Biosignatures and Bacterial Diversity in Hydrothermal...

Geomicrobiology Journal, 21:529–541, 2004Copyright C© Taylor & Francis Inc.ISSN: 0149-0451 print / 1362-3087 onlineDOI: 10.1080/01490450490888235

Biosignatures and Bacterial Diversity in HydrothermalDeposits of Solfatara Crater, Italy

Mihaela Glamoclija,1 Laurence Garrel,1 Jonathan Berthon,2

and Purificacion Lopez-Garcıa2

1International Research School of Planetary Sciences, Universita d’Annunzio, Viale Pindaro 42,65127 Pescara, Italy2Unite d’Ecologie, Systematique and Evolution, CNRS UMR 8079, Universite Paris-Sud,batimet 360, France

We have combined mineralogy, organic geochemistry and mole-cular microbiology to study hydrothermal deposits from SolfataraCrater, a geologically young volcanic formation (∼4,000 yearsold) displaying hot (45–95◦C) and acidic (pH 1.7) mud pools andfumaroles. The search for inorganic (mineral) biosignatures re-vealed the presence of delicate structures, most likely mineral-ized extracellular polymers (EPSs), and the presence of potentialbiologically induced minerals: sulfides, sulfates (barite and alu-nite), elemental sulfur, and iron oxides. Geochemical analyses re-vealed a low total organic carbon content, 0.13 to 0.53%, displayingδ13C values from −17.09 to −27.39‰, and total nitrogen con-tents from 0.03 to 0.12%, which are characteristic of hydrother-mal systems and suggest the presence of autotrophic carbon fixa-tion. Lipid biomarker analysis showed the presence of hopanoidsand linear alkanes, and the absence of detectable steroids, imply-ing the occurrence of bacteria in our samples. We constructed16S rRNA gene libraries from the environmental samples. Mostenvironmental sequences obtained were affiliated to the Alpha-and Betaproteobacteria (Hydrogenophilus-like), the Acidobacteria,and to a lesser extent, the Gammaproteobacteria and Actinobacte-ria. When known, the closest cultivated relatives were often ther-mophilic or thermotolerant bacteria oxidizing iron, hydrogen, or

Received 1 July 2004; accepted 9 September 2004.We thank A. Baliva for XRD analysis, A. Traini for the possibility of

using SEM, and L. Tonucci for NMR analysis. The FT-IR analysis wasdone by G. de Matia, “Parco Scientifico e Tecnologico d’Abruzzo,”Chieti. Special thanks are given to Prof. R. Barbieri, University ofBologna, for his hospitality and useful advisory to M. Glamoclija dur-ing two months. We want to thank A. P. Rossi, IRSPS, for help duringfieldwork, as well as D. Moreira for assistance during purification ofsamples for biological purposes. A special thanks to Vulcano Solfataras.r.l. for the hospitality and interest in this research. This work wasfunded by the Agenzia Spaziale Italiana (ASI), the exobiology pro-gram, and the French CNRS-INSUE program Geomicrobiologie desenvironnements extremes (GEOMEX).

Address correspondence to Mihaela Glamoclija, International Re-search School of Planetary Sciences, Universita d’Annunzio, VialePindaro 42, 65127 Pescara, Italy. E-mail: [email protected]

methane/methanol, suggesting an important microbial contribu-tion to the formation of biominerals.

Keywords 16S rRNA, bacterial diversity, biosignatures, hydrother-mal, Solfatara Crater, thermoacidophile

INTRODUCTIONSince life appeared on Earth and for most of its history, mi-

croorganisms have been the lone inhabitants of our planet. Mi-crobes can live in a wide variety on environments, includingthose exhibiting the most extreme conditions (Rothschild andMancinelli 2001). Microbial genetic diversity is huge, as hasbeen increasingly revealed by molecular ecology surveys overthe past fifteen years (Pace 1997; Hugenholtz et al. 1998), yetprokaryotic microorganisms display a restricted number of mor-photypes. This, together with the fact that microorganisms arerarely preserved in fossil form, has hampered the reconstructionand timing of early evolutionary diversifications. The search fordiagnostic biosignatures from past microorganisms is not onlycrucial to understand early evolution on our planet, but mightalso help to reveal traces of ancient biological activity on planetssuch as Mars, where physical–chemical conditions were similarto those of the Archaean Earth.

In addition to fossils discernible by their morphology, mi-croorganisms and microbial communities influence and modifytheir environment during their lifetime, both at the micro andmacroscale, and may thus leave traces of their existence. Themost easily recognizable are stromatolites and permineralizedbiofilms. At a smaller scale, remnants of mineral-microbe in-teractions may remain but, unfortunately, surface chemistry canoften yield a wide variety of mineral alterations that can beeasily misinterpreted as derived from biological activities. Thedifficulty of finding unmistakable microbial biosignatures is evi-denced by two recent controversies. The first concerns the nature

529

530 M. GLAMOCLIJA ET AL.

of carbon in Isua’s rocks, claimed initially to be of biologicalorigin because of its light isotopic composition (Schidlowski1988). This isotopic fractionation may also be the consequenceof hot fluids reacting with older crustal rocks (metasomatism),however (van Zuilen et al. 2002). The second concerns the na-ture of the “earliest microfossils” (3.4–3.5 Ga) described bySchopf and coworkers in the Australian Warrawoona and ApexFormations as cyanobacteria (Schopf and Packer 1987; Schopf1993), which have been later reinterpreted as possible artifacts(Brasier et al. 2002). The origin of usually microbially derivedminerals, such as magnetite (Schuler and Frankel 1999), hasalso been put into question (Buseck et al. 2001; Treiman 2003;Weiss et al. 2004). Organic fossilized biomarkers, such as fos-sil lipids (e.g., hopanes, steranes) are certainly biogenic, butthese are not exempt of contamination problems. To overcomeall these difficulties, the identification of nonambiguous tracesof ancient microbial activity will most likely demand the com-bination of several concurrent biosignatures. In this sense, thestudy of contemporary model system analogous to past envi-ronments is essential to understand the fossilization process andcorrelate present-day biosignatures with old putative biogenictraces.

Various recent models on the origin of life propose that it orig-inated some time between 4 and 3.7 Ga ago in moderately hot tohot environments, possibly linked to hydrothermal fluid activity(Kasting and Ackerman 1986; Wachtershausser 1988; Nisbetand Sleep 2001; Martin and Russell 2003). Modern hydrother-mal biotopes colonized by thermophilic and hyperthermophilicmicroorganisms constitute therefore potential model systems toidentify biosignatures and link them to a particular microbialdiversity and activity. Among the earliest studied contemporary

Figure 1. Location of Solfatara Crater and sampling sites.

