ORIGINAL RESEARCH
published: 15 January 2019
doi: 10.3389/fmicb.2018.03350
Microbial Biomarker Transition in
High-Altitude Sinter Mounds From
El Tatio (Chile) Through Different
Stages of Hydrothermal Activity
Laura Sanchez-Garcia 1 , Miguel Angel Fernandez-Martinez 1 ,
Miriam García-Villadangos 1 , Yolanda Blanco 1 , Sherry L. Cady 2 , Nancy Hinman 3 ,
Mark E. Bowden 2 , Stephen B. Pointing 4 , Kevin C. Lee 5 , Kimberly Warren-Rhodes 6,7 ,
Donnabella Lacap-Bugler 5 , Nathalie A. Cabrol 6,7 , Victor Parro 1 and Daniel Carrizo 1*
1
Edited by:
Mónica Sánchez-Román,
VU University Amsterdam,
Netherlands
Reviewed by:
Eloi Camprubí Casas,
Utrecht University, Netherlands
Ulrike Kappler,
The University of Queensland,
Australia
Xiaoguo Yu,
State Oceanic Administration, China
*Correspondence:
Daniel Carrizo
dcarrizo@cab.inta-csic.es
Specialty section:
This article was submitted to
Microbiological Chemistry
and Geomicrobiology,
a section of the journal
Frontiers in Microbiology
Received: 21 September 2018
Accepted: 31 December 2018
Published: 15 January 2019
Citation:
Sanchez-Garcia L,
Fernandez-Martinez MA,
García-Villadangos M, Blanco Y,
Cady SL, Hinman N, Bowden ME,
Pointing SB, Lee KC,
Warren-Rhodes K, Lacap-Bugler D,
Cabrol NA, Parro V and Carrizo D
(2019) Microbial Biomarker Transition
in High-Altitude Sinter Mounds From
El Tatio (Chile) Through Different
Stages of Hydrothermal Activity.
Front. Microbiol. 9:3350.
doi: 10.3389/fmicb.2018.03350
Centro de Astrobiología (CSIC-INTA), Madrid, Spain, 2 Environmental Molecular Sciences Laboratory, Pacific Northwest
National Laboratory, Richland, WA, United States, 3 Department of Geosciences, University of Montana, Missoula, MT,
United States, 4 Yale-NUS College, National University of Singapore, Singapore, Singapore, 5 School of Science, Auckland
University of Technology, Auckland, New Zealand, 6 SETI Institute, Mountain View, CA, United States, 7 NASA Ames
Research Center, Moffett Field, CA, United States
Geothermal springs support microbial communities at elevated temperatures in an
ecosystem with high preservation potential that makes them interesting analogs for
early evolution of the biogeosphere. The El Tatio geysers field in the Atacama Desert
has astrobiological relevance due to the unique occurrence of geothermal features with
steep hydrothermal gradients in an otherwise high altitude, hyper-arid environment. We
present here results of our multidisciplinary field and molecular study of biogeochemical
evidence for habitability and preservation in silica sinter at El Tatio. We sampled three
morphologically similar geyser mounds characterized by differences in water activity (i.e.,
episodic liquid water, steam, and inactive geyser lacking hydrothermal activity). Multiple
approaches were employed to determine (past and present) biological signatures and
dominant metabolism. Lipid biomarkers indicated relative abundance of thermophiles
(dicarboxylic acids) and sulfate reducing bacteria (branched carboxylic acids) in
the sinter collected from the liquid water mound; photosynthetic microorganisms
such as cyanobacteria (alkanes and isoprenoids) in the steam sinter mound; and
archaea (squalane and crocetane) as well as purple sulfur bacteria (cyclopropyl acids)
in the dry sinter from the inactive geyser. The three sinter structures preserved
biosignatures representative of primary (thermophilic) and secondary (including endoliths
and environmental contaminants) microbial communities. Sequencing of environmental
16S rRNA genes and immuno-assays generally corroborated the lipid-based microbial
identification. The multiplex immunoassays and the compound-specific isotopic analysis
of carboxylic acids, alkanols, and alkanes indicated that the principal microbial pathway
for carbon fixation in the three sinter mounds was through the Calvin cycle, with a
relative larger contribution of the reductive acetyl-CoA pathway in the dry system. Other
inferred metabolic traits varied from the liquid mound (iron and sulfur chemistry), to the
steam mound (nitrogen cycle), to the dry mound (perchlorate reduction). The combined
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results revealed different stages of colonization that reflect differences in the lifetime
of the mounds, where primary communities dominated the biosignatures preserved in
sinters from the still active geysers (liquid and steam mounds), in contrast to the surviving
metabolisms and microbial communities at the end of lifetime of the inactive geothermal
mound.
Keywords: lipid biomarkers, microbial transition, hydrothermal activity, sinter mounds, high altitude geyser field,
biogeochemical reconstruction
of hydrothermal systems can be abundant. In these extreme
environments, only the microorganisms particularly resistant
and adapted to adverse conditions are able to thrive. Their
distribution and community structures appears to be mostly
determined by factors such as temperature, pH, or the content
of hydrogen sulfide (e.g., Purcell et al., 2007), as demonstrated by
diverse studies on geothermal springs from Yellowstone National
Park (Cady and Farmer, 1996), Thailand (Purcell et al., 2007),
California and Nevada (Zhang et al., 2007), or New Zealand
(e.g., Pantcost et al., 2005; Kaur et al., 2015). Temperature is
limiting for certain groups such as phototrophic bacteria (e.g.,
cyanobacteria, green sulfur and non-sulfur bacteria), as their
growth is seriously hampered at temperatures greater than 73◦ C
(Ward et al., 1998; Miller and Castenholz, 2000). Conversely,
chemolithoautotrophs such as Aquificales are well adapted to
thrive in such high temperatures (Reysenbach et al., 1994;
Kato et al., 2004). Moreover, hydrogen sulfide is a well-known
inhibiting factor of cyanobacteria (Castenholz, 1973; Giovannoni
et al., 1987), while it enhances or necessitates the growth of
phototrophic sulfur bacteria (Madigan, 1986; van der Meer
et al., 2000) or certain species of Aquificales (Skirnisdottir et al.,
2000; Nakagawa and Fukui, 2003; Kato et al., 2004). Taken
together, geothermal springs are ideal settings for investigating
habitability and adaptability of extremophiles in relation to
various environmental conditions (Lacap et al., 2007; Purcell
et al., 2007; Lau et al., 2008).
Geothermal springs continuously release mineral-rich fluids
that precipitate sinters (typically opaline silica, bicarbonate,
or iron oxide) with morphologically and microstructurally
distinct attributes and they preserve evidence of the microbial
communities that thrived at that location when the sinter
precipitated (i.e., primary communities) (Cady and Farmer, 1996;
Renaut et al., 1998; Farmer and Des Marais, 1999; Fouke et al.,
2000; Parenteau and Cady, 2010; Campbell et al., 2015). Once
hydrothermal fluids vent at the surface, the dynamic combination
of evaporation and cooling of thermal waters precipitates sinter
deposits via heterogeneous and homogeneous nucleation and
polymerization (Fournier and Rowe, 1977). Sinter precipitates
begin to dry out along the hydrothermal terraces or cones as
the distance to the spring vent increases and the hot fluids
cool and evaporate. Sinter accretion typically encrusts, emtombs,
and replaces these biological remnants (Cady and Farmer, 1996;
Renaut et al., 1998; Campbell et al., 2015). The rapid, kinetically
driven precipitation of mineraloids and minerals preserves
organic biomarkers over geological time as distinct hydrothermal
lithofacies (Schopf and Packer, 1987; Campbell et al., 2015;
INTRODUCTION
Geothermal springs are natural environments of scientific
interest because of their significance in the early evolution of
the biogeosphere (Walter and Des Marais, 1993; Konhauser
et al., 2003; Cady et al., 2018). Despite their apparent in
hospitability, terrestrial geothermal springs are recognized
habitats for microbial life on Earth (Brock, 1978). Indeed, they
are considered some of the candidate sites where life began (Van
Kranendonk et al., 2017), in contrast to the classical sub-marine
hydrothermal-vents theory including the alkaline hydrothermal
vent model (Russell, 2018). Though surficial hydrothermal
vents are characterized by steep geothermal gradients and a
perpetual supply of nutrients, geothermal springs also provide
an environment in which intermittent wetting and drying of
hydrothermal precipitates occurs due to the stochastic nature
of surface geothermal activity (Damer and Deamer, 2015).
Alternating wet and dry periods of hydrothermal activity
promotes the interaction of simple molecular building blocks
to form complex molecules (Deamer and Georgiou, 2015).
Although not consensus exists on whether wet-dry cycling played
a role during abiogenesis (Russell, 2018), the possibility of a landbased origin of life strengthens the relevance of such settings for
astrobiological exploration (Van Kranendonk et al., 2017).
Geothermal springs and geysers are manifestations of volcanic
or impact activity on a wet rocky planet (Sillitoe, 2015).
