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UV-resistant yeasts isolated from a high-altitude volcanic area on the Atacama Desert as eukaryotic models for astrobiology


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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., Holtermanniella 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 temperatures, 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 pigmentation, 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. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
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UV-resistant yeasts isolated from a high-altitude volcanic
area on the Atacama Desert as eukaryotic models for
e A. Pulschen
, Fabio Rodrigues
, Rubens T. D. Duarte
, Gabriel G. Araujo
, Iara F. Santiago
Ivan G. Paulino-Lima
, Carlos A. Rosa
, Massuo J. Kato
, Vivian H. Pellizari
& Douglas Galante
Chemistry Institute, Universidade de S~
ao Paulo, S~
ao Paulo, Brazil
Microbiology, Immunology and Parasitology Department, Universidade Federal de Santa Catarina, Florian
opolis, Brazil
Interunities Graduate Program in Biotechnology, Universidade de S~
ao Paulo, S~
ao Paulo, Brazil
Brazilian Synchrotron Light Laboratory, Campinas, Brazil
Department of Microbiology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
NASA Postdoctoral Program Fellow at NASA Ames Research Center, Moffett Field, California
Oceanographic Institute, Universidade de S~
ao Paulo, S~
ao Paulo, Brazil
Astrobiology, Atacama Desert, eukaryote,
extremophiles, UV radiation, yeast.
Douglas Galante, Brazilian Synchrotron Light
Laboratory, Av. Giuseppe MaximoScolfaro,
10000 Campinas, S~
ao Paulo CEP 13083-100,
Tel: +551935175081; Fax: +551935121004;
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
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 (0.752.25 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.
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.
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
, 0.4 g L
0.5 g L
; CaCl
, 0.25 g L
; MgSO
, 0.5 g L
, and FeSO
, 0.01 g L
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,
; tryptone, 5 g L
and glucose, 1 g L
) with pH
adjusted to 4.8.
Yeast identification was carried out by sequencing the
D1D2 variable domains of the large rRNA subunit gene
using the primers NL1 (50GCATATCAATAAGCGGAG-
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
; yeast extract,
; malt extract, 5 g L
) 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
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
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 Z15) of the soil
1. Slopesoil
5047 m
2. Slopesoil
3981 m
3. Sulfur-rich
soil 4823 m
pH 4.25 5.36 3.34
Conductivity 11.6 lS/cm 83.9 lS/cm 376 lS/cm
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
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
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
of about 0.60.8, 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
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
. 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
ID Primer Result top BLAST (access no. GenBank)
No. of bp
Species or proposed taxonomic group
(GenBank access no.)
Max. temperature
with observable
Min. temperature
with observable
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.
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
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
~0.480 (after 1:20 dilution), for Exophi-
ala sp.15Lv1, OD
~0.900 (after 1:20 dilution) and for
D. radiodurans OD
~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
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
for UV-B
and 131.5 W/m
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
=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
=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
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
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
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
for UV-A and
15.6 W/m
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
for UV-A
and 11.6 W/m
for UV-B; in Brazil (S~
ao Paulo), under
subtropical conditions, the measurements were 26.4 W/
for UV-A and 9.3 W/m
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
and Ca(ClO
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
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=CC 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
, 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.
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 (280320 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 (320400 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.
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
, while S. cerevisiae lost viability
with smaller fluences (almost three logs drop of CFU
counts after 0.2 kJ/m
of exposure). The yeasts C. fried-
mannii and H. watticus also scored greater survival, losing
90% of viability only after 0.6 kJ/m
. 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, 400700 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
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
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
(280320 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.
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.
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.
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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 ~9099% of CFU/mL count
(600 J/m
for H. watticus and for C. friedmannii, 700 J/
for Exophiala sp. 15Lv1 and R. toruloides) under a flux
of 14.5 W/m
and then subjected to three different treat-
ments: (1) Photoreactivation with PAR (400700 nm);
(2) UV-A photoreactivation (320400 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
F filter to cut the UV-B portion of the spectrum (Fig. S3
for the spectrum), under the flux of 2.5 W/m
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°C10°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
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
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
(~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
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
... Members of the genus Rhodosporidium also proved to be excellent producers of carotenoides, as is the case of the marine yeast Rhodosporidium babjevae (Golubev), isolated from marine Copepod, Norway, the bright red colony produced torularhodin, torulene, b-Carotene, and g-Carotene (Sperstad et al., 2006). In agreement, Pulschen et al. (2015) were able to isolate Rhodosporidium toruloides from soil samples from sairecabur volcano, Atacama Desert, Chile. ...
