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ORIGINAL RESEARCH
UV-resistant yeasts isolated from a high-altitude volcanic
area on the Atacama Desert as eukaryotic models for
astrobiology
Andr
e A. Pulschen
1
, Fabio Rodrigues
1
, Rubens T. D. Duarte
2
, Gabriel G. Araujo
3,4
, Iara F. Santiago
5
,
Ivan G. Paulino-Lima
6
, Carlos A. Rosa
5
, Massuo J. Kato
1
, Vivian H. Pellizari
7
& Douglas Galante
3,4
1
Chemistry Institute, Universidade de S~
ao Paulo, S~
ao Paulo, Brazil
2
Microbiology, Immunology and Parasitology Department, Universidade Federal de Santa Catarina, Florian
opolis, Brazil
3
Interunities Graduate Program in Biotechnology, Universidade de S~
ao Paulo, S~
ao Paulo, Brazil
4
Brazilian Synchrotron Light Laboratory, Campinas, Brazil
5
Department of Microbiology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
6
NASA Postdoctoral Program Fellow at NASA Ames Research Center, Moffett Field, California
7
Oceanographic Institute, Universidade de S~
ao Paulo, S~
ao Paulo, Brazil
Keywords
Astrobiology, Atacama Desert, eukaryote,
extremophiles, UV radiation, yeast.
Correspondence
Douglas Galante, Brazilian Synchrotron Light
Laboratory, Av. Giuseppe MaximoScolfaro,
10000 Campinas, S~
ao Paulo CEP 13083-100,
Brazil.
Tel: +551935175081; Fax: +551935121004;
E-mail: douglas.galante@lnls.br
Funding Information
This work was sponsored by FAPESP (Project
2012/18936-0), CAPES, CNPq – Proantar,
USP, through the Brazilian Research Unity in
Astrobiology – NAP/Astrobio and the NASA
Postdoctoral Program.
Received: 5 October 2014; Revised: 18
March 2015; Accepted: 27 March 2015
doi: 10.1002/mbo3.262
Abstract
The Sairecabur volcano (5971 m), in the Atacama Desert, is a high-altitude
extreme environment with high daily temperature variations, acidic soils,
intense UV radiation, and low availability of water. Four different species of
yeasts were isolated from this region using oligotrophic media, identified and
characterized for their tolerance to extreme conditions. rRNA sequencing
revealed high identity (>98%) to Cryptococcus friedmannii,Exophiala sp., Hol-
termanniella watticus, and Rhodosporidium toruloides. To our knowledge, this is
the first report of these yeasts in the Atacama Desert. All isolates showed high
resistance to UV-C, UV-B and environmental-UV radiation, capacity to grow at
moderate saline media (0.75–2.25 mol/L NaCl) and at moderate to cold tem-
peratures, being C. friedmannii and H. watticus able to grow in temperatures
down to 6.5°C. The presence of pigments, analyzed by Raman spectroscopy,
correlated with UV resistance in some cases, but there is evidence that, on the
natural environment, other molecular mechanisms may be as important as pig-
mentation, which has implications for the search of spectroscopic biosignatures
on planetary surfaces. Due to the extreme tolerances of the isolated yeasts, these
organisms represent interesting eukaryotic models for astrobiological purposes.
Introduction
The Atacama Desert (Chile) is an extreme environment
on Earth, classified as a hyper arid desert, with high UV
radiation incidence, scarce sources of organic carbon,
large daily temperature variations and low water availabil-
ity (Navarro-Gonzalez et al. 2003). As an example, de
los Rios and collaborators registered a variation in the
temperature from 46.5°Cto8.00°C, during 1 year, and
a minimum of humidity of 1.40% at the Salar de Yungay
(De los R
ıos et al. 2010). These harsh conditions make
the Atacama Desert a challenging place for life and a
good analog of extraterrestrial environments, such as
Mars (Navarro-Gonzalez et al. 2003; Cabrol et al. 2007).
The Sairecabur volcano, located on the San Pedro de Ata-
cama region, near the Salar de Atacama, is an example of
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
1
such distinct regions, being oligotrophic, arid, with high
daily temperature variations and occurrence of only occa-
sional snowfalls. The absence of permanent snow cover or
glaciers is indicative of the exceptionally dry climate of
such high-altitude regions (Costello et al. 2009; Lynch
et al. 2012). In addition, the incidence of Solar radiation
is higher at greater altitudes (Cabrol et al. 2014), includ-
ing deleterious UV radiation, thus producing an environ-
ment restrictive to most living organisms (Lynch et al.
2012). Indeed, several works concerning the microbiology
of high altitudes focus on the effect of UV radiation
(Zenoff et al. 2006; Libkind et al. 2009; Ordonez et al.
2009). In addition, microorganisms living at high altitude
must also deal with low temperatures, around the freezing
point of water, and desiccation stress, due to the low
atmospheric pressure and humidity.
The microbial diversity of the Atacama Desert has been
studied over the last years, focusing mainly on the bacte-
rial (Drees et al. 2006; Connon et al. 2007; Okoro et al.
2009; Neilson et al. 2012) and cyanobacterial diversities
(Warren-Rhodes et al. 2006; Wierzchos et al. 2006;
Azua-Bustos et al. 2012). Prokaryotes have been studied
regarding their tolerances to several stressors present in
the Atacama, including the inactivation by environmen-
tal-UV (Dose et al. 2001; Cockell et al. 2008), growth at
several concentrations of salt (Rivadeneyra et al. 1999;
Cockell et al. 2010) and desiccation (Billi 2009). In addi-
tion, some isolates from the Atacama were shown to be
resistant to some stressors that are not found on the ter-
restrial environment, such as UV-C and ionizing radiation
(Billi et al. 2000; Paulino-Lima et al. 2013). The resistance
of those organisms to extreme conditions makes them
interesting candidates for astrobiological studies, as
model-organisms to survive on harsh extraterrestrial envi-
ronments. For this purpose, the high-altitude regions in
the Atacama have a strong potential to be further
explored (Cabrol et al. 2009).
However, even considering all the efforts in characteriz-
ing the microbial diversity of the Atacama, few works
have investigated the presence and adaptive mechanisms
of eukaryotes at the desert (Conley et al. 2006). There-
fore, the goal of this work was to isolate and characterize
yeast strains from soil samples collected at the Sairecabur
volcano, and to submit the isolates to different labora-
tory-simulated extreme conditions. More specifically, the
strains were exposed to different fluences of environmen-
tal-UV, UV-B and UV-C radiation; photoprotective pig-
ments were characterized by Raman spectroscopy and
analyzed for their role on the UV resistance, as well as
the capability of the strains to grow in moderate to high
salt concentrations and at different temperatures. The
results demonstrate the adaptive competence of such
organisms to conditions found in the Atacama Desert,
and even harsher ones, which could be present on extra-
terrestrial environments, contributing to expand our
knowledge of the adaptations of eukaryotes in extreme
conditions, and proposing new models to be used in as-
trobiological studies.
Material and Methods
Site and soil characterization
Samples were collected from the top layer of soil at three dif-
ferent sites of the Sairecabur volcano, Atacama Desert
(Fig. 1), in January 2012, using sterile tools, placed in sterile
50 mL tubes, sealed and kept refrigerated until the analysis.
The three soil samples used in this work were collected in
the following sites (latitude, longitude and altitude, respec-
tively): S5047 (soil from volcano slope) =22.716945°S/
67.923690°W/5047 m; S3981 (soil from volcano
slope) =22.706917°S/67.996050°W/3981 m; S4823 (sulfur-
rich soil) =22.715898°S/67.933632°W/4823 m.
Measurements of UV-A and UV-B fluxes at the vol-
cano were made at 5091 m using a portable radiometer
(Vilber Lourmat VLX-3W, Marne-la-Vall
ee, France). For
comparison, additional measurements were made with
the Sun on the zenith, on the same season, at the Salar
Yungay (Atacama, Chile –24.115012°S/69.880035°W/Alti-
tude: 948 m) and in a region of similar latitude but of
sub-tropical climate, in Brazil (S~
ao Paulo state, Brazil –
23.00464°S/46.964807°W/850 m).
The collected samples were characterized by measuring
the pH using the methodology described by Barrett et al.
(2004). Salinity was evaluated by measuring the conduc-
tivity, as described by Okoro et al. (2009). X-ray fluores-
cence (XRF) was used to determine the elemental
composition of the soil samples (Table 1). The XRF mea-
surements were made at the XRF beamline of the Brazil-
ian Synchrotron Light Laboratory (LNLS) (Perez et al.
1999) in the microbeam mode, using polychromatic exci-
tation and an elliptical capillary for focusing the beam on
a spot of about 50 lm diameter, with the final spectra
being averages of at least five different points to minimize
intrinsic inhomogeneity. All data were treated using the
PyMCA software (Sole et al. 2007) for the calculation of
the absolute values of concentration for each element.
