Every day, the dream to colonize Mars is closer to becoming a reality. However, the challenges for human survival on Mars are huge, and one of the key issues is ensuring low-to-no dependence on Earth’s resources. Therefore, several bioregenerative life-support systems for recycling food, water, and gasses are under development (e.g., Nelson et al., 2010; Häder, 2020), and one pursued solution is the use of Martian resources to support life. Indeed, Mars can possibly provide water, solar energy, carbon, nitrogen, and other nutrients (Cockell, 2014).

In this context, cyanobacteria are pointed out as potentially suitable organisms for use in basic life-support systems (Arai et al., 2008; Arai, 2009; Kimura et al., 2015; Verseux et al., 2016, 2021). This is because cyanobacteria are simple (i.e., prokaryotic) organisms, easy to culture with low nutritional requirements, and able to live in a wide range of conditions and ecosystems (e.g., from freshwater to salt and brackish waters, rainforests, deserts and other terrestrial habitats, and even in the air), including under extreme environmental conditions (Gaysina et al., 2018). Furthermore, cyanobacteria are oxygenic photosynthetic bacteria and hence produce oxygen, which is a remarkably limited resource on Mars. Indeed, this critical element to support human survival only represents 0.13% of the Mars atmosphere, while it represents 21% of the Earth’s atmosphere (Verseux et al., 2016). On Mars, oxygen is expected to be produced on-site by photosynthetic organisms and/or through physicochemical methods, such as the electrolysis of the regolith brine (Gayen et al., 2020).

Compared to plants, photosynthetic microorganisms are better at capturing solar energy on a volume-to-output basis and therefore are very efficient oxygen producers. For example, an industrially cultivated Arthrospira strain has been found to produce ca. 16.8 tons of O₂ ha^–1 year^–1, which suggests a much higher O₂ production rate per unit area than trees that have been shown to release ca. 2.5–11 tons of O₂ ha^–1 year^–1 (Verseux et al., 2016). The production of oxygen by cyanobacteria is so impressive that estimates pointed out that the marine cyanobacterium Prochlorococcus alone can be responsible for ca. 5% of global photosynthesis (Pennisi, 2017). Moreover, plants are more demanding with regard to environmental tolerance ranges and require more space and resources; thus, they are more costly than cyanobacteria, particularly when Mars culturing is equated. Besides oxygen, these organisms are able to produce a wide range of other interesting compounds with several applications, e.g., amino acids, pigments, lipids, polysaccharides, and proteins, with applications in the food and pharmaceutical industries (Eriksen, 2008; Sekar and Chandramohan, 2008; Macário et al., 2021). Different species can also be used directly as food sources (e.g., Arthrospira and Nostoc sp.; Gao, 1998; Johnson et al., 2008), to produce biofuels (Raheem et al., 2018), in bioremediation (Fawzy and Mohamed, 2017), and as biofertilizers (Grzesik and Kalaji, 2017). These features render cyanobacteria highly promising for culturing and exploitation in harsh environments under resource scarcity conditions, such as on Mars.

Indeed, cyanobacteria (e.g., Chroococcidiopsis sp.) were already proven to be able to survive in space, i.e., on the outer surface of the International Space Station at extreme temperature fluctuation, vacuum conditions, and high UV and cosmic radiation (de Vera et al., 2019). In addition, besides the production of oxygen, their biomass can be used to supply different commodities, thus contributing to the development of sustainable and less Earth-dependent life-support systems. Therefore, the primary objective of the present study was to evaluate the ability of several species of cyanobacteria (Anabaena cylindrica, Nostoc muscorum, and Arthrospira platensis) to grow using only the resources existing on Mars (i.e., water and Martian regolith). For performance comparison with the cyanobacteria, we also tested the green microalga Chlorella vulgaris, an eukaryote that can be also studied for most of the previously mentioned applications (Mostafa, 2012). In this work, we also addressed the suitability of end-of-life cyanobacteria/microalgae as biofertilizing agents for vegetable cultivation, considering that plants are an essential component of long-term, bioregenerative life-support systems in Mars (Heinse et al., 2007). For this purpose, we tested cyanobacteria/microalgae cultures grown on Mars regolith extract as a biofertilizer using the macrophyte Lemna minor as a preliminary model.

The forecasts highlight the decade of the 2030s as the year of the launch of a human space flight mission to Mars (United States Congress, 2017). However, Martian conditions challenge human survival, and cyanobacteria can thrive using Martian resources (under an Earth-like atmosphere), supporting Mars colonization by contributing to oxygen production, as well as by becoming a feedstock for other applications (e.g., food supplements, biofertilizers, and biofuel production). Several works already proved that these organisms are able to survive by utilizing the resources available on Mars, namely, water and Martian regolith (Arai et al., 2008; Arai, 2009; Kimura et al., 2015; Verseux et al., 2021). On Mars, water can be found in the form of ice in the northern polar region (Shotwell, 2005) and in regolithic brines (Martínez and Renno, 2013; Lauro et al., 2021). Therefore, water can be “mined” and extracted for exploitation (e.g., Hoffman et al., 2016; Lordos et al., 2021). While having water is the first step for survival, the following could be the use of Martian soil. The composition of Mars soil is known, and simulants of Martian regolith are available for experiments (e.g., MGS-1).

