Human exploration of Mars is one of the key scientific and technological undertakings of our time, providing critical information enabling the discovery and settlement of another world while also facilitating the development of technologies on Earth. Future human space exploration may include returning to the moon, as well as missions to Mars (Henn, 2013; Martinez et al., 2013; National Academies of Sciences, Engineering, and Medicine, 2016), with NASA aiming to send humans to Mars by the 2030s (Miranda, 2020). Current research and planning to send crewed missions to Mars for long term space exploration has underscored the critical need for advanced Bio-regenerative Life Support Systems (BLSS), which are complex mixtures of biological and engineering systems that include atmosphere revitalization, water recycling, food production, and organic waste recycling (Revellame et al., 2021). Algae, which produce much of the oxygen on Earth, can similarly be used to recycle CO₂ and provide O₂ and food to astronauts (Häder, 2020), and therefore, have previously been proposed for space life support systems (Averner et al., 1984).

Since the beginning of human spaceflight missions, algae have been considered promising candidates for space life support systems due to their rapid growth rates, the fact that they are straightforward to grow, and edible biomass (Powell et al., 1961; Rangel-Yagui et al., 2004; Soletto et al., 2005; Ganzer and Messerschmid, 2009; Wells et al., 2017). In the late 1960s, a bio-regenerative system utilizing the algae Chlorella was first studied in ground experiments for CO₂ cleansing and O₂ provision (Eckart, 1996). Since then, various experiments have been conducted to study the effects of radiation, microgravity, space vacuum, and temperature extremes on algae growth for space exploration missions (Horneck et al., 2003; Thirsk et al., 2009), and it has been shown that algae are capable of surviving exposure to spaceflight conditions (Niederwieser et al., 2018).

The atmospheric pressure at the surface of Mars ranges from 1 mbar to 14 mbar depending on the location and season, which is very low compared to the 1013 mbar on average at sea level on Earth (Forget, 2009). One of the goals in space exploratory missions and on Mars is to minimize the amount of mass and energy required to launch and maintain life support systems. Low-pressure is sought by the human spaceflight programs to decrease the engineering cost associated with space vehicles, as it allows a reduction in their size and in the quantity of accompanying consumable materials (Paul and Ferl, 2006). The ability to grow photosynthetic organisms under low pressure conditions is therefore an important step toward establishing advanced life support systems for long-term space missions.

Early studies of algae growth at low pressures ranging from 250 to 500 mbar suggested that low atmospheric pressures have no inhibitory effect and might slightly stimulate growth (Orcutt et al., 1970). Some algae have also developed anoxic metabolisms to adapt to low oxygen conditions (Yang et al., 2016). Limited studies have examined cyanobacterial growth under low atmospheric pressures relevant to potential growth on Mars (Kanervo et al., 2005; Murukesan et al., 2015; Verseux et al., 2021). These studies reported the successful growth of cyanobacteria at pressures as low as 100 mbar achieved with the continuous replenishment of CO₂ and nitrogen. However, the experiments were conducted for relatively short duration (7–10 days) and detailed observations of growth dynamics such as carrying capacities and growth trends at reduced pressures were not reported (Murukesan et al., 2015; Verseux et al., 2021). To the best of our knowledge from the existing literature, including a review of over 50 space studies examining algal growth (Niederwieser et al., 2018), few studies have examined the growth of extremophilic algae, with high nutritional potential, at low pressures relevant to Mars.

