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1 DECOS, Università degli Studi della Tuscia, Largo
dell'Università, Viterbo, Italy
2 German Aerospace Center (DLR), Institute of Aerospace Medicine, Cologne,
Germany
3 Heinrich-Heine-University, Düsseldorf, Germany
4 ESA-ESTEC (HME-GA) Noordwijk, The Netherlands
*
Correspondence: Silvano Onofri,
onofri{at}unitus.it
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Keywords Astrobiology / cryptoendolithic community / fungi / ground-based experiments / lithopanspermia / panspermia / space conditions / stress resistance / viability
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Antarctica is a continent where a combination of dry, cold, and oligotrophic extremes exists, and huge fluxes of dangerous radiations such as UV radiation can be present as well. These conditions become harsher in the ice-free McMurdo Dry Valleys area in continental Antarctica where cryptoendolithic microbial communities are almost the only life-form possible. Black meristematic microfungi of these communities were already suggested as eukaryotic models for the biological exploration of Mars (Onofri et al. 2004). The McMurdo Dry Valleys area, also known as Ross Desert, is the largest ice-free area in Antarctica. It is located within the Transantarctic Mountains in the Southern part of Victoria Land. Winter air temperature fluctuates between –20 and –50 °C (occasionally lower), rising to mean daily values of about –15 °C in the summer, up to 15 °C or higher values at ground surfaces. Wide and repeated thermal fluctuations are a stress factor, more than the minimum values reached. Dryness is also extreme: water is mainly supplied by snow (less than 100 mm water equivalent / yr), that mostly sublimes without visibly wetting the ground or it is blown away (Nienow & Friedmann 1993). High evaporation leads to high salt concentration on rock surfaces and a poor nutrient soil is occasionally present because of the scarcity of organic matter. Finally, UV radiation is high, mainly in the springtime, as a consequence of stratospheric ozone depletion during this period. As for it's oxygenic atmosphere no place on Earth is truly comparable to what is present on Mars, the McMurdo Dry Valleys could be called the closest terrestrial analogue. Therefore, they are one of the best investigated areas as a model environment for astrobiological studies since the 1970ies (Horowitz et al. 1972, Wynn-Williams & Edwards 2000, Onofri et al. 2004).
Friedmann and co-workers (Friedmann & Ocampo-Friedmann 1976, Friedmann 1982, Nienow & Friedmann 1993) discovered various microbial communities in the Ross Desert. The communities live sheltered under the rock surface, where they find a more favorable nanoclimate in the rock pore-spaces. Among these, the "lichen dominated cryptoendolithic community" is the most studied and known (Friedmann 1982). Under a reddish superficial crust, eukaryotic and prokaryotic autotrophic and heterotrophic microorganisms form a clearly stratified community. Antarctic black non-lichenised rock fungi, showing meristematic growth, are recurrent components of this community and common inhabitants of the black zone immediately below the lichen crust. Their biodiversity has been studied only recently. Sampling is still limited, yet some new genera were discovered in these habitats, viz. Friedmanniomyces, Cryomyces, Recurvomyces and Elasticomyces (Onofri et al. 1999, 2004, Selbmann et al. 2005, 2008).
The results from freeze-thaw experiments, after UV exposures and osmotic stress tolerance of two isolates of C. antarcticus (CCFEE 515 and CCFEE 534 = CBS 116301), and one isolate of C. minteri (CCFEE 5187 = CBS 116902), revealed an unusual ability to survive under these pressures (Onofri et al. 2007b).
The opportunity to expose these isolates to space conditions by the "EXPOSE-E" facility of the European Space Agency (ESA) on the EuTEF platform (part of the European Columbus Laboratory) outside of the International Space Station (ISS) inspired ground-based experiments that will be described in this contribution. The experiments have been carried out at the German Aerospace Center (DLR, Köln, Germany), where we tested single and combined simulated space and Martian conditions. Furthermore, we extended the experiments to the entire Antarctic crytptoendolithic community as well, also to test the potential protective role of the rock substratum.
We considered this as a preliminary and essential step before launching Antarctic black fungi and cryptoendolithic communities to outer space and for exposure on the ISS in the framework of the Lichens and Fungi Experiments (LIFE, European Space Agency).
