CHAPTER 2
From Rıo Tinto to Mars:The Terrestrial andExtraterrestrial Ecologyof Acidophiles
R. Amils,*,†,1 E. Gonzalez-Toril,† A. Aguilera,†
N. Rodrıguez,† D. Fernandez-Remolar,† F. Gomez,†
A. Garcıa-Moyano,‡ M. Malki,* M. Oggerin,†
I. Sanchez-Andrea,*,§ and J. L. Sanz§
Contents I. Introduction 42
II. Extremophiles 43
III. Acidophiles 44
IV. Geomicrobiology of Rıo Tinto 46
A. Water column 46
B. Sediments 49
C. Eukaryotes 52
V. Subsurface Geomicrobiology of the
Iberian Pyrite Belt 55
VI. Methanogenesis in Nonmethanogenic Conditions 59
VII. Rıo Tinto as a Geochemical Analogue of Mars 62
VIII. Future Trends 64
References 65
Advances in Applied Microbiology, Volume 77 # 2011 Elsevier Inc.ISSN 0065-2164, DOI: 10.1016/B978-0-12-387044-5.00002-9 All rights reserved.
* Centro de Biologıa Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid,Madrid, Spain
{ Centro de Astrobiologıa (CSIC-INTA), Madrid, Spain{ Department of Biology, University of Bergen, Bergen, Norway} Departamento de Biologıa Molecular, Universidad Autonoma de Madrid, Madrid, Spain1 Corresponding author: e-mail address: [email protected]
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Abstract The recent geomicrobiological characterization of Rıo Tinto, Ibe-
rian Pyrite Belt (IPB), has proven the importance of the iron cycle,
not only in generating the extreme conditions of the habitat (low
pH, high concentration of toxic heavy metals) but also in maintain-
ing the high level of microbial diversity, both prokaryotic and
eukaryotic, detected in the water column and the sediments. The
extreme conditions of the Tinto basin are not the product of
industrial contamination but the consequence of the presence
of an underground bioreactor that obtains its energy from the
massive sulfide minerals of the IPB. To test this hypothesis, a drilling
project was carried out to intersect ground waters that interact
with the mineral ore in order to provide evidence of subsurface
microbial activities and the potential resources to support these
activities. The oxidants that drive the system appear to come from
the rock matrix, contradicting conventional acid mine drainage
models. These resources need only groundwater to launch micro-
bial metabolism. There are several similarities between the vast
deposits of sulfates and iron oxides on Mars and the main
sulfide-containing iron bioleaching products found in the Tinto.
Firstly, the short-lived methane detected both in Mars’ atmosphere
and in the sediments and subsurface of the IPB and secondly, the
abundance of iron, common to both. The physicochemical proper-
ties of iron make it a source of energy, a shield against radiation and
oxidative stress as well as a natural pH controller. These similarities
have led to Rıo Tinto’s status as a Mars terrestrial analogue.
I. INTRODUCTION
One of the major goals of microbiology is to find the limits of life andidentify the mechanisms that set these limits. The exploration of extremeenvironments has led to the discovery of numerous habitats that had beenconsidered uninhabitable only a few years earlier. As a consequence,interest in the diversity and ecology of extreme environments hasgrown for a variety of reasons. Some are fundamental and search for thelimits of life. Others are more practical and study the potential use ofextremophiles and their components in biotechnological processes (e.g.,biomining, bioremediation).
Extremophiles have also had an important role in the development ofastrobiology. According to the NASA Astrobiology Roadmap (http://astrobiology.arc.nasa.gov), one of the main goals of this interdisciplinaryarea of research is to characterize extreme environments, the organismsthriving in them, and the mechanisms by which these organisms are ableto cope with the extreme conditions of the system in which they develop.The evaluation of the first astrobiological experiments performed by the
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Viking missions on Mars in the 1970s concluded that life had little chanceof developing there due to the extreme conditions detected on its surface:intense UV radiation, presence of strong oxidant compounds, absence ofwater, and extremely low temperatures (Margulis et al., 1979). In the past40 years, different advances in science, in general, and microbiology, inparticular, have challenged this rather pessimistic point of view. Researchon extremophiles has increased the chances of finding life in other parts ofthe universe and shown that life is not bound, as we thought previously,to the mild environmental conditions required by the complex eukaryotesthat had been used as reference systems. Although we are still unable todefine life (Margulis, 2000), we know that it is extremely robust andcapable of adapting to many different conditions.
In this chapter, we review the concept of extremophiles, paying specialattention to the acidophilic microorganisms because unlike many otherextremophiles that can adapt to diverse geophysical constrains (tempera-ture, radiation, ionic strength, pressure, etc.), acidophiles actually thrivein the extreme conditions their chemolithotrophic metabolisms generate.In addition, the inorganic products of this metabolism may play animportant part in the formation of specific minerals which are, in turn,extremely important biosignatures that verywellmay lead to the detectionof similar microorganisms in remote locations.
II. EXTREMOPHILES
One of the first observations of extremophiles took place over 100 yearsago when microorganisms able to spoil salt-preserved codfish werediscovered. Salting food was a very common food preservation methodat that time, so the presence of these extremophiles posed a seriousproblem. These peculiar microorganisms were named halophiles becausethey were able to proliferate at extremely high concentrations of salt.Interest in this type of microorganism decreased after the fish conserva-tion industry solved the problem by replacing the salt obtained frommarine water evaporation with salt extracted from continental mines,which contained far fewer viable halophilic microorganisms.
The systematic study of extremophiles started in the 1970s as a resultof the pioneering work of Brock and collaborators, who were able toisolate microorganisms growing at the high temperatures of differentvolcanic features of Yellowstone (Brock, 1995), and Brierley, who isolateda hyperthermophilic sulfur-oxidizing microorganism from the same area(Brierley and Brierley, 1973). The interest in extremophiles was dovetailedwith the Woese and colleagues’ new phylogenetic concepts based onsequence comparison of the ribonucleotides from the small ribosomalsubunits (16–18S rRNA) (Woese and Fox, 1977). This methodology led
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to the discovery of a new group of prokaryotic microorganisms (kingdomArchaebacteria), different from the classical members of the bacterial andeukaryotic kingdoms. The kingdom Archaebacteria (renamed later asdomain Archaea) included the previously mentioned halophiles, togetherwith hyperthermophiles (microorganisms able to grow at extremely hightemperatures) and methanogens (methane-producing microorganismsthat require strict anaerobic conditions to grow). The term ‘‘Archaebac-teria’’ implied a status of evolutionary antiquity due to the extremophiliccharacter of most of the members of the group. This concept waschallenged after the demonstration, using complete sequences of rRNAgenes, that Archaea were evolutionarily closer to eukaryotes than tobacteria, in spite of their phenotypic prokaryotic properties (Woeseet al., 1990). In addition, thorough microbial characterizations of differentextreme environments showed that some bacteria are also able to developunder the same extreme conditions as archaea.
