ARTICLE IN PRESS

Planetary and Space Science 58 (2010) 12791285

Contents lists available at ScienceDirect

Planetary and Space Science

journal homepage: www.elsevier.com/locate/pss

Use of cyanobacteria for in-situ resource use in space applications

Karen Olsson-Francis n, Charles S. Cockell
Centre for Earth, Planetary, Space and Astronomical Research, The Open University, Walton Hall, Milton Keynes MK7 6AA, UK

a r t i c l e in fo abstract

Article history: The regolith of other planetary bodies, such as the Moon and Mars, is rich in inorganic elements that
Received 9 December 2009 could potentially be exploited for space applications. Lithotrophic microorganisms that are capable of
Received in revised form utilising rocks as a growth substrate, and facilitate the extraction of elements, are ideal candidates for
10 May 2010
in-situ resource use. Of particular interest are the cyanobacteria, which have been suggested for
Accepted 14 May 2010
Available online 24 May 2010
applications, such as oxygen, fuel and biomass production, nutrient acquisition, and feedstock
provisions. In this study, Gloeocapsa strain OU_20, isolated from a rock-dwelling community exposed to
Keywords: low Earth orbit; Leptolyngbya strain OU_13 and Phormidium strain OU_10, both isolated from a rock-
Space applications dwelling community exposed to Mars simulated conditions; Chroococcidiopsis 029; Arthrospira
Cyanobacteria
platensis; Synechococcus elongatus; and Anabaena cylindrica, were examined as potential organisms
Lithotrophic
for space in-situ resource use. Volcanic rocks, including basalt (low in SiO2) analogous to martian and
Regolith
Mineral acquisition lunar basalt, rhyolite (high in SiO2), and anorthosite analogous to lunar regolith were used as growth
substrates. The growth rate and rock dissolution were signicantly lower with rhyolite demonstrating
the importance of silica content in dening the potential for in-situ resource use. Biological weathering
resulted in the release of bio-essential elements from the rock matrix, highlighting the potential of
cyanobacteria for applications such as bio-mining and nutrient acquisition, on other planets.
A. cylindrica produced the maximum biomass with the three rock-types and the optimal value was
obtained with the basalt. Exposure experiments demonstrated that A. cylindrica, Chroococcidiopsis 029,
Gloeocapsa strain OU_20, Phormidium strain OU_10, and Leptolyngbya strain OU_13 were able to survive
28 days of exposure to desiccation and Mars simulated conditions, which is benecial in case of system
malfunction and for storage. The results from this study indicate that cyanobacteria can potentially be
used for in-situ planetary resource acquisition.
& 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Microorganisms are known to facilitate the extraction of
elements from rocks and to be capable of growing on rock
substrates, causing bioweathering (Baneld and Nealson, 1997;
Gorbushina and Broughton, 2009). They may therefore have
applications in extraterrestrial in-situ resource use. Of particular
interest are cyanobacteria, oxygenic photosynthetic organisms
that play a signicant role in the production of organic matter and
oxygen. In extraterrestrial environments, such as the Moon and
Mars, the regolith is rich in inorganic elements and could
potentially be exploited by autotrophic cyanobacteria (Brown
et al., 2008).
The use of in-situ resources as a source of nutrients,
particularly by cyanobacteria, has applications in oxygen, fuel
and biomass production, nutrient acquisition, bio-mining and
feedstock provision (Lehto et al., 2006; Hendrickx and Mergeay,
n
Corresponding author. Tel.: + 44 1908 655382.
E-mail address: k.olsson-francis@open.ac.uk (K. Olsson-Francis).
2007; Liu et al., 2008). One such application is bioregenerative life
support systems, for example the MELiSSA system, developed
with support from the European Space Agency (ESA), which is
designed for long-term space ight and extraterrestrial settle-
ments (Lehto et al., 2006; Hendrickx and Mergeay, 2007).
Cyanobacteria are ubiquitous in nature and have adapted to
live in some of the most extreme environments on Earth, varying
from the hyper-arid, cold Antarctic Dry Valleys to the hot, dry
Atacama Desert (Friedmann, 1980; Wierzchos et al., 2006).
Furthermore, cyanobacteria have been isolated after exposing a
rock-dwelling community to low Earth orbit and Mars simulated
conditions. The rock-dwelling Gloeocapsa strain OU_20 survived
exposure to low Earth orbit and Mars simulated conditions
(no UV); whilst, the Leptolyngbya strain OU_13 and the Phormi-
dium strain OU_10 survived exposure to Mars simulated condi-
tions (no UV) (Olsson-Francis et al., 2010).
Although extremophilic cyanobacteria are potential candidates
for extraterrestrial in-situ resource use, they are not ideal as they
are generally slow growing; for example, Chroococcidiopsis has a
generation time of 16 days (Billi and Caiola, 1996). Whereas,
cyanobacteria that are used commercially in food and fertiliser

