evidence for a biogenic, microorganismal origin of rock varnish from the gangdese belt of tibet

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Micron 42 (2011) 401–411 Contents lists available at ScienceDirect Micron journal homepage: www.elsevier.com/locate/micron Evidence for a biogenic, microorganismal origin of rock varnish from the Gangdese Belt of Tibet Xiaohong Wang a,b,, Lingsen Zeng c , Matthias Wiens b , Ute Schloßmacher b , Klaus Peter Jochum d , Heinz C. Schröder b , Werner E.G. Müller b,∗∗ a National Research Center for Geoanalysis, 26 Baiwanzhuang Dajie, CHN-100037 Beijing, China b Institute for Physiological Chemistry, Johannes Gutenberg University, Medical School, Duesbergweg 6, D-55099 Mainz, Germany c Institute of Geology, Chinese Academy of Geological Sciences, CHN-100037 Beijing, China d Max Planck Institute for Chemistry, J.J. Becherweg 27, D-55128 Mainz, Germany article info Article history: Received 4 November 2010 Received in revised form 2 December 2010 Accepted 2 December 2010 Keywords: Varnish Tibet Biogenic mineralization Microorganisms Biofilm Mars abstract In the present study we examined material from the Ashikule Basin of Tibet. Chemical analyses were performed by use of energy dispersive X-ray spectroscopy and electron probe microanalysis to clarify whether the varnish layers that had developed on the surface of the rhyolite are indeed composed of varnish bodies and silica glaze. Electron microscopic analyses revealed that the surface of the varnish is covered both by filamentous hyphae bacterial and cocci-shaped forms. Within the varnish mineral layer in those samples two forms of bacteria-like microorganisms exist; cocci as tightly packed bacterial aggregates [within varnish bodies], and bacillus-like microorganisms [within the varnish matrix, that surrounds the varnish bodies]. The bacillus-like forms are embedded in a network of filaments that have diameters between 35 and 45 nm. These bacilli with the surrounding filaments are assumed to have been involved in biofilm formation (synthesis of extracellular polymeric substances) prior to their live burial. We concluded that the formation of the varnish layers was for the most part biogenically driven by microorganisms. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Rock varnish has interested geo-biologists since Humboldt (1807) for two reasons, first to understand mineral formation in adverse climates and second to obtain further insights into the potential biogenic basis for mineral formation on Mars. Rock var- nish, a term established by Dorn and Oberlander (1980), consists of thin coatings on rock surfaces, measuring usually between a few micrometer and 3 mm (reviewed in Laudermilk, 1931; Engel and Sharp, 1958; Broecker and Liu, 2001; Hodge et al., 2005; Perry et al., 2003a, 2004). Varnish is a brown-black layered veneer of clay mineral on rocks that is rich in oxides and hydroxides of man- ganese [Mn] and iron [Fe] (Potter and Rossman, 1977; McKeown and Post, 2001; Garvie et al., 2008). In spite of intensive light- and electron-microscopic investigations coupled with geochemical analyses the mechanism of nucleation and growth of rock var- Corresponding author at: Institute for Physiological Chemistry, Johannes Guten- berg University, Medical School, Duesbergweg 6, D-55099 Mainz, Germany. Tel.: +49 6131 39 25910; fax: +49 6131 39 25243. ∗∗ Corresponding author. Tel.: +49 6131 39 25910; fax: +49 6131 39 25243. E-mail addresses: [email protected] (X. Wang), [email protected] (W.E.G. Müller). nish remains uncertain (Israel et al., 1997; Dorn, 1998; Perry et al., 2003a, 2004). The main components of rock varnish, Fe and Mn are widely used in the organic world as “cofactors” for electron transfer pro- cesses (see Ehrlich, 2002; Tebo et al., 2005; Edwards et al., 2005). Two mechanisms of oxidation have been distinguished: first the assimilatory metabolism of Mn and Fe, which involves cellular uptake and subsequent function in the cell metabolism, and sec- ond, the dissimilatory metabolism in which Mn and Fe serve either as energy source or as terminal electron acceptor, depend- ing on the oxidation state (Ehrlich, 2002). While in the assimilatory metabolism, small quantities of Mn and Fe are involved per cell, in the dissimilatory metabolism, much larger quantities of Mn and Fe are involved and consumed per cell. Moreover, in the assimila- tory metabolism, Mn and Fe need to be taken into the cell while in the dissimilatory metabolism (energy metabolism) Mn and Fe appear to be acted upon on or in the cell envelope. During those metabolic processes the metal can be deposited in the terres- trial and aquatic environment on organic templates (see Glasby, 2006; Wang and Müller, 2009), as has been determined for man- ganese/polymetallic nodules (Wang et al., 2009c) or crusts (Wang et al., 2009a, 2009b). These forms of organic template-driven min- eralization have been termed biomineralization by Lowenstam and Weiner (Lowenstam and Weiner, 1989; see also Weiner and Dove, 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.12.001