Algae are found in almost all ecosystems on Earth (Rajkumar and Yaakob, 2013; Malavasi et al., 2020). Algae are diverse organisms with specialized adaptations that enable them to survive under extreme environmental conditions including hot or cold deserts (Lewis and Lewis, 2005; Schmidt et al., 2011), hypersaline habitats (Vinogradova and Darienko, 2008; Oren, 2014a; Leena et al., 2018; Cycil et al., 2020), extreme concentrations of heavy metals (Garbayo et al., 2012; Malavasi et al., 2020), deep-sea hydrothermal vents (Edgcomb et al., 2002; Malavasi et al., 2020), and extreme elevations such as the highest volcanoes on Earth (Schmidt et al., 2018; Vimercati et al., 2019). Some preliminary studies also indicated the ability of cyanobacteria and algae to grow using Martian Regolith Simulant (MRS) demonstrating their ability for in-situ resource utilization (Arai et al., 2008; Cycil et al., 2021). In this study, we selected algae species that are ecologically diverse and may have adaptations to thrive under extreme environmental conditions that may help them to grow under conditions relevant to Mars. Snow algae, for example, are the primary oxygen producers in challenging high UV, low temperature, and low nutrient snow environments in lower atmospheric pressures up to 6,000 m above sea level (Painter et al., 2001; Schmidt et al., 2018; Solon et al., 2018; Vimercati et al., 2019; Hoham and Remias, 2020), and can reach concentrations of over one million cells/ml (Hoham, 2001). Halophilic algae are salt-loving algae that are the main or only primary producer in most light-exposed hypersaline environments approaching salt saturation (Banciu et al., 2020). The halophilic algae Dunaliella salina, similar to snow algae, are the primary oxygen producers in hypersaline environments such as The Great Salt Lake, Dead Sea, Lake Tyrell, solar salterns, and brine inclusions (Oren, 2014b).

In this study, we utilized five algae strains to study their growth under Mars-relevant low-pressure conditions: three extremophilic algae, the snow algae Chloromonas brevispina (Hoham et al., 1979) and Kremastochrysopsis austriaca (Remias et al., 2020), and the halophilic algae Dunaliella salina (Teodoresco, 1905), in addition to two well-studied edible algal strains Chlorella vulgaris and Spirulina plantensis that have been used in multiple spaceflights and ground-based studies (Lee et al., 2001; De Morais and Costa, 2007; Daliry et al., 2017; Niederwieser et al., 2018; Detrell et al., 2019; Häder, 2020). Edible microalgae are a source of potentially healthy and sustainable nutrients. D. salina, C. vulgaris, and S. plantensis have been reported to have commercial applications as food supplements due to their rich protein content, presence of vitamins A and B12 and the abundance of β-carotene which is an antioxidant (Mokady et al., 1989; Panahi et al., 2015; Kumudha and Sarada, 2016; Lupatini et al., 2017; Canelli et al., 2020; Sui and Vlaeminck, 2020). Some reports also indicate potential applications of snow algae metabolites in the pharmaceutical industry (Sathasivam et al., 2019; Hans et al., 2021). Therefore, these algae species also have the potential to serve as healthy food sources.

Human exploration of Mars is one of the key scientific and technological undertakings of our time. Current research is ongoing to successfully support astronauts’ food and oxygen needs for long-term space exploration journeys including to Mars (Massa et al., 2017). The results of this research underscore the critical need for advanced BLSS to support human life during extended space flight and on long planetary surface expeditions (Monje et al., 2003; Revellame et al., 2021). Algae are considered an excellent food source for astronauts because they (1) contain all the essential amino acids, (2) are more digestible than traditional plant protein and (3) grow faster than traditional crops (wheat, rice, corn, etc.) (Bleakley and Hayes, 2017; Koyande et al., 2019; Yang et al., 2019). Algal oil also contains substantial contents of poly-unsaturated fatty acid (PUFA) and algal-specific super-antioxidants, both of which may have beneficial effects for astronauts exposed to harsh space environments (Harwood, 2019; Yang et al., 2019). Recent results also signified the biomedical applications of astaxanthin, a pigment produced by algae, in preventing certain cancers, aging, macular degeneration, and inflammation (Grimmig et al., 2017). All these reports suggests that algae can be a competitive food option for food and oxygen production in long-term space exploratory missions (Yang et al., 2019).