0032-0633/$ - see front matter & 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pss.2010.05.005
 ARTICLE IN PRESS
1280 K. Olsson-Francis, C.S. Cockell / Planetary and Space Science 58 (2010) 12791285
production, such as Arthrospira platensis, Synechococcus elongatus,
and Anabaena cylindrica, are faster growing and could potentially
generate sufcient biomass and oxygen for space applications.
In this study we examined the growth of extremophilic and
commercially used cyanobacteria, as potential organisms for
exploiting extraterrestrial in-situ resources. The terrestrial volca-
nic rocks, basalt, and anorthosite, were used as nutrient supplies
as they are analogues for extraterrestrial regolith material. The
more silica-rich rock, rhyolite, was also used to investigate the
effect of high silica content on biomass production. The work
presented in this paper is the rst comprehensive study of
lithotrophic cyanobacteria as potential candidates for in-situ
utilisation of planetary resources.
2. Material and methods
2.1. Rock samples
In this study anorthosite (an analogue to lunar highland
regolith), basalt (an analogue to martian and lunar volcanic
basalt), and rhyolite (a high silica end member), were used as
growth substrates. The anorthosite was collected from Bushveld,
South Africa (24115S, 29155E), the rhyolite from outcrops in
Iceland (6411N, 1916W), and the basalt from near the Torfajokull
volcano in Iceland (6415N, 1913W). The rocks were crushed using
a hollow metal cylinder and a metal plunger (Herrera and Cockell,
2007). To form a powder, the rocks were ground using a Tema
swing mill, for 8 min ( o100 mm in diameter).
2.2. Characterisation of the rock samples
Whole-rock major element compositions for each of the rock-
types were obtained using an ARL 8420+ dual goniometer
wavelength-dispersive XRF spectrometer. XRF analysis was
carried out on glass discs prepared by fusing one part of a
powdered sample with ve parts of a FluXana ux (20% lithium
tetraborate/80% lithium metaborate mix). The analysis procedure
was carried out as previously described (Ramsey et al., 1995).
The elemental composition and inferred minerology of each of
the rock-types was determined by electron microprobe analysis.
Analysis was carried out with a carbon coated thin section using a
Cameca SX100 instrument. The following standards and X-ray
lines were used: jadeite (Na Ka), forsterite (Mg Ka), feldspar (Al, Si
and Ka), uorapatite (P Ka), synthetic KCl (Cl Ka), crocoite
(Cr Ka), rutile (Ti Ka), bustamite (Mn and Ca Ka), hematite
(Fe Ka), and Ni metal (Ni Ka). The conditions were an accelerating
voltage of 20 kV, a probe current of 20 nA, and a beam diameter of
10 mm for individual minerals within the rock and 20 mm for the
matrix. Data was corrected using a PAP correction procedure
(Pouchou and Pichoir, 1991).
2.3. Microorganisms
The cyanobacteria, Phormidium strain OU_10, Gloeocapsa strain
OU_20, and Leptolyngbya strain OU_13 were isolated from a
limestone cliff in Beer, Devon (UK) using low Earth orbit or Mars
simulation conditions as a selection pressure (Olsson-Francis
et al., 2010). Chroococcidiopsis 029 was isolated from cryptoendo-
lithic growth in Nubian sandstone in the Negev desert (obtained
from the University of Rome Tor Vergata). S. elongatus (PCC
6301), A. cylindrica (PCC 6309), and A. platensis (PCC 8005) were
obtained from the Pasteur Culture Collection of Cyanobacteria
(Paris, France).
2.4. Media and growth conditions
For routine growth, Gloeocapsa strain OU_20, Leptolyngbya
strain OU_13, Phormidium strain OU_10, S. elongatus, A. cylindrica,
and Chroococcidiopsis 029 were grown in modied BG-11
medium, pH 7.4. A. platensis was grown in BG-11+ ASNIII (1:1)
medium, pH 9.0 (Olsson-Francis et al., 2009; Rippka et al., 1979).
To study cyanobacterial growth under lithotrophic conditions,
media were prepared using volcanic rock and sterilised dd H2O.
The media either had no amendments or was supplemented with
10 mM (NH4)2SO4, 10 mM NaNO2, or 10 mM (NH4)2SO4 and
10 mM NaNO2. The media were supplemented with 10 mM
(NH4)2SO4 because cyanobacteria benet from low concentration
of sulphates and on Mars gypsum could be used as a supplement.
We also supplemented the media with 10 mM NaNO2 because
the non-nitrogen xers, A. platensis, Chroococcidiopsis 029, and S.
elongatus required nitrogen. The rocks were autoclaved at 121 1C
for 20 min and added aseptically prior to use. For A. platensi, the
dd H2O was replaced with 10 mM sodium carbonate buffer,
pH 9.0 (Vonshak et al., 1996). Preliminary experiments demon-
strated that the optimal rock/dd H2O ratio was 1 g of rock
( o 100 mm in diameter)/5 ml of dd H2O. Therefore, for the
lithotrophic experiment we used 25 ml of liquid media and 5 g of
volcanic rock. The cultures were grown in polymethylpentene
asks that had been soaked overnight in 1% HNO3 and rinsed in
dd H2O.
All of the cultures were incubated for 45 days, at 25 1C, under
ambient atmospheric conditions, in natural sunlight and a
day/night cycle (Olsson-Francis et al., 2010).
2.5. Lithotrophic growth experiment
Prior to inoculating the lithotrophic media, 5 ml of culture
(grown in BG-11 or BG-11+ ASNIII media) was extracted and
centrifuged at 5000 rpm for 10 min. The cell pellet was washed
three times with sterilised dd H2O (or 10 mM sodium carbonate in
the case of A. platensi). The nal cell suspension was resuspended
to a cell density of approximately 109 cells ml  1. A 100 ml aliquot
was used to inoculate the lithotrophic media. Non-biological
controls, which consisted of rock with no added inoculum, were
sampled in an identical manner to the biological experiments.
Each of the growth experiments was conducted in triplicate.
To monitor microbial growth and to determine the specic
growth rate constant k (the number of generations per unit of
time), cell counts were conducted (Pirt, 1975). The cultures were
mixed and a 100 ml aliquot was aseptically withdrawn when
required. Due to the lamentous nature of Phormidium strain
OU_10, Leptolyngbya strain OU_13, A. cylindrica, and A. platensis
the samples were briey vortexed before counting the cells with a
Leica DMRP microscope (1000  ), equipped with epiuorescence.
2.6. Final dry weight biomass
To determine the nal dry weight biomass, a 5 ml sample was
extracted from the culture, after 45 days, and centrifuged for 5 s at
3000 rpm, to pellet the rock. The supernatant, containing the
microbial cells, was centrifuged for 5 min at 16,000 rpm and
the pellet was transferred onto a pre-weighed microscope slide.
The slide was placed in a desiccator containing silica gel beads, for
8 h. After this time, the slide was removed and washed with 5 ml
of dd H2O. The slide was then replaced in the desiccator and dried
for 24 h. The dry weight was calculated as the difference in weight
before and after.
 ARTICLE IN PRESS
K. Olsson-Francis, C.S. Cockell / Planetary and Space Science 58 (2010) 12791285 1281