... Other melanin-type pigments were reported by Okoli et al. (2007), they collected encapsulated strains of Cryptotrichosporon anacardii (CBS 9549, CBS 9550, CBS 9551, CBS 9552, and CBC 9553) that were isolated from fresh cashew flowers at Nnobi in Idemili South Local Government, Nigeria. For more, melanins were found in the species Exophiala sp, and H. werneckii, the first species being recovered from Sairecabur Volcano, Atacama Desert, Chile, and the second of saline waters, in Marakkanam, India, standing out for its antimicrobial activity against Salmonella typhi, Vibrio parahaemolyticus, and Klebsiella pneumoniae, the authors used the plate diffusion method, adding 10 mL of pigment extract (Pulschen et al., 2015;Rani et al., 2013). ...
Synthetic chemical compounds are the main sources of industrial dyes, but due to the recent demand for procedures highlighted in “Sustainable Biotechnology,” a search for natural products from renewable sources such as microorganisms has been stimulated. Microorganisms are particularly important in the production of so-called biopigments, which are compatible with industrial-scale processes, and not dependent on seasonal variations, such as those pigments from plant sources. In this regard, yeasts belonging to the phylum Basidiomycota such as Cystobasidium, Rhodotorula, Rhodosporidium, Sporidiobolus, Sporobolomyces, and Hortaea recovered from environments can be a rich field for the prospect of new molecules such as carotenoids and melanin with biotechnological applications such as antioxidant, antimicrobial, and photoprotection activities, mainly. From this perspective, yeasts isolated from marine and terrestrial samples may be strategically interesting in the production of pigments with biotechnological differentials.
... Our results showed UV-C survival of the non-pigmented N. kalamii and the pigmented pink-colored C. onofrii at up to 3000 J/m 2 UV-C irradiation. Multiple species from the non-pigmented Naganishia species are highly resistant organisms to UV radiation (Pulschen et al. 2015;Schmidt et al. 2017) and our results are in line with this finding. Indeed, we found that the polar yeast N. onofrii is more resistant to UV-C radiation compared to the pigmented C. onofrii, which is capable of synthesizing carotenoids with UV shielding properties (Moliné et al. 2010). ...
Full-text available
During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB 1, RPB2 , CYTB and TEF1 ), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3 T , isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1 T , isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium ( Cystobasidiales, Cystobasidiomycetes ) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3 T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1 T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.
... The Atacama Desert and the Antarctic continent host many extremophiles due to selection pressure applied by harsh environmental challenges such as exposure to long periods of desiccation or freezing, intense UV, high and low temperatures, or high-salt concentrations [33,34]. As a consequence, it was not surprising to find microorganisms presenting a high resistance to UV radiation or ionizing radiation such as Deinococcus species in those samples [35][36][37][38]. However, how such habitats have been colonized by metazoans and how such environments have shaped desiccation and radioresistance remains poorly studied. ...
