Article

Sustainable life support on Mars – the potential roles of cyanobacteria

Abstract and Figures

Even though technological advances could allow humans to reach Mars in the coming decades, launch costs prohibit the establishment of permanent manned outposts for which most consumables would be sent from Earth. This issue can be addressed by in situ resource utilization: producing part or all of these consumables on Mars, from local resources. Biological components are needed, among other reasons because various resources could be efficiently produced only by the use of biological systems. But most plants and microorganisms are unable to exploit Martian resources, and sending substrates from Earth to support their metabolism would strongly limit the cost-effectiveness and sustainability of their cultivation. However, resources needed to grow specific cyanobacteria are available on Mars due to their photosynthetic abilities, nitrogen-fixing activities and lithotrophic lifestyles. They could be used directly for various applications, including the production of food, fuel and oxygen, but also indirectly: products from their culture could support the growth of other organisms, opening the way to a wide range of life-support biological processes based on Martian resources. Here we give insights into how and why cyanobacteria could play a role in the development of self-sustainable manned outposts on Mars.
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Sustainable life support on Mars the
potential roles of cyanobacteria
Cyprien Verseux
1,2
, Mickael Baqué
1
, Kirsi Lehto
3
, Jean-Pierre P. de Vera
4
, Lynn
J. Rothschild
5
and Daniela Billi
1
1
Department of Biology, University of Rome Tor Vergata, Rome, Italy
e-mail: cyprien.verseux@gmail.com
2
NASA EAP Associate, NASA Ames Research Center, Moffett Field, California, USA
3
Department of Plant Physiology and Molecular Biology, University of Turku, Turku, Finland
4
German Aerospace Center (DLR), Institute of Planetary Research, Berlin, Germany
5
Earth Sciences Division, NASA Ames Research Center, Moffett Field, California, USA
Abstract: Even though technological advances could allow humans to reach Mars in the coming decades,
launch costs prohibit the establishment of permanent manned outposts for which most consumables
would be sent from Earth. This issue can be addressed by in situ resource utilization: producing part or all of
these consumables on Mars, from local resources. Biological components are needed, among other reasons
because various resources could be efficiently produced only by the use of biological systems. But most
plants and microorganisms are unable to exploit Martian resources, and sending substrates from Earth to
support their metabolism would strongly limit the cost-effectiveness and sustainability of their cultivation.
However, resources needed to grow specific cyanobacteria are available on Mars due to their photosynthetic
abilities, nitrogen-fixing activities and lithotrophic lifestyles. They could be used directly for various
applications, including the production of food, fuel and oxygen, but also indirectly: products from their
culture could support the growth of other organisms, opening the way to a wide range of life-support
biological processes based on Martian resources. Here we give insights into how and why cyanobacteria
could play a role in the development of self-sustainable manned outposts on Mars.
Received 24 March 2015, accepted 22 June 2015, first published online 3 August 2015
Key words: cyanobacteria, in situ resource utilization (ISRU), life-support systems, Mars exploration, space technologies,
synthetic biology.
Introduction
Sending humans to Mars within a few decades is now a realistic
goal (e.g., Baker & Zubrin 1990; Horneck et al. 2006; Drake
2009; Zubrin & Wagner 2011). However, even though leaving
a footprint and planting a flag could be achieved with not much
more than the current state-of-the-art of engineering, a definite
pay-back is still in doubt. On the other hand, if a Mars mission
can allow extensive human scientific activity and yield mean-
ingful scientific data, the effort is justified. In such a case, scien-
tists will need to spend a considerable period on site, and
multiple short-term missions are not a viable option. Given
the time, costs and challenges associated with the journey,
long-term human bases will likely be needed.
But while the vision of long-term human presence on Mars is
compelling, providing consumables to sustain crews is a chal-
lenge. Even though launch costs vary depending on mission
scenarios and might be reduced in the coming decades, they
have been estimated to be in the order of $300 000 kg
1
(Massa et al. 2007). Sending from Earth all the needed re-
sources does not seem financially sustainable. Should Mars
colonization be consequently deemed too expensive to be real-
istic? Maybe not. There are alternatives.
One is recycling using regenerative systems. Such systems
should have biology-based components: various life-support
functions can be provided by physicochemical processes, but
some valuable products such as high-protein food cannot cur-
rently be produced by the latter (Drysdale et al. 2003;
Montague et al. 2012). In addition, many components of
physicochemical life-support systems are heavy, highly energy-
consuming and/or require high temperatures. Even in the case
where these are the backbones of life-support systems, bio-
logical modules could both complement them and provide
safe redundancies. Consequently, various bioregenerative life-
support systems (BLSS) are or have been under development
for recycling food, water and gases both in space (e.g., Godia
et al. 2002; Gitelson et al. 2003; Drysdale et al. 2004; Lobascio
et al. 2007; Nelson et al. 2010; Giacomelli et al. 2012) and with-
in lunar and Martian outposts (e.g., Gitelson 1992; Blüm et al.
1994; Tikhomirov et al. 2007; Nelson et al. 2010). This may
sound promising: instead of sending resources in amounts
almost proportional to the mission length, only a few weeks
worth of consumables would be sent and recycled. The issue
is: 100% recycling efficiency cannot be reached and losses are
unavoidable. For quantitative information regarding the the-
oretical recycling efficiencies of the Micro-Ecological Life
Support System Alternative (MELiSSA), for example, see
Poughon et al. (2009).
A regenerative systems running time without re-supply is
consequently limited. It also cannot be expanded, as the
International Journal of Astrobiology 15 (1): 6592 (2016)
doi:10.1017/S147355041500021X ©Cambridge University Press 2015
mass of cycling components is, at a given time, at most equal to
their initial mass. In addition to this, most current BLSS pro-
jects represent a large initial volume and mass, as well as a high
power consumption. For instance, the mass of consumables
needed to sustain a crew of 6 using the MELiSSA system has
been assessed to be about 30 metric tons (mt), hygiene water
not included, for a 1000-day mission (Langhoff et al. 2011).
Re-supply is thus needed and the most advanced BLSS projects
heavily depend on materials imported from Earth although a
theoretical physicochemical/biological resource production
system relying on Martian resources has recently been patented
(Cao et al. 2014). These projects are consequently not suitable
for autonomous, long-term human bases on Mars: the mass
problem is reduced, but not solved.
There is however a promising solution: producing resources
from local materials. Like all human settlers of previous gen-
erations, the Martian pioneers must live off the land. A crit-
ical question this fact raises is whether it is possible to feed
biological systems with local resources. On the one hand,
water (H
2
O), solar energy, carbon (C), nitrogen (N) and
many other life-supporting nutrients are found on Mars
(Meyer & McKay 1989; Olsson-Francis & Cockell 2010;
Cockell 2014). But on the other hand, they cannot be directly
exploited in the form in which they are found there by key or-
ganisms of current BLSS projects. This limitation may lead one
to think that BLSS based on local resources are irrelevant for
Mars exploration. What if, however, Martian resources could
be exploited and processed into suitable forms by an additional
biological module? What if there was a biological link between
on-site resources and BLSS? This link might be created by
cyanobacteria. All the inputs needed to sustain a diazotrophic
(N-fixing), bioleaching cyanobacteriums metabolism could in
theory be obtained from Marss mineral resources, water, at-
mospheric gases and incident solar energy. Firstly, some (e.
g., Anabaena spp. and various desert species) have the ability
to extract and biologically fix nutrients from analogues of
Martian rocks (Brown & Sarkisova 2008; Olsson-Francis &
Cockell 2010). Owing to these abilities and to their useful pro-
ducts, cyanobacteria have already been suggested as a basis for
creating planetary BLSS relying on local resources (Brown
2008a; Brown et al. 2008; Stanford-Brown 2011 iGEM team
2011). They are also able to fix C from carbon dioxide (CO
2
)
and some species can fix N from dinitrogen (N
2
), both of which
are found in the Martian atmosphere, leading to organic mat-
ter and dioxygen (O
2
) production without dependency on
carbohydrate feedstock. Some species are highly tolerant to ex-
treme environments (e.g., Rothschild & Mancinelli 2001); con-
sequently their culture is less demanding in terms of hardware
than that of more environmentally sensitive microbial species.
Cyanobacteria could be used directly to produce resources
such as food and O
2
, but products from their cultures could
also be used to feed other living organisms, so opening the
way to a wide range of life-support biological processes.
Cyanobacteria could thus provide the key link between
BLSS and Martian resources, making the former sustainable
and expandable in human bases on Mars. An artists rendering
of such a cyanobacterium-based BLSS (CyBLiSS) is given in
Fig. 1. The present paper is not intended as a specific mission
design, but rather as an overview of how and why cyanobac-
teria could be grown on Mars. We first outline their potential
applications there. Then we discuss the associated challenges
and possible ways of facing them. Finally, we outline the re-
search efforts needed to design functional, cyanobacterium-
based BLSS for human outposts on Mars. A summary of
this papers contents is given in Fig. 2.
Potential roles of cyanobacteria in Mars-specific
BLSS
Feeding other microorganisms
Heterotrophic microorganisms have been used throughout
human history and would be highly useful on Mars. Potential
applications include the production of drugs, food, biomaterials
and various industrially useful chemicals, metal leaching
and food processing for taste improvement (Cumbers &
Rothschild 2010; Langhoff et al. 2011;Montagueet al. 2012;
Menezes et al. 2014; Verseux et al. 2016). As most of these ap-
plications require relatively small culture volumes and no solar
light, cultures could be performed under Earth-like conditions
with reasonable costs. However, heterotrophic microorganisms
rely on organic materials whose availability on Mars remain
very poorly known and are not expected to be abundant there
(Ming et al. 2014). Could the local resources be processed into
suitable substrates by cyanobacteria? Related phenomena nat-
urally occur on Earth, where cyanobacteria are known to sup-
port different heterotrophic communities. They can for
instance be the earliest colonizers of desert habitats and allow
the development of local ecosystems (including heterotrophic
bacteria) through the production of organic compounds, N fix-
ation and rock leaching (e.g., Eldridge & Greene 1994;Danin
et al. 1998;Herreraet al. 2009). In aquatic ecosystems as well,
cross-feeding and metabolite exchange occur between cyano-
bacteria and heterotrophic microorganisms (see, for instance,
Stevenson & Waterbury 2006 and Beliaev et al. 2014).
The question of water is addressed in subsection Water.
Heterotrophic organisms also need, first of all, organic com-
pounds as a source of C and energy. Lysed cyanobacterial bio-
mass could be used as such (and as a source of other critical
macronutrients such as hydrogen [H], oxygen [O], phosphorus
[P] and sulphur [S]; for N, see the following paragraph).
Consistently, a filtrate of grinded Anabaena sp. PCC7120
(100 mg of dried biomass ml
1
before filtration) has been
used as the only source of organic compounds and fixed N to
grow Escherichia coli K-12 MG1655 in phosphate-buffered sa-
line, reaching more than 10
9
cells ml
1
within 24 h (Verseux,
unpublished data). Note that these results are preliminary
and that no optimization step (e.g., choice of a strain that
can metabolize sucrose, alteration of culture conditions to
modify cyanobacterial biomasss composition and/or more ef-
ficient extraction method) has yet been performed. Lysed
cyanobacterial biomass has also been shown to be a suitable
substrate for ethanol production in yeasts (Aikawa et al.
2013; Möllers et al. 2014). In lysogeny broth (LB), the most
66 Cyprien Verseux et al.
common growth medium for heterotrophic bacteria in labora-
tories, the concentration of fermentable sugars and sugar
equivalents (sugar phosphates, oligosaccharides, nucleotides,
etc.) utilizable by E. coli is very low (<100 µM), constraining
bacteria to use amino acids as C sources (McFall & Newman
1996; Sezonov et al. 2007). However, non-destructive ways of
harvesting nutrients could lead to more efficient processes.