III. ACIDOPHILES
As mentioned, acidic environments are especially interesting because, ingeneral, the extreme low pH of their habitats is the result of microbialmetabolism and not a condition imposed by the environment in whichthey live, as is the case for the other extremophiles. Acidic environmentshave two major origins. The first is associated with volcanic activity. Theacidity in this case derives from the microbial oxidation of the elementalsulfur produced as a result of the condensation reaction between oxidizedand reduced volcanic gases
2S0 þ 3O2 þ 2H2O ! 2SO42 " þ 4Hþ. (1)
Acidic, metal-rich environments can also be found associated tomining activities. Coal andmetalminingoperations expose sulfidemineralsto the combined action of water and oxygen, which facilitate microbialdevelopment, generating acid mine drainage (AMD) or acid rock drainage,which are the cause of important environmental problems ( Johnson andHallberg, 2003).
The mechanism by which microbes obtain energy by oxidizing sulfideminerals, a process of biotechnological interest (biohydrometallurgy),was controversial for many years (Ehrlich, 2002) but the demonstrationthat the ferric iron present in the cell envelopes of leaching microorgan-isms is responsible for the electron transfer from insoluble sulfidic mineralsubstrates to the electron transport chain has done much to clarify thesituation (Sand et al., 1995). The differences observed using various sulfideminerals are determined by the chemical oxidation mechanism, whichdepends on the structure of the mineral substrate. Three metal sulfides,
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pyrite, molybdenate, and tungstenite, undergo chemical ferric iron oxida-tion through the so-called thiosulfate mechanism:
FeS2 þ 6Fe3 þ þ 3H2O ! S2O32 " þ 7Fe2 þ þ 6Hþ (2)
S2O32 " þ 8Fe3 þ þ 5H2O ! 2SO4
2 " þ 8Fe2 þ þ 10Hþ (3)
in which sulfuric acid is the main product of the reaction (Sand et al.,2001). The rest of the sulfides (e.g., chalcopyrite, sphalerite, and galena)are susceptible to ferric iron oxidation through another pathway, thepolysulfide mechanism:
8MSþ 8Fe3þ þ 8Hþ ! 8M2þ þ 4H2Sn þ 8Fe2þ n # 2ð Þ; (4)
4H2Sn þ 8Fe3 þ ! S8o þ 8Fe2 þ þ 8Hþ. (5)
In this case, elemental sulfur is the final product, and the metabolicactivity of sulfur-oxidizing microorganisms is needed to generate sulfuricacid. The reduced iron produced in these reactions can then be reoxidizedby iron-oxidizing microorganisms:
4Fe2 þ þ O2 þ 2Hþ ! 2Fe3 þ þ 2H2O. (6)
The main role of acidophilic chemolithotrophic microorganisms is tomaintain a high concentration of ferric iron, the chemical oxidant. Theacidophilic strict chemolithotroph Acidithiobacillus ferrooxidans (formerlyThiobacillus ferrooxidans) was first isolated from a coal mine AMD in the1940s (Colmer et al., 1950). Although A. ferrooxidans can obtain energyoxidizing both reduced sulfur and ferrous iron, bioenergetic considera-tions gave much more importance to the sulfide oxidation reaction (Amilset al., 2004; Ehrlich, 2002; Pronk et al., 1992). The discovery that some strictchemolithotrophs like Leptospirillum ferrooxidans can grow using ferrousiron as their only source of energy and that they have an important role inbioleaching operations and in the generation of AMD, has completelychanged this point of view (Edwards et al., 2000; Golyshina et al., 2000;Rawlings, 2002). Further, it is now well established that iron can beoxidized anaerobically, coupled to anoxygenic photosynthesis or toanaerobic respiration using nitrate as an electron acceptor (Benz et al.,1998; Widdel et al., 1993).
Most of the characterized strict acidophilic microorganisms have beenisolated from volcanic areas or AMD from mining activities. Rıo Tinto(Fig. 2.1) is an unusual ecosystem due to its acidity (mean pH 2.3, bufferedby ferric iron), length (92 km), high concentration of toxic heavy metals(Fe, As, Cu, Zn, Ni. . .), and an unexpected level of microbial diversity,mainly eukaryotic (Aguilera et al., 2006a, 2007a,b; Amaral-Zettler et al.,2002; Lopez-Archilla et al., 2001). It has recently been proved that theextreme acidic conditions of the Tinto system are much older than the
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oldest mining activities in the area, strongly suggesting that they arenatural and not the product of industrial contamination (Fernandez-Remolar et al., 2003, 2005). Due to its size and easy access, Rıo Tinto isconsidered an excellent model for the study of the microbial ecology ofextreme acidic environments.
Although molecular ecology methods allow rapid characterization ofthe diversity of complex systems, isolation of the different constituents isessential to study their phenotypic properties in order to evaluate theirrole in the system and their biotechnological potential. Acidic environ-ments are poorly characterized due to the physiological peculiarities ofthe microorganisms associated to them. Further, strict acidophilic chemo-lithotrophs are, in general, difficult to grow, especially in solid media, andas a consequence difficult to isolate (Hallberg and Johnson, 2001; Johnsonand Hallberg, 2003).
IV. GEOMICROBIOLOGY OF RIO TINTO
A. Water column
The combined use of conventional and molecular microbial ecologymethodologies has led to the identification of the most representativemicroorganisms of the Tinto basin (Gonzalez-Toril et al., 2003, 2006,2010). Eighty percent of the water column diversity corresponds to
FIGURE 2.1 Rıo Tinto at Berrocal, in the middle section of the river.
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microorganisms belonging to three bacterial genera, Leptospirillum, Acid-ithiobacillus, and Acidiphilium, and all members of the iron cycle(Gonzalez-Toril et al., 2003). All Leptospirillum isolated from Rıo Tintoare aerobic iron oxidizers. A. ferrooxidans can oxidize ferrous iron aerobi-cally and reduce ferric iron in anaerobic conditions (Malki et al., 2006).All Acidiphilium isolates can oxidize organic compounds using ferric ironas electron acceptor. Interestingly enough, some Acidiphilium isolates cando so in the presence of oxygen (Coupland and Johnson, 2008; Malki et al.,2008). Although other iron oxidizers (like the archaea Ferroplasma spp.and Thermoplasma acidophilum) or iron reducers (Ferrimicrobium spp.) havebeen detected in the Tinto system (Gonzalez-Toril et al., 2003, 2010), theirlow numbers suggest that they play a minor role in the operation of theiron cycle, at least in the water column.
