the growth and development of Arabidopsis thaliana (ectotype Columbia) and Brassica nigra (cv. Black Mustard). Seeds were surface sterilized for 5 min with 70% (v/v) ethanol + 0.1% (v/v) Tween-20 followed by double rinses with 100% ethanol for 10 sec each. The seeds were then gently suspended in 100% ethanol and ejected onto an autoclaved Whatman filter paper 113 to air dry prior to insertion in hardware. Other methods of seed-handling procedures are summarized in Kitto et al. (2021).

For the experiments reported in this paper, we constructed a 1.2 L Lexan chamber with a clear glass/Lexan top for unobstructed camera view and light penetration. The LED lights and the camera were outside the plant chamber. As in the Hydra 1 unit, and as shown in Figure 1, the camera views the entire seedling platform from the top (only). The LED ring illuminates downward from the top. The LED lights were from Digi-Key Electronics (Thief River Falls, MN, USA) and consisted of 12 warm white (#516-3969-1-ND) and 4 deep red (#L152L-LWC, wavelength = 660nm) LEDs. The light level in the photosynthetically active radiation (400–700 nm wavelength range) was 70.5 μmol m⁻² s⁻¹ at the seed level.

All components in the habitat interior were tested for long-term (up to 6 months) biocompatibility. The root module was constructed from a Delrin plastic block with furrows cut for the seeds. While others (Schuerger et al., 2022) have suggested that Delrin may be biotoxic to bacteria, in all of the trials performed in this study, no indication of poor biocompatibility resulting from use of Delrin was observed. Within each furrow, strips of blotter papers on edge supported by small polypropylene combs held the seed and provided a path for water penetration to the seeds. The seeds were mounted with guar gum on one blotter edge (Figure 3), a method previously used in many spaceflight experiments (Vandenbrink and Kiss, 2016).

Figure 3

The root module sits atop a manifold in the middle of the chamber. Between the root module and the manifold is a single sheet of cheesecloth. Distilled water was used to water the seedlings in the chamber. In these experiments, minerals (MS salts or other nutrients) were not added to the water.

During the run, and depending on the temperature in the chamber, water evaporates from the upper surface of the blotters and the seedlings. Water vapor condenses on the upper inner surfaces of the chamber (gravity driven, even on the Moon). Condensed droplets flow down the inside chamber walls to the bottom, where it is again recycled up to the root module. Only 90 ml of water is needed for each trial run. We note that there was no problem with water distribution in the microgravity Hydra1 experiment on the ISS (Kitto et al., 2021).

Temperature within the chamber is controlled at 22.5 ± 1.5°C by use of an external loop water system. Temperature control is a key issue in flight experiments. On a lunar mission, temperature control must be provided by an external system interfaced between the habitat and the lander. In the Hydra 1 experiment, the temperature control was provided by the CubeSat rack, which held the unit. The lack of precision control resulted in temperatures warmer than optimal and may have negatively affected the experiment (Kitto et al., 2021).

The CO₂ content of the gas in the habitat was measured using a Senseair Sunrise sensor (CO₂ meter.com, Ormond Beach, FL), which has a range of 0–5000 ppm with a resolution of ±20 ppm. Images were obtained with a GoPro (San Mateo, CA, USA) Hero 3 camera. Individual images were combined into videos using Apple iTunes. Individual frames at selected time intervals were analyzed by Easy Leaf Area (Easlon and Bloom, 2014) to generate cotyledon areas of selected seedlings. Circumnutation patterns were generated at selected time intervals using the Circumnutation Tracker (Stolarz et al., 2014).

As discussed above, in the minimal LPX system of interest here, the only two datasets generated are CO₂ concentration over time and images of the plant growth platform over time. While specific flight units may incorporate other sensors, it is of interest to focus on these two datasets and the relevance to plant science that derives from these results.

Carbon balance and CO₂ profile

In the closed system, monitoring CO₂ in the gas phase provided important information on the germination of the seeds and the development of the shoots. It is important to note that over an 8-day experiment, CO₂ does not limit plant growth of Arabidopsis seedlings (Figure 4).

Figure 4

The average mass of our Arabidopsis seeds is about 0.03 mg, or 3 mg for the 100 seeds. As a rough indication of the equivalent CO₂, if we assume a stoichiometry of CH₂O (atm weight 30 g/mole) for this organic material, this implies about 10⁻⁴ moles of C in the 100 seeds. The complete respiration of the organic matter with O₂ yielding CO₂ and H₂O would produce 10⁻⁴ moles of CO₂. In a 1 liter chamber, this would produce a CO₂ level of slightly over 2000 ppm. The reduction in O₂ is negligible.

