Growing plants in Mars: not so easy

Abstract

Future manned missions to Mars or the colonization of the red planet require systems to provide food, oxygen and water for human beings. Plants can produce them in case they could be cultivated in Mars. We have carried out a project with High School Students to assess the capacity of terrestrial plants to grow in a soil with nearly similar features to that of Mars. We have prepared that simulant soil from volcanic scoria, with a chemical and mineralogical composition similar to soils studied by rovers. Organic matter and microorganisms were eliminated. Arabidopsis thaliana and pepper (Capsicum annuum) seeds were cultivated in this soil. Plants showed a lower development when they were cultivated in our simulant soil and irrigated only with distilled water. The addition of a nutrient solution had inconsistent effects on our plants; while the growth of pepper was improved, Arabidopsis plants had a decline in the development and, eventually, they died. Growing plants successfully in Mars requires to study both nutritional requirements and tolerance to potential toxic substances.

Full text

High School Students for Agricultural Science Research
Volume 8
September 2019
Ba
56
137.327
Se
34
78.09
Ciencia
Programa científico para
Bachillerato y Secundaria
“Project Mars”

High School Students for Agricultural Science Research
Volume 8 September 2019
EDITORIAL BOARD
Juan de Dios Alché
Manuel Espinosa-Urgel
Francisco Martínez-Abarca
José Manuel Palma
Antonio Quesada
ISSN: 2340-9746
Published in Granada by Estación Experimental del Zaidín. CSIC
High School Students for Agricultural Science Research. Vol. 7 by Estación Experimental del Zaidín is licensed
under a Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional License.

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Growing plants on Mars: not so easy
Antonio Quesada Ramos1, Carmelo Ruiz2, Manuel Espinosa-Urgel2,
José Manuel Palma2
1IES Zaidín Vergeles. Granada; 2Estación Experimental del Zaidín, CSIC. Granada
Summary
Future manned missions to Mars or the colonization of the red planet require systems to provide
food, oxygen and water for human beings. Plants can produce them in case they could be
cultivated in Mars. We have carried out a project with High School Students to assess the capacity
of terrestrial plants to grow in a soil with nearly similar features to that of Mars. We have prepared
that simulant soil from volcanic scoria, with a chemical and mineralogical composition similar to
soils studied by rovers. Organic matter and microorganisms were eliminated. Arabidopsis thaliana
and pepper (Capsicum annuum) seeds were cultivated in this soil. Plants showed a lower
development when they were cultivated in our simulant soil and irrigated only with distilled
water. The addition of a nutrient solution had inconsistent effects on our plants; while the growth
of pepper was improved, Arabidopsis plants had a decline in the development and, eventually,
they died. Growing plants successfully in Mars requires to study both nutritional requirements and
tolerance to potential toxic substances.
KEYWORDS: Mars, soil analogue, volcanic scoria, Arabidopsis thaliana, Capsicum annuum,
Pseudomonas putida, Pseudomonas stutzeri.
Introduction
Mars is an astrobiological target not only by its similarity or proximity to our planet, but
because of the fact that billions years ago Mars had liquid water and an environment similar
to that of the Earth in which life appeared. Nevertheless, at present life is almost impossible
to exist in the red planet. The scarcity of liquid water, extreme low temperatures, a low
pressure atmosphere with high levels of carbon dioxide, a gravity that is one third of that in
the Earth, the high levels of radiation on the surface, and a lack of organic nutrients are
factors that make very difficult to survive in Mars.
Because of this interest, manned mission to Mars and, furthermore, the hypothetical
colonization of the red planet require systems to provide mainly oxygen, water and food for
the metabolic needs of humans beings,. On the Earth, these functions are basically
facilitated by plants; either through CO2 absorption and O2 emission, water purification
through transpiration, waste product recycling via mineral nutrition or as a food source,
plants key an important role in this context (Monje et al., 2003; Wolff et al., 2014).
Future human missions to Mars generate a special interest in the study of the response of
plants to its extreme conditions. Accordingly, the investigation of the plant growth in