Herein, the two components (i.e., water and Mars regolith) plus atmospheric CO₂ and nitrogen that are also available on Mars (1.9% of N₂ and 98% CO₂;¹ assessed on May 2022) were proven to be enough to support the survival and growth of cyanobacteria, on the basis of optical density and the fluorimetric parameter F_v/F_m. However, not all the tested species were able to grow satisfactorily in Mars regolith extract. A. platensis did not show quantifiable growth even in a synthetic medium, although the data from F_v/F_m showed that the cells were alive and photographic documentation (Supplementary Figure 1) showed low but visible growth. The poor nutrient supply in the treatments supports this limited growth outcome. C. vulgaris was unable to grow, while N. muscorum and A. cylindrica thrived in Mars regolith extract. Verseux et al. (2021) also proved that the cyanobacterium Anabaena sp. was able to grow in MGS-1. Indeed, in the present study, the values of F_v/F_m for N. muscorum were higher in Mars regolith extract than in synthetic medium, suggesting a better photosynthetic activity in this treatment. In this way, considering the main perspective of using cyanobacteria for oxygen production on Mars, N. muscorum can be highlighted among the tested species as a stronger candidate for further studies. Moreover, the use of the same biomass to produce multiple commodities is ideal in the context of low resource availability. Indeed, several species of Nostoc (e.g., N. flagelliforme and N. commune) are considered edible and have been used as food items for a long time in China, other Asian countries, and South America (Gao, 1998; Johnson et al., 2008). Studies on the nutritional profile of N. muscorum would be very important to evaluate if this species could be considered as a food resource for Martian crews.

Further studies are fundamental to understanding the suitability of the species with regard to other critical factors limiting their use on Mars, for instance, their ability to survive the journey and hold very low pressures and vacuum conditions (Arai et al., 2008) and their capacity to thrive under different atmospheres and lower pressures (see the studies of Verseux et al. (2021) with the cyanobacterium Anabaena sp. PCC 7938). It is worth mentioning in this context that specific devices are being developed to create a controlled and Earth-like environment, e.g., the A’MED (Arai’s Mars Eco-systems Dome), which is a closed dome where temperature, humidity, and light can be controlled, and protection is ensured against ultraviolet radiation, cosmic rays, and low pressures (Arai et al., 2008). This is an essential step for the culturing of these organisms, since in an unprotected Mars-like atmosphere, cyanobacteria would not likely thrive due to low pressure, absence of stable liquid water, and low nitrogen levels for diazotrophic growth (Verseux et al., 2021). Also, although water could be “mined,” this would require considerable effort and specific technology (e.g., Hoffman et al., 2016; Lordos et al., 2021). Therefore, it would be relevant to also evaluate the growth performance of cyanobacteria in a solid medium supplemented with MGS-1 (similar to what was made by Ye et al., 2021) in the near future.

Concerning the perspective of maximizing the use of Martian resources, only organisms with low nutritional requirements should be considered, given the poor composition of the Mars regolith. Indeed, the microalga C. vulgaris and the cyanobacterium A. platensis did not grow expressively in Mars regolith extract, suggesting that nutritional requirements for these organisms are higher than for the other two diazotrophic cyanobacteria (e.g., N. muscorum and A. cylindrica). Another possible explanation could be that these species have naturally slower growth dynamics or a higher lag phase, which would require an extended period of time to better observe changes.

Our results show that despite long-term survival and growth being unknown, poor nutrition provided by Mars regolith extract supports cyanobacterial growth in the short term (i.e., 1 month). Carbon, nitrogen, phosphorus, sulfur, potassium, and iron are the critical nutrients for cyanobacteria growth (Markou et al., 2014). Carbon can be obtained by cyanobacteria autonomously from atmospheric CO_2, and nitrogen can be obtained by some species from atmospheric nitrogen fixation (Markou et al., 2014). Diazotrophic species, such as A. cylindrica and N. muscorum, are able to fix atmospheric nitrogen through specialized structures (Issa et al., 2014), while other cyanobacterium species may also fix nitrogen through different mechanisms (Flores et al., 2015). On the other hand, A. platensis is not a nitrogen-fixing organism (Flores et al., 2015), which can explain the lack of growth in Mars regolith extract, similar to C. vulgaris. No traces of phosphorus were found in the Mars regolith extract analysis, but both cyanobacteria and microalgae can accumulate phosphorus reserves as polyphosphate granules, and these reserves can be used in cases of phosphorus shortage (Markou et al., 2014). As the cultures were established from laboratory stocks maintained under sub-optimal growth conditions, the mobilization of reserves should have been the mechanism supporting the appropriate phosphorous supply for cyanobacteria growing in Mars regolith extract.