The CO₂ in the gas phase at time of sealing was ~ 2000 ppm (Figure 4) due to enhanced CO₂ in the indoor environment where the chamber was assembled and sealed. Once the seeds are hydrated, CO₂ release from the respiration in the seeds results in an increase of CO₂ in the chamber, reaching a peak of 5000 ppm after 3 days. Eventually, the development of shoots and the onset of photosynthesis causes a decline in CO₂, reaching 4000 ppm on day 8. Because the duration of these experiments is short, at no time during these experiments is plant growth restricted by CO₂ availability. In all trials, CO₂ drawdown resulting from photosynthesis did not drop CO₂ concentrations below the initial starting level. The CO₂ dataset provides a powerful indication that respiration due to germination has occurred and that subsequently CO₂ uptake due to photosynthesis follows.

Plant growth

The plant growth of Arabidopsis Columbia wild-type seedlings growing in the sealed laboratory LPX unit after 5 and 11 Earth-days after hydration is shown in Figure 5 and Figure 6, respectively. As clearly illustrated in the figures, there is a high rate (98%) of germination. The standard-sized module can support growth of 131 seedlings. The red dot in the center is a size calibration standard.

Figure 5

Figure 6

In the base LPX design, the primary measurement used to quantify individual seedling growth is the exposed cotyledon area determined from the images. Plant height could also be determined from the images using set posts in the growth platform of fixed height or by measuring shadows from asymmetric lighting. Thus, seedling height, inferred from the overhead camera using reference markers, reached 15 ± 5 mm.

Figure 7 illustrates the average increase of seedling photosynthetic area based on analysis of 10 seedlings. Apparent fluctuations in cotyledon areas are caused by diurnal cotyledon movements and circumnutations that impact accuracy of measurements, as viewed from above. After 10 days with illumination from LEDs, the photosynthetic area of Arabidopsis cotyledons per seedling reached 300 mm².

Figure 7

Circumnutations

Circumnutations, the endogenous, non-random, helical, organ movements exhibited by seedlings, can clearly be observed in time-lapse videos (Johnsson et al., 2009). From approximately 3,000 separate images collected at 5-minute intervals spanning initial hydration to the end of the run, we assembled time-lapse videos of germinating and growing Arabidopsis seedlings (see Supplemental Figure 1). Aspects of seedling movements, including direction of rotation, number and timing of direction reversals, and magnitude of deflections, have been studied extensively in Arabidopsis (Schuster and Engelmann, 1997; Wu et al., 2020; Tolsma et al., 2021). Influence of partial gravity on Arabidopsis circumnutations is discussed by Johnsson et al. (2009) and Solheim et al. (2009). Circumnutation movements are influenced by environmental conditions experienced by the seedling, including gravity, light intensity, temperature, photoperiod, and other physical parameters, making it difficult to interpret circumnutation patterns. By comparing circumnutations of plants grown on the Moon with ground controls, it may be possible to discern impacts of the lunar environment on seedling growth.

The focus of the LPX development has been Arabidopsis, an important model organism for plant science. However, the LPX unit can also support growth of other species, or combinations of plants. To illustrate this concept, we show in Figure 8 a mixed population of Arabidopsis and Brassica after 11 days. For both Arabidopsis and Brassica, the percent germination in several different trials averages over 98%.

Figure 8

The Artemis Program at NASA aims to send humans back to the Moon (Creech et al., 2022). The plans include sending robotic landers to the surface of the Moon, building the Gateway outpost in lunar orbit, and, finally, sending humans to the Moon in this decade. NASA's goal is to develop the technologies during Artemis that will eventually allow for human missions to Mars.

Related to the goals of Artemis, the study of plants in space is an important topic for the future long-term spaceflight missions and travel to other planets. Two important aspects of plant space biology are understanding their use in bioregenerative life support systems and the use of microgravity as a unique environment in order to study the fundamental aspects of plant biology. In the area of bioregenerative life support, key discoveries include the demonstration of the growth of multiple generations of plants seed-to-seed and the discovery that microgravity does not have harmful effects on the plant life cycle (Musgrave and Kuang, 2003; De Micco et al., 2014). In the area of basic research, molecular biology experiments on the International Space Station demonstrated that plants at reduced gravity levels have altered expression for multiple genes and biosynthetic pathways when compared to 1g controls (Herranz et al., 2019; Vandenbrink et al., 2019; Hughes and Kiss, 2022). For example, in microgravity, light-associated pathways related to photosynthesis and the chlorophyll metabolism were significantly downregulated (Vandenbrink et al., 2019). Genes related to cell wall and membrane structure were shown to be differentially expressed at the Moon-g levels (Herranz et al., 2019).