Substrates could for instance be secreted. This solution
has been investigated in Lynn Rothschilds laboratory (at
NASA Ames Research Center, Moffett Field, CA) since the
2011 Stanford-Brown iGEM team engineered Anabaena sp.
PCC7120 to secrete sucrose (Stanford-Brown 2011 iGEM
team 2011), which was then used as a C source to grow
Bacillus subtilis (Ryan Kent et al., unpublished data). Prior
to this, Synechococcus elongatus PCC7942 was engineered to
produce and secrete either glucose and fructose, or lactate,
then used as a substrate for E. coli (Niederholtmeyer et al.
2010). In this kind of system, a major limitation arises from
low sugar yields, which are due to relatively low synthesis
rates and to the consumption of sugars by the cyanobacteria
themselves. Production rates could be increased by further gen-
etic engineering to either increase synthesis or decrease pro-
cessing of these products by the producer strains. External
conditions could also be modified; for instance, osmotic stress
induces sucrose accumulation in many cyanobacteria, especially
when synthesis of other osmoprotectants isimpaired(Miao et al.
2003).
Then, fixed N is needed. Heterotrophic bacteria can obtain N
from various organic and inorganic sources such as single amino
acids (e.g., Crawford et al. 1974) and amino acid chains (e.g.,
Hollibaugh & Azam 1983; Coffin 1989), nucleic acids (Paul
et al. 1989) and ammonium (NH
4
+
). Diazotrophic cyanobacteria
can produce all these compounds after N fixation and, here
again, a simple way of making this N available to heterotrophic
bacteria is to lyse cyanobacteria. But NH
4
+
can be naturally re-
leased by some diazotrophic cyanobacteria, without cell lysis;
for instance, extracellular NH
4
+
can reach several mM in cultures
of Anabaena spp. (mutants or wild-type, depending on species)
relying on atmospheric N
2
as a sole N source (Spiller et al. 1986;
Subramanian & Shanmugasundaram 1986). NH
4
+
is the pre-
ferred N source for most microorganisms and becomes limiting
at extremely low concentrations (e.g., belowa few μMforE. coli;
see Kim et al. 2012), several orders of magnitudes below the
above-mentioned concentrations. Released NH
4
+
could be
used for feeding not only heterotrophic microorganism, but
also some phototrophic species of interest which cannot fix
N. Within the MELiSSA loop (e.g., Godia et al. 2002), NH
4
+
(there resulting from human and plant waste processing by
thermophilic anaerobic bacteria) is converted into nitrates by ni-
trifying bacteria before being transferred to Arthrospira sp. cul-
tures. Even though nitrate is considered the preferred N source
for this species (as for most non-N fixing filamentous cyanobac-
teria), using NH
4
+
instead of nitrates has been shown not to re-
duce growth rates (Filali et al. 1997). A drawback is that NH
4
+
becomes growth-limiting from relatively low concentrations
(a few mM) and should consequently, if limiting below these
concentrations, be regularly (or continuously) added to the cul-
ture medium. Semi-separated cultures, where cyanobacterias
and heterotrophic microorganismsculture vessels communicate
through membranes that allow medium but not cell exchange,
can also be considered. In such a case, extensive system charac-
terization is needed to predict outputs, and processes could be
optimized by improving the culture setup and by performing
evolutionary selection of the co-culture to improve metabolic
Fig. 1. Artists rendering of a cyanobacterium-based biological life-support system on Mars. Figure design: Cyprien Verseux and Sean McMahon
(Yale University). Layout: Sean McMahon.
Sustainable life support on Mars 67
interactions. In any case, the rates of NH
4
+
release by strains of
interest under Mars-like constraints, as well as the use of this
NH
4
+
as an N source for non-N fixers, should be further
investigated.
Many heterotrophic microorganisms also need O
2
.E. coli,
for example, roughly needs 1 g of O
2
per gram of dry weight
(gdw) (Shiloach & Fass 2005). O
2
consumption and cell mass
vary according to strain and cultures conditions, but this figure
corresponds to approximately 1 g of O
2
l
1
to reach a culture of
2×10
9
cells ml
1
. Cyanobacteria produce O
2
through photo-
synthesis; details are given in subsection Producing oxygen.
Finally, metals must be provided. Some (macronutrients)
are needed in relatively large amounts; these are mostly potas-
sium (K), magnesium (Mg) and iron (Fe) and, for some species,
sodium (Na) and calcium (Ca). Others (micronutrients) are
needed in trace amounts by some microorganisms and include,
for instance, chromium (Cr), manganese (Mn), nickel (Ni) and
zinc (Zn) (Madigan et al. 2000). As an example, E. coli needs
about 10
8
atoms cell
1
of K and Mg; 10
5
atoms cell
1
of Ca,
Zn and Fe; and 10
4
atoms cell
1
of Mn, molybdenum (Mo)
and selenium (Se) (Finney & OHalloran 2003). All the needed
elements seem to be present in Martian regolith (Cockell 2014),
but some of them may be poorly available to most organisms
with no leaching abilities. However, rock-dwelling cyanobac-
teria can extract metal nutrients from a wide range of rocks
(see Olsson-Francis et al. 2012). Besides mobilizing them in
their own cells, they help release them in the aqueous phase, in-
creasing their concentration there (Olsson-Francis & Cockell
2010; Olsson-Francis et al. 2012) and availability for non-
leaching organisms. Anabaena cylindrica has for instance
been shown to release elements including K, Mg, Na, Ca,
Fe, Mn, Ni and Zn from a Mars basalt analogue
(Olsson-Francis & Cockell 2010). The metals needed in highest
amounts by E. coli, namely K and Mg, reached concentrations
of 125 and 55 µM, respectively, in large excess compared with
E. colis needs (roughly, 0.3 µM to reach 2 × 10
9
cells ml
1
).
Even though lysing cyanobacterial biomass is the most
straightforward option for transferring nutrients from cyano-
bateria to heterotrophic microorganisms, it may thus not be ne-
cessary. What solution minimizes the cost-to-productivity ratio
is still to be determined.
Supporting plant growth
The majority of BLSS projects include plants for air and water
regeneration and food production. Interestingly, all of the nu-
trients needed to grow plants (C, H, N, O, P, S,K, Mg, Fe, Na,
Ca and micronutrients) seem to be present on Mars. Banin
(1989) proposed using Martian regolith to grow plants and
this approach is still under investigation (e.g., Silverstone
et al. 2003,2005; Nelson et al. 2008; Maggi & Pallud 2010;
Wamelink et al. 2014). However, even though Martian regolith
is mostly basaltic and weathered basalt can yield extremely
productive soils on Earth (Dahlgren et al. 1993), regolith will
require physicochemical and/or biological treatment before it
can be used as a growth substrate for plants. Besides excess
salts, oxides and toxins, the main limiting factors are its low nu-
trient bioavailability and poor water-holding potential caused
by low organic C contents (Maggi & Pallud 2010; Cockell
2011). Enrichment of N, in particular, is critical: most plants
cannot fix atmospheric N (a few, mainly legumes, benefit
from symbiotic N fixation by specific bacteria harboured in
their root tissues).
Some elements could be obtained by recycling human waste
and inedible biomass from previous crops. However, relying
solely on this would strongly limit sustainability and scalability
of the system: without adding new components to the loop, the
pool of supplies can only decrease over time. Again, inputs for
plant cultivation should rather be provided from local re-
sources. An interesting approach has been proposed in the con-
text of lunar exploration. Following this approach, plant
species which are tolerant to harsh growth conditions would
be grown in local regolith, after inoculating seeds with a care-
fully chosen consortium of bioleaching bacteria to deliver nu-
trients to plant roots and to protect them against excessive
accumulation of toxic elements. Once these first-generation
plantswould have been grown, their biomass would be con-
verted by microorganisms into a fertile protosoil used to sup-
port the growth of more demanding plants (Kozyrovska et al.
2006; Zaets et al. 2011). An alternative strategy could rely on
cyanobacteria to process elements from rocks and fix N. Even
though no plant cultures are currently produced using cyano-
bacteria as exclusive nutrient sources, the latter are used in
agriculture to improve soil fertility, balance mineral nutrition
and release biologically active substances that promote plant
growth and increase plant resistance (Singh 2014). In the con-
text of space exploration, cyanobacterial cultures have already
been proposed as a way of releasing chemical elements from
rocks to hydroponic solutions (Brown & Sarkisova 2008).
Fig. 2. Visual table of contents.
68 Cyprien Verseux et al.
N fixed by cyanobacteria could also be used in hydroponic
and/or soil-based systems. It could come from two main
sources: from processed biomass containing for instance
amino acid chains, and from released NH
4
+
.
Within the MELiSSA loop, NH
4
+
is converted into nitrates
before being transferred to plants, but plants can also efficient-
ly uptake NH
4
+
(see, e.g., Howitt & Udvardi 2000). In theory,
plant growth rates can even be higher with NH
4
+
than with ni-
trates due, first, to the energy needed to reduce nitrates to NH
4
+
before its incorporation into organic compounds (Bloom et al.
1992) and, second, to a reduced need for photons, water and
catalytic metal use per unit of C fixed when relying on NH
4
+
(see Raven et al. 1992). However, experimental data often do
not support this hypothesis (e.g., Raven et al. 1992). NH
4
+
in-
duces toxicity at lower concentrations than nitrate, with a
threshold which varies widely among species (Britto &
Kronzucker 2002). As a consequence, preference for NH
4
+
over nitrate is species-dependent (Barker & Mills 1980; Zhou
et al. 2011). Even though NH
4
+
toxicity is not fully understood,
it can presumably be explained in large part by the effect of the
absorbed Ns form on the uptake of other ions: NH
4
+
leads to
higher anion uptake and lower cation uptake. It also affects
pH: nitrate and NH
4
+
absorption induce, respectively, a release
of hydroxyl ions (HO
) and a release of protons (H
+
), and for
some plants NH
4
+
s negative effects can be reversed by buffer-
ing the medium (Britto & Kronzucker 2002). Harmful effects
could consequently be mitigated by controlling pH and adjust-
ing the supply of other nutrients (Muhlestein et al. 1999), and
by keeping NH
4
+
concentrations below toxic levels. Further
studies could ascertain to what extent the growth-limiting ef-
fects of high NH
4
+
concentrations can be mitigated; more gen-
erally, work is needed to determine whether the benefits
brought by nitrifying bacteria would justify the complexity
and costs associated with their culture. Besides NH
4
+
, organic
N from cyanobacterium biomass can be considered as an
N source: plants can uptake amino acid chains (e.g., Lipson
& Näsholm 2001; Näsholm et al. 2009). Uptake of single
amino acids and short peptides, and possibly di- and tri-
peptides, occurs via membrane transporters in root cells
(Rentsch et al.2007). Some plants can also uptake proteins
after degradation by fungal symbionts (e.g., Bajwa et al.
1985) and at least some can do it without prior degradation
by other organisms, either directly (presumably by endocyto-
sis) or via the secretion of proteolytic enzymes (Godlewski &
Adamczyk 2007; Paungfoo-Lonhienne et al. 2008).
Symbioses between plants and diazotropic cyanobacteria nat-
urally occur. The best-known instances involve aquatic ferns
from the genus Azolla and their symbionts from the genus
Anabaena (Peters & Meeks 1989), and angiosperms from the
genus Gunnera and their symbionts from the genus Nostoc
(Bergman et al. 1992), but interactions naturally occur between
cyanobacteria and representatives of most plant groups.
