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Ancient Photosynthetic Eukaryote Biofilms in an Atacama Desert Coastal Cave

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Caves offer a stable and protected environment from harsh and changing outside prevailing conditions. Hence, they represent an interesting habitat for studying life in extreme environments. Here, we report the presence of a member of the ancient eukaryote red algae Cyanidium group in a coastal cave of the hyperarid Atacama Desert. This microorganism was found to form a seemingly monospecific biofilm growing under extremely low photon flux levels. Our work suggests that this species, Cyanidium sp. Atacama, is a new member of a recently proposed novel monophyletic lineage of mesophilic "cave" Cyanidium sp., distinct from the remaining three other lineages which are all thermo-acidophilic. The cave described in this work may represent an evolutionary island for life in the midst of the Atacama Desert.
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Ancient Photosynthetic Eukaryote Biofilms in an Atacama
Desert Coastal Cave
A. Azúa-Bustos &C. González-Silva &R. A. Mancilla &
L. Salas &R. E. Palma &J. J. Wynne &C. P. McKay &
R. Vicuña
Received: 18 November 2008 /Accepted: 6 February 2009 /Published online: 5 March 2009
#Springer Science + Business Media, LLC 2009
Abstract Caves offer a stable and protected environment
from harsh and changing outside prevailing conditions.
Hence, they represent an interesting habitat for studying life
in extreme environments. Here, we report the presence of a
member of the ancient eukaryote red algae Cyanidium
group in a coastal cave of the hyperarid Atacama Desert.
This microorganism was found to form a seemingly
monospecific biofilm growing under extremely low photon
flux levels. Our work suggests that this species, Cyanidium
sp. Atacama, is a new member of a recently proposed novel
monophyletic lineage of mesophilic caveCyanidium sp.,
distinct from the remaining three other lineages which are
all thermo-acidophilic. The cave described in this work may
represent an evolutionary island for life in the midst of the
Atacama Desert.
Many cave ecosystems on Earth are known to host
heterotrophic bacteria serving as the food base [5,6,32,
39]. Although much is known about these microorganisms
[6,34], less has been published on photosynthetic micro-
organisms proliferating in caves. Recently, taxonomic
surveys of lamp-associated flora algae and cyanobacteria
in electrically lit passages within show caves have been
reported [14,44]. However, these studies were focused on
their control rather than on their biology. Other groups have
reported bioactive metabolites isolated from cyanobacteria
found in caves, with no further details on the description or
the ecology of the microorganisms [2,3].
A similar trend is found for other phototrophs like
unicellular algae, as it is the case of the Cyanidiales, a
group of asexual, unicellular red algae usually found in
acidic (pH 0.53.0) high-temperature (5055°C) sites
around hot springs [11,36]. Most of these algae are in fact
bluegreen due to the pigments c-phycocyanin and
chlorophyll-a [45]. The Cyanidiales are classified into three
genera, Cyanidium,Cyanidioschyzon, and Galdieria [9,40,
55], of which Cyanidium caldarium is the most studied
member. This species of alga is characterized by rounded
cells with thick walls, nucleus, a single chloroplast, a
vacuole, and one mitochondrion [1,16]. Cyanidium cells
reproduce by internal divisions forming daughter cells or
Microb Ecol (2009) 58:485496
DOI 10.1007/s00248-009-9500-5
A. Azúa-Bustos (*):R. A. Mancilla :L. Salas :R. Vicuña
Departamento de Genética Molecular y Microbiología, Facultad
de Ciencias Biológicas, Pontificia Universidad Católica de Chile,
Alameda 340,
Santiago, Chile
C. González-Silva
Departamento de Ciencias Químicas y Farmaceúticas,
Universidad Arturo Prat,
Iquique, Chile
J. J. Wynne
USGS, Southwest Biological Science Center, and Department
of Biological Sciences, Northern Arizona University,
Flagstaff, AZ 86011, USA
C. P. McKay
NASA-Ames Research Center,
MS 245-3,
Moffett Field, CA 94035, USA
A. Azúa-Bustos :R. Vicuña
Millennium Institute for Fundamental and Applied Biology,
Santiago, Chile
R. E. Palma
Departamento de Ecología, Facultad de Ciencias Biológicas,
Pontificia Universidad Católica de Chile,
Santiago, Chile
autospores in the form of tetrads inside the mother cells
[16]. Both the 16S ribosomal RNA (rRNA) [47,53] and the
rbcL and psbA genes codified in the chloroplast [10], as
well as the 18S rRNA in the chromosomal DNA [45], have
been previously used for identification and phylogenetic
classification of environmental samples. These phylogenet-
ic studies suggest that the Cyanidiales are one of the most
ancient groups of algae, having diverged about 1.3 billion
years ago at the base of the Rhodophyta [19,42,54].
Nevertheless, it has been reported that species of the
Cyanidium genera have also been found growing inside
caves [9,17]. These aerophytic epilithic cave Cyanidium
are the mesophilic members of the clade, and current
phylogenetic analyses support the existence of four
distinct Cyanidiales lineages: the Galdieria spp. lineage,
the C.caldarium lineage, the Cyanidioschyzon merolae
plus Galdieria maxima lineage, and the novel monophy-
letic lineage of mesophilic cave Cyanidium spp. [9].
Previous analyses suggest that there is a high level of
sequence divergence among Cyanidiales species based on
environmental conditions, and, although they have a long
evolutionary history, only a few recognized morphological
species are known [9]. Based on morphological character-
istics alone, Schwabe [41] reported in 1936 a species which
he assigned to the Cyanidium genera in two caves on the
central coast of Chile, which are located about 1,200 and
1,500 km south of our study site. On the other hand, the
Atacama Desert is located between 17° and 27° S latitude
in northern Chile. It is constrained on the east by the front
ranges of the Andes Mountains and on the west by the
Coastal Range. The Atacama is one of the driest and
probably the oldest extant desert on Earth [25], having
experienced hyperaridity for at least three million years and
probably 150 million years [24]. To survive in these
hyperarid conditions, life forms have adapted to very low
air humidity levels, an almost complete absence of rain
events, highly saline soils, and high solar radiation [4,12,
29]. These harsh environmental factors may explain why
parts of the Atacama Desert are almost devoid of microbial
life [12,18,33]. Interestingly, the Atacama Desert has been
established as a prime analog model for the planet Mars,
and many research teams have conducted research on a
diversity of astrobiological-oriented topics [43,48].
