1. Introduction
In recent years, future long-term manned missions in deep space and the possibility to explore other planets of the solar system have been seriously considered by government space agencies and private commercial companies. However, the prolonged human permanence on orbital stations and planetary colonies will need specific technologies to regenerate essential resources, like air and water, and to exploit materials available in situ, while producing food and recycling waste [
1].
In bioregenerative life support systems (BLSSs), selected organisms are combined on the basis of their metabolic paths in successive steps of crew waste recycling (feces, urine, carbon dioxide, and food residues) into oxygen, edible biomass, and potable water [
2]. Accordingly, BLSSs for space will be realized through the integration of compartments hosting living organisms, integrated with physicochemical processes, to realize secure and reliable regeneration processes [
3,
4]. Moreover, the in situ resource utilization (ISRU), including the use of local soils, will contribute to achieve the self-sustenance of space colonies.
Edible plants are efficient bioregenerators, able to perform essential functions for human survival in extra-terrestrial environments, such as air renovation through photosynthesis, water purification through transpiration, and waste recovering through mineral uptake, while supplying fresh food and wellbeing to space crews [
5]. Several crops, including fruit and leafy vegetables, cereals, and tuberous species, have been assessed as possible candidates for space cultivation, after considering specific constraints and technical and dietary requirements of the different mission scenarios. Agronomical features include compact plant size, a fast growing rate, elevated productivity and nutritional and nutraceutical value, and a high harvest index (HI), implying a limited volume of waste [
6,
7]. Among candidate crops, both potato (
Solanum tuberosum L.) and sweet potato (
Ipomoea batatas Poir.) are included as geophyte plants, producing edible underground organs.
Potato is highly productive, showing a good HI (0.7–0.8), offering several advantages, including a great number of genotypes with different features, and the staggered production of tubers, rich in carbohydrates and proteins and suitable for several food preparations [
8]. However, it is typically grown in the field, and knowledge about plant physiology and productivity mainly refers to the outdoor farming on soil, while only a few studies concern the soilless cultivation in growth chambers. Particularly, the plant response to different hydroponic systems, nutrient solution composition, and environmental conditions was studied by the National Aeronautics and Space Administration (NASA) [
8,
9] and the European Space Agency (ESA) [
10], in potato genotypes suitable for BLSSs. In addition, the success of the tuberization process has been verified in ground conditions in a modular system prototype for the cultivation of tuberous crops in microgravity [
11].
Substrate is a crucial factor for potato cultivation since the habitability for the root system is pivotal not only for the proper plant growth but also for the optimal tuberization process. In general, non-arid loam and sandy-loam soils, with a low mechanical resistance to the tuber growth, and a pH interval of 5.5–8.0 (optimal value 6.0–6.5) are the most suitable [
12]. However, in future plant cultivation on Mars, fertile soils could be unavailable and the in situ materials, including the Martian regolith and the organic waste of the mission, could be the only resources for assembling growing substrates [
13].
In Mars-oriented research, since the real regolith cannot be used, investigations on Earth are carried out on commercial simulants, obtained from crushed terrestrial rocks, mimicking the geotechnical and physicochemical features observed in true regolith samples collected in robotic missions [
5]. The MMS-1 Mojave Mars regolith simulant contains plagioclases, amorphous materials and zeolite, releasing essential nutrients (e.g., K, Ca, Mg, and Fe) for plant nutrition, but lacking those sourced from organic matter (organic C, N, P, and S) [
14,
15]. Obviously, the MMS-1 simulant does not represent the complexity of the entire surficial layer of Mars regolith, which shows high heterogeneity and spatial variability, analogously to Earth’s crust [
5]; nevertheless, its chemical composition and mineralogical patterns is as much as similar to those of Mars regolith collected by rovers and robotic spacecrafts. Moreover, the MMS-1 simulant was found to be a coarse-textured and alkaline (pH 8.86) substrate, with scarce content of fine colloidal particles and low water-holding capacity. Consequently, to properly support the growth of plants, it requires an appropriate organic amendment (i.e., plant residues, human waste) to enhance the nutrient availability and the water retention, while contributing to dispose the organic effluents of the mission.
