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Survival of Rock-Colonizing Organisms
After 1.5 Years in Outer Space
Silvano Onofri,
1
Rosa de la Torre,
2
Jean-Pierre de Vera,
3
Sieglinde Ott,
4
Laura Zucconi,
1
Laura Selbmann,
1
Giuliano Scalzi,
1,5
Kasthuri J. Venkateswaran,
5
Elke Rabbow,
6
Francisco J. Sa´ nchez In˜ igo,
2
and Gerda Horneck
6
Abstract
Cryptoendolithic microbial communities and epilithic lichens have been considered as appropriate candidates
for the scenario of lithopanspermia, which proposes a natural interplanetary exchange of organisms by means of
rocks that have been impact ejected from their planet of origin. So far, the hardiness of these terrestrial organisms
in the severe and hostile conditions of space has not been tested over extended periods of time. A first long-term
(1.5 years) exposure experiment in space was performed with a variety of rock-colonizing eukaryotic organisms
at the International Space Station on board the European EXPOSE-E facility. Organisms were selected that are
especially adapted to cope with the environmental extremes of their natural habitats. It was found that some—
but not all—of those most robust microbial communities from extremely hostile regions on Earth are also
partially resistant to the even more hostile environment of outer space, including high vacuum, temperature
fluctuation, the full spectrum of extraterrestrial solar electromagnetic radiation, and cosmic ionizing radiation.
Although the reported experimental period of 1.5 years in space is not comparable with the time spans of
thousands or millions of years believed to be required for lithopanspermia, our data provide first evidence of the
differential hardiness of cryptoendolithic communities in space. Key Words: Astrobiology—Lithopanspermia—
Radiation resistance—Survival—Vacuum. Astrobiology 12, 508–516.
1. Introduction
The lithopanspermia hypothesis suggests that impact-
ejected rocks could transfer living organisms through
space from one planet to another. This scenario implies that
rock-embedded organisms need to survive the following
three phases: first (phase-I), the ejection into space inside rock
fragments due to an impact of a cosmic projectile on one
planet (Melosh, 1984); second (phase-II), the journey through
space for a long time (hundreds, even thousands or millions,
of years) (Gladman et al., 1996); and last (phase-III), the cap-
ture by and landing on another planet (Mileikowsky et al.,
2000; Horneck et al., 2008; Nicholson, 2009). This hypothesis
dates back to Lord Kelvin’s presidential address to the British
Association in 1871 (Thomson, 1871). However, it was dis-
missed by most contemporary scientists at the time because
the general opinion was that outer space would kill any living
being exposed to it and there was no way to test the idea
experimentally. Another severe criticism was that such a hy-
pothesis just shifts the problem of the origin of life to another
planet. Only in the last decade, after the detection of several
meteorites that originated from Mars (Nyquist et al., 2001;
Fritz et al., 2005; Shuster and Weiss, 2005; The Meteoritical
Society, 2011), has lithopanspermia been seriously considered
again (Sancho et al., 2007; Sto
¨ffler et al., 2007; Horneck et al.,
2008, 2010; Nicholson, 2009; de la Torre et al., 2010).
Shock recovery experiments performed to test phase-I of
lithopanspermia showed that spores of Bacillus subtilis and
the lichen Xanthoria elegans could survive pressures up to
40 GPa, which are comparable to those experienced by the
martian meteorites (Sto
¨ffler et al., 2007; Horneck et al., 2008).
Space technology provided the opportunity to study a vari-
ety of biological specimens after exposure to space. Among
the systems tested, bacterial spores (B. subtilis) and the
1
Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
2
Department of Earth Observation, Spanish Aerospace Research Establishment-INTA, Torrejo
´n de Ardoz, Madrid, Spain.
3
Institute of Planetary Research, German Aerospace Center (DLR), Berlin, Germany.
4
Institut fu
¨r Botanik, Heinrich-Heine-Universita
¨t, Du
¨sseldorf, Germany.