Full-text available
Background Bdelloid rotifers are micro-invertebrates distributed worldwide, from temperate latitudes to the most extreme areas of the planet like Antarctica or the Atacama Desert. They have colonized any habitat where liquid water is temporarily available, including terrestrial environments such as soils, mosses, and lichens, tolerating desiccation and other types of stress such as high doses of ionizing radiation (IR). It was hypothesized that bdelloid desiccation and radiation resistance may be attributed to their potential ability to repair DNA double-strand breaks (DSBs). Here, these properties are investigated and compared among nine bdelloid species collected from both mild and harsh habitats, addressing the correlation between the ability of bdelloid rotifers to survive desiccation and their capacity to repair massive DNA breakage in a phylogenetically explicit context. Our research includes both specimens isolated from habitats that experience frequent desiccation (at least 1 time per generation), and individuals sampled from habitats that rarely or never experienced desiccation. Results Our analysis reveals that DNA repair prevails in somatic cells of both desiccation-tolerant and desiccation-sensitive bdelloid species after exposure to X-ray radiation. Species belonging to both categories are able to withstand high doses of ionizing radiation, up to 1000 Gy, without experiencing any negative effects on their survival. However, the fertility of two desiccation-sensitive species, Rotaria macrura and Rotaria rotatoria, was more severely impacted by low doses of radiation than that of desiccation-resistant species. Surprisingly, the radioresistance of desiccation-resistant species is not related to features of their original habitat. Indeed, bdelloids isolated from Atacama Desert or Antarctica were not characterized by a higher radioresistance than species found in more temperate environments. Conclusions Tolerance to desiccation and radiation are supported as ancestral features of bdelloid rotifers, with a group of species of the genus Rotaria having lost this trait after colonizing permanent water habitats. Together, our results provide a comprehensive overview of the evolution of desiccation and radiation resistance among bdelloid rotifers.
... The LAB-07 strain was identified taxonomically by 26 S rRNA and ITS sequencing (Fig. 1B). BLAST result of ITS showed 98% and 100% identity to ITS sequence of Rhodotorula toruloides 16Lv3 (KM243313) and PYCC 5081 (JN246549) strains, and 26 S rRNA D1∼D3 expansion segment domain sequence showed 99% identity to the same 16Lv3 strain [60,61]. Based on the analysis, we could identify that the isolated strain was R. toruloides. ...
Full-text available
Rhodotorula toruloides is a non-conventional yeast with a natural carotenoid pathway. In particular, R. toruloides is an oleaginous yeast that can accumulate lipids in high content, thereby gaining interest as a promising industrial host. In this study, we isolated and taxonomically identified a new R. toruloides LAB-07 strain. De novo genome assembly using PacBio and Illumina hybrid platforms yielded 27 contigs with a 20.78 Mb genome size. Subsequent genome annotation analysis based on RNA-seq predicted 5296 protein-coding genes, including the fatty acid production pathway. We compared lipid production under different media; it was highest in the yeast extract salt medium with glycerol as a carbon source. Polyunsaturated α-linolenic acid was detected among the fatty acids, and docking phosphatidylcholine as a substrate to modeled Fad2, which annotated as Δ12-fatty acid desaturase showed bifunctional Δ12, 15-desaturation is structurally possible in that the distances between the diiron center and the carbon-carbon bond in which desaturation occurs were similar to those of structurally identified mouse stearoyl-CoA desaturase. Finally, the applicability of the extracted total lipid fraction of R. toruloides was investigated, demonstrating an increase in filaggrin expression and suppression of heat-induced MMP-1 expression when applied to keratinocytes, along with the additional antioxidant activity. This work presents a new R. toruloides LAB-07 strain with genomic and lipidomic data, which would help understand the physiology of R. toruloides. Also, the various skin-related effect of R. toruloides lipid extract indicates its potential usage as a promising cosmetic ingredient.
Full-text available
Nakazawaea is a genus of ascomycetous yeasts in the order Saccharomycetales. Currently, 15 species of the Nakazawaea genus have been isolated from diverse habitats, with a predominant association with plant materials . In this study, we describe a novel species, Nakazawaea atacamensis f. a.,sp. nov., isolated from Neltuma chilensis (synonym Prosopis chilensis , Algarrobo) samples in the Atacama Desert of Chile. The novel species was identified based on morphological, physiological, biochemical, and molecular characteristics. The phylogenetic analysis using concatenated sequences of the SSU rRNA gene, ITS region, and D1/D2 domains of LSU rRNA revealed that N. atacamensis sp. nov. formed an early diverging cluster closely related to Nakazawaea siamensis and Nakazawaea odontotermitis . The sequence identity of N. atacamensis differed from closely related species by 3.3% to 20.4% in the investigated regions. Phenotypic comparisons demonstrated that N. atacamensis sp. nov. exhibited distinct carbon assimilation patterns compared to related species. Genome sequencing of the type strain ATA-11A-B revealed a genome size of approximately 12.4 Mbp, similar to other Nakazawaea species, with 5,116 protein-coding genes annotated using InterProScan. In addition, N. atacamensis exhibited the capacity to ferment synthetic wine must, representing a potential new yeast for mono or co-culture wine fermentations. This comprehensive study expands our understanding of the Nakazawaea genus and highlights the ecological and industrial potential of these yeasts in fermentation processes and diverse habitats. The holotype is N. atacamensis sp. nov. (CBS 18375).