Concerning the sulfur cycle, only A. ferrooxidans is found in significantnumbers in the water column. This bacterium can oxidize both ferrousiron and reduced sulfur compounds. Reduced sulfur compounds can beoxidized aerobically and anaerobically. Certain sulfate-reducing micro-organisms have been detected in the sediments in some locations alongthe river (Garcıa-Moyano et al., 2009; Malki et al., 2006; Sanchez-Andreaet al., 2011).
The characterization of macroscopic filamentous structures from RıoTinto (Garcıa-Moyano et al., 2007) has shown that they are made upmainly of prokaryotic cells enmeshed in a matrix of exopolysaccharidesand some mineral particles. Typical representative organisms from AMDdominate these communities, although they differ in microbial composi-tion, and probably in origin, from acid streamers present in other habitats(Hallberg et al., 2006). Most of the prokaryotic diversity can be attributedto the main bacterial genera found in the water column: A. ferrooxidans,L. ferrooxidans, and Acidiphilium spp. A minority of bacterial and archaealgroups are also represented, some of them detected recently in the anoxicsediments of the river (Garcıa-Moyano et al., 2009; Sanchez-Andrea et al.,2011), which suggest that these peculiar filaments could originate in thedeeper parts of the river as a typical acid streamer attached to the rocks ofthe sediments and be pulled up toward the surface when they reachcertain buoyancy (Garcıa-Moyano et al., 2007). The most important pro-karyotic microorganisms detected so far in the water column of the Tintobasin are listed in Table 2.1 and their phylogenetic ascription shown inFig. 2.2.
Iron has different properties of ecological relevance, which give to theTinto ecosystem an interesting astrobiological perspective: (i) iron is agood electron donor, so it can be used to obtain energy through respira-tion; (ii) also, it is a good electron acceptor, so it can be used for anaerobicrespiration using different electron donors; (iii) the hydrolysis of ferriciron is responsible for the maintenance of a constant pH in the system;
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TABLE 2.1 Phylogenetic affiliation of sequences obtained from clones from Rıo Tinto
and metabolic relationship with the iron and sulfur cycles
Affiliation (phylum/class/family/genus/species) and metabolic relationship with
the iron and sulfur cycles
Proteobacteria
Alfaproteobacteria
Acetobacteraceae Acidisphaera IRB
Acidisphaera rubrifaciens
Acidiphilium IRB
All speciesAcidocella IRB
Acidocella facilis
Betaproteobacteria
Unclassified
Betaproteobacteria
Ferrovum IOB
Ferrovum myxofaciens
Gammaproteobacteria
Xanthomonadaceae Frateuria-like (WJ2 cluster) IRBAcidithiobacillaceae Acidithiobacillus IRB, IOB, SOB
Acidithiobacillus ferrooxidans
Acidithiobacillus ferrivorans
Acidithiobacillus thiooxidans
Acidiferrobacter IRB, IOB, SOB
Acidiferrobacter thiooxidans
Deltaproteobacteria Uncultured bacterium related with
this classPlanctomycetes
Planctomycetacia
Planctomycetaceae Uncultured bacterium
Acidobacteria
Acidobacteria
Acidobacteriaceae Acidobacterium IRB
Acidobacterium capsulata
Acidobacterium spp.Nitrospirae
Nitrospira
Nitrospiraceae Leptospirilum IOB
All species
Cyanobacteria Uncultured bacterium related with this
phylum
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(iv) it has been recently demonstrated that ferric iron and iron mineralsare effective protectors against harmful UV irradiation and oxidativestress (Gomez et al., 2007, 2010). Figure 2.3 shows the integrated geomi-crobiological model of the water column of the Tinto basin, in which theiron cycle plays a central role.
B. Sediments
The characterization of the anoxic sediments from acidic environments,like those from Rıo Tinto, had been neglected up to now, with fewexceptions (Lu et al., 2010) because most of the applied interest of theseecosystems was centered on the aerobic iron- and sulfur-oxidizing micro-organisms. But it is clear that the sediments have to be considered if wewant a thorough understanding of the integrated microbial ecology ofthese peculiar extreme environments. Our group has recently used clon-ing and hybridization techniques to carry out a careful comparative
TABLE 2.1 (continued )
Affiliation (phylum/class/family/genus/species) and metabolic relationship with
the iron and sulfur cycles
FirmicutesBacilli
Alicyclobacillaceae Alicyclobacillus
Alicyclobacillus acidiphilus
Clostridia
Unclassified Clostridiaceae Uncultured bacterium. Probably IRB
Clostridiaceae Uncultured bacterium. Probably IRB
Peptococcaceae Desulfosporosinus SRB
Desulfosporosinus spp.Actinobacteria
Actinobacteria
Acidimicrobiaceae Ferrimicrobium IRB, IOB
All species
TRA2-10 cluster. Probably IRB, IOB
Euryarchaeota
Thermoplasmata
Thermoplasmataceae Ferroplasma IOAFerroplasma spp.
Uncultured archaea. Could be IOA
IRB, iron-reducing bacteria; IOB, iron-oxidizing bacteria; SOB, sulfur-oxidizing bacteria; SRB, sulfur-reducingbacteria; IOA, iron-oxidizing archaea.
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analysis of the sediments and the water column of different samples alongthe physicochemical gradient of Rıo Tinto (Garcıa-Moyano et al., 2009).The main conclusions drawn from this study showed: (i) a significantlyhigher biomass and diversity detected in the sediments when comparedto its water column counterpart from the same sampling station and (ii)the existence of a diversity gradient, most probably a consequence of thegeochemical gradient existing along the course of the river. Nearly all themicroorganisms detected and identified in this study are, in one way oranother, related to the iron cycle. Most were previously detected and/orisolated in AMD sites (Gonzalez-Toril et al., 2003; Johnson and Hallberg,2003) or biohydrometallurgical operations (Rawlings, 2005). Nonetheless,some microorganisms, such as members of Actinobacteria, Firmicutes,Acidobacteria, Cyanobacteria, Planctomycetes, and Chloroflexi, have beenidentified for the first time in the Tinto basin.