geothermal areas, together with Yellowstone in the U.S., is theSolfatara Crater close to Naples, Italy (Figure 1), where the ther-moacidophilic archaea Sulfolobus solfataricus and S. brierleyiwere first isolated (DeRosa et al. 1974, 1975; Zillig et al. 1980).Later, other hyperthermophilic archaea belonging to the gen-era Acidianus, Pyrobaculum and Metallosphaera have been iso-lated from Solfatara as well (Huber et al. 2000a, 2000b). TheSolfatara Crater, characterized by its subareal activity, is locatedin the Mid–Eastern part of the Campi Phlegrei Caldera, whichis a nested, 12-km wide structure, formed by two main col-lapses corresponding to Campanian Ignimbrite and NeapolitanYellow Tuff eruptions (37,000 and 12,000 years ago, respec-tively) (Rosi and Sbrana 1987). Early studies revealed a majorupheaval of the mantle beneath the area (Ferrucci et al. 1989).The top of the Campi Phlegrei Caldera magma chamber liesat 5 km depth, probably within carbonate sequences (Rosi andSbrana 1987). Volcanological, petrological and geophysical datasuggest that the activity at Campi Phlegrei Caldera was oncefed by a large magmatic reservoir (Panichi and Volpi 1999).Solfatara’s volcanism belongs to the last volcanic epoch of theCampi Phlegrei Caldera. In past times, both explosive and effu-sive eruptions occurred within short-time intervals, but extensiveexplosive volcanism finished ∼4,000 years before present. To-day, the Solfatara activity is marked by continuous hydrothermalemissions within mud pools in the central sector of the craterand fumarolic activity in the Northern side, possibly fed by a1.5-km deep, low-permeability, geothermal aquifer of mixedmagmatic-meteoric origin (Chioni et al. 1984). The geother-mal system is vapor dominated, and temperatures range fromvery high (the hottest fumaroles ∼160◦C; Tedesco et al. 1988) tomoderate (40–50◦C for areas around emission points). The pH of

BACTERIAL BIOSIGNATURES AND DIVERSITY IN SOLFATARA CRATER 531

Solfatara fluids is ∼1.7, making this environment not only ex-treme by its temperature but also by its acidity.

Despite the isolation of a few thermoacidophilic archaea fromSolfatara hot mud, systematic studies on the prokaryotic diver-sity have not been carried out yet in this biotope. In this study, wehave combined mineralogy, organic geochemistry and molecu-lar ecology techniques to identify possible biosignatures andto characterize the bacterial community associated to Solfatarafumarole-adjacent crusts and mud samples. We have identified anumber of minerals, including barite and alunite crystals as prob-able inorganic biosignatures, hopanoids as organic biomarkers,and a relative wide diversity of putative meso- to thermoaci-dophilic bacteria.

MATERIALSSamples from the Solfatara Crater were collected on May

2001 from both types of emission points (fumaroles and mudpools). A total of 25 samples were selected to study their petrol-ogy, mineralogy and organic chemistry. Two of these were addi-tionally used to characterize the associated microbial diversity.Samples for microbiological and biogeochemical analysis werecollected following protocols to avoid contamination. Plasticgloves were worn all throughout the sampling procedure, andelementary precautions, such as avoiding any physical contactwith samples and sealing the collected material immediately af-ter removal, were taken. Surface crust samples were selectedfrom intact well preserved areas. The rest of samples were col-lected after the removal of ∼10 cm of surface deposits asepti-cally. Samples were collected and stored in sterile 50-ml Falcontubes and/or, in the case of larger volumes, in sterile Ziplockplastic bags. Collected samples were placed in a thermal-isolatedcontainer for their transport to the laboratory. Temperature wasmeasured at each sampling point. pH values of water sampleswere first estimated in situ using pH strips (Carlo Erba), and sub-sequently determined in the laboratory using a pH-meter (GLP22 Crison) equipped with a pH-electrode (n◦ 52-09 Crison). Cal-ibration of the electrode has been done by Crison standard buffersolutions 7.0 and 9.0 pH respectively. Both buffer solutions andwater samples that we measured were at the room temperature(25◦C).

METHODS

Petrology and MineralogyThe identification of minerals in thin sections was done us-

ing a petrological microscope (Nikon E400). X-Ray diffrac-tion (XRD; Siemens D 5005) analysis was performed on pow-dered samples of consolidated crusts collected around mud poolsand fumaroles. The search for possible mineral biosignatureswas first done by optical microscopy (Orthoplan LeitzWetzlar) and, for potential interesting structures, by scanningelectron microscopy (SEM; LEO 435VP). Energy-dispersiveX-ray spectrometry (EDS) coupled to SEM (Philips XL-30; X-

EDS EDAX ECON IV, Microanalytical system 9900) was usedto determine the elementary composition of identified minerals.Electron microscopy observations were done on thin sectionsprepared from intact samples and from samples that had beenetched by 1% and 10% HCl, and 1% and 3% HF for differ-ent time periods. Thin sections for SEM and SEM/EDS weregold-coated previous to their observation.

Organic Chemistry and Lipid Biomarker AnalysisAnalyses of total organic carbon (TOC), isotopic δ13C and

total nitrogen content of Solfatara samples were done at theCNR (Bologna, Italy). Organic carbon and nitrogen were deter-mined on duplicate samples using a FISONS NA2000 ElementalAnalyzer (EA) after removal of the carbonate fraction by disso-lution in 1.5N HCl. Stable isotope analyses of organic C werecarried out by using a FINNIGAN Delta Plus mass spectrom-eter, which was directly coupled to the FISONS NA2000 EAby means of a CONFLO interface. The IAEA standards NBS19were used as calibration materials for carbon. Stable isotopicdata are expressed in the conventional delta (δ) notation in whichthe 13C/12C isotopic ratios are reported relative to the interna-tional PDB standard. The content of organic elements N, C, H,S and O in samples SOL7 and SOL18 used for microbial di-versity analysis was done by the Service d’Analyse des Rocheset Mineraux du CNRS (Nancy, France).

Lipid biomarker analyses were done on samples correspond-ing to non-consolidated crust from a mud pool rim and consol-idated crust. All samples were initially crushed to small piecesand carefully cleaned by repeated washing with 3M HCl andacetone, dilute HCl, methanol and dilute NaClO. After eachstep, samples were again carefully cleaned with organic-freeUV-oxidized water dispensed by a Milli-Q Gradient instrument(Millipore). All glassware used was also treated by soakingin a 10% HCl bath for 24 h, rinsed thoroughly with waterand dried at 200◦C for 24 h. Fragmented samples were driedand then crushed into fine powder. Nuclear Magnetic Reso-nance (NMR) and Fourier-Transform Infrared (FT-IR) analyseswere performed after demineralization of the samples using themethod described by Gelinas et al. (2001). Solid state 13C NMRspectroscopy was performed on demineralized fractions with aBrucker Avance 300 MHz spectrometer using power decoupling,cross polarization and magic angle spinning (CPMAS).

Spectra were recorded at different spin rates in order to dis-criminate spinning side bands. FTIR spectra were recordedon a Perkin Elmer Spectrum 2000 equipped with an MIRsource and a MIR-TDGS detector as 5 mm KBr pellets. ForGas Chromatography-Mass spectrometry (GC/MS) analyses,cleaned samples were saponified in 6% KOH in methanol, thesupernatant was decanted and the residue subjected to Soxhletand/or ultrasonically extracted with different dichloromethane/methanol solutions. The combined supernatants were extractedwith dichloromethane vs. water (pH 2). After concentration ofthe organic phase by rotatory evaporator under reduced pressure,sulfur was removed by freshly prepared activated copper.