The interaction of groundwater with solidified but still-hot
country rock at shallow depths provides a variety of potential
niches for heat-loving microbes. In such geothermal systems,
groundwater percolates through fractures in igneous rock deep
underground, where heat from the nearby magma chamber heats
the pressurized fluid to a temperature above its boiling point
at surface pressure. Rising superheated fluid emerges at surface
effluents associated with hot spring pools and geysers as a column
of hot water and steam that erupts episodically as steam and
fluid separate as they rise to surface. Fumaroles are characterized
exclusively by steam-driven hydrological activity. Regardless of
the surface expression of subsurface hydrothermal activity, the
environmental conditions of such settings are extreme. Water
and steam temperatures are typically several tens of degrees
(that extend to the local temperature of boiling) above mean
air temperatures, and the pH of the fluid can range from acidic
to alkaline. When the elemental content of hydrothermal fluids
is high (i.e., high concentration of dissolved silica, calcium, or
carbonates), sinter precipitation occurs. The metals contents
(Fe, Mn, and Mg), including toxic metals (e.g., Sb, B, or As)
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inactivity. This episodic nature of thermal activity is dictated
by the subsurface fracture system, which is maintained through
earthquake activity (Fournier, 1989). Consequently, any study of
geysers and hot springs represents a snapshot in time. We use this
snapshot to document and compare the microbial community
and its associated biogeochemistry of each feature. The nature of
the hydrothermal activity (or lack thereof) of these three mounds
represented different stages in the lifetime of such features and
ranged from abundant water discharged during geyser activity
to steam to complete dryness. Because the system is dynamic,
there is the potential for population shifts in the microbial
communities as environmental conditions change (Smith et al.,
2003; Lynne, 2015). This snapshot, therefore, combines the
signatures of the original microbial community with those of any
opportunistic communities that might arise as conditions change.
We use visual and petrographic observations to control for such
changes. We combined the use of lipid biomarkers and organic
isotopic composition with immunological (sandwich microarray
immunoassay) and genomic (DNA sequencing) techniques to
investigate the microbial community and functionality in the
three sinter mounds. Characterization of the morphological
features and geochemistry of the sinter provided an ecological
framework for interpreting the molecular and isotopic results.
Compound-specific isotopic analysis of lipid biomarkers
was employed to obtain information about different carbon
cycling pathways; DNA sequencing was used to characterize the
phylogenetic groups; and antibody microarrays were interrogated
to demonstrate the presence of certain microbial strains and
proteins involved in some metabolic and/or environmental traits.
Mineralogy was identified with X-ray diffraction, and lithofacies
traits preserved in the sinters were revealed by optical and
electron microscopy. This multi-analytical (molecular, isotopic,
genomic, mineralogical, geochemical, and paleobiological)
approach was successful in explaining the influence of the degree
of hydrothermal activity on the biomarkers record (i.e., from
past and present) in sinter deposits from high altitude mounds at
El Tatio. This is the first multidisciplinary molecular study of the
biogeochemical evidence preserved in the sinter formations at
El Tatio, which reveals more about the habitability, adaptability,
and preservation of biosignatures in this type of Mars analog
environment.
Westall et al., 2015). In addition, new microbial populations
colonize (secondary communities) when the environmental
conditions change along the hydrothermal activity lifetime. As
a consequence, a variety of microbial communities thrive in
distinct biofacies of geothermal springs. Hot spring deposits
and the preservation of their biosignatures provide insights into
the evolution of early life (Cady et al., 2018) and can inform
astrobiological search strategies (Walter and Des Marais, 1993;
Farmer and Des Marais, 1999).
The preservation of organic compounds in sinter deposits
has been studied in geothermal springs of the major geyser
fields worldwide, including Yellowstone National Park (e.g.,
Jahnke et al., 2001; Pepe-Ranney et al., 2012), Iceland (e.g.,
Konhauser et al., 2001; Tobler and Benning, 2011); Kamchatka
(Goin and Cady, 2009), Tibet (Lau et al., 2008), or New Zealand
(e.g., Jones et al., 2001; Pantcost et al., 2005; Kaur et al.,
2015). In comparison to most other hydrothermal settings, our
understanding of organic preservation in sinter deposits at El
Tatio (Chile), the third largest geyser field in the world and the
largest in the southern hemisphere, is limited. Located within the
Andes Mountains, El Tatio is one of the highest hydrothermal
systems (4,320 mamsl), which subjects it to unique conditions
such as intense UV-A and UV-B radiation, an unusually low
water-boiling point (86◦ C), and severe climatic changes including
large daily thermal oscillation and high atmospheric dryness
(Fernandez-Turiel et al., 2005). These and other specific limiting
factors for life at El Tatio, including a toxic chemistry of the
geothermal-springs water (B, As, or Sb), make the geothermal
field an extreme environment of interest for understanding the
development and persistence of life under severe conditions,
as well as a terrestrial model of a Martian environment. This
model includes, apart from the extreme aridity, high solar
radiation, salinity and oxidant conditions during the last 10–
15 millions of years characteristic of the Atacama Desert
environments (e.g., Navarro-González et al., 2003), presence of
volcanic and hydrothermal activity such as that in the ancient
Mars. El Tatio serves as a natural laboratory for the study of
the biogeochemical processes involved in the deposition and
alteration of siliceous sinter and the potential for preserving
microbial biosignatures. Yet, the few existing microbiological
studies on El Tatio are focused on petrographic and mineralogical
examinations (Fernandez-Turiel et al., 2005), thermal imaging
(Dunckel et al., 2009), electron microscopy and UV-spectroscopy
(Phoenix et al., 2006), or optical/scanning electron microscopy
and molecular (DNA) methods (Barbieri et al., 2014). To the
best of our knowledge, no studies have integrated microbiological
and biogeochemical approaches for exploring the preservation
of microbial biosignatures on sinter deposits from geothermal
springs at El Tatio.
In this work, we investigated the presence and potential to
preserve molecular biomarkers in sinter deposits from three
geothermal-spring mounds at El Tatio. The springs are within
75 m of each other in the Upper Basin of El Tatio. Thermal
features in the basin tap a ∼ 200–220 C hydrothermal reservoir
at <250 m in the subsurface (Giggenbach, 1978; Muñoz-Saez
et al., 2018). As in geyser basins worldwide, individual thermal
features transition between actively emitting water or steam, and
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MATERIALS AND METHODS
Field Settings
El Tatio geysers field (22◦ 20′ S and 68◦ W) is located in the
Andean highlands (i.e., Altiplano) near the Atacama Desert,
northern Chile (Figure 1). This hydrothermal area consists of
three distinct basins: Upper, Middle, and Lower Geyser Basins.
The Middle Basin is composed of pools, fountain-type geysers,
and the runoff streams from geothermal springs and pools
(Glennon and Pfaff, 2003). The El Tatio geysers field, located
along the Salado River Valley, contains more than 80 active
geysers, fumaroles, geothermal springs, and mud volcanoes and
is surrounded by extensive sinter terraces and aprons that
spread over an area of approximately 10 km2 . El Tatio is in
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FIGURE 1 | Site of study and sampling. Map of northern Chile (A), showing the location of the El Tatio geysers field (B), including the three geysers studied here
(New Betsy, NB; Old Betsy, OB; and Orange Myrtle, OM) (C). Both pictures in B,C are satellite images from Google Maps. The general appearance of the three sinter
mounds (liquid NB, steam OB, and dry OM) is shown in D–F, respectively.
a geological region composed of Jurassic marine sediments,
Jurassic-Cretaceous andesites, Cretaceous sediments, Miocene
ignimbrites and andesites, and Plio-Holocene lavas, domes,
dacitic and rhyolitic ignimbrites (Lahsen and Trujillo, 1976).
This geological sequence is overlain by glacial and alluvial
deposits, which are locally covered by silica sinter deposits
(Fernandez-Turiel et al., 2005). The extensive siliceous sinter
formations at El Tatio are the result of silica precipitation from
near-neutral thermal waters with a SiO2 concentration of 147–
285 mg/l (Nicolau et al., 2014). Sedimentary microtextures in
the sinter deposits suggested that the microbial community
at El Tatio is moderately diverse, with a variety of extreme
biological communities of thermophilic bacteria (Chloroflexuslike), cyanobacteria, and diatoms (Fernandez-Turiel et al., 2005).
collected; and the inactive mound known as Orange Myrtle (OM)
lacked both hydrothermal water and steam. For convenience,
and to emphasize the distinct differences in the hydrological
regime, we refer to the collected sinter samples as belonging
to the liquid, steam, and dry mounds (Figures 1d–f). About
100 g of sinter sample were collected from equivalent sampling
spots (i.e., half way down) from the three sinter mounds with
a geological hammer and broken samples were gathered with a
solvent-cleaned (DCM and MeOH) stainless-steel spatula. They
were wrapped in aluminum foil and transported in solvent-clean
containers for biogeochemical analysis at the CAB (Centro de
Astrobiología). A sample of hydrothermal fluid was also collected
from New Betsy for geochemical analysis. Physical splits of the
samples were distributed to collaborators at different locations in
the United States.
Sample Collection
Lithofacies and Scanning Electron
Microscopy
Samples were collected from El Tatio geysers field (Figure 1)
in October 2016, during a NASA Astrobiology Institute NAICAN 7 project (“Changing Planetary Environments and the
Fingerprints of Life”) sampling campaign. Sinter samples were
collected from three sinter mounds that appeared similar based
on their size and the shape of the mounds, though differed in
terms of their hydrological environment. As shown in Figure 1,
the active-geyser mound known as New Betsy (NB) had abundant
liquid water (∼84◦ C) that flowed from the mound, episodically
covering the mound surface; the morphologically similar mound
known as Old Betsy (OB) had a supply of steam (∼75◦ C) that
enveloped various surfaces of the mound where samples were
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The lithofacies of the sinter samples were described using
terminology consistent with the lithofacies model for silica sinters
provided by Walter (1976), based on the gross morphological
features of laminated sinters. The sinter samples were air dried
during transport and analyzed as bulk fragments to identify the
distribution of morphological features consistent with silicified
biological remnants in the context of their biofabrics. The
biofabrics were identified with conventional stereoscopic and a
scanning electron microscopic methods on fractured and sawn
surfaces oriented perpendicular to the lamination. The use of the
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Microbial Biomarkers in Altitude Geysers
(tetracosane-D50 , myristic acid-D27 , 2-hexadecanol). The total
lipid extracts were concentrated to ca. 2 ml by rotary evaporation
and elemental sulfur removed overnight with activated copper.