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MATERIALS AND METHODS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Cryomyces antarcticus CCFEE 515 was isolated by R. Ocampo-Friedmann from sandstone collected at Linnaeus Terrace (Southern Victoria Land) by H. Vishniac, in the Antarctic expedition 1980-81; C. antarcticus CCFEE 534 was isolated by R. Ocampo-Friedmann from weathered rock collected at Linnaeus Terrace by E.I. Friedmann, during the Antarctic expedition 1981–82; C. minteri CCFEE 5187 was isolated by S. Pagano from weathered rocks collected by S. Onofri at Battleship Promontory (Southern Victoria Land) on Dec 28, 1996. For the EVTs, dehydrated fungal colonies were prepared as follows: cell suspensions were spread on MEA (malt extract agar: malt extract, powdered 30 g/L; peptone 5 g/L; agar 15 g/L; Applichem, GmbH) medium (5 mm thick) in Petri dishes and, once grown, maintained at 15 °C for 1 yr. Agar disks (12 mm diameter) containing 1–3 colonies each were then drilled and used for tests.
The colonised sandstone sample, with a well developed and stratified colonisation, was collected by L. Zucconi at Battleship Promontory (76°54'37.6"S 160°55'27.5"E), Southern Victoria Land, on Jan. 24, 2004. Colonised rock fragments (11 mm wide, maximum 6 mm thick) were obtained by hitting the rock lengthwise, and then dehydrated at room temperature.
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The aim of EVT-E2 was to test the responses of rock fungi and cryptoendolithic community to simulated space vacuum, simulated CO2 Martian atmosphere and pressure, simulated space vacuum combined with polychromatic UV radiation, and simulated CO2 Martian atmosphere combined with polychromatic UV radiation (Table 1). All tests were performed in triplicate.
Vacuum (E1 and E2)
The pressure was set at the value of
10–5 Pa as expected to prevail during the
space flight. Samples were accommodated in a vacuum facility, called PSI 6,
and exposed to vacuum for 1 h and 1 wk for E1, and 22 d for E2, after reaching
10–5 Pa (monitored by a Pirani cold
cathode inserted into the vacuum chamber).
Temperature fluctuation (E1)
Temperature fluctuations (–20 / +20 °C) are expected during the
space flight. 50 cycles were therefore performed inside a facility, called PSI
2, within 2 wk, programming 2 h heating, 2.5 h cooling, and maintenance at
–20 °C and +20 °C respectively for 1 h. The temperature was
monitored with a sensor attached to the inner side of a sample carrier.
UV Radiation conditions and fluences (E1 and E2)
E1. The monochromatic UV-C at 254 nm was obtained using a Hg-low-pressure
lamp, the spectral irradiance was measured using a Bentham spectroradiometer
(Fig. 1) giving a total
irradiance of 0.7 W/m2; in addition the irradiance was controlled
before and after each exposure with a calibrated UVX-meter at the sample site.
Samples were arranged within the homogeneously irradiated area. Due to the low
infrared (IR) output of this lamp, no cooling was required. The exposure was
performed additively, by covering the samples that received their assigned
UV-C fluence of 10, 100 and 1000 Jm–2,
with UV opaque filters.
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E2. A combination of polychromatic UV irradiation and vacuum or simulated Martian atmosphere was applied (Table 1). For the polychromatic UV irradiance in vacuum (10–5 Pa) as well as in a simulated CO2 Martian atmosphere (composed of argon 1.56 %, oxygen 0.16 %, nitrogen 2.72 %, carbon dioxide 95.56 %; pressure of 600 Pa), the samples were accommodated on the cold plate kept at 10 °C inside the homogeneous irradiated area inside the facility PSI 2. Temperature was monitored during the irradiation by a sensor attached to the sample carrier. The IR radiation of the solar simulators leads to heating the irradiated samples in the low pressure and hence low-convection environment. Because the SOL2000 caused a high heating of the samples, for more prolonged irradiation times the weaker SOL1000 solar simulator was used (Table 1).
The measured and integrated irradiance of the SOL2000 for the polychromatic UV range 200–400 nm was 479.2 W/m2, and 170.4 W/m2 for the SOL1000, at the sample site. The selected final dose of 1.5 x 105 kJm–2 (full polychromatic irradiation) was reached after a total irradiation time of 18 min with SOL2000 plus 243 h with SOL1000 in simulated CO2 Martian atmosphere, and after a total irradiation time of 244.5 h with SOL1000 irradiation in simulated space vacuum.