Isolation and molecular identification
Yeast colonies were isolated using a mineral, organic-poor
medium, composed of (NH
4
)
2
SO
4
, 0.4 g L
1
;KH
2
PO
4
,
0.5 g L
1
; CaCl
2
, 0.25 g L
1
; MgSO
4
, 0.5 g L
1
;Na
2
S
2
O
3
,
5gL
1
, and FeSO
4
, 0.01 g L
1
;15gL
1
DifcoBacto
Agar; pH adjusted to 4.8 (all reactants were purchased
from Synth (Diadema, S~
ao Paulo, Brazil)). To extract yeast
2ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Extremophilic Yeasts from the High-Altitude Atacama A. A. Pulschen et al.
cells from the soil, 2 g of the sample was transferred to
flasks with 20 mL of sterile 0.9% w/v (roughly 0.15 mol/L)
NaCl saline solution (pH 4.8) and shaken at 150 rpm for
24 h at 30°Cor4°C. Aliquots of 100 lL were spread on
mineral medium and incubated at 30°C and 4°C. Yeast
colonies were then transferred to TGY plates (yeast extract,
5gL
1
; tryptone, 5 g L
1
and glucose, 1 g L
1
) with pH
adjusted to 4.8.
Yeast identification was carried out by sequencing the
D1–D2 variable domains of the large rRNA subunit gene
using the primers NL1 (50GCATATCAATAAGCGGAG-
GAAAAG) and NL4 (50GGTCCGTGTTTCAAGACGG),
as described by Rosa et al. (1999) and were confirmed by
the amplification of the internal transcribed spacer (ITS)
and ITS4. After amplification, the purified PCR fragments
were sequenced with an ABI 3130 Genetic Analyzer auto-
mated sequencing system (Life Technologies, Carlsbad,
CA). The obtained sequences were analyzed using the
GenBank database and the nearest species found were
used in the molecular analysis (Table 2). Yeasts were
stored on GYMP broth (glucose, 20 g L
1
; yeast extract,
5gL
1
; malt extract, 5 g L
1
;Na
2
PO
4
,2gL
1
) with
20% of glycerol at 80°C and deposited in the Culture
Collection of Microorganisms and Cells at the Universid-
ade Federal de Minas Gerais –UFMG.
Raman spectroscopy and characterization of
pigments
To investigate the presence of photoprotective pigments,
mainly melanin and carotenoids, the isolates were analyzed
by Raman spectroscopy, using a Renishaw (Renishaw PLC,
Chile Argentina
Bolivia
Sairecabur
Volcano
Atacama
Desert
Ocean
N
(A)
(C)(B)
Figure 1. Location of Sairecabur volcano, in the Atacama Desert, Chile (A). At the time of the sample collection, temperatures were below the
freezing point and some areas of the volcano were covered with snow (B and C).
Table 1. pH, conductivity and elementary composition (in mass con-
centration, obtained with X-ray fluorescence, for Z≥15) of the soil
samples.
1. Slopesoil
5047 m
(S5047)
2. Slopesoil
3981 m
S3981)
3. Sulfur-rich
soil 4823 m
(S4823)
pH 4.25 5.36 3.34
Conductivity 11.6 lS/cm 83.9 lS/cm 376 lS/cm
S––15.8%
Cl –– –
K 0.41% 0.02% 5.32%
Mn 0.02% –0.05%
Fe 1.07% 0.15% 3.48%
Cu –– –
Trace elements (–) are here defined as those with concentrations
lower than 0.01%.
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 3
A. A. Pulschen et al.Extremophilic Yeasts from the High-Altitude Atacama
Wotton-under-Edge, UK) inVia micro-Raman Spectrome-
ter with HeNe laser line (633 nm), 209objective and CCD
detector, and a Bruker (Bremen, Germany) RFS 100/S FT-
Raman with incident radiation of 1064 nm and a LN
2
-
cooled Ge detector. The samples were analyzed without fur-
ther preparation, by removing the colonies from the plates
and depositing them on glass slides. Low laser power was
used to avoid thermal or photochemical damage.
UV-C and UV-B resistance experiments
The UV-C resistance of the yeasts was evaluated in com-
parison to Saccharomyces cerevisiae, strainBY4742, used as
low-radiation resistant model. Since there is no consen-
sual UV-resistant model for yeasts, it was used the radio-
resistant bacterium Deinococcus radiodurans (R1 type
strain, obtained from Instituto de Radioprotec
ß
~
ao e Do-
simetria –IRD, Rio de Janeiro, Brazil) to validate the
experiment. The yeasts were grown in liquid TGY med-
ium, pH 4.8, under 150 rpm shaking, at 10°C for Exophi-
ala sp. 15Lv1, Cryptococcus friedmannii, and
Holtermanniella watticus,at30°C for S. cerevisiae and
Rhodosporidium toruloides, and at 30°C and no pH adjust-
ment for D. radiodurans.
Cells were grown to OD
595
of about 0.6–0.8, washed
twice with 0.9% w/v NaCl solution (pH corrected to 4.5
for the yeasts, and neutral for D. radiodurans). The pellet
was dispersed in sterile saline solution and adjusted to a
final concentration of 10
6
–10
7
cells/mL. A volume of
10 mL of this cell suspension was transferred to a 10 cm
diameter sterile Petri dish and irradiated under orbital
shaking with a Philips (Philips, Eindhoven, The Nether-
lands) TUV-20W low-pressure Hg lamp (253.7 nm). The
irradiation was monitored during the experiment using a
calibrated radiometer (Vilber Lourmat RMX-3W) and a
UV-C photocell (CX-254, Vilber Lourmat). The UV-C
flux measured during the irradiation was 6.0 W/m²and
the sample was placed at 25 cm from the lamp. No sub-
stantial temperature variation that could affect cell sur-
vival was detected in the solution during the experiment.
After the different fluences, the colony-forming units
(CFU) were evaluated by incubation on TGY agar plates
at 10°Cor30°C in the dark. All experiments were made
in triplicates.
The UV-B experiment was carried out in a similar way,
using the same radiometer with an UV-B photocell (CX-
312, Vilber Lourmat) to monitor the fluence. Two Light-
Tech Narrow Band UV-B 20 W Hg lamps and one Phi-
lips TL20W Hg lamp with major line-emission at 312 nm
were used to generate the UV-B radiation, with a mea-
sured intensity of 16.5 W/m
2
. Since more time under
UV-B radiation was needed to generate enough biological
damage, 7 cm diameter Petri dishes, surrounded by wet
Table 2. Growth range, halotolerance, and molecular identification of the isolated yeasts used on the resistance experiments, using BLASTn and ITS region
Isolate
ID
Sample
ID Primer Result top BLAST (access no. GenBank)
Identity
(%)
No. of bp
analyzed
Species or proposed taxonomic group
(GenBank access no.)
Halotolerance
(NaCl)
Max. temperature
with observable
growth
Min. temperature
with observable
growth
16Lv2 S5047 NL Cryptococcus friedmannii (JX092255) 100 599 Cryptococcus friedmannii (KM243310) 1.75 mol/L 25°C (weak) 6.5°C
ITS Cryptococcus friedmannii (AF145322) 99 549 Cryptococcu sfriedmannii (KM243311)
15Lv1 S4823 NL Exophiala capensis (JF499861) 99 587 Exophiala sp. (KM243304) 1.25 mol/L 25°C0°C
ITS Exophiala sp. (HQ452332) 99 509 Exophiala sp. (KM243305)
16Lv1 S3981 NL Holtermanniella watticus (KC006859) 99 601 Holtermanniella watticus (KM243308) 2.25 mol/L 25°C (weak) 6.5°C
ITS Holtermanniella watticus (JQ857031) 99 501 Holtermanniella watticus (KM243309)
16Lv3 S3981 NL Rhodosporidium toruloides (GQ169736) 99 587 Rhodosporidium toruloides (KM243312) 0.75 mol/L 30°C10°C
ITS Rhodosporidium toruloides (JN246549) 98 524 Rhodosporidium toruloides (KM243313)
ITS, internal transcribed spacer.
1
Coverage of 100% for all sequences analyzed.
4ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Extremophilic Yeasts from the High-Altitude Atacama A. A. Pulschen et al.
cotton, were used to counterbalance the evaporation rate.
On each plate, 6 mL of cell suspension were added, and
no variation on the temperature of the solution was
observed during the exposure. The survival was evaluated
by CFU counting, and the experiments were performed in
triplicates. The lamps, as measured with the UV-C photo-
cell, emitted no UV-C.
Environmental-UV radiation tolerance
experiment
The environmental-UV experiment was designed to
approximate the conditions found on the natural envi-
ronment on Earth. The exposures were performed directly
over agar, since the microorganisms were collected from
solid substrate, not from a solution. In addition, in natu-
ral environments (specially the more restrictive ones),
microorganisms, including yeasts, are likely to exist in a
low metabolic, quiescent state, more similar to the sta-
tionary phase (Gray et al. 2004; Navarro Llorens et al.