The clean extract was separated into two fractions of different
polarity (neutral and acidic) using Bond-elute (bond phase NH2 ,
500 mg, 40 µm particle size) chromatography columns. A neutral
lipid fraction was obtained by eluting with 15 ml DCM/2propanol (2:1, v/v) and an acidic fraction with 15 ml of acetic acid
(2%) in diethyl ether. Further separation of the neutral fraction
into non-polar and polar sub-fractions was done with 0.5 g of
alumina (activated, neutral, 0.05–0.15 mm particle size) in a precombusted Pasteur pipet. The non-polar fraction was obtained
by eluting 4.5 ml of hexane/DCM (9:1, v/v) and the polar fraction
with 3 ml of DCM/methanol (1:1, v/v). The acidic fraction was
derivatized with BF3 in methanol and the polar fraction with
N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA).
The three lipid fractions (non-polar, acid, and polar fraction)
were analyzed with gas chromatography mass spectrometry using
a 6850 GC system coupled to a 5975 VL MSD with a triple
axis detector (Agilent Technologies) operating in conditions
previously described elsewhere (Sánchez-García et al., 2018). For
the non-polar fraction, the oven temperature was programmed
from 50 to 130◦ C at 20◦ C min−1 and then to 300◦ C at 6◦ C
min−1 (held 20 min); for the acidic fraction, from 70 to 130◦ C
at 20◦ C min−1 and then to 300◦ C at 10◦ C min−1 (held 10 min);
and, for the polar fraction, the oven temperature program was
the same as that for the acidic fraction, except that the oven
was held for 15 min at 300◦ C. The injector temperature was
290◦ C, the transfer line was at 300◦ C, and the MS source at
240◦ C. Compounds identification was based on the comparison
of mass spectra with reference materials, and their quantification
on the use of external calibration curves of n-alkanes (C10 to
C40 ), fatty acids methyl esters (FAME; C8 to C24 ), n-alkanols (C10 ,
C14 , C18 , and C20 ), and branched isoprenoids (2,6,10-trimethyldocosane, crocetane, pristane, phytane, squalane, and squalene).
All chemicals and standards were supplied by Sigma Aldrich. The
recovery of the internal standards averaged 69 ± 18%.
biological holder of a Phenom Pro scanning electron microscope
(SEM) eliminated the need for carbon coating, though smaller
fractured fragments of sinter were often carbon coated to reduce
artifacts in the SEM images that were created by regions of higher
porosity.
Mineralogical and Geochemical Analyses
X-ray diffraction analysis was performed on different splits of the
sinter samples at CAB and PNNL (Pacific Northwest National
Laboratory). At CAB, the three sinter samples were analyzed
using a Bruker X-Ray diffractometer (Eco-D8 advance, XRD)
to determine the silica phase and associated mineralogy. Dry
samples of the three sinter mounds were ground, mounted on
a PMMA specimen holder and scanned between 5◦ to 60◦ 2θ,
with a scanning step size of 1 s and 0.05◦ , operated at 40 kV
and 25 mA with a Cu X-ray source (Cu Kα1,2, λ = 1.54060
Å). At PNNL, X-ray diffraction data were collected with the use
of a Panalytical MPD Bragg-Brentano goniometer fitted with
a Cu X-ray source operated at 45 kV and 40 mA, fitted with
variable divergence slits (10 mm illuminated length) and a postdiffraction monochromator. The powders were loaded into the
cavity of a zero-background holder and patterns were collected
between 5 and 100◦ 2θ with 4 s counts at 0.04◦ intervals.
Inorganic anions and organic acids of low molecular weight
were determined by ion chromatography in the water-extractable
phase of the three sinter samples (liquid, steam, and dry), or in the
water sample from New Betsy, according to previous descriptions
(Parro et al., 2011a; Sánchez-García et al., 2018). Briefly, 2 g of
the sinter samples and 1 ml of the water sample were sonicated
(3 min × 1 min cycles) and diluted in 10 mL of deionized
water; then filtered (0.22 µm GFF), and analyzed in a Metrohm
861 Advanced compact ion chromatographer (Metrohm AG,
Herisau, Switzerland), using 3.6 mM sodium carbonate (NaCO3 )
as eluent.
Stable isotopes of organic carbon (δ13 C) and total nitrogen
(δ15 N) were measured on the bulk sinter samples with isotoperatio mass spectrometry (IRMS), following USGS methods
(Révész et al., 2012). Briefly, sinter samples (2 g) were
homogenized by grinding with a corundum mortar and pestle.
Subsequently, HCl was added to the samples to remove
carbonates, equilibrated for 24 h, and adjusted to neutral pH with
ultrapure water. The residue was then dried in an oven (50◦ C)
for 72 h or until a constant weight was achieved and analyzed
in the IRMS (MAT 253, Thermo Fisher Scientific). δ13 C and
δ15 N values were reported in the standard per mil notation using
three certified standards (USGS41, IAEA-600, and USGS40) with
an analytical precision of 0.1h. The content of total organic
carbon (TOC %) and total nitrogen (TN %) was measured with an
elemental analyzer (HT Flash, Thermo Fisher Scientific), during
the stable isotope measurements.
Compound Specific Isotope Analysis
Carbon isotopic compositions of individual lipid compounds
(n-alkanes, carboxylic acids as FAMEs, and n-alkanols) were
performed coupling the gas chromatograph (Trace GC 1310
ultra) to the isotope-ratio mass spectrometry system (MAT 253
IRMS, Thermo Fisher Scientific). The conditions for the GC
analysis were identical to those used for the polar fraction
analysis, whereas the conditions for the IRMS analysis were as
follows: electron ionization 100 eV, Faraday cup collectors m/z
44, 45, and 46, and a temperature of the CuO/NiO combustion
interface of 1000◦ C. The samples were injected in splitless mode,
with inlet temperature of 250◦ C, and helium as a carrier gas
at constant flow of 1.1 ml min−1 . The isotopic values of the
individual lipids separated by GC were calculated using CO2 spikes of known isotopic composition, introduced directly into
the MS source, three times at the beginning and end of every
run. Reference mixtures (Indiana University, United States) of
known isotopic composition of n-alkanes (A6) and FAMEs (F8)
were run after every four samples to check accuracy of the
Geolipids Extraction, Fractionation, and
Analysis
About 50 g of the sinter samples were Soxhlet extracted (24 h)
with a mixture (ca. 250 ml) of dichloromethane/methanol
(DCM/MeOH, 3:1, v/v), after addition of internal standards
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isotopic ratio determined by the GC-IRMS. The δ13 C data for
individual carboxylic acids (i.e., n-carboxylic acids, iso/anteiso
and unsaturated) were calculated from the obtained FAME
values, by correcting them for the one carbon atom added in the
methanolysis (Abrajano et al., 1994).
binding with biological polymers and microbes from extant
or well-preserved extinct life structures (Rivas et al., 2008).
The LDChip200 used in this study contained 181 antibodies
(purified IgG fraction) produced using as immunogens: (i) whole
microbial cells from bacteria and archaea, (ii) spores from Grampositive bacteria, (iii) extracellular polymeric substances from
cultures and environmental samples, (iv) environmental extracts
(from soils, water, sediments, rocks, and biofilms) from extreme
environments (Parro et al., 2011a; Blanco et al., 2017), (v)
conserved proteins and peptides involved in key metabolisms
(e.g., nitrogen fixation, nitrogen and sulfur reduction, energy
metabolisms, iron storage, or PHAs (poly-hydroxyalkanoates)
synthesis), and (vi) 36 preimmune sera (IgG fraction) as negative
controls (for a detailed antibody information see Supplementary
Table S1). The targets for the 181 antibodies used in this work
are described elsewhere (Supplementary Table S1 in SánchezGarcía et al., 2018) and complemented with the antibodies
listed in Supplementary Table S1 in this work. The purified
immunoglobulin (IgG) fraction of each antibody was printed in
a triplicate spot-pattern, fluorescently labeled with Alexa 647,
checked, titrated and used as reported elsewhere (Rivas et al.,
2008).
The LDChip200 is a shotgun antibody microarray
immunosensor produced to increase the success of detecting any
microbial remain in natural samples, either for environmental
monitoring and/or for detecting signs of life in planetary
exploration (Rivas et al., 2008; Parro et al., 2008b, 2011b,
2018). Limitations related to the presence of relatively complex
molecules from abiotic origin in other planetary bodies, are
being presently achieved by adding new antibodies for detecting
molecules such as aromatic amino acids or polyaromatic
hydrocarbons (Moreno-Paz et al., 2018). In continuous process
of improvement, the LDChip is the core sensor of the already
high TRL (Technology Readiness Level) instrument called
SOLID (Signs of Life Detector), specially conceived for missions
concept as the IceBreaker drilling of the Martian permafrost
(McKay et al., 2013). The detailed protocol for the analysis of
the sinter samples at El Tatio with the LDChip200 is described
in Blanco et al. (2017). Briefly, up to 0.5 g of each sample were
resuspended in 2 mL of TBSTRR buffer (0.4 M Tris–HCl pH 8,
0.3 M NaCl, 0.1% Tween 20), ultrasonicated and filtered through
5 µm. The filtrates were used as a multianalyte-containing
sample for the FSMI as described in preceding works (Rivas et al.,
2008; Blanco et al., 2012, 2017). The LDChip200 microarray
images were analyzed and quantified by GenePix Pro Software
(Molecular Devices, Sunnyvale, CA, United States). The final
fluorescence intensity (F) of each antibody spot was calculated
as reported by Rivas et al. (2011). To minimize the probability
of false positives, we increased the stringency by applying to all
spots an additional cutoff value of 2.5-fold the average of F of the
whole array (Blanco et al., 2015; see details on Supplementary
Text S1).
