Biomarkers and Metabolic Patterns in the Sediments of Evolving Glacial Lakes as a Proxy for Planetary Lake Exploration Vı ´ctor Parro, 1 Yolanda Blanco, 1 Fernando Puente-Sa ´ nchez, 1 Luis A. Rivas, 1 Mercedes Moreno-Paz, 1 Alex Echeverrı ´a, 2 Guillermo Chong-Dı ´az, 2 Cecilia Demergasso, 2 and Nathalie A. Cabrol 3 Abstract Oligotrophic glacial lakes in the Andes Mountains serve as models to study the effects of climate change on natural biological systems. The persistent high UV regime and evolution of the lake biota due to deglaciation make Andean lake ecosystems potential analogues in the search for life on other planetary bodies. Our objective was to identify microbial biomarkers and metabolic patterns that represent time points in the evolutionary history of Andean glacial lakes, as these may be used in long-term studies as microscale indicators of climate change processes. We investigated a variety of microbial markers in shallow sediments from Laguna Negra and Lo Encan ˜ado lakes (Regio ´n Metropolitana, Chile). An on-site immunoassay-based Life Detector Chip (LDChip) revealed the presence of sulfate-reducing bacteria, methanogenic archaea, and exopolymeric substances from Gammaproteobacteria. Bacterial and archaeal 16S rRNA gene sequences obtained from field samples confirmed the results from the immunoassays and also revealed the presence of Alpha-, Beta-, Gamma-, and Deltaproteo- bacteria, as well as cyanobacteria and methanogenic archaea. The complementary immunoassay and phylogenetic results indicate a rich microbial diversity with active sulfate reduction and methanogenic activities along the shoreline and in shallow sediments. Sulfate inputs from the surrounding volcanic terrains during deglaciation may explain the observed microbial biomarker and metabolic patterns, which differ with depth and between the two lakes. A switch from aerobic and heterotrophic metabolisms to anaerobic ones such as sulfate reduction and methanogenesis in the shallow shores likely reflects the natural evolution of the lake sediments due to deglaciation. Hydrodynamic deposition of sediments creates compartmentalization (e.g., sediments with different structure and composition surrounded by oligotrophic water) that favors metabolic transitions. Similar phenomena would be expected to occur on other planetary lakes, such as those of Titan, where watery niches fed by depositional events would be surrounded by a ‘‘sea’’ of hydrocarbons. Key Words: Glacier lakes—Sedimentation—Prokaryotic metabolisms and biomarkers—Deglaciation—Life detection—Planetary exploration. Astrobiology 18, 586–606. 1. Introduction W ith global temperature rising, ice worldwide re- treats and thins. It is projected that many low-altitude glaciers could disappear within 20 years (IPCC, 2007). Long- term, multiproxy studies in regions between 33ŶS and 36ŶS in Chile and Argentina have shown a mean frontal retreat of between -50 and -9my -1 , thinning rates between 0.76 and 0.56 m y -1 , and a mean ice area reduction of 3% since 1955 (Roig et al., 2000; Le Quesne and Acun ˜a, 2003; Lara et al., 2005; Vuille, 2006; Le Quesne et al., 2009). The IPCC (2007) lists the Central and Southern Andean countries as particu- larly vulnerable (Painter, 2007). Among those, Chile has one of the world’s largest sources of glacial water, which includes mountain glaciers, ice, and snowfields that are receding rap- idly (Haeberli et al., 2002; Le Quesne and Acun ˜a, 2003; Coudrain et al., 2005; Bradley et al., 2006; Rivera et al., 2007; Vuille et al., 2008; Le Quesne et al., 2009). Glacial lakes and their sediments are highly sensitive temporal markers of en- vironmental variability, which in turn affects their biota. Microbiological studies of oligotrophic Andean lakes have shown that changes in the water column occur in association with fluctuations in water transparency or turbidity during de- glaciation (Modenutti et al., 2012). During early deglaciation (phase 1), the high silt content of water protects microorgan- isms from the high UV that occurs at the higher altitudes of 1 Department of Molecular Evolution, Centro de Astrobiologı ´a (INTA-CSIC), Madrid, Spain. 2 Centro de Biotecnologı ´a ‘‘Profesor Alberto Ruiz,’’ Universidad Cato ´lica del Norte, Antofagasta, Chile. 3 The SETI Institute, Carl Sagan Center, Mountain View, California, and NASA Ames Research Center, Moffett Field, California, USA. ASTROBIOLOGY Volume 18, Number 5, 2018 ª Mary Ann Liebert, Inc. DOI: 10.1089/ast.2015.1342 586 Downloaded by Consejo Superior De Investigaciones Cientificas CSIC from www.liebertpub.com at 07/22/18. For personal use only.
Transcript
Biomarkers and Metabolic Patterns in the Sedimentsof Evolving Glacial Lakes as a Proxy
for Planetary Lake Exploration
Vıctor Parro,1 Yolanda Blanco,1 Fernando Puente-Sanchez,1 Luis A. Rivas,1 Mercedes Moreno-Paz,1
Alex Echeverrıa,2 Guillermo Chong-Dıaz,2 Cecilia Demergasso,2 and Nathalie A. Cabrol3
Abstract
Oligotrophic glacial lakes in the Andes Mountains serve as models to study the effects of climate change on naturalbiological systems. The persistent high UV regime and evolution of the lake biota due to deglaciation makeAndean lake ecosystems potential analogues in the search for life on other planetary bodies. Our objective was toidentify microbial biomarkers and metabolic patterns that represent time points in the evolutionary history ofAndean glacial lakes, as these may be used in long-term studies as microscale indicators of climate changeprocesses. We investigated a variety of microbial markers in shallow sediments from Laguna Negra and LoEncanado lakes (Region Metropolitana, Chile). An on-site immunoassay-based Life Detector Chip (LDChip)revealed the presence of sulfate-reducing bacteria, methanogenic archaea, and exopolymeric substances fromGammaproteobacteria. Bacterial and archaeal 16S rRNA gene sequences obtained from field samples confirmedthe results from the immunoassays and also revealed the presence of Alpha-, Beta-, Gamma-, and Deltaproteo-bacteria, as well as cyanobacteria and methanogenic archaea. The complementary immunoassay and phylogeneticresults indicate a rich microbial diversity with active sulfate reduction and methanogenic activities along theshoreline and in shallow sediments. Sulfate inputs from the surrounding volcanic terrains during deglaciation mayexplain the observed microbial biomarker and metabolic patterns, which differ with depth and between the twolakes. A switch from aerobic and heterotrophic metabolisms to anaerobic ones such as sulfate reduction andmethanogenesis in the shallow shores likely reflects the natural evolution of the lake sediments due to deglaciation.Hydrodynamic deposition of sediments creates compartmentalization (e.g., sediments with different structure andcomposition surrounded by oligotrophic water) that favors metabolic transitions. Similar phenomena would beexpected to occur on other planetary lakes, such as those of Titan, where watery niches fed by depositional eventswould be surrounded by a ‘‘sea’’ of hydrocarbons. Key Words: Glacier lakes—Sedimentation—Prokaryoticmetabolisms and biomarkers—Deglaciation—Life detection—Planetary exploration. Astrobiology 18, 586–606.