The Antarctic continent is an extreme environment recognized mainly by its subzero temperatures. Fungi are ubiquitous microorganisms that stand out even among Antarctic organisms, primarily due to secondary metabolites production with several biological activities. Pigments are examples of such metabolites, which mainly occur in response to hostile conditions. Various pigmented fungi have been isolated from the Antarctic continent, living in the soil, sedimentary rocks, snow, water, associated with lichens, mosses, rhizospheres, and zooplankton. Physicochemical extreme environments provide a suitable setup for microbial pigment production with unique characteristics. The biotechnological potential of extremophiles, combined with concerns over synthetic pigments, has led to a great interest in natural pigment alternatives. Besides biological activities provided by fungal pigments for surviving in extreme environments (e.g., photoprotection, antioxidant activity, and stress resistance), it may present an opportunity for biotechnological industries. This paper reviews the biotechnological potential of Antarctic fungal pigments, with a detailed discussion over the biological role of fungal pigments, potential industrial production of pigments from extremophilic fungi, pigments toxicity, current market perspective and published intellectual properties related to pigmented Antarctic fungi.
Due to their extraordinary genetic and phenotypic plasticity, yeast and yeast-like fungi have been able to adapt and colonize a wide range of ecological niches. Pigmented and nonpigmented extremophilic yeasts have been discovered in areas on Earth characterized by physical and chemical conditions similar to those found in extraterrestrial environments. Thus, these "simple" eukaryotic life forms have evolved unique genetic, metabolic, and phenotypic characteristics for coping with extreme conditions, existing in both natural (polar continents, deep sea, stratosphere, etc.) and manmade environments such as the cleanrooms where spacecraft are built. This makes them ideal test organisms for astrobiology research. All of the results from the numerous experiments in which they have been tested are helping us to understand what to look for and where in space missions searching for signs of present and/or past life. Meanwhile, we must continue to explore the most inhospitable places on Earth to discover new promising extremophiles that could be used as model organisms for astrobiology research.
Chemical processes are the main sources of industrial colorants, but the recent appeal for green procedures and natural products has stimulated the search for molecules obtained from plants or microorganisms. The latter are particularly important, since the so-called biopigments are compatible with industial-scale processes and not dependent on seasonal variations. In this regard, extreme environments are a rich field for prospection of new molecules as the conditions observed in those places (e.g., low/high pH and temperatures, UV radiation, salinity) induces diverse adaptation mechanisms such as genetic and biochemical variations responsible to induce the production of “protective” secondary metabolites, such as coloring molecules. Thus, this chapter aims to describe the pigments produced by bacteria, yeast and filamentous fungi living in extreme environments such as polar, hypersaline, high altitude, desertic and volcanic. The number of studies employing extemmophilic fungi is more limited than the reports with bacteria from these environments, but both types of microorganisms are promising sources for prospecting new pigments as well as for investigating their potential to be used in industrial scale.
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Anthropogenic activities have been increasing Polycyclic Aromatic Hydrocarbons (PAHs) release, promoting an urgent need for decontamination methods. Therefore, anthracene biodegradation by endophytic, extremophilic, and entomophilic fungi was studied. Moreover, a salting-out extraction methodology with the renewable solvent ethanol and the innocuous salt K2HPO4 was employed. Nine of the ten employed strains biodegraded anthracene in liquid medium (19–56% biodegradation) after 14 days at 30 °C, 130 rpm, and 100 mg L−1. The most efficient strain Didymellaceae sp. LaBioMMi 155, an entomophilic strain, was employed for optimized biodegradation, aiming at a better understanding of how factors like pollutant initial concentration, pH, and temperature affected this process. Biodegradation reached 90 ± 11% at 22 °C, pH 9.0, and 50 mg L−1. Futhermore, 8 different PAHs were biodegraded and metabolites were identified. Then, experiments with anthracene in soil ex situ were performed and bioaugmentation with Didymellaceae sp. LaBioMMi 155 presented better results than natural attenuation by the native microbiome and biostimulation by the addition of liquid nutrient medium into soil. Therefore, an expanded knowledge about PAHs biodegradation processes was achieved with emphasis to the action of Didymellaceae sp. LaBioMMi 155, which can be further employed for in situ biodegradation (after strain security test), or for enzyme identification and isolation aiming at oxygenases with optimal activity under alkaline conditions.