2010). Previous authors, working with high-altitude envi-
ronments and performing UV experiments have also pre-
ferred not to irradiate the cells during active growth
(Zenoff et al. 2006). With that in mind, the cells were
grown in TGY media to the beginning of the stationary
phase. For C. friedmannii, H. watticus, R. toruloides and S.
cerevisiae, OD
595
~0.480 (after 1:20 dilution), for Exophi-
ala sp.15Lv1, OD
595
~0.900 (after 1:20 dilution) and for
D. radiodurans OD
595
~0.670 (after 1:10 dilution). The
suspension of washed cells was spread on TGY pH 4.8
agar plates (or TGY pH 7 for D. radiodurans) at different
dilutions (10
7
–10
3
cells/mL) and the plates were exposed
(without lid) to simulated environmental-UV radiation,
using an Oriel
(California, USA) Sol UV-2 Solar simula-
tor (85.7% UV-A, 11% UV-B and 3.3% of visible light).
The flux during the irradiation was 96.0 W/m
2
for UV-B
and 131.5 W/m
2
for UV-A measured with a Vilber Lour-
mat radiometer with UV-B and UV-A photocells (CX-312
and CX-365, Vilber Lourmat). The plates were exposed
for 10, 20, 30, and 40 min, at room temperature (~20°C)
and then incubated. The survival was evaluated by CFU
counting, in triplicates. Once again, using the UV-C pho-
tocell, we certified that the simulator emitted no signifi-
cant UV-C during the irradiation procedure (see Fig. S3
for the complete spectrum).
Temperature growth range, halotolerance,
and oligotrophy
The temperature experiment was performed with cultures
on TGY pH 4.8 agar plates incubated at 0, 4, 10, 15, 20,
25, and 30°C and in TGY pH 4.8 liquid medium under
agitation with anti-freezing solutes for temperatures rang-
ing from 0 to 6.5°C (Chin et al. 2010). The growth
curves were recorded at 0°C and 3°C with the flasks
initially inoculated to OD
595
=0.2 from a previous
growth performed at 0°C. For the growth curve at
6.5°C, flasks were inoculated also to an initial
OD
595
=0.2 from a previous growth at 3°C with glyc-
erol 0.4 mol/L (w/v). The high initial optical density was
used to allow a faster evaluation of the yeasts’ develop-
ment, since flasks with small initial optical densities
would take longer times to show evidences of growth of
the organism, especially at low temperatures. To minimize
freezing, the experiments at 3°C, were performed using
either 0.5 mol/L of NaCl (w/v), a kosmotropic solute, or
0.4 mol/L of glycerol (w/v), a chaotropic solute (Chin
et al. 2010). At 6.5°C, it was used 0.6 mol/L (w/v) of
glycerol.
For the studies concerning the halotolerance, agar
plates of saline TGY medium pH 4.8 were prepared with
different NaCl concentrations: 0.50, 0.75, 1.00, 1.25, 1.50,
1.75, 2.00, 2.25, and 2.50 mol/L. To ensure proper solidi-
fication, it was used 24 g L
1
of agar. The inoculum on
the saline medium was made from recently cultured
nonsaline plates (TGY medium), and CFU was evaluated.
Finally, since the yeasts were isolated in culture media
without the addition of any carbon source, the growth
capacity in low nutrient conditions of the isolates was
confirmed with a similar methodology to the one used by
Uetake et al. (2012), using ultra-pure water agar medium
(UWA). The medium was prepared with 1.5% w/v of
Difco Bacto Agar and ultrapure water (Milli-Q
, from
Millipore (Molsheim, France) Direct-Q system), with pH
adjusted to 4.8 with a 0.5 mol/L H
2
SO
4
solution. The
cells were spread on the UWA plates, incubated and the
CFU (and the size of the colonies) was evaluated after
20 days.
Results and Discussion
Soil samples and environmental conditions
Some of the physicochemical characteristics of the sam-
pling sites are shown in Table 1. The data obtained from
XRF corroborates the observation that S4823 consists in a
sulfur-rich soil. In addition, this was the most acidic sam-
ple studied, with the low pH of volcanic soils being
defined by many factors, as age, precipitation, presence of
organic matter, volcanic, and biological activity (Flierman
and Brock 1972; Ugolini and Dahlgren 2002; Dahlgren
et al. 2004). Since yeasts can grow in acidic media, this
might represent an advantage to those organisms at this
particular environment.
Concerning the conductivity, Drees et al. (2006),
reported a large variation in conductivity values in soils
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 5
A. A. Pulschen et al.Extremophilic Yeasts from the High-Altitude Atacama
of the Atacama, with lower ones of about 10 lS/cm and
higher ones greater than 2000 lS/cm. In the same way,
the values found by Okoro et al. (2009) vary from 126 to
1540 lS/cm, being the last one from a salt-rich soil col-
lected at the Valle de la Luna site. In our work, the values
found in S5047 and S3981 are comparable with the lower
values, while that for S4823 is an average value. The low
conductivity obtained in S5047 and S3981 are in accor-
dance with the elemental composition measured, since it
was observed a low content of ion-forming species such
as chloride, potassium, calcium and sulfur. In S4823 that
had a greater value of conductivity, a higher concentra-
tion of potassium (5.32%) was measured. In addition, the
presence of sulfur-oxidizing microorganisms could be
responsible for the increase in conductivity, due to sulfate
production, which is coherent with the lower pH,
although this was not evaluated on the present work.
It has to be considered, however, that the macroscopic
conductivity values and mineral composition may not be
exactly the ones existing on the aqueous milieu where
microorganisms can be thriving. Although microniches
can be present and macroscopic measurements cannot
properly analyze these small environments, this data is
still useful for a general characterization of the sample.
Considering the environmental radiation measure-
ments, the intensity of UV-B and UV-A increases with
the altitude and absence of clouds or water vapor (Blumt-
haler et al. 1997), hence the dry conditions of the desert
and the altitude of the volcano contribute for the intense
UV flux. The value measured at 5091 m at the time of
the sampling (at noon) was 36.4 W/m
2
for UV-A and
15.6 W/m
2
for UV-B. For comparison, the radiation val-
ues measured at Salar Yungay (on the hyper arid region
of the Atacama, at 948 m) were 29.5 W/m
2
for UV-A
and 11.6 W/m
2
for UV-B; in Brazil (S~
ao Paulo), under
subtropical conditions, the measurements were 26.4 W/
m
2
for UV-A and 9.3 W/m
2
for UV-B radiation.
Molecular identification, growth and
pigment characterization of the isolates
Four different species were identified from the isolated
yeasts (Table 2). To our knowledge, this is the first report
of those species at the Atacama Desert. The yeast C. fried-
mannii and H. watticus were both first isolated from the
Antarctica samples (Vishniac 1985; Guffogg et al. 2004),
and also found in other cold environments, as Iceland
and Russia (Vishniac 2006). It is known that the genus
Cryptococcus is commonly found and adapted to live in
deserts (Vishniac 1998), being tolerant to UV, desiccation
and nutrient-poor conditions, with some species already
described on the Atacama Desert (Vishniac 2006). The
yeast R. toruloides has been suggested to be adapted to
some extreme environments, with low pH and high con-
centrations of toxic metals (Gadanho et al. 2006), but this
is the first time that it is reported in a cold, high-altitude
environment. A black yeast species was isolated from
S4823, however, the analysis of the NL and ITS could
only state that it belongs to Exophiala genus, being other
primers at different regions necessary for the identifica-
tion at the species level. More experiments are being per-
formed by the group and will be presented in a future
work. Although some of the same yeast species were iso-
lated from different samples of the volcano (R. toruloides
in S5047, (Genbank KM243302 for NL and KM243303
for ITS), C. friedmannii in S3981, (Genbank KM243300
for NL andKM243301 for ITS) and also in S4823
(KM243306 for NL andKM243307 for ITS), the full set of
resistance experiments was performed only with one
strain of each species (described on Table 2). Preliminary
experiments demonstrated that there were no major dif-
ferences on the biological responses between the same
species isolated from different samples.
The qualitative results concerning halotolerance, oligo-
trophic growth, and growth temperatures are presented in
Table 2. Although the volcanic soils presented low to
moderate salinity in a macroscopic scale, the salt toler-
ance of the isolates was evaluated to assess their potential
for astrobiological studies, since salt deposits were discov-
ered on Mars (Osterloo et al. 2008). Concentrations of
0.6 mol/L NaCl are already toxic for S. cerevisiae, for
example (Prista et al. 1997). Rhodosporidium toruloides
was shown to possess the lowest NaCl tolerance among
the yeasts isolated from the volcano, being 0.75 mol/L of
NaCl already enough to diminish greatly the formation of
colonies. The other yeasts have shown greater tolerance,
being H. watticus the most salt-tolerant one, capable of
growth even at 2.25 mol/L of NaCl. According to Vishni-
ac (1998), there is no obvious correlation between yeasts
found in arid soils and deserts and osmotolerance, since
the low organic content in these soils is not normally
compatible with the high energy requirements for halotol-
erance in yeasts. Low growth rates have also been shown
to have an anti-correlation with halotolerance.
