Survival of Antarctic Cryptoendolithic Fungi in Simulated Martian Conditions On Board the International Space Station

Abstract

Dehydrated Antarctic cryptoendolithic communities and colonies of the rock inhabitant black fungi Cryomyces antarcticus (CCFEE 515) and Cryomyces minteri (CCFEE 5187) were exposed as part of the Lichens and Fungi Experiment (LIFE) for 18 months in the European Space Agency's EXPOSE-E facility to simulated martian conditions aboard the International Space Station (ISS). Upon sample retrieval, survival was proved by testing colony-forming ability, and viability of cells (as integrity of cell membrane) was determined by the propidium monoazide (PMA) assay coupled with quantitative PCR tests. Although less than 10% of the samples exposed to simulated martian conditions were able to proliferate and form colonies, the PMA assay indicated that more than 60% of the cells and rock communities had remained intact after the ''Mars exposure.'' Furthermore, a high stability of the DNA in the cells was demonstrated. The results contribute to assessing the stability of resistant microorganisms and biosignatures on the surface of Mars, data that are valuable information for further search-for-life experiments on Mars.

Full text

Research Article
Survival of Antarctic Cryptoendolithic Fungi
in Simulated Martian Conditions On Board
the International Space Station
Silvano Onofri,
1
Jean-Pierre de Vera,
2
Laura Zucconi,
1
Laura Selbmann,
1
Giuliano Scalzi,
1
Kasthuri J. Venkateswaran,
3
Elke Rabbow,
4
Rosa de la Torre,
5
and Gerda Horneck
4
Abstract
Dehydrated Antarctic cryptoendolithic communities and colonies of the rock inhabitant black fungi Cryomyces
antarcticus (CCFEE 515) and Cryomyces minteri (CCFEE 5187) were exposed as part of the Lichens and Fungi
Experiment (LIFE) for 18 months in the European Space Agency’s EXPOSE-E facility to simulated martian
conditions aboard the International Space Station (ISS). Upon sample retrieval, survival was proved by testing
colony-forming ability, and viability of cells (as integrity of cell membrane) was determined by the propidium
monoazide (PMA) assay coupled with quantitative PCR tests. Although less than 10% of the samples exposed
to simulated martian conditions were able to proliferate and form colonies, the PMA assay indicated that more
than 60% of the cells and rock communities had remained intact after the ‘‘Mars exposure.’’ Furthermore, a
high stability of the DNA in the cells was demonstrated. The results contribute to assessing the stability of
resistant microorganisms and biosignatures on the surface of Mars, data that are valuable information for further
search-for-life experiments on Mars. Key Words: Endoliths—Eukaryotes—Extremophilic microorganisms—
Mars—Radiation resistance. Astrobiology 15, xxx–xxx.
1. Introduction
W
ith the exploration of Mars by orbiters, landers,
and rovers, evidence of long-lasting periods of liquid
water during the first billion years after the planet’s formation
has been substantiated (Poulet et al., 2005; Ehlmann et al.,
2011; NASA Release 14-326, 2014; Mahaffy et al., 2015),
attesting to the presence of a dense atmosphere and a more
clement and wet climate before about 3.5 billion years ago.
Such long-lasting periods of liquid water on the surface of a
planet are considered to be a presupposition for habitability
(Kasting et al., 1993). Therefore, future missions to Mars will
include the search for biosignatures as evidence for extinct or
even extant life (Parnell et al., 2007; Aerts et al., 2014).
The current surface climate of Mars is characterized by (i)
high temperature variations, depending on the time of day,
season, and geographical location—the Mars Science La-
boratory rover, for example, measured diurnal variations of
up to 80C, from about -70C at night to up to 10C at noon
(Go
´
mez-Elvira et al., 2014)—(ii) an anoxic atmosphere of
95% CO
2
at a pressure that varies, depending on season and
time of day, between 600 and 900 Pa (Go
´
mez-Elvira et al.,
2014); (iii) cosmic radiation at a mean dose rate of 0.2 mGy/
day (Hassler et al., 2014); (iv) and intense solar electro-
magnetic radiation at wavelength k > 200 nm at an UV ir-
radiance up to 20 W/m
2
at midday (Kuhn and Atreya, 1979;
Cockell et al., 2000; Go
´
mez-Elvira et al., 2014). It has been
suggested that these harsh conditions could rarely support
microbial growth or even survival over extended periods of
time (Horneck, 2000; Cockell et al., 2005; Kminek et al.,
2010; Rummel et al., 2014).
In preparation of life-detection experiments on Mars
(Parnell et al., 2007), terrestrial analogues of putative hab-
itats on Mars have been identified, and the tolerance limits
of their microbial communities have been studied. Due to its
cold, arid climate and seasonally enhanced UV radiation,
Antarctica has been considered an ideal terrestrial model in
the quest for life on Mars (Wynn-Williams and Edwards,
2000). In this hostile environment, fungi and cyanobacteria
have adopted a strategy to escape most of the stress
1
Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
2
Institute of Planetary Research, German Aerospace Center (DLR), Berlin, Germany.
3
Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California, USA.
4
Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany.
5
Department of Earth Observation, Spanish Aerospace Research Establishment-INTA, Torrejo
´
n de Ardoz, Madrid, Spain.