Analyses of responses to tested parameters
Responses to test parameters were analysed both by cultural and staining
methods. Viability of isolates of C. antarcticus (CCFEE 515 and CCFEE
534) and of C. minteri (CCFEE 5187) after EVTs by cultural methods
was evaluated as number of formed colony. Tested colonies were collected,
preliminarily treated in a rotator (9 g/min) with 1 mL of a sterile solution
of Tween 20 (0.35 %) for 15 min to remove eventual external contaminants, and
then washed with 1 mL of sterile physiological solution (4 times), for 15 min,
to remove Tween 20. Fungal suspensions were obtained by crumbling colonies
with a sterile needle, followed by serial dilutions up to a final
concentration of about 104 cells per mL of inoculum in
physiological solution (0.9 % NaCl). Cultural tests were performed by
spreading 0.15 mL of standardised inocula on MEA (Malt Extract Agar, Applichem
Gmbh), with the addition of chloramphenicol (100 ppm) to prevent bacterial
growth. Cultures were then incubated at 15 °C. The number of growing
microcolonies was recorded after 1 and 2 mo of incubation. Data are reported
as percentage of colonies compared to the untreated control. All tests were
performed in triplicate.
Viability of samples from rocks was demonstrated by directly spreading small fragments of rocks on 5 different cultural media: MEA (Malt Extract Agar; Applichem Gmbh) and DRBC (Oxoid) media (Dichloran Rose-Bengal Chloramphenicol agar: peptone 5 g/L; dextrose 10 g/L; biacid potassium phosphate 1 g/L; magnesium sulphate 0.5 g/L; dichloran 0.002 g/L; rose-bengal 0.025 g/L; agar 15 g/L) for fungal growth (filamentous and black fungi respectively); TM medium (Trebouxia Medium: Bold's Basal Medium 970 mL; proteose peptone 2.5 g/L; glucose 5 g/L; agar 15 g/L) for algal growth; TY and BG11 media (Trypton Yeast Medium: trypton 5 g/L; yeast extract 3 g/L; CaCl2 anhydrous 0.4 g/L; agar 17 g/L. Blue Green Algae Medium: NaNO3 1.5 g/L; K2HPO4 0.04 g/L; MgSO4 x 7H2O 0.075 g/L; CaCl2 x 2H2O 0.036 g/L; citric acid 0.006 g/L; ammonium ferric citrate 0.006 g/L; EDTA 0.001 g/L; metal traces 1 mL/L: H3BO3 2.86 g/L, MnCl2 x 4H2O 1.81 g/L, ZnSO4 x 7H2O 0.222 g/L, NaMoO4 x 2H2O 0.39 g/L, CuSO4 x 5H2O 0.079 g/L, Co(NO3)2 x 6H2O 49.4 mg/L; agar 10 g/L) for bacterial and cyanobacterial growth, respectively. The occurrence / absence of growing colonies was simply recorded after 3 mo of incubation in the dark for fungi and bacteria, and in the light for algae and cyanobacteria.
Viability was also evaluated by staining methods using two dyes, the LIVE / DEAD dye FUN 1 (30 µM) and SYTOX Green (10 µM) in PBS (Dulbecco's Phosphate Buffered Saline: NaCl 8 g/L; KCl 0.2 g/L; Na2HPO4 anhydrous 1.15 g/L; KH2PO4 0.259 g/L). This enabled us to detect viable and metabolically active cells as well as non-viable or damaged cells. Staining methods were applied only to Cryomyces minteri isolate.
Each tested colony was rehydrated with a PBS solution for 1 h, included in a polyethylene glycol mixture (Killik, Bio-Optica) inside a cryostat chamber (Leica, CM1510 S) at –20 °C for 10 min, cut into sections about 25 µm thick by a microtome, and coloured on microscope slides. The best dye penetration for FUN 1 and SYTOX Green was obtained with an incubation of 1–3 h and 1.5 h respectively, under dark conditions at room temperature.
A fluorescence microscope Axioskop 2 plus (Zeiss), provided with long-pass
filters (488 nm with emission 
530 nm for fluorescein isothiocyanate, 546
nm with emission 580 nm for the rhodamine), was used to analyse the
viability of the C. minteri samples after exposure to test
parameters. Red (viable and metabolically active) and green (viable but not
metabolically active) emitting images of stained samples were separately
acquired by a mounted Axiocam using AxioVision v. 4 software. The combined
image obtained by overlapping both images allows to distinguish clearly the
viability state. The SYTOX Green dye (S-7020, Molecular Probes) stains the DNA
of non-viable cells green when excited at 450–500 nm, and using an
appropriate filter (504 nm with emission 524 nm). The imagines were
imported in Adobe Photoshop whereas the quantitative analyses were performed
using KS300 software and expressed as percentage of live / dead cells.