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conditions similar to those of Mars could indicate the feasibility of cultivating them in that
planet. Wamelink et al. (2014) have investigated the possibility of growing plants in a Mars
simulant soil. They propose that all essential minerals for the growth of plants appear to be
present in sufficient quantities with the exception of nitrogen in reactive forms (NO3, NH4).
In fact, in their experiments, several species of plants were able to germinate and complete
their life cycle for a period of 50 days without the addition of nutrients.
The main objective of this research was to assess the capacity of terrestrial plants for
growing in a soil as similar as possible to that of Mars. In our laboratory we have studied the
growth of Arabidopsis thaliana and pepper (Capsicum annuum) in a Mars simulant soil. We
have prepared the analogue from volcanic scoria, with a mineralogical and chemical
composition similar to that of some soils of Mars. Organic matter has been eliminated by
washing the rocks in running water. As there are no known life forms in martian soil, pots
were sterilized to eliminate microorganisms.
Material and methods
Mars soil simulant
Material similar to the Martian regolith was prepared from volcanic scoria adquired from a
commercial brand used in gardening (“Greda volcánica”, Batllé). Chemical and mineralogical
composition of these rocks were analysed by X-ray fluorescence and X-ray diffraction at the
Instituto Andaluz de Ciencias de la Tierra (CSIC, Granada, Spain). The results showed that our
samples had similar characteristics to some soils studied in Mars by rovers (Castillo Tejada et
al, 2019). The chemical composition of the soil can be consulted in that reference in this
same volume.
To prepare the soil for plant culture and growing, rocks were crushed with an iron mortar
and washed with tap water at least for two hours and then dried in an oven at 80ºC. Pots
with 750 g of the so obtained soils containing volcanic fragments of different sizes (from fine
powder to about 0.125 mm3) were prepared. Then, they were sterilized by autoclaving (two
cycles, 121ºC, 15 minutes each).
Plant species
We have studied germination and growth of two plant species in our Mars soil simulant
under different conditions.
Arabidopsis thaliana, cv. Columbia is a small flowering plant used as a model organism in
plant biology and it has been used by several authors in experiments on Astrobiology
(Richards et al., 2006; Wolff et al. 2014). It has not agronomic significance (it is rather a
weed) but it offers advantages for basic research in genetics and molecular biology. It has a
rapid life cycle: about six-eight weeks from germination to mature seeds. A. thaliana
produces very small seeds; it provided an advantage for our research as its internal nutrient
pool is quickly metabolized and the plant becomes totally dependent of light and whatever

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is available in the soil. Columbia ecotype has been used for these experiments since is the
one which is commonly reported as wild type for in most laboratories.
The second plant used has important agricultural and nutritional values. Pepper (Capsicum
annuum L. cv. Padrón) receives its name from the municipality of Padrón (A Coruña) where
they are widely cultivated since the XVII century. These are small peppers, about 5 cm long,
with elongated conic shape and a colour ranging from bright green to yellowish green while
they are still unripe, which is the common consumed appearance. Peppers are not only
important as a food, but the fruits are also an excellent source of health-related compounds,
such as ascorbic acid (vitamin C), carotenoids (pro-vitamin A), and other antioxidants such as
tocopherols, flavonoids and capsaicinoids (Wahyuni et al.2013). Peppers seeds were surface-
sterilized with NaClO at 5% (w/v) for five minutes, and then rinsed several times with
deionized water.
Figure 1 shows a comparison between seeds from A. thaliana and pepper, variety Padrón.
Figure 1. Comparative size of
Capsicum annum and Arabidopsis
thaliana (lower right corner)
seeds observed through a
stereomicroscope. The scale is
shown on the lower left corner.
Microorganisms
In Mars, it has been proposed the presence in the soil of toxic substances, like heavy metals
and metalloids, that could prevent plants from thriving. Furthermore, Martian regolith is
poor in assimilable nitrogen like nitrates or ammonia and so volcanic soils are. In order to
improve the conditions of our Mars-simulating soil to grow plants, it has been considered
the addition of microorganisms to our samples. For that purpose, species of the genus
Pseudomonas are specially interesting as they are able to metabolize toxic compounds from
soils, and even to fix dinitrogen.
Pseudomonas putida is a Gram negative, saprotrophic soil bacterium. It has a diversified
metabolism which allows it to degrade organic compounds and it also has a great capacity to
tolerate heavy metals and metalloids (Canovas et al., 2003). In fact, it has been used in
bioremediation as this microorganism is able to degrade environmental pollutans.