In addition, the molecular abundance, richness, diversity, and
evenness were also estimated. As all the immunoassays were
performed on the same amount of sample and upon similar
experimental and scanning conditions, the sum of fluorescence
intensity in every positive antigen-antibody reaction for each
DNA Extraction, PCR Amplification, and
DNA Sequencing
Genomic DNA was extracted from the three sinter samples,
using the CTAB genomic DNA extraction method (WarrenRhodes et al., 2018). Bacterial 16S rDNA V3-V4 gene region
from all DNA extracts was then PCR amplified, using the
primer pairs 341-F/805-R (Herlemann et al., 2011). Archaeal
16S rDNA V3-V4 region was PCR amplified using the primer
pair Arch1F/Arch1R (Cruaud et al., 2014) only from the dry
extract, due to the little amount of archaeal biomass in the
liquid and steam samples. Both bacterial and archaeal PCR
amplifications were carried out according to standard procedures
(Warren-Rhodes et al., 2018). Microbial communities were then
identified by the construction of a paired-end amplicon library by
means of Illumina MiSeq sequencing (Illumina Inc., San Diego,
CA, United States). Raw sequence data were deposited at the
NCBI Sequence Read Archive (SRA1 ), under accession number
PRJNA507699.
Raw sequences were processed either in the MOTHUR
software v.1.39.5 (Schloss et al., 2009), using a custom script
based upon MiSeq SOP (Kozich et al., 2013), or in R package
‘phyloseq’ (McMurdie and Holmes, 2013). Sequence reads were
clustered into OTUs (Operational Taxonomic Units) at the 97%
similarity level. Datasets were rarefied independently by random
selection to even sequencing depth, corresponding to the lesser
number of sequences found in the samples (i.e., 40264 reads).
Taxonomic affinities for the reads were assigned by comparison
of OTUs representative sequences against RDP database (RDP
reference files v.16; release 11, Cole et al., 2014). OTU’s affinities
reported as “cyanobacteria/chloroplast” were further assigned
to a taxonomic identity by comparing them against nr/nt
(NBCI), EMBL, Greengenes and SILVA databases for more
precise cyanobacteria taxonomic identification. The sequences
assigned to “mitochondria” or “chloroplast” were removed from
further analyses. The total number of OTUs was provided as an
estimate of phylogenetic richness. Shannon’s diversity index (H’)
and Pielou’s Evenness (J’) based on OTU data were calculated
on the three samples by means of R package ‘vegan’ (Oksanen
et al., 2017). The same package was also used to perform a
Correspondence Analysis (CA) between microbial classes and
sinter samples.
Multiplex Fluorescent Sandwich
Microarray Immunoassay (FSMI)
Powdered sinter samples were analyzed by fluorescent sandwich
microarray immunoassays (FSMI) with the LDChip200 (i.e.,
Life Detector Chip; Parro et al., 2008a, 2011a), to interrogate
a panel of about 200 polyclonal antibodies produced for
1
http://www.ncbi.nlm.nih.gov/sra
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Microbial Biomarkers in Altitude Geysers
that were surrounded by the remnants of silicified biofilms
and detrital grains. The downward parabolic laminations of the
spicules were observed in this orientation as concentric laminae,
once again distinguishable as variations in color (white, tan,
and gray). A SEM image of the abrupt interface between the
highly porous silicified biofilm and massive vitreous opal-A
(Supplementary Figure S2B) suggested that once environmental
conditions supported biofilm growth on the periphery of a
spicule, that growth continued unabated until the structure
was intermittently immersed in hydrothermal fluid, allowing
spicular growth to resume. The pore space between closely
spaced spicules in the steam sinter was completely filled with a
combination of silicified biofilm and detrital grains of opal-A and
accessory minerals. A SEM image of a spicule separated from the
sample (Supplementary Figure S2C) showed relicts of silicified
biofilm attached to the spicule surface and, occasionally, cellular
remnants of bacteria that either colonized the spicular surfaces
(Supplementary Figure S2D) or infiltrated the pore space of
previously silicified remnants (Supplementary Figure S2E).
Supplementary Figure S3A showed a cross-sectional
stereoscopic view of wavy laminations of opal-A in the sinter
sample from the dry mound. A stereoscopic plan view of the dry
mound sample (Supplementary Figure S3B) showed evidence
of an irregular network of ridges. The wavy lamination visible
in cross-section (Supplementary Figure S3A) is due to the
ridge-like nature of the accretionary surface. An SEM image of
this surface showed the well-preserved silicified remnants of a
biofilm that consisted primarily of short filaments, rods, and the
fibrils typical of dehydrated EPS (Supplementary Figure S3C).
Heavily entombed filaments (Supplementary Figure S3D) and
organically preserved remnants of a biofilm that consisted of
cocci (Supplementary Figure S3E) were also found on the
surfaces and in fractures of the dry sinter sample.
sample was used as an estimation of the molecular abundance
(Parro et al., 2011c), whereas the number of positive antigenantibody reactions was employed as an indirect measure of the
sample richness. The molecular diversity was then determined
through the Shannon Index (H’), according to the following
equation:
s
X
′
H =−
pi log2 pi
(1)
i=1
where H’ defines the molecular diversity in the samples analyzed
by the LDChip200; s corresponds to the molecular richness
(i.e., sample richness above) and pi is the partial diversity
calculated as a rate between the fluorescence for each positive
immuno-detection and the total fluorescent for each sample
under analysis. Finally, the molecular evenness at each sample
was then determined through the Pielou’s evenness index (J’),
according to the following equation:
.
′
′
J = H lnS
(2)
where J’ defines Pielou’s evenness index; H’ corresponds to the
molecular diversity in the samples analyzed by the LDChip200;
and S matches to the molecular richness.
RESULTS
Silica Sinter Lithofacies in the Three
Sinter Mounds at El Tatio
Stereoscope and SEM images of the liquid mound and steam
mound sinter samples in different orientations illustrated
they consisted primarily of spicules characterized by parabolic
laminations of vitreous clear or opaque opal-A. Supplementary
Figure S1A shows a cross-sectional stereoscopic view of closely
spaced spicules in the liquid mound sample. The inner core
of the spicules comprised dense vitreous opal-A, which did
not always appear laminated on fractured surfaces of the
structures. The outermost opaque and tan laminations of the
spicules, which contrasted with the clear inner core of the
structures, revealed their downward parabolic orientation that
was visible due to differences in their color. The more porous
and bright white precipitate located between the spicules was
the remnants of silicified microbial biofilms. SEM images of the
top surface of the liquid mound sample revealed the presence
of heavily silicified and intertwined filaments (Supplementary
Figure S1B) and less-silicified remnants of short filaments and
rods on the most recent accretionary surface of the sample
(Supplementary Figure S1C). SEM images from the inside of this
sample illustrated entombment of microbial remnants in opalA nanocolloids (Supplementary Figure S1D) and the remnants
of exopolysaccharides (EPS) that are often preserved with a
honeycomb-like structure on surfaces where microorganisms
attached reversibly (leaving only EPS remnants) and irreversibly
(leaving intertwined cellular remnants and EPS relicts) on
exposed sinter surfaces (Supplementary Figure S1E).
Supplementary Figure S2A showed a top-down stereoscopic
view of closely spaced spicules in the steam mound sample
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Mineralogy and Bulk Geochemistry of
the Three El Tatio Sinter Mounds
The X-ray diffraction patterns produced by fragments of spicules
and columns that were separated from the liquid and steam
mound samples and from a non-porous, white region from
the top of the dry sinter (Supplementary Figure S4) revealed
that they consisted almost entirely of opal-A (Rodgers et al.,
2004). As an aqueous precipitate, the mineraloid opal-A does not
have a three-dimensional crystalline structure, yet the ubiquitous
presence of randomly ordered silica tetrahedra produces a
diffraction pattern that consists of a single broad feature centered
on 23◦ 2θ (ca. 3.9 Å), typical of X-ray amorphous materials. The
matrix of the liquid and steam mounds consisted of fine-grained
detrital material primarily formed from opal-A (Supplementary
Figure S4) and the accessory phases quartz, halite, feldspar, clay
minerals, and iron oxides (data from analysis at CAB, not shown).
Distinct detrital layers with a similar accessory mineral inventory
were also found in the dry mound sinter sample.
Ion chromatography (IC) showed the presence of diverse
inorganic anions (Figure 2A), in the three sinter samples with
variable content depending on the hydrothermal system (i.e.,
liquid, steam, or dry). Chloride was present in the three sinter
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FIGURE 2 | Geochemical analysis. Concentration (µg g−1 ) of inorganic (A) and organic anions (B) in the three sinter mounds at El Tatio (liquid, steam, and dry).
extracts, with similarly high concentrations in the liquid (612 µg
g−1 ) and steam (596 µg g−1 ) mound samples, and an even higher
concentration in the dry one (831 µg g−1 ). Nitrate was present in
the three systems, with the steam sample containing the largest
concentration of all mounds and ions (1497 µg g−1 ) and the
dry sample the lowest (20 µg g−1 ). In fact, nitrate and chloride
concentrations were inversely related among the three mounds.
Sulfate was measured in low concentrations (<45 µg g−1 ) in
all samples, as well as fluoride (<20 µg g−1 ) (Supplementary
Table S2). The water collected from the liquid mound contained
higher concentrations of chloride (7068 µg g−1 ), sulfate (346 µg
g−1 ), fluoride (12 µg g−1 ), and bromide (56 µg g−1 ) compared
to its sinter deposits (Supplementary Table S2).