However, optimization of algal growth for their use in self-sustaining BLSS is needed. Therefore, the goal of this study was to grow algae for potential oxygen and food production under low pressure conditions, such as might be possible in an enclosed low-pressure environment made with flexible materials on Mars. Flexible materials, such as those used for the extravehicular mobility unit (EMU) spacesuit that enables pressurized oxygen, ventilation, as well as carbon dioxide, water vapor, and trace contaminant removal, maintains a pressure of 296 mbar (4.3 psi) (National Research Council, 1997). Low pressure (∼200–300 mbar) martian or lunar greenhouses and inflatable structures have already been conceptualized and designed (e.g., Cadogan et al., 1999; Wheeler and Martin-Brennan, 2000). The use of flexible materials to make inflatable structures would considerably reduce the mass and volume of any martian greenhouse structure making it more viable for deployment. Therefore, the ability to grow photosynthetic organisms under low pressures (<296 mbar) facilitates the use of BLSS that could be utilized on Mars. Furthermore, pressurizing such a structure initially with the martian atmosphere would reduce transported oxygen/air resource requirements.

In this study, the maximum growth for each species was observed at the pressures of 330 mbar for C. brevispina with 19.8 ± 0.9 × 10⁵ cells/ml and at 160 mbar for D. salina with 30.0 ± 5.6 × 10⁵ cells/ml and for C. vulgaris with 13.0 ± 1.5 × 10⁵ cells/ml. To put these cell concentrations into context, here we compare them with optimum growth conditions as reported in both natural and laboratory conditions. For C. brevispina, under optimum conditions, cell concentrations were observed reaching 10⁶ cells/ml (Hoham et al., 1979; Harrold et al., 2018), and for D. salina, the highest cell concentrations reached 10⁶ –10⁷ cells/ml under laboratory conditions (García et al., 2006; Hadi et al., 2008; Ahmed et al., 2017). For C. vulgaris, the maximum growth under optimum conditions was also observed to range from 10⁶ to 10⁷ cells/ml under laboratory conditions (Mandalam and Palsson, 1995; Adamczyk et al., 2016; Sánchez-Saavedra et al., 2020). The ability of our candidate algae species to grow under low pressure conditions and reach cell concentrations close to maximum cell counts observed for these species under optimum conditions, makes them excellent candidates to be used for BLSS.

The results of our experiments also show that three species showed substantial growth at 80 mbar and 160 mbar (Supplementary Figures 27, 28), well below the 200–300 mbar lower limit generally proposed for flexible materials on Mars, and well below the value thought to be the limit for vascular plant growth (Hublitz et al., 2004; Paul and Ferl, 2006; Richards et al., 2006). Under these conditions of very low pressure (80 mbar), the growth rates of the cultures C. brevispina, D. salina, and C. vulgaris were relatively slow (with doubling times of ∼5–9 days, although these are comparable to C. brevispina growth under optimum conditions (Harrold et al., 2018).

According to previous estimates, each astronaut performing 2 h of intense physical activity each day would consume approximately 1 kg of O₂ per day (Horneck et al., 2003), which can be photosynthetically produced by bio-fixation of 1.3 kg of CO₂ (Verseux et al., 2016). Previous work indicates that for C. vulgaris species, a maximal bio-fixation rate of 1.4 g CO₂ /L/d was observed at a cell concentration of 1.3 × 10⁷ cells/ml under optimum conditions (Adamczyk et al., 2016) and for D. salina species, under optimum conditions, the CO₂ bio-fixation rate was observed to range from 0.71 g CO₂/L/d to 1.102 g CO₂/L/d at maximum biomass concentrations (Mortezaeikia et al., 2016). Based on these values of CO₂ bio-fixation rates, it can be estimated that the cell counts of C. vulgaris and D. salina measured in our experiments reaching 13.0 ± 1.5 × 10⁵ cells/ml and 30.0 ± 4.6 × 10⁵ cells/ml at 160 mbar, respectively, could potentially generate enough oxygen for astronaut consumption. Snow algae C. brevispina is also known to be an important CO₂ sink in snow environments (Williams et al., 2003) and their cell counts reaching 19.8 ± 0.9 × 10⁵ cells/ml at 330 mbar in our experiments indicate their potential to photosynthetically generate substantial oxygen via CO₂ bio-fixation. These calculations, however, are simply estimates as the photosynthesis machinery of algae can be influenced by various environmental factors [light exposure, pressure, activity of reactive oxygen species (ROS), pH fluctuations, etc.]. Therefore, even though the biomass data suggest that these strains could be pursued as food and oxygen producers on Mars, further research is needed to directly optimize and quantify CO₂ fixation and O₂ generation under these low-pressure settings.