The organic components of the CH2Cl2 phase were fraction-ated by column chromatography (i.d. 15 mm, length 35 mm,Merck Silica gel 60, 70–230 mesh) and eluted with 2 columnvolumes of n-hexane (“total hydrocarbon”) and 3 column vol-ume of CH2Cl2 (alcohol/ketone fraction). The latter was deriva-tized with acetic acid anhydride in an equal volume of pyridine(14 h at room temperature) and fractions were analyzed by aFisons Instruments MD 800 GC 8000 series GC/MS spectrom-eter equipped with a 50-m fused silica capillary column (DB5-HT, 0.32 mm i.d. 0.25-µm film thickness) using He as carriergas. Temperature program: 5 min 80◦C to 310◦C at 4◦C/min:20 min at 310◦C.

Nucleic Acid Extraction and 16S Ribosomal RNAGene Libraries

Samples (water from mud pool, hot mud, and nonconsoli-dated crusts) devoted to the biological study of microbial di-versity by molecular methods were kept in 80% ethanol at 4◦Cuntil DNA extraction. The DNA used in this study was extractedfrom nonconsolidated black crust at the edge of a mud pool in thecentral part of the crater (SOL7, 40◦C) and from white, melteddeposits adjacent to a chimney in the Northeast fumarole area ofthe crater (85◦C, SOL18) by two methods, the SoilMaster DNAextraction kit (Epicentre) and a classical phenol-chloroform ex-traction. For the latter, prior to DNA extraction, samples wererehydrated with phosphate saline buffer (130 mM NaCl, 10 mMphosphate buffer, pH 7.7, PBS). PBS was also added to thesediment to a same final volume of 0.5 mL. Samples were thensubjected to 6 freezing/thawing cycles in liquid nitrogen to facili-tate cell lysis. Subsequently, 80 µg mL−1 proteinase K, 1% SDS,1.4 M NaCl, 0.2 β-mercaptoethanol and 2% hexadecyltrimethy-lammonium bromide (CTAB) (final concentrations) were addedsequentially.

Lysis suspensions were incubated overnight at 55◦C. Lysateswere extracted once with hot phenol (65◦C), once with phenol-chloroform-isoamylalcohol, and once with chloroform-isoamyl-alcohol. Nucleic acids were concentrated by ethanol prec-ipitation. 16S rRNA genes were amplified by PCR using thebacterial-specific primer 63F (CAGGCCTAACACATGCAA-GTC) and the prokaryote-specific reverse primer 1397R (GGG-CGGWGTGTACAAGGC). Polymerase chain reactions (PCR)were performed under the following conditions: 30 cycles (de-naturation at 94◦C for 15 s, annealing at 50◦C for 30 s, extensionat 72◦C for 2 min) preceded by 2 min denaturation at 94◦C, andfollowed by 7 min extension at 72◦C. Dimethyl sulfoxide wasadded to a final concentration of 3–5% to the PCR reactionmix. rDNA clone libraries were constructed using the Topo TACloning system (Invitrogen) following the instructions providedby the manufacturers. After plating, positive transformants werescreened by PCR amplification of inserts using flanking vectorprimers.

Sequence and Phylogenetic AnalysesA total of 63 (34 from SOL18 and 29 from SOL7) expected-

size amplicons from these libraries was partially sequenced

(lengths from 849 to 1050 nucleotides) with the primer 1387R(Genome Express). Closest relatives to our sequences were iden-tified in databases by BLAST (Altschul et al. 1997) and retrievedfrom GenBank 〈http://ncbi.nlm.nih.gov/〉. Sequences were au-tomatically aligned using the program BABA (H. Philippe, per-sonal communication) to a 16S rRNA gene alignment containing∼17,000 sequences. The multiple alignment was then manuallyedited using the program ED from the MUST package (Philippe1993). A preliminary phylogenetic analysis of all partial se-quences was done by distance methods (neighbor-joining, NJ)using the program MUST, allowing the identification of iden-tical or nearly identical sequences and the selection of repre-sentative clones for subsequent analysis. We then selected 15representative sequences to be included in a phylogenetic tree,together with their closest relatives in GenBank and some culti-vated species. A total of 710 positions were used in our analysisafter removal of gaps and ambiguously aligned positions. Themaximum likelihood (ML) tree was done using TREEFINDER(Jobb 2002) applying a general time reversible model of se-quence evolution (GTR), taking among-site rate variation intoaccount by using an eight-category discrete approximation of a�gistribution (invariable sites are included in one of the cate-gories). The α parameter of the � distribution estimated fromthe sequence set was 0.27. ML bootstrap proportions were in-ferred using 500 replicates. The sequences reported in this studywere submitted to GenBank with accession numbers AY629323to AY629339 (see also Figure 6).

RESULTS AND DISCUSSION

Petrological Characteristics of Solfatara SamplesThe studied material from the Solfatara Crater is geologi-

cally young (∼4,000 years) which, in principle, should facilitatethe identification of unaltered biosignatures left by recent andpresent-day microbial communities. Hydrothermal springs inthe center of the crater have their foundation on local faults thatrun radially through the crater, whereas the fumarolic activity ismainly dependent on the regional fault along the Northeast craterwall (Figure 2A, 2B). Products of Solfatara activity consistedmainly of breccia and stratified deposits with layers of pisoliticand coarse ashes, the basal surge structure being composed ofbeds of well-sorted pumice lapilli that were a few decimeters insize. The surges covered the East-Northeast wall of the craterand overflowed the Eastern and Western crater edges. Most vol-canic products in the area were hydrothermally altered, givingraise to disordered trachytic deposits. Samples collected for thisstudy included highly porphyritic scoria with trachytic appear-ance composed of sanidine, plagioclase, clinopyroxene, biotite,and opaque phenocrystals. Samples of emitted material aroundmud pools were mainly fine-grained clays, consolidated to someextent. All samples were very rich in sulfur, sulfates, and a vari-ety of iron minerals. In the Northeastern zone of the crater, wherefumarolic activity is intense, gypsum was also present. Fromthe petrological and mineralogical characteristics, the observed


Figure 2. Sampling sites inside Solfatara Crater: (A) mud pool with boiling water in the central part of the crater; (B) one of theassociated fumaroles chimney, close to the North-Eastern wall of the crater.

and sampled material represent an environment with reducingchemistry.

Inorganic (Mineral) BiosignaturesIn many extreme environments, such as hot and acid biotopes,

microbial communities derive their energy from oxidation-reduction reactions made possible by the coexistence of ade-quate electron donors and acceptors along a geochemical gradi-ent. Different types of surfaces and cracks in mineral depositswhere electron donors and acceptors co-exist are suitable envi-ronments for microbial colonization. Biological redox reactionscan lead to the direct or indirect formation of “bio-minerals,”which are potential indicators of microbial metabolic activity(Banfield et al. 2001). Among known minerals that can have abiologically induced origin, elemental sulfur, sulfides, sulfates,and iron oxides/hydroxides are highly abundant in Solfatara.Aiming at establishing the biogenic nature of some mineralsfound in small cracks within Solfatara samples, we looked forthe co-occurrence of bio-alteration signs in different samples.Two types of crack samples in Solfatara material were foundto be biologically-altered and associated to likely biologicallyinduced mineral precipitation.