In addition to the inorganic anions, three light organic acids
were detected by IC (Figure 2B). Acetate and formate were
present at similarly low concentrations (<2.5 µg g−1 ) in the
extracts from the steam and dry sinter samples, but not detected
in that of the liquid mound sample. Conversely, extracted tartrate
was measured at higher concentration in the steam (7.2 µg g−1 )
than the liquid (1.1 µg g−1 ) samples, but was not detected in
the dry sample. The water sample from the liquid mound was
measured to contain only tartrate, which was present in very high
concentration (286 µg ml−1 ) (Supplementary Table S2).
The content of TOC (0.07–0.10%) and TN (0.01–0.04%)
varied little between the three sinter samples (Table 1), with
the dry mound showing the lowest values of both elements.
The resulting TOC over TN ratios (C/N) varied from 3 to 7.
The biomass isotopic ratios δ13 C and δ15 N ranged from −15.7
to −24.0h and from −0.9 to 5.4h, respectively (Table 1).
The steam sinter sample showed the most depleted (δ13 C) and
enriched (δ15 N) isotopic composition.
TABLE 1 | Bulk geochemical composition of the sinter samples from the three
mounds at El Tatio.
Liquid
Dry
TOC (% dw)
0.10
0.10
0.07
TN (% dw)
0.02
0.04
0.01
δ13 C OC (h)
−15.9
−24.0
−15.7
δ15 N TN (h)
−0.9
5.4
−1.1
5
3
7
n-alkanes
0.05
0.08
0.05
Branched alkanesa
0.11
0.14
0.04
Octadecene (C18:1 )
n.d.
0.04
n.d.
Hentriacontatriene (C31:3 )
n.d.
0.01
n.d.
n-carboxylic acids
2.53
2.71
2.35
Unsaturated carboxylic acidsb
0.39
0.34
0.23
Dicarboxylic acidsc
0.06
0.05
0.03
Iso-/anteiso carboxylic acidsd
0.71
0.38
0.24
Other branched carboxylic acidse
0.11
0.02
0.01
Cyclopropyl acidsf
n.d.
n.d.
0.03
n-alkanols
1.40
1.12
1.81
Stigmastanol
0.12
0.13
0.06
β-sitosterol
0.01
0.04
n.d.
Cholesterol
n.d.
0.01
0.17
Pristane
0.05
0.12
n.d.
Phytane
0.21
0.35
n.d.
Squalane
n.d.
n.d.
0.003
n.d.
n.d.
C/N
Crocetane
g−1 )
0.002
a Sum
Concentration (µg
and compositional distribution of lipid biomarkers.
of
mono-methyl, di-methyl and tri-methyl alkanes. b Sum of mono- and di-unsaturated
carboxylic acids between 16 and 19 carbon units. c Sum of dicarboxylic acids
between 6 and 10 carbon units. d Sum of iso and anteiso carboxylic acids between
15 and 19 carbon units. e Sum of other mono-methyl (MM) carboxylic acids. f Sum
of cyclopropyl C17 and C19 acids. Please go to Supplementary Figures S5–S8 for
consulting individual concentrations of the (normal, branched, and other) carboxylic
acids, alkanols, and alkanes families.
Molecular Distribution of Lipid
Biomarkers in the Three Sinter Mounds
Several lipid families were detected in the three sinter samples,
with a generalized larger abundance of the functionalized lipid
groups. The most abundant class of lipids were the linear
carboxylic acids (i.e., n-carboxylic acids), which concentration
ranged from 2.35 to 2.71 µg g−1 (Table 1). The molecular
distribution of the n-carboxylic acids showed chain lengths
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Steam
ranging from C8 to C30 , a clear dominance of the even carbons,
maximum at C16 or C18 , and secondary groups at C22 to
C26 (Supplementary Figure S5). After the straight-chained
congeners, the most abundant carboxylic acids were those with
branches at iso- and/or anteiso- positions (0.24–0.71 µg g−1 ),
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were present in the liquid and steam mounds, whereas the dry
sinter contained only small traces of squalane and crocetane
(Table 1). Low concentrations of octadecene (C18:1 ) and
hentriacontatriene (C31:3 ) were also found in the steam sinter
sample (Table 1).
followed by the unsaturated (0.23–0.39 µg g−1 ), dicarboxylic
(0.03–0.06 µg g−1 ), and cyclopropyl (0.03 µg g−1 ) congeners
(Figure 3). Iso and anteiso carboxylic acids of 13 to 19 carbon
units were found in the three sinter samples (Supplementary
Figure S6), with more of the iso/anteiso-C18 in the liquid and
dry mounds. Carboxylic acids with one or two unsaturations
were generally measured at chain lengths of C16 (16:1 ω7, and
16:1 ω9), C17 (17:1 ω6), C18 (18:1 ω9, and 18:2 ω6,9), and C19
(19:1 ω9) (Supplementary Figures S6D–F). Dicarboxylic acids
of short chain (C6 to C10 ) and branched carboxylic acids other
than iso/anteiso congeners (i.e., monomethyl acids, MM) were
found to be minority congeners in the acidic fractions (Figure 3).
Cyclopropyl C17 and C19 acids were only detected in the dry
sinter sample at a total concentration of 0.03 µg g−1 (Table 1).
Other functionalized lipids, the straight chain alkanols (i.e.,
n-alkanols), were found at chain lengths between C10 and C29 ,
and concentrations ranging from 1.12 to 1.81 µg g−1 (Table 1).
Similarly to the n-carboxylic acids, the molecular distribution
of the n-alkanols showed a markedly even character dominated
by the C16 and C18 congeners (Supplementary Figure S7).
Together with the linear alkanols, two phytosteroids (β-sitosterol
and stigmastanol), and cholesterol were identified within the
polar fraction (Table 1). The largest amount of cholesterol was
measured in the sinter sample from the inactive, dry mound.
In contrast to the functionalized lipids, saturated
hydrocarbons were less abundant in the three sinter samples
(Table 1). Branched alkanes including mono-, di-, and trimethyl ramifications were detected at one order of magnitude
larger concentration (0.11–0.14 µg g−1 ) than the n-alkanes
(0.05–0.08 µg g−1 ) in the liquid and steam sinter samples. In
the dry mound, the concentration of branched and n-alkanes
was similar. The n-alkanes distribution ranged in the three
sinter samples from C10 to C33 , and showed different maximum
peaks at C17 (liquid mound), C15 (steam mound), or C25 (dry
mound) (Supplementary Figures S8A–C). Among the branched
alkanes, the mono-methyl congeners (C15 , C17 , and C18 )
were the most abundant in all sinter samples (Supplementary
Figures S8D–F), especially in the steam mound. Other branched
alkanes of isoprenoid configuration were detected at variable
concentrations among the sinter samples. Pristane and phytane
Isotopic Distribution (δ13 C) of Lipid
Biomarkers in the Three Sinter Mounds
The compound-specific carbon isotopic ratio of alkanes,
carboxylic acids and alkanols showed certain trends between
the samples (Figure 4). The n-alkanes δ13 C values ranged
from −22.5 to −31.3h in the three sinter samples, with a
general depletion with increasing number of carbon units,
and from the liquid to the dry mound (Figure 4A). In the
liquid sinter sample, a clear enrichment was observed for the
C17 (−22.9h) and C19 (−22.5h) relative to the remaining
n-alkanes. The δ13 C composition of the n-carboxylic acids ranged
from −20.5 to −36.1h (Figure 4B), with an even clearer
depletion with increasing carbon units and a marked enrichment
of the odd relative to the even carbons. The compoundspecific isotopic ratio of other acids could only be achieved
for a few iso/anteiso, unsaturated, and cyclopropyl congeners
(Supplementary Table S3). The iso/anteiso and unsaturated
acids contained generally homogeneous δ13 C ratios, whereas the
cyclopropyl C17 and C19 acids only found in the dry mound
were relatively enriched (Figure 4D). Only three members of the
n-alkanols family (C14 , C16 , and C18 ) were detectable for their 13 C
content (Supplementary Table S3). The three samples showed
similar δ13 C values for the C14 (−27.4 to −27.6h) and C16
(−27.7 to −28.4h) n-alkanols, whereas a distinct composition
was observed for the C18 congener in the liquid (−24.8h), steam
(−27.7h), and dry (−30.2h) samples (Figure 4C). Overall, all
lipid groups showed a depleting trend in the 13 C composition
from the liquid to the dry mound.
Microbial Diversity Based on DNA
Analysis
Phylogenetic analysis of environmental 16S rRNA gene amplicon
sequences showed that almost 70% of total bacterial reads
FIGURE 3 | Relative amount (%) of carboxylic acids (linear and saturated, or normal; unsaturated; dicarboxylic; iso/anteiso; other branched; and cyclopropyl) in the
three sinter mounds at El Tatio. “Unsaturated” includes mono- and di-unsaturated carboxylic acids, whereas “other branched” covers middle chain mono-methyl
carboxylic acids.
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FIGURE 4 | Compound-specific isotopic composition (δ13 C) of specific lipid families in the three sinter mounds (liquid, steam, and dry); n-alkanes (A), n-carboxylic
acids (B), n-alkanols (C), and iso/anteiso-, unsaturated, and cyclopropyl (Cyc 17:0 and Cyc 19:0) acids (D). The bulk isotopic ratio of the total biomass (i.e., relative
to TOC) was also depicted for the three samples as full circles. A dashed line was provided in each panel to facilitate the visualization of the δ13 C shifts observed
(mostly) in the n-carboxylic acids.