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Page 1: Evidence for a biogenic, microorganismal origin of rock varnish from the Gangdese Belt of Tibet

Journal Identification = JMIC Article Identification = 1592 Date: March 10, 2011 Time: 5:1 pm

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Micron 42 (2011) 401–411

Contents lists available at ScienceDirect

Micron

journa l homepage: www.e lsev ier .com/ locate /micron

vidence for a biogenic, microorganismal origin of rock varnish from theangdese Belt of Tibet

iaohong Wanga,b,∗, Lingsen Zengc, Matthias Wiensb, Ute Schloßmacherb,laus Peter Jochumd, Heinz C. Schröderb, Werner E.G. Müllerb,∗∗

National Research Center for Geoanalysis, 26 Baiwanzhuang Dajie, CHN-100037 Beijing, ChinaInstitute for Physiological Chemistry, Johannes Gutenberg University, Medical School, Duesbergweg 6, D-55099 Mainz, GermanyInstitute of Geology, Chinese Academy of Geological Sciences, CHN-100037 Beijing, ChinaMax Planck Institute for Chemistry, J.J. Becherweg 27, D-55128 Mainz, Germany

r t i c l e i n f o

rticle history:eceived 4 November 2010eceived in revised form 2 December 2010ccepted 2 December 2010

eywords:

a b s t r a c t

In the present study we examined material from the Ashikule Basin of Tibet. Chemical analyses wereperformed by use of energy dispersive X-ray spectroscopy and electron probe microanalysis to clarifywhether the varnish layers that had developed on the surface of the rhyolite are indeed composed ofvarnish bodies and silica glaze. Electron microscopic analyses revealed that the surface of the varnishis covered both by filamentous hyphae bacterial and cocci-shaped forms. Within the varnish mineral

arnishibetiogenic mineralizationicroorganisms

iofilm

layer in those samples two forms of bacteria-like microorganisms exist; cocci as tightly packed bacterialaggregates [within varnish bodies], and bacillus-like microorganisms [within the varnish matrix, thatsurrounds the varnish bodies]. The bacillus-like forms are embedded in a network of filaments that havediameters between 35 and 45 nm. These bacilli with the surrounding filaments are assumed to havebeen involved in biofilm formation (synthesis of extracellular polymeric substances) prior to their liveburial. We concluded that the formation of the varnish layers was for the most part biogenically driven

arsby microorganisms.

. Introduction

Rock varnish has interested geo-biologists since Humboldt1807) for two reasons, first to understand mineral formation indverse climates and second to obtain further insights into theotential biogenic basis for mineral formation on Mars. Rock var-ish, a term established by Dorn and Oberlander (1980), consistsf thin coatings on rock surfaces, measuring usually between a fewicrometer and 3 mm (reviewed in Laudermilk, 1931; Engel and

harp, 1958; Broecker and Liu, 2001; Hodge et al., 2005; Perryt al., 2003a, 2004). Varnish is a brown-black layered veneer oflay mineral on rocks that is rich in oxides and hydroxides of man-

anese [Mn] and iron [Fe] (Potter and Rossman, 1977; McKeownnd Post, 2001; Garvie et al., 2008). In spite of intensive light-nd electron-microscopic investigations coupled with geochemicalnalyses the mechanism of nucleation and growth of rock var-

∗ Corresponding author at: Institute for Physiological Chemistry, Johannes Guten-erg University, Medical School, Duesbergweg 6, D-55099 Mainz, Germany.el.: +49 6131 39 25910; fax: +49 6131 39 25243.∗∗ Corresponding author. Tel.: +49 6131 39 25910; fax: +49 6131 39 25243.

E-mail addresses: [email protected] (X. Wang), [email protected]. Müller).