The results of the present study show that plant biology experiments on one of the robotic lunar landers would be feasible in a CubeSat-type habitat system for plant growth (Kitto et al., 2021). Our current experiments supplement the CubeSat with our previous experiments performed on the International Space Station in which seedlings of Arabidopsis thaliana were successfully grown in similar hardware. In our previous study on the ISS (Kitto et al., 2021), we showed that synthetic biology has potential spaceflight applications. Thus, an exogenously inducible biological circuit for protein production in Arabidopsis thaliana, pX7-AtPDSi, was flown and was functional in the microgravity environment of spaceflight. The first robotic lunar landers have severe limitations on mass and power (Creech et al., 2022), but our current hardware design can accommodate these restrictions. The present study shows that seedlings can exhibit robust growth in a sealed laboratory analog to the type of chamber that would be used in robotic lunar experiments. Experiments are needed to understand plant growth on the Moon. There are very little data to show how plants will grow in the partial gravity and high radiation environment on the surface of the Moon or Mars (reviewed in Kiss, 2014). The vast literature on the growth of plants on Earth has been augmented by a rapidly increased knowledge of plant growth in microgravity (Vandenbrink and Kiss, 2016; Hughes and Kiss, 2022). In comparison, we know little about plant behavior in reduced (sometimes termed fractional) gravity environments (less than the nominal 1g that occurs on Earth). How biology responds to partial gravity remains relatively unexplored. Only recently have studies with small centrifuges on ISS begun investigations at partial gravity to test response of plants (Kiss et al., 2012; Vandenbrink et al., 2019).

The choice of which plant species to grow in lunar experiments is driven by sample size and suitable growth parameters. As demonstrated, Arabidopsis, the preferred test species of many plant experiments, is well suited for growth in a microhabitat. Small durable seeds provide a large sample size. Additionally, Arabidopsis performs well in a temperature regimen suited for electronics, making it convenient to include within the lander with other temperature-sensitive components, all maintained similarly. In addition, we can also grow Brassica in these chambers.

Growth of plants in early lunar landers will require plant performance in extreme conditions of small habitat weight, power demands, and limited data bandwidth. This study exemplifies how Arabidopsis and other species can be utilized for early lunar experiments. Visual data streams and CO₂ dynamics, alone, can provide a wealth of information for assessment of plant growth on the Moon. Each of the data streams, all by itself, can confirm experiment success since the possible failure of one does not necessarily disrupt the other.

The technology we have developed will allow us to grow plants on the surface of the Moon and conduct some basic experiments in plant biology. For instance, we will be able to test the combined effects of high radiation and reduced gravity found on the lunar surface on plant growth and development for the first time. In the longer term, once we move past these basic experiments, we will be able to expand the use of the hardware to perform experiments of greater complexity. For instance, if we are able to have sample return, then we can do a series of gene-profiling experiments to understand how gene expression in plants changes relative to the lunar environment (Vandenbrink et al., 2019; Hughes and Kiss, 2022). In the long term, this knowledge would help us to genetically modify plants to optimize growth and development on the lunar surface. Thus, we would be closer to our goal of using plants in bioregenerative life support systems to generate oxygen and food to support a sustained human presence on the Moon.

A lunar day light regimen of 14 Earth-days of sunlight followed by 14 Earth-days of darkness may hold for a near-equatorial landing site. A lunar landing at other latitudes can lengthen the duration of sunlight exposure suitable for extending the plant growth period before darkness and plummeting temperatures end the experiment. Any extension of the lunar day will provide additional data for characterizing plant growth on the Moon since the habitat will continue to function so long as light and power are available. Depending on lander configuration and capabilities, the lander may also be able to provide keep-alive power during lunar darkness, thus shielding the seedlings from freezing. Long-term (several lunar days) growth of plants offers the prospect of seed-to-seed experiments wherein seeds are generated in space from dry seeds sent from Earth and grown to sexual maturity on the Moon. Production of seeds in space, on the Moon or elsewhere, opens the possibility of endless space travel where there is no need to return to Earth to replenish old Earth-produced seed stocks.

We have constructed a functional laboratory model of the LPX. The LPX is a sealed system (~ 1 liter volume) for plant growth. Based on our results, we can answer the four questions listed above related to plant growth in a small, sealed chamber.

With Arabidopsis, there is no depletion of CO₂ in the sealed chamber over a 10-day experiment due to CO₂ released by respiration during seed germination.

These studies show that plants such as Arabidopsis and Brassica can be grown well in a small (~1 L) habitat well suited for deployment as part of a lunar lander. Moreover, the plants will continue to grow well over the duration of a lunar day (equivalent to 14 Earth-days) and will exhibit growth parameters capable of being compared to normal plants grown on the surface of Earth. The design of the microhabitat is sufficiently flexible to accommodate changes that might be imposed by a specific lander configuration.