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Home , Acidimicrobium, Methanococcus

Biotechnological application of

R. Amils CBMSO and CAB

Gran Sasso, November 2019 extreme environments geophysical constrains - high temperature: hipertehermophiles - low temperature: psicrophiles - high ionic strength: - high pressure: barophiles - high radiation (adaptation) Charles Darwin Yellowstone submarine hydrothermalism Uyuni salt flat, Bolivia radiation + desecation Volcan Illimani, Bolivia Acidic hypersaline lake (SW- Australia) radiation (nuclear plant Japan) Russian drilling base at Vostok subsurface extreme environments geophysical constrains - high temperature: hipertehermophiles - low temperature: psicrophiles - high ionic strength: halophiles - high pressure: barophiles - high radiation (adaptation)

geomicrobiology of metallic sulfides pyrite, molibdenite, tungstenite (thiosulfate mec.) 3+ 2- 2+ + FeS2+6Fe +3H2O → S2O3 +7Fe +6H 2- 3+ 2- 2+ + S2O3 +8Fe +5H2O → 2SO4 +8Fe +10H rest of sulfides (polisulfide mec.) 3+ + 2+ 2+ 8MS+8Fe +8H → 8M +4H2Sn+8Fe (n≥2) 3+ o 2+ + 4H2Sn+8Fe → S8 +8Fe +8H o 2- + S8 +4H2O (S oxidizers) → SO4 +8H come-meteoritos role of the microbial activity in the leaching of pyrite

chemical 3+ reaction Fe

Fe2+ microbial activity

2- + SO4 + H

isolation is fundamental to study the physiological properties of microorganisms

A B Step 4 Step 1

+ Fe(II) + 4 O2 + 4H2O Fe(III) + 8H oxidation and reduction of Fe by At. ferrooxidans

2- + Sº + Fe(III) + + 4H2O SO4 + Fe(II) + 8H

Step 3 Step 2 DGGE phylogeny of cloned 16SrRNAs of Acidithiobacillus spp.

Group II

Acidithiobacillus spp. AF339743 At. caldus Z29975 At. caldus AF137369 Acidithiobacillus spp. AF359940 At. caldus AB023405 Group I Acidithiobacillus DSM612 AJ459802

T1

Group III At. ferrooxidans AF465607 Tinto 3 Acidithiobacillus spp. AF023264 Acidithiobacillus spp. AF407402 Acidithiobacillus spp. AF376020 Thermithiobacillus tepidarius spp. At. ferrooxidans AJ457808 At. ferrooxidans AJ278719 AF023264 At. ferrooxidans X75268 At. ferrooxidans AF465604

Leptospirillum ferrooxidans X86776 0.10 fluorescence in situ hybridization (FISH)

DAPI-stained cells Cells hybridized with LEP636 probe (Cy3-labeled) specific for L. ferrooxidans confocal microscopy of pyrite colonized with Acidithiobacillus ferrooxidans (CARD-FISH) phylogeny of acidophilic microorganisms detected in Rio Tinto

Actinobacteria

Spirochaetes

Acidobacteria Planctomycetacia

Chlamydiae Verromicrobiae /Flavobacteria/ Chlorobia Sphingobacteriaa OP3 Thermomicrobia OP8 OP9 e - OP2 OP10 d -Proteobacteria Deinicocci

OP1

Koraarchaeota a-Proteobacteria

Aquificae

0.1 b/g -Proteobacteria geomicrobiological model of the water

Oxic column of Río Tinto Anoxic [O ] 2 [O2] S2-

At. ferrooxidans At. ferrooxidans At. thiooxidans SRB (CH O) At. caldus 2 n Acidiphilium spp. SO 2- Acidimicrobium spp. 4 Ferromicrobium spp. (CH O) 2 n CO2 Acidiphilium spp.

CO2 2+ 3+ + Fe Fe +H2O Fe(OH)3+H

At. ferrooxidans

L. ferrooxidans Fe2O3 ( ) Ferroplasma spp. Acidimicrobium spp. Ferromicrobium spp. ecological paradox Chlamidom onas sp. Bodo sp. Stichococcus sp.

Mesotaenium sp. Chl orella sp.