Although much has been published about the hyperarid
areas of the Atacama Desert [10,28,29,50], fewer reports
exist on the microbial life present in areas of the Atacama
with a little more water availability. These areas could give
valuable information to be compared with that of other sites
located elsewhere in the hyperarid regions of the desert.
The arid Coastal Range that separates the hyperarid
Atacama Desert plateau from the Pacific Ocean, acts as a
topographic barrier to clouds and moisture-rich marine air
moving eastwards from the ocean. Consequently, the dry
hills of the Pacific Coastal Range have more benign sites
for the development of microbial life, differing from the
typical desert conditions associated to the Atacama. Only
recently, caves in the arid areas of the Atacama have begun
to be explored, and to date no microorganisms of any type
have been described living inside them [51].
Here, we describe the presence of biofilms growing at
mesophilic conditions at the dimmest lighted zone of a
coastal cave of the Atacama Desert. Our microscopy and
molecular biology data suggest that the photosynthetic
member of these biofilms is represented mainly, if not
solely, by a new species of the Cyanidium genus,
Cyanidium sp. Atacama, closely related to the only two
other known cave Cyanidium previously described.
Biofilm Sampling
Biofilm samples were taken in situ by scraping the walls of
the cave with a sterile scalpel and depositing the material
obtained into sterile 50-ml Falcon tubes. When working at
the dimmest lighted areas of the cave, care was taken in not
disturbing the light conditions of the site by using strong
sources of artificial lights.
Temperature, RH, and pH Measurements
Temperatures of rocks inside the cave were measured with
a Raytek infrared thermometer (Raytek Corporation, Santa
Cruz, CA, USA). Three readings per measurement were
made by placing the thermometer about 5 cm from the rock
being analyzed. Measurements were taken every 2 m from
the cave bottom in both the western and eastern walls. The
pH of the cave walls where biofilms develop was measured
in situ by collecting water droplets from the rocks and using
pH strips papers. Relative humidity (RH) and air temper-
ature inside the cave were measured with a DO 9406
thermometerhygrometer data logger (Delta Ohm, Padua,
Italy). Measurements were taken at the bottom of the cave,
near the entrances, and close to the eastern and western
walls. Measurements were taken in March and June of
Transmission Electron Microscopy
Biofilm samples were centrifuged at 3,000 rpm on a
tabletop centrifuge. The resulting pellet was fixed with
3% glutaraldehyde in sodium cacodylate buffer 0.1 M
pH 7.2 during 18 h at room temperature. After three
consecutive 20-min washes with sodium cacodylate buffer
0.1 M pH 7.2, the samples were treated with 1% aqueous
486 A. Azúa-Bustos et al.
osmium tetroxide during 60 min. The samples were then
dehydrated with a graded acetone series (50% to 100%),
15 min for each concentration used. The samples were pre-
embedded overnight with eponacetone 1:1 and then
embedded in pure epon. The polymerization process was
done at 60°C during 24 h. Sixty-nanometer-thin sections
were obtained with a Sorvall MT-5000 ultramicrotome, and
stained with 4% uranyl acetate in methanol and lead citrate.
Observations were made with a Philips Tecnai 12 trans-
mission electron microscope operated at 80 kV.
Scanning Electron Microscopy
A small rock sample covered with biofilm was fixed with
2% glutaraldehyde in cacodylate buffer overnight and then
washed with the same buffer three times, 20 min each.
After critical point drying and gold coating, the samples
were observed with a scanning electron microscope Jeol
JSM 25S-II at 30 kV.
Confocal Laser Scanning Microscopy
Biofilm samples of the dimmest lighted areas of the cave
were suspended in water and immediately observed using
an Olympus FV 1000 confocal laser scanning microscope
equipped with a ×100 oil immersion objective (Olympus,
Hamburg, Germany). The images were analyzed with the
Fluoview 5.0 software.
Bright Field Microscopy
Biofilms samples were suspended in water, placed in a
Neubauer cell counting chamber, and immediately observed
with a Nikon Optiphot-2 microscope equipped with a
Qimaging Micropublisher 3.3 RTV digital camera.
Photosynthetic Photon Flux Density Measurements
For photosynthetic photon flux density (PPFD) measure-
ments, an Apogee Quantum Meter QMSW-SS calibrated
for sunlight was used as instructed by the manufacturer. For
the wall biofilms inside the cave, the sensor was placed
parallel to the biofilms and then pointed to the entrances to
measure the amount of light reaching each sector. Each
reading was recorded when the sensor showed a stable
value for at least 30 s.
Isolation and Sequencing of 16S rRNA, rbcL, and psbA
Gene Sequences
Approximately 100 mg of biofilm material were aseptically
collected and total genomic DNA was extracted using a Soil
DNA Isolation Kit (MoBio Laboratories, Solano Beach,
CA, USA) according to the manufacturers instructions.
The 16S rRNA plastid gene present in the extracted DNA
was amplified using the Cyanidium-specific oligonucleotide
forward primer Cyan100F (5-TATAATGGAGAGTTT
GATCCTGGCT-3) and the reverse primer Cyan1450R
l,5-bisphosphate carboxylase/oxygenase (RuBisCO) rbcL
plastid gene present in the extracted DNA was amplified
using the rbcL-specific oligonucleotide forward primers
and the reverse primer rvrub1063 (5-GCCTCTAAAGC
TACCCTATTAGC-3). These primers were designed on the
basis of sequence comparisons of known 16S rRNA and
rbcL Cyanidium genes. The psbA gene was amplified as
previously reported [9].
For amplification of the DNA templates, the Go Taq
green and colorless Master Mix (Promega Corporation,
Madison, WI, USA) were used according to the manufac-
turers instructions, using a 1:100 dilution of the extracted
Polymerase chain reaction (PCR) conditions consisted
on an initial denaturing step at 94°C for 2 min followed by
four sequential cycles of 94°C for 1 min, n°C for 1 min,
and 72°C for 1 min, in which n=46474849°C. This was
then followed by 31 cycles of 94°C for 1 min, 50°C for
1 min, 72°C for 1 min, and a final extension step at 72°C
for 15 min, thus totaling 35 cycles of amplification.