Although phosphorus was not present in the tested Mars regolith extract, other important elements, such as potassium, manganese, calcium, and sulfur, were available. Potassium is, along with nitrogen and phosphorus, the main macronutrient for the growth of photosynthetic organisms (Markou et al., 2014). Cyanobacteria thylakoids bear ion channels where potassium flows to allow a maximal efficiency of photosynthesis (Checchetto et al., 2012). Potassium also plays a significant role as an activator of several enzymes involved in photosynthesis and respiration, protein and carbohydrate synthesis, and the regulation of the osmotic potential inside the cells (Markou et al., 2014).

While manganese is required as a micronutrient, it can be toxic in high (micromolar range) concentrations to cyanobacteria, thus negatively affecting photosynthesis, growth rates, and nitrogen fixation (Rueter and Petersen, 1987). Manganese is more toxic to cyanobacteria than to microalgae (Rueter and Petersen, 1987), thus it is unlikely that this element can explain the limited growth of C. vulgaris in Mars regolith extract. Calcium has a central role as a secondary messenger and signaling molecule in all living organisms (Clapham, 2007). It is also involved in atmospheric nitrogen fixation (Allen and Arnon, 1955), and its concentration has an influence on biomass and protein building (Walter et al., 2016). Sulfur is important for protein synthesis in general and in the biosynthesis of coenzymes (Schmidt, 1988), but contrarily to animals, for example, cyanobacteria are not dependent on the external supply of reduced sulfur compounds, since they can autonomously reduce sulfate for protein and coenzyme synthesis. In these organisms, sulfur is needed for sulfolipid biosynthesis, which is found in the photosynthetic membranes (Schmidt, 1988). Although surprising, the presence of strontium in Mars regolith extract was also detected in similar circumstances in other works (e.g., Olsson-Francis and Cockell, 2010), which suggests that this element is a common contaminant in MGS-1. The presence of this element is not beneficial to cyanobacteria, as it negatively affects their growth rates and chlorophyll a content (Foster et al., 2020). Although the composition of Mars regolith extracts allowed cyanobacterial growth in the present study, it is important to remark that extracts can be prepared under different protocols (e.g., Verseux et al., 2021), possibly allowing optimization of bioavailability of some elements and rendering it more suitable as a culture medium for defined species. In addition, there are several types of Mars regolith with different compositions that can perform better for cyanobacteria/microalgae growth (² assessed on 21 February 2022).

Besides the main task of producing oxygen, a claimed application for cyanobacteria is utilization as biofertilization agents (Chittora et al., 2020). Diazotrophic cyanobacteria (e.g., Anabaena sp. and Nostoc sp.) are particularly pointed out as eco-friendly biofertilizers due to their ability to fix atmospheric nitrogen in specialized structures and deliver it to plants (Rashid et al., 2016). Furthermore, cyanobacteria improve soil porosity and water holding capacity, produce adhesive substances and excrete phytohormones, vitamins, and amino acids (Chittora et al., 2020). Indeed, their successful application as biofertilization agents was already reported for several vegetable cultures (e.g., barley, oat, tomato, radish, cotton, sugarcane, maize, chili, and lettuce) (Chittora et al., 2020). The nutrients necessary to grow plants are present is Mars (Verseux et al., 2016), and several crop cultures (e.g., tomato, rye, carrot, and garden cress) were already proven to grow on Mars regolith, developing at similar rates on Martian and low-nutrient Earth soil (Wamelink et al., 2014). Although these are exciting results, there are few organic molecules on Mars (e.g., Heinz and Schulze-Makuch, 2020) that can support overall soil fertility in the long term, and plants cannot fix atmospheric nitrogen, which indicates that the enrichment with nitrogen, like that operated by diazotrophic cyanobacteria, is very important (Verseux et al., 2016). Arai et al. (2008) demonstrated that a terrestrial Nostoc sp. HK-01 can successfully grow in Mars regolith, releasing polysaccharides that contributed to converting regolith into organic soil. The ability of A. cylindrica to release K, Mg, Na, Ca, Fe, Mn, Ni, Sr, Cu, Li, and Zn from a basalt analog was also shown (Olsson-Francis and Cockell, 2010), and this feature can increase the odds for successful plant growth on Martian regolith. Also, the use of the lysate of diazotrophic cyanobacteria previously grown on Mars and Moon regoliths for the growth of Lemna sp. was already reported as successful (Ramalho et al., 2020). Despite this encouraging evidence, it is fair to recognize that the culturing conditions influence the composition of cyanobacteria, mainly the carbohydrate composition (Depraetere et al., 2015). The mechanism that limits the biofertilizer role of cyanobacteria/microalgae should be considered in future research. Another constraint could be the lack of a beneficial rhizosphere, facilitating nutrient uptake by the plants. For example, for Lemna, several studies showed that the association with specific bacteria not only increases plant growth but also prevents oxidative stress (Ishizawa et al., 2017a,b). In the case of poor culture media, such as the Mars regolith extract, the presence of these beneficial rhizobacteria would exert a major influence.