1. Introduction
W ith global temperature rising, ice worldwide re-treats and thins. It is projected that many low-altitude
glaciers could disappear within 20 years (IPCC, 2007). Long-term, multiproxy studies in regions between 33�S and 36�S inChile and Argentina have shown a mean frontal retreat ofbetween -50 and -9 m y-1, thinning rates between 0.76 and0.56 m y-1, and a mean ice area reduction of 3% since 1955(Roig et al., 2000; Le Quesne and Acuna, 2003; Lara et al.,2005; Vuille, 2006; Le Quesne et al., 2009). The IPCC (2007)lists the Central and Southern Andean countries as particu-larly vulnerable (Painter, 2007). Among those, Chile has one
of the world’s largest sources of glacial water, which includesmountain glaciers, ice, and snowfields that are receding rap-idly (Haeberli et al., 2002; Le Quesne and Acuna, 2003;Coudrain et al., 2005; Bradley et al., 2006; Rivera et al., 2007;Vuille et al., 2008; Le Quesne et al., 2009). Glacial lakes andtheir sediments are highly sensitive temporal markers of en-vironmental variability, which in turn affects their biota.
Microbiological studies of oligotrophic Andean lakes haveshown that changes in the water column occur in associationwith fluctuations in water transparency or turbidity during de-glaciation (Modenutti et al., 2012). During early deglaciation(phase 1), the high silt content of water protects microorgan-isms from the high UV that occurs at the higher altitudes of
1Department of Molecular Evolution, Centro de Astrobiologıa (INTA-CSIC), Madrid, Spain.2Centro de Biotecnologıa ‘‘Profesor Alberto Ruiz,’’ Universidad Catolica del Norte, Antofagasta, Chile.3The SETI Institute, Carl Sagan Center, Mountain View, California, and NASA Ames Research Center, Moffett Field, California, USA.
ASTROBIOLOGYVolume 18, Number 5, 2018ª Mary Ann Liebert, Inc.DOI: 10.1089/ast.2015.1342
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glacial lakes. With time, however, as glaciers continue to re-cede, the meltwater discharge and sediment load into the lakesdecrease (phase 2), and the transparency and UV levels of thewater column increase. A comparative study of six Andeanultra-oligotrophic lakes characterized by low-phosphorusconcentrations revealed that high photosynthetically activeradiation (PAR), UVA, and UVB forced planktonic organismsinto deeper layers of the water column (Callieri et al., 2007). Incontrast, the microbial diversity (i.e., picocyanobacterial as-semblages) of such lakes may remain high, a finding attributedto habitat fragmentation generated by geographic barriers,which resulted in rapid speciation (Caravati et al., 2010).
The combination of environmental and climate factors canalso affect the biodiversity of microbial communities in gla-cial lake sediments. Leon et al. (2012) reported differences inthe metabolic activity and structural and functional compo-sition of bacterial communities between the sediments ofthree Patagonian Chilean oligotrophic lakes of quaternaryglacial origin. They attributed these differences to the uniquegeomorphological pattern of each lake due to both local (e.g.,volcanic activity) and global (climate change) disturbances.
Such examples illustrate how the microbial communitiesthat live in the water column and sediments of glacial lakes andreservoirs reflect their environmental setting and geochemicalinput. Although lower and more acidic precipitation, increasedtemperature, and snow melting that accompanies deglaciationstrongly affect the dissolved organic carbon concentration ofglacial lakes (Beniston et al., 1997; Sala et al., 2000; Wil-liamson et al., 2009), microbes ultimately control the carboncycle of glacial lakes through metabolic processes, such asfermentation of complex organic matter or the production ofmethane (Wadham et al., 2008).
Oligotrophic water, characterized by low organic carbonand microbial biomass, is a general feature of glacial Andeanlakes. Although some works have reported on the microbialcontent, photosynthetic activity, and geochemistry of the watercolumn of these lakes, very little is actually known about theirsediments, which may reveal key aspects of the evolution ofthese important ecosystems with regard to ecology, water re-sources (e.g., for human consumption), and the evolution ofglacier lakes at planetary scales. Determining whether there arepredictable microbial successions or key biomarkers that mightbe indicative of the progressive evolution of glacial lake sys-tems, and whether those biomarkers are preserved in theirsediments, could aid in our understanding of the evolutionaryhistory of these lakes and reservoirs.
Here, we report the results of a geomicrobiological studythat included biomarker profiling via the use of an on sitemultiplex immunological assay and additional laboratory-based analyses of sediments from two oligotrophic Andeanlakes. Immunological techniques are advantageous for fieldstudy because sample preparation is relatively easy and canbe performed in the field. Conventional molecular ecologytechniques used for massive DNA sequencing of environ-mental samples also require time-intensive and specializedexpertise, which is not required for immunological assays.
The Life Detector Chip (LDChip) is an antibody microarray-based biosensor designed for in situ life-detection studies ofanalog deposits in preparation for planetary exploration andfor monitoring microbial diversity and metabolisms in ex-treme environments (Rivas et al., 2008; Parro et al., 2008,2011a, 2011b). It has been developed over the course of the
past 10 years and has been implemented—along with theSigns of Life Detector (SOLID) instrument—for in situ detec-tion of biomarkers (Parro et al., 2008, 2011b). The LDChip hasbeen used for in situ detection of prokaryotes and biomarkerprofiling in different extreme environments that include theacidic iron-rich sediments of the Rıo Tinto in Spain (Parro et al.,2008, 2011c; Puente-Sanchez et al., 2014), subsurface sedi-ments (down to 5 m depth) cored in the hypersaline AtacamaDesert (Parro et al., 2011a; Fernandez-Remolar et al., 2013),and the surface and permafrost (down to 4.2 m) sedimentsdrilled on Deception Island in Antarctica (Blanco et al., 2012).The LDChip results reported here reveal a rich geochemistry inAndean lakes that is capable of sustaining an active anaerobicmetabolism and broad microbial diversity. Such data alsoprovides a baseline for further monitoring to understand theevolution of deglaciation as recorded in glacial lake sediments.
2. Geological Setting
Laguna Negra and Lo Encanado are located on the south slopeof the Echaurren glacier watershed in the Central Andes of Chile(33.65S, 70.13W; Fig. 1). Monitoring these lakes over timewould allow for characterization of their prokaryotic diversity,physical processes, and spatiotemporal changes. The two lakesare part of a complex of freshwater resources in the Santiago areathat includes El Yeso lake to the east, which is damned (on itssouthern end). Though Laguna Negra and Lo Encanado wereconnected by a human-made overflow tunnel, the lake level ofLaguna Negra has decreased enough that the two lakes havebeen isolated from each other for at least the past 5 years.
Both lakes are located in the same catchment area (up to4600 m in elevation), yet they are fed by two distinct streamsystems and have contrasting physical characteristics: LagunaNegra is a large (6.1 · 1.7 km) and deep (276 m in 2013) lakelocated at 2700 m above sea level, whereas Lo Encanado isa smaller, 980 · 635 m wide, and 45 m deep lake located at2492 m elevation.