The PCR products were ligated to the pGEM-T Easy
Vector System (Promega Corporation, Madison, WI, USA)
andclonedinEscherichia coli XL1-Blue cells. The
resulting plasmid vectors were isolated and purified using
the Invisorb Spin Plasmid Mini Two (Invitek GmbH,
Berlin, Germany) according to the manufacturers instruc-
tions. The automated sequencing of the clones and three
PCR products was done by Macrogen DNA Sequencing
Inc. (Seoul, Korea) using the M13 forward primer site of
the pGEM-T Easy Vector.
Phylogenetic Analysis
The nucleotide sequence of the Cyanidium sp. Atacama
16S rRNA, rbcL,andpsbA genes was analyzed using the
Megablast option for highly similar sequences of the
BLASTN algorithm against the National Center for
Biotechnology Information non-rebundant database (www. A multiple alignment of the orthologous
16S rRNA, psbA, and rbcL genes sequences of related
Cyanidiales species was then performed using CLUS-
TALW [ 27]. Accession numbers for the plastid genes of
previously sequenced rhodophytes used for phylogenetic
comparisons were: 16S rRNA: AB002583, AY882672,
AY391361, AY391359, AY391360, AF170718, X52985,
EU586032, and X81840, rbcL: AB002583, AY119765,
Atacama Cave Cyanidium 487
AY391370, AY541297, AY541298, X53045, AF022186,
AY391368, and AY391369, psbA: AY391365, AY391366,
AB002583, AY391367, AF022186, and X14667.
Accession numbers for the 16S rRNA, rbcL, and psbA
genes of Cyanidium sp. Atacama are FJ390400, FJ402842,
and FJ447339, respectively.
Phylogenetic reconstructions were performed through
maximum parsimony using PAUP* 4.0b8. All characters
were analyzed as unordered with four possible states (A, C,
G, and T), excluding phylogenetically uninformative
characters. The most parsimonious trees were found
through an exhaustive search. Phylogenetic analyses were
also accomplished using the neighbor-joining and
maximum-likelihood algorithms available in PAUP. For
the latter analysis, we first obtained the best fitting model of
sequence evolution, using the Akaike Information Criterion
in Modeltest 3.06. For the 16S rRNA genes, the AIC
indicated that the GTR+G model (gamma: 0.2328) was the
best model to describe the evolutionary process. Values
were: ln L=4,847.07952 (base frequencies: A 0.2713, C
0.2176, G 0.2957). In the case of the psbA genes, the AIC
indicated that the GTR+G+I model (alpha 3.3312) was the
best model to describe the evolutionary process. Values
were: ln L=3,887.80561 (base frequencies: A 0.25730, C
0.18620, G 0.21670). Consistency on the nodes was
evaluated via bootstrap, performing 1,000 replicates for
each of the optimality criterion analysis. Trees were rooted
with the out-group criterion using the published sequence
of the Cyanophora paradoxa 16S rRNA gene [9].
Site Description
The cave is located in the coastal range of the Atacama
Desert, about 20 km north of the city of Antofagasta, Chile.
It is placed at the base of a 50-m-high cliff, facing south
towards the Pacific Ocean. The cave is approximately 35 m
in depth and 6 m wide, with an average height of 3 to 4 m,
although in parts its height exceeds 6 m (Fig. 1). It has two
entrances; the one facing east is about 3 m wide and 3.5 m
tall (Fig. 2a). The other entrance point south, confronts the
Pacific Ocean, and is about 4 m wide and 3 m tall. The sea
enters the cave through this entrance covering no more than
one third of the ground during high tides. The walls of the
cave are formed by Jurassic breccias of basaltic andesite,
and the ceiling is formed by an overlaying conglomerate
bed of fossilized marine sandstone and fossil shells dating
35 to two million years ago (Fig. 2b) [20]. The cave has a
high RH that varies dynamically in time and is relatively
homogenous along its interior (Table 1). The temperature of
the rock walls inside the cave is around 15°C during most
part of the day and is the same at similar distances from the
entrance of the cave in both the eastern and western walls
(Table 2). Water droplets collected from the walls where
biofilm is located have a pH of 4.5. These water droplets
seem to be evenly distributed along the cave and appear to
be periodic since they were observed during March of 2008
but not in June, when the walls of the cave were drier.
Figure 1 Map and profile view
of the Atacama Desert cave
488 A. Azúa-Bustos et al.
Biofilm Distribution
The studied biofilms have an intense greenemerald color
and are dispersed throughout the cave, always away from
direct light coming from the two entrances. The zone
showing most biofilm development is located on the north
western wall, facing the east entrance but not directly
confronting it. This zone is located about 13 m from the
east entrance, and the biofilm is located about 1.5 m from
the cave floor, extending 1.5 m towards the cave ceiling.
Part of the ceiling above the main development zone also
shows biofilm but to a lesser extent (Fig. 2c). On this same
wall, about 23 m from the east entrance and towards the
bottom of the cave, there is also biofilm development,
although it is placed closer to the ground and on adjacent
ground rocks (Fig. 2d). The eastern wall shows no
development of biofilms (Fig. 2b).