A first type of fractures (Type 1) was observed within par-tially consolidated stratified deposits of pisolitic ashes from theNortheastern sector of the crater close to fumarola emissions(Figure 2B). Most ground minerals in this area displayed differ-ent levels of alteration. In particular, these deposits contained∼1 mm-wide fractures, which were traversed by fluids rich inH2S, CO2, and, to a lesser extent, HCl, CH4, and H2, while theywere still unconsolidated (Valentino et al. 1999). A narrow, red-dish altered zone was observed under the microscope on oneof the fracture walls in the sample shown in Figure 3B. Sincethe appearance of this alteration could be suggestive of biologi-cal activity, rather than thermal alteration induced by hot fluids,we used electron microscopy to detect any further indicationof biogenic origin. A closer look revealed that the altered zoneexhibited irregular masses of elemental sulfur (S0) accompa-

nied by a layer of pyrite crystals and, in lower quantity, smallbarite crystals (Figure 3B, C, D). S0 is unstable in nature andusually gets readily oxidized. It can precipitate around rims offumaroles in orthorhombic and monoclinic form. However, thecharacteristic widespread sulfur interlayering generally associ-ated to abiotic crystallization, which is otherwise observed onfumarola edges, was not seen in this case (Figure 3A and B).One possible explanation for the presence of S0 along this frac-ture is, therefore, the occurrence of sulfur-metabolizing bacteria(sulfide-oxidizers or sulfur disproportionating bacteria) whoseactivity may at some point lead to the accumulation of locallyimportant amounts of S0.

SO2−4 ⇐ S0 ⇒ S2−sulfur-disproportionating bacteria [1]

SO2−4 ⇐ S0 ⇐ S2−sulfur-oxidizing bacteria and

phototrophic sulfur bacteria [2]

Pyrite (FeS2) was also present along the altered area, but juston one of the fracture walls (Figure 3B). When sulfide oxidationoccurs in the presence of adsorbed oxidized metal ions, there is aconcomitant reduction and precipitation of these metal species.Several chemolithotrophic and mixotrophic acidophiles use ele-mental sulfur and reduced sulfur components as electron donors,and ferric iron (Fe3+), as electron acceptor, to support growth(Johnson 1998). This may lead to pyrite formation as follows:

S0 + 6Fe3+ + 4H2O ⇒ HSO−4 + 6Fe2+ + 7H+

Fe2+ + S2− ⇒ FeS (soluble)

FeS (soluble) + S2O2−3 ⇒ FeS2 + SO2−

3

Since pyrite was only observed on one of the fracture walls,processes involving sulfide oxidation by microorganisms differ-entially attached to one side could have influenced this asym-metric distribution of pyrite.

Small (a few microns in size) barite (BaSO4) grains were alsoobserved associated to this same crack (Figure 3C and D). Barite


Figure 3. Layered sulfur- and iron-rich crust sample collected near the associated fumarol chimney (Figure 2B). (A) Overall viewof the layered sulfur-iron crust. (B) Micrograph of a thin section showing a ∼1-mm wide crack with dark, iron-rich bio-alteration(arrows). “S” indicates the position of elemental sulfur precipitates and “Py” pyrite crystals. (C) Scanning electron micrograph frombio-altered zone showing barite crystals (ba) and mineralized extracellular polymers (EPS). (D) Scanning electron micrograph ofirregular elemental sulfur (S) and barite (ba) precipitates in the same area. The inset between panels C and D corresponds to EDSspectra of sulfur and barite obtained from this samples. Scale bars: 100 µm (B), 1 µm (C), and 10 µm (D).

frequently results from hydrothermal alteration by substitutionof calcium by barium in gypsum (CaSO4), and precipitates at lowpH (∼1.2) in the presence of large amounts of sulfates (Africanoand Bernard 2000). Though the precipitation of barium and sul-fates is clearly an abiotic reaction, sulfide- and sulfur-oxidizersmay have contributed to increase the local sulfate concentration.Microbial production of sulfate is known to occur in Solfatara,as different archaeal (Sulfolobales) species able to oxidize ele-mental sulfur (Segerer and Stetter 1999) have been isolated fromSolfatara mud pools, and it is also suggested by isotopic S frac-tionation (Valentino et al. 1999). Since barite minerals appearedto co-occur with extracellular polymer-like structures (see later),a direct induction of BaSO4 crystallization might have occurredin association with microbial cells. The precipitation of bariteindirectly produced by some bacterial species has been docu-mented (Gonzalez-Munoz et al. 2003).

In addition to these minerals, delicate reddish net-like struc-tures were also observed in this type of fractures. In most cases,they were associated with irregular sulfur masses and barite min-erals (Figure 3C, D). The reddish rust color is due to the presenceof iron oxy-hydroxide compounds which may replace microbial

soft parts during fossilization (Butterfield et al. 1996; Provencioand Polyak 2001). The location of these delicate iron-rich struc-tures in the fracture, together with their morphology (irregularnet appearance) and morphometry (size in the nanometer range),suggests that they correspond to permineralized extracellularpolymers (EPS). Many microorganisms synthesize EPSs to at-tach to the substrate and to other biofilm-forming microbes. Be-cause of their negative charge, EPSs are among the first macro-molecules to permineralize (Butterfield et al. 1996; Barker et al.1998; Gehrke et al. 1998; Karthikeyan and Beveridge 2002;Hockin and Gadd 2003). The presence of net-like structure rem-iniscent of EPS is one of the (morphological) criteria employedto identify ancient microfossils (Cady et al. 2003).

A second type of fractures (Type 2) displayed alunite min-erals that were always covering one of the walls (Figure 4).Opaque pyrite minerals usually followed the alunite distribu-tion. This type of mineralization was observed in samples ofconsolidated crusts collected both near the hydrothermal springsand fumaroles. Alunite is a hydrous potassium aluminosulfate,from the jarosite mineral group, forming irregular masses andeuhedral crystals. In Type 2 cracks (Figure 4), pyrite constituted


Figure 4. Thin section taken under optical microscope showing a fracture inside a crust sample collected close to Solfatara mudpool. The fracture contains alunite minerals (indicated by arrows) that have precipitated on the upper edge along with secondarypyrite “Py.” X-ray spectrometry (EDS) spectrum of alunite is at the bottom.

a secondary mineral phase, i.e., it was formed after precipitationof alunite. In contrast to Type 1 cracks, morphologically iden-tifiable microbial structures were not observed. Alunite formsby sulfuric acid solutions acting on deposits (rocks) contain-ing potassium feldspar at acid pH (∼1.3) in deep subsurfacehydrothermal anoxic environments. The formation of alunitedecreases the concentration of potassium (K+) and aluminum(Al3+) cations in solution. The concomitant rise in pH is com-pensated by the production of H+ during the reaction.

K+ + 3Al3+ + 2HSO−4 + 6H2O ⇔ KAl3(SO4)2(OH)6 + 8H+.

This has been also documented in other settings displayinghydrothermal alteration (Bove and Hon 1990). Although aluniteis abiotically formed, sulfide oxidizers might have contributedto increase local concentrations of sulfuric acid (H2SO4), thustriggering the formation of this mineral. Indeed, the latter issupported by the fact that alunite and pyrite were specificallyformed on the same wall in this crack.