Uetake et al. (2012) have recently reported the isolation
of oligotrophic yeasts from the Gulkana glacier (Alaska,
USA) using UWA media, suggesting that those yeasts
were capable of oligotrophic growth on sites where liquid
water was present on the glacier. Using the same organic-
poor medium, H. watticus, C. friedimannii, and Exophiala
sp.15Lv1 showed the capacity to generate 1 mm colonies
after 20 days. The yeast R. toruloides was also capable of
developing colonies at UWA media, but they were smaller
when compared with the other yeasts, no bigger than
0.5 mm. The capacity of development at low nutrient
conditions is interesting for astrobiological purposes, but
6ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Extremophilic Yeasts from the High-Altitude Atacama A. A. Pulschen et al.
it can also represent an advantage for prevailing at
organic poor soils, which constitutes one of the limiting
factors for life at volcanic areas at the Atacama Desert
(Lynch et al. 2012).
Three of the yeasts have shown growth at low tempera-
tures (shown in Fig. S1), including at 0°C. However, only
H. watticus and C. friedmannii have shown growth at
3°C and 6.5°C. It was also observed a slow growth of
Exophiala sp.15Lv1 at 0°C, but not at lower temperatures.
Using NaCl, a kosmotropic solute (which disfavors growth
of fungi at lower temperatures [see Chin et al. 2010]), it
was observed a diminishment of the optical density for
longer periods of incubation. Using the chaotropic solute
glycerol, there was no increase at the optical density after
14 days, but after this period, the Exophiala flasks started
to freeze (for unknown reasons). As it was possible to
have good growth with the tested solutes, on these con-
centration, at the optimum temperature for the yeasts
(although with a small delay for NaCl, see Fig. S1), the
observed effects at the experiment were mostly due to the
temperature, discarding interferences from the anti-freez-
ing agents. To confirm the capability of Exophiala
sp.15Lv1 to grow at temperatures below 0°C, higher con-
centrations of glycerol and longer incubations periods
might be needed, due to the slow growth behavior of this
isolate. Although the R. toruloides strains isolated from the
volcano did not develop at 4°C and 0°C, and it was
observed that colony growth at 10°C induced a change on
the pigmentation from orange to an intense pink, which
might indicate a stress-response strategy of R. toruloides to
the high altitude and cold environment of the volcano.
The Atacama Desert presents great daily temperature
variations, but due to the high altitude, the temperatures
are mainly colder than in lower areas, being the capacity
to develop at temperatures as low as 0°C a possible
advantage to such organisms. In addition, as has been
already pointed out by Chin et al. (2010), the presence of
chaotropic salts found on Mars may favor the survival of
cold, tolerant microorganisms at lower temperatures.
Recently, Fischer et al. (2014), studying the chaotropic
salts NaClO
4
and Ca(ClO
4
)
2
have demonstrated that
regions where salts and ice coexist in Mars might form
liquid brines temporarily, which could allow microbial
growth to thrive, even on the surface of that planet. Also,
according to Osterloo et al. (2008), chlorides salts are
globally spread on Mars. Thus, the capability of thriving
at temperatures below the freezing point in chaotropic
solutes (as glycerol) or in kosmotropic solutes (as NaCl)
as observed by C. fredmannii and H. watticus, as well the
salt tolerance of the isolates, are also important when
considering these yeasts by an astrobiological perspective.
In order to further characterize each isolate, pigment
analysis using Raman spectroscopy was performed. This
technique is being used in the literature in the search of
biosignatures in the context of astrobiology (Ellery and
Wynn-Williams 2003; Edwards et al. 2005) and it can be
a simple analytical method to analyze the presence of
some categories of photoprotective pigments with minor
preparation of the samples. In the present work, the spec-
tra (Fig. 2) were obtained directly from colonies growing
on solid culture media, without further processing.
The two strong peaks at ca. 1150 and 1510 cm
1
in the
Raman spectra, as observed in Figure 2B, are well
described on the literature as corresponding to carote-
noids, more specifically to the C=C–C stretch modes (De
Oliveira et al. 2010). Since carotenoids are widespread in
different living systems, including several kinds of micro-
organisms, and their Raman bands are very intense, they
are being proposed as one potential spectroscopic biosig-
nature for the detection of extant life on Mars (Parnell
et al. 2007). The two broad bands at ca. 1320 and
1570 cm
1
, as observed in Figure 2C, can be attributed to
the class of melanin (Perna et al. 2013). The laser power
was kept low to avoid damaging the samples, and a
threshold test was performed to ensure that the 1300/
1600 bands are not caused by the presence of D and G
carbon bands normally observed in the decomposition of
organic matter. The failure to find well-defined bands on
the spectra of the other samples, as on Figure 2A, should
not be interpreted as the total absence of pigments, since
they can be present in minor quantities, below the detec-
tion limit of this technique in the conditions of the
Figure 2. Raman spectra of the yeast cultures: (A) Cryptococcus
friedmannii. Similar spectra with no evident peaks were acquired for
Holtermanniella watticus and Saccharomyces cerevisiae, not shown
here; (B) Rhodosporidium toruloides. Similar spectra were acquired for
Deinococcus radiodurans and therefore not shown; (C) Exophiala sp.
15Lv1.
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 7
A. A. Pulschen et al.Extremophilic Yeasts from the High-Altitude Atacama
measurement (Jehlicka et al. 2014). In all the cases, the
interpretation of the Raman spectra is in agreement with
the color of the colonies grown on TGY media: orange
for R. toruloides, black to Exophiala sp. and pale-beige for
C. friedmannii and for H. watticus.
Carotenoids and melanins are known as UV protective
pigments (Singaravelan et al. 2008; Libkind et al. 2009)
since they are capable of absorbing UV radiation at differ-
ent wavelengths, including on the UV-C (Wynn-Williams
and Edwards 2002). In addition, carotenoids can scavenge
free radicals generated by UV light, protecting the cell
against oxidative damage (Moore et al. 1989; Schroeder
and Johnson 1993). It is important to state, however, that
other pigments or photoprotective compounds without
intense Raman bands can also be present in the samples,
without being detected by this technique, due to charac-
teristics of the samples (such as strong fluorescence).
UV exposure experiments and survival
profile of the isolates
Any microorganism that could inhabit the Martian sur-
face would be exposed to intense ultraviolet radiation
from the Sun, including UV-C radiation, due to the lack
of an ozone layer (Paulino-Lima et al. 2013), unless it
was shielded by soil or dust. UV-C radiation (200–
280 nm) was probably present on the environment of the
Archaean Earth, before the rise of oxygen in the atmo-
sphere (Cockell 1998). It is still abundant above the ozone
layer in the present day Earth’s stratosphere, where some
microorganisms have been detected since 1936 (Smith
2013). UV-C radiation is very effective to inactivate
microorganisms, being commonly used for sterilizing sur-
faces, as used on most of the germicidal lamps. Therefore,
it is an easily accessible tool to select extremely radiation-
resistant microorganisms. UV-B radiation (280–320 nm)
is the most biologically active form of UV radiation
reaching the surface of present-day Earth (Bj€
orn 2008),
however, it is easily scattered by clouds in the atmo-
sphere. Since UV-A radiation (320–400 nm) is the most
abundant of the Solar UV spectrum, it is usually consid-
ered the most important for practical purposes on natural
environments (Bj€
orn 2008).
Both UV-C and UV-B portions of the electromagnetic
spectrum are capable of generating cyclobutane pyrimi-
dine dimers (CPDs) and (6-4) pyrimidine-pyrimidine
(Douki et al. 1997), which can lead to DNA breaks due
to inefficient repair (Santos et al. 2013). UV-C radiation
is more efficient than UV-B in generating DNA photo-
products (Mitchell et al. 1991; Ravanat et al. 2001). How-
ever, UV-B has a higher efficiency when compared to
UV-C radiation in generating reactive oxygen species
(ROS) (Santos et al. 2013), which leads to cell death by
damaging not only DNA, but also other cell components,
as lipids and proteins (Daly 2012; Santos et al. 2013).
Although UV-A radiation can also induce DNA photo-
products, like CPDs, it is less efficient than UV-C and UV-
B (Cadet et al. 2012) with its lethal biological effects occur-
ring mainly due to generation of ROS species (Hoerter
et al. 2005; Santos et al. 2013). An important oxidative
base modification generated under UV-A irradiation, 7,8-
dihydro-8-oxoguanine (8-oxoG), occurs through oxidative
damage of the DNA and has great impact on mutagenesis
and cell survival of S. cerevisiae, for example. In this sce-
nario, CPDs have minor importance when compared to 8-
oxoG (Kozmin et al. 2005), although experiments with
several mammalian cells suggests that CPDs plays impor-
tant roles in UV-A genotoxicity in such models (Cadet
et al. 2012). Also, the combined exposure of UV-A and
UV-B radiation can lead to the photoisomerization of 6,4-
PPs into Dewar valence isomers, a third class bipyrimidine
photoproduct, and important when considering microbial
survival under environmental UV exposure (Meador et al.
2014).
In our experiments, it was observed that all isolates
were highly resistant to UV-C and UV-B, especially when
compared to S. cerevisiae, being some of the survival pro-
files similar to D. radiodurans (Figs. 3, 4).