The watershed area of the lakes is composed primarily ofvolcanic constructs and basalt and andesite deposits thatwere covered by modern glacial deposits from the recedingEchaurren glacier, which now lies above the lakes at 3500 melevation. Lahar deposits mark recent to modern interactionsbetween volcanic and glacial activity on the west shore ofLaguna Negra. Residual frontal moraines enclose the basinon its south and southeast shore and separate Laguna Negrafrom Lo Encanado to the south and El Yeso to the east.Moraine deposits, as well as large glacial erratics at thebottom of the lake, consist of residual granodiorite that wasexcavated by glacial erosion from the basement of the basin.
Both lakes are monomictic and oligotrophic and in dif-ferent phases of the deglaciation process: Laguna Negra ishighly transparent (i.e., phase 2), while the latter is turbid asa consequence of the larger biomass content (i.e., phase 3),which translates into distinct orbital spectral signatures invisible (black and green, respectively) spectra.
3. Materials and Methods
3.1. Sampling and environmental parameters
Sediments were sampled from the shoreline of LagunaNegra (LNS, coordinates 33�66061S, 70�12161W) and LoEncanado (LES, coordinates 33�674168S, 70�1278W) and
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at 4 m water depth in Laguna Negra (LN4) during a fieldcampaign in 2011. Sample LN4 was collected by diving to4 m depth below the water surface some meters away from theshore. Sediments along the shorelines of both lakes appearedsimilar, that is, highly granulated and low in organic material,whereas the sediment collected from the lake bottom con-tained significantly more decomposing matter. All the sedi-ments were collected in 50 mL sterile Falcon tubes and keptrefrigerated during transport until they reached the laboratoryand were analyzed. Conductivity, pH, temperature, and redoxpotential were measured at the time of sample collection witha multiparametric portable instrument (WTW, Germany).The sediment samples were analyzed in the field by the im-munological biosensor LDChip (see below) and then in thelaboratory by ion chromatography and DNA sequencing.
3.2. Geochemical analysis
Ion chromatography was used to measure the concentrationof inorganic anions (Cl-, SO4
and oxalate) of interest. All samples were run on a Metrohm861 Advanced Compact IC (Metrohm AG, Herisau, Switzer-land) as described previously (Parro et al., 2011a).
Two types of samples were extracted for geochemicalanalysis via the ion chromatograph (IC): the interstitial fluidsof the sediments and supernatants prepared by physical agi-tation of the sediments after they had been separated from theinterstitial fluids. To separate the interstitial water (IW) fromthe sediments, 2 g of wet sediment from each sample wascentrifuged at 2000 g, and 1 mL of the IW was removed fromthe tube with the sediment ‘‘pellet.’’ The 1 mL of IW was thendiluted in 10 or 20 mL of distilled water (analytical grade) tolet the IC values fit into the calibration curves and to havedifferent IC measurements. To extract the small organicmolecules and anions of interest that were associated with thesurfaces of the sediment materials (minerals and particulateorganic matter), any remaining IW supernatant was removedfrom the centrifuged sediment samples so that the sedimentscould be resuspended in 10 mL of sterile distilled water,mixed in a vortex, and then physically agitated for 1 h. Afterthe solid particles were removed by centrifugation at 2000 g
FIG. 1. ASTER image showing (1) Laguna Negra, (2) Lo Encanado, and (3) El Yeso, and their respective spectral orbitalsignatures in the visible. El Yeso is the richest in glacial flour, which gives its aqua color. Topographical profiles A–B andC–D are shown.
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for 10 min, the supernatants of these samples were thenanalyzed by ion chromatography to measure the small or-ganic molecules and inorganic anions in the same manner asthe IW, as noted above.
3.3. Antibody microarrays: printing LDChipand fluorescent labeling of antibodies
In this work, the LDChip antibody microarray contained 193antibodies (see Appendix Table 1 on page 000) and was designedto enable the identification of bacterial strains from each mainphylogenetic group of bacteria and archaea; a variety of uni-versal proteins, peptides, and biological polymers (nucleic acids,lipopolysaccharides, exopolysaccharides) used in modern (andpresumably ancient) metabolisms (e.g., nitrogen fixation, sulfatereduction, iron homeostasis, and nitrate metabolism); see alsoRivas et al. (2008, 2011) and Parro et al. (2008, 2011a, 2011c).The immunoglobulin (IgG) fraction of each of the 193 protein A-purified antibodies was printed in a spot pattern in duplicate onthe surface of epoxy-activated glass slides (Arrayit, CA, USA)with a MicroGrid II TAS arrayer (Biorobotics, Genomic Solu-tions, UK) as reported previously (Rivas et al., 2008; Parro et al.,2011a). For this particular study, 141 out of the 193 antibodies inAppendix Table 1 (52 antibodies produced against Rıo Tintonatural samples were excluded to avoid bias in the results) werefluorescently labeled with Alexa 647 (Molecular Probes) astracers for fluorescent immunoassay detection.
To reveal the immunoreactions on the antibody micro-array, we produced a homogenate that consisted of 141 fluo-rescent antibodies (final concentration was 100 lg/mL). Theconcentration of the individual antibodies in the mixture(inferred via titration, data not shown) averaged 0.7 lg/mL.This mixture was frozen and then lyophilized for transportand use in the field at a 1/10 final working dilution.
Six new antibodies have been used in this work for the firsttime (Appendix Table 1, bottom): one genus-specific anti-body produced to recognize Polaromonas sp., two strain-specific antibodies produced to recognize Planococcusspp. (kindly supplied by Dr. Lyle Whyte, McGill University,Montreal, Canada), and three antibodies produced as de-scribed previously (Rivas et al., 2008) that reacted positivelyto environmental extracts of samples collected from (a) 2 mdepth in the Atacama Desert, (b) a permafrost sample fromDeception Island, Antarctica, and (c) a biofilm growing on aconcrete wall on a building structure next to Centro de As-trobiologıa (Madrid, Spain). The titration and detailed per-formance of these six antibodies will be reported elsewhere.
3.4. Sandwich microarray immunoassays
Sandwich-type microarray immunoassays were performedas described previously (Parro et al., 2011a; Blanco et al.,2012). In summary, printed microscope slides with the anti-body microarray (LDChip with 193 antibodies) were blockedwith 0.5 M Tris-HCl in 5% BSA for 5 min. Slides were thenimmersed in 0.5 M Tris-HCl with 2% BSA and gently agitatedfor 30 min. After washing with TBSTRR buffer (0.4 M Tris-HCl pH 8, 0.3 M NaCl, 0.1% Tween 20) and drying the chip byquick centrifugation, the slides were mounted in a portablemulti-array analysis module (MAAM) device (Rivas et al.,2008; Parro, 2010) composed of nine incubation chambersthat isolated and sealed each antibody microarray and allowedfor the processing of all samples simultaneously.