2.7. Elemental concentrations Table 1

Comparison of the chemical composition of the terrestrial volcanic rocks and
extraterrestrial regolith.
Elemental release from the volcanic rock was measured at the
end of the experiment. A 10 ml aliquot of sample was ltered Element Basalt Wedgea Rhyolite Anorthosite 12032, 44b
through a 0.2 mm nylon syringe lter and acidied with (Mars) (Moon)
concentrated HNO3 (nal 5% acid). The samples were stored at
SiO2 51.36 52.2 69.41 47.45 46.8
4 1C until analysis.
TiO2 1.75 1.0 0.29 0.04 N/A
The elemental concentrations were measured by Inductively Al2O3 14.22 10.0 14.28 29.82 33.2
Coupled Plasma Atomic Emission Spectrometry (ICP-AES) analysis Fe2O3 13.36 15.4 3.07 1.64 0.34
(Prodigy, high dispersion ICP). The following standards and MnO 0.21 N/A 0.07 0.02 N/A
wavelengths were used: Al (396.152 nm), Ca (422.673 nm), Cu MgO 6.31 4.9 0.43 2.40 N/A
CaO 10.86 7.4 1.19 14.97 17.7
(324.754 nm), Fe (239.563 nm), K (766.491 nm), Li (670.784 nm), Na2O 2.46 3.1 5.34 1.99 1.44
Mg (279.553 nm), Mn (259.372 nm), Na (589.592 nm), Ni K2O 0.46 0.7 4.46 0.11 0.01
(231.604 nm), Sr (407.771 nm), Zn (213.856 nm), and Si (251.611). P2O5 0.18 N/A 0.04 0.01 N/A
Each of the measurements was conducted in triplicate and the mean Cl N/A 0.5 0.31 0.53 N/A
SO3 N/A 2.8 N/A N/A N/A
was reported.
Total 100.94 97.1 98.92 98.99 99.5

N/A not available.