The observed decrease in duration of the estimated lag phase with decreasing pressure for D. salina and C. vulgaris, and the increasing carrying capacities with decreasing pressure observed for C. vulgaris (Table 2) suggest that the cultures may be acclimatizing to the decreasing pressure conditions. Few studies have explored the mechanisms of adaptation of microorganisms under low pressure conditions (Kanervo et al., 2005; Nicholson et al., 2013; Murukesan et al., 2016; Schuerger and Nicholson, 2016; Verseux, 2020). Previous research on prokaryotes growing at low pressures, including transcription analysis, revealed that owing to their specialized adaptations to thrive in extreme environments, extremophiles are most likely to be better suited to survive under low pressures conditions (Schuerger et al., 2013; Verseux, 2020). This could explain the successful growth at low pressure of the extremophilic candidate strain D. salina and C. brevispina, where their natural adaptations to cope with high salinity, cold temperatures, and high irradiance might be responsible for their tolerance to low pressures as well. Some reports also suggest that microorganisms could evolve toward higher tolerance if exposed to low pressure over multiple generations (Verseux, 2020). This may explain the trend observed in the C. vulgaris species where the increasing trend in carrying capacity was observed with decreasing pressure. Additionally, C. vulgaris is globally distributed in both aquatic and terrestrial habitats (Bock et al., 2011; Aigner et al., 2020) suggesting that the species has adaptations to survive in these contrasting habitats. The molecular mechanisms of such adaptations are not clearly understood (Aigner et al., 2020) but may also contribute to the tolerance to low pressures as well.

Further analysis of the molecular basis of low-pressure adaptations will be required to understand different growth dynamics of algae species under low pressure conditions and may reveal key genes or quantitative trait loci that are involved in growth at low pressure. Such key genes or quantitative trait loci could potentially be selected for use in breeding studies, resulting in more useful algal strains. In addition, long-term growth under low pressure on Earth should lead to the development of strains with elevated productivities under low pressure. Together, such studies would accelerate the development of an algal-based BLSS for Mars.

Life support represents one of the most critical technologies needed for successful and safe long-term deep space human exploration missions and will require substantial amounts of both oxygen and food production. The results from this study contribute to the development of a BLSS by demonstrating the potential contributions of three candidate species C. brevispina, D. salina, and C. vulgaris. All three of the candidates showed exponential growth at low pressures of 80 mbar and 160 mbar, which indicates the possibility of using inflatable greenhouses to produce oxygen on the surface of Mars. If these cultures produce approximately similar O₂ yields per unit of dry biomass as recorded previously (Kirensky et al., 1968; Gitelson, 1992), the biomass of the algae used as food could also generate enough O₂ for the astronauts’ use. In addition, the lag phases of D. salina and C. vulgaris decreased with decreasing pressure, and the carrying capacity of C. vulgaris increased with decreasing pressure, which suggests that the cultures may be acclimatizing to the decreasing pressure conditions and may be increasingly useful in BLSS. Together these results indicate that these species may be able to contribute to potential BLSS on Mars using low pressure (∼200–300 mbar) greenhouses and inflatable structures that have already been conceptualized and designed.

The data supporting the conclusions of this paper are available in the Supplementary Material.

LC, EH, DM, and WR contributed to the conception and design of the study. LC conducted the experiments. EH and LC analyzed the data and performed the statistical analysis. CA contributed to the experimental design and wrote a section of the manuscript. JR and DR contributed to sample collection and experimental setup. All authors contributed to manuscript revision, read, 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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