Artificial symbioses between diazotrophic cyanobacteria and
plants which normally do not harbour cyanobacterial sym-
bionts, where the former provide fixed N to the latter, have
also been successfully created with various plants including cer-
eals (Gusev et al. 2002). In a hydroponic system, an Anabaena
variabilis strain has even been shown to provide fixed N to
wheat grown in an otherwise N-free medium, yielding plant
growth comparable with that of the control plants grown in a
nitrate-containing medium (Spiller & Gunasekaran 1990).
Assessments of the cyanobacterium-to-plant biomass conver-
sion efficiency, when all substrates besides CO
2
and water
come from cyanobacteria, require further experiments.
Even though experimental research is needed to quantify the
nutrient composition of culture supernatants (with and with-
out cell lysis), to determine the most efficient way of transfer-
ring nutrients from cyanobacterial cultures to other organisms
and to get quantitative estimates of the systems requirements,
cyanobacteria could thus be considered as a useful tool for pro-
cessing local inorganic resources into a form which is available
to other organisms (for a visual summary, see Fig. 3). But
cyanobacteria could also be used directly for various applica-
tions. The most critical are outlined below.
Producing food
If all food came from Earth and assuming the easiest option
of providing shelf-stable, prepackaged food similar to
International Space Stations provisions, the amount to be
sent would be about 1.8 kg day
1
crewmember
1
(Allen et al.
2003). Adding the needed vehicle and fuel weight to carry it
and assuming a 10:1 vehicle-to-payload ratio (Hoffman &
Kaplan 1997), 1-year food supply for a crew of 6 would add
more than 29 mt to the initial mass of the transit vehicle.
Worse, the load needed for a healthy diet would be much higher:
even though convenient due to reduced need for storage facil-
ities and contamination risks, a diet composed exclusively of
this type of food would be nutritionally incomplete and thus
not adequate in the long term. Frozen food could be seen as
an interesting complement but would be extremely demanding
in terms of storage facilities, could not be kept at commercial
temperatures for more than about 1 year without losing palat-
ability and would be unreliable due to potential freezing failure.
Thus, even though current space food systems are relevant for
short-term space missions and missions close to Earth (in low
Earth orbit and possibly on the Moon), they must be developed
further to meet the requirements of a manned mission to Mars
(Perchonok et al. 2012). Establishing long-term human settle-
ment on Mars seems unrealistic without local food production
systems.
Growing plants might appear as an obvious option, even
though creating an adequate environment on Mars would be
particularly challenging at the first steps of colonization (de-
tails are given in the Plants or cyanobacteria?section
below). However, cyanobacteria could be an interesting alter-
native. Some species are edible, can be grown at a large scale
and are expected (based on comparisons of metabolic path-
ways and human nutritional requirements) to be a nearly com-
plete nutritional source, lacking only vitamin C and possibly
essential oils (Way et al. 2011). Some species, notably from
the genus Arthrospira, have consequently been studied as a po-
tential food source in life-support systems (Hendrickx et al.
2006; Lehto et al. 2006). Arthrospira platensis is already used
on Earth as a food supplement, and its nutritional value and
Sustainable life support on Mars 69
lack of toxicity are well-characterized. Its dried biomass has
been categorized Generally Recognized As Safe(GRAS) for
human consumption by the Food and Drug Administration
(FDA) of the United States (FDA GRAS Notice No. GRN
000127). It has high protein contents (roughly, 5070% of the
dry weight) and a high productivity: cultures in outdoor
ponds under non-optimized conditions typically yield from 1
to 3 × 10
7
gdw ha
1
yr
1
(Jiménez et al. 2003) and produce
20 times more proteins per hectare than soyas (Hendrickx
et al. 2006; Henrikson 2009). Yields can be much enhanced
in more controlled conditions. Cultures in photobioreactors
can produce about 1 gdw l
1
day
1
and, with such yields and
assuming energetic contents of 3.75 kcal gdw
1
(Tokusoglu &
Unal 2003), 2000 kcal day
1
can be produced with about
0.53 m
3
. That being said, this figure is a very rough approxima-
tion of the actual needs: even though it covers mean caloric
needs for a 3050-year-old adult, it does not represent the
amounts needed to cover nutritional needs. On the other
hand, production rates can be further increased; for instance,
a productivity of 16.8 gdw m
2
h
1
(there corresponding to
1.2 gdw l
1
h
1
) has been attained for a short period of time
using high A. platensis cell densities, a very short light path
(14 mm), a very high photon flux (8000 µmol m
2
s
1
; about
four times the solar flux on Earth when the Sun is directly over-
head) and highly turbulent mixing (Qiang et al. 1998).
Arthrospira spp. have health-promoting properties, including
antiviral and antimutagenic functions which are especially rele-
vant in Marss highly irradiated environment (Lehto et al.
2007). They are also much more digestible than eukaryotic mi-
croalgae from the genus Chlorella, their main competitor as a
photosynthetic microorganism-based food source, which have
poorly digestible cellulose-containing cell walls.
However, Arthrospira spp. biomass currently cannot be used
as a staple food: in addition to a taste that very few people
would qualify as appealing, its carbohydrate-to-protein ratio
is low, it contains very high levels of vitamin A, and it lacks
vitamin C and possibly essential oils. Due to this nutritional
imbalance, it is generally not recommended to consume more
than 10 g day
1
. Cyanobacteria could be mixed with other mi-
croalgae and plants to optimize nutrient and fibre contents, as
well as to diversify taste and texture, but limitations could also
be addressed using synthetic biology (Way et al. 2011).
Modifying the sugar, protein and lipid proportions, as well as
introducing molecules that humans require (e.g., vitamin C)
could be achieved using metabolic engineering and other gen-
etic manipulations. Preliminary work has been done in this dir-
ection; for instance, mutant strains of A. platensis have been
selected that are richer than the wild-type in compounds in-
cluding essential amino acids, phycobiliproteins and carote-
noids (Brown 2008b). Novel taste, smell and colour
molecules could be produced by cells to increase palatability,
attractiveness and meal diversity. It should also be taken into
account that, depending on its adjusted pressure, Martian at-
mosphere can strongly affect the composition of cyanobacter-
ial cultures: A. cylindrica cells grown under low pressure and
high CO
2
concentrations showed decreased protein contents
Fig. 3. Using cyanobacteria to process Martian resources into substrates for other organisms. In this scheme, cyanobacteria are fed with various
nutrients obtained from the regolith, gaseous carbon and nitrogen from the atmosphere, energy from solar radiation, and water from various
possible sources including ice caps, subsurface ice, atmosphere and hydrated minerals. Additional organic material, CO
2
and water could be
provided from metabolic and manufacturing waste resulting from human activity. Products from cyanobacterial cultures are then used as a
substrate for heterotrophic microorganisms and plants.
70 Cyprien Verseux et al.
and increased sugar contents compared with cells grown under
ambient conditions (Lehto et al., unpublished data), shifting
the protein/carbohydrate ratio closer to humansneeds.
Research has been extensively focused on Arthrospira spp.,
but other cyanobacterial species such as Nostoc commune,
Nostoc flagelliforme and Anabaena spp. in symbiosis with
Azolla spp. are traditionally consumed and more are edible.
Arthrospira cultures require extensive nutrient supply (includ-
ing fixed N), a high temperature with an optimum around 35°C
and an alkaline pH. Other species which are less demanding in
terms of culture conditions might thus be more suitable on
Mars and, even within the genus Arthrospira, species could
be screened for the highest productivity under on-site
constraints.
Cyanobacteria could also be used for food complementation
without being directly eaten or even inactivated, as they can be
engineered to secrete nutritional compounds (Way et al. 2011).
As mentioned above, the possibility of engineering them to
produce and secrete sugars has already been demonstrated
(Niederholtmeyer et al. 2010; Stanford-Brown 2011 iGEM
team 2011).
Finally, high-protein food could be suggested to come from
animals. Granted, it is unrealistic to envision growing large
species on Mars during its earliest colonization steps, due for
instance to the need for an Earth-like environment, to low
protein yields-to-allocated resources (area, water, vegetal-
originated proteins, working time, etc.) ratios and to the risk
of pathogen transmission to humans. However, aquaculture
of densely growing fish species (e.g., Tilapia spp.), crustaceans
and shellfish may be considered (McKay et al. 1993) and
cyanobacteria could be used for feeding them. Cyanobacteria
are already used on Earth as a main food source for larvae of
many species of fish, zooplankton (itself used for feeding fish
larvae), crustaceans and shellfish (Pulz & Gross 2004).
Producing oxygen
O
2
will be one of the most critical resources in human bases on
Mars, the most obvious reasons being human respiration and
fuel oxidation. But it represents only 0.13% of Marss atmos-
phere, against 21% of Earths. Given the total pressures
of both atmospheres, its partial pressure on Mars is more
than 20 000 times lower than on Earth. O
2
thus needs to be ei-
ther brought to Mars or produced there and, as for other re-
sources, the second option is likely the most viable in the
long term. On-site O
2
production could be performed using
physicochemical methods: by processing regolith, water and/
or atmospheric CO
2
(e.g., by combining a CO
2
scrubber, a
Sabatier reactor and an electrolyser). However, cyanobacter-
ium-based O
2
production from H
2
O and CO
2
could provide
a safe redundancy (Sychev et al. 2003) and complement
physicochemical methods. It could also be less energy demand-
ing, and faster to set up and expand to face unexpected needs.
How much O
2
is needed? Each crewmember consumes
about 1 kg of O
2
per day for respiration, assuming 2 h a day
of intensive physical exercise (Horneck et al. 2003). If produced
photosynthetically, this requires the fixation of more than 1.3
kg of CO
2
a day, part of which could be provided, if useful, by
recycling CO
2
produced by the crews metabolic activity (about
1 kg day
1
crewmember
1
) and manufacturing activity in add-
ition to using atmospheric CO
2
.
Cyanobacteria are very efficient O
2
producers: whereas trees
release about 2.511 t of O
2
ha
1
yr
1
, industrial cultivation in
open ponds of Arthrospira species in Southeastern California
release about 16.8 t of O
2
ha
1
yr
1
. Cultures have been stated
to be about 2.5 times more productive in the tropics
(Henrikson 2009); under these conditions, about 80 m
2
of cul-
ture per crewmember would be needed to cover human respir-
ation needs. However, O
2
production rates can be dramatically
increased by photobioreactor-like culture systems which opti-
mize for instance temperature, nutrient flow rates, cell density
and illumination. Photosynthetic microorganisms from a eu-
karyotic microalgal genus, Chlorella, have been well-studied
in the context of O
2
production for life-support systems. For
instance, an experimental system (BIOS-I) was designed
and tested in the 1960s where a man living in a sealed volume
had his atmosphere and water regenerated by a Chlorella
vulgaris culture. This work was preceded by experiments
aimed at investigating the potential use of Chlorella spp.
for air regeneration, performed by Y. Y. Shepelev and
G. I. Meleshko at the Institute of Aerospace Medicine,
Moscow, in 19601961 (Salisbury et al. 1997). In BIOS-I,
CO
2
excreted by the man and biogenous elements from his
urine (N, P, S and K) were fed into the algal culture, which
in turn produced O
2
and purified water. Investigators showed
that producing 500 g of dry algal biomass per person and per
day was enough for water and air generation, and could be
achieved in 20 litres only (with a 0.5 cm thick cultivation com-
partment between 8 m
2
plane parallel walls to maximize light
absorption), using one-side illumination with photosynthetic-
ally active radiation at 250300 W m
2
(Kirensky et al. 1968;
Gitelson 1992). The system was further developed, plants were
added and various manned closure experiments were per-
formed within BIOS-2and BIOS-3, where Chlorella sp.
was used to recycle part of the air (even though the Chlorella
compartment was later replaced with additional plant com-
partments, mainly for food-related issues; see Salisbury et al.