5
Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California, USA.
6
Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany.
ASTROBIOLOGY
Volume 12, Number 5, 2012
ªMary Ann Liebert, Inc.
DOI: 10.1089/ast.2011.0736
508
lichens Rhizocarpon geographicum and X. elegans stood out
due to their high resistance to the hostile space environment
(Sancho et al., 2007; de la Torre et al., 2010; Horneck et al.,
2010). However, eukaryotes have never been studied after
long-term exposures to space conditions.
To investigate the fate of lithic organisms and communi-
ties during long-term travel in space, we used ESA’s
EXPOSE-E facility (Rabbow et al., 2009, 2012). This facility
was attached to the balcony of the Columbus module of the
International Space Station (ISS) (Fig. 1A). EXPOSE-E was
designed to expose a variety of biological systems to selected
parameters of space over time spans of one year and more.
All biological test systems of the LIFE (Lichens and Fungi
Experiment) experiment were rock-dwelling organisms from
hostile regions: Antarctic cryptoendolithic (dwelling inside
rocks) communities in their natural sandstone, microcolonial
black cryptoendolithic fungi (Cryomyces antarcticus and
Cryomyces minteri) isolated from Antarctic sandstone, and
high mountain epilithic lichens (R. geographicum and X. ele-
gans) (de Vera et al., 2003, 2008; Selbmann et al., 2005; Sancho
et al., 2007; de la Torre et al., 2010). These lichens were se-
lected as test systems due to their high resistance to space
conditions demonstrated during short-term (10–16 days)
exposures (Lichens experiment on ESA’s Foton-M2 Mission
2005 and Lithopanspermia experiment on ESA’s Foton-M3
Mission 2007) (Sancho et al., 2007; de la Torre et al., 2010). The
biological samples were accommodated in small chambers
(1.4 cm in diameter) of the EXPOSE-E facility (Fig. 1B).
During the space mission, they were exposed either to the
full space environment (vacuum from 10
-7
to 10
-4
Pa,
fluctuations of temperature between -21.5C and +59.6C,
cosmic ionizing radiation up to 190 mGy, and solar extra-
terrestrial electromagnetic radiation up to 6.34 ·10
8
Jm
-2
)or
they were shielded from insolation. After 1.5 years in space,
the samples were retrieved and their viability investigated.
During the mission, the Sun-exposed LIFE samples were
exposed to 1879 estimated solar constant hours (Rabbow
et al., 2012).
2. Material and Methods
2.1. Experiment hardware and biological
samples of the LIFE experiment on board
the International Space Station
The EXPOSE-E facility is part of the European Technology
Exposure Facility (EuTEF) (Fig. 1A), which was designed for
testing different materials under selected parameters of
space. On 7 February 2008, EuTEF with EXPOSE-E accom-
modating the biological samples of the LIFE experiment (Fig.
1B) was launched on board Space Shuttle STS-122 for the ISS.
On 15 February 2008, EXPOSE-E was mounted onto the
outside balcony of the Columbus module by extravehicular
activity. EXPOSE-E was decommissioned on 1 September
2009, retrieved by extravehicular activity on 2 September
2009, and returned to Earth on 12 September 2009 with STS-
128. During the 1.5 years mission, the samples were exposed
to space vacuum (10
-7
to 10
-4
Pa) (Horneck et al., 2010),
galactic cosmic radiation ( £190 mGy) (Berger et al., 2012),
and the full spectrum of solar extraterrestrial electromagnetic
radiation (k>110 nm) with fluences of 9.19 ·10
5
Jm
-2
(below
a 0.1% transmission neutral density filter) and 6.34 ·10
8
Jm
-2
(100% transmission insolated samples). All fluences were
calculated for the biologically active UV range of 200 nm <
k>400 nm, depending on the orientation of the ISS to the Sun.