An in-depth analysis of two anoxic sediments from Rıo Tinto hasrecently shown that the distribution of major phylogenies differedamong sample sites (Sanchez-Andrea et al., 2011). In one of the sediments,JL Dam, the most numerous group of Bacteria corresponded to the phy-lum Firmicutes (56.6%), followed by the phylum Acidobacteria (27.3%), andthe class Deltaproteobacteria (11.6%). Organisms from the phylum
Bacilli/Clostridia
Actinobacteria
OP2
OP9
OP8OP3
OP10
Aquificae
Therm
otog
ae
Cop
roth
erm
obac
ter
Therm
odesu
lfobact
eria
Chloroflexi
Thermomicrobia
Deinicocci
Bacteroidetes/Flavobacteria/
Sphingobacteriaa
Fibrobacteres
Nitrospira
Spirochaetes
Fusobacteria
Chlorobia
Planctomycetacia
VerromicrobiaeChlamydiae
Acidobacteria
Cyanobacteria
e-Proteobacteria
Euryarchaeota
Crenarchaeota
Koraarchaeota
OP1
d-Proteobacteria
0.1
b/g-Proteobacteria
a-Proteobacteria
Nitr
ospi
na
Defe
rrib
acte
res
Am
inobacte
rium
et al.
FIGURE 2.2 Prokaryotic phylogenetic affiliation of acidophilic microorganisms identi-
fied in the Tinto basin.
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Actinobacteria and the Gammaproteobacteria class were much less abundant(0.4%). In the SN Dam, the phylum Proteobacteria was the most repre-sented: Alfaproteobacteria (39.6%) and Gammaproteobacteria (30.4%),followed by Actinobacteria (20.4%). Organisms of the Firmicutes (5.3%)and Acidobacteria (1.7%) phyla were present in low percentages. Thisstudy also showed a differential pattern of distribution in the sedimentsbased on depth. In the surface layers of JL Dam, sequences belonging tothe phylum Acidobacteria were found, while in the intermediate layer,sequences of the phylum Firmicutes were detected, and in the deepestlayers, extremely anaerobic organisms were found, including sulfate-reducing bacteria such asDesulfosporosinus andDesulfurella. In the surfacelayer of the SN Dam, sequences belonging to the Acidithiobacillaceae family(Gammaproteobacteria) were identified. In the deepest layers, organismsrelated to the spore-forming sulfate-reducing bacteria Desulfosporosinuswere detected. Some of these microorganisms have been identified previ-ously in the floating macroscopic filaments of the river (Garcıa-Moyanoet al., 2007).
Bioleaching processes and high evaporation rates induce the formationof concentrated acidic brines (Fernandez-Remolar et al., 2003). Iron oxidesassociated to sulfates are the characteristic minerals that are formed in the
SRBA. ferrooxidansA. thiooxidans
A. caldus(CH2O)n
Acidiphilium spp.Acidimicrobium spp.
Ferromicrobium spp.
So
A. ferrooxidans
Acidiphilium spp.
Oxic[O2]
Fe2+
A. ferrooxidansL. ferrooxidans
Ferroplasma spp.
Acidimicrobium spp.
Ferromicrobium spp.
Anoxic[O2]
SO42-
CO2
Fe3+ + H2O Fe(OH)3+H+
Fe2O3
(CH2O)n
CO2
FIGURE 2.3 Geomicrobiological model of the iron and sulfur cycles operating in the
water column of the Tinto basin.
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modern sediments and young terraces: hydronium jarosite, schwertman-nite, copiapite, coquimbite, natronojarosite, gypsum, and other sulfateminerals, while gothite and hematite are the predominant minerals inthe old terraces of the Tinto basin (Fernandez-Remolar et al., 2005).
C. Eukaryotes
It is usually assumed that the toxicity of high metal concentrations inacidic habitats limits eukaryotic growth and diversity (Gross, 2000). How-ever, colorful biofilms covering large surfaces of the Tinto basin as well asfilamentous microbial communities and macroscopic algae are commonfeatures of acidic environments (Aguilera et al., 2006a,b, 2007a). In fact,eukaryotic algae contribute over 60% of the river biomass (Lopez-Archillaet al., 2001). The eukaryotic biodiversity in the ecosystem includes speciesof most of the major lineages (Aguilera et al., 2006b, 2007a,b; Amaral-Zettler et al., 2002; Lopez-Archilla et al., 2001). Most of the eukaryoticspecies thriving in Rıo Tinto are photosynthetic. Among them, chloro-phytes related to different genera such as Chlamydomonas, Dunaliella,Chlorella, as well as Euglena are the dominant eukaryotic microorganismspresent in the river, and they form large green patches all along the riverbed. These species are known for their high metal tolerance (Aguilera andAmils, 2005; Fisher et al., 1998; Olaveson and Nalewajko, 1994). Filamen-tous algae, represented by the genera Zygnemopsis and Klebsormidium,have also been found. The occurrence of both filamentous species ishigher during the dry summer months, when most physicochemicalparameters are more extreme. Other chlorophytes, such as species of thegeneraMesotaenium and Stichococcus, have been also detected, although inlow numbers.
The most acidic part of the river is inhabited by a eukaryotic commu-nity dominated by two species related to the genera Dunaliella (Chloro-phyta) and Cyanidium (Rhodophyta). The genus Dunaliella includes some ofthe most extreme acidophiles reported so far (Gimmler and Weis, 1992).Pennate diatoms are also present in the river forming large brown bio-films. These biofilms are usually dominated by only one species related tothe genus Pinnularia, although some other minority genera have beenidentified, including Nitzschia or Cyclotella.
In addition to photosynthetic species, heterotrophic protists are alsowidely distributed along the river. The mixotrophic flagellates are domi-nated bymembers of the generaBodo andOchromonas. At least, two speciesof ciliates are members of the community. The dominant ciliate taxabelong to the order Hypotrichida. Although two different species havebeenmicroscopically observed, only clones related toOxytrichia granuliferahave been molecularly identified. Amoebas are frequently found feedingon large diatoms, even in the most acidic part of the river. Vahlkampfia
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species have been identified microscopically as well as other species,including lobosea-like and acanthamoeba-like amoebas. Other species ofheliozoan belonging to the genus Actinophyris are also present in the river.Heliozoa seem to be characteristic top predators of the benthic food chainin the river. The only animal found in the river is a species of bdelloidrotifer related to the genus Rotifera (Amaral-Zettler et al., 2002). Thispioneer rotifer species can persist because of their high physiologicaltolerance to severe acidic stress and the lack of other more efficient com-petitors. The genus of the main protists identified up to now in the Tintobasin are listed in Table 2.2. A display of some acidophilic eukaryotes isshown in Fig. 2.4.