Photosynthetic Photon Flux Density Measurements
The PPFD measured outside the southeast entrance of the
cave on a clear summer day was of 1,668 μmol m
Two different measurements were made, namely, at 11 AM
on a summer day (March) and at 14:30 PM on a winter day
(June) of 2008, showing similar trends in the light profile
inside the cave at the dimmest lighted zones. Five meters
inside this entrance, the PPFD values on the western wall
were of 25 μmol m
. The main biofilm development
Figure 2 Atacama cave de-
scription. aEast entrance. b
View towards the bottom (north)
of the cave. The biofilm area
studied is located on the left side
wall. cDetail of Cyanidium
biofilm. dBiofilm on rocks at
the bottom of the cave. Note the
biofilm development only on
the rock face oriented towards
the east entrance. In both a,b,
and c, the different geological
origin of the walls and ceiling of
the cave can be observed
Time Air temperature (°C) Air RH (%) IR t° (C)
Eastern wall Western wall
14:00 At main biofilm 21.7 77 ––
Cave entrance 22 80 ––
Cave bottom 22 80 ––
14:20 25.5 59 14 14
14:50 19.1 85 15 15
15:00 77 ––
15:40 18.8 89 15
Table 1 Variations in time of
air temperature and RH and
infrared temperature of walls of
the cave interior (March 2008)
Atacama Cave Cyanidium 489
zone showed values of 1 to 3 μmol m
, that is, 0.06%
to 0.17% of the outside incident light. The bottom of the
farthermost zone of the cave, where a thin biofilm could
still be found, had values of 1 μmol m
(Table 3). The
eastern wall had much lower or received no light, as
measured by the used PPFD sensors. Thus, the areas of the
cave where the biofilm develops are subject to extremely
low light intensities, close to measuring sensitivity of the
PPFD equipment.
Light microscopy examination of aqueous resuspended
samples of biofilm scraped from the cave walls revealed a
homogeneous and possibly monospecific population of
photosynthetic primary producers. The spherical cells are
36μm in diameter (Fig. 3a). Some autospore-containing
cells are also seen (Figs. 3a and 4b). The cells single
chloroplasts emit an intense chlorophyll autofluorescence
red signal under the confocal microscope, as observed in
Fig. 3b. Transmission electron microscopy (TEM) micro-
graphs unveiled the typical ultrastructural elements previ-
ously described for these unicellular red algae (Fig. 4a).
The chloroplast, nucleus, and mitochondria are clearly
distinguished, as well as their corresponding membranes.
The thylakoid concentric membranes show the embedded
phycobillisomes that have been described for this species.
Several electron-dense bodies are also observed associated
to the cell membrane (Fig. 4a), which in some cells appear
to form two different chains at the opposite poles of the
cell. On the other hand, scanning electron microscopy
(SEM) micrographs show the Cyanidium biofilm attached
to its parent rock. Two different aggregates can be
observed: one in which the cells are close together with
no supporting matrix (Fig. 5a) and another aggregate type
where the cells are well embedded in matrix of (probably)
exo-polysaccharides (EPS; Fig. 5b).
Phylogenetic Analysis of 16S rRNA, psbA, and rbcL
Chloroplast Genes
Using 16S rRNA, psbA,andrbcL Cyanidium-specific
primers, we amplified the 16S rRNA, psbA, and rbcL
genes consisting of 1,461-, 920-, and 548-bp products,
respectively. Automated sequencing of eight of the 16S
rRNA clones and three PCR products showed that all where
identical and matched (97% identity) with a partial
sequence of the 16S ribosomal RNA gene of Cyanidium
sp. Monte Rotaro, isolated from a cave in Italy. The next
most similar sequence match (93% identity) corresponded
Table 2 Infrared temperatures of cave interior walls (June 2008)
Distance from bottom of the cave (m) IR temperature eastern wall (°C) IR temperature western wall (°C)
1 15.5 15.7
3 15.3 15.3
5 15.2 15.2
7 15.1 15
9 14.8 14.8
11 14.6 14.5
13 14.7 24.8
Table 3 Lighting profile along the length of the cave. The light available for photosynthesis was determined with a photosynthetic photon flux
density measuring device outside and inside the cave at different places in both the western an eastern walls of the cave
March 2008 June 2008
Distance from
cave entrance (m)
PPFD (μmol m
) Percentage of outside light PPFD (μmol m
) Percentage of outside light
Western wall Eastern wall Western wall Eastern wall Western wall Western wall
0 1,668 1,668 100 100 1,250 100
5 25 12 1.50 0.72 158 12.64
10 6 3 0.36 0.18 54 4.32
15 6 0 0.36 0 21 1.68
20 3 0 0.18 0 7 0.56
25 2 0 0.12 0 2 0.16
30 1 0 0.06 0 1 0.08
490 A. Azúa-Bustos et al.
to the 16S rRNA gene of Cyanidium sp. Sybil cave, also
located in Italy. For the 16S rRNA gene, a single
maximum-likelihood tree was obtained through the heuris-
tic search option of PAUP, and the Modeltest software
suggested that the best fitting model of sequence evolution
to reconstruct the likelihood tree was as observed in Fig. 6.
The analysis of the 16S rRNA gene show that cave
Cyanidiumspecies were recovered as a monophyletic
group that included Cyanidium sp. Atacama and Cyanidium
sp. Monte Rotaro as sister species with 100% of bootstrap
support, whereas Cyanidium sp. Sybil cave was basal to
this relationship with a bootstrap support of 91%. Similar
results were obtained when using the psbA gene, and
automated sequencing of three of the psbA clones showed
that they matched (91% identity) with a partial sequence of
the psbA gene of Cyanidium sp. Monte Rotaro. The next
most similar sequence match (86% identity) corresponded
to the psbA gene of Cyanidium sp. Sybil cave. In the case
of the psbA gene, the cave Cyanidium species also were
recovered as a monophyletic group that included Cyanidium
sp. Atacama and Cyanidium sp. Monte Rotaro as sister
species with 99% of bootstrap support, whereas Cyanidium
sp. Sybil cave was basal to this relationship with a
bootstrap support of 92% (Fig. 7). In the case of the rbcL
gene, the most similar sequence match (89% identity)
corresponded to the rbcL gene of Cyanidium sp. Monte
Rotaro. The maximum-likelihood tree obtained for the rbcL
gene did not provide sufficient resolution, as Long Branch
attraction persisted for the rbcL gene of Cyanidium sp.
Atacama (data not shown).
