A fundamental challenge in human missions to Mars is producing consumable foods efficiently with the in situ resources such as soil, water, nutrients and solar radiation ailable on Mars. The low nutrient content of martian soil and high salinity of water render them unfit for direct use for propagating food crops on Mars. It is therefore essential to develop strategies to enhance nutrient content in Mars soil and to desalinate briny water for long-term missions on Mars. We report simple and efficient strategies for treating basaltic regolith simulant soil and briny water simulant for suitable resources for growing plants. We show that alfalfa plants grow well in a nutrient-limited basaltic regolith simulant soil and that the alfalfa biomass can be used as a biofertilizer to sustain growth and production of turnip, radish and lettuce in the basaltic regolith simulant soil. Moreover, we show that marine cyanobacterium Synechococcus sp. PCC 7002 effectively desalinates the briny water simulant, and that desalination can be further enhanced by filtration through basalt-type volcanic rocks. Our findings indicate that it is possible to grow food crops with alfalfa treated basaltic regolith martian soil as a substratum watered with biodesalinated water.
IntroductionHuman exploration of Mars is a future mission goal for the U.S. National Aeronautics and Space Administration, and is driving technology development to support such an endeor. While the vision to sustain human activity on Mars is compelling, providing consumables to the crews is a fundamental challenge due to transport costs and need for continuous replenishment. Consequently, long-term human missions to Mars will rely on using martian resources ailable in situ to develop food production system. Yet, many resources such as soil, water, solar radiation, carbon and nitrogen found on the martian surface [1] cannot be directly exploited by plants for growth in the form which they are found.
Mars’ surface is mostly basaltic in composition as a consequence of past volcanism [2–4]. Martian regolith soil is largely weathered basalt, which contains salts such as sulfates and perchlorates [4]. Analyses he shown that basaltic regolith soil contain basic macro elements (C, H, O, N, P, S, K, Mg, Na and Ca) and minor elements (Mn, Cr, Ni, Mo, Cu, Fe and Zn) essential for life [5–7]. However, the main limiting factors of the basaltic regolith soil are its low nutrient bioailability and poor water-holding capacity due to absence of organic carbon [8–10]. Most surface water on present Mars is in the form of polar ice sheets, and subglacial liquid has been detected in these areas [11,12]. Several lines of evidence also suggest the occurrence of deliquescence processes caused by hygroscopic salts or by permafrost melts resulting in the formation of liquid brines or saline seeps [13–17]. Due to the geochemical properties of martian basaltic regolith soil and briny water, these in situ resources are unsuitable for growing plants for food. Hence, there is a critical need for suitable resources for growing plants on Mars.
A first step towards enabling habitation of Mars is the optimization of in situ resources as suitable resources for growing plants as part of a bioregenerative food system [18]. Consumable plants are an essential part of bioregenerative systems but also he secondary benefits such as contributing to generation of oxygen and organic waste recycling [19,20]. Although there is no location on Earth exactly reproducing martian environmental conditions, the harsh martian climate and geological features are comparable to some places on Earth [21–23]. The use of terrestrial analogues has therefore lead to the understanding of the physical, geochemical and environmental conditions that occur on Mars and help to resolve limitations to habitation relying on martian resources [8,24–33]. Microbes and higher plants he the potential to adapt to extreme terrestrial environments that simulate martian conditions [7,8,10,24,25,27,34–37]. Photosynthetic bacteria that are commonly employed in seawater biodesalination technologies allow cost and energy-efficient desalination [38,39]. Exploitation of such microbes in desalination of martian briny water may provide a usable water resource. Application of such potential biologicals in treating basaltic regolith soil and briny water for suitable resources for raising food crops would benefit future Mars missions. Here, we aimed to find strategies for treating basaltic regolith simulant soil and briny water simulant as suitable resources for growing food crops.
Materials and methods Basaltic regolith soil simulantNatural basalt-type volcanic rocks (Pleasant Hearth and Ace Hardware Corporation) were crushed in a Bico pulverizer (Department of Geological and Atmospheric Sciences, Iowa State University) to particles of 3 mm diameter or smaller and used as basaltic regolith simulant soil. Element composition and physical properties of the simulant soil were analyzed by Solum Inc. (Ames, Iowa, USA). The simulant soil was subjected to Mehlich 3 extraction [40] and mineral element content was analyzed by inductively coupled plasma spectrometry. A continuous flow system (Timberline TL 2900) was used for analyzing nitrogen content according to the manufacturer’s instructions. Water retention capacity of the simulant soil was determined from difference between constant weight of water saturated soil and dried soil. Two weeks after seed germination in basaltic regolith simulant soil, the soil (100 g) was treated with 15 ml chemical fertilizer (16.55% total nitrogen, 4.93% phosphate, 16.29% potash, 1.51% magnesium, 0.016% boron, 0.007% copper, 0.082% iron, 0.04% manganese, 0.007% molybdenum and 0.04% zinc; Jack’s Professional LX) or 100 mg of powder of alfalfa (grown in bare basaltic regolith soil) on a weekly basis. As plant species enriched in macronutrients, micronutrients, minerals, vitamins, amino acids and hormones could potentially be used as fertilizers in soil management [41], we assessed growth of alfalfa in bare basaltic regolith simulant soil and used powder of those alfalfa plants to treat the basaltic regolith soil. Garden soil was included as a control. In each plant growth experiment, at least 5 pots per soil type were used as replicates.