Euglena mutabilis Dunaliella bardawil

Cyanidium Vahlkampfia caldarium ustiana Colpidium sp. Oxytricha granulifera Rotaria sp.

Eolimna m inima

Actinophryis sp.

Pinnularia sp. 5 Viridiplantae Cercozoa Stramenopiles Red Algae

Fungi Alveolata

Animals Amoebae

Euglenozoa acidophilic fungi from Río Tinto endemic plants : Erica andevalensis Fe hyperaccumulator plants such as Imperata cylindrica produce jarosite and Fe oxides in the interior of their tissues GEOMICROBIOLOGICAL MODEL OF TINTO RIVER SEDIMENTS

Acidithiobacillus Acidithiobacillus, Sulfobacillus, Acidiphilium Sulfobacillus, Alicyclobacillus, Acidiphilium, Pedobacter, Variovorax, Pseudomonas Ferroplasma, Leptospirillum, E Ferrimicrobium, Ferrithrix O2 2- 2- 2+ 3+ SO4 S Fe Fe (CH2O)n CO2 Acidithiobacillus Acidiphilium, Acidobacterium, Pseudomonas Sulfobacillus Sulfobacillus, Ferrimicrobium, Ferrithrix, Chromatiales Alicyclobacillus, Ferroplasma, Geobacter, - Aciditerrimonas, Desulfosporosinus NO - NO3 3 N2O/N2 NO - N2O/N2 3 N2O/N2 Paludibacter, Staphylococcus, Clostridium Clostridium, Desulfitobacterium Desulfurella, Thermoplasma, Propionibacterium, Propionispora, Bacillus Propionibacterium, Acidovorax Acidithiobacillus Lysinobacillus, Rummelibacillus Organic acids/Alcohols Pseudomonas, Dechloromonas Thermodesulfobium Sedimentibacter, Alcaligenes Desulfotomaculum Syntrophobacter Clostridium, Geobacter Desulfosporosinus Bacilllus, Paenibacillus Syntrophobacter Acetate + CO +H Delftia, Commamonas Desulfobulbus 2 2 Pseudochrobactrum CO + H O Methanobrevibacter 0 2 2 S8 Methanosaeta Methanosarcina CO + CH 2 4 pH Acidophilic bacteriophages from Rio Tinto

Phage ACD-RT1 – a myovirus infecting Acidiphilium sp., both host and phage isolated from Rio Tinto. GEOmicrobiología mineral deposits of Fe preserved in sedimentary rocks iron minerals on sedimentary rocks Fe deposits older than 106 years which is the origin of this peculiar extreme acidic environment? subsurface microbiology Existing woreholes

Artesian wells

Devoted drills

R2

R1 ' & / 3 ' / * / % 2 % "

L1 " L2 $ $ ! "#$%&' Site 1() *+%, *+'- .' 45%/+(6.' Site 2 4

5 45%/&' % / + ( 6

. 45%/+(7.' ! "#$%/' ' (0++%1*+'- .' 45%//(7.' 45%/) ' 45%//(6.' 4 5 % / / ( 6 . ' 8&' 8/'

Core Processing Steps

Cores brought to surface Cores in plastic liners, cut, labeled

Bags filled with N2 Anaerobic chamber

Profiling BH10 After drilling

XRD Analysis cromatografía iónica, BH10

- NO3

- NO2 ppm

= PO4

= SO4

0 613 m Depth cromatografía iónica BH10, ácidos orgánicos

0.2

0.1 Tartrate

0 3 2 Propionate 1 0 100

50 Acetate

ppm 0 10

5 Formate

0 6

3 Oxalate

0 0 613 m Depth cromatografía de gases BH10 1,4

1,2

1

0,8

0,6

0,4

0,2

0

%H2 %CO2 %CH4 Total proteins ans sugars, BH10

100 612 LD300Chip, sample from 538mbs, BH10

7

4 1 2 3 5

6

40000

30000

20000 3 4 10000 6 1 2 5 7

0

1→Sulfobacillus acidophilus 4→NAG-NAM polymers 2→Bacterioferritin protein 6→Shewanella gelidimarina 5→DPS protein 3→ furiosus 7→Poly-Glutamic acid Chip tp detect 16S rRNA genes

enrichment cultures: -pyrite, Fe and S oxidizes - ferric iron and sulfate reducers - - methanotrophs - denitrifiers - acetogenic isolated bacteria from enrichment cultures