To prepare the samples for sandwich microarray immuno-assay analysis, approximately 0.5 g of sediment was collectedfrom the field, resuspended in 2 mL of TBSTRR, and sonicated(3 · 1 min cycle) with a handheld Ultrasonic Processor (UP50H,Hielscher, Germany) at the field site. The sediment was decantedfor 2–5 min, depending the clay content, and 50lL of the crudeenvironmental extracts were injected into each of the nine in-cubation chambers and incubated for 1 h with the LDChip atambient temperature. After a wash with TBSTRR, all chamberswere flooded with the fluorescently labeled antibody mixture for1 h. The slides were then washed, dried, and scanned for fluo-rescence at 635 nm in a GenePix4100A scanner (in a nearbylaboratory). Parallel immunoassays were performed as a blankcontrol by using only buffer as a negative control and treatedwith the same fluorescent antibody mixture. All sets of newlyprinted LDChips are tested in the laboratory with randomly se-lected antibody-immunogen pairs. We have demonstrated thatthe LDChip is fully functional for at least 9 months of storage atambient temperature (de Diego-Castilla et al., 2011). Ad-ditionally, as a positive control sample to be used in the field, wespiked the buffer with the human hepatitis B antigen and ran atest to verify that the antibodies were still functional. The scan-ned images were analyzed in the field with GenePix Pro software(Genomic Solutions) installed on a laptop computer. The finalfluorescence intensity was quantified as previously reported(Parro et al., 2011a; Rivas et al., 2011).
3.5. DNA extraction and sequencing
DNA was extracted from 10 g of sediments with the MoBioDNA extraction kit according to the manufacturer’s instruc-tions. The 16S rRNA gene from Bacteria and Archaea wasPCR amplified, cloned, and sequenced as described previously(Parro et al., 2011a). Additionally, the amplicons were sub-jected to high throughput sequencing by the 454 Roche pyro-sequencing system (Lifesequencing S.L., Valencia, Spain).For pyrosequencing, the V3–V5 region of the 16S rRNA genewas amplified using key-tagged eubacterial primers (Life-sequencing S.L.) based on the design of Sim et al. (2012). PCRreactions were performed with 20 ng of metagenomic DNA,200 lM of each of the four deoxynucleoside triphosphates,400 nM of each primer, 2.5 U of FastStart HiFi Polymerase andthe appropriate buffer with MgCl2 supplied by the manufac-turer (Roche, Germany), 4% of 20 g/mL BSA (Sigma, Dorset,UK), and 0.5 M betaine (Sigma). Thermal cycling consisted ofan initial denaturation at 94�C for 2 min followed by 35 cyclesof denaturation at 94�C for 20 s, annealing at 50�C for 30 s, andextension at 72�C for 5 min. Amplicons were combined in asingle tube in equimolar concentrations. The pooled ampliconmixture was purified twice (AMPure XP kit, Agencourt,Takeley, UK), and the cleaned pool was requantified by usingthe PicoGreen assay (Quant-iT, PicoGreen DNA assay, In-vitrogen). Subsequently, an amplicon was submitted to thepyrosequencing services provided by Lifesequencing S.L.(Valencia, Spain) where EmPCR was performed. Subse-quently, unidirectional pyrosequencing was carried out on a454 Life Sciences GS FLX+ instrument (Roche) following theRoche Amplicon Lib-L protocol.
3.6. 16S rRNA gene phylogenetic analysis
Partial-length bacterial and archaeal 16S sequences weretrimmed, assembled, and screened for vector contamination
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with the CodonCode Aligner (CodonCode Corporation,Centerville, MA) software package. Assemblies longer than795 bp were used as input files for the mothur version 1.21.1(Schloss et al., 2009). Sequences were aligned to the SILVA(Pruesse et al., 2007) reference databases provided by mothurin order to scan them for chimeras with the use of the chimeraslayer tool. The alignment was then used to calculate distancematrices, and the sequences were clustered into operativetaxonomic units (OTUs) by using the average neighbor al-gorithm. Rarefaction curves, richness estimators (Ace,Chao1), and community diversity indexes (Simpson Index)implemented in mothur were also obtained. For each sample,one representative of each 0.03 distance OTU was selected forphylogenetic tree construction. Sequences were aligned withthe SILVA reference alignment using SINA (Pruesse et al.,2012) and imported into the ARB phylogenetic package(Ludwig et al., 2004). ARB parsimony algorithm was used toadd the sequences to the SSUref_NR98 reference tree, ap-plying a filter to exclude the most variable positions.
The 16S rRNA gene sequences obtained by 454 pyro-sequencing were analyzed as follows: Raw GS FLX +454reads were analyzed with mothur version 1.31.2 as rec-ommended by Schloss et al. (2011). Sequences were de-multiplexed, denoized using mothur’s implementation of thePyroNoise algorithm (Quince et al., 2009), aligned to acombination of silva.archaea and silva.bacteria databases,screened for chimeras using UCHIME (Edgar et al., 2011),and clustered by using the average neighbor algorithm. Sincevarious diversity metrics are sensitive to the procedure usedfor sampling, 4979 sequences for each sampling site wererandomly selected for further analysis. Rarefaction curves,richness estimators, community diversity indexes, and com-munity structure were obtained as previously described.
4. Results
4.1. Lake geochemistry
Differences in the concentrations of low-molecular-weight organic acids and anions involved in microbial me-tabolism were observed between both lakes and, in the caseof Laguna Negra, with depth (Table 1). On average, theanion concentration was higher in the IW, except for nitrate,which was preferably attached to the coarse material.Acetate, formate, chloride, phosphate, and sulfate were es-pecially abundant in LN4 IW and coarse material. Thesevalues were lower in the shore sediments, whose visualinspection showed less decomposing organic matter. Pro-pionate and oxalate were only detected in LN4, the former
being associated with the solid material and the latter withIW. The concentration of nitrite was lower than the limit ofdetection of the technique used to analyze all the sedimentsamples. Relatively high concentrations of bromide weredetected but only in the IW in the LN4 sediments, wherevisible decomposing macrophyte algal remains were ob-served. The ratio of Cl-/Br- ions was low (23.95), though theaccumulation of Br- was found to be much higher than thatof many freshwater lakes (Davis et al., 1998).
The presence of relatively high concentrations of acetateand formate, indicative of microbial fermentation processes,supported the growth of sulfate-reducing bacteria and/oracetoclastic methanogenic archaea.