Finally, in addition to the above potential biominerals, highquantities of magnetite were locally observed on several placeswithin samples. Even though the presence of magnetite is oftencorrelated with biological activity, the observed minerals weretoo large (∼4 µm in size) to constitute intracellular products,and magnetite appeared to be the product of biotite oxidation. In-deed, the magnetite observed was associated to oxybiotite min-erals. Oxybiotite is actually a melted biotite that has releasedits iron content (necessary for magnetite formation) while pass-ing through processes of oxidation. Titanium, often found as animpurity in hydrothermal fluids, was detected in oxybiotite andmagnetite by EDS analysis. The precipitation of magnetite re-quires circumneutral pH settings (>5.5) and sufficiently oxidiz-ing conditions (Bell et al. 1987; Tor et al. 2001). The associationof magnetite with oxybiotite suggests that, locally, the redoxpotential may have been relatively high. Although pH valuesare generally very low at Solfatara (∼1.7 on average), circum-neutral pH could have been maintained at microniches by theactivity of particular bacteria (Dong et al. 2000), which would


eventually contribute indirectly to magnetite formation by cre-ating the appropriate environmental conditions for the abioticprocess to occur.

Organic Geochemistry and BiosignaturesTotal organic carbon (TOC) was present at very low concen-

trations, 0.13 to 0.53%, in the different Solfatara samples. δ13Cvalues in those samples were also low, −17.09 to −27.39 ‰,which would be in agreement with chemoautotrophic organicsynthesis. Total N varied from 0.04 to 0.12%, implying a C/N ra-tio between 1.63 and 7, which would also be compatible with mi-crobial organic production (Des Marais et al. 1992; Des Marais1996; Strauss et al. 1992; Meyers 1997; Schidlowski 2000;Twichell et al. 2002; House et al. 2003).

Kerogen, the naturally occurring insoluble organic matterin rocks, may retain primary biogeochemical information inthe form of lipid biomarker compounds (Ourisson et al. 1987;Summons et al. 1996). We looked for the presence of lipidbiomarkers in two Solfatara samples: black crust from the rim

Figure 5. Analysis of organic matter from Solfatara samples: (A) Solid state CPMAS 13C-NMR spectrum (after demineralization).(B) FTIR spectrum (after demineralization). (C) Distribution of products identified by GC/MS (after saponification).

of the central mud pool (corresponding to SOL7, see later), andgray endured crust near the fumarola chimney where type 1cracks were characterized. Nondestructive spectroscopic meth-ods like NMR and IR spectroscopy constitute suitable techniquesfor the examination of organic matter in heterogeneous macro-molecular mixtures and yield good results concerning the grosschemical composition.

The CPMAS 13C NMR (Figure 5A) spectra of Solfatarasamples (after demineralization) exhibited peaks at the samechemical shift. They were dominated by an intense peak due toaliphatic carbon that reached its maximum at 30 ppm (carbonfrom polymethylenic chains CH2). This peak showed shouldersat 15 and 35 ppm due to methyl groups and substituted carbons,respectively. The second characteristic signal (110–160) with amaximum at 130 ppm was due to unsaturated carbon, which canbe, a priori, in olefinic or aromatic units. However, the maxi-mum at 130 ppm suggests that the major part of unsaturated car-bon corresponds to alkenes. In any case, solid state (CPMAS)13C-NMR can highly overestimate the aliphacity of heteroge-neous compounds materials, due to a more efficient transfer of


polarization to protonated aliphatic carbon than to aromatic car-bons, or carbon involved in highly cross-linked structures.

The FT-IR spectra (Figure 5B) of the samples studied (afterdemineralization) showed similar functions. According to NMR,the presence of CH2 and CH3 is confirmed by their stretchingbands at respectively 2,920 and 2,850 cm−1 as well as their asym-metric C H bending bands at 1,455 and 1,375 cm−1. Absorptionof olefinic and aromatic carbon C C stretching vibration fell inthe range 1,570 to 1,680 cm−1. However, the bands centeredat 1,628 and 1,637 cm−1 (C C nonconjugated), and the weakbroad shoulder detected in the 3,000–3,100 cm−1 range (aro-matic C H stretching centered vibration), suggest that alkenesare more abundant than aromatics, in accordance with the NMRdata. Oxygen containing functions were detected as a broadband of great intensity from 3,400 to 3,600 cm−1 (O H). C Ostretching vibration are almost absent (band at 1,707 cm−1 isvery weak). The presence of C O is confirmed by intense bandsfrom 1,050 to 1,150 cm−1.

Thus, the organic matter in Solfatara samples is mainly com-posed of:

• Saturated carbon, mostly formed by secondary (R-CH2-R′) and, in less quantity, tertiary carbon (R-CH-RR′) witha few methyl groups (15 ppm) and few quaternary car-bons. This suggests the occurrence of straight-chain skele-tons with few branched cyclic alkanes skeletons.

• Olefins, constituting the majority of unsaturated carbon.• Oxygen-containing functions, mostly alcohols and ethers.

In accordance with these results, GC-MS analysis of thealkane-alkene soluble fraction showed a major contribution oflinear alkanes (m/z = 57), hopenes C27H43R (m/z = 191) alongwith few branched alkanes (m/z = 43, 57), linear alkenes andlinear alkanols (Figure 5C).

The wide range of alkane carbon numbers and their distri-bution (even/odd number predominance) excludes a terrestrialplant wax origin. Hopanoid lipids are particularly importantbiomarkers for bacteria (De Las Heras et al. 1997; Tritz et al.1999; Farrimond et al. 2000). They derive from the diagenesisof bacterial cell membranes and are extremely stable over geo-logical time. Their presence attests for the presence of contem-porary and/or past bacterial cells. The occurrence of essentiallytwo types of lipids, linear alkanes and especially hopanoids,as well as the absence of steroids, suggests that the microbialcommunity is essentially composed of bacteria.

Bacterial Diversity in Hydrothermal DepositsA first inspection under the optical microscope of different

Solfatara samples showed the presence of various prokaryoticmorphotypes (cocci and short rods), already suggesting a cer-tain microbial diversity. Therefore, in addition to the searchfor biosignatures, we carried out molecular diversity surveys intwo Solfatara samples. One corresponded to the same noncon-solidated black crust collected near the central mud pool used

for lipid biomarker analyses (SOL7, 40◦C), and the second tomelted deposits at a chimney in the Northeast Solfatara fumarolearea, adjacent to the samples in which type 1 cracks were found(SOL18, 85◦C). The concentrations of organic elements in thistwo samples showed the following values for SOL7 and SOL18,respectively: 0.1 and 0.02 (N%), 0.34 and 0.33 (C%), 0.93 and0.32 (H%), 17.38 and 44.50 (S%), and 18.34 and 4.37 (O%).There was a similar weak C content suggesting that biomasswas low. Despite various attempts using different PCR condi-tions and primer pairs specific for archaea, we always failedto amplify 16S rRNA genes for this prokaryotic domain. How-ever, thermoacidophilic archaea have been isolated from centralSolfatara boiling mud pools (DeRosa et al. 1975; Huber et al.2000a, 2000b; Zillig et al. 1980). Biases due to DNA extractionor primer utilization could explain this apparent discrepancy,although DNA was extracted by various methods and differentPCR conditions were used to minimize possible biases. There-fore, this result may actually reflect the absence or a very lowdensity of archaea in these particular samples. This explanationwould be in agreement with lipid biomarker analysis, whichshows a clear dominance of bacterial hopanoids.