Being in an ancient desert and a recognized Mars analog,
caves of the Atacama Desert represent a prime target for the
search of its associated microorganisms. As for photosyn-
thetic microorganisms, to date, mainly cyanobacteria have
Figure 3 Micrographs of Cyanidium sp. cells found in biofilms of the
Atacama cave. aBright field micrograph of Cyanidium sp. composed
of single photosynthetic cells and small fragmented colonies. Scale
bar =10μm. bMerged CLSM micrograph of aqueous suspension of
Cyanidium cells extracted from the cave biofilm. The differential
interference contrast image was merged with the red fluorescence
(excitation/emission 543 nm/long pass filter <570 nm) due to the
autofluorescence emitted by the cell chloroplast containing chloro-
phyll. Scale bar=10 μm. The arrows in bindicate endospore-
containing cells
Figure 4 TEM micrograph of
an ultrathin section of
Cyanidium cells showing cells
in different developmental states
within the same aggregate. a
Note the characteristic organ-
elles and the multilayered enve-
lopes surrounding the cells.
Scale bar = 1.4 μm. bCell in a
dividing state. Scale
bar = 1.7 μm. C: chloroplast, V:
vacuole, N: nucleus, M: mito-
chondria, cw: cell wall, nm:
nuclear membrane, eps: exo-
polysaccharide layer, edb:
electron-dense bodies
Atacama Cave Cyanidium 491
been described elsewhere in the Atacama [48,50]. On the
other hand, the Cyanidiales, an order of the Rhodophyta
algae, are an ancient group of microorganisms dating as far
as 2,000 million years ago [52].
The finding of a member of the Cyanidiales living in a
cave in this area was unexpected, considering that most
known species of this order inhabit acidic thermal springs.
In our case, it was determined that the biofilms remain cool
during most part of the day (15°C measured around noon),
and that water droplets associated to it have a slightly acidic
pH (4.5). The internal ultrastructure of cells scraped from
the cave biofilm and observed under TEM shows the
typical type and amount of organelles (one spherical
chloroplast and one mitochondrion, in addition to the
nucleus) already described for Cyanidium species [16,
30]. As for the SEM micrographs, two modes of cell
aggregations in the biofilm could be observed: one in which
the cells appear to be loosely associated with each other,
with low or no presence of extracellular materials, and
another form of aggregation in which the cells are
embedded in a well-developed extracellular matrix.
Subaerial biofilm species often secrete EPS that facilitate
further adhesion onto the substrate and, in the case of desert
environments, the retention for longer periods of the scarce
water available [22]. In our case, the reasons for the
observed variations on biofilm conformation in an appar-
ently isotropic humid microhabitat are still unclear. It has
been suggested that downregulating EPS production at high
cell densities could allow cells to redirect energy from EPS
production into growth and cell division prior to a dispersal
event [32]. Aside from the presence of EPS for cell
adhesion to the wall matrix, another interesting possibility
Figure 5 SEM micrographs of Atacama cave Cyanidium. aBiofilm
where individual cells are forming loose well-defined aggregates can
be observed. Scale bar =10μm. bBiofilm conformation where the
cells are well embedded in matrix of exo-polysaccharides covering the
parental rock. Scale bar = 10 μm
Figure 6 Maximum-likelihood
tree obtained from the aligned
sequences of the 16S rRNA
chloroplast gene for Cyanidiales
species. Numbers above the
node represent 1,000 replicate
bootstrap values
492 A. Azúa-Bustos et al.
is that EPS could be used as a medium for soluble
molecules involved in intercellular communication and
quorum sensing as previously suggested [26]. Being this
the case, intercellular communication would no longer be
needed previous to a dispersal event, which could explain
the observed lack of an EPS matrix in some cases. One last
possibility was proposed by Bellezza et al. (2006) [7]in
which EPS, being rich in negative charges, may allow the
adsorption of constituent cations from the mineral substrata.
Irrespective of these possibilities, in both cases of biofilm
conformation, a seemingly monospecific Cyanidium bio-
film is morphologically observed. This observation is
supported by the 16S rRNA analysis, which unambiguously
shows only one type of photosynthetic species related to
16S rRNA gene sequence present in the dimmest lighted
areas of the cave. When using cyanobacteria 16S-rRNA-
specific primers [35], no species of this phylum were
detected at this part of the cave after repeated attempts (data
not shown). This is remarkable since photosynthetic micro-
organisms on rock surfaces rarely grow as colonies
comprising a single species, and epilithic biofilms are
commonly composed of several species of cyanobacteria
and algae [22]. Nevertheless, an initial molecular charac-
terization of the non-photosynthetic components of the
biofilm shows that other species of heterotrophic bacteria
can be detected as forming part of the biofilm which cannot
be seen by the microscopy methods used, species of
gammaproteobacteria related to the genera Salinisphaera
(98% identity by 16S rRNA sequencing) and another
bacteria morphologically similar to Saccharomonospora
actinobacteria (data not shown). In the latter case, this
bacterium can only be detected on collected rock samples in
ex situ conditions, in which the water, temperature, and
light conditions have been greatly altered for extended
periods of time.
The maximum-likelihood trees obtained for the 16S
rRNA and psbA genes observed in Figs. 6and 7show the
Cyanidium sp. Atacama forming part of the proposed
Cyanidium cavemonophyletic group that includes the
other two known Cyanidium cave species, Cyanidium sp.
Monte Rotaro being its closest relative. Only two of the
proposed 17 known species of Cyanidiales, namely
Cyanidium sp. Sybil and Cyanidium sp. Monte Rotaro,
appear to inhabit caves, both located near the Vesuvius
volcano [9]. These two species are considered to be the
mesophilicmembers of the group. Thus, the 16S rRNA
and psbA genes data support that the Atacama cave
Cyanidium is part of the cave Cyanidium group, which as
previously proposed [9] may be a novel monophyletic
lineage of mesophilic Cyanidium spp., distinct from the
remaining three other known lineages. In the case of the
rbcL gene, although a much lower identity percentage is
observed, its closest reported relative still is Cyanidium sp.
Monte Rotaro [9]. A maximum-likelihood tree obtained for
the rbcL gene did not provide sufficient resolution, as Long
Branch attraction persisted for the rbcL gene Cyanidium sp.
Atacama (data not shown). It has been previously reported
that, in the Cyanidiales, ribosomal sequences are more
conserved than that of the rbcL gene sequences [9,45,46]
and that rbcL gene sequences are known to be subjected to
higher rates of sequence evolution, leading to saturation
events precluding sufficient resolution in single-gene and
even combined-genes approaches [21,22,45,46]. This
discrepancy may be explained by the lateral transfer of the
rbcL gene [45]. Nevertheless, both the transmission
electron microscopy and molecular data presented in this
paper support the contention that a species of Cyanidium
inhabits this Atacama cave.