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Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the
environment. It has been proposed as a model organism for denitrification studies. Some
strains are able to fix dinitrogen, and others participate in the degradation of pollutans or
interact with toxic metals (Lalucat et al., 2006).
The tolerance of P. putida and P. stutzeri to environmental conditions similar to those of
Mars have been previously studied at our laboratory (Castillo-Tejada et al., 2019; Delgado-
Alaminos et al., 2019).
Experimental design
The main objective of this research was to assess the capacity of Arabidopsis thaliana and
Capsicum annuum to grow in a sterile soil similar to that of Mars and to study simultaneous
treatments that could improve the thriving of plants. For each plant species, we applied four
treatments (T1-T4) and prepared four pots with the same quantity (750 g) of Mars soil
simulant. In the case of Arabidopsis, nine groups of non sterilized seeds (2-3 each group)
were planted in every pot. Six sterilized seeds of Capsicum were planted in every pot.
- Treatment T1: Seeds were deposited directly over the soil and pots were only irrigated with
distilled water to prevent the potential interferences with nutrients that might be present in
the water; in this case, plants only had available for their growth the mains contained in the
Mars soil simulant.
- Treatment T2: Seeds were deposited directly over the soil and and pots were watered with
a nutrient solution. We used the modified Hewitt nutrient solution described in Tortosa et
al. (2018), diluted 1:2 with distilled water.
- Treatment T3: Previous to planting, seed were inoculated with two Pseudomonas species.
100 ml of a liquid culture of bacteria containing either P. putida or P. stutzeri were added to
seeds from both plant species. Some of them were treated with. Plants were only irrigated
with distilled water as in T1.
- Treatment T4: Previous to planting, seed were inoculated as in T3 and then supplied with
the nutrient solution as in T2.
After sowing, pots were covered with aluminum foil during three days for A. thaliana, and
for five days in case of C. annuum seeds. Then, the cultures were maintained for 15 weeks
and pictures were taken after 1, 4, and 8 weeks and at the end of the experiment. Plants
were illuminated for 16 hours every day with compact fluorescent lamps: 2x 8W, 2700K
lamps (Phillips) and 2x 20W 6400K lamps (ROHS). Plants were watered as corresponding to
each treatment three times a week. At the end of the experiment, the presence of
microorganisms in the soil where inoculated seeds were planted was assessed. For this, 50 µl
were taken from the pots and spread in Petri dishes with TSA (tripticase soy agar) medium.
Plates were incubated at 28ºC for two days.

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Results
Almost all the seeds of A. thaliana and C. annuum germinated during the first week.
However, the germination and growth response to the analogue of the Martian soil was
different in both plant species. Figure 2 shows the evolution of A. thaliana plants throughout
fifteen weeks in the four treatments (T1-T4). Although Arabidopsis has a rapid life cycle that
takes about six-eight weeks from germination to mature seeds, growth of the plants in this
assay was very slow. Under the best conditions (T1 and T3), the plant height was about 1 cm
and plants did not reach the flowering stage.
Figure 2. Growth of Arabidopsis thaliana in a Mars soil simulant under different treatments (T1-T4).
Differences in the response of A. thaliana to different treatments were observed. Thus, it
was found that plants irrigated only with distilled water had a higher percentage of survival.
On the contrary, all the plants supplied with nutrient solution were not able to grow until
the end of the experiment. The addition of microorganisms did not have any obvious effect
either in the growth or the survival of plants (Figure 3).