1997).
Productivity will depend on conditions which will be pro-
vided on site. It should also be noted that the needed resources
do not need to be dedicated to O
2
production, as O
2
will any-
way be a side product of other processes relying on cyanobac-
teria and could be coupled with, for instance, food production.
Producing biofuels
If sending it from Earth, fuel would represent most of the loads
mass to be sent from Earth and (for the journey back, assuming
a return mission) from Mars. If it could be produced on-site,
costs and technical challenges would be much reduced.
The next question concerns fuel-type. Dihydrogen (H
2
)canbe
used for reducing local CO
2
to hydrocarbons, and it has been
proposed to bring some to Mars to produce methane (CH
4
)
and H
2
O (the latter can then be hydrolysed into O
2
and H
2
).
H
2
could theoretically be directly used as a fuel: a mixture of li-
quid H
2
and O
2
have suitable propulsion performances. It is
Sustainable life support on Mars 71
however much less attractive than CH
4
,amongotherreasons
because of its very low density which induces a need for specia-
lized combustion apparatus. In the Mars Direct plan (Baker &
Zubrin 1990; Zubrin et al. 1991; Zubrin & Wagner 2011), 6 t of
H
2
would be used to generate enough CH
4
and O
2
to bring the
return vehicle back to Earth and to power ground vehicles on the
Martian surface. Mass could be further reduced if H
2
was also
obtained on Mars. Many cyanobacteria can produce it; among
them, N-fixing heterocystous species such as Nostoc and
Anabaena spp. are the most promising candidates. Their H
2
pro-
duction activity comes from their nitrogenases. These enzymes
mainly catalyse the reduction of N
2
to NH
4
+
but also, in the ab-
senceofN,reduceH
+
to H
2
. On Earth, though, H
2
photopro-
duction is currently too low for it to have practical applications.
As an example, A. cylindrica grown in a standard medium for
cyanobacteria (BG11), at ambientair composition and pressure,
yields only about 0.2 µmol H
2
mg Chl a
1
h
1
(Murukesan et al.
2015). One of its main limitations isthe inhibition by atmospher-
ic O
2
and N
2
: nitrogenases are irreversibly inactivated by O
2
,
while N
2
strongly inhibits H
+
reduction. The high CO
2
,low
O
2
and N
2
composition of the Martian atmosphere could thus
be an advantage here. Consistently, yields above 20 ml l
1
h
1
(about 0.8 mmol l
1
h
1
) were obtained with concentrated cul-
tures of about 50 mg Chl a l
1
of A. variabilis (so, roughly, 16
µmol H
2
mg Chl a
1
h
1
) under a 99% argon (Ar), 1% CO
2
at-
mosphere (Liu et al. 2006). Recently, levels of about 25 µmol mg
Chl a
1
h
1
were also reached with A. cylindrica cultures under
high CO
2
/low N conditions (Murukesan et al. 2015). With a
concentration of 50 mg Chl a ml
1
, these rates could lead to
1.5 mmol l
1
h
1
(about 35 ml l
1
h
1
at room temperature,
with 30 µmol mg Chl a
1
h
1
)ofH
2
. Producing 6 t of H
2
would then require about 228 m
3
yr. It corresponds, for ex-
ample, to 22 days in a culture system the size of 3 m-deep
Olympic swimming pool, or to 2 years and 4 months in
100 m
3
, assuming yields can be maintained at this scale. But
these figures are very rough approximations: rates that would
effectively be obtained on Mars depend on the mission scenario
and technology choices for culture systems. Finally, current
productivity is still far from its maximum, and increases are
regularly obtained by culture conditionsoptimization and
metabolic engineering.
Leaving H
2
aside, cyanobacteria can generate various
biofuel precursors and components including alkanes, ethyl-
ene, hydrogen peroxide (H
2
O
2
; which can be used as a mono-
propellant) and lipids, without relying on organic precursors
(see, for instance, Quintana et al. 2011). They could also pro-
vide organic substrates for production of biofuel precursors by
other organisms, particularly relevant on Mars where import-
ing substrates from Earth would be impractical. For instance,
as mentioned above, lysed cyanobacterial biomass has been
used as a fermentation substrate for ethanol production in
yeasts (Aikawa et al. 2013; Möllers et al. 2014). However, gen-
etic engineering could remove the need for other organisms
even for production of biofuel precursors which are not
naturally produced by cyanobacteria in adequately large
amounts if at all. For instance, some cyanobacteria have
been engineered to produce ethanol. Whereas yeasts rely on a
sugar-based pathway and thus on the availability of agricul-
tural substrates, engineered cyanobacteria produced it directly
from CO
2
and solar energy, following a much simpler process
where pyruvate is first converted to acetaldehyde bya pyruvate
decarboxylase and then to alcohol by an alcohol dehydrogen-
ase (Deng & Coleman 1999; Dalton & Roberto 2008; Dexter &
Fu 2009). Generally speaking, a lot of work has been done to
engineer new biofuel (or biofuel precursor) production path-
ways or to improve existing ones in cyanobacteria (see,
e.g., Ducat et al. 2011). There is still much room for improve-
ment, especially under Mars-like conditions.
Thus, even though extensive development and optimization
is needed, cyanobacterium-based biotechnologies could re-
present original contributions to the production of rocket
fuel for the return flight, for powering surface vehicles and,
more generally, for powering equipment that can be operated
by combustion engines.
Other applications
Various other applications involving cyanobacteria have been
proposed for human outposts on Mars. One, for instance, is
biomining. Microorganisms are used on Earth to extract me-
tals of industrial interest (e.g., copper and gold) from rocks,
and their use on Mars to mine basalt and potential ores has
been suggested (Cockell 2010,2011; Navarrete et al.2012).
Cyanobacteria are known to leach a wide range of rock
types, including terrestrial volcanic rocks with compositions
similar to Marss regolith (Brown & Sarkisova 2008;
Olsson-Francis & Cockell 2010; Olsson-Francis et al. 2012).
The use of cyanobacteria, possibly engineered to increase
their ability to dissolve rocks and harvest specific elements
(Cockell 2011), can thus be considered for simple bioleaching
processes that would not rely on imported C sources (needed
for heterotrophic microorganisms) or toxic elements such
as cyanide (used in non-biological leaching processes).
Extracted elements could be used within a wide range of chem-
ical and manufacturing processes such as, for instance, CO
2
cracking, electroplating, production of alloys and manufactur-
ing of solar cells (Dalton & Roberto 2008; Cockell 2011).
Cyanobacteria have also been suggested for controlling
Marss surface dust, through the production of biological crusts,
in enclosed structures such as greenhouses and habitats (Liu
et al. 2008). Indeed, some can grow and form biofilms within
the interstices of desert minerals and produce extracellular poly-
saccharides that bind the particles together, preventing wind-
induced dust release. Such crusts could also be used as anair fil-
ter to remove dust from the atmosphere (Cockell 2010). Then,
the use of dehydrated Chroococcidiopsis cells as a gene reposi-
tory for on-site molecular biology has been suggested (Billi
et al. 2013), as they can preserve plasmids during long-term des-
iccation (Billi 2012) and presumably repair them when da-
maged. Cyanobacteria could also be used to process human
waste products and recycle their organic C, water, nitrates and
mineral nutrients. Cultures could be used directly (Filali et al.
1997; Godia et al. 2002;Lehtoet al. 2006;Yanget al. 2008)
but also indirectly; for instance, H
2
O
2
generated from
cyanobacterium-produced O
2
and H
2
O could be used to oxidize
72 Cyprien Verseux et al.
human wastes following a physicochemical process developed
by researchers of the Institute of Biophysics of the Siberian
Branch of the Russian Academy of Sciences (Kudenko et al.
2000); nutrients could then be recycled in cyanobacterial
cultures (Tikhomirov et al. 2007). Cyanobacteria have also
been suggested for the production, beyond Earth, of various
chemicals including nutritional molecules, drugs, bioplastics
and cellulosic building materials (Way et al. 2011;Menezes
et al. 2014).
Finally, the ability of cyanobacteria to produce organic ma-
terial from Martian resources, coupled to our increasing abil-
ities in metabolic engineering, make it possible to consider
many other applications ranging from performing basic life-
support functions to generating comfort products.
Plants or cyanobacteria?
Assuming that both plants and cyanobacteria can be grown on
Mars, some functions such as food and O
2
production could be
performed by either or both of them. For these functions,
plants are the most commonly proposed photosynthetic organ-
isms. This choice, however, is mostly due to our historical reli-
ance on and experience with them: they have been a highly
available food source throughout most of human history.
Matters need to be reconsidered where environmental condi-
tions, resources and other constraints are different.
Photosynthetic microorganisms are more efficient, on a vol-
ume basis, at capturing solar energy than plants. Their culture
in photobioreactors could yield much more biomass (especially
proteins) and O
2
for a given volume and light intensity than
greenhouse-type cultures of staple edible plants (Dismukes
et al. 2008;Wayet al. 2011;Wanget al. 2012). This is critical
where resources are scarce and cultivation areas are highly con-
trolled. Moreover, a considerable part of plant biomass (e.g.,
roots or stems, depending on species) is inedible and hard to
recycle: plant cell walls are among the least degradable poly-
mers in BLSS (Hendrickx & Mergeay 2007).
Plants are also much more demanding in terms of environ-
mental conditions. For instance, they are harmed by anoxia
(roughly, a partial pressure of O
2
of at least 50 hPa is needed
for proper development, mainly for non-photosynthetic tis-
sues; see Thomas et al. 2005) and high concentrations of CO
2
(negative effects have been observed in some plants above
4 hPa [Wheeler 2004], while the partial pressure of CO
2
[pCO
2
] on Mars is above 6 hPa). Photosynthetic cultures on
Mars, be they vegetal or bacterial, will have to be protected
from biocidal environmental conditions. The needed level of
protection and the associated costs will depend on the abil-
ity of cultured organisms to withstand these conditions, and
many cyanobacteria are much more resistant than staple plants
to the Martian surfaces environmental stressors. They thrive
in the most extreme habitable conditions on Earth, such as in
dry deserts and ice lakes of Antarctica, and within ices of high
Arctic seas (Wierzchos et al. 2006; Scalzi et al. 2012). Some
have an outstanding resistance to environmental factors occur-
ring on Marss surfaces, including ultraviolet (UV) and ioniz-
ing radiation (see, for instance, Billi et al. 2013 and Thomas
et al. 2006) and can survive in space when protected from
UV radiation (Olsson-Francis et al. 2010).
In habitable compartments, gas consumed and produced by
crewmembers must be balanced with gas consumed and pro-
duced by an atmosphere regenerating system. Since CO
2
will
be available in the atmosphere and since both CO
2
and O
2
will be generated as by-products of other processes, this system
could be more flexible on Mars than in places where simple
closed systems would be used: losses could be compensated
and excess vented out. However, extensive control of the
atmospheric balance would both increase safety and reduce re-
source consumption. By adjusting culture conditions, control-
ling culturesassimilation quotient (CO
2
consumed/O
2
produced) to match humansrespiratory quotient (CO
2
pro-
duced/O
2
consumed) is much easier with cyanobacteria than
with plants, making the atmospheric O
2
/CO
2
balance much
more manageable with the former (Averner et al. 1984;
Horneck et al. 2003).
As crewmembers should be available for research and col-
ony settlement and maintenance, time spent on culture man-
agement will have to be kept to a minimum. Culturing plants
requires significant manpower; within Biosphere 2, for in-
stance, agricultural and food-related tasks took about 45% of
crew time (Silverstone & Nelson 1996). In this framework,
automation should be extensive. Cyanobacterial cultures are
much more suitable for automation than plantsdue to their
culture homogeneity, growth in liquid medium and lack of in-
edible parts that should be sorted out. They are also much more
manageable; culture parameters could be more easily adjusted
to cover human needs, with a safety margin but without excess.