Among decomposers, fungi are the most abundant, and both unicel-lular and filamentous forms are present (,Lopez- Archilla et al., 2005;Lopez-Archilla et al., 2001). While many species of fungi have beenisolated from the river, one fungus (related to Hobsonia) has been identi-fied in many parts of the river where it forms dendritic macrofilamentsclosely associated with other protists. When the fungus is present, acommunity, embedded in a mucilaginous substance, forms to protect
TABLE.2.2 Eukaryotic protists detected in the Tinto basin
Order Family Genus ID technique
Volvocales Chlamydomonadaceae Chlamydomona LM/DG/18S
Volvocales Dunaliellaceae Dunaliella LM/DG/18SChlorellales Chlorellaceae Chlorella LM/DG/18S
Zygnematales Mesotaeniaceae Mesotaenium M/DG
Zygnematales Zygnemataceae Zygnemopsi LM/DG/18S
Ulotrichales Ulotrichaceae Stichococcus LM/18S
Klebsormidiales Klebsormidiaceae Klebsormidium LM/18S
Naviculales Pinnulariaceae Pinnularia LM/DG/18S
Euglenales Euglenophyceae Euglena LM
Porphyridiales Porphyridiaceae Cyanidium LMSchizopyrenida Vahlkampfiidae Vahlkampfia LM
Schizopyrenida Vahlkampfiidae Naegleria LM
Actinophryida Actinophyridae Actinophrys LM
Kinetoplastida Bodonidae Bodo LM
Ebriida Cercomonadidae Cercomonas LM
Ochomonadales Ochromonadaceae Ochromonas LM
Labyrinthulida Labyrinthulidae Labyrinthula LM/18S
Bdelloidea Philodinidae Rotaria LMStichotrichida Oxythrichidae Oxytricha LM/DG/18S
Hymenostomatida Turaniellidae Colpidium LM
LM, light microscopy; DG, DGGE; 18S, 18S rRNA gene cloning.
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the inner microbial community from the external extreme conditions bycreating differential physicochemical conditions.
Most of these microorganisms form complex photosynthetic biofilmswhich differ in composition and structure along the physicochemicalgradient of the river, most of them attached to the surface of rocks(Aguilera et al., 2007b, 2008a,b; Souza-Egipsy et al., 2011). Fungi seem to
A B
C
E F
D
FIGURE 2.4 Gallery of acidophilic eukaryotes detected in different sampling stations
along the river. (A) Filamentous green algae Klebsormidium sp., (B) Amoebas, (C) Green
algae Chlamydomonas spp., (D) Heliozoa actinophrys sp., (E) Diatoms, and (F) Euglena
mutabilis.
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play a fundamental role in their development, and the most abundantbacteria from the water column can be found associated to them (Souza-Egipsy et al., 2008).
V. SUBSURFACE GEOMICROBIOLOGY OF THEIBERIAN PYRITE BELT
From the results discussed so far, it is clear that the main characteristics ofthe Tinto basin are not the product of industrial contamination but aconsequence of the existence of an underground reactor in which themassive sulfide minerals of the Iberian Pyrite Belt (IPB) are the mainenergy source and the river is the exhaust pipe releasing the products ofthe metabolic reactions occurring in the subsurface. To test this hypothe-sis, a drilling project, MARTE project, was developed to intersect groundwaters interacting with the mineral ore to provide evidence of subsurfacemicrobial activities and the potential resources to support these activitiesin situ (Amils et al., 2008; Fernandez-Remolar et al., 2008a,b).
The main goal of the MARTE project, a collaborative effort betweenNASA and the Centro de Astrobiologıa, was the search for subsurfacemicrobial activity associated to the IPB. The selected study site was Penade Hierro on the north flank of the Rıo Tinto anticline. The hydrothermalactivity in the area is recorded as complex-massive sulfide lenses orstockwork veins of pyrite and quartz, which occur at the upper part ofthe IPB volcanic sequence (Leistel et al., 1998).
The well locations were selected to monitor spatial changes in microbialand hydrogeochemical processes. Coring was carried out using a commer-cial coring rig at three locations designated BH1, BH4, and BH8. The bore-holes were continuously cored by rotary diamond-bit drilling using awireline system that produced 60-mm diameter cores within a plasticliner. Water was used as drilling fluid to refrigerate the bit. NaBr wasused as a chemical tracer for controlling contamination introduced duringthe drilling. Upon retrieval, cores were flushed with N2, sealed and trans-ported to a nearby laboratory for geomicrobiological analysis. Sampleswere prepared aseptically in anaerobic conditions using an anaerobicchamber. After drilling, the wells were completed by installing PVC cas-ings set in clean gravel packing. Underground sampling for water and gasaquifer analysis was done by the installation of multilevel diffusion sam-plers (MLDS) at different depth intervals. Anion and metal concentrationsand dissolved gases were determined by ion and gas chromatography.
The groundwater entering the ore body at Pena de Hierro was char-acterized by analyzing springs upslope. The water from these springs wasaerobic, with a neutral pH and a low ionic strength. The environmentwithin the ore body was sampled by drilling boreholes BH4 and BH8.
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These wells cored around 165 m of pyrite stockwork. The lithology ofborehole BH4 is shown in Fig. 2.5. The water table was encountered atnearly 90 m below the surface. The sulfide ore (ca. 120 m) was a complexmixture of polymetallic sulfide minerals dominated by pyrite (Fernandez-Remolar et al., 2008b).
Rock leachate analyses were performed to detect contamination bydrilling fluids and to estimate resources available to microorganisms fromthe solid phase. Sulfate, as expected, was abundant and a good indicatorof the degree of oxidation of the sulfides. Surprisingly, nitrite and nitratewere present at concentrations higher than 100 ppm in many samples.Both ferrous iron (average concentration 95 ppm) and ferric iron (averageconcentration 22 ppm) could be leached from powdered ore samples.Organic carbon content of the core samples was near the detection limit(0.01%). From the rock leachate experiments, it can be concluded thatelectron acceptors for anaerobic respiration, particularly Fe3þ, SO4
2!, NO2!,
NO3!, and carbonates, are available from the volcanically hosted massive
sulfide (VHMS) deposits of the rock matrix.Borehole fluids from the MLDS were analyzed as a proxy for forma-
tion fluids. Formation water in BH4 was sampled with the MLDS from 85to 105 and from 135 to 150 mbls at different time intervals after drilling.The measured composite pH was ca. 3.5 and has remained acidic for thetwo sampling years after drilling. Dissolve iron ranged from 108 to480 ppm with an average of 188 ppm. The dissolved ferric to ferrousiron ratio ranged from 0.3 to 4.3 and did not appear to correlate with thetotal iron concentration. Sulfate concentration was relatively constant andca. 1000-fold lower than in rock leachates. Neither nitrates nor nitriteswere detected in the water. Small quantities of oxygen and NO2 gas werepresent in some samples, and the two were inversely correlated. Dis-solved methane was detected in many of the MLDS samples, indicatingactive methanogenic activity within the ore body.