The survival of the black yeast Exophiala sp.15Lv1 and
R. toruloides was higher than the negative control S. cere-
visiae, even matching that of D. radiodurans. Using our
experimental protocol, Exophiala sp.and R. toruloides
only lost one log of viability (measured by CFU counting)
after exposure to 1 kJ/m
2
, while S. cerevisiae lost viability
with smaller fluences (almost three logs drop of CFU
counts after 0.2 kJ/m
2
of exposure). The yeasts C. fried-
mannii and H. watticus also scored greater survival, losing
90% of viability only after 0.6 kJ/m
2
. Regarding the UV-B
Figure 3. Survival curves for UV-C radiation of the tested organisms.
The error bars indicate the variance between the triplicates.
8ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Extremophilic Yeasts from the High-Altitude Atacama A. A. Pulschen et al.
radiation, Exophiala sp.15Lv1 and R. toruloides have also
shown similar resistances to that of D. radiodurans and
greater survival when compared to S. cerevisiae. However,
the difference of survivability in this wavelength range
among the isolated yeasts and the negative control (S. ce-
revisiae) appears to be smaller than on the UV-C. These
differences might reflect the biological effects caused by
UV-C and UV-B radiation.
Since the intensity and effects at the cell level are differ-
ent, the yeast cells must display different mechanisms to
deal or avoid those damages, being one of them the pres-
ence of photoprotective pigments. In our experiments,
the two pigmented yeasts (Exophiala sp.15Lv1, melanin
rich and R. toruloides, carotenoid rich) have shown the
greatest survival under UV-B and UV-C irradiation, when
compared to the yeasts without detectable bands attrib-
uted to pigments on the Raman spectra (C. friedmannii
and H. watticus). Moline et al. (2009), comparing pig-
mented and natural albino strains of yeasts, have pro-
posed that carotenoids enhanced the UV-B resistance on
those organisms. Regarding melanin, Wang and Casadev-
all (1994) have found that the presence this pigment has
enhanced the UV-C resistance of melanized Cryptococcus
neoformans, when compared with the nonmelanized cells.
However, Schiave et al. (2009) reported that there is small
or no difference between melanized and nonmelanized
cells of C. neoformans and Cryptococcus laurenti after UV-
B exposure, questioning the capacity of melanin to pro-
vide protection against UV-B.
Although the pigmented yeasts tested in this work were
more resistant to UV-C and UV-B than the nonpig-
mented ones, the presence of carotenoids and melanin
itself cannot ensure the survival against radiation, as it
does not completely block UV from affecting the DNA
(Sinha and Hader 2002). Thus, complementary cellular
mechanisms should be present. This is corroborated by
our data, as on the survival curves of H. watticus and C.
friedmannii (Figs. 3, 4), which, despite the lack of signal
of the bands attributed to pigments on Raman spectra in
our growth conditions, still presented a significant resis-
tance to UV-B and, especially, to UV-C radiation.
All the isolated yeasts have presented significant resis-
tance for simulated environmental-UV (Fig. 5). Concern-
ing the presence of melanin or carotenoids, the
pigmented ones have not presented the best survival rates,
suggesting that, at least for the tested species, other pro-
tection or repair mechanisms could be more important
for environmental-UV resistance than pigmentation.
The presence of other photoprotective molecules like
mycosporine (Libkind et al. 2009) or the expression of
antioxidant enzymes that can efficiently scavenge ROS,
may also be important in preventing the cellular malfunc-
tion generated by UV radiation (Hoerter et al. 2005). In
addition, photoreactivation mechanisms might contribute
to the repair of photoreaction damages to DNA, gener-
ated by UV light and constitute an important adaptation
to endure such radiations (Zenoff et al. 2006).
Photoreactivation is generally performed by photolyas-
es, enzymes that can repair CPDs and 6,4-PPs generated
by UV-B and UV-C light. The repair can be induced by
photosynthetically active radiation (PAR, 400–700 nm) or
UV-A radiation and it has been demonstrated to be an
important adaptive mechanism in high-altitude microor-
ganisms (Zenoff et al. 2006; Albarrac
ın et al. 2012). It is
also known that Dewar lesions, generated under Solar
irradiation, can be repaired by (6,4) DNA photolyases,
although not very efficiently (Glas et al. 2010). In fact, all
Figure 4. Survival curves for UV-B radiation of the tested organisms.
The error bars indicate the variance between the triplicates.
Figure 5. Survival curves for environmental-UV radiation of the
tested organisms. The error bars indicate the variance between the
triplicates.
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 9
A. A. Pulschen et al.Extremophilic Yeasts from the High-Altitude Atacama
of the isolated yeasts have shown photorepair activity
under the tested conditions in a qualitative experiment
(Fig. S2). Using bioinformatics’ approaches, Lucas-Lled
o
and Lynch (2009) have already demonstrated that some
fungi species seems to completely lack photolyase genes
(C. neoformans and Candida albicans, e.g.) and argued
that the low efficiency of natural selection on eukaryotic
photolyases might be due to environments with low UV-
incidence. Thus, the presence of such repair mechanism
in our isolated yeasts can represent an adaptive advantage
at the UV-rich Sairecabur environment. In addition, the
capability of greater UV-C endurance via photolyases is
interesting from an astrobiological point of view.
When exposed to environmental radiation, even the
nonpigmented S. cerevisiae presented a better survival
than the pigmented and radioresistant bacteria D. radio-
durans. Indeed, it is known that D. radiodurans, even pos-
sessing carotenoids, is sensitive to UV-A radiation, mainly
because of the singlet oxygen species generated by this
kind of radiation (Slade and Radman 2011). This result
suggests that even an organism that presents resistance to
UV-B irradiation when tested with a lamp emitting spec-
tral lines, may not necessarily be adapted to environmen-
tal-UV radiation. To ensure that this behavior was not
caused due our experimental procedure, different growth
phases of D. radiodurans were tested in saline solution,
showing that is was consistently more sensitive than S. ce-
revisiae to environmental-UV in all cases (Fig. S4).
It should be noted that the differences in the biological
response for the same fluence with the UV-B lamps and
Solar simulator (as can be observed on the experiment
depicted on Figs. 3, 5) might reflect different underlying
processes. As already pointed, the UV lamps produce
intense spectral lines, in contrast to the broad continuous
emission of the Solar simulator (Fig. S3). This can induce
nonequivalent biological responses when both sources are
compared, even for the same measured fluences, as the
UV-B radiometer sensor cells used in this work, and on
many others, have a range of sensitivity around 312 nm
(280–320 nm). The UV-B lamps used have a peak emis-
sion at 312 nm, so most of the measured intensity corre-
spond to this wavelength. The UV spectrum provided by
the Solar simulator ranges from 280 nm to 400 nm, with
a large proportion at the longer wavelengths, which have
lower biological effectiveness (Horneck et al. 2006).
This explains why the UV-B radiation from the lamp
was more effective than the UV-B radiation provided by
the Solar simulator in inactivating the microorganisms. In
any case, the direct comparison of the response to a line
source, as the low-pressure Hg lamps for UV-B, to a
broadband source, as the Solar simulator or natural Solar
light, should be made with caution, as it has been already
demonstrated that there are significant differences in
microbial inactivation when comparing irradiated cells
with polychromatic and monochromatic UV light (Zim-
mer and Slawson 2002). The Solar simulator represents
better (and thus was used to mimic) the environmental
conditions of Solar illumination on the surface of Earth,
avoiding some of the bias of low-pressure lamps.
Melanized fungi isolated from the Antarctica samples
have already been submitted to space and Martian-simu-
lated conditions, presenting great resistance to such con-
ditions (Onofri et al. 2008). Recently, a work by
Zakharova et al. (2014) showed that black microcolonial
fungi, including an Exophiala strain, were capable of
maintaining metabolic activity under a simulated Martian
environment, which shows the capability of eukaryotic
microorganisms to be also good models for astrobiology,
together with the prokaryotes.
Conclusions
The capability of enduring high fluences of UV radiation
is important when considering the potential for life as we
know thriving on exposed surfaces of planetary bodies
(Onofri et al. 2008; Abrevaya et al. 2011), such as Mars,
icy moons or asteroids. In this work, we have isolated
and characterized yeasts from a high-altitude area of the
Atacama Desert, which are as resistant to UV radiation as
the model organism D. radiodurans, one of the best can-
didates to survive in extraterrestrial conditions (Diaz and
Schulze-Makuch 2006; Paulino-Lima et al. 2010). It was
shown that the presence of melanin and carotenoids, as
measured by Raman spectroscopy, does not always corre-
late with UV-resistance for the tested yeasts, especially for
the case of environmental-UV, which is an important
finding when considering the search for biosignatures on
planetary surfaces, demanding additional studies. As the
Atacama Desert is commonly considered a good Mars
analog and the isolated extremophilic yeasts have demon-
strated significant radiation and cold tolerance (growth at
0°C for three isolates and down to 6.5°C for two), these
organisms can be used as model microorganisms to better
understand the response of eukaryotes to extreme condi-
tions, similarly to what has been proposed for the eukary-
otes isolated from the Antarctica (Onofri et al. 2007). In
this way, they can complement the existing models con-
sidered for astrobiological studies, and are already being
applied in new multiparametric simulations of planetary
surfaces and biosignature detection projects, with dedi-
cated systems for this purpose (Rodrigues et al. 2012). In
addition, although the objective of this work was not to
characterize the full diversity of yeasts at the volcano, the
species found at the samples are evidence of the potential
of such sites for studying the diversity and global disper-
sion of cold-adapted yeasts.