Although Schwabe [41] reported a species which he,
based on morphology, proposed as belonging to the
Cyanidium genera, it was found in two caves on the central
coast of Chile located over 1,300 km south of the Atacama
cave. It is known that, under the microscope, species of red
algae are difficult to identify based on morphology alone
[38]. In particular, species of the genera Cyanidium and
Galdieria are indistinguishable [15,37]. On the other hand, a
high level of sequence divergence is systematically observed
among Cyanidiales species with the partitioning of taxa,
allopatric divergence, and speciation based on environmental
conditions, suggesting that new species diverge after long-
term geographic isolation [9,45,49]. Thus, considering the
distant latitudes, it is likely that the species described in this
work and that reported by Schwabe are not the same. This is
consistent with the discrepancies found for the rbcL gene
molecular data reported in this work. Schwabe did not
provide more specific coordinates of the exact location of the
caves he studied, but efforts are now being made to locate
them, allowing future comparisons.
Figure 7 Maximum-likelihood tree obtained from the aligned
sequences of the psbA chloroplast gene for Cyanidiales species.
Numbers above the node represent 1,000 replicate bootstrap values
Atacama Cave Cyanidium 493
As for the general water environment internal and
external to the cave, water relations are critical and
probably the most limiting factor for life in the hyperarid
Atacama desert. The coastal area where the cave is located
is exposed to fogs that usually arrive at late afternoon and
night. Although their influence as a source of water for the
cave Cyanidium biofilms cannot be dismissed, the more
direct and constant influence of the nearby ocean spray and
mist probably accounts for the long-term most stable humid
environment inside the cave. The presence of the seawater
at the cave entrance does not seem to create a differential
bottom to entrance water gradient inside the cave since RH
is homogenously high along the cave interior (Table 1).
Since high humidity values (89%) were measured inside the
cave after midday of clear days, it may be assumed that
these are the minimum humidity values encountered at this
location. Thus, the possible condensation of periodical fogs
inside the cave at night should not have an appreciable
impact on biofilm development in its interior.
Interestingly, the habitat description of the caves further
south made by Schwabe [41] on this matter is almost
identical to the reported site in this work: The air at the
observed locations exhibit continuously high moisture. The
site however show only moderate damp to dry areas and
apparently does not seem to be under continuing water
Also, the habitat description on terms of the light
conditions made by Schwabe [41] is again identical to the
one described in this work: the best development was
found several meters from the cave entrance, at the walls
and ceilings. However, areas exposed to the light (cave
entrance) show no growth. This may be understood if the
relatively low temperatures and high light conditions of the
areas close to the entrances of the cave are taken into
consideration. It is well known that the concurrence of
these two conditions determine an important stress upon the
photosynthetic machinery [31], causing the production of
highly deleterious reactive oxygen species and photo-
inhibition. In our case, we observe a sharp transition in
the presence of Cyanidium biofilms in relation to the
availability of light. As seen in Fig. 2d, only the entrance-
facing side of a rock located on the ground shows biofilm
development. A similar transition is also observed from the
western to the eastern wall. Even at places where PPFD
values reach 1 μmol m
,Cyanidium biofilms can still
be observed. However, as soon as no light is measured at
the eastern wall, Cyanidium biofilms are no longer
detected. To find areas with only one photosynthetic
species inhabiting a cave where water availability seems
not to be the limiting factor could be explained by the very
low light levels found towards the bottom, which could be
preventing the sustained growth of other species of photo-
trophs. Thus, at the bottom of the cave, only Cyanidium
biofilms can be found due to their highly efficient
photosynthetic machinery, which in turn may not be able
to cope with the higher levels of light found closer to the
entrance. It would be interesting to understand the
mechanisms of photosynthetic quantum efficiency of this
type of cave Cyanidium. Near the cave entrances, the
higher light levels may allow the colonization of the walls
by other phototrophic microorganisms which may outcom-
pete the growth of the Cyanidium biofilms. In fact, we do
observe the appearance of cyanobacteria on the walls
directly in front of the entrances but not at the bottom of
the cave where the Cyanidium biofilm are located.
biological context of a Martian cave [8,13,23], where a
hypothetical phototrophic microorganism like the reported
cave Cyanidium could be found inside a cave well
protected from the harsh outside conditions using minimum
photon flux levels coming from a nearby entrance, but high
enough for enabling the photosynthetic processes critical
for survival.
Acknowledgments This work was supported by the Millennium
Institute of Fundamental and Applied Biology (Chile). We also thank
the members of Rafael Vicuñas Laboratory for critical comments and
insights which helped to improve this manuscript.
Disclosure Statement No competing financial interests exist in
connection with the submitted manuscript. This applies to all authors
of this paper.
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... Sybil Cave, inhabitants of Italian caves [34,35], and the third, Cyanidium sp. Atacama, in a cave on the Chilean coast [36]. ...
... inhabitants of Italian caves [34,35], and the third, Cyanidium sp. Atacama, in a cave on the Chilean coast [36]. ...
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... As to uncultured populations related to phyla Bacillariophyta and Rhodophyta, results also showed the presence of these populations in biofilms not containing salt efflorescence. Both phyla have previously been identified in historic stone monuments and show caves (Ortega-Calvo et al., 1995;Azúa-Bustos et al., 2009;Del Rosal et al., 2014;Pfendler et al., 2018). ...