Until recent times, the use of Mars simulants amended with organic matter for plant cultivation in BLSSs was scarcely investigated [
16,
17,
18]. In the last years, our team started a series of experiments focused on the characterization and exploitation of Mars simulants as plant growth media [
5], also in mixture with organic materials of a different nature (i.e., green compost, peat, horse manure), mimicking the possible waste of a Mars mission (urine, feces, plant residues). In the first studies, lettuce was used as a model for leaf vegetables with a short cycle [
14,
15,
19,
20,
21]. Later, experiments started also in tuber plants (potato) [
22] and seed species (soybean). Overall, our results demonstrated that the organic amendment reduced the alkalinity and increased the nutrient availability, making the Mars regolith simulant suitable for plant growth.
In this experiment, we assessed the plant performance of potato plants grown in pots in an unheated glasshouse, on six substrates: the MMS-1 Mojave Mars simulant, pure and amended with a green compost (70:30
v:
v), a fluvial sand, pure and mixed with compost at the same rate, a red soil, and a volcanic soil. We reported the results on the physicochemical properties of substrates before cultivation, and plant physiology and growth parameters in Caporale-Paradiso et al. [
22]. In this paper, we show detailed data on the (i) nutrient bioavailability and chemical/physical fertility of the substrates after the plant growth cycle (to study their evolution from the starting point); and (ii) the elemental profile and nutritional quality of potato tubers yielded in the pot trial.
Our research questions were whether and to what extent the growth of potato plants and the production of tubers could modify the chemical features (i.e., content of essential nutrients, pH, EC) and the most relevant structural properties affecting the water-holding capacity (i.e., porosity) of regolith-based substrates could be reused in consecutive cultivations, specifically, whether the in-depth analysis of the evolution of growing media as an effect of plant cultivation provides information on the substrate fertility over time is useful in potato and, more in general, in long-cycle crop rotation. This knowledge is of crucial importance in the specific scenario, since relying on fertile substrates, able to sustain the plant growth in successive growing cycles, is a fundamental requirement to develop stable and reliable crop systems, suitable to fulfil vital regeneration functions (i.e., air renovation and water purification) and to produce food in a predictable and durable way. In addition, the insight of the influence of different substrates on the plant food quality (i.e., potato tubers) is decisive to define balanced diets with proper nutrient intakes (based on the actual concentration in the plant product) and to ensure the food security in terms of anti-nutritional compounds (e.g., potato glycoalkaloids) to guarantee the astronauts’ survival in space, as well as to exploit nutraceutical properties of fresh food as a countermeasure to human diseases related to space factors acting as stressors on the human body (e.g., antioxidants and other health-promoting compounds).
3. Discussion
To grow properly and complete a normal life cycle, edible plants need essential nutrients sourced by soils or growth media, except for carbon, oxygen, and hydrogen which are obtained from air and water. Previous characterization studies of MMS-1 Mars simulant [
14,
20] demonstrated that the total content of many vital elements in the simulant could be adequate to fulfil the plant requirements. However, plants commonly absorb only the bioavailable fractions of mineral nutrients (i.e., the readily soluble and exchangeable forms), while they cannot use the elements integrated in mineral lattices, released only after mineral (bio)weathering [
5]. Nutrient bioavailability and plant nutrition in the rhizosphere soil are governed by many dynamic processes and the pseudo-equilibrium between the water and the solid phases, rather than by the total concentrations of mineral nutrients. Several properties, including texture, clay content, pH, and EC, have a pivotal role in the regulation of the nutrient bioavailability and the root uptake [
5]. Hence, the extraction and the quantification of bioavailable nutrient pools, matched with the measurement of soil pH and EC, are fundamental to understand the (bio)chemical rhizosphere processes, strongly affecting the nutrient uptake and the plant nutritional status.