968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.micron.2010.12.001

© 2010 Elsevier Ltd. All rights reserved.

nish remains uncertain (Israel et al., 1997; Dorn, 1998; Perry et al.,2003a, 2004).

The main components of rock varnish, Fe and Mn are widelyused in the organic world as “cofactors” for electron transfer pro-cesses (see Ehrlich, 2002; Tebo et al., 2005; Edwards et al., 2005).Two mechanisms of oxidation have been distinguished: first theassimilatory metabolism of Mn and Fe, which involves cellularuptake and subsequent function in the cell metabolism, and sec-ond, the dissimilatory metabolism in which Mn and Fe serveeither as energy source or as terminal electron acceptor, depend-ing on the oxidation state (Ehrlich, 2002). While in the assimilatorymetabolism, small quantities of Mn and Fe are involved per cell,in the dissimilatory metabolism, much larger quantities of Mn andFe are involved and consumed per cell. Moreover, in the assimila-tory metabolism, Mn and Fe need to be taken into the cell whilein the dissimilatory metabolism (energy metabolism) Mn and Feappear to be acted upon on or in the cell envelope. During thosemetabolic processes the metal can be deposited in the terres-trial and aquatic environment on organic templates (see Glasby,

2006; Wang and Müller, 2009), as has been determined for man-ganese/polymetallic nodules (Wang et al., 2009c) or crusts (Wanget al., 2009a, 2009b). These forms of organic template-driven min-eralization have been termed biomineralization by Lowenstam andWeiner (Lowenstam and Weiner, 1989; see also Weiner and Dove,
Page 2: Evidence for a biogenic, microorganismal origin of rock varnish from the Gangdese Belt of Tibet

Journal Identification = JMIC Article Identification = 1592 Date: March 10, 2011 Time: 5:1 pm

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of high-K rhyolite mineral formed at 49.85 ± 0.92 Ma, as dated by

02 X. Wang et al. / Mi

003). Biomineralization refers to those mineralization processeshat occur in close association with organic molecules or matri-es or is even (micro)biologically mediated. While mineralizationollows exclusively chemical and physical principles and results inhe accumulation of inorganic materials from solution without anyarticipation of organic molecules, the processes of biomineraliza-ion are induced on the surfaces of organic templates [biologicallynduced mineralization], or are almost entirely controlled by anrganic scaffold [biologically controlled mineralization]. Examplesf biologically induced mineralization are the formations of poly-etallic nodules (Wang et al., 2009c) or crusts (Wang et al., 2009a,

009b) whereas biologically controlled mineralization processesre the major principle in the formation of the hard, inorganic skele-on of large-sized animals (see Weiner and Dove, 2003; Schrödert al., 2008; Müller et al., 2009).

Rock desert varnish coatings are formed in numerous arid andemi-arid regions of the world, including the Sonoran and Mojaveeserts of the United States and Mexico (Engel and Sharp, 1958;otter and Rossman, 1977), the Negev Desert in the Middle EastKrumbein and Jens, 1981), the Gibson and Great Victoria Desertsf Western Australia (Beard, 1970), and the Gobi Desert as well ashe Gangdese Belt region in Tibet of China (Krinsley et al., 2009). Forhe formation of varnish abiogenic as well as biogenic origins haveeen proposed. The assumption of an inorganic, abiogenic depo-ition of the varnish coatings (Potter and Rossman, 1977; Blumet al., 1985) is based on experiments suggesting that deposition ofhe ferromanganese oxides within the clay matrix of varnish is dueo capillary movement of varnishing solutions from the rocks. Fur-hermore, the minerals have been proposed to originate from dustnd rain, as well as from the surrounding soils (Allen, 1978; Engelnd Sharp, 1958; Scheffer et al., 1963; Krumbein, 1969; Potter andossman, 1979). In a series of thorough studies the group of PerryPerry et al., 2003b) provided strong evidence for the participa-ion of microorganisms in growth of these rock coatings, e.g. bynalysis of the amino acid composition of varnish. Furthermore,trong evidence for the existence of bacteria within the varnishas been provided by Krumbein and Jens (1981) who observedhe presence of microorganisms with a potential for iron and/or