FIGURE 5 | Comparison of microbial groups in the sinter samples from the three mounds at El Tatio (A), and Correspondence Analysis (CA) between the main
microbial groups (red) and the sampling sites (black) (B). In a, the microbial groups inferred from DNA analysis are expressed as relative abundance. Bacillales and
Clostridiales (Firmicutes), Rhodobacteriales, Rhizobiales, Burkholderiales and Desulfuromonadales (Proteobacteria), as well as Actinomycetales (Actinobacteria) were
the most represented orders within the most abundant bacterial phyla. In b, CA1 (X axis) accounted for 70.90% of total variability explained by the model, and CA2
(Y axis) for 29.02%. The closer clustering of microbial groups to a sampling site is, the more characteristic these groups are at the site. Distance among sampling
sites depicts compositional differences in them.
Clostridiales (Clostridia), together accounting for more than
80% of the Firmicutes’ sequences, were the most frequently
represented orders. Proteobacterial classes were generally more
abundant in the liquid mound, especially Alphaproteobacteria
(17.6% in liquid mound versus 9.2 and 2.5% in steam and
in the El Tatio sinter geysers belonged to the Firmicutes and
Proteobacteria phyla (Figure 5A). The low GC Gram-positive
Firmicutes were mainly found in the liquid and steam sinter
samples, which showed a closer correspondence than the dry
sample to that phylum (Figure 5B). Bacillales (Bacilli) and
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metal (mainly iron) reducers occupied anaerobic microregions
with organic matter available for oxidizing in parallel to the metal
reduction.
A heat map showing the fluorescence intensity of the positive
immuno-detections (Figure 7) displayed relative differences in
the composition of the microbial community in the three sinter
mounds. Chemolithoautotrophs involved in the oxidation of
sulfur (Acidithiobacillus spp.), and oxidation (Leptospirillum sp.
and Acidimicrobium sp.) or reduction of iron (Acidocella sp.,
Acidiphilium sp., and Shewanella spp.) were more abundant
in the liquid and steam samples. These sinter samples (mostly
the steam one) were also richer than the dry sample in
primary producers such as benthic (Anabaena sp., Leptolyngbya
sp., and Tolypothrix sp.) and endolithic (Chroococcidiopsis sp.)
cyanobacteria, consistent with the higher detection of nitrogen
fixation proteins (i.e., Nif and chaperon HscA; Figure 7). Sulfate
reducers were also detected in higher intensity in the steam and
(mostly) liquid samples, through antibodies to Desulfovibrio sp.
and Desulfosporosinus sp., as well as to the DsrA protein. In
addition, positive immuno-detections of archaeal strains such
as Methanobacterium sp., Pyrococcus sp. or Halorubrum sp.,
accounting for methanogenic and heterotrophic metabolisms,
were only observed in the liquid and steam samples. In contrast,
the dry sinter recorded the highest immuno-signals against (i)
bacteria capable of perchlorate reduction (Magnetospirillum sp.,
Ideonella sp. and Dechlorobacter sp.), (ii) an aquifical bacterium
(Hydrogenobacter thermophilus), and (iii) proteins related to the
synthesis of PHAs (Figure 7).
The sum of fluorescence intensity decreased from the liquid
to the dry mound by a factor of ca. 5 (Figure 6A), indicating
that the dry sample contained lower biomass or that target
microbial markers were transformed or degraded with time.
The declining number of positive immuno-signals from the
liquid (n = 39) to the dry (n = 13) sample resulted in parallel
decreasing trends of the molecular richness (Figure 6B) and
diversity (Figure 6C) toward the less active mound. In contrast,
the Pielou’s evenness showed a somewhat increasing trend with
the loss of hydrothermal activity (Figure 6D).
dry mounds, respectively). In contrast, Gammaproteobacteria
were more represented in the dry (22% of total sequences)
than in the liquid (9.9%) and steam (3.9%) mounds, as the
correspondence analysis illustrated (Figure 5B). The orders
Rhodobacteriales and Rhizobiales (Alphaproteobacteria),
Burkholderiales and Hydrogenophilales (Betaproteobacteria),
Desulfuromonadales (Deltaproteobacteria), as well as Vibrionales
and Xanthomonadales (Gammaproteobacteria) together
accounted for 70% of the total proteobacterial sequences.
The Actinobacteria (mainly Actinomycetales), Bacteroidetes
(Cytophagales and Flavobacteriales), and Cyanobacteria together
accounted for the 22% of the total number of sequences found in
the sinter samples (Figure 5A). Actinobacteria and Bacteroidetes
were particularly abundant in the dry mound (23.7 and 13.9%
of sample sequences, respectively), whereas Cyanobacteria was
more prevalent (5.9%) in the steam mound sample (associations
illustrated in Figure 5B). In contrast, Acidobacteria, Chloroflexi,
and Planctomycetes each accounted for >1% of the sequences
only in the liquid sinter (Figure 5B). The Deinococcus-Thermus
class is of special interest as it was observed to be similarly
representative of both the liquid (1.7% of total sequences) and
dry (2.9%) mounds (Figure 5B), with sequences only belonging
to Thermales and Deinococcales orders, respectively.
Archaeal sequences were recovered only in the dry sample
(Figure 5A). They accounted for <2% of overall diversity in these
samples and comprised taxa from just two classes: Halobacteria
(Euryarchaeota) and Thermoprotei (Crenarchaeota) (Figure 5B).
The OTU-richness varied from 78 in the steam sample to 142
in the dry sample (Figure 6B). The Shannon diversity index (H’)
of the microbial communities at OTU level showed increasing
values from the liquid (1.7) to the steam (2.3) and dry (3.3)
samples (Figure 6C), concurrently with the Pielou’s Evenness
index (J’ of 0.38, 0.52, and 0.67, respectively) based on the OTU
data (Figure 6D).
Microbial Mass and Biomarkers
Detected by a Multiplex Immunoassay
The LDChip200 revealed the presence in the sinter samples of
biomolecules recognized by antibodies produced against iron
and sulfur oxidizing bacteria and crude environmental extracts
from biofilms of the Río Tinto area (Supplementary Figure S9)
such as Leptospirillum ferrooxidans, Acidithiobacillus spp. and
others (Amils et al., 2002). The immunoassays also detected
positive immuno-signals against metal, sulfate and perchlorate
reducers, bacteroidetes, actinobacteria including spore-forming
bacteria, cyanobacteria, the thermo-aquifical sulfur oxidizer
Hydrogenobacter thermophilus as well as methanogenic,
thermophilic and halophilic archaea. In addition, positive
immuno-detections were revealed with antibodies to proteins
related to nitrogen fixation, energy metabolisms, thermal and
hydric stress, poly-hydroxyalkanoates (PHAs) synthesis and
sulfate and nitrate reduction (Supplementary Figure S9).
Detection of chemolithoautotrophs involved in the use of sulfur
and iron (Figure 7 and Supplementary Figure S9) revealed the
occurrence of sulfur-iron metabolism, where sulfur and iron
oxidizers could proliferate in oxygen-rich microniches, while
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DISCUSSION
Mineralogical, Geochemical, and
Morphological Features in the Three
Sinter Mounds at El Tatio
The mineralogical composition of the sinter samples was similar
in the three geyser mounds, with amorphous silica phases (i.e.,
opal-A) dominating the three XRD spectra (Supplementary
Figure S4). The shift of the strongest feature (3.9 Å) to slightly
higher d-spacing (3.93 Å) for the dry sinter relative to the
liquid and steam sinters suggested a more dense packing of
SiO4 tetrahedra in the dry sinter, consistent with dehydration.
In the liquid and steam mounds, the sinter deposit was
composed of spicular or columnar geyserite, which typically
forms under conditions of intermittent inundation and splashing
at temperatures >80◦ C (Walter, 1976; Cady and Farmer, 1996;
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FIGURE 6 | Estimation of the molecular abundance, richness, diversity, and evenness in the three sinter samples at El Tatio, based on the DNA sequencing (blue)
and LDChip200 (orange) results. Molecular abundance (A) was only considered for the LDChip technique, as the sum of the fluorescence intensity in all positive
immuno-detections in the LDChip200. Richness (B) was based on the total number of OTUs (DNA sequencing) and on the number of positive immuno-detections
(LDChip200). The diversity (C) was based on the Shannon index in both cases. Community evenness (D) was based on Pielou’s evenness index. Please note that
the opposite trend of richness and diversity in the LDChip200 and OTUs estimates were attributed to the different approach employed by both techniques (i.e., close
versus open methods, respectively).
mounds displayed gross morphological and biofabric evidence of
having formed in an intermittently wetted environment. Under
such conditions, fluid is typically drawn by capillary action to
topographical highs on the sinter due to rapid evaporation at
the apices of spicular protrusions (Cady and Farmer, 1996).
Walter (1976) was the first to describe spicular and columnar
geyserites formed along the rims and edges of hot springs in
Yellowstone National Park (United States) and he attributed their
morphology to episodic splashing at the nearby pool. At El Tatio,
spicular geyserite structures occurred on the tops and sides of the
liquid and steam mounds in distinct regions that appeared to be
associated with either (i) intermittent fluid flow from the top of
the erupting liquid geyser mound or (ii) from a region within the
base of the steam mound that was engulfed in steam from hot
fluid inside the sinter cone at the base of the structure. Regardless
of the distribution of these structures on the top, side, or base of
the mounds, the liquid and steam spicules grew outward from the
mounds with their apices pointing away from and perpendicular
to their accretionary surfaces. In contrast, the wavy laminated
biofabric of the dry mound sinter lacked well-developed spicules.