Outputs could thus be much more controllable and
predictable.
The time needed to establish cultures also matters. Even
though shelf-stable food can be sent to sustain crews before cul-
tures are first set up, it is important to be able to re-establish
cultures in case of accidental loss likely enough, and some-
thing with catastrophic consequences if not rapidly fixed.
Being able to quickly extend cultures to cover unexpected
needs (e.g., to compensate an O
2
loss) can also be critical.
Even though some plants can be grown faster, staple crops
can take 34 months to mature even under favourable condi-
tions (Drysdale et al. 2003). On the other hand, microbial cul-
tures can be quickly expanded and re-deployed from very small
amounts.
Finally, cyanobacteria are much easier to engineer than
plants due to their rapid division times, compatibility to trans-
formation, unicellularity and relatively simple genetic back-
ground (Koksharova & Wolk 2002;Wayet al. 2011; Berla
et al. 2013). They could therefore be much more easily modi-
fied for new functions and adaptation to Martian conditions
(see section Engineering cyanobacteriabelow).
It should however be noted that plants have some advan-
tages over cyanobacteria: they could provide tasty and
carbohydrate-rich comfort food, and have beneficial psycho-
logical impacts on crewmembers (Allen 1991). Establishing
small-scale cultures is not obviously unrealistic, especially if
nutrients are provided from local resources as described above.
Sustainable life support on Mars 73
Besides plants, one may wonder why the present paper fo-
cuses on cyanobacteria rather than eukaryotic microalgae,
which could also perform some of the functions described
above. Reasons include cyanobacterias overall better abilities
to use Martian resources (e.g., by N fixation and regolith
leaching) and to withstand Martian conditions, the higher di-
gestibility of their edible species and their higher growth and
photosynthetic rates. They are also more suitable for genetic
engineering, in part due to current transformation systems
which are much simpler and well developed for cyanobacteria
than for eukaryotic microalgae (Wang et al. 2012; Wijffels
et al. 2013).
What about non-photosynthetic (chemotrophic) micro-
organisms? Heterotrophs may be useful on Mars, but not as
primary producers (see subsection Feeding other micro-
organisms). Resources needed to feed the metabolism of
some chemoautotrophs, on the other hand, may be found on
Mars; for example, reduced iron could be used as an energy
source by iron-oxidizing bacteria (Nixon et al. 2013).
Chemoautotrophs could thus be considered for some applica-
tions, such as the extraction of industrially useful minerals
(Cockell 2010,2011). However, none has the versatility of
cyanobacteria to be the basis for BLSS: none combines, for in-
stance, high N fixation rates, high growth rates, ability to rely
exclusively on Martian resources, O
2
production, H
2
produc-
tion, amenability to genetic manipulation and edible biomass.
Growing cyanobacteria on Mars
All organisms we currently know have evolved on Earth and
none of them would be able to grow efficiently on the
Martian surface. Cyanobacteria must be provided with shield-
ing and an environment suitable for metabolism and growth.
Elaborated hardware systems providing Earth-like conditions
have been proposed but they rely on complex technology and
require accurate control of all the process parameters (e.g.,
gases, temperatures and pressures in each compartment), are
very demanding in terms of construction materials and energy
consumption, need to be constructed on Earth, are very mas-
sive and expensive to carry to Mars, and can consequently be
applied to small-scale cultures only. In order to be cost-
effective and reliable, culture hardware for large-scale, long-
term, sustainable BLSS on Mars should be much simpler.
Fortunately, reproducing Earth-like conditions is not
needed: cyanobacteria can grow under conditions which are
much closer to Marss. In addition, most inputs if not all
needed for growing cyanobacteria can be found on-site. An ad-
equate culture system could thus provide a set of parameters
(radiation shielding, atmospheric composition and pressure,
gravity, nutrient supply, etc.) resulting from a compromise be-
tween (i) efficient support to growth and metabolism, and (ii)
system feasibility and substrate availability on Marss surface.
This culture system should be able to resist an inside/outside
pressure difference, fine dust, large temperature gradients and
strong radiation fluxes. Whatever its final design, efforts
should be made to keep its weight, cost and energy consump-
tion as low as reasonably possible given the other
requirements. Ideally, the design should allow manufacturing
from on-site compounds (e.g., regolith-based materials for ra-
diation shielding, glass manufactured using silicon dioxide
from Martian soil, and metallic parts derived from metal oxi-
des mined in the regolith), assuming that equipment needed for
processing is available on site. The potential of ultimately cre-
ating many of these facilities with local resources is currently
being explored in Lynn Rothschilds laboratory.
Nutrient sources
Most elements needed for feeding plants and microorganisms
can be found in Martian regolith.
Data on the composition of Martian soils and rocks have
been obtained from analyses of the SNC (Shergottites,
Nakhlites, Chassignites) group of meteorites (McSween
1994), the Viking (Clark et al. 1982), Pathfinder (Rieder
1997) and Phoenix (Hecht et al. 2009) landers, the Spirit
rover (Morris et al. 2004), instruments operated from orbiters
noteworthy the Thermal Emission Spectrometer on Mars
Global Surveyor (see Christensen et al. 2001), the Gamma
Ray Spectrometer on Mars Odyssey (Boynton et al. 2007)
and the Compact Reconnaissance Imaging Spectrometer for
Mars (CRISM) on the Mars Reconnaissance Orbiter
(Mustard et al. 2008)and, recently, using X-ray spectro-
meters aboard the Opportunity and Curiosity rovers. In par-
ticular, the latter two have allowed detailed mineral
compositions to be deduced at multiple sites in the
Endeavour crater (Arvidson et al. 2014) and Gale crater
(Grotzinger et al. 2014; McLennan et al. 2014; Ming et al.
2014; Vaniman et al. 2014). On Earth, basalt is the dominant
rock type on the surface. It harbours much of the biosphere
and, as an abundant source of redox couples and macronutri-
ents, provides an efficient support to microbial life ( for a review
in the context of the search for life on Mars, see McMahon
et al. 2013). Martian regolith is also in large part composed
of basaltic minerals; more generally, Marss surface seems to
be mostly basaltic (McSween et al. 2009; Taylor &
McLennan 2009). All basic elements needed for cyanobacteria
and other organisms (C, H, O, N, P, S, K, Mg, Na, Ca, Fe), as
well as other elements needed in smaller amounts (Mn, Cr, Ni,
Mo, Cu, Zn, etc.), have been detected there.
The most convenient sources of C and N will probably be at-
mospheric CO
2
and N
2
(see Atmospheric pressure and compos-
itionsection below). Additional C can be found in the CO
2
ice
caps, in the surface andsubsurfaceregolith due to exchange with
the atmosphere, and possibly in reservoirs formed when the at-
mosphere was thicker (Kurahashi-Nakamura & Tajika 2006). It
has also been suggested that fixed N, derived from Marssat-
mospheric N
2
, may be buried in the regolith (Mancinelli &
Banin 2003;Boxeet al. 2012). Consistent with this, N-bearing
compounds have been detected there (Ming et al. 2014).
However, the exact nature and bioavailability of these com-
pounds has not yet been determined.
Thus, all elements needed to support life seem to be present
in Marss rocks (Cockell 2014) and atmosphere. These nutri-
ents can be directly made available to cyanobacteria, as mul-
tiple species thrive in a lithotrophic lifestyle, extracting all
74 Cyprien Verseux et al.
their needed mineral nutrients from rocky substrates (including
basalt) and obtaining their whole N and C supply via photo-
synthesis and biological N fixation. Accordingly, some strains
(e.g., A. cylindrica) have been grown in distilled water contain-
ing only powdered Mars basalt analogues (experiments were
performed under terrestrial atmosphere; considerations on
what atmospheric conditions are suitable are given in the
Atmospheric pressure and compositionsection below).
Non-N fixing cyanobacteria could also grow when NaNO
2
was added. On Mars, fixed N could come from N fixers (see
above sections), while the possibility that nitrate beds are pre-
sent cannot be ruled out. Supplementing the media with a sul-
phate source, (NH
4
)
2
SO
4
, had a positive impact on some of the
tested species. The authors suggested that, on Mars, gypsum
(NaSO
4
·2H
2
O) could be used as such a supplement
(Olsson-Francis & Cockell 2010). Gypsum dunes have indeed
been found in the northern polar region of Mars by the
OMEGA instrument on ESAs Mars Express orbiter
(Langevin et al. 2005) and the CRISM and High Resolution
Imaging Science Experiment instruments on NASAs Mars
Reconnaissance Orbiter (Roach et al. 2007), and later con-
firmed by the rover Opportunity (Squyres et al. 2012). Other
studies showed that the growth of several siderophilic cyano-
bacterial species isolated from iron-depositing hot springs in
Yellowstone National Park was stimulated by the presence of
Martian soil analogues in culture media (Brown & Sarkisova
2008) and that Nostoc sp. HK-01 could grow on a Mars rego-
lith stimulant for at least 140 days, without other nutrient
source besides atmospheric gas (Arai et al. 2008). Other cyano-
bacteria have been grown using other Martian soil analogues
as substrates, in distilled water or spread on gelified water
plates. DLRs P-MRS and S-MRS simulants (Böttger et al.
2012), and NASA JSCs Mars-1A simulant (Allen et al.
1997), for instance, efficiently supported the growth of
Matteia sp. and Anabaena sp. PCC7120 (see Fig. 4), respec-
tively (Verseux et al., unpublished data). The need for provid-
ing regolith might be an issue for automation. However, using
drilling photobioreactors that extract raw materials from the
surface and directly bring them to cultures has been suggested
(Cumbers & Rothschild 2010) and technologies have been de-
signed to excavate large amounts of regolith with minimal
weight and time (see, e.g., Mueller & Van Susante 2011).
Thus, even though it might be relevant to adequately mix
rock types to have all nutrients in appropriate proportions
and suitable pH, and even though some salts, oxides and toxins
might need to be removed, all nutrients and micronutrients
needed to support cyanobacterial metabolism seem to be pre-
sent on Mars. Additional nutrients could come from human
waste. If some micronutrients (e.g., some cofactors) could
not be mined or produced on site, bringing them from Earth
would add a negligible mass to the initial payload as they are
needed in trace amounts only. Methods for physicochemical
preprocessing of Martian regolith and atmosphere (to gener-
ate, for instance, a broth of pre-leached regolith and nitric
acid in which CO
2
is bubbled; see Cao et al. 2014) could be con-
sidered, if the increased productivity outweighed the increased
running cost and complexity.
Atmospheric pressure and composition
Use of the minimal suitable pressure would greatly lower con-
struction weight and cost of cyanobaterial culture systems on
Mars, and would minimize the risk of organic matter leakage
(Lehto et al. 2006). Relying on a gas composition which is as
close as possible to Marss would make the establishment of
such systems even simpler and cost-effective.
What is this minimal suitable pressure? No clear answer has
been given to this question, as little work has been focused on
microbial growth at low pressure. The lowest pressure at which
biological niches are naturally present on Earth is about
330 hPa (at the top of the Mount Everest), way above Marss
surface pressure of about 511 hPa (Fajardo-Cavazos et al.
2012). Although viable bacteria have been sampled a few
times from stratospheric air above 30 km (e.g., Wainwright
et al. 2003), where atmospheric pressure goes down to Marss
surface pressure, no microbial growth there was indisputably
evidenced. Some methanogens can keep metabolic activity
(shown by detectable CH
4
production) at 50 hPa of pressure
under simulated Martian environmental conditions (Kral
et al. 2011; Schirmack et al. 2014), and a few bacteria such as
Serratia liquefaciens (Schuerger et al. 2013) and isolates from
Siberian permafrost samples (Nicholson et al. 2013) have
been grown under 7 hPa of CO
2
-enriched anoxic atmospheres,
but a wide range of microorganisms have been shownto be un-
able to grow on a semisolid agar medium at pressures below25
hPa of ambient air (Nicholson et al. 2010).