10 ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Extremophilic Yeasts from the High-Altitude Atacama A. A. Pulschen et al.
Acknowledgments
This work was sponsored by FAPESP (Project 2012/
18936-0), CAPES, CNPq –Proantar, and USP, through
the Brazilian Research Unity in Astrobiology –NAP/As-
trobio. The authors thank Luiz Henrique Rosa (UFMG)
for contributing in the discussion of the results, Mario H.
Barros (USP) for donating the S. cerevisiae BY4742 strain,
Lydia F. Yamaguchi (USP) for helping in the samples col-
lection, and the local support in the Atacama from Benito
Gomez-Silva (Universidad de Antofagasta, Chile). The
authors acknowledge the Brazilian Synchrotron Light Lab-
oratory –LNLS, for the use of the X-Ray Fluorescence
beamline (XRF) under the proposal XAFS1 –13689, the
Laboratory of Molecular Spectroscopy (IQ-USP) for the
use of the Bruker FT-Raman and the staff of the Brazilian
Astrobiology Laboratory –AstroLab, where most of the
experiments were conducted. We also thank the National
Council for the Improvement of Higher Education in
Brazil (Capes) and the NASA Postdoctoral Program
administered by Oak Ridge Associated Universities for
providing IGPL’s postdoctoral fellowships.
Conflict of Interest
None declared.
References
Abrevaya, X. C., I. G. Paulino-Lima, D. Galante, F. Rodrigues,
E. C
orton, and C. A. S. Lage. 2011. Comparative survival
analysis of Deinococcus radiodurans and the haloarchaea
Natrialba magadii and Haloferax volcanii exposed to vacuum
ultraviolet irradiation. Astrobiology 11:1034–1040.
Albarrac
ın, V. H., G. P. Pathak, T. Douki, J. Cadet, C. D.
Borsarelli, W. Gartner, et al. 2012. Extremophilic
acinetobacter strains from high-altitude lakes in Argentinean
Puna: remarkable UV-B resistance and efficient DNA
damage repair. Orig. Life Evol. Biosph. 42:201–221.
Azua-Bustos, A., C. Urrejola, and R. Vicuna. 2012. Life at the
dry edge: microorganisms of the Atacama Desert. FEBS Lett.
586:2939–2945.
Barrett, J. E., R. A. Virginia, D. H. Wall, A. N. Parsons, L. E.
Powers, and M. B. Burkins. 2004. Variation in
biogeochemistry and soil biodiversity across spatial scales in
a polar desert ecosystem. Ecology 85:3105–3118.
Billi, D. 2009. Subcellular integrities in Chroococcidiopsis sp.
CCMEE 029 survivors after prolonged desiccation revealed
by molecular probes and genome stability assays.
Extremophiles 13:49–57.
Billi, D., E. I. Friedmann, K. G. Hofer, M. G. Caiola, and R.
Ocampo-Friedmann. 2000. Ionizing-radiation resistance in
the desiccation-tolerant cyanobacterium Chroococcidiopsis.
Appl. Environ. Microbiol. 66:1489–1492.
Bj€
orn, L. O. 2008. Photobiology: the science of life and light.
Springer, New York, NY.
Blumthaler, M., W. Ambach, and R. Ellinger. 1997. Increase in
solar UV radiation with altitude. J. Photochem. Photobiol. B
39:130–134.
Cabrol, N. A., D. Wettergreen, K. Warren-Rhodes, E. A. Grin,
J. Moersch, G. C. Diaz, et al. 2007. Life in the Atacama:
searching for life with rovers (science overview). J. Geophys.
Res. Biogeosci. 112, G04S02, doi: 10.1029/2006JG000298
Cabrol, N. A., E. A. Grin, G. Chong, E. Minkley, A. N. Hock,
Y. Yu, et al. 2009. The high-lakes project. J. Geophys. Res.
Biogeosci. 114, G00D06. doi: 10.1029/2008JG000818
Cabrol, N. A., U. Feister, D. H€
ader, H. Piazena, E. A. Grin,
and A. Klein. 2014. Record solar UV irradiance in the
tropical Andes. Front. Environ. Sci. doi: 10.3389/
fenvs.2014.00019
Cadet, J., S. Mouret, J. Ravanat, and T. Douki. 2012. Photoinduced
damage to cellular DNA: direct and photosensitized reactions.
Photochem. Photobiol. 88:1048–1065.
Chin, J. P., J. Megaw, C. L. Magill, K. Nowotarski, J. P.
Willams, P. Bhaganna, et al. 2010. Solutes determine the
temperature windows for microbial survival and growth.
Proc. Natl. Acad. Sci. USA 107:7835–7840.
Cockell, C. S. 1998. Biological effects of high ultraviolet
radiation on early earth –a theoretical evaluation. J. Theor.
Biol. 193:717–729.
Cockell, C. S., C. P. Mckay, K. Warren-Rhodes, and G.
Horneck. 2008. Ultraviolet radiation-induced limitation to
epilithic microbial growth in and deserts –dosimetric
experiments in the hyperarid core of the Atacama Desert.
J. Photochem. Photobiol. B 90:79–87.
Cockell, C. S., G. R. Osinski, N. R. Banerjee, K. T. Howard, I.
Gilmour, and J. S. Watson. 2010. The microbe-mineral
environment and gypsum neogenesis in a weathered polar
evaporite. Geobiology 8:293–308.
Conley, C. A., G. Ishkhanova, C. P. Mckay, and K. Cullings.
2006. A preliminary survey of non-lichenized fungi cultured
from the hyperarid Atacama Desert of Chile. Astrobiology
6:521–526.
Connon, S. A., E. D. Lester, H. S. Shafaat, D. C. Obenhuber,
and A. Ponce. 2007. Bacterial diversity in hyperarid Atacama
Desert soils. J. Geophys. Res. Biogeosci. 112, G04S17.
doi:10.1029/2006JG000311
Costello, E. K., S. R. Halloy, S. C. Reed, P. Sowell, and S. K.
Schmidt. 2009. Fumarole-supported islands of biodiversity
within a hyperarid, high-elevation landscape on Socompa
Volcano, Puna de Atacama, Andes. Appl. Environ.
Microbiol. 75:735–747.
Dahlgren, R. A., M. Saigusa, and F. C. Ugolini. 2004. The
nature, properties and management of volcanic soils. Adv.
Agron. 82:113–182.
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 11
A. A. Pulschen et al.Extremophilic Yeasts from the High-Altitude Atacama
Daly, M. J. 2012. Death by protein damage in irradiated cells.
DNA Repair 11:12–21.
De los R
ıos, A., S. Valea, C. Ascaso, A. Davila, J. Kastovsky, C.
P. McKay, et al. 2010. Comparative analysis of the microbial
communities inhabiting halite evaporites of the Atacama
Desert. Int. Microbiol. 13:79–89.
De Oliveira, V. E., H. V. Castro, H. G. M. Edwards, and L. F.
C. de Oliveira. 2010. Carotenes and carotenoids in natural
biological samples: a Raman spectroscopic analysis. J.
Raman Spectrosc. 41:642–650.
Diaz, B., and D. Schulze-Makuch. 2006. Microbial survival
rates of Escherichia coli and Deinococcus radiodurans under
low temperature, low-pressure, and UV-irradiation
conditions, and their relevance to possible Martian life.
Astrobiology 6:332–347.
Dose, K., A. Bieger-Dose, B. Ernst, U. Feister, B. Gomez-Silva,
A. Klein, et al. 2001. Survival of microorganisms under the
extreme conditions of the Atacama Desert. Orig. Life Evol.
Biosph. 31:287–303.
Douki, T., T. Zalizniak, and J. Cadet. 1997. Far-UV-Induced
dimeric photoproducs in shot oligonucleotides: sequence
effects. Photochem. Photobiol. 66:171–179.
Drees, K. P., J. W. Neilson, J. L. Betancourt, J. Quade, D. A.
Henderson, B. M. Pryor, et al. 2006. Bacterial community
structure in the hyperarid core of the Atacama Desert,
Chile. Appl. Environ. Microbiol. 72:7902–7908.
Edwards, H. G., C. D. Moody, S. E. Jorge Villar, and D. D.
Wynn-Williams. 2005. Raman spectroscopic detection of key
biomarkers of cyanobacteria and lichen symbiosis in
extreme Antarctic habitats: evaluation for Mars Lander
missions. Icarus 174:560–571.
Ellery, A., and D. Wynn-Williams. 2003. Why Raman
spectroscopy on Mars? –a case of the right tool for the
right job. Astrobiology 3:565–579.
Fischer, E., G. M. Mart
ınez, H. M. Elliott, and N. O. Renn
o.
2014. Experimental evidence for the formation of liquid
saline water on Mars. Geophys. Res. Lett. 41:4456–4462.
Flierman, C. B., and T. D. Brock. 1972. Ecology of sulfur-
oxidizing bacteria in hot acid soils. J. Bacteriol. 111:343–350.