4.2. Biomarker profiling with the LDChipimmunosensor in the field
Samples LNS, LES, and LN4 were analyzed in thefield with the LDChip antibody microarray immunosensor(Materials and Methods). Sediment samples (ca. 0.5 g) werehomogenized by a handheld ultrasonicator, filtered, andincubated with the LDChip. Positive immunoreactions wererevealed with a fluorescent antibody mixture and scannedfor fluorescence, and the digital data was plotted (Fig. 2).Although one might expect that the immunograms from thesediments collected along the shoreline of both lakes (LNSand LES) would be more similar, they showed meaningfuldifferences. The immunogram of LNS had a lower numberof positive immunoreactions, while that of LES exhibited aricher inmunoprofile (Fig. 2 and Table 2). For example,spots/bars 11 and 24, which correspond to antibodies pro-duced to Desulfotalea psychrophila and Desulfosporosinusmeridiei (two sulfate-reducing bacteria) or spots/bars pro-duced to Shewanella gelidimarina and Geobacter spp. (metalreducers), were absent in LNS. Notwithstanding, positivereactions with antibodies produced to extracellular poly-meric substances and whole cell extracts from Gammapro-teobacteria were obtained along the shoreline of both lakes.Interestingly, the immunopattern obtained with the LN4sample was more similar to that of LES than to LNS. An-tibodies produced to a psychrophilic sulfate-reducing bac-terium (D. psychrophila), other sulfate-reducing bacteria(Desulfovibrio vulgaris, Geobacter sulfurreducens, or D.meridiei), and methanogenic archaea (Methanosarcina ma-zeii, Methanobacterium formicicum) showed clear positivereactions. Similarly, antibodies produced to heterotrophicbacteria, such as Burkholderia fungorum, Acidiphilium sp., orAcidocella aminolytica also showed positives in both LN4and LES samples. Somewhat unexpectedly, the LN4 sample
Table 1. Geochemical Analysis of the Laguna Negra and Lo Encanado Sediments Showing
Low-Molecular-Weight Organic Acids and the Main Inorganic Anions (lg/mL)
LES, Lo Encanado shore sediments; LNS, Laguna Negra shore sediments; LN4, Laguna Negra sediments from 4 m below the surfacewater; _w, interstitial water; _ex, extract from coarse material from the centrifuged sediments, with ultrapure water.
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Table 2. List of the Antibodies Showing Positive Immunoreactions after On-Site Analysis
of the Sediment Samples from Laguna Negra (LNS and LN4) and Lo Encanado (LES)
Phylogenetic group Ab to Ab name* LNS LN4 LES References
Alfaproteobacteria Acidocella aminolytica IVG1C1 Rivas et al., 2011Acidiphillium spp. IVG2C1 Parro et al., 2011a
Betaproteobacteria,Burkholderiales
Polaromonas IVF15C1 This paperBurkholderia fungorum IVI4C2 Rivas et al., 2008B. fungorum IVI4S100 Rivas et al., 2008B. fungorum IVI4S2 Rivas et al., 2008
Gammaproteobacteria Acidithiobacillus ferrooxidans A183 Parro et al., 2005A. ferrooxidans IVE3C1 Rivas et al., 2008A. ferrooxidans IVE3S100 Rivas et al., 2008A. thiooxidans A184 Rivas et al., 2008A. thiooxidans IVE4C1 Rivas et al., 2008A. thiooxidans IVE4C2 Rivas et al., 2008A. thiooxidans IVE4S100 Rivas et al., 2008A. albertensis IVE5C1 Rivas et al., 2008A. albertensis IVE5C2 Rivas et al., 2008A. albertensis IVE5S100 Rivas et al., 2008A. caldus IVE6S100 Rivas et al., 2008Halothiobacillus neapolitanus IVE7C1 Rivas et al., 2011Methylomicrobium capsulatum IVI15C1 Rivas et al., 2008Pseudomonas putida IVI1C2 Rivas et al., 2008Shewanella gelidimarina IVF2S2 Rivas et al., 2008
Deltaproteobacteria Desulfotalea psychrophila IVF18C1 Rivas et al., 2011Desulfovibrio vulgaris IVI10C1 Rivas et al., 2008Geobacter sulfurreducens IVI11C1 Rivas et al., 2008G. metallireducens IVI12C1 Rivas et al., 2008
Actinobacteria Acidimicrobium ferrooxidans IVE8C1 Parro et al., 2011aA. ferrooxidans IVE8S2 Parro et al., 2011aCryobacterium psychrophilum IVF6S1 Rivas et al., 2008
Firmicutes Desulfosporosinus meridiei IVI19C1 Parro et al., 2011aBacillus spp. (environ. isol.) IVI2S2 Rivas et al., 2008Bacillus subtilis 3610 IVI8C1 Rivas et al., 2008Planococcus or2 IVF31C1 This paperPlanococcus IVF34S2 This paper
Nitrospiraceae Leptospirillum ferrooxidans A139 Parro et al., 2005L. ferroxidans A186 Rivas et al., 2008L. pherrifilum (LPH2) IVE1S100 Rivas et al., 2008L. pherrifilum spp. IVE2S1 Rivas et al., 2008
Bacteroidetes Psychroserpens burtonensis IVF4S2 Rivas et al., 2008Salinibacter ruber PR1 IVI21C1 Rivas et al., 2011
Verrumicrobia Verrucomicrobium spinosum IVI14C1 Rivas et al., 2008Euryarchaeota Haloferax mediterranei IVJ1C1 Rivas et al., 2008
Methanobacterium formicicum IVJ4C1 Rivas et al., 2008Methanosarcina mazeii IVJ5C1 Rivas et al., 2008Halorubrum spp. IVJ8C1 Parro et al., 2011a
and extracellularsubstances (S2)from environmentalsamples
Atacama Extract VID1C1 This paperSolar saltern EPS A-EPS_SP Parro et al., 2011aBiomass from concrete VIID3BF This paperRıo Tinto (3.2 water dam) IA1S1 Rivas et al., 2008Rıo Tinto IA2C1 Rivas et al., 2008Rıo Tinto IA2S1 Rivas et al., 2008Rıo Tinto (Arroyo 3.1) IA3C1 Rivas et al., 2008Rıo Tinto (Arroyo 3.1) IA3S1 Rivas et al., 2008Rıo Tinto (3.2 water dam) IA3C1 Rivas et al., 2008Rıo Tinto (3.0 Stream) A140 Rivas et al., 2008Rıo Tinto (‘‘Nacimiento’’) A138 Rivas et al., 2008Rıo Tinto (Playa 3.1) IC1C1 Rivas et al., 2008Rıo Tinto (Playa 3.2) IC1S1 Rivas et al., 2008Rıo Tinto (Arroyo 3.1) IC2S2 Rivas et al., 2008Rıo Tinto (3.1 water dam) IC2C3 Rivas et al., 2008Rıo Tinto (‘‘Nacimiento’’) IC3S2 Rivas et al., 2008Rıo Tinto (3.2 water dam) IC4C1 Rivas et al., 2008Rıo Tinto (Playa 3.1) IC6C1 Rivas et al., 2008Rıo Tinto (3.2 water dam) IC7C1 Rivas et al., 2008Rıo Tinto (3.1 stream bank) IC8C1 Rivas et al., 2008Rıo Tinto (3.1 stream bank) IC8S1 Rivas et al., 2008Rıo Tinto (3.1 mine ruins) IC9C1 Rivas et al., 2008Pena de Hierro (93m deep) ID18S2 Rivas et al., 2008Pena de Hierro (154m deep) ID4S2 Rivas et al., 2008Antarctic (DI) permafrost IIIC3C1 This paper
Shaded boxes indicate positive detection, that is, the presence of the corresponding strain/compound or a highly similar one. *SeeAppendix Table 1 for Ab details.
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gave positive reactions to halophilic archaea (Haloferaxmediterranii and Halorubrum sp.) and other halophilic bac-teria (Salinibacter ruber). The number of positive signalsfrom aromatic compounds and nucleotide derivatives washigher in sediments from LES than those in LNS or LN4.