2.8. Exposure experiments
The cyanobacteria were exposed to simulated Mars conditions
(  27 1C, 0.8 kPa, CO2), with and without UV radiation, vacuum
(5  10  3 kPa) to represent the high vacuum on the Moon, and
desiccation. Each of the experiments was carried out in triplicate
with a set of replicas stored under ambient conditions in the dark,
as controls. A 100 ml of cells (1  109 cells ml  1) was used for each
experiment. The cells were washed in dd H2O and dried onto a
microscope coverslip.
Samples, including the controls, were collected aseptically
after 1, 4, 7, 21, and 28 days (unless stated differently below) of
exposure and incubated in 5 ml of BG-11 media (BG-11+ ASNIII
for A. platensi), under natural day/light cycles at 25 1C. After 28
days, the cultures were analysed and the viability of the cells in
the exposed samples was determined as a percentage (compared
to the controls).
A simulation chamber was used to expose the cells to Mars
simulation conditions, with and without UV radiation, and
vacuum as previously described (Olsson-Francis et al., 2009).
The microscope coverslips were positioned inside the chamber
within a sterilised Petri dish. For the desiccation experiment, the
microscope slips were placed in a desiccator containing silica gel
beads, at room temperature.
3. Results
3.1. Characterisation of the rock samples
The major elements of the rocks were obtained using XRF
analysis. The basalt had a typical basaltic composition, rich in
MgO and CaO, and low in SiO2. The composition was similar to the
martian basalt, measured at Half Dome and Wedge, by the Alpha
Proton X-ray Spectrometer on board the Mars Pathnder mission,
as seen in Table 1 (Rieder et al., 1997). All of the major oxides,
except Al2O3 and CaO were comparable. In contrast, the rhyolite
was high in SiO2, Na2O, and K2O, compared to the basalt, but was
depleted in MgO and CaO. Microprobe analysis detected
plagioclase (Na, Ca, Al, SiO4), pyroxyene (Fe, Mg, Al, Si, Ca) and
irontitanium (Fe, Ti) within the glassy (high SiO2) groundmass
(data not shown).
The anorthosite consisted of plagioclase, chromite, and
pyroxene (data not shown). The calcium-containing plagioclase
(CaAl2  Si2O8) was identied as bytownite, which typically
contains 7090% of calcium in the crystal structure. The
composition was similar to sample 12032, 44 from the Woods
group of gabbroic anorthosites that may have originated from the
a
Composition taken from Rieder et al. (1997).
b
Composition taken from Wenk and Nord (1971).
lunar highlands, as seen in Table 1 (Wenk and Nord, 1971). In
comparison to the basalt, the anorthosite was high in Al2O3 and
low in TiO2.
3.2. Lithotrophic growth experiment
Preliminary experiments were conducted to optimise the
lithotrophic media. The optimal rock/dd H2O ratio and the size
of the rock were determined. The same effect was observed for all
of the cyanobacteria; the specic growth rate increased with the
amount of rock, but plateaued at a rock/H2O ratio of 1:5. For
example, the specic growth rate for A. cylindrica was 0.363 and
0.342 day  1 for cells grown in a rock/H2O ratio of 1:5 and 1:2.5,
respectively (data not shown). Also, the specic growth rate
increased as the size of the rocks decreased. For example, the
specic growth rate of A. cylindrica decreased from 0.363 to 0.121
day  1, as the diameter of the basalt rock increased from
o100 mm to 41 cm. Overall, the addition of both (NH4)2SO4
and NaNO2 to the lithotrophic media had a benecial effect on
the cyanobacteria, as seen in Table 2. The non nitrogen-xing
cyanobacteria, Chroococcidiopsis 029, A. platensis, and S. elongatus,
required supplementary NaNO2 for growth.
The rock-type affected cyanobacterial growth. The utilisation
of rhyolite, as a growth substrate, was challenging; this was
demonstrated by the low specic growth rates. The maximum
specic growth rates were obtained with the basalt, as seen in
Table 2.
The nal dry weight biomass values were concurrent with the
specic growth rates, as shown in Table 3. For example, the
maximum specic growth rate for A. cylindrica was obtained with
basalt as a growth substrate. This correlated with a maximum
total dry biomass of 0.4970.02 g/kg of basalt, as shown in
Table 3. The values were signicantly lower than those obtained
with the BG-11 (BG-11+ ASNIII) media.
3.3. Dissolved elemental concentrations
The elemental concentrations were measured in the litho-
trophic media with no amendments and with supplementary
NaNO2 and (NH4)2SO4, after 45 days. The elemental concentra-
tions of the dd H2O and the supplementary media were used as
blanks. The non-biological controls demonstrated that the
elemental leaching from the rhyolite was lower than with basalt
 1282
Table 2
Effect of rock-type on the specic growth rates of the cyanobacteria (day  1). The cyanobacteria were grown in dd H2Oa, or supplemented with 10 mM (NH4)2SO4, 10 mM NaNO2, or 10 mM (NH4)2SO4 and 10 mM NaNO2, with
volcanic rock.