Bacterial 16S rRNA genes were successfully amplified fromthe two Solfatara samples, and 63 clone inserts were sequencedfrom the generated 16S rDNA libraries. The diversity in terms ofmajor bacterial taxa was relatively low (Figure 6 andTable 1). In the case of SOL7, phylotypes identified belongedonly to the alpha and beta subdivisions of the Proteobacteria, andto the Acidobacteria, whereas in SOL18 phylotypes affiliated tothe Gammaproteobacteria and the Actinobacteria were found aswell (Figure 6). Some phylotypes displayed sequences nearly100% identical to those of known thermophilic or thermotoler-ant cultivated species, such as Hydrogenophilus thermoluteolus,a facultative hydrogen-oxidizer growing optimally at 50–52◦C

Figure 6. Taxonomic distribution of bacterial phylotypes in16S rDNA libraries corresponding to soft deposits adjacent toa chimney (fumarolic Northeast area), and to iron-sulfur-richcrust adjacent to a mud pool in the center of the Solfatara crater.


Table 1Phylogenetic affiliation of representative bacterial 16S rDNA clones obtained from Solfatara crust and mud samples as deduced

from BLAST searches

PhylotypeSequence

length (bp)

Numberof similar

sequences∗Phylogenetic

ascription

Closest relativein database, environmental

origin (GenBank accession number)% identity(BLAST)

SOL18-11 997 11 Acidobacteria Uncultured bacterium WD257,polychlorinated biphenyl-polluted soil(AJ292583)

94


98


98


97

SOL18-51 1,001 1 Actinobacteria Agrococcus jenensis (X92492) 99SOL7-22 1,025 5 Alphaproteobacteria Paracoccus marcusii (Y12703) 99SOL7-1 1,050 13 Alphaproteobacteria Iron-oxidizing acidophilic methylotrophic

isolate Y005, Yellowstone (AY140237)92

SOL18-52 1,001 2 Alphaproteobacteria Iron-oxidizing acidophilic methylotrophicisolate Y005, Yellowstone (AY140237)

92

SOL18-16 1,000 5 Alphaproteobacteria Methylosinus sp. NCIMB 13214,methane-utilizing bacterium from abacterial consortium that rapidly degradestrichloroethylene (AB007840)

95

SOL7-40 993 1 Betaproteobacteria Delftia acidovorans (AY367028) 99SOL7-4 1,030 12∗∗ Betaproteobacteria Hydrogenophilus thermoluteolus (AB009828) 99SOL18-39 1,025 Betaproteobacteria Hydrogenophilus thermoluteolus (AB009828) 98SOL18-21 985 1 Gammaproteobacteria Acinetobacter sp. (Z93446) 96SOL18-37 984 1 Gammaproteobacteria Pseudomonas thermotolerans (AJ311980) 98SOL18-38 1,018 1 Gammaproteobacteria Pseudomonas thermotolerans (AJ311980) 99

∗Number of sequences >98% identity. ∗∗12 corresponds to the total number of Hydrogenophilus-like sequences.

(Hayashi et al. 1999) or Pseudomonas thermotolerans, growingoptimally at 47◦C (max 55◦C) (Manaia and Moore 2002). In-deed, most betaproteobacterial clones from both SOL samplesaffiliated to the genus Hydrogenophilus (Table 1 and Figure 7).Together betaproteobacteria, alphaproteobacteria and acidobac-teria were the most represented in our libraries. Interestingly,within the alphaproteobacteria, a majority of clones were mostclosely related to the iron-oxidizing strain Y005, which wasisolated from acidic geothermal areas in Yellowstone NationalPark. Y005 is related to methylotrophic species and, indeed, itcan also be grown in ferrous iron/methanol medium (Johnsonet al. 2003).

Finally, members of the Acidobacteria were profuse in bothSOL7 and 18 libraries (Figure 6). The Acidobacteria/Holophagarepresents a broad bacterial division occupying various environ-ments from activated sludge to marine sediments where most

members remain uncultivated so far (Ludwig et al. 1997). Mem-bers of this division have been identified in high proportion inshallow submarine vents near Milos (Greece) (Sievert et al.2000) and around deep-sea vents (Lopez-Garcıa et al. 2003).Remarkably, members of the Acidobacteria were also found tobe very abundant in soils following a geothermal heating event atYellowstone. Their amount increased in soils incubated at 50◦Cindicating that various members of this group are thermophilic(Norris et al. 2002). The Solfatara acidobacterial sequences arerelatively varied, but they are all more closely related to thegenus Acidobacterium and quite distant from Holophaga. Thefew available cultivated members of this group are heterotrophs,and this has also been suggested for lineages found in shallowthermal areas. However, not only the Solfatara samples are verypoor in organic content, but it is very difficult to predict thephysiology of the Solfatara lineages on the basis of sequence


Figure 7. Maximum likelihood (ML) phylogenetic tree showing the position of representative bacterial 16S rDNA sequencesfrom iron-sulfur crust close to a mud pool (SOL7) and melted deposits in the Northern fumarolic area (SOL18) samples. OnlyML bootstrap values above 50% are shown at nodes. The scale bar corresponds to 10 substitutions for a unit branch length. CFB,Cytophaga-Flexibacter-Bacteroides group.

comparison, since they are quite distant from the few currentlyisolated species (Figure 7). Furthermore, the lack of more iso-lated strains from this broad division may imply that currentculture media are not suitable for their growth and that these or-ganisms may display novel metabolic strategies. Since many ofthese lineages are identified in metal-rich, acidic thermal areas,they may possibly rely on some type of strict or chemolithoau-totrophic metabolism.

CONCLUDING REMARKSWe have applied a multidisciplinary analysis to the study

of hydrothermal deposits from the Solfatara crater, involvinggeology, micropaleontology, organic chemistry and molecularmicrobiology methods. The hydrothermal deposits were young(∼4,000 years old), mildly hot to hot (40–95◦C) and acidic(pH ∼1.7). Chemical conditions were predominantly anoxicand reducing, although oxic/anoxic transition zones existed in


surface areas, and rock fractures. These features could favorthe development of chemolithotrophic microbes by allowingaccess to different electron and acceptor donors along physical-chemical gradients. Potential biomineral signatures were iden-tified in altered fractures within sulfur-rich Solfatara deposits,along with lipid biomarkers. These included particles of ele-mental sulfur, pyrite and barite crystals, which were sometimesassociated to permineralized EPS net-like structures. Thoughbarite minerals are usually affiliated with marine or with deep un-derground hydrothermal environments, here they seem to haveformed in the surface settings.