In this study, the bioavailability of the nutrients was assessed by chemical extractants which simulate the solutions circulating in the rhizosphere environment. The single-step 1 M NH
4NO
3 soil extraction is widely used to quantify the readily soluble and easily bioavailable fractions of the elements in the soil system [
24]. Soil extraction with 0.05 M EDTA at pH 7, instead, allows to quantify the potentially bioavailable pools of elements in the soil, since EDTA can chelate several metal ions [
25] and can partially extract metals organically bound or occluded in secondary minerals and oxides [
26]. Our extractions demonstrated that all the substrates provided bioavailable fractions of essential nutrients to potato plants (
Table 2 and
Table 3); however, when not amended with compost, both Mars simulant and fluvial sand cannot fulfil the plant requirements if not adequately supported by fertigation. This aspect was also addressed in a growth experiment on Mars and Lunar simulants amended with a horse and swine manure [
20], through a comparison between the nutrient requirements of lettuce plants and the bioavailable nutrients extracted by extraterrestrial simulants. The lower bioavailability of nutrients in non-amended substrates had an evident negative impact on potato plant physiology and productivity [
22]. Moreover, the significant reduction of pH values in the RH vs. BK soils (
Figure 1A) also evidenced a greater effort (likely, exudation of acid organic compounds) by the plant to mobilize vital nutrients from these poorly fertile soils.
The addition of stable organic matter to mineral substrates made them more similar to the terrestrial soils, where humified organic matter and related microbiota interact with the mineral moiety to form a porous and highly dynamic environment. Martian regolith, and its terrestrial simulants, in fact, lack the essential microbial communities commonly present in the organic matter of the terrestrial soils, which have a key role in the rhizosphere nutrient cycling and plant growth processes [
5]. Thus, when mixed with a quality compost, a Martian regolith simulant such as MMS-1 can be able to provide similar ecosystem services to terrestrial soils. Accordingly, the overall nutrient bioavailability in the amended substrates reached the same order of magnitude of VS and RS terrestrial soils (
Table 2 and
Table 3,
Figure 2). Hence, the addition of composted organic material to the alkaline and poorly fertile R100 and S100 basically enhanced their (bio)chemical fertility and lowered the alkaline pHs. Additionally, the growth of potato roots and tubers and the intense release of exudates increased the pool of organic C and total N in the S100 and R100 substrates (
Table 1), if compared with the starting point ([
22]: Table 1); indeed, the supply of nutrient solution contributed to enriching the two substrates of N as well. The transformation of fresh organic material into high-quality compost to recycle as crop amendment is a key step to assure a sustainable use of scarce resources in extraterrestrial BLSSs [
5].
The primary constraints of regolith are the limited availability of nutrients for biological processes and the inadequate ability to retain water, which is attributed to the absence of organic carbon and fine-grained colloidal particles [
27,
28]. Adding compost to regolith can improve several properties, including water-holding capacity, medium structure, porosity, and permeability. We quantified how the compost improved the water retention capacity of the regolith within the physiological limits suitable for potato root water uptake. Furthermore, compost can improve soil and regolith structure by promoting aggregation of soil particles and increasing porosity, allowing for a better water infiltration and gas exchange between the solid phase and the atmosphere.
The analysis of the pore area distribution (
Figure 3) revealed distinct water retention characteristics among the different soil types, with sandy soil (S100) showing a sharp peak at low suction values, while red soil (RS) and volcanic soil (VS) showed a well-graded distribution, indicating better water retention over a wider range of intake values. The addition of compost to both sandy soil and regolith demonstrated significant improvements in total readily extractable water (TAW) and pore frequency distribution. The physical properties of MMS-1, including particle size distribution, particle density, and bulk density, influenced their overall porosity and saturated water content [
20]. This suggests that the compost improves the water-holding capacity, particularly for suction values below 25 cm. This finding, in addition to its intrinsic value, allows for more efficient irrigation management from an energy point of view, since the irrigation objective can be achieved with a reduced number of irrigation events.