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High elevation, thin ozone layer, and clear sky produce intense ultraviolet (UV) radiation in the tropical Andes. Recent models suggest that tropical stratospheric ozone will slightly decrease in the coming decades, potentially resulting in more UV anomalies. Data collected between 4300 and 5916 m above sea level (asl) in Bolivia show how this trend could dramatically impact surface solar irradiance. During 61 days, two Eldonet dosimeters recorded extreme UV-B irradiance equivalent to a UV index (UVI) of 43.3, which is the highest ground value ever reported. If they become more common, events of this magnitude may have societal and ecological implications, which make understanding the process leading to their generation critical. Our data show that this event and other major UV spikes were consistent with rising UV-B/UV-A ratios in the days to hours preceding the spikes, trajectories of negative ozone anomalies (NOAs), and radiative transfer modeling.
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In this paper, it is demonstrated how Raman spectroscopy can be used to detect different carotenoids as possible biomarkers in various groups of microorganisms. The question which arose from previous studies concerns the level of unambiguity of discriminating carotenoids using common Raman microspectrometers. A series of laboratory-grown microorganisms of different taxonomic affiliation was investigated, such as halophilic heterotrophic bacteria, cyanobacteria, the anoxygenic phototrophs, the non-halophilic heterotrophs as well as eukaryotes (Ochrophyta, Rhodophyta and Chlorophyta). The data presented show that Raman spectroscopy is a suitable tool to assess the presence of carotenoids of these organisms in cultures. Comparison is made with the high-performance liquid chromatography approach of analysing pigments in extracts. Direct measurements on cultures provide fast and reliable identification of the pigments. Some of the carotenoids studied are proposed as tracers for halophiles, in contrast with others which can be considered as biomarkers of other genera. The limits of application of Raman spectroscopy are discussed for a few cases where the current Raman spectroscopic approach does not allow discriminating structurally very similar carotenoids. The database reported can be used for applications in geobiology and exobiology for the detection of pigment signals in natural settings.
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Evidence for deliquescence of perchlorate salts has been discovered in the Martian polar region while possible brine flows have been observed in the equatorial region. This appears to contradict the idea that bulk deliquescence is too slow to occur during the short periods of the Martian diurnal cycle during which conditions are favorable for it. We conduct laboratory experiments to study the formation of liquid brines at Mars environmental conditions. We find that when water vapor is the only source of water, bulk deliquescence of perchlorates is not rapid enough to occur during the short periods of the day during which the temperature is above the salts’ eutectic value, and the humidity is above the salts’ deliquescence value. However, when the salts are in contact with water ice, liquid brine forms in minutes, indicating that aqueous solutions could form temporarily where salts and ice coexist on the Martian surface and in the shallow subsurface.
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Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.
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High elevation, thin ozone layer, and clear sky produce intense ultraviolet (UV) radiation in the tropical Andes. Recent models suggest that tropical stratospheric ozone will slightly decrease in the coming decades, potentially resulting in more UV anomalies. Data collected between 4,300-5,916 m above sea level (asl) in Bolivia show how this trend could dramatically impact surface solar irradiance. During 61 days, two Eldonet dosimeters recorded extreme UV-B irradiance equivalent to a UV index (UVI) of 43.3, which is the highest ground value ever reported. If they become more common, events of this magnitude may have societal and ecological implications, which make understanding the process leading to their generation critical. Our data show that this event and other major UV spikes were consistent with rising UV-B/UV-A ratios in the days to hours preceding the spikes, trajectories of negative ozone anomalies (NOAs), and radiative transfer modeling.