Briny water simulantBG-11 medium containing a mixture of inorganic salts was prepared as described in the University of Texas Algal Culture Collection (UTCC) recipe [42]. Sodium chloride was added to the BG-11 medium to 0.343 mol L-1 (briny water simulant) and autocled at 121°C and 15 psi for 20 min. Salinity (32.5 practical salinity unit, PSU) and electrical conductivity (EC; 48 mS/cm) of the briny water simulant were determined using a Thermo Electron-Orion Conductivity Benchtop Meter.
Growth of Synechococcus sp. strain PCC 7002 in briny water simulantMarine cyanobacterium Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum) desalinates high salinity water [39] and we tested the efficacy of Synechococcus sp. PCC 7002 to desalinate the briny water simulant. Synechococcus sp. PCC 7002 was obtained from the UTCC and maintained on BG-11 agar medium containing 0.171 mol L-1 NaCl and 1.5% agar. To the autocled BG-11 agar medium, 1 mmol L-1 sodium thiosulphate pentahydrate and 0.1% vitamin B12 in 50 mmol L-1 HEPES buffer (pH 7.8) were added prior to use. A dense lawn of Synechococcus sp. PCC 7002 was inoculated into a 250 ml Erlenmeyer flask containing 100 ml of briny water simulant and grown statically in a growth chamber (Percival-Scientific) under white soft light ≤650 μmol photons/m2/s for 16 h at 26°C and dark for 8 h at 24°C. Growth of Synechococcus sp. PCC 7002 in the briny water simulant over time was determined by measuring absorbance of the culture at 730 nm in a Beckman spectrophotometer. Salinity and EC of the briny water simulant were measured during Synechococcus sp. PCC 7002 growth.
Filtration of Synechococcus sp. PCC 7002 grown in briny water simulantSynechococcus sp. PCC 7002 cultured briny water simulant (100 ml) (Fig 1A) was filtered by gritational flow through a bed of basalt-type volcanic rocks packed tightly in a 1 L Pyrex glass column (Fig 1B and 1C). Filtrate was collected and re-filtered twice in the same column. Salinity and EC were measured on the final filtrate.
Fig 1. Grity filtration of Synechococcus sp. PCC 7002 cultured in briny water simulant through basalt-type volcanic rocks.
(A) Flasks containing Synechococcus sp. PCC 7002 grown briny water simulant. (B and C) Image of basalt-type volcanic rocks packed in a glass column with largest particles (~60 mm) at the top, smallest particles (~ 6 mm) at the bottom and intermediate particles in between. Final filtrate was collected and used to grow plants.
Plant growth experimentsTurnip (Brassica rapa), radish (Raphanus sativus), lettuce (Lactuca sativa) or alfalfa (Medicago sativa) seeds were sown in pots containing basaltic regolith simulant soil or garden soil and germination was assessed after a week. Plants were grown in a growth chamber (Percival-Scientific) under controlled condition [16 h white soft light (650 μmol photons/m2/s) at 26°C and 8 h dark at 24°C] and watered once a week. Plant shoot height and root length were measured at 1 week interval. To determine biomass, individual (whole) plants were dried at 37°C in an incubator until it reached a constant weight and dry weight was measured, or fresh weight of individual plant bulbs was measured. Alfalfa was grown in bare basaltic regolith simulant soil, dried at room temperature and hand crushed into powder.
In each experiment, at least three replicates were used. Data represent erage ± standard error (SE). Significance between treatments were statistically analyzed by paired t-test or one-way ANOVA with JMP16 (SAS Institute, NC, USA) and post-hoc tests were determined by Tuckey’s HSD.
Results Seed germination and plant growth in basaltic regolith simulant soilAnalysis of basaltic regolith simulant soil showed a low content of nitrate, ammonium, phosphorous, potassium, sulfur, zinc, manganese, boron, calcium, magnesium, iron and copper relative to garden soil (Table 1). The simulant soil was poor in organic matter content and cation exchange capacity, and alkaline as compared to garden soil.
Table 1. Properties of basaltic regolith simulant soil. Property Basaltic regolithsimulant soil Gardensoil* NO3-N (mg g-1) 0.001 0.0174 NH4-N (mg g-1) 0.0002 0.0037 Mineral element (mg g-1)PKSZnMnBCaMgFeCu 0.01770.08450.00220.00070.0190.00060.47560.15650.45190.0033 0.09110.44630.010.00540.05520.00193.6250.56510.56510.0024 Organic matter (% mass) 0.2 6.9 Cation exchange capacity(meq/100g) 4.0 21.8 pH 8.6 7.0 Open in a new tab*control.