Gadanho, M., D. Libkind, and J. P. Sampaio. 2006. Yeast
diversity in the extreme acidic environments of the Iberian
Pyrite Belt. Microb. Ecol. 52:552–563.
Glas, A. F., E. Kaya, S. Schneider, K. Heil, D. Fazio, M. J.
Maul, et al. 2010. DNA (6-4) photolyases reduce Dewar
isomers for isomerization into (6-4) lesions. J. Am. Chem.
Soc. 132:3254–3255.
Gray, J. V., G. A. Petsko, G. C. Johnston, D. Ringe, R. A.
Singer, and M. Werner-Washburne. 2004. “Sleeping beauty”:
quiescence in Saccharomyces cerevisiae. Microbiol. Mol. Biol.
Rev. 68:187–206.
Guffogg, S. P., S. Thomas-Hall, P. Holloway, and K. Watson.
2004. A novel psychrotolerant member of the
hymenomycetous yeasts from Antarctica: Cryptococcus
watticus sp. nov. Int. J. Syst. Evol. Microbiol. 54:275–277.
Hoerter, J. D., A. A. Arnold, D. A. Kuczynska, A. Shibuya, C.
S. Ward, M. G. Sauer, et al. 2005. Effects of sublethal UVA
irradiation on activity levels of oxidative defense enzymes
and protein oxidation in Escherichia coli. J. Photochem.
Photobiol. B 81:171–180.
Horneck, G., P. Rettberg, R. Facius, and K. Scherer. 2006.
Quantification of biological effectiveness of UV radiation.
Environ. UV Radiat. Impact Ecosyst. Hum. Health Predict.
Models 57:51–69.
Jehlicka, J., H. G. Edwards, K. Osterrothov
a, J. Novotn
a, L.
Nedbalov
a, J. Kopecky, et al. 2014. Potential and limits of
Raman spectroscopy for carotenoid detection in
microorganisms: implications for astrobiology. Philos.
Trans. R. Soc. A 372:20140199.
Kozmin, S., G. Slezak, A. Reynaud-Angelin, C. Elie, Y. de
Rycke, S. Boiteux, et al. 2005. UVA radiation is highly
mutagenic in cells that are unable to repair 7,8-dihydro-8-
oxoguanine in Saccharomyces cerevisiae. Proc. Natl. Acad.
Sci. USA 102:13538–13543.
Libkind, D., M. Moline, J. P. Sampaio, and M. van Broock.
2009. Yeasts from high-altitude lakes: influence of UV
radiation. FEMS Microbiol. Ecol. 69:353–362.
Lucas-Lled
o, J. I., and M. Lynch. 2009. Evolution of mutation
rates: phylogenomic analysis of the photolyase/cryptochrome
family. Mol. Biol. Evol. 26:1143–1153.
Lynch, R. C., A. J. King, M. E. Farias, P. Sowell, C. Vitry, and
S. K. Schmidt. 2012. The potential for microbial life in the
highest-elevation (>6000 m.a.s.l.) mineral soils of the
Atacama region. J. Geophys. Res. Biogeosci. 117, G02028.
doi:10.1029/2012JG001961.
Meador, J. A., A. J. Baldwin, J. D. Pakulski, W. H. Jeffrey, D.
L. Mitchell, and T. Douki. 2014. The significance of the
Dewar valence photoisomer as a UV radiation-induced
DNA photoproduct in marine microbial communities.
Environ. Microbiol. 16:1808–1820.
Mitchell, D. L., J. Jen, and J. E. Cleaver. 1991. Relative
induction of cyclobutane dimers and cytosine
photohydrates in DNA irradiated in vitro and in vivo
with ultraviolet-C and ultraviolet-B. Photochem.
Photobiol. 54:741–748.
Moline, M., D. Libkind, M. D. Dieguez, and M. van
Broock. 2009. Photoprotective role of carotenoids in
yeasts: response to UV-B of pigmented and naturally-
occurring albino strains. J. Photochem. Photobiol. B
95:156–161.
Moore, M. M., M. W. Breedveld, and A. P. Autor. 1989. The
role of carotenoids in preventing oxidative damage in the
pigmented yeast, Rhodotorula mucilaginosa. Arch. Biochem.
Biophys. 270:419–431.
Navarro Llorens, J. M., A. Tormo, and E. Martinez-Garcia.
2010. Stationary phase in gram-negative bacteria. FEMS
Microbiol. Rev. 34:476–495.
Navarro-Gonzalez, R., F. A. Rainey, P. Molina, D. R. Bagaley,
B. J. Hollen, J. de la Rosa, et al. 2003. Mars-like soils in the
12 ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Extremophilic Yeasts from the High-Altitude Atacama A. A. Pulschen et al.
Atacama Desert, Chile, and the dry limit of microbial life.
Science 302:1018–1021.
Neilson, J. W., J. Quade, M. Ortiz, W. M. Nelson, A. Legatzki,
F. Tian, et al. 2012. Life at the hyperarid margin: novel
bacterial diversity in arid soils of the Atacama Desert, Chile.
Extremophiles 16:553–566.
Okoro, C. K., R. Brown, A. L. Jones, B. A. Andrews, J. A.
Asenjo, M. Goodfellow, et al. 2009. Diversity of culturable
actinomycetes in hyper-arid soils of the Atacama Desert,
Chile. Antonie Van Leeuwenhoek 95:121–133.
Onofri, S., L. Selbsmann, G. S. de Hoog, M. Grube, D.
Barreca, S. Ruisi, et al. 2007. Evolution and adaptation of
fungi at boundaries of life. Adv. Space Res. 40:1657–1664.
Onofri, S., D. Barreca, L. Selbmann, D. Isola, E. Rabbow, G.
Horneck, et al. 2008. Resistance of Antarctic black fungi and
cryptoendolithic communities to simulated space and
Martian conditions. Stud. Mycol. 61:99–109.
Ordonez, O. F., M. R. Flores, J. R. Dib, A. Paz, and M. E.
Farias. 2009. Extremophile culture collection from Andean
lakes: extreme pristine environments that host a wide
diversity of microorganisms with tolerance to UV radiation.
Microb. Ecol. 58:461–473.
Osterloo, M. M., V. E. Hamilton, J. L. Bandfield, T. D. Glotch,
A. M. Baldridge, P. R. Christensen, et al. 2008. Chloride-
bearing materials in the southern highlands of Mars. Science
319:1651–1654.
Parnell, J., D. Cullen, M. R. Sims, S. Bowden, C. S. Cockell, R.
Court, et al. 2007. Searching for life on Mars: selection of
molecular targets for ESA’s aurora ExoMars mission.
Astrobiology 7:578–604.
Paulino-Lima, I. G., S. Pilling, E. Janot-Pacheco, A. N. de
Brito, J. A. R. G. Barbosa, A. C. Leita
˜o, et al. 2010.
Laboratory simulation of interplanetary ultraviolet radiation
(broad spectrum) and its effects on Deinococcus radiodurans.
Planet. Space Sci. 58:1180–1187.
Paulino-Lima, I. G., A. Azua-Bustos, R. Vicuna, C. Gonzalez-
Silva, L. Salas, L. Teixeira, et al. 2013. Isolation of UVC-
tolerant bacteria from the hyperarid Atacama Desert, Chile.
Microb. Ecol. 65:325–335.
Perez, C. A., M. Radtke, H. J. Sanchez, H. Tolentino, R. T.
Neuenshwander, W. Barg, et al. 1999. Synchrotron radiation
X-ray fluorescence at the LNLS: beamline instrumentation
and experiments. X-Ray Spectrom. 28:320–326.
Perna, G., M. Lasalvia, C. Gallo, G. Quartucci, and V. Capozzi.
2013. Vibrational characterization of synthetic eumelanin by
means of Raman and surface enhanced Raman scattering.
Open Surf. Sci. J. 5:1–8.
Prista, C., A. Almagro, M. C. LoureiroDias, and J. Ramos.
1997. Physiological basis for the high salt tolerance of
Debaryomyces hansenii. Appl. Environ. Microbiol. 63:4005–
4009.
Ravanat, J., T. Douki, and J. Cadet. 2001. Direct and indirect
effects of UV radiation on DNA and its components. J.
Photochem. Photobiol. B 63:88–102.
Rivadeneyra, M. A., G. Delgado, M. Soriano, A. Ramos-
Cormenzana, and R. Delgado. 1999. Biomineralization of
carbonates by Marinococcus albus and Marinococcus
halophilus isolated from the Salar de Atacama (Chile). Curr.
Microbiol. 39:53–57.
Rodrigues, F., D. Galante, I. G. Paulino-Lima, R. T. Duarte,
A. Friaca, C. Lage, et al. 2012. Astrobiology in Brazil:
early history and perspectives. Int. J. Astrobiol. 11:189–
202.
Rosa, C. A., M. A. Lachance, W. T. Starmer, J. S. F. Barker, J.
M. Bowles, and B. Schlag-Edler. 1999. Kodamaea
nitidulidarum,Candida restingae and Kodamaea anthophila,
three new related yeast species from ephemeral flowers. Int.
J. Syst. Bacteriol. 49:309–318.
Santos, A. L., V. Oliveira, I. Baptista, I. Henriques, N. C. M.