ASTROBIOLOGY
Volume 15, Number 12, 2015
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ast.2015.1324
1
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parameters by colonizing the inside of rocks (Friedmann,
1982). We have selected the Antarctic rock-inhabiting
meristematic fungi Cryomyces antarcticus and C. minteri as
well as fragments of rocks colonized by the Antarctic
cryptoendolithic community (Selbmann et al., 2005, 2011)
to test their tolerance under simulated martian conditions, as
provided by the EXPOSE-E mission on board the Interna-
tional Space Station (ISS) (Rabbow et al., 2012). Cryomyces
antarcticus and C. minteri demonstrated resistance to ex-
treme desiccation under 10
-5
Pa vacuum and UV radiation
(200–400 nm) during preparatory ground-based tests (Ex-
periment Verification Tests, EVT) for this space experiment
(Onofri et al., 2008); they also survived space conditions in
low-Earth orbit in the frame of the Lichens and Fungi Ex-
periment (LIFE) on board the ISS (Onofri et al., 2012).
These black yeast strains that were exposed to lethal doses
of UVC survived, while Saccharomyces pastorianus died
(Selbmann et al., 2011).
2. Material and Methods
2.1. Spaceflight data of LIFE
In LIFE, Antarctic black fungi were exposed for 1.5 years to
simulated martian environmental conditions during the orbital
flight of the EXPOSE-E facility of the European Space
Agency (ESA) (Rabbow et al., 2009, 2012). EXPOSE-E was
launched on 7 February 2008 with Space Shuttle STS-122 to
the ISS. It was mounted on 15 February 2008 by extravehic-
ular activity (EVA) to the balcony of the Columbus module of
the ISS as part of the European Technology Exposure Facility
(EuTEF), and samples were exposed to a simulated martian
environment (Table 1). EXPOSE-E was decommissioned on
1 September 2009, retrieved by EVA on 2 September 2009,
and returned to Earth on 12 September 2009 with STS-128.
During the 1.5-year mission, the samples were kept in a
pressurized, simulated martian atmosphere (1.6% argon,
0.15% oxygen, 2.7% nitrogen, 370 ppm H
2
O, in CO
2
at a
pressure of 10
3
Pa) and exposed to simulated martian UV
radiation (Table 1) such that the spectrum of solar extrater-
restrial electromagnetic radiation was cut off with optical
filters at a wavelength of k = 200 nm (Rabbow et al., 2012).
Some samples were insolated with a reduced irradiance by 3
orders of magnitude by neutral density filters. In addition, dark
flight samples were located beneath the insolated ones. Cos-
mic radiation dose and temperature were recorded as provided
under the conditions of the EXPOSE-E mission (Table 1).
2.2. Biological test systems of LIFE
The psychrophilic, UV-resistant, and desiccation-tolerant
black meristematic fungi Cryomyces antarcticus CCFEE
515 and C. minteri CCFEE 5187 are known to be good
eukaryotic models for astrobiological investigations (Onofri
et al., 2004, 2012). They were isolated from the McMurdo
Dry Valleys of Antarctica (Southern Victoria Land), which
is one of the most hostile environments on Earth (Selbmann
et al., 2005) and considered the closest terrestrial analogue
for Mars. These fungi excel by an extraordinary ability to
survive a number of stresses, such as extreme temperature
and high radiation levels, especially when dehydrated
(Onofri et al., 2008; Selbmann et al., 2011, 2014).
Two test systems were used in LIFE, as follows:
The first is comprised of dried sandstone fragments that
were colonized by a stratified cryptoendolithic microbial
community and collected in January 2004 at Battleship
Promontory (7654¢37.6†S, 16055¢27.5†E), Southern Vic-
toria Land, Antarctica. The community develops within
10 mm depth below the rock crust and is organized in dif-
ferent colored and biologically distinct bands (Fig. 1). Fungi
occupy the dark and white zones of the community.
The second test system consists of dried colonies of the
microcolonial black yeastlike fungi Cryomyces antarcticus
CCFEE 515 and C. minteri CCFEE 5187, both of which
dwell cryptoendolithically. They were isolated from sand-
stone collected in the McMurdo Dry Valleys, Antarctica
(Selbmann et al., 2005) and cultivated on malt extract agar.
Colonies were nearly spherical with a diameter of approx-
imately 1 mm. Single cells are 10 lm in diameter on aver-
age, with a thick black melanized outer wall (Fig. 1).
Colonies and colonized rock fragments were obtained and
dehydrated at room temperature in silica-gel desiccator ac-
cording to Onofri et al. (2008, 2012).
2.3. Viability assays of the LIFE biological
test systems
After flight and before performing viability assays, col-
onies were rehydrated for 72 h in 1.5 mL tubes containing
1 mL of 0.9% NaCl solution, at 5C. For growth tests, the
suspension of a single colony was opportunely diluted to
reach a concentration of 5 · 10
4
cells/mL. A 100 lL portion
of the suspension, containing 5000 cells, was seeded on malt
agar Petri dishes (five replicates) and incubated at 15C for
2 months, and the colonies formed were counted.
Table 1. Exposure Conditions during LIFE Lasting 1.5 Years On Board the ISS
Environmental parameter
Sample Sample type Atmosphere
Solar UV radiation
200–400 nm MJ/m
2
Cosmic
radiation mGy Temperature C
Flight Simulated Mars Dark 95% CO
2
atmosphere,
1000 Pa
0 220 -21.7 to +42.9
Flight Simulated Mars 0.1%
solar UV radiation
95% CO
2
atmosphere,
1000 Pa
0.63 238 -21.7 to +42.9
Flight Simulated Mars 100%
solar UV radiation
95% CO
2
atmosphere,
1000 Pa
475 238 -21.7 to +42.9
Data are from Rabbow et al. (2012) and Berger et al. (2012).