In 2013, UNESCO classified the ‘University of Coimbra – Alta and Sofia’ (Portugal) as World Heritage site. Some of the limestone monuments that compose the site, such as the Old Cathedral of Coimbra, show clear visual effects of biological colonization by cyanobacteria and microalgae. The aim of this study was to thoroughly characterize the structural diversity of the photoautotrophic community at this site, combining isolation and molecular identification with next-generation sequencing techniques. Culture-dependent approaches resulted on the identification of green microalgae belonging to Prasiolales, Chlorellales, Watanabea, Chlamydomonadales and Sphaeropleales clades, and cyanobacteria belonging to Nostocales and Synechococcales clades. Culture-independent methods using Illumina Platform resulted in the identification of 610 Operational Taxonomic Units (OTUs), revealing the presence of populations within phyla Chlorophyta, Cyanobacteria, Rhodophyta and Stramenopiles/Bacillariophyta. In addition, principal component analysis (PCA) and a network analysis were generated and successfully related the different populations to specific biodeterioration scenarios observed at this monument. The use of culture-dependent and -independent techniques together with PCA and network analysis allowed us to understand the distribution patterns of the photoautotrophic community among the different biofilms observed at this monument, as well as their ecological specifications. This study highlights that both cultivation and metagenomics methodologies should be employed synergistically in similar biodeterioration studies.
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From 2.5 to 2.0 billion years ago, atmospheric oxygen concentration [O2] rose thousands of times, leading to the first mass extinction. Reactive Oxygen Species (ROS) produced by the non-catalyzed partial reduction of O2 were highly toxic eliminating many species. Survivors developed different strategies to cope with ROS toxicity. At the same time, using O2 as the final acceptor in respiratory chains increased ATP production manifold. Thus, both O2 and ROS were strong drivers of evolution, as species optimized aerobic metabolism while developing ROS-neutralizing mechanisms. The first line of defense is preventing ROS overproduction and two mechanisms were developed in parallel: 1) Physiological uncoupling systems (PUS), which increase the rate of electron fluxes in respiratory systems. 2) Avoidance of excess [O2]. However, it seems that as avoidance efficiency improved, PUSs became less efficient. PUS includes branched respiratory chains and proton sinks, which may be proton specific, the mitochondrial uncoupling proteins (UCPs) or unspecific, the mitochondrial permeability transition pore (PTP). High [O2] avoidance also involved different strategies: 1) Cell association, as in biofilms or in multi-cellularity allowed gas-permeable organisms (oxyconformers) from bacterial to arthropods to exclude O2. 2) Motility, to migrate from hypoxic niches. 3) Oxyregulator organisms: as early as in fish, and O2-impermeable epithelium excluded all gases and only exact amounts entered through specialized respiratory systems. Here we follow the parallel evolution of PUS and O2-avoidance, PUS became less critical and lost efficiency. In regard, to proton sinks, there is fewer evidence on their evolution, although UCPs have indeed drifted in function while in some species it is not clear whether PTPs exist.
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The Atacama Desert is by far the driest and oldest desert on Earth, showing a unique combination of environmental extremes (extreme dryness, the highest UV radiation levels on Earth, and highly saline and oxidizing soils), explaining why the Atacama has been largely investigated as a Mars analog model for almost 20 years. Based on the source and the amount of water available for life and its analogy with Mars, two ecosystems are of interest in the Atacama: its Coastal Range and the much drier hyperarid core, which we here review in detail. Members of the three domains of life have been found across these ecosystems living at the limit of habitability, suggesting the potential dry limits for each domain and also unveiling the highly patchy distribution of microbial life in its most extreme regions. The thorough study of the Atacama has allowed us to understand how life has adapted to its extreme conditions, the specific habitats that life occupies in each case (thus suggesting the most likely places in which to search for evidence for life on Mars), and the number of biosignatures detected across this desert. Also, the characterization of west-to-east transects across this desert has shown to be of significant value to understand the potential adaptations that Martian microorganisms may have followed in an ever-drying planet. All of this explains why the Atacama is actively used as the testing ground of the technologies (detection instruments, rovers, etc.) that were sent and will be sent to Mars. We also highlight the need to better inform the exact locations of the sites studied to understand general trends, the need to identify the true native microbial species of the Atacama, and the impact of climate change on the most arid and most Martian desert of Earth.
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Galdieria sulphuraria is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that G. sulphuraria cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon. ●Here, we show that G. sulphuraria SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO2 supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO2. ●Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO2 concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Rubisco and decreasing photorespiration. ●We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.
Phlegrean Fields is a large volcanic area situated southwest of Naples (Italy), including both cave and thermoacidic habitats. These extreme environments host the genus Cyanidium; the species C. chilense represents a common phototrophic microorganism living in anthropogenic caves. With a view to provide a comprehensive characterization for a correct taxonomic classification, morpho‐ultrastructural investigations of C. chilense from Sybil's cave (Phlegren Fields) was herein carried out and compared with the thermoacidophilic C. caldarium. The biofilm was also analyzed to define the role of C. chilense in the establishment of a biofilm within cave environments. Despite the peculiar ecological and molecular divergences, C. chilense and C. caldarium shared all the main diacritic features, suggesting morphological convergence within the genus; cytological identity was found among C. chilense strains geographically distant and adapted to different substrates, such as the porous yellow tuff of Sybil cave, and calcyte, magnesite and basaltic rocks from other caves. C. chilense is generally dominant in all biofilms, developing monospecific islets, developing both superficially or between fungal hyphae and coccoid cyanobacteria. Extracellular polymeric substances (EPS) were recorded in C. chilense biofilms from Sybil cave, confirming the role of EPS in facilitating cells adhesion to the surface, creating a cohesive network of interconnecting biofilm cells.
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The Karst Waters Institute Breakthroughs in Karst Geomicrobiology and Redox Geochemistry conference in 1994 was a watershed event in the history of cave geomicrobiology studies within the US. Since that time, studies of cave geomicrobiology have accelerated in number, complexity of techniques used, and depth of the results obtained. The field has moved from being sparse and largely descriptive in nature, to rich in experimental studies yielding fresh insights into the nature of microbe-mineral interactions in caves. To provide insight into the changing nature of cave geomicrobiology we have divided our review into research occurring before and after the Breakthroughs conference, and concentrated on secondary cave deposits: sulfur (sulfidic systems), iron and manganese (ferromanganese, a.k.a. corrosion residue deposits), nitrate (a.k.a. saltpeter), and carbonate compounds (speleothems and moonmilk deposits). The debate concerning the origin of saltpeter remains unresolved; progress has been made on identifying the roles of bacteria in sulfur cave ecosystems, including cavern enlargement through biogenic sulfuric acid; new evidence provides a model for the action of bacteria in forming some moonmilk deposits; combined geochemical and molecular phylogenetic studies suggest that some ferromanganese deposits are biogenic, the result of redox reactions; and evidence is accumulating that points to an active role for microorganisms in carbonate precipitation in speleothems.