Responses to high temperatures
Thermal model calculations by ESA estimated that during the space flight
the temperature may reach high values (up to 90 °C without any coverage).
Therefore we tested the resistance of C. antarcticus CCFEE 515 and
CCFEE 534 and C. minteri CCFEE 5187 at 60 °C (selected as limit
value for closing automatically the EXPOSE sample carrier lids) and of C.
antarcticus CCFEE 515 and C. minteri CCFEE 5187, previously
selected for flight, also at 80 and 90 °C. Six-mo-old dried colonies of
all the isolates were incubated at 60 °C for 15 and 60 min (3 replicates
for each test) and six-mo old dried colonies of C. antarcticus CCFEE
515 and C. minteri CCFEE 5187 were incubated for 60 min at 80 and 90
°C (5 replicates). All colonies were subsequently maintained overnight in
a desiccator, to allow a slow cooling to room temperature. Survival was tested
by cultural methods, as described above, as number of colonies growing after
two mo of incubation on MEA at 15 °C, for the tests at 60 °C, and as
presence / absence of growing colonies for tests at 80 and 90 °C.
Viability after exposure at 80 and 90 °C was also evaluated in C.
minteri by staining methods.
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Freeze and thawing cycles
Results of the viability of dried isolates after 50 repeated freeze and
thawing cycles, recorded as above and compared to the untreated controls,
showed different responses: C. antarcticus CCFEE 515 was the most
negatively affected with a strong reduction of growth ability, C.
minteri CCFEE 5187 showed a 75 % reduction, while C. antarcticus
CCFEE 534 seemed to be almost unaffected.
UV radiation at different spectral ranges
With respect to the capability of dried fungal isolates to form colonies
after increasing monochromatic or polychromatic radiation doses, respectively,
C. antarcticus isolates were both negatively affected by the higher
doses of polychromatic UV, whereas the species showed diversified response to
the monochromatic irradiation (Fig.
6). CCFEE 515 remained practically unaffected, and CCFEE 534 was
totally inhibited after the highest exposure only. Cryomyces minteri
CCFEE 5187 showed a good survival after both radiation types and doses, with a
certain decrease only at the highest doses.
UV radiation plus vacuum or Martian atmosphere and pressure
Figure 6 shows the results
of exposure of dried samples to polychromatic UV spectrum in space-simulating
vacuum as well as in simulated Martian atmosphere compared to vacuum and
Martian atmosphere exposure, respectively. Growth obtained with both isolates
of C. antarcticus was scarce, with a complete inhibition of isolate
CCFEE 534 and a substantial reduction in viability in CCFEE 515. This response
was consistent with negative results obtained after vacuum and 1.5 x
105 kJm–2 polychromatic irradiation. Propagules of
C. minteri CCFFEE 5187 showed a higher survival than both C.
antarcticus strains after exposure either to vacuum or Martian
CO2, and a reduction, but not complete inhibition, at combined UV
radiation and Martian atmosphere. A total absence in viability apparently
appears in the combined test of vacuum and maximum UV (200–400 nm) dose
of 1.5 x 105 kJm–2, but this result was
disproved by the staining techniques.
The LIVE / DEAD dye FUN1 (F-7030, Molecular Probes) passively penetrates
the cytoplasm and stains cells with a yellowish green to green fluorescence:
the intravacuolar enzymatic activity of viable and metabolically active cells
is indicated by the appearance of compact cylindrical intravacuolar structures
(CIVS) with an orange to red fluorescence (when a 
ex
between 470 and 590 nm is used) and a concomitant reduction of the green to
yellow cytoplasmatic fluorescence.
The staining procedure failed with the isolates of C. antarcticus because the thickness of cell wall prevented the penetration of the dyes, while the same procedure was successful with C. minteri. Good viability percentages obtained in all EVTs with the LIVE / DEAD dye FUN 1 (Fig. 7) confirmed the positive responses obtained by the cultural methods and the high resistance of C. minteri to simulated space and Martian conditions in both EVTs, while combined vacuum and maximum UV (200–400 nm) exposure gave ambiguous results when the responses were tested with cultural or staining approach. In fact, even though C. minteri was unable to grow after this treatment, cell viability in a 25 µm section of a colony was suggested by the red and green fluorescence shown in Fig. 8. Data (not shown) obtained by the SYTOX Green dye, confirmed results reported in Fig. 7.