It might be possible to decrease the lowest suitable pressure.
As no biological niche is naturally present on Earth at pres-
sures close to Marss, selective pressure is virtually non-existent
for current terrestrial microorganisms and the full potential for
Fig. 4. Anabaena sp. PCC7120 growing in distilled water containing
JSC Mars-1A regolith simulant, in Lynn Rothschilds laboratory.
Sustainable life support on Mars 75
growth at low pressures is probably far from being reached.
There might thus be much room for improvement by artifi-
cially evolving cyanobacteria to grow faster under low (and
to grow at lower) pressures, including low pressures of
Mars-like gas compositions. Consistently, an isolate of
B. subtilis showed increased fitness at 50 hPa after a 1000 gen-
eration culture at this pressure (Nicholson et al. 2010). The
minimal suitable pressure can also be dependent on atmos-
pheric composition, as described below, and on medium type
(liquid or solid). It should however be noted that a physical
limitation derives from the need to maintain a liquid phase at
growth-permissive temperatures.
In recent experiments, a decreased atmospheric pressure
(50 kPa instead of the ambient 100 kPa) negatively affected
the growth of cyanobacteria from several genera (Qin et al.
2014). But this was performed under ambient gas composition
and, like atmospheric pressure, atmospheric composition mat-
ters. CO
2
and N
2
are present in Marss atmosphere but their
partial pressures differ from Earths (see Table 1). Marss
higher-than-Earth pCO
2
(6.67 versus 0.38 hPa) might actually
be beneficial: elevated levels of CO
2
can have a fertilizing effect
on cyanobacterial cultures (Murukesan et al. 2015). Below one
atmosphere of pressure, CO
2
seems to become the limiting fac-
tor and cyanobacteria benefit from much higher-than-normal
CO
2
concentrations; Synechocystis sp. PCC 6803 was shown to
grow more than three times faster under 100 hPa with 5% CO
2
(as well as under 1 bar with 5% CO
2
) than under 1 bar of am-
bient air (0.04% CO
2
), and to grow under 33 hPa of 100% CO
2
with growth rates close to those obtained under 1 bar of ambi-
ent air (Lehto et al. 2007; see the culture system in Fig. 5). Later
results showed that an increase in CO
2
concentration (at least
up to 20%, even though growth rates started to decrease after
10% for the 1-bar samples) under either 1 bar or 100 hPa leads
to higher growth rates than ambient air composition at the cor-
responding pressure (Murukesan et al. 2015; unpublished
data). Thus, this strain seems to benefit from higher-than-usual
pCO
2
, with a saturation about 4 hPa, at which level an around
3.5-fold increase in growth rates is observed for cells previously
grown in ambient terrestrial atmosphere. When CO
2
is not lim-
iting, its growth rates are not negatively affected by a 10-fold
reduction in atmospheric pressure. Similarly, cultures of A. pla-
tensis and A. cylindrica were shown to benefit from
higher-than-ambient CO
2
concentrations (Murukesan et al.
2015). Finally, at least some cyanobacteria are able to survive
(Synechococcus PCC7942, Anabaena sp.) and even to grow
(Plectonema boryanum) in liquid culture under 1 bar (1000
hPa) of pure CO
2
, at least in the short term, when pCO
2
is grad-
ually increased by 150 hPa day
1
(Thomas et al. 2005). If
needed, bacterial resistance to high CO
2
levels could probably
be further increased by preventing pH decrease in the medium,
which happens due to carbonic acid (H
2
CO
3
) formation when
CO
2
dissolves in water.
The most striking shortage of substrates on Mars derives
from the low partial pressure of N
2
(pN
2
) in the atmosphere
and the presumed low availability of N in the regolith. Some
work performed with Azotobacter vinelandii and Azomonas
agilis showed that microbial N fixation is possible at a pN
2
of 5 hPa, even though below 400 hPa growth rates decreased
with decreasing pN
2
(Klingler et al. 1989). The lower limit
for N fixation might vary among species and is still to be
defined, but it has been proposed to be within the range of
110 hPa (McKay & Marinova 2001). Crossing this limit
using Marss air composition would require at least 550
times the local ambient pressure (reaching a total pressure of
approximately 40 hPa to 40 kPa). However, pN
2
would still
be limiting and higher values are needed for efficient processes.
Rather than only increasing the total pressure to reach ad-
equate pN
2
values, N
2
could be concentrated by separating
CO
2
from the other atmospheric gases (mainly N
2
and Ar)
and mixing them in different proportions to reach an appropri-
ate pN
2
value in an otherwise optimized total pressure. Gas
separation techniques are well developed (see, for instance,
Meyer & McKay 1996 and Zubrin & Wagner 2011) and
could be based on processes routinely used by industry on
Earth. Assuming similar pN
2
needs for cyanobacteria as for
A. vinelandii and A. agilis (see Klingler et al. 1989), a pN
2
of
95 hPa would only slightly limit growth (experiments are
planned to define the lowest pN
2
allowing efficient growth of
diazotrophic cyanobacteria). For wild-type, diazotrophic
cyanobacteria, a 100 hPa atmosphere with 95% N
2
and non-
limiting (5%) CO
2
could thus lead to higher growth rates
than an Earth-like atmosphere. Cultures could also be supple-
mented with N recycled from human and biomass waste, even
though this should not be the only N source as sustainability
and expandability would be compromised.
Depending on its adjusted pressure, a Martian-like atmos-
phere can strongly affect other aspects of the lithotrophic
growth of cyanobacteria through indirect effects (e.g., a low-
ered pH due to H
2
CO
3
formation can influence both cell via-
bility and nutrient release from substrates). As forall processes
suggested here, extensive and faithful simulations of culture
conditions expected to be provided on-site are needed.
Atmosphere of selected gas composition and pressure
could be provided within inflatable, tunnel-like containments
(Lehto et al. 2006). Tight sealing should allow the desired
pressure to be maintained, and adjusted gas supply systems
could allow CO
2
and some N
2
to be provided from Martian
Table 1. Environmental parameters on Mars and Earth surfaces
(adapted from Graham [2004] and Kanervo et al. [2005])
Parameter Mars Earth
Surface gravity 0.38 g 1.00 g
Mean surface temperature 60°C +15°C
Surface temperature range 145 to +20°C 90 to +60°C
Mean PAR photon flux 8.6 × 10
19
photons
m
2
s
1
2.0 × 10
20
photons
m
2
s
1
UV radiationspectral range >190 nm >300 nm
Atmospheric pressure 511 hPa 1013 hPa (mean at
sea level)
Atmospheric composition (average)
N
2
0.189 hPa, 2.7% 780 hPa, 78%
O
2
0.009 hPa, 0.13% 210 hPa, 21%
CO
2
6.67 hPa, 95.3% 0.38 hPa, 0.038%
Ar 0.112 hPa, 1.6% 10.13 hPa, 1%
76 Cyprien Verseux et al.
atmosphere possibly after N enrichment. An onion-like struc-
ture, with a pressure gradient throughout layers, has also been
proposed. This would offer the additional advantages of a bet-
ter filtering of the Martian dust and a better thermal insulation
(Lehto et al. 2006).
Water
One of the most critical resources to provide to cyanobacterial
cultures will be water. As water is needed within human out-
posts, regardless of the use of cyanobacteria, various hardware
systems for its extraction and processing on Mars have been
suggested elsewhere. There follow a few examples of water
sources and mining techniques under consideration. This list
is not exhaustive.
Water could be generated on Mars by importing H
2
from
Earth and combining it to O from local CO
2
(Zubrin et al.
1991) but, even though H represents only 11% by weight of
pure H
2
O, relying on imported H
2
would strongly limit the au-
tonomy and sustainability of human outposts. However, water
can be found in various forms throughout Mars (reviewed in
Tokano 2005; Rapp 2007 and Cockell 2014). It is present in
large amounts as ice at the north polar ice cap and under the
south carbon ice cap, and throughout the planet as near-
surface deposits of water ice. The presence of liquid water
has to date not been unquestionably established, but several
lines of evidence strongly suggest transient liquid brines (Zisk
& Mouginis-Mark 1980; McEwen et al. 2011; Martín-Torres
et al. 2015). It might also exist below the cryosphere, in areas
where temperatures and pressures are high enough (e.g.,
Clifford et al. 2010), possibly within drilling range. The abun-
dance, location and nature of near-surface water deposits is still
to be accurately determined, but mining these to generate us-
able water may be practical (e.g., Rapp 2013). If it appears
that mining liquid water or large ice deposits cannot be done,
water could be extracted from the soils hydrated minerals.
Energy requirements for heating soil at 500°C in an oven,
before collecting steam, have been assessed to be about
5.2 kWh kg
1
of water in a 2%-water soil, plus a small amount
(below 0.1 kWh kg
1
of water) for excavating and conveying
the soil (Stoker et al. 1993). Other processes where soil is heated
for water extraction are under consideration, including the use
of focused light and of microwaves, the latter being one of the
most promising regarding energy requirements, mass and
reliability (Wiens et al. 2001; Ethridge & Kaulker 2012). A
team of the Colorado School of Mines designed a system, re-
ferred to as the Microwave Pizza Oven, aimed at extracting
water from the soil of Mars using microwaves (generated
using about 12 kWh kg
1
of water, provided by silicon solar
cells) in conjunction with a conveyor belt mechanism to pro-
cess soil and extract bound water. Expected yields are in the
order of 1 kg of water per 2.5 h in a 2%-water soil, for a
systems mass below 20 kg (Wiens et al. 2001). The use of
microwave heating could also reduce dramatically excavation
needs, as microwaves could be inserted down bore holes to heat
at desired depths (Ethridge & Kaulker 2012). But energy and
excavation needs for water extraction from the soil could be
further reduced: according to Zubrin & Wagner (2011), a
0.1 mm thick polyethylene dome could be used to farm-
selected soil sites by increasing temperature and collecting
volatilized water in a cold trap device. Using this approach, a
25 m, 100 kg dome ringed by reflectors could allow about
150 kg of water to be farmed in an 8 h day from a soil with
2% water (Zubrin & Wagner 2011).
There is also water vapour in the atmosphere. Relying on it
rather than on soil water would remove the need for processing
regolith and/or to move to new locations once a site has been
dried up. Concentrations vary through time and location and
are very low, but total atmospheric water has been assessed to
amount to approximately 1.3 km
3
(1.3 × 10
9
litres), in very
large excess compared with human need for a research base,
and extracted water would be renewed naturally by exchange
with regolith and polar caps (McKay et al. 1993). In spite of
the large volumes of atmosphere that need to be processed,
water extraction from the Martian atmosphere could be done
with an energy consumption below 100 kWh kg
1
using a pro-
cess of atmosphere cooling/compression and water condensa-
tion designed by Meyer & McKay (1984) and improved by
Clapp (1985). Note that energy used in this process would
not be dedicated to water extraction, as atmospheric gas
could be isolated using the same setup. Energy needs for
water extraction from the atmosphere could be brought further
down, as shown by a system referred to as the Water Vapour
Fig. 5. Underpressurized culture vials used in Kirsi Lehtos laboratory (at the University of Turku, Finland) to grow cyanobacteria in
low-pressure/high pCO
2
atmospheres.
Sustainable life support on Mars 77
Adsorption Reactor (WAVAR). In WAVAR, atmosphere is
filtered and drawn through a regenerative adsorbent bed of
zeolite molecular sieve, from which water is later desorbed
using microwave radiation (Williams et al. 1995; Coons et al.