The organic C content of Solfatara deposits was low, yetlipid biomarkers including bacterial hopanoids were identified.Finally, a molecular diversity survey carried out on melted de-posits in a fumarolic area and on iron-rich crust, for which lipidand mineral signatures were detected, allowed the detection ofdifferent bacterial phylotypes. The diversity was relatively low,and the phylotypes detected affiliated to known thermophilic orthermotolerant bacteria, some of which are iron- and hydrogen-oxidizers, or methane/methanol consumers. This is in agreementwith the substrate’s nature and with the content of fumarolicemissions. Nevertheless, many phylotypes, such as the acidobac-terial lineages detected, are too distant from cultivated speciesto safely conclude anything about their metabolic capabilities.Given the nature of the habitat and the low content in organ-ics, some of these could correspond to chemolithotrophic bacte-ria obtaining energy from redox reactions involving species ofiron or sulfur, similarly to the sulfur-oxidizing archaea that havebeen isolated from the hottest Solfatara mud pools. The mineraland biochemical signatures observed could therefore attest forthe activity of past (recent) microbial communities, which werelikely very similar to those observed today, as well as contempo-rary microorganisms. A better understanding of the genesis ofthese potential biosignatures from the autochthonous microbialcommunities will be further needed to interpret most of ancientsignals from similar environments.

REFERENCESAfricano F, Bernard A. 2000. Acid alteration in the fumarolic environment of

Usu volcano, Hokkaido, Japan. J Volcan Geoth Res 97:475–495.Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ.

1997. Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs. Nucleic Acids Res 25:3389–3402.

Banfield JF, Moreau JW, Chan CS, Welch SA, Little B. 2001. Mineralogicalbiosignatures and the search for life on Mars. Astrobiology 1:447–465.

Barker WW, Welch SA, Chu S, Banfield JF. 1998. Experimental observations ofthe effects of bacteria on aluminosilicate weathering. Amer Mineral 83:1551–1563.

Bell PE, Mills AL, Herman JS. 1987. Biogeochemical conditions favoring mag-netite formation during anaerobic iron reduction. Appl Environ Microbiol53:2610–2616.

Bove DJ, Hon K. 1990. Compositional changes induced by hydrothermal alter-ation at the red mountain alunite deposit, Lake City, CO. US Geol Surv Bull1936:1–21.

Brasier MD, Green OR, Jephcoat AP, Kleppe AK, Van Kranendonk MJ, LindsayJF, Steele A, Grassineau NV. 2002. Questioning the evidence for Earth’s oldestfossils. Nature 416:76–81.

Buseck PR, Dunin-Borkowski RE, Devouard B, Frankel RB, McCartney MR,Midgley PA, Posfai M, Weyland M. 2001. Magnetite morphology and life onMars. Proc Natl Acad Sci USA 98:13490–13495.

Butterfield NJ, Knoll AH, Swett K. 1996. Paleobiology of the NeoproterozoicSvanbergfjellet formation, Spitsbergen. Fossils Strata 34:3–83.

Cady SL, Farmer JD, Grotzinger JP, Schopf JW, Steele A. 2003. Morphologicalbiosignatures and the search for life on Mars. Astrobiology 3:351–368.

Chioni R, Corazza E, Marini L. 1984. The gas/steam ratio as indicator of heattransfer at the Solfatara fumarole, Phlegrean Fields (Italy). Bull Volcanol47:295–302.

De Las Heras FXC, Grimalt JO, Lopez JF, Albaiges J, Damste JSS, SchoutenS, De Leeuw JW. 1997. Free and sulphurized hopanoids and highly branchedisoprenoids in immature lacustrine oil shales. Org Geochem 27:41–63.

DeRosa M, Gambacorta A, Bu’Lock JD. 1975. Extremely thermophilic aci-dophilic bacteria convergent with Sulfolobus acidocaldarius. J Gen Microbiol86:156–164.

DeRosa M, Gambacorta A, Millonig G, Bu’Lock JD. 1974. Convergent char-acters of extremely thermophilic acidophilic bacteria. Experientia 30:866–868.

Des Marais DJ. 1996. Stable light isotope biogeochemistry of hydrothermal sys-tems. In: Bock GR, Goode JA, editors. Evolution of Hydrothermal Ecosystemson Earth (and Mars?), New York: John Wiley and Sons. p. 83–98.

Des Marais DJ, Bauld J, Palmisano A, Summons RE, Ward DM. 1992. Thebiogenicity of carbon in modern microbial mats. In Schopf JW, Klein C, edi-tors. The Proterozoic Biosphere, A Multidisciplinary Study. NY: CambridgeUniversity Press. P. 299–308.

Dong H, Fredrickson JK, Kennedy DW, Zachara JM, Kukkadapu RK, OnstottTC. 2000. Mineral transformation associated with the microbial reduction ofmagnetite. Chem Geol 169:299–318.

Farrimond P, Head IM, Innes HE. 2000. Environmental influence on thebiohopanoid composition of recent sediments. Geochim Cosmochim Acta64:2985–2992.

Ferrucci F, Gaudiosi G, Pino NA, Luongo G, Hirn A, Mirabile L. 1989. Seismicdetection of a major upheaval beneath the Campanian volcanic area, Naples,Southern Italy. Geophys Res Lett 16:1317–1320.

Gehrke T, Telegdi J, Thierry D, Sand W. 1998. Importance of extracellularpolymeric substances from Thiobacillus ferrooxidans for bioleaching. ApplEnviron Microbiol 64:2743–2747.

Gelinas Y, Baldock JA, Hedges J. 2001. Demineralization of marine and fresh-water sediments for CP/MAS 13C NMR analysis. Org Chem 32:677–693.

Gonzalez-Munoz MT, Fernandez-Luque B, Martınez-Ruiz F, Chekroun KB,Arias JM, Rodrıguez-Gallego M, Martınez-Canamero M, De Linares C,Paytan A. 2003. Precipitation of barite by Myxococcus xanthus: possible im-plications for the biogeochemical cycle of barium. Appl Environ Microbiol69:5722–5725.

Hayashi NR, Ishida T, Yokota A, Kodama T, Igarashi Y. 1999. Hydrogenophilusthermoluteolus gen. nov., sp. nov., a thermophilic, facultatively chemolithoau-totrophic, hydrogen-oxidizing bacterium. Int J Syst Bacteriol 49:783–786.

Hockin SL, Gadd GM. 2003. Linked redox precipitation of sulfur and sele-nium under anaerobic conditions by sulfate-reducing bacterial biofilms. ApplEnviron Microbiol 69:7063–7072.

House CH, Schopf WJ, Stetter KO. 2003. Carbon isotopic fraction by Archaeansand other thermophilic prokaryotes. Org Geochem 34:345–356.

Huber R, Huber H, Stetter KO. 2000a. Towards the ecology of hyperther-mophiles: biotopes, new isolation strategies and novel metabolic properties.FEMS Microbiol Rev 24:615–623.

Huber R, Sacher M, Vollmann A, Huber H, Rose D. 2000b. Respiration ofarsenate and selenate by hyperthermophilic archaea. Syst Appl Microbiol23:305–314.

Hugenholtz P, Pitulle C, Hershberger KL, Pace NR. 1998. Novel division levelbacterial diversity in a Yellowstone hot spring. J Bacteriol 180:366–376.

Jobb G. (2002). TREEFINDER, Distributed by the author at 〈www.treefinder.de〉.

Johnson DB. 1998. Biodiversity and ecology of acidophilic microorganisms.FEMS Microbiol Ecol 27:307–317.