• Tessarococcus profundi (-139m) Tessarococcus lapidicaptus (-284m, -336m)) • Shewanella hafniensis (-121m) Desulfivibrio oxamicus (-450m) • Rhodoplanes piscinae (-420m) Pseudomonas stutzeri (-420m) • Rhizobium selenitireducens (-538m) Microbacterium saccharophilum (-284m) • Acetoanaerobium notareae (-45m) Citrobacter amalonaticus (-336m) • Cellulomonas fimi (-450m) Pleomorphomonas oryzae (-63m) • Macelibacteroides fermentans (-63m) Oerskovia turbata (-414m) Parabacteroides chartae (-450m) Nocardiodes pyridinolyticus (-420m) Propinicimonas paludícola (-414m) BH10-414,80 BET42a GAM42a

THIO820

IPBSL CARD-FISH SONDAS SAMPLE EUB338 I-III EUB338 II ALF968 ACD840 BET42a ACI145 GAM42a THIO820 THIO1 SBR385 DSS658 LGC354a LGC354b SUL228 HGC69a SS_HOL1400 CF319a LF655 CYA361 ARC915 MSSH859 MC1109 MG1200 MEB859 BH10-50,00 - - - BH10-75,00 - - - BH10-90,00 + + + ------+ - + + + + - - + - --- BH10-102,60 - + ------+ - --- - + - - BH10-103,50 + + - - - - - + - - - + - + + + - - - + - + - - BH10-121,80 - + - - + - - - - + - - - + ------BH10-130,80 - - - - + ------BH10-139,40 + + - - + + - - - + - + - + + ------BH10-206,60 + - + - - + - - + + + + - + + + - - + + + - - + BH10-228,60 + + ------+ - - - + - --- BH10-249,80 + - + - + - + + - -+ - - - + - - + + - - - - + BH10-266,30 + + ------+ - - + BH10-284,00 + + + + + + + + - + + - - - + + + + - + +/++ - - - BH10-294,45 + + + - - - + - - - - + - + + + ------BH10-294,65 ------+ + ------+ BH10-311,10 + - + - + - - - - -+ + ------BH10-352,65 + ------+ - + - - - - - + - --- BH10-353,15 ------+ - - --- BH10-355,70 + + ------+ + - - + - --- + - - + BH10-392,90 + ------+ + +/+ - - - BH10-401,90 ------+ - - - - + + + --- BH10-409,70 - - - + - - + ------+ ------BH10-414,00 + ------+ - --- BH10-414,80 + - + - + + + + ------+ ------BH10-415,30 ------+ + - BH10-415,97 ------+ - - - - ?------BH10-416,55 - + ------+ ------+ BH10-420,00 + + ------+ + - - + - + --- BH10-426,15 + - - + - - + - - + - - - - + - - - - + - --- BH10-450,30 + + + - + - + - - - - + + + ------BH10-468,80 + + ------+ - - - + ------+ - - BH10-477,45 + + ------+ ------BH10-487,20 + + - + ------+ - - + + + - --- + --- BH10-492,60 + + ------+ - + - + + + + --- + --- BH10-496,75 + + + - + - + ------+ + + - - - + +/-- - - BH10-519,05 + + ------+ - - - - - + + - - + BH10-520,00 + + - -- - - + - -+ + - + - - - - - + - --- BH10-544,00 + + - + + ------+ - + - --- + --- BH10-568,60 + + + ------+ + + ------+ + BH10-607,60 + + - - - - + ------+ + ------+ BH10-612,94 + + - - - - - + - - - + - - + - - - - + +/+ - - - Fe and S cycles

Fe2+

Fe3+

Fe2+ Fe3+ Acidovora Sulfobacill 10μm S S x us red ox

-139.4mAcidiphilli SRB 10μ um m Fermentatio Pseudomonasn 2- Rhodococcus SO4 Organic acids