2 ONOFRI ET AL.
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For comparison, the same test was also performed on
dehydrated, untreated colonies that were kept in the dark in
the laboratory (Dark control, Fig. 2). Viability was ex-
pressed as percentage of colony-forming units (CFU). Pro-
pidium monoazide (PMA) assay (Mohapatra and La Duc,
2012) was used for evaluating percentage of cells with un-
damaged cell membranes by quantifying fungal DNA ex-
tracted from colonies of C. antarcticus and C. minteri or
colonized sandstone fragments. This was performed by
adding PMA (Biotium, Hayward, CA), at a final concen-
tration of 200 lM, to both fungal colonies, rehydrated as
above, and powdered rock suspensions in phosphate-
buffered saline (PBS) solution. PMA penetrates only
damaged cell membranes, cross-linking to DNA after light
exposure and thereby preventing PCR. Following DNA
extraction and purification (Maxwell 16 automatic DNA
extraction instrument, Promega, Madison, WI), quantitative
polymerase chain reaction (qPCR; BioRad CFX96 real-time
PCR detection system) was employed to quantify the
number of fungal ribosomal DNA fragments present in
samples either treated or not treated with PMA. Genomic
DNA was added at a concentration of 0.1 ng to 23 lLof
PCR cocktail containing 1X Power Sybr-Green PCR Master
Mix (Applied Bios, Foster City, CA), and 1 lL of 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 M final concentration.
These primers amplify a 203 bp product spanning the 18S/
ITS1 region of rRNA encoding genes.
A standard qPCR cycling protocol consisting of a hold
at 95C for 10 min, followed by 40 cycles of denaturing at
95C for 15 s, annealing at 58C for 20 s, and elongation at
72C for 15 s was followed. Fluorescence measurements
were recorded at the end of each annealing step. At the
conclusion of the 40
th
cycle, a melt curve analysis was
performed by recording changes in fluorescence as a func-
tion of raising the temperature from 60Cto95C in 0.5C
per 5 s increments. All tests were performed in triplicate.
Statistical analyses were performed by one-way analysis of
variance (ANOVA) and pairwise multiple comparison pro-
cedure (Tukey test), carried out by using the statistical
software SigmaPlot.
3. Results
After a stay of 1.5 years in space under simulated martian
conditions, the samples did not change in shape or color
FIG. 1. (A) Section of a dried sandstone fragment, colonized by a stratified cryptoendolithic microbial community. The
community develops within 10 mm deep below the rock crust and is organized in different colored and biologically distinct
bands. Black fungi occupy the dark zone of the community. (B) SEM micrograph showing growth of Cryomyces on quartz
crystals of fractured sandstone. The micro-colonial black yeast-like fungi Cryomyces antarcticus CCFEE 515 (C) and
C. minteri CCFEE 5187 (D).
ANTARCTIC BLACK FUNGI ON THE ISS 3
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compared to their preflight appearance. Viability of the
black meristematic fungi C. antarcticus and C. minteri and
the endolithic fungal communities was tested via colony-
forming ability test (Fig. 2) and PMA assay (Fig. 3).
Cryomyces antarcticus and C. minteri, even if kept in the
dark, showed a very low survival (as CFU%) after exposure
to the simulated martian atmosphere (gas composition and
pressure) with 1.48 – 0.26% and 0.08 – 0.06% CFU, re-
spectively (Fig. 2); the PMA assay gave 66.47 – 6.15% of
cells with undamaged membranes for C. antarcticus and
17.29 – 4.85% for C. minteri (Fig. 3).
Different results were obtained for irradiated samples that
were exposed to the simulated (full or attenuated) martian
UV radiation spectrum (k > 200 nm).
Survival of fungi, which were exposed beneath neutral
density filters (0.1% transmission), was more than 4–5 times
higher than that of the dark samples (CFU% of 8.40 – 1.65
for C. antarcticus and 2.07 – 0.33 for C. minteri; Fig. 2),
while the PMA assay gave a survival of about 3 times higher
for C. minteri (51.12 – 3.34) and nearly identical for C.
antarcticus (65.02 – 3.54; Fig. 3).
Without attenuation (100% solar electromagnetic radia-
tion at k > 200 nm), survival was in the range of the dark
samples: CFU% of 0.87 – 0.18 for C. antarcticus and of
0.30 – 0.02 for C. minteri (Fig. 2). In the PMA assay, am-
plified DNA from cells with undamaged membranes was
66.32 – 6.75% and 45.66 – 1.07% for C. antarcticus and C.
minteri, respectively (Fig. 3); these values did not signifi-
cantly differ from those of samples that received 1000 times
less solar irradiance.
Colonized sandstone exposed to the simulated martian
atmosphere in the dark yielded the highest amount of fungal
DNA amplified from intact cells, 75.3 – 6.40%, while a
strong decrease was recorded for insolated samples, for
which 17.90 – 13.52% and 10.72 – 9.24% were measured for
samples exposed to attenuated and full radiation, respec-
tively (Fig. 3).
Survival of the controls was much higher, though it was
not 100%; cultivation tests from dried colonies, kept in the
dark in the laboratory (Dark controls, Fig. 2), gave
46.50
– 7.89 and 16.76 – 2.78 CFU in C. antarcticus and C.
minteri, respectively.
4. Discussion
The EXPOSE-E mission on board the ISS provided, for
the first time during a space mission, environmental condi-
tions that mimicked most of the parameters of the martian
surface:
(i) Atmospheric composition and pressure.