BG-11 Rhyolite Anorthosite Basalt

dd H2O N SO4 N +SO4 dd H2O N SO4 N+ SO4 dd H2O N SO4 N + SO4

Anabaena cylindrica 0.792 7 0.012 0.123 7 0.041 0.152 70.034 0.083 7 0.014 0.213 70.047 0.2477 0.140 0.212 7 0.141 N.D. 0.3567 0.054 0.315 7 0.101 0.097 70.008 0.125 70.042 0.363 7 0.014
Chroococcidiopsis 0.114 7 0.005 N.D. 0.0547 0.011 N.D. 0.0417 0.008 N.D. 0.026 7 0.014 N.D. 0.0247 0.014 N.D. 0.013 70.035 N.D. 0.0827 0.012
029
Arthrospira platensi 0.312 7 0.041 N.D. N.D. N.D. N.D. N.D. 0.057 7 0.004 N.D. 0.0277 0.015 N.D. 0.012 70.004 N.D. 0.0887 0.007
Synechococcus 1.581 7 0.414 N.D. N.D. N.D. 0.0437 0.012 N.D. 0.219 7 0.031 N.D. N.D. N.D. 0.124 7 0.007 N.D. 0.269 7 0.034
elongatus

K. Olsson-Francis, C.S. Cockell / Planetary and Space Science 58 (2010) 12791285

Phormidium strain 0.332 7 0.147 0.012 70.005 0.0157 0.004 0.017 7 0.004 0.0357 0.009 0.0467 0.014 0.063 7 0.007 0.043 70.001 0.0187 0.004 0.056 7 0.014 N.D. 0.0847 0.004 0.0877 0.011
OU_10
Leptolyngbya strain 0.255 7 0.054 0.015 70.004 0.0137 0.004 0.086 7 0.014 0.0277 0.007 0.0657 0.014 0.043 7 0.006 0.027 70.021 0.1137 0.014 0.084 7 0.014 0.123 7 0.064 0.0967 0.014 0.0727 0.021
OU_13
Gloeocapsa strain 0.098 7 0.014 N.D. N.D. N.D. 0.0187 0.004 N.D. N.D. N.D. 0.0127 0.007 0.013 7 0.001 N.D. N.D. 0.0227 0.008
OU_20

ARTICLE IN PRESS
N.D. none detected.
a
No growth was detected with only dd H2O.

Table 3
Effect of rock-type on the nal dry biomass of the cyanobacteria (g/kg of rock). The cyanobacteria were grown in dd H2O, or supplemented with 10 mM (NH4)2SO4, 10 mM NaNO2, or 10 mM (NH4)2SO4 and 10 mM NaNO2, with
volcanic rock.

BG-11 Rhyolite Anorthosite Basalt

dd H2O N SO4 N + SO4 dd H2O N SO4 N +SO4 dd H2O N SO4 N+ SO4

Anabaena cylindrica 3.087 0.21 0.44 70.02 0.12 7 0.08 0.38 70.04 0.29 7 0.08 0.227 0.06 0.35 70.11 N.D. 0.35 70.12 0.497 0.09 0.147 0.06 0.42 7 0.01 0.497 0.02
Chroococcidiopsis 029 1.087 0.14 N.D. 0.45 7 0.05 0.017 0.01 0.39 7 0.05 N.D. 0.097 0.01 N.D. 0.23 70.09 N.D. 0.207 0.06 N.D. 0.337 0.11
Arthrospira platensi 2.107 0.24 N.D. N.D. N.D. 0.017 0.04 N.D. 0.047 0.02 N.D. 0.047 0.05 N.D. 0.127 0.07 N.D. 0.107 0.02
Synechococcus elongatus 1.58 7 0.41 N.D. N.D. N.D. 0.047 0.01 N.D. 0.21 70.03 N.D. N.D. N.D. 0.127 0.07 N.D. 0.697 0.03
Phormidium strain OU_10 1.407 0.32 N.D. N.D. N.D. 0.047 0.01 N.D. 0.067 0.02 N.D. N.D. N.D. 0.337 0.05 N.D. 0.117 0.03
Leptolyngbya strain OU_13 1.85 7 0.21 0.15 70.08 0.22 7 0.11 0.307 0.01 0.17 7 0.03 0.477 0.02 0.35 70.08 0.147 0.08 0.36 70.05 0.227 0.07 N.D. 0.19 7 0.08 0.467 0.01
Gloeocapsa strain OU_20 0.657 0.10 N.D. N.D. N.D. 0.037 0.01 N.D. N.D. N.D. 0.027 0.01 0.01 7 0.03 N.D. N.D. 0.027 0.01

N.D. none detected.