The average temperatures occurred in greenhouse throughout the experiment were greater than the optimal level for potato for tuber sprouting (15 °C), and close to the optimal values during tuberization (18 °C) [
29].
The ‘Colomba’ potato plants showed a good growth performance in pots in the fall–winter period, in the unheated glasshouse in a Mediterranean climate, and all the studied plant growth media (MMS-1 simulant as well) allowed the development of a good amount of biomass and the process of tuberization. This confirms the good adaptability to different root environments known for the crop, as observed in previous space-oriented experiments in phytotron comparing several possible substrates and containers, such as a peat-based mixture in cylindrical boxes [
30], a peat–vermiculite mix in trunk conical pots [
8], a cellulosic sponge in rectangular trays [
11], and in different cultivation systems, including the nutrient solution only (NFT) with Molders et al. [
10].
The growth of the plant epigeal part was promoted by volcanic soil and both sand and Mars regolith simulant mixed with green compost, compared to the same non-amended substrates, confirming the need for soil organic matter for potato cultivation [
31] (p. 928). Accordingly, the development of the hypogenous organs (i.e., roots, stolons, and tubers) was considerable on the same substrates, and also on pure sand. On the other hand, the Mars regolith simulant MMS-1 was unable to properly support the plant growth, presumably because of the scarce water retention, the high pH, and the scarcity of organic carbon and essential mineral nutrients. However, the addition of compost improved the physical and chemical properties (structure, water, and nutrient availability) and the overall fertility of the regolith, with positive effects on the crop productivity, as previously observed in lettuce on the same media [
15].
Potato plants on regolith simulant developed the shortest roots and the greatest percentage of thicker roots, hence a root system impairing the plant resource acquisition [
32]. In fact, the finer roots increase the root surface, as well as the explored soil volume, and usually allow a higher nutrient uptake capacity per unit of root mass [
33]. In plants on the regolith simulant, the reduced percentage of fine roots and the higher proportion of ticker roots could indicate a possible mechanism of resource conservation [
34].
Our plants completed the tuber-to-tuber cycle and produced healthy tubers on all the studied substrates, in a predictable time for the Colomba potato cultivar. This result agrees with that obtained in ‘Colomba’ in the growth chamber from pre-sprouted tuber seeds, on both a peat-based mixture [
30] and a cellulosic sponge [
11]. However, in this experiment, the final tuber yield was lower than that expected for mini-tuber potato plants (data provided by the breeder for the cultivar;
www.hzpc.com; accessed on 8 January 2024), while the percentage of dry matter was in line with the reference value for the cultivar. The potato productive performance depends on several factors, including genotype, climate, and soil features [
31] (p. 928). In this experiment, temperatures experienced by plants during the early developmental stages, together with the relatively low solar radiation due to the greenhouse frame and cover, and the cloudy weather of the period might have limited the growth rate and anticipated the senescence. Nevertheless, the tuber number is indicative of the potential plant productivity, as the tubers developed in the initial 3 weeks of tuberization will determine the majority of the final yield [
31].