(ii) Cosmic radiation (maximum dose rate at the sample
site: 368 – 27 lGy/d according to Berger et al.,
2012). This dose rate was slightly higher than the
210 – 40 lGy/d measured by Hassler et al. (2014) in
Gale Crater on Mars during the Mars Science La-
boratory mission. In addition, the spectra are dif-
ferent; whereas on Mars galactic cosmic rays
prevail, protons and electrons of the radiation belts
must be added to the galactic radiation source in the
orbit of the ISS (Dachev et al., 2012).
(iii) UV radiation (maximum 1572 solar constant hours
of solar electromagnetic radiation at k > 200 nm, re-
sulting in a fluence at the sample site of 475 MJ/m
2
for 200 nm < k < 400 nm UV, or 630 kJ/m
2
beneath a
FIG. 2. Viability of C. antarcticus and C. minteri expressed as percentage of CFU. Test was performed in five replicates.
Statistical significance was calculated by using the Tukey test comparing dataset from the same organism. ** and * indicate
that values are or are not significantly different, respectively, according to the Tukey test (P < 0.05).
4 ONOFRI ET AL.
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neutral density filter of 0.1% transmission). Because
the martian solar constant amounts to 45% of Earth’s
solar constant, the applied radiation would be equal
to 3493 Mars solar constant hours.
(iv) Long-term exposure (559 days in operation, total
time in space 583 days). This period corresponds to
nearly 1 martian year (687 days).
Temperature was not actively controlled and oscillated
between -21.7C and +42.9C with a one-time peak at 62C
for a few hours (Rabbow et al., 2012). This range differed
substantially from the temperatures on the surface of Mars,
which can reach about 20C as a maximum at noon at the
equator and -153C as a minimum at the poles.
It should be noted that the technical conditions of the ISS
did not allow simulating diurnal changes of the environ-
mental parameters as they prevail on Mars. Temperature and
insolation varied with the orbit of the ISS, and there was
interim shadowing by parts of the ISS, especially the solar
panels. In spite of these technical restraints, the EXPOSE-E
mission provided a workable tool with which to assess the
limits of microbes and microbial communities as well as the
stability of biomolecules at the surface of Mars, especially
in preparation of future search-for-life ventures.
This Lichens and Fungi Experiment of the EXPOSE-E
mission provided, for the first time, data on the viability of
rock-dwelling organisms and microbial communities after
long-term exposure to martian conditions simulated in
space. Even if the ‘‘Mars exposed’’ black fungi Cryomyces
antarcticus CCFEE 515 and C. minteri CCFEE 5187
showed fewer than 10% survivors in growth tests, their
colony-forming ability was maintained to a certain extent in
both strains. This demonstrates that test organisms survived
the simulated martian conditions that were applied for 1.5
years and were later able to propagate. In the same experi-
ment, Scalzi et al. (2012) reported that they grew one green
alga and one fungus from colonized rock samples after ex-
posure to simulated martian surface conditions. These or-
ganisms were identified as representatives of Stichococcus
sp. and the lichenized genus Acarospora. In another ex-
periment during the EXPOSE-E mission with Bacillus
subtilis spores that were exposed to simulated martian
conditions, postflight analysis showed that up to 20% of the
spores were able to germinate, grow out, and form colonies,
if arranged in multilayers (Horneck et al., 2012).
Although the majority of the ‘‘Mars-exposed black fun-
gi’’ were not able to form colonies, the resulting high
quantities of DNA, originating only from cells with un-
damaged membranes, suggested that cellular integrity was
not completely destroyed by the treatments. A similar high
fraction of intact cells was found by Brandt et al. (2015) for
the lichen Xanthoria elegans, which was exposed to simu-
lated martian conditions in LIFE; after LIVE/DEAD stain-
ing, more than 80% of metabolically active mycobiont cells
(fungi) were observed, and more than 60% metabolically
active photobiont cells (algae).
Survival rates in the growth tests were surprisingly higher
in both strains exposed to 0.1% of the radiation than in sam-
ples kept in the dark. This is difficult to explain because the
increase of 3 orders of magnitude of the radiation (100%)
results in a net decrease of survival, as expected (Fig. 2); yet
the same result was obtained from the same strains exposed to
solar UV radiation under full space conditions in the same
experiment LIFE (Onofri et al., 2012).
Remarkably, differences in vitality between C. antarc-
ticus and C. minteri are very similar in all the conditions
FIG. 3. Percentage of intact and damaged cells measured with PMA coupled with qPCR. Test was performed in three
replicates. Statistical significance was calculated by using the Tukey test comparing dataset from the same organism or from
rock. ** and * indicate that values are or are not significantly different, respectively, according to the Tukey test (P < 0.05).
ANTARCTIC BLACK FUNGI ON THE ISS 5
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tested, including control dried colonies that were not
exposed.
There was a substantial discrepancy in the viability data
obtained by PMA assay or colony test. The PMA assay gave
nearly identical values, around 65% cells with undamaged
membrane, for all samples exposed to simulated martian
conditions in C. antarcticus, regardless of whether they
were insolated or not. Similar high values—around 50%
cells with undamaged membrane—were found for insolated
samples of C. minteri, whereas more than 80% of the dark
samples were damaged. Significantly lower was the survival
of samples exposed to simulated martian conditions based
on cultivation tests where all values remained below 10% of
CFU, regardless of whether they were insolated or kept in
the dark (Figs. 2 and 3). The differences between the sur-
vival rates, measured by the growth test, and the damage to
the membranes, measured by PMA test, may be due to the
lower sensitivity of the membranes to UV radiation, com-
pared to the ability to multiply; possibly cosmic radiation
could be involved in resulting differences.