Turnip seed germination in basaltic regolith simulant soil and garden soil (watered with fresh water) was compared. In basaltic regolith simulant soil, 62% of the seeds germinated within a week, while 55% of the seeds germinated in garden soil. Turnip plants grown in basaltic regolith simulant soil were stunted and produced fewer and smaller discolored lees than plants grown in garden soil (Fig 2A). Overall, the growth of turnip plants in the basaltic regolith simulant soil was unhealthy as compared to that grown in garden soil.
Fig 2.
(A) Growth of turnip plants (after 3 weeks) in basaltic regolith simulant soil or garden soil watered with fresh water. (B and C) Growth of turnip plants (after 4 weeks) in chemical fertilizer treated or untreated basaltic regolith simulant soil watered with fresh water. Shoot height of at least 5 individual plants in each treatment was measured. Data represent erage ± SE and significant difference is indicated by an asterisk (p < 0.01) as determined by paired t-test.
Growth of turnip plants in basaltic regolith simulant soil was then assessed after a chemical fertilizer treatment. Shoot height of turnip plants in fertilizer treated simulant soil was significantly increased (163%) relative to plants in unfertilized simulant soil (Fig 2B and 2C). Chemical fertilization of the simulant soil resulted in improvement in overall growth and normal phenotype of the turnip plants.
Growth of food crops in alfalfa treated basaltic regolith simulant soil watered with fresh waterAs plant species enriched in macronutrients, micronutrients, minerals, vitamins, amino acids and hormones could potentially be used as fertilizers in soil management [41], in this context, we assessed growth of alfalfa in bare basaltic regolith simulant soil watered with fresh water. Given the robust growth of alfalfa in the bare simulant soil (Fig 3A), alfalfa was further tested if it can serve as a nutrient source in simulant soil for growing food crops. Growth and biomass of turnip, radish and lettuce plants in alfalfa (grown in bare simulant soil) treated simulant soil (watered with fresh water) was evaluated. Growth of all three type of plants was boosted in the alfalfa treated simulant soil as compared to that grown in untreated simulant soil (Fig 4A, 4C, 4D and 4F). Growth of turnip plants increased to 190% in alfalfa treated simulant soil (Fig 4B). Turnips plants produced healthy bulbs in alfalfa treated simulant soil (Fig 4C). Biomass of radish bulbs (311%) (Fig 4E) and lettuce lees (79%) (Fig 4G) was significantly improved as compared to that when grown in untreated simulant soil.
Fig 3.
Growth of alfalfa in bare basaltic regolith simulant soil or garden soil watered with (A) fresh water or (B) fresh water or filtered biodesalinated water.
Fig 4.
Growth of (A, B and C) turnip, (D and E) radish and (F and G) lettuce in alfalfa treated or untreated basaltic regolith simulant soil watered with fresh water. Shoot height of 5 individual turnip plants (after 4 weeks), fresh weight of 5 radish bulbs (after 7 weeks), and dry weight of 5 whole lettuce plants (after 7 weeks) per treatment were measured. Data represent erage ± SE and significant difference is indicated by an asterisk (p ≤ 0.02) as determined by paired t-test.
Growth of food crops in alfalfa treated basaltic regolith simulant soil watered with biodesalinated waterAbsorbance of Synechococcus sp. PCC 7002 inoculated simulant water increased with time and growth was maximum after 4 weeks (Fig 5A). During the growth of Synechococcus sp. PCC 7002, salinity and EC of the simulant water gradually declined (Fig 5B). Within 4 weeks, salinity of the simulant water was reduced to about 32%, which demonstrated effective biodesalination mediated by Synechococcus sp. PCC 7002.
Fig 5.
(A) Growth, (B) salinity and EC of Synechococcus sp. PCC 7002 inoculated briny water simulant. Measurements were made in cultures from at least 5 individual flasks. Data are the erage ± SE and significant difference in salinity or EC (B) between briny water simulant starting material and simulant at each time point was analyzed by paired t-tests.
We then assessed growth of turnip plants in alfalfa treated basaltic regolith simulant soil watered with biodesdalinated water. Turnip plants did not produce healthy shoots when watered with biodesalinated briny water as compared to that grown with fresh water (Fig 6A). Following these results, Synechococcus sp. PCC 7002 cells grown in the biodesalinated water were removed by filtration through basalt-type volcanic rocks. Absorbance of the final filtrate was reduced to 0.25 (initial absorbance, 1.45) (Fig 6B). Both salinity and EC of the final filtrate was significantly reduced to 91%, as compared to the starting material (Fig 6C and 6D).
Fig 6.
(A) Growth of turnip plants (after 3 weeks) in alfalfa treated basaltic regolith simulant soil watered with unfiltered biodesalinated water or fresh water (control). (B) Flasks containing biodesalinated water before or after filtration. (C) Salinity and (D) EC of unfiltered and filtered biodesalinated water, and starting briny water simulant material. (E) Salinity and (F) EC of deionized water and fresh water (controls). At least three replicates per water type were used for salinity and EC measurements. Data represent erage ± SE. (C and D) Average salinity or EC of samples that do not share a letter above the bar are significantly different (P