CO2 Chemoheterotrophy Organic matter (Anaerobic (buried or produced respiration) chemolithoautotrophically) Methanosarcina Tessaracoccus e- donors e- aceptors Pseudomonas H2 CO2 2- SO S2Fe SO4 2- - 2- S SM NO3 4 Desulfovibrio Fe2+ Fe3+ Sulfobacillus - Methanotrophy Desulfosporosinus NO2 Acidiphillium CH4 Shewanella Organic acids 2- At. ferrooxidans S 2+ 0 2- CO2 Fe S SO4 Nitrate At. ferrooxidans reductio Desulfovibrio Acidovorax Sulfobacillus n 2- 2- SO4 Desulfosporosinus S sulfides Secondary H 2+ 2- 2 Fe SO4 S subsurface biofilms

-355.7m

-420m

-519.1m

LESSONS LEARNED

- It has been detected a high level of diversity and functional activity in the deep subsurface of the IPB (up to -610m) - Iron can be efficiently oxidized in anaerobic conditions - The detected subsurface microbial activities allow to explain the extreme characteristic conditions of Río Tinto

- H2 has an important role as a source of energy in the deep subsurface of the IPB (possible origin: water radiolysis, geochemical, biological) - The most important biogeochemical cycles ( C, N, S, Fe) are operative along the different depths of the solid matrix of the IPB - Subsurface biofilms have been detected in situ for the first time. Biofilms seems to be common in the subsurface eventhough it is considered a high energy consuming strategy not recommended for oligotrophic environment like the subsurfaces and which is the interest of these extreme acidic environment ? Biomining (biohydrometallurgy)

3+ 2- 2+ + FeS2+6Fe +3H2O → S2O3 +7Fe +6H 2- 3+ 2- 2+ + S2O3 +8Fe +5H2O → 2SO4 +8Fe +10H

3+ + 2+ 2+ 8MS+8Fe +8H → 8M +4H2Sn+8Fe (n≥2) 3+ o 2+ + 4H2Sn+8Fe → S8 +8Fe +8H Mars exploration biohydrometallurgy heap bioleaching Bio-reactor the most extreme condition that chemolithotrophic microorganisms have to deal with is the high concentration of toxic heavy metals generated by their metabolism - Fluorescence in situ hybridization (CARD- FISH) is a technology ready to be applied to biohydrometallurgical operations Cells hybridized with Lep154 DAPI probe specific for L.ferriphilum. Alexa488. L. ferriphilum in MGM cobaltiferrous

concentrate tank reactor 100 90 80

70 60 50 40 30

Cells detected (%) 20 10 0 Feed R1 R2 R3 Continuous Bioleaching reactors

EUB33 GAM42a NTR12 THIO1 LEP154 SUL1238 FER656+TMP65454

endemic plants: Erica andevalensis future: combination of bio- techniques. Biomining + specific sequester of metals using acidophilic fungi acidophilic fungi • Tabla 2. Eficiencia y especificidad de secuestro de metales pesados • ______• • Aislados concentración de metal secuestro % • • Bahusacala sp. O66 1mM Ag(I) 66 • Scytalidium sp. P65 10mM Cd(II) 90 • Penicillium sp. I25 200mM Zn(II) 93 • Penicillium sp. P34 100mM Cu(II) 35 • 10mM As(V) 68 • Penicillium sp. V80 100mM Cr(III) 75 • Alternaria sp. I14 0.1mM Hg(II) 95

Mars exploration habitability

Misión MER, crater Endurance, 2005

SPIRIT IN GUSEV CRATER blueberries in Columbia Hills sulfates exhumed in crater Gusev silica exhumed (hidrotermal?) MEX, wáter vapor (SPICAM) Fe oxides distribution (MEX)

Curiosity Crater Gale MSL, arm with instruments MSL ChemCam,elemental analysis

Mars Express

it can be concluded that on Mars there are sedimentary rocks that were formed in acidic conditions (acidic lakes or oceans) terrestrial analogues: - acidic environments - hydrothermal activities comparison between MARS and RIO TINTO