4.3. Prokaryotic diversity by 16S rRNA gene sequencing
Total environmental DNA was extracted from all thesamples, and the universal 16S rRNA gene was amplified and
subjected to massive sequencing by 454-pyrosequencing.More than 4900 bacterial sequences were retrieved from eachsample, and they showed nearly saturated rarefaction curves(Fig. 3), while very few archaeal sequences were obtained.Regarding the bacterial sequences, the LNS sediments fromLaguna Negra showed a lower number of OTUs (827 fromLNS and 884 from LN4) than that of LES (1549) for a similarnumber of sequences. The Simpson index for an OTU defini-tion of 0.03 was 18.58, 41.62, and 145.58 for LNS, LN4, andLE, respectively, confirming that the bacterial diversity was
FIG. 2. On-site microbial biomarker detection with the LDChip, an antibody microarray-based life-detector chip. (A)LDChip fluorescent image corresponding to the on-site analysis of the Laguna Negra 4 m deep sediment (LN4) sample (topfigure) and the negative control where only buffer was used (lower figure). (B) The image in (A) and those corresponding tothe other samples (not shown) were quantified and the relative fluorescence units plotted: Laguna Negra shore sample(LNS), Laguna Negra 4 m deep (LN4), and Lo Encanado shore sample (LES). The antibodies showing positive im-munodetection were clustered (a–f) and numbered as follows: a, antibodies to acidic environmental extracts enriched inGammaproteobacteria (e.g., 1, IC4C1, a cellular extract from a biofilm); b, bacterial cell extracts from acidic environment,mostly Gammaproteobacteria and Nitrospira (2, Leptospirillum ferrooxidans; 3, L. pherrifilum; 4, Acidithiobacillus fer-rooxidans; 5, A. thiooxidans; 6, A. albertensis; 7, A. caldus; 8, Halothiobacillus neapolitanus; and 9, Acidimicrobiumferrooxidans); c, psychrophiles (10, Polaromonas spp.; 11, Desulfotalea psychrophila; 12, Shewanella gelidimarina; 13,Planococcus; 14, Planococcus spp.; 15, Psychroserpens meridei; 16, Cryobacterium psychrophilum); 17, Acidocellaaminolytica; 18, Acidiphillium spp.; 19, Desulfovibrio vulgaris; 20, Geobacter sulfurreducens; 21, G. metallireducens; 22,Verrucomicrobium spinosum; 23, Methylomicrobium capsulatum; 24, Desulfoporosinus meridiei; 25, Pseudomonas putida;26, Salinibacter ruber PR1; 27, Bacillus spp.; 28, Burkholderia fungorum; 29, Bacillus subtilis 3610; d, Archaea (30,Haloferax mediterranei; 31, Methanobacterium formicicum; 32, Methanosarcina mazeii; 33, Halorubrum spp.); e, peptidesand proteins; f, nitroaromatic compounds and nucleotide derivatives; 34, extracts from a biofilm on a concrete wallpavement; 35, whole extracts from 3–4 m deep cores of permafrost (Deception Island, Antarctica); 36, extracts from a 2.5 mdepth sulfate- and halite-rich core sample from the Atacama Desert; 37, EPS from a solar saltern.
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much higher in the sediments from Lo Encanado than in thesediments from Laguna Negra. Members of the main groups ofProteobacteria (Alpha-, Beta-, Gamma- and Deltaproteo-bacteria) were present in all samples (Fig. 4), with a sig-nificant increase in the number of Deltaproteobacteria in theLN4 and LES samples. Two of the most abundant phylo-genetic groups, Actinobacteria and Acidobacteria, werepreferentially abundant in the shoreline sediments of bothlakes (Fig. 4), while the groups of Firmicutes, Planctomy-cetes, Chloroflexi, Bacteroidetes, and Chlorobi dominatedand showed similar patterns to those of the LN4 and LESsamples, with very few representatives in LNS. Members ofthe Deinococcus-Thermus group were only found in LNSand, by contrast, Cyanobacteria were absent in this samplethough they appeared in LN4 and LES. No 16S rRNA geneamplicons related to the domain Archaea were obtained inLNS with the universal primers used. However, most of the13 archaeal sequences obtained by pyrosequencing from theother two samples corresponded to members of the metha-nogenic Archaea (10 sequences from Methanosaeta =Methanothrix genus in LN4 and 1 from Methanosarcina inLES), while two sequences from Halobacteria were re-trieved from LN4.
Additionally, a full-length 16S rRNA gene was amplifiedby PCR, cloned, and sequenced, and the retrieved sequences
were analyzed for accurate phylogenetic affiliations (Fig. 5).Again, no amplicons were obtained with archaeal primersfrom LNS. Of the 72 clones retrieved from LN4 and 78 fromLES, 70 clones (in each case) corresponded to the Metha-nosarcinales order (mostly Methanosarcina genus) in LN4sample and Methanosaeta = Methanothrix in LES sample.The phylogeny of bacterial clones (46 for LNS, 46 fromLN4, and 18 from LES) was generally in agreement withmassive sequencing, although with a different proportionbetween the groups. For example, Proteobacteria were ma-jor components in the libraries (Fig. 5) from the shoreline ofboth lakes (LNS and LES), and in particular, Betaproteo-bacteria representatives from the Gallionellaceae, Hydro-genophilaceae, and Comamonadaceae families dominated inthe LNS sample. Further, we obtained more clones in thissample that belong to Deltaproteobacteria (one unculturedDesulfuromonadales, one Desulfobacteraceae bacterium,two Geobacter strains) than in the other two samples,and a Gammaproteobacteria (Thiofaba tediphila) of theHalothiobacillaceae family was also detected. DifferentDeltaproteobacteria, from the order Myxococcales (Ha-liangium spp), as well as bacteria from the order Gemmati-monadetes and the phylum Acidobacteria were found in theLES sample. In the LN4 sample, a different Deltaproteo-bacteria was identified, corresponding to the Syntrophaceaefamily, while the Firmicutes group (mostly Clostridia) ac-counted for more than 20% of the sequences in LN4, which isin close agreement with the massive sequencing data. Theonly cloned sequence within the phylum Cyanobacteria wasfound in the LN4 sample. Archaeal sequences were obtainedfrom LN4 and LES, mostly corresponding to the Methano-sarcina genus in the former and the Methanosaeta =Methanothrix genus in the latter (Fig. 5), which is in agree-ment with the pyrosequencing data (see above). No archaealclones and sequences were obtained after several trials fromthe LNS sample.
We previously demonstrated that the LDChip is a usefultool for environmental monitoring (Rivas et al., 2008; Parroet al., 2011a; Blanco et al., 2012, 2014; Fernandez-Remolaret al., 2014; Puente-Sanchez et al., 2014). Biomarker andmicrobial profiles in Laguna Negra and Lo Encanado wereobtained by sandwich antibody microarray immunoassayswith an LDChip (Fig. 2 and Table 2) with 193 immobilizedantibodies and a mixture of 141 fluorescent ones as tracers ofthe immunoreactions (Materials and Methods). We did notinclude the 52 antibodies developed to recognize cells andpolymeric materials for Rıo Tinto samples to avoid any po-tential bias on the LDChip results. Positive immunologicalinteractions were identified with several antibodies, such asthose from sulfate-reducer biomarkers detected in the LN4sample, which is consistent with a higher amount of sulfate inthis sample. Exopolymeric material from Gammaproteo-bacteria, such as exopolymeric substances (EPS) and wholecell extracts, was detected in all samples, but preferentially inthe LN4 and LES samples where the largest number of DNAsequences were retrieved for this phylum. Although individ-ual compound and derivative confirmation would be needed,
FIG. 3. Rarefaction curves for bacterial diversity by 454-pyrosequencing in the three analyzed samples (a) and for thediversity of Archaea by gene cloning and sequencing in LES(b) and LN4 (c). See main text for explanation.