Propidium monoazide assay, applied to the colonized
sandstones, revealed a significant increase in cell membrane
damage in treated samples, yet a comparable damage was
recorded both in screened and fully exposed samples. This
seems to indicate that sandstone does not represent a sig-
nificant protection to lithobionts. Besides, the rock is mainly
composed of quartz, which is the component of the Suprasil
filters (>200 nm) used in the experiment; therefore, it is
possible that the same wavelengths penetrate the screen and
the rock crystals. Furthermore, it should be taken into ac-
count that data from rock samples include responses of the
whole fungal community and not just the black fungi, which
are much more protected by melanins.
The qPCR analyses (performed with the PMA assay)
revealed that biomolecules such as DNA are still detectable
after 1.5 years of exposure to simulated martian conditions.
These results may qualify this biomolecule as a biosignature
for future search-for-life extraterrestrial missions; moreover,
photoproduct analysis by high-performance liquid chroma-
tography coupled with electrospray ionization–tandem mass
spectrometry of DNA extracted from Bacillus subtilis spores
flown on EXPOSE-E to simulated martian conditions re-
vealed the presence of a high amount of the 5,6-dihydro-
5(a-thyminyl)thymine as the only DNA photoproduct found
(Panitz et al., 2015).
It was recently observed that actively growing C. ant-
arcticus (strain CCFEE 534) that was exposed for a short
period of time to thermophysical Mars-like conditions re-
sponded with a decrease in protein number but recovered the
normal metabolic activity within 1 week only (Zakharova
et al., 2014).
Therefore, Cryomyces is able to withstand short-term,
Mars-simulated ground-based exposition when actively
growing and long-term ground-based exposition (up to 1.5
years) when dehydrated.
5. Conclusions and Outlook
The European Space Agency’s EXPOSE facility on board
the ISS has proven to be an ideal platform for astrobiology
investigations in low-Earth orbit. Research areas studied
include

chemical evolution of organics in space (Cottin et al.,
2012, 2015; Carrasco et al., 2015),

stability of biomolecules in space (Bertrand et al.,
2012, 2015),

stability of biomolecules and biosignatures under Mars-
like conditions (Noblet et al., 2012; Vergne et al.,
2015; and this article),

likelihood of the lithopanspermia hypothesis (Onofri
et al., 2012; Tepfer et al., 2012; Wassmann et al., 2012;
Panitz et al., 2015),

survival of microorganisms under present-day condi-
tions of Mars (Horneck et al., 2012; Baque
´
et al.,
2013a; Brandt et al., 2015; and this article),

planetary protection requirements (Horneck et al.,
2012).
With EXPOSE-R2, the third EXPOSE mission is cur-
rently underway on the ISS (ESA, 2014). One of its goals is
to extend the current studies by determining the protective
role of ‘‘martian regolith’’ on the stability of resistant mi-
croorganisms and biosignatures under simulated martian
conditions (de Vera et al., 2012; Baque
´
et al., 2013b).
Acknowledgments
The European Space Agency is acknowledged for the
provision and operations of the EXPOSE-E facility. We also
thank the Italian National Program of Antarctic Researches
(PNRA) and Italian National Antarctic Museum for funding
the collection of Antarctic samples, the preservation of the
Culture Collection of Fungi from Extreme Environments
(CCFEE), and sample analyses. Special thanks are also due
to the German Ministry of Economy and Technology BMWi
as well as to the HGF-Foundation in the frame of the
Helmholtz-Alliance ‘‘Planetary Evolution and Life,’’ which
have partly supported the German Co-Is of this work.
Disclosure Statement
No competing financial interests exist for Silvano Onofri,
Jean-Pierre de Vera, Laura Zucconi, Laura Selbmann,
Giuliano Scalzi, Kasthuri J. Venkateswaran, Elke Rabbow,
Rosa de la Torre, and Gerda Horneck.
References
Aerts, J.W., Ro
¨
ling, W.F.M., Elsaesser, A., and Ehrenfreund, P.
(2014) Biota and biomolecules in extreme environments on
Earth: implications for life detection on Mars. Life 4:535–565.
Baque
´
, M., Scalzi, G., Rabbow, E., Rettberg, P., and Billi, D.
(2013a) Biofilm and planktonic lifestyles differently support
the resistance of the desert cyanobacterium Chroococci-
diopsis under space and martian simulations. Orig Life Evol
Biosph 43:377–389.
Baque
´
, M., de Vera, J.-P., Rettberg, P., and Billi, D. (2013b)
The BOSS and BIOMEX space experiments on the EXPOSE-
R2 mission: endurance of the desert cyanobacterium Chroo-
coccidiopsis under simulated space vacuum, martian atmosphere,
UVC radiation and temperature extremes. Acta Astronaut
91:180–186.
Berger, T., Hajek, M., Bilski, P., Ko
¨
rner, C., Vanhavere, F., and
Reitz, G. (2012) Cosmic radiation exposure of biological test
systems during the EXPOSE-E mission. Astrobiology 12:
387–392.
6 ONOFRI ET AL.
AST-2015-1324-ver9-Onofri_3P.3d 11/26/15 1:04am Page 6

Bertrand, M., Chabin, A., Brack, A., Cottin, H., Chaput, D., and
Westall, F. (2012) The PROCESS experiment: exposure of
amino acids in the EXPOSE-E experiment on the Interna-
tional Space Station and in laboratory simulations. Astro-
biology 12:426–435.