1997; Schneider & Bruckner 2003). Energy consumption
has been assessed as ranging from approximately 3 to 30
kWh kg
1
of water, depending on time and location (Grover
& Bruckner 1998). Additional water could be recycled from
human metabolism (about 0.4 kg of transpiration water and
respiration moisture a day per crewmember) and human
waste (Meyer & McKay 1989; Tikhomirov et al. 2007). It
will also be a by-product of physicochemical processes such
as on-site production of biofuels and materials (e.g., Zubrin
et al. 1997). The systems described above are still in their in-
fancy; increasing knowledge of Marss resources, coupled to
engineering effort, will likely support the development of
more efficient water mining technologies and processes.
Strategies can also be developed to minimize water needs
for cyanobacterial culture systems, in addition to recycling
water from culture effluents. For the production of some pro-
ducts where cells lysis is not needed, water requirements (as
well as requirements for maintenance, nutrients and energy)
could be reduced by immobilizing cyanobacteria within poly-
meric matrices. Such entrapment can preserve cyanobacter-
ias metabolic activities for prolonged periods of time, up to
several years (Lukavský 1988; Hertzberg & Jensen 1989;
Chen 2001). Water requirements could be further reduced
by growing terrestrial cyanobacteria as biofilms, directly on
the surface of Martian rocks, in a semi-closed environment
where suitable temperature, pressure and moisture are pro-
vided. Such a growth system could be relatively close to the
natural lifestyle of rock-dwelling cyanobacteria, noteworthy
in terrestrial deserts (see, e.g., Friedmann & Ocampo 1976;
de la Torre et al. 2003; Warren-Rhodes et al. 2006 and Billi
et al. 2013) but with more favourable moisture, UV protection
and temperatures.
Finally, a reduced pressure at growth-permissive tempera-
tures, as envisioned in the culture system, will foster water
evaporation. However, if pN2 sets the lower limit for atmos-
pheric pressure and the culture system provides about a tenth
of Earths atmospheric pressure at sea level (see subsection
Atmospheric pressure and composition), evaporation can
easily be reduced by saturating the incoming air with water va-
pour (Kirsi Lehto, personal communication). Besides, as the
system needs to be air-tight (apart from controlled gas ex-
change), water loss by evaporation can be minimized.
Solar energy and harmful radiations
On Earth, the solar flux is in large excess compared with the
needs of cyanobacteria and green plants, which cannot use
more than about 1020% of maximal sunlight on the surface
(Way et al. 2011). Some cyanobacterium species such as
Arthrospira spp. can utilize higher light densities but, at least
for A. platensis, the radiation level for maximal photosynthesis
activity is well below terrestrial mean day values provided
agitation intensity and cell density allow sufficient exposure
(Hoshino et al. 1991). Even though Mars is on average 1.52
times farther from the Sun than Earth and consequently re-
ceives about 43% as much sunlight at comparable latitude
and time of day, this is in excess compared with photosynthesis
needs, even in the case of a dust storm (McKay et al. 1993). The
light flux needed for optimal photosynthesis of many cyano-
bacterial species is about 3 × 10
19
photons m
2
s
1
, which is
only about 10% of Marss average ambient light flux (Lehto
et al. 2007). At lower light levels photosynthesis is still possible,
albeit at lower efficiencies. It has for example been assessed
that, at midday at vernal equinox, light levels on Mars are
about 5000 times greater than the minimum required for
photosynthesis (Cockell & Raven 2004).
It might be thought challenging to let photosynthetically ac-
tive radiation (PAR light; 400700 nm) reach cultures while
protecting the latter from harsh radiation namely UV radi-
ation, solar energetic particles (SEP) and galactic cosmic rays
(GCR). First, the fraction of biologically effective UV radi-
ation reaching the surface of Mars is much greater than that
reaching the surface of Earth and includes UV-C radiation
(<280 nm). The models of Cockell et al. (2000) and
Schuerger et al. (2003) predict a maximum flux of around
50 W m
2
of UV
200400 nm
irradiance on the equatorial
Martian surface at the mean orbital distance. Recent data
from the Curiosity rover suggest lower values, with a max-
imum UV
200380 nm
irradiance of about 20 W m
2
recorded
at midday at Gales crater (Gómez-Elvira et al. 2014).
Despite being strongly germicidal on the surface, this radiation
can be blocked by a few millimetres of Martian dust coverage
(Mancinelli & Klovstad 2000; Dartnell & Patel 2014) or, to let
PAR light in, by mere transparent covers such as glass filters.
Besides UV, ionising radiations of SEP and GCR can reach
the Martian surface and subsurface with high energies due to
Marss lack of intrinsic magnetic field and thin atmosphere.
Pavlov et al.(2012) and Dartnell et al. (2007) assessed absorbed
dose rates at the surface to be in the range of 50150 mGy yr
1
,
and data from the Curiosity rover indicate an absorbed dose
rate of 76 mGy yr
1
at Gale crater (Hassler et al. 2013).
Could cells be protected from this radiation? Even though
the primary cosmic radiation component decreases with
shielding, secondary particles (lighter particles including neu-
trons and gamma-rays) form when radiation penetrates sub-
strates and dose rates increase with shielding until the
Pfotzer maximum, before decreasing due to energy loss, ab-
sorption and decay processes. From 76 mGy yr
1
at the sur-
face, for instance, they reach 96 mGy yr
1
under 10 cm of
Martian rock with a density of 2.8 g cm
3
(Hassler et al.
2013). The design of culture chambers and the choice of ma-
terial used for shielding should take into account their interac-
tions with primary radiations on the Martian surface (see Le
Postollec et al. 2009).
Approximately 5 m of Mars dirt would confer a protection
to radiation equivalent to Earths atmosphere (McKay et al.
1993). However, bacterial cells are overall much more
radiation-resistant than human cells and such a protection is
unlikely to be needed. In addition, cyanobacteria are known
to have high ploidy levels (Griese et al. 2011), pigments and
specific morphological features which tend to increase radio-
78 Cyprien Verseux et al.
resistance. The resistance of cyanobacteria to photon irradi-
ation (gamma-rays and X-rays) has been studied as early as
in 1951 (Bonham & Palumbo 1951), and the high resistance
of some species was observed in the early 1960s (Shields
et al. 1961; Godward 1962). In the following decade, the resist-
ance of a wide range of species from various genera was tested.
Resistance appeared to be highly variable among species, ran-
ging from sensitive ones with D
10
(dose required to reduce the
viable number of cells by 90%) below 1 kGy to highly resistant
ones with D
10
above 10 kGy (e.g., Shields et al. 1961; Godward
1962; Bruce 1964; Kumar 1964; Kraus 1969; Asato 1971).
These results were confirmed and deepened by recent studies.
Synechococcus and Synechocystis spp. were shown to be rela-
tively sensitive to gamma rays, with D
10
of about 0.3 kGy
(Domain et al. 2004; Agarwal et al. 2008) and 0.7 kGy
(Domain et al. 2004), respectively. Two Anabaena strains
were shown to tolerate a 5 kGy gamma-ray dose without loss
of survival, to have a GI
50
(dose where growth is inhibited by
50%) of 611 kGy and to survive doses of 15 kGy (Singh et al.
2010; Singh et al. 2013). Arthrospira sp. PCC 8005 cells were
shown to be able to survive exposure to doses of at least
6.4 kGy of gamma irradiation, 1 kGy of He particle radiation
and 2 kGy of Fe particle radiation (Badri et al. 2015).
Chroococcidiopsis spp. cells were shown to withstand 2.5 kGy
of X-ray irradiation in liquid culture with small to medium
viability loss: 2065%, depending on the strain, with a D
10
of
25 kGy (Billi et al. 2000a). Viable Chroococcidiopsis spp. cells
were also recovered after exposure to 15 kGy of X-rays (Billi
et al. 2000a) and to 12 kGy of gamma-rays (Verseux et al.
manuscript in preparation). Moreover, no significant DNA
or membrane damage was detected after exposure to 1 kGy
of He particle radiation, 2 kGy of Fe particle radiation or 1
kGy of Si particle radiation (Verseux et al. manuscript in prep-
aration). A dose of 1 kGy roughly corresponds to a thousand
years on the Martian surface, a timeframe way beyond that of
division and repair of metabolically active cyanobacteria; cells
are therefore not expected to be affected in BLSS cultures. The
biological effect of ionizing radiation cannot be assessed based
on the dose only: other parameters are to be taken into account
for instance, the composition of the radiation flux and its
modification when interacting with the environment and the
cellsproperties but the studies mentioned above show the or-
ders of magnitude involved. Even if the effect of radiation on
Mars was, at equivalent dose, more damaging by several orders
of magnitude than the radiation flux used in these studies,
the replication time and repair dynamics of even slow-
growing strains would be way below the time needed to re-
ceive a sterilizing dose. Consistently, it has been estimated
that even vegetative cells of the bacterium E. coli could sur-
vive the ionizing radiation dose that would be received after
more than a thousand years on the surface of Mars (Dartnell
et al. 2007). More generally, ionizing radiation on Mars is not
expected to prevent microbial life (Dartnell et al. 2007;
Horneck 2008).
Cultures could be buried inside regolith or covered with
regolith-based material: although not expected to be needed
for radiation protection, regolith shielding could be relevant
for temperature control (taking advantage of the regoliths
thermal insulating properties) and protection from the wind
and dust. Manufacturing processes could be simple and cost-
effective, taking for instance advantage of: (i) the cement-like
properties of unprocessed regolith mixed with water (McKay
& Allen 1996); (ii) the ubiquity of clay-like materials on
Mars; (iii) the large fraction (about 40% by weight of
Viking 1 and 2 soil samples) of Martian soil represented by
silicon dioxide (the basic constituent of glass); and (iv) the
fact that plastics may be derived from local C and H
(Zubrin & Wagner 2011). In this case, artificial lighting, mir-
rors or fibre optics could be used to bring PAR light to the
cultures. Lighting could also be electrically powered, using
for instance solar, wind or geothermal energy sources.
Various studies have been performed regarding electrical
lighting for plants in BLSS (easily adaptable to cyanobacter-
ial culture), light-emitting diode (LED)-based systems being
the most promising (Massa et al. 2007). However, even
though electrical lighting would allow an accurate control
on light intensity and photoperiods, energy and mass require-
ments could be greatly reduced compared with electrical
lighting by using systems based on fibre-optics technology
to harvest and transmit selected wavelengths from solar en-
ergy. Several of such systems have been developed in the con-
text of space exploration, among others for the cultivation of
microbial phototrophs (Mori et al. 1987) and plants (Jack
et al. 2002; Nakamura et al. 2009). Equivalent system mass
calculations showed a net benefit of using solar lighting
rather than electric lighting in this context, under realistic
mission assumptions (Drysdale et al. 2008). The cost effect-
iveness of such systems may be limited by temporary de-
creases in light availability (duetodiurnalandseasonal
light cycles, variable distance from the Sun and global dust
storms), which drives a need for increasing collector size rela-
tive to culture areas, and may consequently be inadequate for
growing plants in Mars BLSS (Massa et al. 2007). They could
however represent a solution for growing cyanobacteria,
which use light more efficiently and suffer less than plants
from temporary reduction of light and from changes in illu-
mination patterns. That being said, the simplest and most
cost-effective way of providing PAR could be to directly ex-
pose cultures to solar light while protecting them from lethal
levels of radiation. A transparent but UV-blocking and ther-
mal insulating material, which should also be resistant to
Mars surface conditions in the long term, could be used.
Radiation coming from straight above could also be stopped
by regolith-based materials, while allowing light to come
from the sides at adequate intensities for photosynthesis to
occur (de Vera et al. 2014).