Temperature varied between -21.5Cand +59.6C (Rabbow
et al., 2012).
2.2. Test systems of the LIFE experiment
The lichen Xanthoria elegans was collected from alpine
habitats between 2000 and 3000 m altitudes at Zermatt,
Switzerland (4559¢N, 748¢E) (de Vera et al., 2003, 2008);
FIG. 1. Experiment hardware and biological samples of the LIFE experiment. (A) EXPOSE-E facility (arrow) attached to the
Columbus module of the ISS during orbital flight, accommodating the LIFE samples. (B) Samples of the LIFE experi-
ment accommodated in one of the compartments of the EXPOSE-E facility (well diameter 1.4 cm). Vertical rows show
X. elegans (b1) and R. geographicum (b2) on their natural rock habitat; dried cultures of the lichen fungus (mycobiont) of X.
elegans (1
st
and 3
rd
sample from the top in b3); sandstone fragments colonized by a stratified cryptoendolithic microbial
community (2
nd
and 4
th
samples from the top in b4), and the fungi C. antarcticus and C. minteri (from the top 2
nd
and last
sample in b3, upper and 3
rd
sample in b4). Color images available online at www.liebertonline.com/ast
ROCK-COLONIZING ORGANISMS IN SPACE 509
Rhizocarpon geographicum was collected from the Plataforma
de Gredos, Spain (4017¢N, 514¢W) at an altitude of 2020 m
(de la Torre et al., 2010); sandstone fragments colonized by a
stratified cryptoendolithic microbial community were col-
lected by L. Zucconi at Battleship Promontory (7654¢37.6†S,
16055¢27.5†E), Southern Victoria Land, Antarctica, in Janu-
ary 2004; and the microcolonial black yeast-like fungi Cryo-
myces antarcticus CCFEE 515 and Cryomyces minteri CCFEE
5187, both dwelling cryptoendolithically, were isolated from
sandstone collected in McMurdo Antarctic Dry Valleys
(Selbmann et al., 2005). The survival of those cryptoendolithic
organisms is of special interest in terms of lithopanspermia
because rocks may supply an additional external protection
to face the impact-driven ejection into space (Horneck et al.,
2008; Meyer et al., 2011) and transfer from one planet to
another.
2.3. Viability assays of the LIFE test systems
The photosynthetic activity of the lichens X. elegans and
R. geographicum was measured after reactivating the samples
in a climatic chamber under the following controlled con-
ditions: constant temperature of 10C, 12 h light and 12 h
dark cycles for 96 h (X. elegans)or72h(R. geographicum).
Irradiation with photosynthetically active light was per-
formed by using a mercury lamp with a 100 lmol m
-2
s
-1
photosynthetic photon flux density. For rehydration, sam-
ples were sprayed twice a day with deionized water. After
reactivation, the activity of the photosystem II (PSII) of the
photobiont was measured with a Mini-PAM fluorometer
(Heinz Walz GmbH), as described previously (Sancho et al.,
2007; de Vera et al., 2010). Lichens were rewetted immedi-
ately before each measurement. The optimum quantum
yield of chlorophyll awas determined by fluorescence
measurements after 20 min of dark adaptation, according to
the equation
Fv=Fm ¼(Fm Fo)=Fm
with Fv =variable fluorescence yield, Fm =maximal fluores-
cence yield, and Fo =minimal fluorescence yield. This opti-
mum quantum yield of PSII was taken as an indication of the
PSII activity of the photobiont of the lichen system after the
exposure to space parameters. The percentage (n=2 for space
100% insolated and for space 0.1% insolated, and n=4 space
dark samples) of PSII activity was determined from the ratio
of the Fv/Fm of the same flight sample before and after
flight. Control data are given for the same sample measured
before spaceflight (preflight).