anganese precipitation in these coatings on the surface of thearnish as well as within these minerals. Subsequently, biogenicnitiation of varnish formation was frequently suggested when of aarge variety of bacteria was isolation (reviewed by Kuhlman et al.,006). In addition, the isolation of fungi from the surfaces of varnishas been described (Staley et al., 1982; Staley et al., 1983) whoseole has also been implicated in varnish mineral formation (Taylor-eorge et al., 1983; Gorbushina et al., 1993). Finally, it had beenroposed that varnish or varnish-like materials may exist on MarsAllen et al., 2001; Guinness et al., 1997; Israel et al., 1997; Probstt al., 2002), and hence varnish might be a niche for the coloniza-ion by extraterrestrial life forms, such as bacteria. Often, especiallyn older varnishes a layered botryoidal structure has been observedPerry and Adams, 1978) which displayed similarities to the deepea manganese nodules, a view that had already been discussed byoussingault (1882). Also similarity of the varnish with cyanobacte-ial stromatolites (Monty, 1973; Krumbein and Lange-Giele, 1979)as been described.

The growth rate of the varnish is slow and amounts to 1 tobout 40 �m per 1000 years (Liu and Broecker, 2000). The lami-ated layering of the varnish is surely an indicator for past climaticituations, mirroring especially the past alterations in environmen-al moisture (Liu and Broecker, 2000; Broecker and Liu, 2001).

n turn rock varnish harbors historical records for environmen-al processes, such as long-term climate changes (Liu, 2003). Even

ore, varnish has been considered to preserve atmospheric signa-ures and varnish deposits may provide clues to the biogeochemicalycle of sulfur (Bao et al., 2001). It should also be mentioned that

2 (2011) 401–411

rock varnish has attracted archeologists to date petroglyphs thatwere etched into varnish by ancient cultures (Dragovich, 2000;Watchman, 2000).

In the present study we investigated rock varnish from theAshikule Basin of Tibet. This region has been characterized to showsimilarity to the Mars environment (Krinsley et al., 2009). It isespecially the sediments, which consist of products from eolianabrasion of lava that occurs at a cold, sulfate-rich, high elevation,low-pressure, and very dusty environment. Such a location resem-bles the Martian surface (Arocena et al., 2003), even though theannual precipitation of 300 mm (Dorn, 1998) is at the upper limitfor a terrestrial analogue of Mars. Varnish material from this regionhas recently been described and was found to comprise distinctrock varnish bodies, oval shaped accretions that are surrounded bysilica glaze (Krinsley et al., 2009). Interestingly, the surfaces of theTibetan varnish comprise (living) Mn-enhancing bacteria of the fol-lowing three morphotypes: (i) coccoid forms, (ii) filamentous formsof bacterial size, and (iii) budding bacterial forms (Krinsley et al.,2009). No distinct microorganisms could be resolved in the sub-surface regions of those samples. In the present study we show thatinside the varnish two assemblages of fossilized microorganismsexist; firstly, cocci occurring in aggregates that are localized in thevarnish bodies and secondly, bacillus-like [rod shaped] microor-ganisms that exist outside the varnish bodies but within the varnishmatrix. Scanning electron microscopic inspection revealed that thebacteria are often found embedded in a matrix of extracellular poly-meric substances (EPS), displaying a filamentous morphology. EPSare known to be produced by biofilm-forming bacteria (O’Tooleet al., 2000), and are abundantly produced by microorganisms liv-ing in soils (Or et al., 2007). This EPS contributes to 0.1–1.5% to theorganic matter of soils (Chenu, 1995). Since the same bacterial mor-photypes, like those found fossilized within the mineral material,also exist on the surface of the varnish sample, as documented byscanning electron microscopic imaging, we postulate that varnishformation can (at least partially) be ascribed to a biogenic origin.