MP RTsurf RTss • - hematite ++ ++ + • - jarosite ++ ++ + • - goethite ++ ++ + • - ionic strength ++ ++ ++ • - temperature suf low 4-35oC • - temperature subs ? 25-30oC • - methane + - + • - oxygen +/- ++ - • - µorganisms ? ++ + the actual conditions on the surface of Mars, intens radiation and very oxidant conditions, do not seems to be the ideal place for life development (mechanisms of protection, methodological problems). Life on the subsurface has much more possibilities. It is important to develop a martian drilling mission in a near future. . the exploration and characterization of the Tinto ecosystem is important to understand the properties of microorganisms that could develop on Mars. Also is a good bench to test instruments designed to detect life on Mars… and to better understand the period in which life appeared on Earth (Archean)

banded iron formations of Pilbara (Australia) THANK YOU

isolation in acidic waters from a coal mine of Thiobacillus ferrooxidans terrestrial acidic environments natural acidic environments: - areas with volcanic activity 0 SO2 + H2S → S + H2O Yellowstone natural acidic environments: - metal mining activities 3+ 2- + FeS2 + H2O —> Fe + SO4 + H in these cases the extreme acidic conditions are promoted by biological activity Biomining, the future of

»mining R. Amils CBMSO and CAB

Bolonia, november 2018 isolation in acidic waters from a coal mine of Thiobacillus ferrooxidans metabolisms involved

la irrupción de técnicas moleculares al estudio de la ecología microbiana ha sido una auténtica revolución DGGE mesocosms hibridación in situ con marcadores fluorescentes (FISH)

DAPI-stained cells Cells hybridized with LEP636 probe (Cy3-labeled) specific for L. ferrooxidans 16S rRNA gene-based oligonucleotide microarray filogenia de los microorganismos acidófilos detectados en la cuenca del Tinto

Actinobacteria Cyanobacteria

Spirochaetes

Acidobacteria Planctomycetacia Fibrobacteres

Chlamydiae Verromicrobiae Bacteroidetes/Flavobacteria/ Chlorobia Sphingobacteriaa OP3 Thermomicrobia OP8 OP9 e -Proteobacteria Chloroflexi OP2 OP10 d -Proteobacteria Fusobacteria Deinicocci

OP1

Koraarchaeota a-Proteobacteria Firmicutes

Aquificae

0.1 Crenarchaeota b/g -Proteobacteria Euryarchaeota UV radiation protection with Fe3+ radiation protection by ferric iron

Dunaliella control, 0 hours

7 Dunaliella control, 96 h 6 5 Dunaliella RT media, 5 mW cells x10+5/ml 4 cm-2 3 Dunaliella RT media, 10 mW 2 cm-2 1 Dunaliella A media, 5 mW 0 cm-2 96 hours incubation Dunaliella A media, 10 mW cm-2 Fe meteorites (irons) chemolithoautotrophy “irons” as a source of energy

t0 meteorite oxidation by chemolithoautotrophic 1 día microorganisms

3 días

7 días phyllosilicates can be generated in acidic conditions (Río Tinto) basic knowledge is needed to improve the efficiency of bioleaching

Probes used in Petiknas Zn 35ºC, Petiknas Zn 35ºC and Aguablanca

Targe Probe Sequence (5’ to 3’) (%) FMa35ºC Specificity Reference tet

EUB338 16S GCT GCC TCC CGT AGG AGT 0-35 Bacteria domain Amann, 1990

EUB338-II 16S GCA GCC ACC CGT AGG TGT 0-35 Planctomyces Daims, 1999

EUB338-III 16S GCT GCC ACC CGT AGG TGT 0-35 Verrumicrobia (and others) Daims, 1999

THC642 16S CAT ACT CCA GTC AGC CCG T 35 Acidithiobacillus caldus Bond, 2000

González-Toril, LEP154 16S TTG CCC CCC CTT TCG GAG 35 Leptospirillum ferriphilum 2003

SUL141 16S CGG CCC GAT ATC CCC CAC 35 Sulfobacillus spp. Diez et al. BIOMINE

FER656 16S CGT TTA ACC TCA CCC GAT C 35 Ferroplasma spp. Edwards, 2000

NON338 ----- ACT CCT ACG GGA GGC AGC 35 Negative control Amann, 1990 Cells hybridized with Sul141 DAPI probe specific for Sulfobacillus sp. Alexa488. Tabla 3. Resistencia constitutiva e inducible a metales pesados ______