We interpret the absence of the spicules on this part of the dry
mound as an indication that, when the geyser was more active,
fluid flow was likely more persistent and flowed over the sinter
for longer periods of time during its formation.
Braunstein and Lowe, 2001). Evaporative salts and accessory
minerals contribute to the tan and gray color of the matrix. The
accessory mineral inventory is most likely wind-blown material
from the volcanic country rock that surrounds the hydrothermal
basin (Nicolau et al., 2014). Differences in the amounts of such
detrital phases incorporated during accretion, often give these
layers the appearance of being laminated. Yet these interlayers
have an important role in defining the environmental conditions
that define the habitat in each mound.
A geochemical difference among the mounds complements
the mineralogical observations. The driest mound would be
expected to have the most evaporative salt and, indeed, a higher
concentration of chloride was found in the dry mound than in
the other two samples (Figure 2A). The progressive wettingdrying cycles that would occur during waning geyser activity
would likely concentrate salts evaporatively from the chloriderich hydrothermal fluid. The biofabric of the dry sinter sample
was consistent with this interpretation of prolonged periods of
dryness at this inactive mound. During periods when the geyser
mound was active, the relatively flat-to-wavy laminated sinter
fabric would have developed when accretionary surfaces were
perpetually wet and immersed in liquid for long periods of time.
Continuous inundation would preclude spicule formation, which
occurs on splashy, intermittently dry surfaces. A comparison of
the random network of ridges on the outer surface of the dry
sinter (Supplementary Figure S3) developed during the waning
stages of geyser activity. That is, spicules develop in evaporative
settings, ridges develop when there is intermittent wetting, and
flat and wavy laminations develop when flow is continuous. These
three environments are visible in the organic compositions and
microbial populations as well.
The morphological characteristics of the biofabrics of the
three sinter samples confirmed that the liquid and steam
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Microbial Transition Through Different
Stages of Hydrothermal Activity at
El Tatio
The different geochemical, mineralogical, and morphological
features of the three sinter mounds were accompanied by
distinct molecular (lipids and immuno-detections) and genomic
biopatterns, which allowed us to describe a biogeochemical
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FIGURE 7 | Heat map showing the results obtained with LDChip200 immunoassays. The antibodies (see Supplementary Table S3) were reorganized on the basis
of main phylogenetic groups and metabolic traits of the target immunogens and protein functions in 19 different categories. The averaged fluorescence intensity of
those positive immuno-detections within the same category (Supplementary Figure S9), obtained from three replicates per sample (that means nine spots per
antibody), is plotted in a color scale from white (negative results) to red (maximum of 17452). Please note that 0 stands for those values under the limit of detection.
transition (prokaryotic-signatures based) along the hydrothermal
activity gradient (Figure 8). Despite the ubiquitous dominance
of Firmicutes and Proteobacteria in the three systems, the
relative abundance of certain phylogenetic groups and specific
biomarkers made a difference in the microbial composition
at the three stages of hydrothermal activity. For instance,
in the hydrothermally active liquid mound, the microbial
community was observed to show a strong correspondence with
phylogenetic groups such as Chloroflexi or Deltaproteobacteria.
The detection of chlorophyll-derived lipids, such as pristane and
phytane (Table 1), supported the contribution of photosynthetic
microorganisms such as Chloroflexi to the community. Pristane
and phytane are isoprenoid compounds mainly originating
from phytol (Brocks and Summons, 2003), the esterifying
alcohol of phototrophic chlorophylls (Didyk et al., 1978),
although additional sources such as biphytane, archaeols or
even tocopherol in the case of phytane, have been also
described (Brocks and Summons, 2003). As for Proteobacteria,
the detection by the LDChip200 of certain members such
as Desulfuromonadales and Desulfovibrio sp., coincided with
other against the Firmicutes Desulfosporosinus sp., suggesting
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the occurrence of sulfate reduction in the liquid, as well
as steam, mound. In the liquid sample in particular, that
detection agreed with the greater signal from sulfate reduction
protein DsrA (Figure 7) and a larger detection of iso/anteisocarboxylic groups (Figure 3) also associated with sulfate
reducing bacteria (SRB), particularly the C15 and C17 pairs
(Langworthy et al., 1983). In the absence of measurement of
other sulfur species in the samples, the removal of sulfate
by sulfate reducers (and/or less sulfate in the hydrothermal
fluid at different times) would explain the lower concentration
of this anion in the liquid sample (Figure 2A), accompanied
by metabolic processes that utilized the only organic anion
available to donate electrons in this geothermal system (i.e.,
tartrate; Figure 2B). Low molecular-weight organic acids such
as tartrate are excellent energy sources for anaerobic microbial
metabolisms (e.g., Menes and Muxí, 2002; Maune and Tanner,
2012) such as sulfate reduction and methanogenesis (Parro et al.,
2018).
Another distinctive molecular feature of the liquid mound was
the larger concentration of dicarboxylic acids relative to the other
two systems (Table 1), which may be attributed to the presence
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Microbial Biomarkers in Altitude Geysers
FIGURE 8 | Schematic of the biogeochemical reconstruction of the three geothermal systems (liquid, steam, and dry) at El Tatio as a function of the hydrothermal
activity based on bulk elemental and isotopic geochemistry, characteristic lipid biomarkers (source diagnosis in brackets), compound-specific isotopic analysis
(metabolism), DNA sequencing (phylogeny), and LDChip immunoassays (phylogeny, metabolism, and biogeochemical traits). SRB means Sulfur Reducing Bacteria,
GnSB Green non-Sulfur Bacteria, and PSB Purple Sulfur Bacteria. Red, acetyl-CoA stands for the reductive acetyl-CoA pathway for autotrophic CO2 fixation.
In the steam mound, the strongest correspondence of
the microbial community was observed with Cyanobacteria
(Figure 5B). The relative enrichment of these microorganisms
in this mound was consistently suggested by the relatively larger
concentration of different cyanobacteria lipidic markers, such
as mid-chain-length mono- and di-methyl alkanes (Dembitsky
et al., 2001), octadecene (Shiea et al., 1991; Campbell et al., 2015),
or unsaturated carboxylic acids (i.e., 16:1 ω7, 18:1 ω9, or 18:2 ω6;
Cohen et al., 1995; Allen et al., 2010; Pagès et al., 2015; Table 1
and Supplementary Figures S6, S8). In agreement, the LDChip
detected in this sample a relatively higher signal of biomaterial
immunologically associated with Anabaena sp., Leptolyngbya
sp., Tolypothrix sp., and Chroococcidiopsis sp. (Figure 7 and
Supplementary Figure S9). In the steam system, Cyanobacteria
seem to play an important role in the metabolism that supports
the microbial community and possibly in the structures that
formed under intermittent drier conditions.
Metabolically speaking, the steam sinter mound appeared
largely influenced by the nitrogen cycle. The relatively larger
immuno-signals of nitrogen fixation proteins (Nif and related
Chaperons such as the HscA protein; Figure 7), together with
the highest concentration of nitrate in the steam mound extract
(Figure 2A), indicated that organisms capable of N2 fixation
and subsequent initiation of nitrification, such as Cyanobacteria
or the alphaproteobacterial order Rhizobiales, were active in the
hydrothermally intermittent system. Additionally, the presence
of the nitrate reductase protein (NRA in Figure 7) suggested that
nitrate-reducing microorganisms could be making the most of
the nitrate as an energetic source to thrive in the steam mound.
Oxidation of organic matter (Chin and Janssen, 2002) or other
of thermophiles or hyperthermophiles, typically containing
additional external protective membranes. Comparative
distributions of dicarboxylic acids of slightly longer (C16 to C22 )
or much longer (C30 to C32 ) chains were described by Carballeira
et al. (1997) in cultures of hyperthermophiles such as Pyrococcus
furiosus and Thermotoga maritima, respectively. In the present
study, the somewhat decreasing trend in concentration of the
dicarboxylic acids from the liquid to the dry mound sample
(Figure 3) was consistent with the selective disappearance of
thermophiles from the hottest (i.e., intermittent water at ∼80◦ C)
to the coolest (i.e., dry) setting.
Microbial metabolism in the liquid mound sample was
dominated by the Calvin cycle, along with lesser sulfur and iron
chemolithotrophic pathways. Autotrophic metabolism (Hayes,
2001) was supported by a large fractionation (i.e., 11 to 26h;
Quandt et al., 1977; Preuβ et al., 1989) of δ13 C values relative
to atmospheric CO2 (ca. −8h; Graven et al., 2017). Indeed,
relatively light δ13 C values (−21 to −36h) were measured
in the liquid sample for the carboxylic acids, n-alkanes, and
n-alkanols (Figure 4). Complementarily, chemolithoautotrophs
using sulfur-iron metabolism (Figure 7) were detected in
different microenvironments, where heterotrophic metal
(mainly iron) reducers would occupy anaerobic micro-regions,
while sulfur and iron oxidizers would proliferate either in
anaerobic or oxygen-rich microniches. In this case, the accessory
minerals and dissolved ions would provide the metal and
sulfur needed to support this community. Biofilms and EPS
contributing to layering in the spicules observed in the sample
(Supplementary Figure S1) may have aided to the isolation of
these microenvironments.
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desiccation and exposure to higher radiation in the inactive sinter
geyser.