The survival of C. antarcticus and C. minteri was determined
from their colony-forming ability as percentages of colony-
forming units (CFU). Growth tests were performed by sus-
pending fungal cells from rehydrated colonies in a 0.9% NaCl
solution, inoculating them on malt agar Petri dishes (five
replicates), and incubating them at 15C for 30 days. Control
data were obtained from fresh colonies. Statistical analyses
were performed by one-way analysis of variance (ANOVA)
and pairwise multiple comparison procedure (Tukey test),
which was carried out by using the statistical software Sig-
maStat 2.0 ( Jandel, USA). The means (n=5) –standard devi-
ation (s.d.) are plotted. *P=0.001; power of performed test
with a=0.050: 1.000.
The propidium monoazide (PMA) assay was used to
check the integrity of the cell membranes after spaceflight.
Fractions of DNA extracted from intact cells of cryptoendo-
lithic fungi C. antarcticus and C. minteri colonies were com-
pared with the fraction of DNA extracted from cells isolated
from the space-exposed colonized sandstone fragments. It
was performed by adding PMA (Biotium, Hayward, CA) at a
final concentration of 200 lMto the rehydrated fungal
colonies or to powdered rock suspensions in phosphate-
buffered saline solution. PMA penetrates only damaged cell
membranes, cross-links then to DNA after light exposure,
and thereby prevents polymerase chain reaction (PCR). Fol-
lowing DNA extraction and purification (Maxwell 16 auto-
mated DNA extraction instrument, Promega, Madison, WI),
quantitative PCR (Bio-Rad CFX96 real time PCR detection
system) was used to quantify the number of fungal internal
transcribed spacer (ITS) ribosomal DNA fragments present
in both PMA treated and non-treated samples. For all reac-
tions, 1 lL of purified genomic DNA was added to 23 lLof
PCR cocktail containing 1X Power Sybr-Green PCR Master
Mix (Applied Bios, Foster City, CA), as well as NS91 forward
(5¢-gtc cct gcc ctt tgt aca cac-3¢) and ITS51 reverse (5¢-acc ttg
tta cga ctt tta ctt cct c-3¢) primers, each at 0.02 Mfinal con-
centration. These primers amplify a 203 bp product spanning
the 18S/ITS1 region of rRNA-encoding genes.
A standard quantitative PCR cycling protocol consisting of a
hold at 95C for 10 min, followed by 40 cycles of denaturing at
95C for 15 s, annealing at 58C for 20 s, and elongation at 72C
for 15 s, was performed. Fluorescence measurements were re-
corded at the end of each annealing step. At the conclusion of
the 40
th
cycle, a melt curve analysis was performed by re-
cording changes in fluorescence as a function of raising the
temperature from 60Cto95Cin0.5C per 5 s increments.
These protocols were applied to the processing of both fungal
colonies and cryptoendolithic sandstone samples.
Control data were obtained for an identical sample stored on
ground in the laboratory during the mission (Control). Because
cryptoendolithic communities are not uniformly distributed
within the rocks, different quantities of total fungal DNA were
obtained from different rock samples.
Space data are given for samples shielded from extraterres-
trial UV radiation (Space Dark), exposed to k>110 nm at a
fluence of 9.19 ·10
5
Jm
-2
beneath a 0.1% transmission MgF
2
filter (Space 0.1% insolated), or at a higher fluence of 6.34·10
8
J
m
-2
(Space 100% insolated). Statistical analyses were per-
formed by one-way ANOVA and pairwise multiple compari-
son procedure (Tukey test), which was carried out by using the
statistical s oftware SigmaStat 2.0 ( Jandel, USA) *P=0.001;
**P>0.05. Power of performed test with a=0.050: 1.000. The
means (n=3) and –s.d. are plotted.