2. Materials and methods

2.1. Rock varnish from the Ashikule Basin of Tibet

The Linzizong Formation, that is located along the southern partthe Gangdese Belt, was formed during the Late Cretaceous to EarlyTertiary (70–40 Ma). The samples analyzed were collected in theSouth-Central Gangdese Belt, that is located in the Sangsang area(Kapp et al., 2007; Xie et al., in press); Fig. 1. This region is locatedalong the Yarlung Tsangpo suture zone in the south. The Tanggulathrust system is located in the north (Wang et al., 2008). It is embed-ded in the Gangdese retroarc thrust belt (Kapp et al., 2007). TheGangdese Belt was shaped by magmatic events and tectonic driftsof the Indian and the Asia Plate. The Sangsang volcanic rocks devel-oped by melting of the metasomatic lithospheric mantle, inducedeither by rolling-back or by breaking off from the Indian slab. Southof the study area in the Gangdese lies the fore-arc basin, a locationdominated by the accumulation of the Shigatse flysch-like mate-rial formed during the Albian-Cenomanian age (Burg, 1983). To theNorth, the research area is bordered by the Linzizong volcanic rocksand the Oligocene granite. The collection site (Ashikule Basin) islocated at 29◦37′N and 86◦41′E at an altitude of 4900 m. Volcanicrocks are widely distributed in that area and are composed mainly

the SHRIMP zircon U/Pb age technique (Xie et al., in press).Varnish samples from the Ashikule Basin were studied with

respect to element distribution and zonation as well as the pres-ence of recent bacterial communities and the fossilized traces ofthem within the mineral layer.

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Journal Identification = JMIC Article Identification = 1592 Date: March 10, 2011 Time: 5:1 pm

X. Wang et al. / Micron 42 (2011) 401–411 403

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ig. 1. Collection site of varnish samples in the Ashikule Basin, close to Sangsanglong the Southern part the Gangdese Belt. South of the study area the Gangdese fore marked by dashed lines; TTS, Tanggula thrust system [North] and YZS, Yarlungegend, the reader is referred to the web version of the article.)

.2. The climate condition in Sangsang area

Sangsang is located within a plateau, characterized by a sub-rigid-sub-temperate and semi-arid monsoon climate zone. Thelimate is relatively dry and cold with a marked day/night temper-ture shift and a peak temperature of 10 ◦C to 15 ◦C. The frost-freeeriod lasts about 105 days per year; the annual average sunshineime is >3000 h. The wet and dry seasons are distinct. The averagennual rainfall is about 250–300 mm allowing only sparse vegeta-ions (Kapp et al., 2007).

.3. Chemical analysis

The varnish samples were ground to -200 mesh powder.liquots of 0.5 g of the sample were thoroughly mixed with 5 gf Li2B4O7 and fused into a glass bead. Finally the material wasnalyzed by the X-ray fluorescence (XRF) technique following therocedure described by Wu et al. (2008).

.4. Elemental analysis: EDX and EPMA

The varnish samples were sliced (50–300 �m thick) with a LeicaM2500/SP2600 sliding microtome with ultramilling attachmentLeica Microsystems, Nußloch, Germany) and polished with 1 pmiamond paste (Engis Corp., Morton Grove, IL, USA). With theselices energy dispersive X-ray spectroscopy (EDX) was performedith an EDAX Genesis EDX System attached to a scanning electronicroscope (Nova 600 Nanolab; FEI, Eindhoven, The Netherlands)

perating at 10 kV with collection time of 30 s. X-ray spectra wereecorded and analyzed using a compilation manual (Wang and Gao,

994).

Then, electron probe microanalysis (EPMA) was performed witholished thin sections through the varnish/rhyolite samples for O,, Fe, Mn, P, Ca, K, Mg, Si and Al with a Jeol Jxa 8200 electron probe atax Planck Institute of Chemistry (Mainz University) as described

); marked [red circle]. This area is embedded in the Linzizong Formation, locatedbasin is located disclosing Shigatse flysch-like formation. The major suture zones

po suture zone [South]. (For interpretation of the references to color in this figure

(Sobolev et al., 2007). The slices were coated with a thin gold layer(≈10 nm). Each sample was analyzed in the geometrical center at20 kV accelerating voltage and probe current of 300 nA with longcounting times for both peak and background. Where indicatedEPMA microanalysis was performed in combination with HR-SEM.

2.5. Optical and electron microscopy

Cross sections or small lithic flakes were analyzed. For digi-tal light microscopic analyses a VHX-600 Digital Microscope fromKeyence (Neu-Isenburg, Germany), equipped with a VH-Z25 zoomlens [magnification from 25× to 175×], was used. Fluorescenceoptical microscopic images were taken with a BZ-8000K Keyenceepifluorescence microscope using the objective lenses APO 2 X, orApo 20× 0.75 in combination with a green filter (excitation wave-lengths: 400–418 nm) or a red filter (632 nm). The images wereanalyzed with the BZ-Analyzer software BZ-H1AE/BZ-H1TLE/BZ-H1M3E (Keyence).