Aislados resistencia constitutiva máximo nivel de resistencia

Cladosporium sp. Y18 < 1mM Cr(III) 400mM Cr(III) Nigrospora sp. V12 1mM Ag(I) 1mM Ag(I) Penicillium sp. P34 < 1mM Cu(II) 200mM Cu(II) Penicillium sp. Y22 < 1mM Zn(II) 400mM Zn(II) Tabla 4. Eficiencia del secuestro específico de Cr(III) de Penicillium V80 ______tipo de crecimiento biomasa tiempo de exposición (días) medio secuestro %

crecimiento activo 0.93g (final) 7 YEPD 75.7 crecimiento activo 0.8g (final ) 7 50% YEPD 34.9 crecimiento activo 0.76g (final) 7 C 39.8 fase estacionaria 1g 1 YEPD 1.2 células rotas 1g 1 YEPD 0.9 Tabla 5. Comparación de eficiencias de recuperación de V80 utilizando distintos tratamientos ______número de ciclos [Cr(III)] (mM) inóculo tiempo (días) eficiencia% Cr (moles)

tratamiento #1 un ciclo (A) 100 fresco1/100 7 73.9 73.9 dos ciclos (A+B) 26.1 ½ A 2 55.1 87.5 tres ciclos (A+B+C) 12.5 ½ B 2 11.1 88.9 volumen total tratado: 100ml

tratamiento #2 un ciclo (M) 100 fresco 1/100 7 79.9 79.9 dos ciclos (M+N) 100 ½ A 2 40.2 120.1 tres ciclos (M+N+L) 100 ½ B 2 3.1 123.2 volumen total tratado: 300 ml Themal area of Gunhuver, Iceland contact/no-contact (direct/indirect) confocal microscopy of pyrite colonized with Acidithiobacillus ferrooxidans (CARD-FISH) Fe content of the core 8,68c MARTE project, CARD-FISH of sample from core 8,50a (-107m)

Immunoprofiling by Sandwich Microarray Immunoassay (SMI)

Multianalyte Incubation with the Sample Ab microarray (50-150 800 spots m spot diameter)

10 mm

5 mm Wash Addition and incubation with fluorescent antibodies

Wash Scanning

Image Quantification of the signal

C1- Burnt sample Sample C2- Buffer Intensity 0 0 0 Antibodies Antibodies Antibodies CARD-FISH: Catalyzed reporter deposition Fluorescence In Situ Hybridization

Alexa-488

Nature Reviews Microbiology 6, 339-348

IPBSL anaerobic methanogenic consortium

IPBSL Sulfolobus acidocaldarius

100,0

3,9 90,0 28,5 1,8

80,0

22,9 70,0 3,9 70,9 60,0 EUB338+II+III 92,1 % of microorganisms microorganisms of% 25,5 50,0 THC642 LEP154

40,0 SUL141 FER656

30,0

20,0 62,8 41,2 10,0

0,0

Petiknas Zn 35 Petiknas Zn 45 Samples heap leaching Tabla 1. Perfiles de resistencia a metales exhibidas por hongos acidófilos ______

Aislados valores máximos de resistencia otras resistencias

Scytalidium sp. O64 1mM Hg(II), 400mM As(V) 200mM Cr(III) Cladosporium sp. I18 400mM Zn(II) y Cr(III) 50mM Cu(II) Cladosporium sp. P72 400mM As(V) 100mM Zn(II), 200 mM Cr(III) Alternaria sp. I14 1mM Ag(I) --- Aspergillus sp. P37 --- 50mM Cu(II), As(V) y Cr(III) Aspergillus sp. P51 100mM Cr(III) --- Bahusakala sp. O62 1mM Ag(I) --- Bahusakala sp. O66 400mM As(V), 1mM Ag(I) y Hg(II) 10mM Ni(II) y Cd(II) Penicillium sp. P54 1mM Ag(I), 400mM As(V) 50mM Cu(II), 100mM Cr(III) Penicillium sp. V80 200mM Cr(III) --- Penicillium sp. P34 200mM Cu(II), 1mM Ag(I) 50mM As(V), 100mM Cr(III) Hormonema sp. I12 400mM As(V), 1mM Ag(I) 200mM Cr(III) Hormonema sp. I17 1mM Ag(I) 50mM Zn(II) Nodulisporium sp. V56 400 mM As(V), 10mM Ni(II) 50mM Cr(III), 10mM Zn(II) Nodulisporium sp. V58 400mM As(V) 100mM Cr(III) Trichoderma viride O6 1mM Ag(I), 10mM Ni(II) y Cd(II) 200mM As(V) Trichoderma viride CECT 2423 ------