2.4. Confocal laser scanning microscopy (CLSM)
imaging: viability of X. elegans analyzed by LIVE/
DEAD staining kit FUN 1
Adult lichen thallus, young thallus, and the isolated my-
cobiont of X. elegans were stained by FUN 1 to determine
their viability. A green and yellow color of the cells indicates
that they still maintain vitality. A change from green to
yellow in the cytoplasm and from green to red in the vacu-
oles is an indication of physiological activity expressed by
accumulation of the dye in the vacuoles. Dead cells cannot
510 ONOFRI ET AL.
be stained; therefore, red crystals are not formed in the
vacuoles.
Instruments used for imaging are as follows: LSM 510
META of Carl Zeiss Mikroskopsysteme Jena GmbH with
objective lenses 10 ·and 40 ·/oil immersion, scanning res-
olution 1024 ·1024 pixels and time 64 ls to 1.76 ls, scan
zoom 0.7 ·-2.8, image bit depth 12 bit, experimental tem-
perature condition 22C, UV-laser with emission wavelength
488/561/633 nm and VIS-laser HeNe with 633 nm excitation
and 5 mW power, filters with ChS1 679–754; Ch2 BP 505–550;
Ch3 BP 575–615. For operation of the instrument, the Carl
Zeiss Jena GmbH software system at the Institute of Genetics
of the Heinrich-Heine-Universita
¨tDu
¨sseldorf was used.
3. Results
First visual inspections assured that the ‘‘space samples’’
had not changed in shape and color compared to their pre-
flight appearances. Tailored to each test system, different
viability assays were applied: (i) photosynthetic activity of
the lichenized alga (photobiont) of X. elegans and R. geo-
graphicum (Fig. 2); (ii) colony-forming ability of C. antarcticus
and C. minteri (Fig. 3A and 3B); (iii) fraction of DNA am-
plified from intact cells of C. antarcticus and C. minteri and of
cryptoendolithic communities inside sandstone fragments
(Fig. 3C, 3D, and 3E); and (iv) viability of X. elegans and the
fungus of the lichen (mycobiont, cultured without the algal
symbiont and dried) by means of vital staining (Fig. 4).
Among those space samples that were shielded from ex-
traterrestrial insolation but exposed to space vacuum, cosmic
radiation, and temperature fluctuations (Space Dark in Figs.
2 and 3), the lichen X. elegans excelled by a PSII activity of
99.35 –0.59% compared to the preflight data of the same
samples (Fig. 2A). This high viability was not reached by the
other space test systems kept in the dark during the mission:
2.46 –1.39% for the PSII activity of the lichen R. geographicum
(Fig. 2B), and 8.04 –3.05% and 0.13 –0.07% surviving cells for
C. antarcticus and C. minteri, respectively (Fig. 3A and 3B).
PMA assay showed 98 –4.67% and 8.14 –0.35% of DNA
amplified from intact cells from colonies of C. antarcticus and
C. minteri, respectively (Fig. 3C and 3D), in comparison to
total extracted DNA; the percentage of DNA amplified from
intact fungal cells in sandstone fragments was 18 –0.18%
(Fig. 3E).
The viability of culturable cells was not further decreased
in the black fungi (12.5 –4.11% for C. antarcticus and
0.46 –0.24% for C. minteri) that were insolated with the full
extraterrestrial spectrum (k>110 nm) of the Sun, having
AB
FIG. 2. Viability of the lichens (A)X. elegans and (B)R. geographicum, determined by fluorescence measurements of the
photosynthetic activity of the photobiont. Control data are given for the same sample measured before spaceflight (preflight), as
100% viability. Space data are given for samples shielded from extraterrestrial UV radiation (Space Dark), and insolated
(k>110 nm) at a fluence of 6.34 ·10
8
Jm
-2
(Space 100% insolated). Color images available online at www.liebertonline.com/ast
ROCK-COLONIZING ORGANISMS IN SPACE 511
*
*
*
*
AB
FIG. 3. Viability of the fungi (A)C. antarcticus and (B)C. minteri, as percentage of survival, determined as CFU. Control data
were obtained from fresh colonies. No CFU were obtained from the samples ‘‘Space 100% insolated.’’ The means–s.d. are
plotted. *P=0.001. Power of performed test with a=0.050: 1.000. ITS rDNA fragments amplified from intact cells of C.