High-resolution scanning electron microscopy (HR-SEM) anal-yses were carried out with a Gemini Leo 1530 high resolution fieldemission scanning electron microscope (Oberkochen; Germany) ata voltage of 3–5 kV. Samples of broken splints from the rock varnishsample were mounted onto aluminum stubs (SEM-Stubs G031Z;Plano, Wetzlar; Germany).

3. Results

3.1. Rock varnish: surface

The varnish/rock samples from the Ashikule Basin, which we

studied, had an average area of 8–10 cm × 5–10 cm. The thickness ofthe varnish was 300 �m at maximum and thin down to 20–50 �m(Fig. 2). It had been formed on the surface of rhyolite that originatedfrom extruded lavas. Its chemical composition was similar to thatof granite, and contained as dominant minerals besides of quartz,
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404 X. Wang et al. / Micron 42 (2011) 401–411

Fig. 2. Rock varnish from the Ashikule Basin (Tibet). (A and B) The rock samples, composed of rhyolite (rh; double-headed arrow), are overlaid with 10–500 �m thick amber todark brown varnish coatings (va; arrow; double-headed arrow). (B and C) The surface of the varnish (va) is smooth and comprises coccoid to spiral/lobate structures. (D) Lightmicroscopic inspection of a cross section through the varnish layer, displaying the characteristic rock varnish bodies (va) and the silica glaze (sg); overlay of simultaneouslyr yellowm s (va;s gnal foi

abbso

ecorded green and red fluorescence images. The rock varnish bodies highlight inicroscopy images likewise allows the distinction between the rock varnish bodie

ilica glaze matrix. (G and H) EDX analyses of the silica glaze (G), showing a high sin varnish bodies (the intensities of the signals are given on the ordinate).

lkali feldspar, and plagioclase feldspar, the minor components ofiotite and pyroxene. Varnish layers that have an amber to darkrown color formed on the surface of the light gray rhyolite. Theurface of the varnish was largely smooth (Fig. 2A). The diameterf the lobate structures, found on the surface of the varnish was in

, while the silica glaze appears in red. (E and F) Back-scattered scanning electronbright) and the silica glaze (sg; dark). The rock varnish bodies are surrounded byr Si, and of the rock varnish bodies (H) with the signals for Mn and Fe, not present

the range 200–800 �m (Fig. 2B and C). A close inspection of the sur-face by SEM revealed slightly undulating nests (Fig. 3B). They weresurrounded and embedded in a mineral matrix (Fig. 3A). The evagi-nations/bulgings/nests on the surface of the varnish (Fig. 3B) werecomposed of 2 �m spheres, first signs for fossilized microorgan-

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X. Wang et al. / Micron 42 (2011) 401–411 405

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ig. 3. Outer surface of the varnish; SEM. (A) The major surface area appears as a ntructures (co).

sms. These tightly packed, coccus-like fossils did not show signsf a mycelial framework. Similar nests on surfaces of varnish rocksave been described as surface-associated “nests of microcolonial

ungi” (Taylor-George et al., 1983).

.2. Rhyolite rock: the substrate of varnish layers

The substrate of the varnish layers, the rhyolite rock, was ana-yzed by X-ray fluorescence (XRF). Its chemical composition wasiO2 70.40%, TiO2 0.42%, Al2O3 14.36%, FeO 2.52%, MnO 0.08%, MgO.82%, CaO 2.26%, Na2O 3.58%, K2O 4.30% and P2O5 0.11%.

.3. Rock varnish: cross sections

Cross sections through the varnish layer were performed tonalyze its texture. The Tibet varnish sample did not show the dis-inct micro-lamination, as documented for the Sonoran and Mojaveeserts material (Perry et al., 2003b). However, the characteristicorphology of the Tibetan rock varnish, described by Krinsley et al.