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Figure 3. Survival of A. thaliana plants in a Martian-like soil under different treatments (T1-T4).
The presence of microorganisms in the soil simulant was monitored at the end of the
experiment. Figure 4 shows colonies grown after liquid samples taken from the soil of
cultivation were spread onto TSA Petri dishes. There was bacterial growth in all the samples,
the nutrient solution showing the higher proliferation of microorganisms. Although bacteria
could not be totally identified, some colonies of P. putida were recognizable by the presence
of pioverdine in the dishes.
Figure 4. Bacterial growth after incubation
of liquid samples obtained from soils of
treatments T1-T4 appplied to Arabidopsis
thaliana.
T1
T2
T3
T4
time (weeks)
T1
T4
T2
T3

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On the other hand, almost all pepper seeds germinated. Figure 5 shows the evolution of
pepper plants in the martian soil simulant. Plants experienced a development decline when
they were irrigated only with distilled water. From six plants germinated in every pot only
two developed real leaves after 15 weeks, both in pots with and without microorganisms.
It was also observed that nutrient solutions favoured the plants growth, but bacteria did not
have a great influence on the growth of pepper plants.
Figure 5. Growth of C. annuum plants in a Mars soil simulant under different treatments (T1-T4).
As with A. thaliana, there were also differences between the concentration of
microorganisms in the soil where pepper plants grew after fifteen weeks of cultivation
(Figure 6). The higher number of colonies was observed in dishes inoculated with soil water
from pots treated with microorganisms.

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Figure 6. Bacterial growth after incubation
of liquid samples obtained from soils of
treatments T1-T4 appplied to C. annuum.
Discussion
Environmental conditions are harsh for life in Mars. An atmosphere with low pressure and
high concentration of carbon dioxide, low temperatures, high levels of radiation, scarcity of
liquid water and a soil that lacks of organic material and contains toxic compounds like
perchlorates and iron oxides seems to draw an impossible scenario for growing plants in the
red planet.
In this research we have studied the potential effect of the martian regolith on the
germination and development of two plant species: Arabidopsis thaliana and Capsicum
annuum. We have simulated a martian soil from volcanic scoria and have eliminated its
organic matter and microorganisms. Chemical composition of soil is similar to that indicated
by analyses made in Mars; also as in Mars, our soil lacks assimilable forms of nitrogen but
has certain levels of iron oxides.
Our experiments showed that our soil simulant, and probably the soil of Mars, are perhaps
inappropriate for growing plants. We have observed a lack of development in plants in that
soil only irrigated with distilled water. Our soil lacks essential nutrients for plants like
nitrogen and has elements like iron or aluminum oxides which in high concentrations might
be toxic and probably inhibited plant growth. These results disagree with those described by
Wamelink et al. (2014) who reported that plants were able to germinate, grow and flower in
a Mars regolith simulant handled by the NASA. The lack of nutrients probably causes the
observed lack of development both in A. thaliana and C. annuum. However, the addition of
the same nutrients to the pots through the supplementation of nutrient solution showed
inconsistent results. Arabidopsis plants died in those cases, while peppers improved their
T1
T4
T2
T3

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development. Probably some compound of the nutrient solution is toxic for Arabidopsis per
se or combined with some chemical present in the soil.
Microorganisms and organic matter are essential compound of soils. We have not observed
an improvement in the development of plants grown in soil added with both species of
Pseudomonas. However, we have noticed the presence of fungi on pots treated with
nutrient solution, revealing the presence of organic matter, probably with a positive effect
on pepper plants.
From our research we can conclude that it is not easy to cultivate plants neither in a martian
soil analogue nor in Mars. Every plant has specific nutritional requirements and it is
necessary to study them as well as their tolerance to possible toxic substances present in
those soils in order to be successful in a future hypothetical colonization of the Red Planet.
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