Bertrand, M., Chabin, A., Colas, C., Cade
`
ne, M., Chaput, D.,
Brack, A., Cottin, H., and Westall, F. (2015) The AMINO
experiment: exposure of amino acids in the EXPOSE-R ex-
periment on the International Space Station and in laboratory.
International Journal of Astrobiology 14:89–97.
Brandt, A., de Vera, J.-P., Onofri, S., and Ott, S. (2015) Viability
of the lichen Xanthoria elegans and its symbionts after 18
months of space exposure and simulated Mars conditions on the
ISS. International Journal of Astrobiology 14:411–425.
Carrasco, N., Cottin, H., Cloix, M., Je
´
rome, M., Be
´
nilan, Y.,
Coll, P., Gazeau, M.-C., Raulin, F., Saiagh, K., Chaput, D.,
and Szopa, C. (2015) The AMINO experiment: methane
photolysis under solar UV irradiation on the EXPOSE-R fa-
cility of the International Space Station. International Journal
of Astrobiology 14:79–87.
Cockell, C.S., Catling, D.C., Davis, W.L., Snook, K., Kepner,
R.L., Lee, P., and McKay, C.P. (2000) The ultraviolet envi-
ronment of Mars: biological implications past, present, and
future. Icarus 146:343–359.
Cockell, C.S., Schuerger, A.C., Billi, D., Friedmann, E.I., and
Panitz, C. (2005) Effects of a simulated martian UV flux on
the cyanobacterium, Chroococcidiopsis sp. 029. Astrobiology
5:127–140.
Cottin, H., Guan, Y.Y., Noblet, A., Poch, O., Saiagh, K., Cloix,
M., Macari, F., Je
´
rome, M., Coll, P., Raulin, F., Stalport, F.,
Szopa, C., Betrand, M., Chabin, A., Westall, F., Chaput, D.,
Demets, R., and Brack, A. (2012) The PROCESS experiment:
an astrochemistry laboratory for solid and gaseous organic
samples in low-Earth orbit. Astrobiology 12:412–425.
Cottin, H., Saiagh, K., Guan, Y.Y., Cloix, M., Khalaf, D.,
Macari, F., Je
´
rome, M., Polienor, J.-M., Be
´
nilan, Y., Coll, P.,
Fray, N., Gazeau, M.-C., Raulin, F., Stalport, F., Carrasco, N.,
Szopa, C., Bertrand, M., Chabin, A., Westall, F., Vergne, J.,
Da Silva, L.A., Maurel, M.-C., Chaput, D., Demets, R., and
Brack, A. (2015) The AMINO experiment: a laboratory for
astrochemistry and astrobiology on the EXPOSE-R facility of
the International Space Station. International Journal of As-
trobiology 14:67–77.
Dachev, T., Horneck, G., Ha
¨
der, D.-P., Schuster, M., Richter,
P., Lebert, M., and Demets, R. (2012) Time profile of cosmic
radiation exposure during the EXPOSE-E mission: the R3DE
instrument. Astrobiology 12:403–411.
de Vera, J.-P., Boettger, U., de la Torre Noetzel, R., Sa
´
nchez,
F.J., Grunow, D., Schmitz, N., Lange, C., Hu
¨
bers, H.-W.,
Billi, D., Baquee
´
, M., Rettberg, P., Rabbow, E., Reitz, G.,
Berger, T., Mo
¨
ller, R., Bohmeier, M., Horneck, G., Westall,
F., Ja
¨
nchen, J., Fritz, J., Meyer, C., Onofri, S., Selbmann, L.,
Zucconi, L., Kozyrovska, N., Leya, T., Foing, B., Demets, R.,
Cockell, C.S., Bryce, C., Wagner, D., Serrano, P., Edwards,
H.G.M., Joshi, J., Huwer, B., Ehrenfreund, P., Elsaesser, A.,
Ott, S., Meessen, J., Feyh, N., Szewzyk, U., Jaumann, R., and
Spohn, T. (2012) Supporting Mars exploration: BIOMEX in
low Earth orbit and further astrobiological studies on the
Moon using Raman and PanCam technology.
Planet Space
Sci 74:103–110.
Ehlmann, B.L., Mustard, J.F., Murchie, S.L., Bibbring, J.-P.,
Meunier, A., Fraeman, A.A., and Langevin, Y. (2011) Sub-
surface water and clay mineral formation during the early
history of Mars. Nature 479:53–60.
ESA. (2014) The worst trip around the world. Available online at
http://www.esa.int/Our_Activities/Human_Spaceflight/Research/
The_worst_trip_around_the_world
Friedmann, E.I. (1982) Endolithic microorganisms in the Ant-
arctic cold desert. Science 215:1945–1953.
Go
´
mez-Elvira, J., Armiens, C., Carrasco, I., Genzer, M., Go
´
-
mez, F., Haberle, R., Hamilton, V.E., Harri, A.-M., Ka-
hanpa
¨
a
¨
, H., Kemppinen, O., Lepinette, A., Martı
´
n Soler, J.,
Martı
´
n-Torres, J., Martı
´
nez-Frı
´
as, J., Mischna, M., Mora, L.,
Navarro, S., Newman, C., de Pablo, M.A., Peinado, V.,
Polkko, J., Rafkin, S.C.R., Ramos, M., Renno
´
, N.O., Ri-
chardson, M., Rodrı
´
guez-Manfredi, J.A., Romeral Planello
´
,
J.J., Sebastia
´
n, E., de la Torre Jua
´
rez, M., Torres, J., Urquı
´
,
R., Vasavada, A.R., Verdasca, J., and Zorzano, M.-P. (2014)
Curiosity’s rover environmental monitoring station: overview
of the first 100 sols. J Geophys Res Planets 119:1680–1688.