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Cryptoendolithic community
Table 2 reports the presence
or absence of colonies grown from rock fragment samples after EVTs. Eleven
verification tests E1 and E2 were carried out, and colony appearance was
recorded in 8 of them. Negative results are those concerning polychromatic
irradiation at higher doses and vacuum plus polychromatic UV-irradiation at
the lower dose. Some samples treated with simulated Martian atmosphere with or
without combined polychromatic UV radiation have been lost and EVT-E2 data for
these experiments are missing.
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DISCUSSION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Cryomyces antarcticus CCFEE 515, which gave better results compared with CCFEE 514 of the same species, was also selected to investigate short and long term resistance to space conditions on the ISS.
The high resistance to space conditions shown by the three isolates tested could be ascribed to the peculiar morpho- and physiological features of black meristematic fungi. These microorganisms produce slowly expanding, cauliflower-like colonies, barely differentiated structures, and thick and heavily pigmented cell walls (Selbmann et al. 2005, Onofri et al. 2007a). These characteristics convey to high tolerance to extreme terrestrial environments and, by coincidence, spacial conditions. Melanin, for instance, is a biological macromolecule, ubiquitous in nature, mainly known for its protective role against UV and ionising radiation, extreme temperatures, and desiccation (Sterflinger 2005). The high tolerance to the UV-B exposure of single cells of the tested fungi has been reported recently (Onofri et al. 2007b). The thickness of the colony itself may represent an additional protection for the cells in the inner layers.
The high resistance of tested isolates to temperatures up to 90 °C is in agreement with literature on survival of dehydrated colonies of other meristematic fungi subjected to high temperatures (Sterflinger & Krumbein 1995, Sterflinger et al. 1999, Sterflinger 2005). The ability to enter a cryptobiotic state under poikilohydric conditions could be aided by the presence of abundant extracellular polymeric substances (EPS) in many species. EPS production may be abundant (Selbmann et al. 2005) and may appear as a gelatinous matrix in lichen thalli (de Vera et al. 2004) which may serve as a water reservoir to survive long dry periods (de los Ríos et al. 2004, 2005). Not surprisingly, many meristematic black fungi are commonly recorded from Mediterranean areas and hot deserts, where substrate surface temperatures can reach very high values. The ability to survive long-term desiccation makes these isolates pre-adapted to the extreme conditions of space, since high-vacuum conditions produce an extreme dehydrating effect.
Recent studies also show that lichens, as well as their isolated photobionts and mycobionts, cope with the extreme conditions of outer space in ground-based experiments (de Vera et al. 2003, 2004). Xanthoria elegans was able to photosynthesise under simulated Martian conditions with light in visible wave-lengths and in the presence of water (de Vera et al. 2007). Finally, samples of the lichens Rhizocarpon geographicum and X. elegans survived 16 d of exposure to space in the BIOPAN-5 facility of the European Space Agency located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite (Sancho et al. 2007).
By means of the NASA Space Shuttle Atlantis flight launched on Feb 7, 2008,
these fungi are now exposed to actual space in the EXPOSE facility (created by
Kaiser-Threde - DE) on the outside platform EuTEF of the International Space
Station, orbiting round the Earth at a height of 
300 km, where space
conditions include pressures of 10–5 pa, temperatures ranges
between –20 and +20 °C, and full solar (including UV-A, UV-B, and
UV-C) and cosmic radiation. These conditions are normally prohibitive for
life.
Lithopanspermia postulates the feasibility of interplanetary transfer of living material, protected against extraterrestrial solar UV and possibly heat within asteroids, comets and meteorites (Nicholson et al. 2000). Our knowledge on the limits of life has largely expanded in the last decades. The discovery of extremophiles, the high survival of Bacillus subtilis spores over six yr in space (Horneck et al. 1994), the survival of lichens after ground-based experiments (de Vera et al. 2003, 2004), as well as in space for 2 wk during the Biopan experiments (Sancho et al. 2007), and our results with the Experiment Verification Tests on meristematic fungi and cryptoendolithic communities give additional support to the idea of lithopanspermia. Considering that 1 yr is the minimum flight time estimated for Martian meteorites landing on Earth (Mileikowsky et al. 2000), the eventual survival after 1.5 yr permanence in space planned in the LIFE experiment represents a further contribution in the scenario of interplanetary transfer of life.
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Acknowledgments |
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