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and anorthosite. For example, the concentration of leached
silicate was 15.241 mM for rhyolite, compared to 75.062 and
85.421 mM for basalt and anorthosite, respectively.
The elemental concentrations in the media were greater in the
biological experiments than the non-biological controls, as seen in
Table 4. The biological effect varied between the rock-types. For
example, the potassium concentration released from the rhyolite
was similar to that obtained for the non-biological control;
whereas the potassium released from the anorthosite and basalt
in the biological experiment was double the concentration
obtained for the non-biological controls.
For each of the rock-types, the elemental concentrations varied
between the cyanobacteria species. A comparison of Tables 2 and
4 indicates that the concentration was dependent on the
cyanobacterial specic growth rate. For example the specic
growth rates and elemental concentrations, for each of the rock-
types, were highest with A. cylindrica.
3.4. Exposure experiment
The conditions during the simulated Mars experiment stayed
constant with the pressure at 0.8 70.2 kPa and the temperature at
2771 1C. Chroococcidiopsis 029, A. cylindrica, Gloeocapsa strain
OU_20, Phormidium strain OU_10, and Leptolyngbya strain OU_13
were able to survive 28 days (the maximum time examined) of
exposure; whereas, S. elongatus and A. platensis were unable to
survive 1 day, as shown in Fig. 1A. UV radiation (325400 nm)

Table 4
Effect of rock-type on the nal elemental concentrations, after 45 days of growth.

Ca (lM) Cu (lM) Fe (lM) K (lM) Li (lM) Mg (lM) Mn (lM) Na (lM) Ni (lM) Sr (lM) Zn (lM) SiO4 (lM)

Rhyolite
N2 + SO4
Arthrospira platensi 0.218 B.D. B.D. 15.006 1.413 9.330 B.D. 0.052 0.001 0.035 0.118 30.912
Synechococcus elongatus 7.632 B.D. B.D. 15.144 3.265 7.947 B.D. 0.084 0.001 0.021 0.069 29.831
Chroococcidiopsis 029 6.510 0.400 0.215 14.137 2.684 8.183 0.100 0.040 0.001 0.010 0.083 22.544
Anabaena cylindrica 11.100 0.420 2.150 21.266 6.510 8.970 B.D. 0.034 0.005 0.034 0.190 35.240
Phormidium strain OU_10 14.500 B.D. 8.150 26.540 2.527 9.543 0.052 0.243 B.D 0.155 2.454 32.214
Leptolyngbya strain OU_13 13.200 B.D. 3.284 27.450 3.983 8.950 0.003 0.160 B.D 0.060 2.583 39.142
Gloeocapsa strain OU_20 8.511 B.D. 2.154 18.512 3.871 9.543 B.D 0.147 0.004 0.047 1.412 40.992
Control (non-biological)a 0.251 0.113 1.213 13.164 2.324 4.061 0.015 0.045 B.D. 0.011 0.213 15.214

dd H2O
Anabaena cylindrica 9.637 0.533 0.394 21.783 3.190 9.520 B.D. 0.028 B.D. 0.026 0.087 32.210
Phormidium strain OU_10 15.065 B.D. 7.585 27.852 3.249 10.042 0.025 0.125 0.002 0.038 2.100 29.541
Leptolyngbya strain OU_13 16.331 B.D. 2.231 36.081 2.884 10.891 B.D 0.161 0.003 0.046 2.100 28.871
Gloeocapsa strain OU_20 7.452 B.D. 2.143 21.432 3.544 11.245 B.D 0.145 0.004 0.024 2.142 18.514
Control (non-biological)b 0.271 0.171 1.214 15.064 2.431 3.985 0.036 0.037 B.D. 0.031 0.211 15.241

Basalt
N2 + SO4
Arthrospira platensi 35.224 0.301 3.100 64.852 11.625 55.261 0.225 0.525 0.868 0.052 0.516 124.210
Synechococcus elongatus 36.800 0.500 3.500 51.653 5.853 56.742 0.351 0.262 0.826 0.064 0.613 132.241
Chroococcidiopsis 029 37.100 0.210 3.445 55.853 6.271 51.736 0.251 0.315 0.772 0.075 0.667 123.251
Anabaena cylindrica 43.500 0.651 7.540 135.240 32.254 61.251 0.022 0.615 0.006 0.242 1.515 141.046
Phormidium strain OU_10 55.250 6.500 6.510 61.250 16.250 54.500 0.541 0.310 0.350 0.161 1.010 131.214
Leptolyngbya strain OU_13 49.500 4.540 7.410 63.250 15.240 59.150 0.487 0.162 0.014 0.147 0.954 124.210
Gloeocapsa strain OU_20 51.242 1.241 4.512 55.241 15.241 54.251 0.645 0.241 0.514 0.098 0.654 111.254
Control (non-biological) a 29.142 0.145 0.344 25.154 0.985 51.214 B.D. 0.097 B.D. 0.012 0.306 72.412

dd H2O
Anabaena cylindrica 61.481 B.D. 6.881 125.042 21.997 55.232 B.D. 0.492 B.D. 0.642 1.369 125.351
Phormidium strain OU_10 42.477 5.126 5.498 56.167 11.025 55.264 0.625 0.214 0.115 0.140 1.115 121.323
Leptolyngbya strain OU_13 54.164 6.136 5.502 54.364 15.036 56.354 0.415 0.232 0.231 0.120 1.234 115.211
Gloeocapsa strain OU_20 49.522 2.211 4.541 56.241 16.241 55.241 0.741 0.254 0.641 0.087 0.741 98.542
Control (non-biological) b 31.210 B.D. 0.265 27.250 1.247 54.254 B.D. 0.148 B.D. 0.032 0.410 75.062