Hassler, D.M., Zeitlin, C., Wimmer-Schweingruber, R.F., Eh-
resmann, B., Rafkin, S., Eigenbrode, J.L., Brinza, D.E.,
Weigle, G., Bo
¨
ttcher, S., Bo
¨
hm, E., Burmeister, S., Guo, J.,
Ko
¨
hler, J., Martin, C., Reitz, G., Cucinotta, F.A., Kim, M.H.,
Grinspoon, D., Bullock, M.A., Posner, A., Go
´
mez-Elvira, J.,
Vasavada, A., and Grotzinger, J.P.; MSL Science Team.
(2014) Mars’ surface radiation environment measured with
the Mars Science Laboratory’s Curiosity rover. Science 343,
doi:10.1126/science.1244797.
Horneck, G. (2000) The microbial world and the case for Mars.
Planet Space Sci 48:1053–1063.
Horneck, G., Moeller, R., Cadet, J., Douki, T., Mancinelli, R.L.,
Nicholson, W.L., Panitz, C., Rabbow, E., Rettberg, P., Spry,
A., Stackebrandt, E., Vaishampayan, P., and Venkateswaran,
K.J. (2012) Resistance of bacterial endospores to outer space
for planetary protection purposes—experiment PROTECT of
the EXPOSE-E mission. Astrobiology 12:445–456.
Kasting, J.F., Whitmire, D.P., and Reynolds, R.T. (1993)
Habitable zones around main sequence stars. Icarus 101:
108–128.
Kminek, G., Rummel, J.D., Cockell, C.S., Atlas, R., Barlow, N.,
Beaty, D., Boynton, W., Carr, M., Clifford, S., Conley, C.A.,
Davila, A.F., Debus, A., Doran, P., Hecht, M., Heldmann, J.,
Helbert, J., Hipkin, V., Horneck, G., Kieft, T.L., Klingel-
hoefer, G., Meyer, M., Newsom, H., Ori, G.G., Parnell, J.,
Prieur, D., Raulin, F., Schulze-Makuch, D., Spry, J.A., Sta-
bekis, P.E., Stackebrandt, E., Vago, J., Viso, M., Voytek, M.,
Wells, L., and Westall, F. (2010) Report of the COSPAR
Mars special regions colloquium. Adv Space Res 46:811–829.
Kuhn, W.R. and Atreya, S.K. ( 1979) Solar radiation incident on
the martian surface. J Mol Evol 14:57–64.
Mahaffy, P.R., Webster, C.R., Stern, J.C., Brunner, A.E.,
Atreya, S.K., Conrad, P.G., Domagal-Goldman, S., Eigen-
brode, J.L., Flesch, G.J., Christensen, L.E., Franz, H.B.,
Freissinet, C., Glavin, D.P., Grotzinger, J.P., Jones, J.H.,
Leshin, L.A., Malespin, C., McAdam, A.C., Ming, D.W.,
Navarro-Gonzalez, R., Niles, P.B., Owen, T., Pavlov, A.A.,
Steele, A., Trainer, M.G., Williford, K.H., and Wray, J.J.; the
MSL Science Team. (2015) The imprint of atmospheric
evolution in the D/H of Hesperian clay minerals on Mars.
Science 347:412–414.
Mohapatra, B.R. and La Duc, M.T. (2012) Rapid detection of
viable Bacillus pumilus SAFR-032 encapsulated spores using
novel propidium monoazide–linked fluorescence in situ hy-
bridization. J Microbiol Methods 90:15–19.
NASA Release 14-326. (2014) NASA’s Curiosity rover finds
clues to how water helped shape martian landscape. Available
online at http://www.nasa.gov/press/2014/december/nasa-s-
ANTARCTIC BLACK FUNGI ON THE ISS 7
AST-2015-1324-ver9-Onofri_3P.3d 11/26/15 1:04am Page 7

curiosity-rover-finds-clues-to-how-water-helped-shape-martian-
landscape
Noblet, A., Stalport, F., Guan, Y., Poch, O., Coll, F., Szopa, C.,
Cloix, M., Macari, F., Raulin, F., Chaput, D., and Cottin, H.
(2012) The PROCESS experiment: amino and carboxylic
acids under Mars-like surface UV radiation conditions on
low-Earth orbit. Astrobiology 12:436–444.
Onofri, S., Selbmann, L., Zucconi, L., and Pagano, S. (2004)
Antarctic microfungi as models for exobiology. Planet Space
Sci 52:229–237.
Onofri, S., Barreca, D., Selbmann, L., Isola, D., Rabbow, E.,
Horneck, G., de Vera, J.-P., Hatton, J., and Zucconi, L. (2008)
Resistance of Antarctic black fungi and cryptoendolithic
communities to simulated space and martian conditions. Stud
Mycol 61:99–109.
Onofri, S., de la Torre, R., de Vera, J.-P., Ott, S., Zucconi, L.,
Selbmann, L., Scalzi, G., Venkateswaran, K.J., Rabbow, E.,
Sa
´
nchez In
˜
igo, F.J., and Horneck, G. (2012) Survival of rock-
colonizing organisms after 1.5 years in outer space. Astro-
biology 12:508–516.
Panitz, C., Horneck, G., Rabbow, E., Rettberg, P., Moeller, R.,
Cadet, J., Douki, T., and Reitz, G. (2015) The SPORES ex-
periment of the EXPOSE-R mission: Bacillus subtilis spores
in artificial meteorites. International Journal of Astrobiology
14:105–114.