Heating will be needed to maintain liquid water and allow
metabolism. Heating systems could be either directly based
on solar energy, or on electricity generated by solar energy
(e.g., using solar panels) and/or based on other power sources
used within Martian outposts (e.g., wind, geothermal activity
or fuels produced on site). This issue is largely documented
elsewhere, as it applies to other components of human colonies
such as habitats, and will not be detailed here.
Sustainable life support on Mars 79
Gravity
Would Marss lower-than-Earth gravity affect cyanobacteria?
The earliest microbiology experiments in space, reported in the
early 1960s, did not show any effect of microgravity on individ-
ual cells (Zhukov-Verezhnikov et al. 1962). Consistently, the-
oretical studies suggested that microgravity would not
directly affect cells of a diameter below 10 µm, in part due to
the density uniformity and smallness of intracellular compo-
nents (Pollard 1965,1967). Later, experiments on-board the
orbital station Salyut 6 and biosatellites Kosmos 1887 and
2044 showed that unicellular algae were not affected in their
development by microgravity (Sychev et al. 2001). However,
a wide range of altered behaviour and growth properties such
as increased virulence, reduced lag phase, increased final cell
population, increased productivity of secondary metabolites
and increased conjugation rates have been reported in later
microbiology experiments in spaceflight and simulated micro-
gravity (see, e.g., Nickerson et al. 2000; Benoit & Klaus 2007
and Wilson et al. 2007), in various prokaryotes including
cyanobacteria (Wang et al.2004,2006; Xiao et al.2010). A
well-supported hypothesis suggests that these effects are
motility-dependent, with non-motile cells being the most af-
fected. This might be explained by the reduced flow of metabo-
lites and nutrients and by the reduced exchanges between
bacteria and the environment (e.g., because mass-driven con-
vection does not occur), which result in a modified chemical
environment around cells that alters biological responses
(Horneck et al. 2010). However, microgravity was shown not
to reduce N fixation abilities, photosynthetic O
2
production
rates or growth in cyanobacteria (e.g., Wang et al. 2006).
Marss reduced gravity (0.38 g) is consequently not expected
to be an obstacle to cyanobacterium-based processes, especial-
ly if stirring generates a non-limiting flux of nutrients and me-
tabolites. Studies aimed at confirming that reduced gravity do
not alter cell processes of interest would however be useful.
Engineering cyanobacteria
In addition to adapting the Martian environment to cyanobac-
teria, work can be done to adapt cyanobacteria to the Martian
environment. Synthetic biology may provide the tools for this.
This field, or set of fields, aroused NASAs interest due to its
potential for engineering microorganisms with useful features
for resource production in space (Cumbers & Rothschild 2010;
Langhoff et al. 2011; Menezes et al. 2014; Verseux et al. 2016).
In the present context, the use of synthetic biology tools and
methods is investigated for optimizing the abilities of selected
cyanobacteria to: (i) withstand environmental stresses faced
during space exploration missions, and (ii) grow and perform
biological functions of interest under on-site constraints (see
Fig. 6).
Increasing cyanobacterias abilities to withstand extreme
conditions would allow failure risk and culture system-related
costs to be minimized (Cockell 2010; Olsson-Francis & Cockell
2010). First, for storage during the journey: cellstolerance to
long periods of dehydration, possibly in a differentiated state
(e.g., akinetes), would allow a storage free of risk of loss by
freezing failure. Secondly, for growth on site: higher tolerance
to Marss environmental factors would reduce the required
level of shielding. It would also increase safety in case of
system malfunction during which cultures could be exposed
to harsher conditions (e.g., desiccation, low pressure, high
radiation levels, altered pH and sudden temperature shift),
either when stored or grown. Thirdly, increasing the abilities
of cyanobacteria to rely on on-site resources would allow
productivity to be increased while relaxing constraints and sup-
plementation needs. In particular, it would be highly beneficial
to increase their abilities to leach Martian mineral resources
and to get most of their nutrients from these, within a wide
range of pH, and to fix N at a lower pN
2
. Finally, new func-
tions and nutritional properties could be engineered and opti-
mized under conditions that would be provided within Mars
bases.
To increase their resistance, cyanobacteria could be trans-
formed with heterologous genes known to increase fitness
under conditions found on the surface of Mars (Cumbers &
Rothschild 2010). This approach has been successful in other
contexts: for instance, E. colis resistance to gamma irradi-
ation, to desiccation and to low temperatures has been in-
creased by expressing, respectively, the PprI protein from
Deinococcus radiodurans (Gao et al. 2003), a sucrose-6-
phosphate synthase from Synechocystis sp. strain PCC 6803
(Billi et al. 2000b) and the chaperonin Cpn60 and co-
chaperonin Cpn10 from the psychrophilic bacterium
Oleispira antarctica (Ferrer et al. 2003). Once specific genes
have been shown to confer an advantage to a targeted stress
in a target organism, they could be improved using various
computational and molecular biology tools and methods.
These are becoming more and more efficient thanks to,
among other factors, a sharp decrease in DNA synthesis
costs, the improvement of automated gene assembly methods,
and the development of biological computer-aided design
(BioCAD) and other computational tools.
However, despite the fact that expressing heterologous genes
(or overexpressing homologous genes) may confera significant
advantage in coping with some environmental stressors, this
approach might be much more challenging for resistance fea-
tures which are highly multifactorial, each individual factor
having a relatively weak impact. There is not a single factor
that confers on D. radiodurans its extreme radiation resistance,
for instance, but a very wide combination of features, including
efficient DNA repair mechanisms, anti-oxidation mechanisms
and specific morphological features (see, for instance, Slade &
Radman 2011). For this kind of feature, dramatic increases
through rational design seem very challenging given
bio-engineerings current state-of-the-art. Instead, directed
evolution iterations of mutagenesis and artificial selection
at the scale of the whole organism can allow complex modifi-
cations, affecting both known and unknown mechanisms. The
dynamics of experimental evolution have been widely studied
in recent decades and have been successfully used to increase
organismsspecific properties (see, e.g., Elena & Lenski 2003
and Conrad 2011), including radiation resistance in bacteria
80 Cyprien Verseux et al.
(Ewing 1995; Harris et al. 2009; Wassmann et al. 2010;
Goldman & Travisano 2011).
For increasing the abilities of cyanobacteria to use resources
found on-site, and for conferring on them abilities (and/or im-
proving these abilities) to produce resources of interest from
local resources, rational genetic engineering might be more ef-
ficient than it is for increasing resistance. Some clues have even
been given regarding the engineering of microorganisms with
increased bioleaching abilities (Cockell 2011). However, des-
pite metabolomicss great advances in the last decade and the
synthetic biology benefiting from it (see, for instance, Ellis &
Goodacre 2012 and Lee 2012), the complex interactions occur-
ring in cells are still hard to predict and whole-cell-scaled direc-
ted evolution will here again be very useful.
One of the main issues when designing an optimization pro-
cess based on directed evolution is the need for either linking
the optimized function (e.g., production of a compound of in-
dustrial interest) to organismsfitness or selecting mutants
after screening. When increasing resistance to environmental
stressors (e.g., low pressure or radiation) or abilities to use a
given nutrient source (e.g., Martian regolith or atmosphere),
the process is more straightforward: selection can be done by
applying increasing levels of the targeted stress (when improv-
ing resistance) or by conducting growth/re-inoculation cycles
to let fast-growing mutants become dominant (when improv-
ing use of a given nutrient source). Directed evolution can be
improved by automation (Dykhuizen 1993; de Crecy et al.
2009; Marlière et al. 2011; Grace et al. 2013; Toprak et al.
2013) and by recent methods such as, for instance, genome
shuffling (Patnaik et al. 2002) and multiplex genome engineer-
ing (Wang et al. 2009). It might also be more efficiently per-
formed on Mars, once a microbial production system is well
established and automated (Way et al. 2011): Earth-based si-
mulations of some of the factors encountered on Mars (and
their combinations) are difficult, expensive and cannot faith-
fully reproduce all of their effects. The most straightforward
use of directed evolution would be to improve cyanobacterial
culturesproductivity by, for example: (i) increasing the abil-
ities of cyanobacteria to grow faster under low (and to grow
at lower) pressures, including low pressures of Mars-like gas
compositions; (ii) improving their efficiency to extract mineral
nutrients from Martian regolith; and (iii) increasing their resist-
ance to potentially toxic elements in the regolith (e.g., perchlo-
rates). As selective pressure for some of these factors is low or
inexistent on Earth, fitness under these conditions is likely far
from its evolutionary potential and could be largely increased
by directed evolution. The amounts of resources allocated to
cyanobacterial cultures for producing a given amount of
other resources could thereby be significantly reduced.
Besides direct applications, performing directed evolution
could give clues on whether and how much organisms can gen-
etically adapt to the Martian environment, and therefore help
assessing the risk of biological contamination in case of acci-
dental release.
Rational genetic engineering and directed evolution are
not mutually exclusive: they can be used in combination
(Rothschild 2010). Rationally engineered organisms can be
submitted to directed evolution for optimization and, con-
versely, data obtained from genome sequencing of evolved or-
ganisms (so as to understand which mutations are responsible
for improved properties) can give gene targets for rational
design.
Besides increased resistance and abilities to thrive on local
substrates, synthetic biology could be used to confer on
Fig. 6. Simplified overview of the potential roles of synthetic biology in the development of Mars-specific, cyanobacterium-based BLSS.
Sustainable life support on Mars 81
cyanobacteria specific functions for Mars-specific BLSS.
Numerous genetic engineering tools and methods have been
developed for enhancing their metabolic capabilities. Various
applications which are considered on Earth (see, e.g., Wang
et al. 2012 and Berla et al. 2013) could be useful on Mars as
well, but others might be developed specifically for this planet
(Verseux et al. 2016). Some resources may be much easier to
obtain using synthetic biology when on Mars, while being eas-
ier to obtain by other means (e.g., by chemical methods, using
the natural host or by simply harvesting rather than producing
them) when on Earth. Finally, metabolic pathways might dif-
fer: the set of potential starting compounds will be much re-
duced on Mars and some substrates that are cheap and
abundant on Earth will be extremely hard to provide there,
changing the cost-effectiveness of a given pathway.
A critical issue to be faced is genetic instability: artificial
gene constructs might be lost. Plasmids are useful for screening
and optimization steps, but are generally too unstable to be
used on a large scale and in the long term. Exogenous genes
should rather be irreversibly inserted in cyanobacterias gen-
omes, which leads to much higher stability and more control
over expression levels (e.g., Herrero et al. 1990; Heap et al.
2012). Genome insertion in cyanobacteria is made harder
than in most model bacteria by their polyploidy and the un-
viability of recA mutants (Murphy et al.1990; Matsuoka
et al. 2001), but specific methods are available to do so (e.g.,
Andersson et al. 2000: Takahama et al.2004; Clerico et al.
2007; Chaurasia et al. 2008). For standard, organism-scaled di-
rected evolution, genome integration will not be an issue as
variability will mostly affect genomes. However, even when in-
tegrated into a genome, modifications may be lost. How com-
monly this phenomenon is encountered among teams
performing cyanobacterium genetic engineering, and how lim-
iting the issue is, is hard to assess due to a tendency not to docu-
ment failures (Jones 2014). Many reports of successful and
stable genetic constructs in cyanobacteria can be found (e.g.,
Guerrero et al. 2012; Bentley et al. 2014; Dienst et al. 2014),
but a few others document instability (e.g., Takahama et al.
2003; Angermayr et al. 2012). In spite of the lack of available
data, it is expected that modifications having a negative effect
on growth (e.g., insertion of a system to produce a chemical of