Anorthosite
N2 + SO4
Arthrospira platensi 32.654 0.842 1.154 41.551 16.284 42.522 0.416 0.162 0.001 0.584 0.010 105.225
Synechococcus elongatus 36.584 2.211 1.524 39.221 19.526 43.584 0.436 0.173 0.016 0.612 0.106 108.540
Chroococcidiopsis 029 25.987 0.312 1.536 35.262 16.516 44.784 0.683 0.013 0.033 0.485 0.002 113.214
Anabaena cylindrica 43.541 0.452 1.541 52.210 24.251 49.854 0.125 0.046 0.028 0.415 2.510 124.592
Phormidium strain OU_10 32.432 0.264 1.655 45.210 14.253 47.435 0.893 0.217 0.032 0.213 1.698 91.241
Leptolyngbya strain OU_13 26.514 0.242 1.798 35.250 13.250 37.351 0.740 0.314 0.048 0.117 1.541 97.210
Gloeocapsa strain OU_20 19.871 0.192 2.144 32.251 16.254 18.432 0.417 0.241 0.041 0.087 1.654 98.214
Control (non-biological) a 8.231 0.142 1.451 15.843 3.731 25.132 0.017 0.014 B.D. 0.014 0.110 82.541

dd H2O
Anabaena cylindrica 36.844 0.312 1.416 41.226 22.125 39.841 0.131 0.023 0.014 0.202 2.183 104.212
Phormidium strain OU_10 22.562 0.212 1.872 41.261 12.214 32.412 0.980 0.120 0.041 0.216 2.143 112.412
Leptolyngbya strain OU_13 10.552 0.322 1.972 39.415 13.152 39.332 1.146 0.212 0.024 0.144 2.114 98.142
Gloeocapsa strain OU_20 17.845 0.221 1.452 33.251 17.451 32.142 0.521 0.187 0.014 0.097 1.871 80.210
Control (non-biological) b 2.541 0.154 1.365 16.711 3.338 26.432 0.040 0.026 B.D. 0.016 0.105 85.421

B.D. below detection.

a
The non-biological control contained rock with 10 mM NaNO2 and 10 mM (NH4)2SO4.
b
The non-biological control contained rock and dd H2O.
 ARTICLE IN PRESS
1284 K. Olsson-Francis, C.S. Cockell / Planetary and Space Science 58 (2010) 12791285

4. Discussion
100
The development of successful techniques to utilise and
exploit resources on the Moon and Mars has been highlighted
80 by the development of the NASA In-Situ Resource Utilization
(ISRU) group and the ESA Geomicrobiology for Space Settlement
and Exploration (GESSE), Topical Team. The use of regolith on
Survival (%)

60 other planetary surfaces, which is rich in inorganic elements, has

major implications for many applied problems, such as oxygen
40 production, fuel production, biomass production, nutrient extrac-
tion, and feedstock (Hendrickx and Mergeay, 2007; Lehto et al.,
2006).
20 The regolith of the Moon and Mars has been extensively
characterised (McSween et al., 2009; Ohtake et al., 2009; Ashwal,
1993). It is widely accepted that the lunar highland regolith is
0
predominately composed of aluminosilicate basic rocks, which
0 10 20 30
are mainly anorthosites, nortic anorthosites, and gabbroic
anorthosites (Ashwal, 1993). The Mars regolith is mostly basalt,
100 dominated by tholeiitic basalts (Fink, 1980; McSween et al.,
2009). Terrestrial anorthosite and basalt were used as anologs for
this study. Furthermore, rhyolite, which is rich in silicate, was
80
employed as an additional rock-type. Although rhyolite is not a
major component of the Moon or Mars regolith, the high silica
Survival (%)