An adequate intake of mineral nutrients is crucial for the health of astronauts, to meet their nutrient needs and to counteract the detrimental effects of the space environment [
35]. According to the multielement profile of tubers (
Table 4 and
Table 5), the consumption of potatoes (adequately cooked) can allow space crews to intake minerals such as K, N (primarily), S, P, Mg, and Ca (secondly), and essential micronutrients (i.e., Na, Fe, Zn, B, Mn, and Cu). A sufficient intake of minerals is crucial for sustaining the astronaut’s wellbeing. Potassium, for instance, abundantly sourced by potato tubers, helps to keep normal fluid levels in human cells (while Na, its counterpart, maintains normal fluid levels outside of cells) and normal blood pressure, and helps with muscle contraction [
36]. It is noteworthy as the nutrient concentrations in potato tubers grown on Mars simulant, sole or amended with compost, are similar to that of tubers grown in our terrestrial soils from Italy (i.e., VS and RS), as well as in Brazilian [
37], Canadian [
38]
, and Chinese [
39] soil environments. The amendment of MMS-1 simulant with compost improved both the productivity of potato plants [
22] and the nutritional value of tubers (in terms of concentrations and contents of healthy elements;
Table 4 and
Table 5). The compost applied as a soil amendment was found to increase the potato tuber yield and size/quality in many studies [
40,
41]. These increases can be attributed to both ‘nutrient’ (i.e., slow-release source of elements) and ‘non-nutrient’ (e.g., increase in soil water retention) benefits related with the organic matter [
42].
Potatoes are rich in a variety of nutrients, including proteins, carbs, vitamins, dietary fibers, minerals, and a number of other health-promoting compounds, and also contain a few compounds with harmful effects, if ingested in excess [
43] (pp. 191–211). To ensure safe human consumption, the content of 20 mg/100 g fw is recommended as the upper limit for glycoalkaloid content in potato [
44]. The prevalent alkaloids in the Solanaceae family (and especially in the Solanum genus), synthesizing a variety of alkaloidal chemicals, are the glycoalkaloids, nitrogen-containing steroidal glycosides. Glycoalkaloids are found in tubers in variable quantities and may represent a source of water and soil contamination [
45]. Their concentrations are influenced by several factors, including geographic location, genotypes and varieties, maturity at harvest, and growth conditions [
46]. In Solanum genus, more than 80 glycoalkaloids were identified, although α-solanine and α-chaconine represent the most abundant [
47]. The tuber content of glycoalkaloids obtained in our experiment is in line with previous studies in potato, which report values ranging from 0.9 to 37 mg/100 g for α-chaconine and from 0.4 to 17 mg/100 g for α-solanine [
48,
49,
50].
5. Conclusions
This experiment was carried out within a wider research program aiming at identifying the best substrate while exploiting in situ resources (i.e., Martian regolith and organic waste of the mission), to realize reliable and sustainable cultivation systems for candidate crops in planetary colonies.
Our analyses showed that the Mojave Mars regolith simulant MMS-1 lacks essential plant nutrients normally generated and geo-chemically regulated by organic matter (i.e., N, P, and S), and presents several features hampering the plant growth (e.g., high pH and Na content, poor physical structure, and low water-holding capacity). Consequently, the growth of potato plants on the regolith simulant alone limited both the epigeal and hypogeal growth compared to the other substrates, even in the presence of fertigation. The low tuber biomass obtained on pure regolith simulant determined a higher content of nutrients in their tissues (concentration effect) in comparison to other substrates, implying that MMS-1 produced a scarce tuber yield but with high nutritional quality. Nevertheless, the amendment with green compost improved the structure and general fertility of the medium, enhancing the plant performance, the overall dry matter accumulation, and the tuber yield and quality. The addition of this source of organic matter led to the best agronomic outcome, combining a high yield with the best tuber quality, and allowed the maintenance of a sufficient level of substrate fertility for successive cultivations.
In conclusion, compost amendment is a successful strategy to create long-lasting fertile substrates from the poor Mars regolith and the organic waste of the mission (here mimicked by green compost). This evidence represents useful information on the performance of potato (as a model of tuberous crop) in containers under protected cultivation and on plant response to the growth medium, which could contribute to develop efficient cultivation systems for resource bioregeneration in future Mars settlements. In this view, further investigations are in progress on the regolith-based substrates retrieved after the potato growing cycle used for cultivation of other candidate crops, including plant species typically improving soil fertility (i.e., Leguminosae performing atmospheric N-fixation).