Dissolved H2 concentration averaged 25 ppm, except in the zone withinthe massive pyrites, just below the water table, from 90 to 100 mbls, whereconcentrations ranged from 100 to 1000 ppm. A similar pattern wasobserved in the second borehole, BH8, with an average H2 concentrationmeasured 12 months after drilling of ca. 25 ppm and with isolated zoneswith higher concentration. Electron donors available in the VHMS formicrobial metabolism included ferrous iron, reduced sulfur, and H2. Labo-ratory experiments showed thatH2 couldbeproduced by reaction ofVHMSrocks with water. It is reasonable to assume that H2 production supportsmethanogenic activities throughout the wet sections of the VHMS.
Microorganisms were detected in different uncontaminated samplesusing both culture-dependent and culture-independent methods. Distri-bution of microbes was heterogeneous along the column, as expected in asystem dominated by fracture flow. Aerobic chemolithoautotrophs using
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0Alteredtuff
Thiosulfates Iron No iron Methanogens LALDAPI [RS]SUB-SURFACE LITHOLOGY
Gossan
FracturedquartzGossanizedchert
Stockworkwith oxidizedpyrite
Pyriticstockworkwithoxides
Pyriticstockworkwithoxidecracks
Pyriticstockworkwith solvedcracks
Dark chert
Chloritizedtuff
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40
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110
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FIGURE 2.5 Core lithology and location of biological indicators for BH4. Blue-shaded
area indicates the water table. Columns left to right: 1, example images of cores from
each lithology; 2, lithology; 3, growth of denitrifying thiosulfate-oxidizing organisms
in anaerobic chemolithotrophic enrichment cultures; 4, detection of microorganisms
by fluorescence microscopy; 5, growth of iron-oxidizing microorganisms in aerobic
chemolithotrophic enrichment cultures with ferrous iron; 6, growth of organisms in
aerobic chemolithotrophic enrichment cultures with sulfide minerals as source of
energy; 7, growth of methanogens in enrichment cultures with added H2; 8, positive
limulus amebocyte lysate (LAL) assay. Solid lines in columns 3–8 indicate positive results
in samples without detectable bromine tracer; empty lines correspond to samples in
which some drilling fluid was detected.
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enrichment cultures, mainly pyrite and iron oxidizers, and anaerobicthiosulfate oxidizers using nitrate as electron acceptor, sulfate reducersand methanogens, were enriched from several samples (Fig. 2.5). Usingfluorescence in situ hybridization (CARD-FISH), we have been able toprove the presence of active microorganisms in different uncontaminatedsamples and to show that in these conditions the cell number wasextremely low. Higher cell numbers could be seen in cracked samples,which were discarded due to the presence of bromide, a signal of possiblecontamination from the drilling fluid.
The environment down-gradient from the ore body was sampled bydrilling borehole BH1. We considered that in this zone, fluids wouldrepresent the end product of subsurface interaction with the VHMS.Well BH1 cored 59 m of the younger dark shales. Core samples fromBH1 consisted of greenish shales derived from volcanic ash with finesandy lenses and lutites bearing organic matter, which were overlaid by7 m of mine tailings (Fernandez-Remolar et al., 2008b).
As expected, sulfate and iron concentrations were lower in the lea-chates from BH1 shales than those from BH4 and BH8 pyrites. Only smallamounts of NO3
were detected in the leachates. Oxygen was not detectedin the aquifer zone. Where present, dissolved sulfate in groundwater wasin much higher concentrations than in groundwater from BH4 and BH8,indicating that these waters had experienced more interaction with theore. Neither NO2
nor NO3 was detected in water samples; however,
dissolved NOx gases were present at concentrations slightly higher thanin water samples from BH4. Dissolved H2, where detected, was at con-centrations lower than in BH4 but still sufficient to make H2 available as amicrobial electron donor. Methane concentrations were several orders ofmagnitude higher than at BH4. These observations are consistent with theplume of groundwater representing the downstream output from reac-tions within the ore body.
Microorganisms were also observed in BH1. Aerobes or denitrifierswere not detected. Sulfate reducers and methanogens were recoveredfrom enrichment cultures, and the methane concentrations that weremeasured near 18 and 50 mbls suggested that H2 produced within the orebody supports thesemicrobial activities down-gradient. At depths between50 and 60m, themethane-bearingwater appears tomixwith sulfate-bearingwater. Decreasing CH4 and H2 was accompanied by increasing SO4
2 andCO2 concentrations. Although nonstequiometric, this relationship suggeststhat anaerobic methane oxidation may occur in this zone.
The alteration of the sulfide ore induced the production of differentgases: CO2, CH4, and H2, all of them participating in the biogeochemicalcycles involved in the IPB decomposition. The observed characteristics ofthe underground mineralogy, dominated by iron oxyhydroxides andsulfates, resulted from the alteration of the abundant sulfides of the IPB
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by chemolithotrophic microorganisms. As both secondary mineralogyand gas by-products are the result of cryptic microbial communities livingin the Rıo Tinto acidic aquifer, they can be used as potential biomarkers toexplore subsurface life in deep regions.
In contrast to well-knownAMD systems, the environments within anddown-gradient from the Pena de Hierro VHMS appear to be anoxic, witha weakly acidic pH and evidence of methanogenic and sulfate-reducingactivities. Any O2 available from inflowing groundwater would initiallybe available as an electron acceptor for microaerophilic microorganisms,but it could be also consumed by abiotic reactions (Chalk and Smith, 1983;Conrad, 1996). Because dissolved nitrate was not detected, quantitiesleached from the rock matrix are apparently consumed rapidly. Enrich-ment culture results suggest that some denitrifiers are present to utilizenitrate whenever it becomes available.
Some of the spring waters down-gradient from the ore body arelargely acidic, high in ferric iron, and red in color, as previously described(Fernandez-Remolar et al., 2003), which is typical of aerobic AMD pro-cesses. However, another group of springs found in the area producesanaerobic acidic waters with high concentration of ferrous iron. Theorigin of these iron-reduced spring waters remains to be determined(Gonzalez-Toril et al., 2011; Lu et al., 2010).