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The High Lakes Project is a multidisciplinary astrobiological investigation studying high-altitude lakes between 4200 m and 6000 m elevation in the Central Andes of Bolivia and Chile. Its primary objective is to understand the impact of increased environmental stress on the modification of lake habitability potential during rapid climate change as an analogy to early Mars. Their unique geophysical environment and mostly uncharted ecosystems have added new objectives to the project, including the assessment of the impact of low-ozone/high solar irradiance in nonpolar aquatic environments, the documentation of poorly known ecosystems, and the quantification of the impact of climate change on lake environment and ecosystem. Data from 2003 to 2007 show that UV flux is 165% that of sea level with maximum averaged UVB reaching 4 W/m2. Short UV wavelengths (260-270 nm) were recorded and peaked at 14.6 mW/m2. High solar irradiance occurs in an atmosphere permanently depleted in ozone falling below ozone hole definition for 33-36 days and between 30 and 35% depletion the rest of the year. The impact of strong UVB and UV erythemally weighted daily dose on life is compounded by broad daily temperature variations with sudden and sharp fluctuations. Lake habitat chemistry is highly dynamical with notable changes in yearly ion concentrations and pH resulting from low and variable yearly precipitation. The year-round combination of environmental variables define these lakes as end-members. In such an environment, they host ecosystems that include a significant fraction of previously undescribed species of zooplankton, cyanobacterial, and bacterial populations.
Cryptococcus friedmannii Vishniac sp. nov. from an Antarctic cryptoendolithic community is a psychrophilic basidioblastomycete characterized by cream-colored colonies of cells with smooth, layered walls, budding monopolarly, producing amylose and extracellular proteinase, utilizing nitrate and D-alanine (inter alia) as nitrogen sources and L-arabinose, arbutin, cellobiose, D-glucuronate, maltose, melezitose, salicin, soluble starch, trehalose, and D-xylose as carbon sources. This species differs from all other basidiomycetous yeasts in possessing the following combination of characters: amylose production (positive), assimilation of cellobiose (positive), D-galactose (negative), myo-inositol (negative), D-mannitol (negative), and sucrose (negative).
Since the publication of the first edition in 2002, there has been an explosion of new knowledge in the field of photobiology. Photobiology: The Science of Life and Light, Second edition, is fully updated and offers eight new chapters for a comprehensive look at photobiology. The chapters cover all areas of photobiology, photochemistry, and relationship between light and biology, each with up-to-date references. The chapter authors (of which seven are new) have very different backgrounds, and have produced a truly cross-disciplinary treatise. The book starts with the physics and chemistry of light, and how to handle light in the laboratory and measure it in the field, the properties of daylight, and new uses of light in research. It deals with the evolution of photosynthesis and with the mechanisms of its primary steps. Four chapters deal with how organisms use light for their orientation in space and time: the biological clock and its resetting by light, the light-dependent magnetic compass, and photoperiodism in animals and plants. There are also several medically oriented chapters and two chapters specifically aimed at the photobiology educator. The book is suitable for biologically interested readers at different levels from undergraduates to professors, researchers and medical doctors. © 2008 Springer Science+Business Media, LLC. All rights reserved.
Induction of pyrimidine dimers in DNA by solar UV radiation has drastic effects on microorganisms. To better define the nature of these DNA photoproducts in marine bacterioplankton and eukaryotes, a study was performed during a cruise along a latitudinal transect in the Pacific Ocean. The frequency of all possible cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (64PPs) and their related Dewar valence isomers (DEWs) was determined by HPLC-mass spectrometry. Studied samples were bacterioplankton and eukaryotic fractions isolated from sea water either collected before sunrise or exposed to ambient sunlight from sunrise to sunset. Isolated DNA dosimeters were also exposed to daily sunlight for comparison purposes. A first major result was the observation in all samples of large amounts of DEWs, a class of photoproducts rarely considered outside photochemical studies. Evidence was obtained for a major role of UVA in the formation of these photoisomerization products of 64PPs. Considerations on the ratio between the different classes of photoproducts in basal and induced DNA damage suggests that photoenzymatic repair (PER) is an important DNA repair mechanism used by marine microorganisms occupying surface seawater in the open ocean. This result emphasizes the biological role of DEWs which are very poor substrate for PER.