Gomes, A. Almeida, et al. 2013. Wavelength dependence of
biological damage induced by UV radiation on bacteria.
Arch. Microbiol. 195:63–74.
Schiave, L. A., R. S. Pedroso, R. C. Candido, D. W. Roberts,
and G. U. L. Braga. 2009. Variability in UVB tolerances of
melanized and nonmelanized cells of Cryptococcus
neoformans and C. laurentii. Photochem. Photobiol. 85:205–
213.
Schroeder, W. A., and E. A. Johnson. 1993. Antioxidant role
of carotenoids in Phaffia rhodozyma. J. Gen. Microbiol.
139:907–912.
Singaravelan, N., I. Grishkan, A. Beharav, K. Wakamatsu, S.
Ito, and E. Nevo. 2008. Adaptive melanin response of the
soil fungus Aspergillus niger to UV radiation stress at
“Evolution Canyon”, Mount Carmel, Israel. PLoS One 3:
e2993.
Sinha, R. P., and D. P. Hader. 2002. UV-induced DNA
damage and repair: a review. Photochem. Photobiol. Sci.
1:225–236.
Slade, D., and M. Radman. 2011. Oxidative stress resistance in
Deinococcus radiodurans. Microbiol. Mol. Biol. Rev. 75:133–
191.
Smith, D. J. 2013. Microbes in the upper atmosphere and
unique opportunities for astrobiology research. Astrobiology
13:981–990.
Sole, V. A., E. Papillon, M. Cotte, P. Walter, and J. Susini.
2007. A multiplatform code for the analysis of energy-
dispersive X-ray fluorescence spectra. Spectrochim. Acta
Part B At. Spectrosc. 62:63–68.
Uetake, J., Y. Yoshimura, N. Nagatsuka, and H. Kanda. 2012.
Isolation of oligotrophic yeasts from supraglacial
environments of different altitude on the Gulkana Glacier
(Alaska). FEMS Microbiol. Ecol. 82:279–286.
Ugolini, F. C., and R. A. Dahlgren. 2002. Soil development in
volcanic ash. Global Environ. Res. Engl. Ed. 6:69–82.
Vishniac, H. S. 1985. Cryptococcus friedmannii, a new species
of yeast from the Antarctic. Mycologia 77:149–153.
Vishniac, H. S. 1998. The ecology of extremophile yeasts.
Instrum. Meth. Missions Astrobiol. 3441:68–74.
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 13
A. A. Pulschen et al.Extremophilic Yeasts from the High-Altitude Atacama
Vishniac, H. S. 2006. A multivariate analysis of soil yeasts
isolated from a latitudinal gradient. Microb. Ecol. 52:90–
103.
Wang, Y. L., and A. Casadevall. 1994. Decreased susceptibility
of melanized Cryptococcus neoformans to UV-light. Appl.
Environ. Microbiol. 60:3864–3866.
Warren-Rhodes, K. A., K. L. Rhodes, S. B. Pointing, S. A.
Ewing, D. C. Lacap, B. Gomez-Silva, et al. 2006. Hypolithic
cyanobacteria, dry limit of photosynthesis, and microbial
ecology in the hyperarid Atacama Desert. Microb. Ecol.
52:389–398.
Wierzchos, J., C. Ascaso, and C. P. McKay. 2006. Endolithic
cyanobacteria in halite rocks from the hyperarid core of the
Atacama Desert. Astrobiology 6:415–422.
Wynn-Williams, D., and H. M. Edwards. 2002. Environmental
UV radiation: biological strategies for protection and
avoidance. Pp. 245–260 in G. Horneck and C. Baumstark-
Khan, eds. Astrobiology. Springer, Berlin, Heidelberg.
Zakharova, K., G. Marzban, J. de Vera, A. Lorek, and K.
Sterflinger. 2014. Protein patterns of black fungi under
simulated Mars-like conditions. Sci. Rep. 4:5114. doi:
10.1038/srep05114
Zenoff, V. F., F. Sineriz, and M. E. Farias. 2006. Diverse
responses to UV-B radiation and repair mechanisms of
bacteria isolated from high-altitude aquatic environments.
Appl. Environ. Microbiol. 72:7857–7863.
Zimmer, J. L., and R. M. Slawson. 2002. Potential repair of
Escherichia coli DNA following exposure to radiation from
both medium- and low-pressure UV sources used in
drinking water treatment. Appl. Environ. Microbiol.
68:3293–3299.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. (A) Growth curves of (◄)Holtermanniella
watticus, (*) Exophiala sp. (●)Cryptococcus friedmannii at
low temperatures with different solute concentrations. (B)
Control growth curves performed at 15°C in: (&) TGY,
(●) TGY +0.5 mol/L NaCl, (N) TGY +0.4 mol/L glyc-
erol, (.) TGY +0.6 mol/L glycerol.
Figure S2. Survival rates for all the isolates in different
environmental conditions: (dark) Incubated in dark after
UV-C irradiation; (PAR) incubated with photossintheti-
cally active radiation after UV-C irradiation; (UV-A)
exposed to UV-A radiation after UV-C irradiation. Meth-
odology: Cells were grown and washed using the same
protocol established for the UV-C and UV-B experiments.
Several dilutions of the cells were then plated on TGY
agar plates, which were pre-incubated for 45 min at 13°C
for Exophiala sp. 15Lv1, Holtermanniella watticus and
Cryptococcus friedmannii, and at 30°C for Rhodosporidium
toruloides, ensuring they were at optimum temperature
during the procedures. After the pre-incubation period,
the plates were exposed to a single fluence of UV-C radia-
tion, enough to diminish ~90–99% of CFU/mL count
(600 J/m
2
for H. watticus and for C. friedmannii, 700 J/
m
2
for Exophiala sp. 15Lv1 and R. toruloides) under a flux
of 14.5 W/m
2
and then subjected to three different treat-
ments: (1) Photoreactivation with PAR (400–700 nm);
(2) UV-A photoreactivation (320–400 nm); (3) Dark
incubation. For the dark repair treatment, the plates were
immediately incubated in the dark after UV-C exposure.
For the PAR photoreactivation treatment, after UV-C
exposure, the plates were immediately transferred to a
photoperiod incubator equipped with three fluorescent
lamps (Osram 765 15W, Fig. S3 for the spectrum) which
remained on during the whole incubation period, until
the colonies were grown. For the UV-A photorepair treat-
ment, after UV-C irradiation, cells were exposed to UV-A
continuous radiation generated by an Oriel
Sol UV-2
Solar simulator equipped with an Oriel
SOL-UV-A-
F filter to cut the UV-B portion of the spectrum (Fig. S3
for the spectrum), under the flux of 2.5 W/m
2
(measured
with the UV-A photocell) for 45 min. During this period,
R. toruloides plates were kept at ~30°C using a heating
plate. For the other yeasts, plates were kept cold, at
~13°C–10°C using a cold bath. The temperature of the
plates during the irradiations was monitored using an
electronic digital thermometer (HI-955502 Pt100, Hanna
Instruments). After the UV-A exposure, plates were
immediately incubated in the dark. We performed the
irradiation of the cells directly on agar plates, since using
liquid suspension implies in the post-manipulation of the
cell suspension (for diluting it and platting), which might
incur on light exposure, interfering on the photorepair
assay. Experiments were performed in triplicates for each
organism.
Figure S3. Spectra produced by the sources of radiation,
as recorded using an Ocean Optics
QE65000 UV-Vis
fiber-optic coupled spectrometer. (A) UV-C lamps, (B)
Set of UV-B lamps, (C) Oriel
Sol UV-2 Solar simulator,
(D) Oriel
Sol UV-2 Solar simulator equipped with
Oriel
SOL-UV-A-F filter, (E) fluorescent lamps used on
photoincubator. Notice the difference between the spectra
(B and C) and the intense peak at 312 nm generated by
the UV-B lamps, in contrast to a broad emission of the
Solar simulator, more similar to the environmental UV
radiation. We also show, in shaded light gray, the Vilber
Lourmat UV-B photocell efficiency range. This illustrates
that even with the same reading of flux at the radiometer,
the UV-B spectra can differ significantly from each UV
source, thus producing different biological responses.
Figure S4. Survival curves of Deinococcus radiodurans and
Saccharomyces cerevisiae at different growth phases. The
14 ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Extremophilic Yeasts from the High-Altitude Atacama A. A. Pulschen et al.
irradiation procedures were performed by growing the
cells to the indicated OD
595
(~0.1, ~0.7 and ~2.3), wash-
ing the cells twice in 0.9% w/v NaCl solution and expos-
ing the cell suspension to Solar irradiation, using the
Oriel
Sol UV-2 Solar simulator. For the exposure,
600 lL of the cell suspension were added to a 24-well
plate and gently shaken during the irradiation procedure.
The experiments were repeated twice, at different days, in
duplicates for each experiment and yielded similar results.
The survival was measured using the same methodology
used on the UV-C, UV-B, and environmental-UV experi-
ments. The data presented here is one of the two different
repetitions and the error bars indicate the variance
between duplicates.
ª2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 15
A. A. Pulschen et al.Extremophilic Yeasts from the High-Altitude Atacama