EL CAPITAN (MP) temperature gradient (Endurence) clauds at Meridiani Planum Viking I 16S rRNA gene-based oligonucleotide microarray enrichment cultures: - pyrite, Fe and S oxidizers - Fe and sulfate reducers -methanogens - metanotrophs - denitrifiers - acetogenic bacteria 4H2 + CO2 → CH4 + 2H2O B 2- + - H2 + SO4 + H → HS + 4H2O B 2+ Fe + H2S→FeS + H2 Q FeS + H2S → H2 + FeS2 Q 3+ 2- 2+ + FeS2+Fe +8H2O→2SO4 + Fe +16H B 2+ - 3+ - Fe + NO3 →Fe + NO2 B 3+ 2 + Cn(H2O)n+Fe +H2O→CO2+Fe +4H B - + - H2 + HCO3 + H → CH3COO + H2O B 2- - 2- C3H6O3 + SO4 → CH3COO + S B H2O on Mars MER OPPORTUNITY AT MERIDIANI PLANUM Crater Eagle rock outcrop at Eagle crater (MP) robotic arm with instruments

blueberries, Endurance crater,

SPIRIT AT GUSEV CRATER blueberries at Columbia Hills exhumed sulfates at Gusev crater exhumed silica (hydrothermal?) MEX, water wapor (SPICAM) Fe oxides distribution on Mars (MEX) HRSC-MRO HRSC-MRO paleo-ocean (K, Th, Fe) Gamma Ray spectrometer (Mars Odissey) MEX, phylosilicates (OMEGA) MEX, H2O-ice in the South Pole (MARSIS) Phoenix landing site, june 2008

MGS craterización reciente, MGS MEX, methane (PFS)

Curiosity Crater Gale meteorito de Fe-Ni marciano MSL, brazo con instrumentación MSL ChemCam, análisis elemental

se puede concluir que en Marte existen rocas sedimentarias formadas en condiciones ácidas (lagos o océanos ácidos) análogos terrestres:

- ambientes ácidos - hidrotermalismo submarino comparación entre Meridiani Planum y Río Tinto MP RTsurf RTss • - hematites ++ ++ + • - jarosita ++ ++ + • - goetita ++ ++ + • - fuerza iónica ++ ++ ++ • - T superficie low 4-35oC • - T subsuelo ? 25oC • - metano + - + • - oxígeno +/- ++ - • - µorganismos ? ++ + Las condiciones actuales de la superficie de Marte, fuerte irradiación UV y condiciones muy oxidantes, no parecen ser el lugar ideal para el desarrollo de la vida (mecanismos de protección, problemas metodológicos). La vida en el subsuelo tiene muchas más posibilidades que en la superficie. Es importante diseñar y desarrollar una misión de perforación si queremos detectar vida en Marte Mars Express

. La exploración y caracterización del ecosistema del Tinto es importante para entender las propiedades de los microorganismos que se podrían desarrollar en Marte. Además es un buen banco de pruebas para probar las prestaciones de los instrumentos que volarán a Marte en futuras misiones de exploración… y para entender mejor el periodo en el que apareció la vida sobre la Tierra (Archean)

formaciones de hierro bandeado de Pilbara (Australia) the irruption of molecular biology techniques into microbial ecology has produced an authentic revolution • Proteobacteria (Acidithiobacillus, Acidiphilium, Acidiferrobacter, Ferrovum) • Nitrospira (Leptospirillum) • Firmicutes (Alicyclobacillus, Sulfobacillus) • Actinobacteria (Ferrimicrobium, Acidimicrobium, Ferritrix) • (Sulfolobus, Acidianus, Metallosphaera, Sulfurisphaera, Ferroplasma) Fungal diversity

Eurotiomycetes

Índice de bootstrap: 100% 90-99% 80-89% XRD and Mössbauer spectra MARTE project, SEM of a sample from core 8,68c (-162m)

MEX: OMEGA meteorito de Fe-Ni marciano MARTE project: geomicrobiological exploration of the Iberian Pyritic Belt subsurface