The preliminary results from the MARTE project indicate that asgroundwater enters in contact with the VHMS system, biotic and abioticprocesses removeO2with the concomitant oxidation of iron and generationof acidity. Electron acceptors available formicrobialmetabolism includeO2,NO2
, NO3 , SO4
2 , Fe3þ, and CO2. Electron donors include Fe2þ, sulfide, andH2 generated by water/rock interaction. This supports a population ofmicroaerophilic and denitrifying autotrophs. As the fluids become morereduced, methanogenesis and sulfate reduction, using H2, become thedominant microbial processes and the pH rises. Oxidants to drive thesystem appear to be supplied by the rockmatrix, in contrast to conventionalAMDmodels. These resources need only groundwater to launch microbialmetabolism. These observations confirmed the hypothesis thatmicroorgan-isms are active in the subsurface of the IPB and are responsible for thecharacteristic extreme conditions detected in the Tinto basin.
VI. METHANOGENESIS IN NONMETHANOGENICCONDITIONS
Althoughmethane can be abiotically generated, 80% of Earth’s methane isbiologically produced as a final product of the degradation of organicmatter in anoxic ecosystems by methanogenic Archaea (Deppenmeler,2002; Thauer et al., 2008). Methanogens are generally found in habitats
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that share two important physicochemical properties: reduced redoxpotentials (under 200 mV) and circumneutral pH (with few exceptionsaround pH 4) (Kotsyurbenko et al., 2007; Taconi et al., 2008). These condi-tions are diametrically opposed to the extreme acidic and oxidative con-ditions existing in Rıo Tinto.
After the detection of methane in the borehole fluids of the MARTEdrilling project, a systematic survey for methanogenic activity wasinitiated in the sediments of the river (Sanz et al., 2011). The first site inwhich methane production was detected in the Tinto basin was Campo deGaldierias. Sediments from this site showed specific positions with nega-tive redox potential, under 200 mV, while in the surrounding sediments,just a few centimeters away, the redox potential values were overþ400 mV, similar to the river water values. Microcosms were establishedusing reduced sediments from this site and spiked by the addition ofdifferent methanogenic substrates (formate, acetate, lactate, methanol, ora volatile fatty acid (VFA) mixture). The best methane stimulation resultwas observed in microcosms spiked with methanol. In all cases, theproduction of methane was associated with a decrease in redox potentialsto negative values and with an increase of pH to values between 5.4 and 6.
A second site, JL Dam, was selected to have access to deeper sediments.Cores from this site showed characteristic well-defined black bandsbetween the otherwise reddish-brown sediments (Fig. 2.6). Black bandswere associated with negative reduced redox potentials and higher pHvalues compared to the positive high redox potentials and acidic pH ofthe adjacent red and brown layers. Total DNA from the black bands wasextracted, preamplified, and sequences corresponding to Methanosaetaconcilii were obtained.
To further explore the methanogenic diversity of the cores, enrichmentcultureswere designed using different substrates. The highest CH4 produc-tion occurred in the presence of lactate–methanol–sucrose mixture. OnlyMethanosaeta concilii was detected in this microcosm, suggesting that thiswas the predominant methanogenic Archaea in environments exposed toorganic substrates. Methanobacterium bryantii and Methanosarcina barkeriwere identified in cultures enriched with H2 or methanol, respectively.
The occurrence of these three types of methanogens deserves somecomment. Methanosarcina barkeri, a methanol-consuming methanogen,was identified in methanol-spiked microcosms and enrichment cultures.In the dam JL sampling site, the predominant Archaea was Methanosaetaconcilii, an acetate-consuming methanogen. A high content of acetatedetected in the black bands could justify its dominance overH2-consumingmethanogens. In addition, the reported inhibition of methanogenesis byferric iron, higher forMethanospirillum hungatei andMethanosarcina barkerigrowing on H2/CO2 than for Methanosaeta concilii and Methanosarcinabarkeri growing on acetate, could explain these results (Bodegom et al.,
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2004; Zhang et al., 2009). Lastly, the occurrence of an exclusiveH2-consum-ing methanogen, Methanobacterium bryantii, was observed only in enrich-ment cultures fed with H2/CO2.
Although the Shelford tolerance law imposes environmental physico-chemical restrictions on the development of life, it seems, at least in thiscase, that they cannot be deduced from the macroscopic properties of thehabitat. The bulk environmental conditions at Rıo Tinto, especially withrespect to pH and redox potential, are far from the conditions required todevelop methanogenic Archaea. This apparent contradiction can beresolved at the microscopic level. The generation of micro-niches,observed in this study at two sampling sites, might lead to the prolifera-tion of microorganisms with very different requirements from those
Deep (cm)
Overlayed water
0
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5.8
5.4
5.9
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-108.6
-33.2
-278.4
-168.6
1141.7
1141.0
pH Eh (mV)
FIGURE 2.6 Core from the anoxic sediments of JL Dam. Eh and pH values at different
depths are shown (Sanz et al., 2011).
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found in the macroscopic habitat. These micro-niches were shown to belocally mildly acidic and reducing environments in which methanogenscould thrive despite the harsh environmental conditions of the surround-ing environment.
If we accept this scenario, new questions arise as to how the unfavorablephysicochemical conditions are modified and which microorganisms areresponsible for the modification. It is well established that iron-reducingbacteria can outcompete methanogenic archaea for acetate and hydrogen.This preferential use of the major methanogenic substrates could lead to aninitial suppression of methanogenesis in iron-rich freshwater sediments(Roden andWetzel, 2003). However, as a consequence of the Fe3þ reductionto Fe2þ, the redox potential decreased, the pH rose (ferric iron is a strongacidic buffer, ferrous iron is not), and the inhibitor ferric iron concentrationdecreased, eventually creating conditions favorable for methanogenesis.Iron reduction in Rıo Tinto is known to be catalyzed by bacteria such as A.ferrooxidans and Acidiphilium spp. Even methanogens themselves could beimplicated in iron reduction (Bodegom et al., 2004).
The presence of methanogens in an environment controlled by oxi-dized iron and sulfur has interesting astrobiological implications since itcould be a scenario for the biological production of the atmosphericmethane that was recently detected on Mars using different methodolo-gies (Formisano et al., 2004; Mumma et al., 2009). The argument that Mars’environmental conditions are not suitable for methanogenesis can bechallenged by the methane production observed in Rıo Tinto. Consider-ing the short lifetime of methane in the Mars atmospheric conditions,there is a possibility that extant methanogens are currently active on thered planet. Future Mars exploration missions should be appropriatelyequipped to test this possibility.