antarcticus and C. minteri (Cand D) colonies and from sandstone fragments (E) after different space conditions, compared to
total extracted DNA. PMA penetrates only damaged cells’ membranes, cross-linking to DNA after light exposure and thereby
preventing PCR amplification. Following DNA extraction and purification, quantitative PCR was used to quantify the
number of fungal ITS rDNA fragments (203 bp) amplifiable in samples treated and non-treated with PMA. The means (n=3)
and –s.d. are plotted. Statistical significance was calculated by using the Tukey test. *P=0.001. **P>0.05. Power of performed
test with a=0.050: 1.000. Color images available online at www.liebertonline.com/ast
512 ONOFRI ET AL.
received an UV irradiation of 9.19 ·10
5
Jm
-2
(Space 0.1%
insolated, Fig. 3A and 3B). The highest fraction of intact
fungal cells (35 –0.15%) occurred in a sandstone sample that
had received the full influx of solar electromagnetic radiation
of 6.34 ·10
8
Jm
-2
(Space 100% insolated, Fig. 3E). When
comparing all test systems exposed to outer space, including
the high influx of full solar extraterrestrial radiation
(6.34 ·10
8
Jm
-2
), again the lichen X. elegans, with a PSII
activity of 45 –2.50% (Fig. 2A) and C. antarcticus with
80 –0.82% of DNA from intact cells (Fig. 3C), were the most
resistant test systems of LIFE. However, black Antarctic
cryptoendolithic fungi lost colony-forming ability after ex-
posure to full insolation; no survivors were detected in the
space 100% insolated samples. Resistance of X. elegans and
its mycobiont after exposure to the combined action of all
space parameters tested, including full insolation, was con-
firmed by vital staining and CLSM. Figure 4 shows the high
capacity of the lichenized fungus (part of the symbiotic li-
chen association) in a young thallus (B), and of the lichen
fungus of X. elegans in pure culture (C), to resist space ex-
posure. The fungal cells in the cortex (Cx) of the young
thallus were still vital, although a protecting mucilage layer
had not been formed, and the parietin layer appeared to be
very thin. The same features appeared to (C), the fungal cells
of the mycelium.
4. Discussion
The LIFE experiment provided for the first time data on
the viability of rock-dwelling organisms and microbial
communities after a long-term exposure to space parameters.
These conditions cannot easily be simulated in the labora-
tory, if at all. The test systems collected from hostile condi-
tions, such as Antarctica and high mountain regions, are
FIG. 3. Continued.
ROCK-COLONIZING ORGANISMS IN SPACE 513
adapted to cope with high radiation intensities, arid phases,
and extreme temperature fluctuations that are in some ways
similar to those experienced in space. In C. antarcticus, for
example, globular cells are enveloped in a thick melanized
cell wall, which protects them from radiation and desicca-
tion. Their meristematic way of producing colonies (i.e., di-
viding in all directions) further supports their resistance
(Onofri et al., 2008, 2009; Selbmann et al., 2005; Sterflinger,
2005). Special protection against environmental extremes is
also granted for the photobiont, the green alga Trebouxia sp.
of the lichen X. elegans. In this symbiotic organization, the
fungus forms a cortex with an upper layer encrusted with
parietin and a mucilage layer that envelopes the algal cells in
a medulla matrix (Fig. 4A and 4B) (de Vera et al., 2003, 2008).
All organisms selected for the LIFE experiment are poi-
kilohydric, that is, they are able to dehydrate till most bio-
chemical activities stop. In this state, they are highly tolerant
to stresses and may resume their metabolism once water
becomes available again. Specifically, exposure to vacuum
should inhibit any oxidative process related to their metab-
olism; this particular effect could protect against other
damaging effects induced, for example, by solar UV radia-
tion, cosmic ray ions, and temperature extremes.