The closest correspondence of the dry sample was with
Euryarchaeota and Crenarchaeota (Figure 5B). The contribution
from archaeal sources solely in the dry mound was consistent
with the detection of squalane and crocetane only in that sample
(Table 1), respectively, attributed to halophylic (ten Haven et al.,
1988) or methanogenic/methanotrophic archaea (Brocks and
Summons, 2003). The lack of archaeal signal in the dry sample
by the LDChip200 was attributed to intrinsic limitations on the
technique, in relation to the absence of antibodies against the
specific archaeal strains detected here by DNA sequencing. The
LDChip200 is an assay interrogating a panel of 181 polyclonal
antibodies (i.e., close method), in contrast to the ability of
the DNA sequencing in detecting any existing phylogenetic
group (i.e., open method). Furthermore, the antibodies used
in the LDChip were produced using whole cell lysates of
particular strains or whole EPS fractions as immunogens. This
means that each polyclonal antibody preparation may contain
subpopulations of antibody molecules recognizing their target
with different specificities and affinities. They can bind epitopes
from a variety of microorganisms, not necessarily the same
species, but related ones, or even to others well conserved among
large phylogenetic groups. Consequently, a direct correlation at
species level between LDChip and DNA sequences should not
be expected. This is a drawback of the LDChip, yet it increases
the chances for detecting any microbial remains in life detection
experiments.
Finally, certain occurrence of perchlorate reduction activity
was considered in the dry mound, as suggested by the LDChip200
detection against Proteobacteria capable of perchlorate reduction
such as Magnetospirillum sp., Ideonella sp, and Dechlorobacter
sp. (Supplementary Figure S9). Despite the detection of markers
by the LDChip, the presence of perchlorate reducing indicators
in the dry sinter should be further investigated, since no
perchlorate anions were detected in the samples. Whether these
microorganisms are indeed using minor amounts of perchlorate
as electron acceptor (Nepomnyashchaya et al., 2012) or they
are only detoxifying photochemically generated chloride species
(Trumpolt et al., 2005) has to be determined. As for CO2 fixation,
the consistent depletion (∼2–4h) of the δ13 C ratios in the
majority of lipid compounds in the dry sample relative to the
other two mounds (Figure 4) suggested a shift in the metabolic
fingerprint that could be related to water scarcity. Even though
the Calvin cycle may still dominate the autotrophic metabolism
in the dry sample (δ13 C values from −24 to −32h), the general
decrease in compound-specific δ13 C ratios may be caused by
some contribution from assimilation pathways with larger 13 C
fractionations (i.e., reductive acetyl-CoA pathway; Fuchs, 1989;
Preuβ et al., 1989).
The microbial community in the three sinter mounds showed
a transition through the different stages of hydrothermal activity,
with the dry mound displaying generally larger alpha-diversity
estimates (Figure 6), that is, a richer, more diverse and even
population. Although the opposite trend was observed in some
of the LDChip-based estimates (i.e., richness and Shannon’s
diversity), the discrepancy was attributed to known factors, such
inorganic compounds (e.g., H2 or sulfides) would assist with
nitrate reduction. The distinctly high nitrate concentration in
the steam sinter extract corresponded to morphological changes
in the biofilms that developed in the interstices between the
spicules. This shift in the microbial population is supported by
the change to highly porous and silicified remnants observed
in the interstices (Supplementary Figure S2). The presence
of nitrate would also serve as a substrate for denitrification
processes, which would be consistent with the highest detection
of nitrate reductase (Figure 7) and the exclusive detection
of nitrite in the steam sinter (Supplementary Table S2). In
either case, the molecular evidence indicated that, together
with autotrophic Calvin pathways (Figure 4), nitrogen cycle
was a crucial metabolic trait in the microbial biofilm of the
steam mound, where nitrate appeared as a central metabolite of
different, co-occurring metabolic pathways.
The microbial community in the dry mound showed a
strong correspondence with Archaea, Gammaproteobacteria,
and Actinobacteria (Figure 5B). The relative abundance of
Actinobacteria, a phylum with great adaptability to aridity and
resistance to hydric stress and UV radiation (Makarova et al.,
2001), coincided with the positive immuno-detections of proteins
related to hydric stress (i.e., DhnA1 peptide from a dehydrin
protein; Supplementary Figure S9), consistent with the limited
access to water in the inactive geothermal system. In addition,
the LDChip detected compounds indicative of nutritional stress
conditions such as PHAs (Figure 7), which are produced upon
need for carbon storage under nutritional stress or high C/N
ratios (Steinbüchel, 1991; Byrom, 1994). The episodic waning
of geyser activity in the history of sinter accretion coincides
with the scarcity of water and bioavailable nutrients, which
would subsequently lead to hydric and nutritional stress. Indeed,
the most abundant Firmicutes orders in the dry mound (i.e.,
Bacillales and Clostridiales) corresponded to microorganisms
characteristically resistant to extreme conditions (e.g., desiccation
and oxidative stress), including those able to form endospores
(Paredes-Sabja et al., 2011) as an adaption for desiccation and
oxidative stress.
Gammaproteobacteria was also particularly representative
of the dry-mound microbial community (Figure 5B). Within
them, purple sulfur bacteria (PSB) appeared to be present,
according to the detection of low concentration (Table 1) of
C17 and C19 cyclopropyl acids (Bühring et al., 2014) with δ13 C
values (Figure 4D) in the range of those assigned to PSB
(from −20 to −29h) in microbial mats from, e.g., Shark Bay
(Pagès et al., 2015). Cyclopropane carboxylic acids are bacterial
membrane components typically transformed from unsaturated
carboxylic acids when exposed to stressful conditions such
as oxidants, starvation, or desiccation (Grogan and Cronan,
1997; Chen and Gänzle, 2016). Their formation decreases the
permeability of bacterial membranes, enhancing their stability
under environmental stress (Poger and Mark, 2015). Wilhelm
et al. (2018) reported detection of these carboxylic acids in surface
soils from hyperarid regions of the Atacama Desert at about
430–480 km southwest of the El Tatio geysers field (Chañaral
and Altamira). In the present study, their detection only in the
dry sample may be a response to an adaptive strategy against
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against desiccation and increased exposure to intense UV
radiation as geyser activity waned. Alpha-diversity estimates
supported the transition toward a richer and more even
microbial population with the waning of the hydrothermal
activity. Our results demonstrated the effectiveness of integrating
microbiological and biogeochemical approaches to document
and understand the microbial community structure and function
in high-altitude geothermal environments with resemblance to
Hesperian surfaces on Mars. Gathering data about the capability
of different analytical techniques to decipher information from
preserved fossil biosignatures is of vital importance for future
astrobiological missions.
as (i) the differences in approach underlying the LDChip200
and DNA sequencing techniques (i.e., closed versus open
methods), and (ii) the fact that the drier the sample, the lower
the biomass to analyze, which can reduce the concentrations of
many target molecules to values below the limit of detection
for the LDChip200. The consistently greatest Pielou’s index
values in the dry sample illustrated the impact of the decreasing
hydrothermal activity in the microbial community structure.
Greater evenness is generally correlated with less active
communities and is consistent with the dormant stage of the
dry sinter mound (i.e., inactive geyser). A hydrothermally
active geyser, such as the liquid water-engulfed sinter mound
at El Tatio, is initially colonized (primary community) by
microorganisms able to endure and thrive at high temperatures
(i.e., thermophiles and hyperthermophiles). The reduced group
of taxa capable to establish in such high temperature results in a
microbial community of low richness, diversity and evenness.
As hydrothermal activity wanes and dry periods lengthen
(i.e., the steam-wetted and dry sinter mounds at El Tatio),
temperature ceases to be a key factor and thermophiles became
less competitive compared to other microbial communities.
Opportunistic microorganisms comprising endoliths and
communities contributed by environmental contaminants
emerge overprinting the primary thermophiles (secondary
community). While approaching dormancy, the inactive, dry
geyser displayed the richest, most diverse and most even
microbial community composed of members and metabolisms
that survived the waning of geyser activity.
AUTHOR CONTRIBUTIONS
DC, NC, KW-R, and NH collected the sinter samples. DC and
LS-G extracted and analyzed the lipidic fractions and wrote the
manuscript. MF-M, SP, KL, KW-R, MB, and DL-B extracted DNA
performed DNA sequencing and discussed phylogenetic results.
MG-V, YB, and VP performed and analyzed the LDChip200
immunoassays. MG-V and VP performed ion chromatography
analysis. VP, SC, NH, KW-R, and SP contributed to improve the
manuscript edition.
FUNDING
This work was funded by the Spanish Ministry of Economy and
Competitiveness (MINECO/FEDER) projects no. RYC-201419446, CGL2015-74254-JIN, and ESP2015-69540-R, and NASA
Astrobiology Institute NAI-CAN7 project no. NNX15BB01A.
CONCLUSION
The multidisciplinary field and molecular study was successful
in explaining the influence of the degree of hydrothermal
activity on the biomarkers record (i.e., including present
and past biosignatures) in the three morphologically similar
geyser mounds at El Tatio. In the context of the ecological
and environmental setting, the phylogenetic, molecular, and
metabolic patterns, in agreement with differences in the micronscale morphology of the geyserite, followed a hydrodynamic
gradient predictable during the lifetime of a geyser mound; the
transition from persistent hydrothermal activity to intermittent
steam exposure to episodic and eventually, continual dryness.
Accordingly, the microbial population shifted from primary
communities adapted to high temperature (thermophiles and
hyperthermophiles) to populations adopting protective strategies
ACKNOWLEDGMENTS
We acknowledge the Complejo Turístico Tatio Mallku (Chile)
for allowing access and sampling. We thank Paloma MartínezSarmiento for stable isotopes analysis and Maria Teresa
Fernández-Sampedro for XRD analysis at CAB.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2018.03350/full#supplementary-material
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Copyright © 2019 Sanchez-Garcia, Fernandez-Martinez, García-Villadangos,
Blanco, Cady, Hinman, Bowden, Pointing, Lee, Warren-Rhodes, Lacap-Bugler,
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