2009) could be resolved. Besides the oval-shaped accreting bod-es, representing the “rock varnish”, silica glaze was observed that

as scattered within the non-structured varnish matrix (Fig. 2D–F).he rock varnish was embedded in the brittle varnish matrix thatarbored the silica glaze islands. Light microscopic inspection ofhe slices revealed that the rock varnish bodies highlighted yel-ow if inspected under red and green fluorescence, followed by anverlay of the images, and hence allowed thereby a simple dis-inction between the silica glaze and the non-structured matrixFig. 2D). Analysis of the samples by back-scattered scanning elec-ron microscopy permitted a similar clear distinction between theock varnish bodies and the silica glaze, as described by Krinsleyt al. (2009). While the rock varnish appeared bright, the silica glazeppeared in dark (Figs. 2E and F). The rock varnish bodies had a pixelexture and were always surrounded by the silica glaze (Fig. 2F). Therevalence of the element Si in the silica glaze was determined byDX analysis (Fig. 2G). In contrast, in the rock varnish bodies thelements Mn and Fe could be additionally recorded, and also higherignals for C are seen (Fig. 2H).

.4. EPMA analysis of elements in varnish

An electron probe microanalysis (EPMA) was performed withinmeasuring area of 200 × 200 �m on slices prepared from min-

ral material taken 100 �m below the surface of the varnish layernd 200 �m above the rhyolite (Fig. 4A). The electron backscatteredmage of the analysis area showed that this selected region com-rised one rock varnish body (Fig. 4B). The varnish bodies wereurrounded by a silica glaze matrix and additional brittle varnish

ructured matrix. (B) Undulating nests (ne) composed of tightly packed coccus-like

material. This zonation, varnish – silica glaze, was impressively sup-ported also by the EPMA elemental maps. The varnish region had asignificantly lower level of O, compared to the silica glaze (Fig. 4C).However, significantly higher concentrations of Fe (Fig. 4D) and Mn(Fig. 4E) were seen. The matching concentration patterns for thesetwo elements, Fe and Mn, within the varnish nodule were signif-icant, suggesting distinct elemental enhancement attributable tobiogenic production. P was homogeneously distributed (Fig. 4F).The concentration of the alkali metal K (Fig. 4G) within the var-nish was comparatively low, however, in the center of the rockvarnish the concentration of K was higher and followed a pattern,measured for Fe and Mn. The levels of the two earth alkali met-als, Ca (Fig. 4H) and Mg (Fig. 4I), were inversely correlated. Whilethe concentration of Ca was high within the varnish region, thatfor Mg in the corresponding areas was low (Fig. 4I). But again, adistinct local increase in these two elements was seen within thevarnish, a pattern that coincided with the local distribution of Feand Mn, as is apparent from the corresponding images. Finally, asexpected, the concentration of Si was significantly higher in the sil-ica glaze region, surrounding the rock varnish body (Fig. 4J). Theoverall distribution pattern for Al was inverse to the one recordedfor Si (Fig. 4K). As was the case with P in silica glaze, the overallconcentration of C within the varnish bodies was also low and didnot exhibit a recognizable pattern (Fig. 4L).

3.5. Microbial forms on the outermost surface of the varnish

The process of varnish formation has often been suggested to bemicrobially mediated (reviewed in Perry et al., 2003b). To supportthe assumption about the samples studied here HR-SEM analyses ofthe surface of the mineral as well as the interior of the varnish wereperformed. Examination of the varnish surface disclosed clustersof bacterial forms (Fig. 5) that were, as expected, almost identi-cal to those described recently from varnish collected in the sameregion in Tibet (Krinsley et al., 2009). Dominant were the hyphalforms (Fig. 5A and B). These rod-shaped bacteria had a diameter of0.5–0.7 �m and a length of 1.0–1.3 �m and were assembled into10–25 �m long chains. Embedded within these filamentous bacte-ria were cocci that were usually associated with clusters (Fig. 5C).The diameter of these coccus-shaped forms was between 1.1 and1.5 �m. In the vicinity of these bacteria (both cocci and rods) rope-like structures existed, especially in regions that are fissured, thatwe interpret as biofilm (Fig. 5C). At a higher magnification these

rope-like structures had a smooth and even surface; they had diam-eters of 90–150 nm (Fig. 5D). In order to support the view that thosebacteria that were found on the surface of the varnish had been aliveand had not undergone extensive mineralization processes an EDXanalysis was performed (Fig. 5E). The spectrum showed that the rel-
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406 X. Wang et al. / Micron 42 (2011) 401–411

Fig. 4. Element distribution within the varnish region. (A) Light microscopic image of the varnish region (va) on top of the granite rock (gr). The varnish layer is marked bythe brown color; cross section. The area in the varnish used for EPMA analyses is marked by crossed double-headed arrows. (B) Secondary electron image of the selectedregion used for EPMA analyses. The varnish (va) area and the silica glaze (sg) region are marked. The distributions of the following elements have been determined; (C) forO, (D) for Fe, (E) for Mn, (F) for P, (G) for K, (H) for Ca, (I) for Mg, (J) for Si, (K) for Al, and (L) for C. The relative concentrations increase with a change in color from dark/blueto yellow to red. Images (B) to (L) have the same magnifications; the size bar is given in (B). (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of the article.)