Parnell, J., Cullen, D., Sims, M.R., Bowden, S., Cockell, C.S.,
Court, R., Ehrenfreund, P., Gaubert, F., Grant, W., Parro, V.,
Rohmer, M., Sephton, M., Stan-Lotter, H., Steele, A., To-
porski, J., and Vago, J. (2007) Searching for life on Mars:
selection of molecular targets for ESA’s Aurora ExoMars
mission. Astrobiology 7:578–604.
Poulet, F., Bibring, J.P., Mustard, J.F., Gendrin, A., Mangold,
N., Langevin, Y., Arvidson, R.E., Gondez, B., and Gomez, D.
(2005) Phyllosilicates on Mars and implications for early
martian climate. Nature 438:623–627.
Rabbow, E., Horneck, G., Rettberg, P., Schott, J.-U., Panitz, C.,
L’Afflitto, A., Heise-Rotenburg, R., Willnecker, R., Baglioni,
P., Hatton, J., Dettmann, J., Demets, R., and Reitz, G. (2009)
EXPOSE, an astrobiological exposure facility on the Inter-
national Space Station—from proposal to flight. Orig Life
Evol Biosph 39:581–598.
Rabbow, E., Rettberg, P., Barczyk, S., Bohmeier, M., Parpart,
A., Panitz, C., Horneck, G., von Heise-Rotenburg, R., Hop-
penbrouwers, T., Willnecker, R., Baglioni, P., Demets, R.,
Dettmann, J., and Reitz, G. (2012) EXPOSE-E: an ESA as-
trobiology mission 1.5 years in space. Astrobiology 12:374–
386.
Rummel, J.D., Beaty, D.W., Jones, M.A., Bakermans, C.,
Barlow, N.G., Boston, P.J., Chevrier, V.F., Clark, B.C., de
Vera, J.-P.P., Gough, R.V., Hallsworth, J.E., Head, J.W.,
Hipkin, V.J, Kieft, T.L., McEwen, A.S., Mellon, M.T., Mi-
kucki, J.A., Nicholson, W.L., Omelon, C.R., Peterson, R.,
Roden, E.E., Sherwood Lollar, B., Tanaka, K.L., Viola, D.,
and Wray, J.J. (2014) A New Analysis of Mars ‘‘Special
Regions’’: Findings of the Second MEPAG Special Regions
Science Analysis Group (SR-SAG2). Astrobiology 14:887–
968.
Scalzi, G., Selbmann, L., Zucconi, L., Rabbow, E., Horneck, G.,
Albertano, P., and Onofri, S. (2012) LIFE experiment: iso-
lation of cryptoendolithic organisms from Antarctic colo-
nized sandstone exposed to space and simulated Mars
conditions on the International Space Station. Orig Life Evol
Biosph 42:253–262.
Selbmann, L., de Hoog, G.S., Mazzaglia, A., Friedmann, E.I.,
and Onofri, S. (2005) Fungi at the edge of life: cryptoendo-
lithic black fungi from Antarctic desert. Stud Mycol 51:1–32.
Selbmann, L., Isola, D., Zucconi, L., and Onofri, S. (2011)
Resistance to UV-B induced DNA damage in extreme-
tolerant cryptoendolithic Antarctic fungi: detection by PCR
assays. Fungal Biol 115:937–944.
Selbmann, L., de Hoog, G.S., Zucconi, L., Isola, D., and Onofri,
S. (2014) Black yeasts from cold habitats. In Yeasts from
Cold Habitats, edited by P. Buzzini and R. Margesi.,
Springer-Verlag, Berlin, pp 173–189.
Tepfer, D., Zalar, A., and Leach, S. (2012) Survival of plant
seeds, their UV screens, and nptII DNA for 18 months outside
the International Space Station. Astrobiology 12:517–528.
Vergne, J., Cottin, H., da Silva, L., Brack, A., Chaput, D., and
Maurel, M.C. (2015) The AMINO experiment: RNA stability
under solar radiation studied on the EXPOSE-R facility of the
International Space Station. International Journal of Astro-
biology 14:99–103.
Wassmann, M., Moeller, R., Rabbow, E., Panitz, C., Horneck,
G., Reitz, G., Douki, T., Cadet, J., Stan-Lotter, H., Cockell,
C.S., and Rettberg, P. (2012) Survival of spores of the UV-
resistant Bacillus subtilis strain MW01 after exposure to low-
Earth orbit and simulated martian conditions: data from the
space experiment ADAPT on EXPOSE-E. Astrobiology 12:
498–507.
Wynn-Williams, D.D. and Edwards, H.G.M. (2000) Antarctic
ecosystems are models for extraterrestrial surface habitats.
Planet Space Sci 48:1065–1075.
Zakharova, K., Marzban, G., de Vera, J.-P., Lorek, A., and
Sterflinger, K. (2014) Protein patterns of black fungi under
simulated Mars-like conditions. Sci Rep 4, doi:10.1038/
srep05114.
Address correspondence to:
Silvano Onofri
Department of Ecological and Biological Sciences
University of Tuscia
01100 Viterbo
Italy
E-mail: onofri@unitus.it
Submitted 3 June 2015
Accepted 8 October 2015
Abbreviations Used
CFU ¼ colony-forming units
EuTEF ¼ European Technology Exposure Facility
EVA ¼ extravehicular activity
ISS ¼ International Space Station
LIFE ¼ Lichens and Fungi Experiment
PMA ¼ propidium monoazide
qPCR ¼ quantitative polymerase chain reaction
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