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positive signals from nitro-aromatic compounds and nucleo-tide derivatives were higher in the LES sediments.
The immunological profiles (Fig. 2) indicate that the LN4and LES samples were closer to each other than those fromthe shorelines (LNS and LES), suggesting that local envi-ronmental conditions (nutrients, geochemistry) control themicrobial diversity regardless of the lake or the depth. TheLDChip detected positive immunoreactions with antibodiesto prokaryotes capable of anaerobic metabolisms (e.g.,sulfate-reducing bacteria) and to methanogens in the LN4and LES samples, which is consistent with the DNA se-quencing data.
5.2. A geomicrobiological model of the LagunaNegra and Lo Encanado sediments
Geochemical and immunological studies combined with16S rRNA gene cloning and massive sequencing identifiedrich prokaryotic communities and allowed us to infer thediverse metabolisms that can operate in two Andean oli-gotrophic lakes. We identified bacteria capable of sulfatereduction, sulfide oxidation, nitrogen oxidation, anaerobicammonia oxidation, nitrate reduction, and methane oxidation(methanotrophic), photosynthesis, anaerobic phototrophy, andmethanogenic archaea (Fig. 6). The fermentative and anaerobic(mainly reduction) metabolisms seemed to dominate in theLN4 and LES samples, while heterotrophic and oxidativeprocesses were significant in the LNS sample. Based on the
geochemical and molecular ecology results, we propose ageomicrobiological model for the shallow sediments, whichapplies to the ecosystem at the shoreline and 4 m underwater at Laguna Negra and Lo Encanado (Fig. 7). Primaryproducers, such as microalgae, macrophytes, and the cya-nobacteria that grow along the shorelines, produce organicmatter that is transformed by fermenters in shallow anaer-obic niches. Key products from fermentation are the small-molecular-weight organic acids such as acetate, formate,propionate, and oxalate, all of them detected by ion chro-matography (Table 1). These compounds are excellent en-ergy sources for anaerobic microbial metabolisms such assulfate reduction and methanogenesis, which is consistentwith our detection of sulfate-reducing bacteria and metha-nogenic archaea with the immunosensor LDChip (Fig. 2)and 16S rRNA gene sequencing (Fig. 4). The presence ofimmunoreactive material from halophilic archaea (withanti-Haloferax and anti-Halorubrum antibodies) and bacte-ria (Salinibacter, from Bacteroidetes group) in LN4 maybe a consequence of the relatively high accumulation(504 ppm) of chloride at the sampling site (Table 1), whichallows proliferation of these microbes. DNA sequencesfrom Halobacteria and Bacteroidetes were actually retrievedfrom this sample. At this stage, further sampling would benecessary to conclude whether this chloride accumulation isthe result of a local phenomenon (e.g., seepage from thelake floor) or if it is widespread and occurs across the entirebottom of the lake basin.
FIG. 4. Prokaryotic diversity (A, archaea and B, bacteria) in the sediments of Lo Encanado (LES) and Laguna Negra(LNS) shores and at 4 m below the water surface in Laguna Negra (LN4). Percentage of the retrieved 16 S rRNA genesequences ascribed to the corresponding phylogenetic taxon. The numbers above the bars indicate the number of sequencesused in the analysis. Sequences from bacteria were obtained by massive sequencing by 454 pyrosequencing and those fromarchaea by gene cloning and Sanger-type sequencing (asterisks). Bars represent the percentage of the retrieved 16 S rRNAgene sequences ascribed to each phylogenetic group (legend to the right) from the total number of sequences analyzed.
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FIG. 5. Panel a (Continued).
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FIG. 5. Panel b (Continued).
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The bacterial diversity was roughly similar in sedimentsfrom the shorelines of both lakes (LNS and LES), withrepresentatives of Alpha-, Beta-, and Deltaproteobacteria(Fig. 4). However, in the LN4 sample the bacterial patternchanged, and the larger number of sequences correspondedto the Gram-positive Firmicutes group, particularly from theClostridiales order. No archaebacterial representatives weredetected in the LNS sample, while in the LES sample mostof the retrieved archaeal DNA sequences correspondedto the methanogenic Methanosarcina genus and to theMethanosaeta = Methanothrix genus that were observed inthe LN4 sample. These differences might be due to thespecific geochemical features in each site. The sulfate con-centration was 0.026, 1.22, and 2.74 mM in the LES, LNS,and LN4 samples, respectively, while that of acetate was
0.055, 0.004, and 11.11 mM in the LES, LNS, and LN4samples, respectively (Table 1). These values rendered ac-etate/sulfate ratios of 2.08, 0.004, and 4.05 for the LES,LNS, and LN4 samples, respectively. It has been shown thatsulfate-reducing bacteria compete with methanogenic ar-chaea for hydrogen and acetate (Lovley and Klug, 1983) atin situ sulfate concentrations of 0.06–0.105 mM. In bothLaguna Negra sediments, the sulfate concentration is higher(see above and Table 1). The absence of archaea in the LNSsample can be explained by the very low acetate/sulfateratio, which indicates that they were completely out-competed by sulfate-reducing bacteria. However, the ace-tate/sulfate ratio is much higher in the LES and LN4samples, which might compensate the sulfate concentrationeffect. The genus Methanosarcina is a highly versatile,
FIG. 5. Maximum parsimony tree based on 16S rRNA sequences showing the relationships of the OTUs retrieved fromsamples LNS (a), LN4 (b), and LES (c) with their closest phylogenetic neighbors. The number of clones retrieved for eachOTU is indicated in parentheses. Bar: substitutions per nucleotide.
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metabolic generalist (Thauer et al., 1989) capable of usingseveral substrates, which include methanol, H2, methyl-amines, and acetate. The presence of Methanosarcina in theLES samples indicates that it most likely proliferated pref-erentially under low organic acid content (e.g., acetate) andlow sulfate concentration. By contrast, the Methanosaeta =Methanothrix strains, which are the most nutritionally re-strictive specialist with a high affinity for acetate (Patel andSprott, 1990), were found to grow at the high concentrationof acetate detected in the LN4 sample. At high sulfateconcentration, sulfate reducers compete for acetate and in-hibit acetoclastic methanogens (Ward and Winfrey, 1985).Therefore, our finding of methanogenic species such asthose from the Methanosaeta = Methanothrix genus, whichhave a high affinity for acetate, were found to proliferate andcoexist with sulfate reducers in the LN4 sample.