60 content would be expected to retard cation release and so it can

be used to explore the effects of silica on nutrient availability to
an added biota.
40 For this investigation, we examined a variety of cyanobacteria
as potential candidates for in-situ resource use. All of the
20 cyanobacteria grew optimally and produced the greatest biomass
with the basalt; whereas, growth was limited with the rhyolite.
The effect of the cyanobacteria on the dissolution of the rhyolite
0 was also low compared to the basalt and anorthosite. This may
0 10 20 30 reect the fact that rhyolitic rocks are not as easily weathered by
microorganisms. High silica content requires greater energy to
100 disrupt the covalent SiO bonds or to break apart the silica
tetrahedra and access the biologically important cations, which
are also present at a lower concentration in rhyolitic rocks
80 compared to rocks of basaltic composition (Herrera et al., 2008).
Elemental dissolution from the anorthosite and the basalt
varied for each element. The release of calcium, iron, potassium,
Survival (%)

60
magnesium, nickel, sodium, and zinc was greatest from the basalt.
However, the dissolution of copper was greatest with the
40 anorthosite. Therefore on planets with anorthosite and basalt,
such as the Moon, basalt would be used predominantly to yield
organic matter; however, anorthosite could be utilised for the bio-
20 mining of bio-essential elements, such as copper.
Of particular interest for applications on other planetary
surfaces are the nitrogen-xing cyanobacteria. Our results
0
0 10 20 30 demonstrate that certain genera of nitrogen-xing cyanobacteria
could grow on locally available regolith, such as basalt or
Exposure (d)
anorthosite, eliminating the requirement for complex growth
Fig. 1. Effect of (A) Mars simulation conditions, (B) desiccation and (C) vacuum on media. Furthermore, the nitrogen and organic matter produced by
the survival of S. elongatus (W), Chroococcidiopsis (&), A. cylindrica (K), A. platensi the cyanobacteria, as well as the elements released from the
(J), Phormidium strain OU_10 (E), Leptolyngbya strain OU_13 (m), and Gloeocapsa regolith, could be utilised by other microorganisms in-situ.
strain OU_20 ().
Although this work was focused on the growth of cyanobac-
teria, the ability to survive exposure to the ambient environment
is benecial in case of system malfunction. Furthermore,
was lethal and none of the cyanobacteria were able to survive desiccated vegetative-state cells are also an ideal method for
1 min of exposure. transportation and storage. The ndings in this paper demon-
The cyanobacteria were also exposed to desiccation and strate that the cyanobacteria, A. cylindrica, Chroococcidiopsis
vacuum conditions. Vacuum was more detrimental to the 029, Gloeocapsa strain OU_20, Leptolyngbya strain OU_13, and
cyanobacteria than desiccation. For example, A. cylindrica sur- Phormidium strain OU_10, are resistant to desiccation. The
vived 1 day of vacuum compared to 28 days (the maximum time desiccated forms survived exposure to Mars simulation condi-
examined) of desiccation, as shown in Fig. 1B and C. Notably, tions, therefore suggesting that on Mars, the desiccated cells could
S. elongatus and A. platensis were unable to survive desiccation or be stored in containers that are only covered by a UV shield;
vacuum. whilst on the Moon, they would need to be stored in a pressurised
 ARTICLE IN PRESS
K. Olsson-Francis, C.S. Cockell / Planetary and Space Science 58 (2010) 12791285
container, to protect them from the effects of vacuum. It must be
noted that certain genera of heterocystous cyanobacteria, such as
A. cylindrica, produce akinetes, specialised spore-like cells, under
stressed conditions that are resistant to adverse conditions such
as desiccation, low temperature, Mars simulation conditions, and
low Earth orbit (Adams and Duggan, 1999; Li and McClane, 2006;
Setlow, 1995; Olsson-Francis et al., 2009).
From the cyanobacteria used in this study, A. cylindrica would
be the ideal cyanobacterium to utilise in space applications. The
cyanobacterium produced the maximum biomass, and the great-
est specic growth rate, on all of the rock-types. The optimal
values were obtained with basaltic rock, which is found on both
the Moon and Mars. This correlated with the element release data.
Overall, the elemental dissolution of all of the rock-types was
greatest with A. cylindrica. Therefore, A. cylindrica would not only
be an ideal candidate for producing organic matter, oxygen, and
nitrogen but also for bio-mining of bio-essential elements.
Furthermore, desiccated cells or akinetes of A. cylindrica could
be used for long-term storage and transportation.
The development of successful techniques to utilise and
exploit resources on the Moon and Mars has major implications
for future missions. The extraction of elements, particularly by
phototrophs, from the regolith is a useful method for exploiting
in-situ resources. In this paper, we demonstrated that cyanobac-
teria can utilise terrestrial volcanic rocks (analogous to lunar and
Mars regolith) as growth substrates resulting in elemental release
and microbial biomass production. We therefore conclude that
cyanobacteria are ideal candidates for space applications.
Acknowledgements
This work was supported by a STFC Grant (PP/E001408/1) to
Prof. C.S. Cockell. We would like to thank Dr. Manish Patel for his
help with the Mars simulation chamber. Finally, we would like to
thank Dr. Paul Buchanan for the anorthosite used in this paper.
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