Johnson DB, Okibe N, Roberto FF. 2003. Novel thermo-acidophilic bacteriaisolated from geothermal sites in Yellowstone National Park: physiologicaland phylogenetic characteristics. Arch Microbiol 180:60–68.

Karthikeyan S, Beveridge TJ. 2002. Pseudomonas aeruginosa biofilms reactwith and precipitate toxic soluble gold. Environ Microbiol 4:667–675.

Kasting JF, Ackerman TP. 1986. Climatic consequences of very high car-bon dioxide levels in the Earth’s early atmosphere. Science 234:1383–1385.

Lopez-Garcıa P, Duperron S, Philippot P, Foriel J, Susini J, Moreira D. 2003.Bacterial diversity in hydrothermal sediment and epsilonproteobacterial dom-inance in experimental microcolonizers at the Mid-Atlantic Ridge. EnvironMicrobiol 5:961–976.

Ludwig W, Bauer SH, Bauer M, Held I, Kirchhof G, Schulze R, Huber I, SpringS, Hartmann A, Schleifer KH. 1997. Detection and in situ identification ofrepresentatives of a widely distributed new bacterial phylum. FEMS MicrobiolLett 153:181–190.

Manaia CM, Moore ER. 2002. Pseudomonas thermotolerans sp. nov., a ther-motolerant species of the genus Pseudomonas sensu stricto. Int J Syst EvolMicrobiol 52:2203–2209.

Martin W, Russell MJ. 2003. On the origins of cells: a hypothesis for the evolu-tionary transitions from abiotic geochemistry to chemoautotrophic prokary-otes, and from prokaryotes to nucleated cells. Phil Trans R Soc Lond B BiolSci 358:59–83.

Meyers PA. 1997. Organic geochemical proxies of paleoceanographic, pale-olimnologic, and paleoclimatic processes. Org Geochem 27:213–250.

Nisbet EG, Sleep NH. 2001. The habitat and nature of early life. Nature409:1083–1091.

Norris TB, Wraith JM, Castenholz RW, McDermott TR. 2002. Soil microbialcommunity structure across a thermal gradient following a geothermal heatingevent. Appl Environ Microbiol 68:6300–6309.

Ourisson G, Rohmer M, Poralla K. 1987. Microbial lipids betrayed by theirfossils. Microbiol Sci 4:52–57.

Pace NR. 1997. A molecular view of microbial diversity and the biosphere.Science 276:734–740.

Panichi C, Volpi G. 1999. Hydrogen, oxygen and carbon isotope ratios of Solfa-tara fumarole (Phlegrean Fields, Italy): further insight into source processes.J Volcanol Geotherm Res 91:321–328.

Philippe H. 1993. MUST, a computer package of management utilities for se-quences and trees. Nucleic Acids Res 21:5264–5272.

Provencio PP, Polyak VJ. 2001. Iron oxide-rich filaments: possible fossil bacteriain Lechuguilla Cave, New Mexico. Geomicrobiol J 18:297–309.

Rosi M, Sbrana A. 1987. Phlegrean fields. Consiglio Nazionale Delle RicercheQuaderni De “La Ricercha Scientifica” 114(9):175.

Rothschild LJ, Mancinelli RL. 2001. Life in extreme environments. Nature409:1092–1101.

Schidlowski M. 1988. A 3,800-million-year isotopic record of life from carbonin sedimentary rocks. Nature 333:313–318.

Schidlowski M. 2000. Carbon isotopes in microbial sediments. In: Riding RE,Awramik SM, editors. Microbial sediments, Berlin: Springer Verlag. P. 84–96.

Schopf JW. 1993. Microfossils of the Early Archean Apex chert: new evidenceof the antiquity of life. Science 260:640–646.

Schopf JW, Packer BM. 1987. Early Archean (3.3-billion to 3.5-billion-year-old)microfossils from Warrawoona Group, Australia. Science 237:70–73.

Schuler D, Frankel RB. 1999. Bacterial magnetosomes: microbiology, biomin-eralization and biotechnological applications. Appl Microbiol Biotechnol52:464–473.

Segerer AH, Stetter KO. 1999. The Order Sulfolobales. In: Dworkin M, editors,The prokaryotes: an evolving electronic resource for the microbiological com-munity. Berlin: Springer-Verlag, 〈http://link.springer-ny.com/link/service/books/10125/〉.

Sievert SM, Kuever J, Muyzer G. 2000. Identification of 16S ribosomal DNA-defined bacterial populations at a shallow submarine hydrothermal vent nearMilos Island (Greece). Appl Environ Microbiol 66:3102–3109.

Strauss H, Des Marais DJ, Hayers JM, Summons RE. 1992. Concentrations oforganic carbon and maturities and elemental compositions of kerogens. In:Schopf, JW, Klein C, editors. The Proterozoic Biosphere, A MultidisciplinaryStudy. NY: Cambridge University Press. P. 95–101.

Summons RE, Jahnke LL, Simoneit BR. 1996. Lipid biomarkers for bacterialecosystems: studies of cultured organisms, hydrothermal environments andancient sediments. Ciba Found Symp 202:174–193.

Tedesco D, Pece R, Sabroux JC. 1988. No evidence of the new magmatic gas con-tribution to the Solfatara volcanic gases during bradiseismic crisis at CampiPhlegrei, Italy. Geophys Res Lett 15:1441–1444.

Tor JM, Kashefi K, Lovley DR. 2001. Acetate oxidation coupled to Fe(III)reduction in Hyperthermophilic microorganisms. Appl Environ Microbiol67:1363–1365.

Treiman AH. 2003. Submicron magnetite grains and carbon compounds in Mar-tian meteorite ALH84001: inorganic, abiotic formation by shock and thermalmetamorphism. Astrobiology 3:369–392.

Tritz JP, Herrmann D, Bisseret O, Connan J, Rohmer M. 1999. Abiotic and bio-logical hopanoid transformation: towards the formation of molecular fossilsof the hopane series. Org Geoch 30:499–514.

Twichell SC, Meyers PA, Diester-Haass L. 2002. Significance of high C/N ratiosin organic-carbon-rich Neogene sediments under Benguela current upwellingsystem. Org Geochem 33:715–722.

Valentino GM, Cortecci G, Franco E, Stanzione D. 1999. Chemical and isotopiccompositions of minerals and waters from Campi Flegrei volcanic system,Naples, Italy. J Volc Geoth Res 91:329–344.

van Zuilen MA, Lepland A, Arrhenius G. 2002. Reassessing the evidence forthe earliest traces of life. Nature 418:627–630.

Wachtershausser G. 1988. Before enzymes and templates: theory of surfacemetabolism. Microbiol Rev 52:452–484.

Weiss BP, Kim SS, Kirschvink JL, Kopp RE, Sankaran M, Kobayashi A,Komeili A. 2004. Magnetic tests for magnetosome chains in Martian me-teorite ALH84001. Proc Natl Acad Sci USA 101:8281–8284.

Zillig W, Stetter KO, Schulz W, Priess H, Scholz I. 1980. The Sulfolobus-“Caldariella” group: taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch Microbiol 125:259–260.

Biosignatures and Bacterial Diversity in Hydrothermal...

Documents

Transcript of Biosignatures and Bacterial Diversity in Hydrothermal...