The sequence data and the number of bacterial OTUsobtained in all the samples indicate that the sediments ofboth lakes are rich and diverse ecosystems (Fig. 3), in spiteof the oligotrophic characteristics of the water. In fact, thegeochemical analysis (Table 1) showed the presence ofrelatively high concentrations of organic acids from fer-mentation or acetogenic processes, which can be used ascarbon and energy sources by microbes, while oxidizedcompounds such as nitrate and sulfate can be used as elec-tron acceptors for respiration. In contrast to Bacteria, thearchaeal diversity was found to be very low, with rarefactioncurves next to saturation with less than 100 clones and a
FIG. 7. Geomicrobiological processes operating in the shallow sediments of the oligotrophic lakes Laguna Negra (LN)and Lo Encanado (LE). Scheme showing the geochemistry and microbial metabolisms as deduced from the geochemicaland biodiversity analysis (immunological and DNA sequencing) from the sediment samples collected at the shore of LN(LNS), at 4 m below the water surface (LN4), and at the shore of Lo Encanado (LES). Basically, in LN4 the highconcentration of small organic acids (such as acetate and formate), obtained from the fermentation of organic matter(OM), and sulfate are the main drivers for sulfate reduction by sulfate-reducing bacteria (SRB). In this scenario, smallorganic acids are mainly consumed by SRB, and only some acetoclastic methanogens, such as Methanosaeta =Methanothrix strains that have high affinity for acetate, may compete for these resources. Contrarily, in LES sediments,where sulfate concentration is much less, other more versatile methanogens, such as Methanosarcina strains, canproliferate. While anaerobic metabolisms dominated in the LN4 and LES samples, the heterotrophic and oxidative ones,including methane oxidation (methanotrophy), dominate in the shore of LNS (see text for further discussions). Yellowarrows indicate the difference in the amount of sulfate supplied by the water stream. SOB, sulfur oxiding bacteria.
FIG. 6. Potential metabolisms in Laguna Negra and LoEncanado lakes. Based on the prokaryotic diversity frommassive 16 S rRNA gene sequence analysis, we inferred thepotential metabolisms associated with the detected micro-organisms. The chart represents the percentage of the re-trieved sequences that can be ascribed to microbes capableof the corresponding metabolism (ordinates). Sample originis LN4 (Laguna Negra 4 m deep), LES (Lo Encanado shore),and LNS (Laguna Negra shore).
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clear predominance of two methanosarcinales genera, Me-thanosarcina and Methanothrix (Fig. 3).
5.3. Deglaciation evolution determines the microbialmetabolisms in the sediments
Lakes are considered sentinels of climate change(Williamson et al., 2009) that record environmental fluctu-ations in their sediments. As their habitats change, it isexpected that their microbial ecosystems will be modified.Therefore, understanding the link between microbial com-munities and their metabolic traits, climate history, andphysicochemical evolution may reveal microbial markersassociated with different phases of deglaciation.
This work provides a baseline to monitor the geomicro-biological evolution of these two oligotrophic Andean lakesin response to climate change. More data will be necessaryalong with time-course monitoring of the microbial com-munities associated with the types of sediments studied;however, regardless of whether they evolve toward eutroph-ication or oligotrophication with time, the geochemistry andmicrobiology of the sediment will play critical roles in theevolution of these communities. Important variables such asthe nutrient inputs from the water catchment, precipitationregime, temperature, lake stratification, and the proliferationof primary producers will affect the evolutionary pathway ofthe microbial communities as a function of time.
Figure 7 illustrates the metabolic processes that we found tooperate in the Laguna Negra and Lo Encanado sediments alongwith a schematic of our hypothesis for the behavior of lakesediments in an advanced deglaciation scenario. In this case,recently deglaciated soils might be colonized by a diversecommunity of cyanobacteria and other microbes during the firstyears following the glacial retreat (Schmidt et al., 2008). Inthese soils, carbon and nitrogen fixation would dominate andincrease the production of organic matter in the watershed.Such organic material would feed the glacial lakes throughrunoff, and depending on the pluviometric regime and glacialmelting, produce an increase in the internal primary productionthat would lead to greater sedimentation of organic matter.Under aerobic conditions, microbial heterotrophs woulddominate organic matter oxidation, as is the case of LNS(Fig. 7). In deeper and anaerobic sediments where sulfate fromthe catchment accumulates, fermenters would oxidize thecomplex organic material and produce small-size organic acidsto fuel sulfate reduction and specialized acetoclastic metha-nogenesis as in LN4 (Fig. 7). However, in lakes at a later de-glatiation stage, with higher organic content and sedimentation,the anaerobic metabolisms already dominate even in the shal-low sediments of the shore as in LES (Fig. 7 right): fermenta-tion, anaerobic ammonia oxidation, sulfate reduction, and theversatile multi-substrate-powered methanogenesis. In fact, themetabolic profiles from LES and the deeper LN4 sediments aremore similar between them than with the LNS (in agreementwith the LDChip results), with the difference that, among themethanogens, only the highly specialized acetoclastic Metha-nosaeta spp. are detected in LN4. Whether similar processesocurred on ancient Mars is unknown; however, deglaciationphenomena might have generated new sedimentary environ-ments where a rich microbial population flourished. Remnantsof such ancient environments are candidate targets for thesearch for remains of life on future planetary missions.
6. Conclusions
Understanding the evolution of high-altitude oligotrophiclakes requires knowledge of their geochemical and geomi-crobiological characteristics. For Laguna Negra and LoEncanado, we have presented critical geochemical featuresand the prokaryotic community compositions of each lake,and inferred their operating microbial metabolisms in shal-low sediments. Although the waters are oligotrophic, thesediments from the shore of Lo Encanado and from 4 mbelow the water surface in Laguna Negra are rich in nutri-ents and contain a great bacterial diversity, with sulfate re-duction and methanogenesis as key anaerobic metabolisms.Although attempting to associate alteration of the lakes toclimate change would be a complex task that would needtime-course monitoring, our data constitute a reference pointfor future studies. Additionally, we demonstrated that on siteimmunosensing techniques such as the LDChip for bio-markers and microbial detection are useful tools for suchstudies. Ongoing geochemical and microbiological work inthe water column at different depths and deeper sedimentsamples will help clarify the effect of deglaciation phases onthe prokaryotic communities.
Acknowledgments
We thank Aguas Andinas Company (Santiago, Chile) fortheir help and issuing of the permits to access to the field sites,Campoalto Operaciones for its support in the field, and MiriamGarcıa-Villadangos for technical assistance. This work wasfunded by the Spanish ‘‘Secretarıa de Estado de InvestigacionDesarrollo e Innovacion’’ from the Economy and Competi-tiveness Ministry (MINECO) grants No. AYA2011-24803 andESP2014-58494-R ‘‘Detection of Biomolecules in PlanetaryExploration,’’ and NASA’s ASTEP grant No. 10-ASTEP10-0011 ‘‘Planetary Lake Lander.’’ F.P. is a JAE-pre fellow fromthe Consejo Superior de Investigaciones Cientıficas (CSIC).
DNA sequences have been deposited in Genebank underaccession number PRJNA256272.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:Victor Parro
Department of Molecular EvolutionCentro de Astrobiologıa (INTA-CSIC)
Carretera de Ajalvir km 4Torrejon de Ardoz, Madrid
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