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The sedimentary succession in the Atacama Desert records deposition under an and to semiarid climate from the late Jurassic (150 Ma) to the present day. Palaeomagnetic data indicate no significant latitudinal movement of this area since the late Jurassic. The present-day location of the Atacama within the dry subtropical climate belt is the principal cause of aridity. This situation is likely to have prevailed since the late Jurassic, supplemented by (1) the continentality effect (enhanced by the Gondwanan landmass), and (2) the presence offshore of a cold, upwelling current (from at least the early Cenozoic onwards and possibly earlier), resulting in conditions promoting climatic stability and desert development. Rapid and extreme climatic fluctuations during the Plio-Pleistocene were not sufficient to destabilize the climate within the Atacama. Comparison with other long-lived deserts (e.g. SW USA, Namib, Sahara and Australia) suggests that the Atacama is the oldest extant desert on Earth.
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A detailed paleomagnetic investigation was carried out on the Jurassic La Negra Formation. A total of 32 consecutive lava flows (about 280 standard paleomagnetic cores) were collected. Several rock-magnetic experiments were carried out in order to identify the magnetic carriers and to obtain information about their paleomagnetic stability. Continuous susceptibility measurements with temperature yield in most cases reasonably reversible curves with Curie points close to that of Ti-poor titanomagnetite. Titanomaghemites seem to be the main magnetic carrier in a few cases. Judging from the ratios of hysteresis parameters, it seems that all samples fall in the pseudo-single domain grain-size region, probably indicating a mixture of multidomain and a significant amount of single-domain grains. Twenty-five flows yielded reliable paleomagnetic results. The mean paleodirection obtained in this study is I = -30.7, D = 11.3°, k = 18, a95 = 6.8°, N = 25. Combining all currently available data, including those obtained in previous studies for the La Negra volcanic sequence, we obtained a well-defined mean paleomagnetic direction with I = -33.9°, D = 10.5°, k = 15, a95 = 6.1°, N = 40. These directions are in good agreement with the expected paleodirections at about 180 Ma, which indicates that studied units do not show evidence for significant tectonic rotation. Fifteen sites yield reverse polarity magnetization and 11 flows are normally magnetized. The tentative direct magnetostratigraphic correlation suggests that La Negra volcanics have been emplaced during a relatively large time span of about 5 m.y.
The presence of nucleus, chloroplasts, mitochondria, microbodies and a vacuole are confirmed in Cyanidium caldarium strain CCAP 1355/1. Chloroplasts usually contain parallel rows of unstacked thylakoids surrounded by a peripheral thylakoid, although the concentric arrangement has also been observed. The chloroplast envelope consists of two closely appressed membranes which can only be resolved after gluteraldehyde-osmium fixation. The chloroplast of Rhodosorus marinus also contains parallel, unstacked thylakoids surrounded by a peripheral thylakoid but, in addition, a prominent, stalked pyrenoid projects into the cytoplasm and is bounded by both the peripheral thylakoid and the chloroplast envelope. This structure is covered by a cap of starch grains and contains membranous vesicles in the matrix. Phycobilisomes are absent from the chloroplasts of both algae. The recognition of C. caldarium as a rhodophyte is supported by these observations.
The taxonomic and systematic chapters (Ott and Seckbach in this volume) gave the following binomials (and where applicable their respective formae) that have been applied at various times throughout the years to material presently recognized as the Cyanidaceae Geitler 1933. After each listed binomial there will be found, in parentheses, the modern name of the respective algal division to which the binomial would have been assigned at the time of its initial circumscription. The names of these respective algal divisions here employed were those recommended by Papenfuss (1955) with the single exception that the name for the blue-green algal division has been taken from Bold (1967).
Obligatory cavernicoles, or troglobites, have traditionally been of special interest to evolutionary biologists for several reasons. The existence of animal life in caves and other subterranean spaces at first attracted attention because of its novelty; intensive biological exploration of caves began little more than a century ago. Although the discovery and description of the cave faunas of the world is far from complete, especially in the Western Hemisphere, so much descriptive information has been compiled that we can safely assert that, at least in unglaciated, temperate parts of the world, the occurrence of numerous species of troglobites in any major limestone region is a common and highly probable phenomenon.
The fine structure of Cyanidium caldarium, as seen in thin sections of KMnO4-fixed cells examined with the electron microscope, is described. This organism, whose taxonomic position among algae is undetermined, contains a single well defined chloroplast, a nucleus, and mitochondria. Studies, with the electron microscope, of Chlorella pyrenoidosa and Nostoc are also reported. Structural differences within cells of Cyanidium, chlorella, and Nostoc are discussed. It is concluded that if Nostoc can be taken as a typical Cyanophyte and Chlorella as a representative Chlorophyte and if the items of fine structure examined are diagnostic, then Cyanidium is certainly not a Cyanophyte and, while it has numerous features in common with Chlorella, is not a green alga similar to Chlorella. Comparisons are also made between Cyanidium and other algae whose fine structure has been described by others.
The cytomorphology and the exopolysaccharides of two heterocystous cyanobacteria, Scytonema ocellatum CP8-2 and Fischerella maior NAV 10 bis, isolated from biofilms collected in two Roman hypogea, were investigated from a cytochemical, ultrastructural and biochemical point of view. Cytochemical stains and observation using light and transmission electron microscopy showed a bi-layered sheath in both cyanobacteria, with a different composition in sulphated and carboxylic groups. RP-HPLC of the extracted polysaccharides forming the mucous matrix showed the neutral sugars glucose and arabinose and the charged galacturonic acid as the main monosaccharides. Circular dichroism spectra recorded at varying pH and temperature showed that these polymers followed a random coil model and did not present a structured organised conformation at ambient temperature. The outcomes of this study increase the understanding of colonization and biodeterioration of important archaeological and cultural sites by cyanobacterial biofilms and the role of the exopolymeric substances involved.