VII. RIO TINTO AS A GEOCHEMICAL ANALOGUE OF MARS
The recent mineralogy described by the MER missions on Mars (ironoxides, iron sulfates, phyllosilicates) is compatible with the geomicrobiol-ogy existing in Rıo Tinto (Fernandez-Remolar et al., 2005). Obviously, theactual conditions in which the Tinto ecosystem operates are differentfrom the ones that might prevail on Mars, but the properties of themicroorganisms isolated so far in this environment allow us to extrapo-late their performance in these systems.
Some considerations concerning water content and environmentaltemperature are required before introducing the Rıo Tinto basin as ananalogue for Mars’ hematite sites (Fairen et al., 2004; Fernandez-Remolaret al., 2004, 2005). As indicated, liquid water is abundant in the Tintobasin, both on the surface and underground. Conversely, due to
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environmental constraints, water appears only in solid or vapor phases onthe current Mars surface. Although we have the orbital technology toreveal the possible existence of liquid water on the subsurface of Mars,there is only indirect evidence of widespread subterranean ice (Boyntonet al., 2002) and direct identification of polar water-ice (Bibring et al., 2005).However, images from Mars, as well as spectral data provided by differ-ent instruments in orbit and on the surface of the planet, give support todistinctive episodes of water release on Mars’ surface in the past, includ-ing ocean-related landforms (Baker, 2001; Clifford and Parker, 2001;Fairen et al., 2003; Head et al., 1998; Parker et al., 1993), massive layeredoutcrops (Malin and Edgett, 2000a), valley networks and accompanyingfluvial redistribution of sediments (Bhattacharya et al., 2005; Craddockand Howard, 2002; Mangold et al., 2004), anastomosing and meanderingrivers and deltas (Malin and Edgett, 2003), cross-stratification in rockoutcrops (Squyres et al., 2005), mineralogies indicating ancient aqueousenvironments over regional scales (Arvidson et al., 2005; Hynek, 2004;Poulet et al., 2005; Squyres et al., 2005), and almost contemporary surfacerunoff (Heldmann and Mellon, 2004; Heldmann et al., 2005; Malin andEdgett, 2000b). Interestingly enough, recent high-resolution images fromthe Mars Reconnaissance Orbiter (McEven et al., 2011) and results fromthe Phoenix landing mission (Smith et al., 2009) suggest the existence ofliquid brines on the surface of Mars theoretically predicted by Fairen et al.(2009) using the ionic conditions reported by different Mars missions.
Climatic studies of the early atmospheric evolution of Mars (Carr,1999) indicate that during the Noachian, the atmospheric pressure washigh enough to sustain substantial amounts of liquid water on its surfacethus answering for the above mentioned water-related features. Thepresence of liquid water is the only constraint on life development onMars given the presence of mineral energy sources (similar to those usedon Earth) and alternative radiation protection mechanisms, like the oneexerted by ferric iron (Gomez et al., 2007, 2010).
The discovery of some Noachian iron lithological units on Mars, thatis, Meridiani Planum (Herkenhoff et al., 2005; Klingelhofer et al., 2005;Rieder et al., 2005; Soderblom et al., 2005; Squyres et al., 2005; Zolotov andShock, 2005), suggests the Rıo Tinto basin as a possible analogue withwhich to better understand those geomicrobiological processes that mayhave driven the generation of iron oxides and sulfates on the NeochianMars. One of the sulfates identified on Mars, jarosite, can only be formedin acidic conditions (Bigham et al., 1996), giving a possible scenario for theformation of sedimentary rocks in Meridiani Planum and explaining thelack of carbonates on Mars due to the acidic conditions of its water bodies(Fairen et al., 2004). The existence of mineral relics in the Tinto basin mighthelp to unravel those rock-forming processes involved in the transforma-tion of iron-rich sediments (Fernandez-Remolar et al., 2003, 2005).
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Although there is only a remote possibility that the Martian hematiticformations are the product of chemolithoautotrophy, the microbial diver-sity found in the Tinto basin, with metabolisms compatible with theconditions prevailing on Mars, allows us to suggest that microorganismsmay have or still growing in places where mineral and water converge(Amils et al., 2007; Fernandez-Remolar et al., 2004). It should be pointedout that we are dealing with an extant ecosystem. Appropriate questionscould facilitate a more detailed characterization of the system, which inturn would help to clarify its origin and the role of the different compo-nents of the habitat in different evolutionary scenarios.
VIII. FUTURE TRENDS
As the genesis of the extreme Rıo Tinto conditions becomes clearer,projects to gain insight into diverse and complementary aspects of thesystem have been undertaken: (i) The systematic study of the anoxicsediments of the river aims to determine the level of microbial diversityin this important phase of the ecosystem, and a high level of microdiver-sity is emerging from the preliminary studies (Garcıa-Moyano et al., 2009;Sanchez-Andrea et al., 2011; Sanz et al., 2011). Understanding its microbialecology, which is probably quite different along the physicochemicalgradient of the river, presents exciting challenges. Some newmicroorgan-isms have already been identified by cloning, and specific probes areunder design to evaluate their cell number using hybridization methodol-ogies (Gonzalez-Toril et al., 2006). (ii) Further exploration of the subsur-face geomicrobiology of the IPB will clarify many aspects of the complexunderground ecosystem that generates the extreme conditions in theTinto basin. The results of the MARTE project have led to a new drillinginitiative to analyze subsurface microbial activity in real time. This proj-ect, known as IPBSL and sponsored by the European Research Councilstarted operations in 2011. Its main challenge is to design probes withwhich to follow the evolution of functional metabolites at different depthsin the rock matrix. (iii) The comparative study of iron bioformationsshould allow us to understand the generation and identification ofbiosignatures, a critical step for the detection of life signatures on Mars(Fernandez-Remolar et al., 2005). (iv) Different omics are being tested tostudy the differential gene expression of the main microorganisms(A. ferrooxidans, L. ferrooxidans, and Acidiphilium sp.) operating along thephysicochemical gradient of the Tinto basin. (v) Preliminary resultsshowed the ability of extreme chemolithoautotrophs to feed on ironmeteorites (Gonzalez-Toril et al., 2005), and since the discovery of differ-ent meteorites of this class on Mars, this interesting ability shouldbe studied in greater detail. (vi) Iron-oxidizing and iron-reducing
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acidophiles are being tested for their aptness for use as anodes andcathodes of microbial fuel cells (Carbajosa et al., 2010; Malki et al., 2008).
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