In the present work, the duckweed L. minor was used as a vegetable model. This family of plants (Lemnaceae) was already pointed out as suitable for space exploration, since they are 100% edible, rich in nutrients, and fast growing (Stewart et al., 2020). Our results show that the photosynthetic efficiency, in some cases, was higher in Mars regolith extract than in the standard culture medium. This suggests that, although L. minor grows at a smaller pace in Mars regolith extract than in optimal medium, its healthy growth can be sustained for longer toward better overall biomass production. The treatment made to the cyanobacteria/microalgae cultures provided as biofertilization agents (filtration or sonication) did not affect the photosynthetic efficiency of L. minor. In addition, it is worth noting that the photosynthetic efficiency of L. minor did not differ among treatments with cyanobacteria and sole Mars regolith extract culturing, while treatments where the microalga was supplied as a biofertilizer harmed the photosynthetic efficiency of the plant. Actually, despite the fact that C. vulgaris is one of the most studied species for space life-support systems, some of its by-products were already proven to be toxic to higher plant crops (Terskov et al., 1979). However, while the number of Lemna fronds increased more throughout the experiment in the control with the synthetic medium than in any of the other treatments, the dry weight yield recorded for plants growing in Mars regolith extract supplemented with S. platensis and C. vulgaris is similar to that recorded in the control. These are the two species that grew worst in Mars regolith extract, which indicates that even low biomass levels may have interesting biofertilization potential. Analysis of the composition of Lemna growth media showed relevant levels of potassium, nitrates, and orthophosphates (major constraints of plant growth; Goopy and Murray, 2003) in A. platensis samples, which concur to explain why these were the treatments better promoting Lemna growth. C. vulgaris samples were not particularly rich in any of these elements, which suggests that the presence of other unmonitored compounds is responsible for their biofertilization potential. Indeed, previous studies, such as the work of Refaay et al. (2021), already showed the positive contribution of A. platensis and C. vulgaris in the culturing of several vegetable species due to their high levels of proteins, carbohydrates, and important phytohormones, such as abscisic, gibberellic, and indoleacetic acid.

In spite of a higher duplication rate (reflected in frond numbers), the plant culture in the synthetic medium did not produce higher biomass (reflected in dry weight) than the cultures in Mars regolith supplemented with these cyanobacteria and the microalga, and certain questions arise regarding the differential nutritional value of the cultured plants. Indeed, Chakrabarti et al. (2018) showed that the nutritional profile depends on the cultivation conditions. Duckweeds have an attractive nutritional profile, bearing high protein levels and essential amino acids in levels within the range recommended by WHO for good human nutrition, beneficial fatty acids (Appenroth et al., 2017), and antioxidant carotenoids (Stewart et al., 2020). A preliminary analysis of our samples by FTIR-ATR separated the nutritional profile (polysaccharides in particular) of the plants grown in regolith and supplemented regolith from those grown in synthetic media and ultrapure water. Plants are important sources of carbohydrates, which are responsible for providing energy to humans. These carbohydrates can be divided into energy storage carbohydrates, like starch, oligosaccharides, and sugars, and structural cell wall polysaccharides (Lovegrove et al., 2017), which differ in features such as their digestibility. Given the importance of these features when considering crop production on Mars to support colonization attempts, differences in the nutritional profiles of different experimental treatments, such as the ones noticed herein, are a critical object for future research. For instance, an in-depth analysis of major nutritional groups (i.e., proteins, carbohydrates, lipids, minerals, and fibers) is required to gain a robust insight into the nutritional value of vegetable species grown in Martian soil. A final note is worth making on the capacity of duckweeds to accumulate protective carotenoids, such as zeaxanthin, which can play an important role in providing protection from high levels of damaging radiations (Stewart et al., 2020). Therefore, future research should also address the production of these compounds.

The original contributions presented in this study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

IM contributed to the conceptualization, methodology, data treatment, and writing. TV did the conceptualization, performed the methodology, and carried out the data treatment. SF did the conceptualization and performed the methodology. JS wrote, reviewed, and edited the manuscript. HP and CS performed the methodology. FG supervised the data and wrote, reviewed, and edited the manuscript. SV and JP did the conceptualization, wrote, reviewed, and edited the manuscript, and supervised the data. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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