The LIFE experiment has demonstrated that some, but not
all, of those most robust microbial communities from ex-
tremely hostile regions on Earth are also partially resistant
against the even more hostile environment of outer space. In
this experiment, the following species stood out as the most
persistent survivors after 1.5 years in outer space: the black
fungus C. antarcticus (as determined from PMA assay) and
the symbiotic X. elegans (as determined from PSII activity)
and its mycobiont (as determined by LIVE/DEAD staining).
However, the CFU test did not yield any survivors of C.
antarcticus flight samples that were exposed to the un-
attenuated solar extraterrestrial spectrum (space 100% in-
solated) and less than 10% survivors for the space dark
samples. This means that even if the cell membrane seemed
to be intact, as indicated by the PMA test, the cells had lost
their ability to grow and divide.
Earlier studies have shown that the circumpolar and al-
pine red lichen X. elegans was able to retain its photosyn-
thetic activity almost completely after 14 days in space
(Sancho et al., 2007; de la Torre et al., 2010). This observation
was confirmed in the present study for a much longer ex-
posure time of 565 days in space, although this high viability
was only observed for the lichen that had been shielded from
solar electromagnetic irradiation, maintaining 45% PSII ac-
tivity after 100% insolation exposure. CLSM, in combination
with the use of specific fluorescent probes, allowed for the
assessment of the physiological state of the cells. Particularly,
the mycobiont seemed to play a fundamental role in main-
taining the viability of the entire lichen system, because 565
days in space appeared not to have any effect on its physio-
logical activity, even after 100% insolation (Fig. 4C). These
observations are in agreement with earlier ones from short-
term space exposures (de la Torre et al., 2010); in those studies
the spores also maintained a high germination capacity, even
after exposure to the full spectrum of solar UV radiation.
However, so far, the ability of X. elegans to reproduce after this
long-term space exposure remains an open question.
Interestingly, it has been earlier shown that X. elegans also
resisted shock pressures comparable to those experienced by
the martian meteorites during impact ejection (Horneck et al.,
2008; Meyer et al., 2011), as required for phase-I of litho-
panspermia.
Although we have demonstrated that some rock-dwelling
species are capable of partially withstanding the harsh en-
vironment of outer space, or certain parameters of it, for at
least 1.5 years, the data are insufficient for drawing any
consequences for the likelihood of lithopanspermia. The
possibility of surviving a much longer journey in space, as
would be required for natural travel from Mars to Earth or
vice versa, still remains an open question. This especially
applies to organisms that dwell at the surface of rocks, like
the lichen X. elegans, which would be fully exposed to the
lethal spectrum of solar extraterrestrial UV radiation during
a hypothetical interplanetary transfer. The only one data point
at an exposure time of 1.5 years, resulting in a viability of
45 –2.50%, as determined by PSII activity, does not allow any
extrapolation over hundreds, thousands, or even millions of
years, as would be required for lithopanspermia (Gladman
et al., 1996). The situation would be much more favorable for
FIG. 4. CLSM imaging: viability of X. elegans analyzed by LIVE/DEAD staining kit FUN I. (A) Adult lichen thallus, (B)
young thallus, and (C) lichen fungus isolated in pure culture (mycobiont) of X. elegans, all dried and exposed to 100%
insolation and space vacuum. Green to yellow cells are stained by FUN 1, indicating vital cells. Turning from green to yellow
and finally to red indicates physiological activity expressed by the accumulation of the dye in the vacuoles. The high degree
of maintained viability is due to a cortex Cx, a mucilage layer Mc, and crystal deposits of parietin P on the surface of the
lichen. These layers are able to protect interior cells of the algal layer A and the medulla M.
514 ONOFRI ET AL.
endolithic organisms, because they would be shielded from
solar UV radiation by the surrounding rock material. How-
ever, the cryptoendolithic organisms tested in this study did
not show a high survival rate in space, even if shielded from
solar UV radiation. Our experiments also demonstrate that
outer space can act as a selection pressure on the composition
of microbial communities (Cockell et al., 2011). Further studies
are needed with more resistant communities. An example is
the vagrant lichen species Aspicilia fruticulosa that, after 10
days in space, showed a complete recovery of space-induced
damage after 72 h of reactivation (Raggio et al., 2011).
Natural transfer of microorganisms between planets via
lithopanspermia could have occurred over the course of
billions of years (Gladman et al., 1996; Nicholson, 2009). In
the last 50 years, human activities in space exploration have
become another potential source of spreading microorgan-
isms between planets. Nearly 40 robotic missions have been
launched with Mars as their destination (Horneck et al.,
2007). To prevent the introduction of microbes from Earth to
another celestial body or vice versa, a concept of contami-
nation control has been elaborated by the Committee on
Space Research (COSPAR) under consideration of specific
classes of mission/target combinations, which have been
recommended to be followed by each space-faring organi-
zation (COSPAR, 2011). Lander missions to Mars require
especially strict measures of cleanliness and partial sterility
of the spacecraft (COSPAR, 2011). The test systems that have
been used in the LIFE experiment, though unlikely to be
found on spacecraft, can be considered as models for dem-
onstrating how life, if accidentally transferred from Earth to
outer space, may resist and contaminate other celestial
bodies and planets (i.e., Mars) and thereby interfere with
future life-detection missions.
Acknowledgments
We thank the staff at the European Space Agency for the
provision and operations of the EXPOSE-E facility and
Thomas Berger for the cosmic ray dosimetry data. We also
thank the Italian National Program of Antarctic Research
and National Antarctic Museum for funding the collection of
Antarctic samples, strains, and sample analyses, as well as
the European Coordination Action for Research Activities on
life in Extreme Environments (CAREX) for one ToK Grant to
G.S. Collection and preparation of samples, and CLSM and
photosynthetic activity analyses of X. elegans have been
supported partly by the Helmholtz Association through the
research alliance ‘‘Planetary Evolution and Life’’ and the
BMWi, 50WB0614; the selection and preparation of the Rhi-
zocarpon geographicum samples have been financed by the
Space Program of the Spanish Ministry of Science and Edu-
cation (ESP2005-25292-E) and INTA. We thank Philipp
Holzwig, Isabella Halezki, Eva-Maria Posthoff, and the In-
stitute of Genetics at the Heinrich-Heine-Universita
¨tDu
¨s-
seldorf for skillful technical assistance. Special thanks also go
to Prof. Giovanni Bignami for his scientific advice.
Author Disclosure Statement
No competing financial interests exist for Silvano Onofri,
Rosa de la Torre, Jean-Pierre de Vera, Sieglinde Ott, Laura
Zucconi, Laura Selbmann, Giuliano Scalzi, Kasthuri J. Ven-
kateswaran, Elke Rabbow, Francisco Javier Sa
´nchez In
˜igo, or
Gerda Horneck.
Abbreviations
ANOVA, analysis of variance; CFU, colony-forming units;
CLSM, confocal laser scanning microscopy; COSPAR, Com-
mittee on Space Research; EuTEF, European Technology
Exposure Facility; ISS, International Space Station; ITS, in-
ternal transcribed spacer; PCR, polymerase chain reaction;
PMA, propidium monoazide; PSII, photosystem II; s.d.,
standard deviation.
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Address correspondence to:
Professor Silvano Onofri
Department of Ecological and Biological Sciences (DEB)
Largo dell’Universita
`snc
01100 Viterbo (Italy)
E-mail: onofri@unitus.it
Submitted 30 September 2011
Accepted 1 April 2012
516 ONOFRI ET AL.
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