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X. Wang et al. / Micron 42 (2011) 401–411 407

Fig. 5. Bacterial forms existing on the surface of the varnish samples. Samples were inspected by HR-SEM analysis. (A and B) General view of the varnish surface, displayingt cocci (t s extrE ich inM

afsoE

3v

eafovaoicd(t1sv

he hyphal forms of bacteria (hy). In addition, to these dominant hyphae forms alsowo cocci (co). Adjacent to them one rope-like structure is seen that we designated aPS structure at higher magnification. (E) EDX analysis of the area (3 �m × 3 �m) rn. Comparatively low is the signal for Al, that had been taken as a reference.

tive peak height for the C signal, with respect to that of Al [markeror a non-biotic element], was high compared to the C/Al ratio mea-ured at the surface of the silica glaze (Fig. 2G), and that measuredn the rock varnish bodies (Fig. 2H), under the same setting of theDAX/EDX System (10 kV).

.6. Fossil microorganisms within the varnish region: cocci in thearnish bodies

Small lithic flakes, obtained from the interior of the varnish lay-rs, approximately 50–150 �m from the surface and 100–150 �mbove the border to the rhyolite were chosen for the analysis. Theracture planes were not flat due to the different breaking strengthf the mineral material. The silica glaze was more resistant than thearnish matrix/varnish bodies that were more brittle (Fig. 6A). Thereas of the varnish bodies were cluttered with coccus-like spheresf uniform sizes (Fig. 6B). Frequently these spheres which we termn view of the bacteria were seen on the surface of the varnish asoccus-shaped bacteria. They were aggregated to clusters that oftenetached from the surrounding basic matrix of the varnish bodies

Fig. 6C). The diameters of these spherical clusters range from 15o 60 �m; they are composed of spheroidal cocci of a diameter of–3 �m (Fig. 6D). Some of these spheroidal cocci showed dividingtructures suggesting that the bacteria had been included in thearnish bodies in the living state. The division structures, follow-

co) are found that are frequently associated to clusters. (C) Higher magnification ofacellular polymeric substance (EPS) with filamentous morphology. (D) FilamentousEPS. A high signal for C is recorded; likewise prominent are the signals for Fe and

ing along planes in an irregular pattern, appeared to form clumps,similar to structures that are known from the genus Staphylococ-cus (e.g. Tzagoloff and Novick, 1977), or even Streptobacillus (e.g.Lee et al., 1999). This finding suggests that the clumps/rods changetheir orientation/direction in accordance with changes in the divi-sion plane. Hence, some of these rod-shaped aggregates have a bentform (Fig. 6F).

3.7. Bacillus-like microorganisms and potential biofilm structurein the varnish

The technique of HR-SEM was applied to demonstrate thepotential existence of biofilms in the varnish matrix, outside thepresumed microbial, sheroidal bacterial cells. The HR-SEM imagesshowed that at this location, the morphotypes of bacilli were dom-inant over those of cocci (Fig. 7). The bacillus-like microorganismswere embedded in a network of filaments that indicated the pres-ence of a biofilm (Fig. 7A and B). None of these microorganismswere seen in aggregates; they occurred at distances between 20and 50 �m. The sizes of these bacillus-like organisms were uni-

form between 2.1 and 0.8 �m. The images showed further that thefilaments were closely attached to, or were even released from, thetermini of the microbial cells (Fig. 7C). The filaments were abun-dant and mostly separated from each other (Fig. 7C and E); emptyimprints of these filaments were rare (Fig. 7D). The diameters of
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408 X. Wang et al. / Micron 42 (2011) 401–411

Fig. 6. Distribution of fossil microorganisms within the varnish layer; SEM images. (A) Small lithic flakes were obtained from the interior of the varnish layer. There thevarnish bodies can be distinguished from the surrounding silica glaze by their brittle fracture surface (va), in contrast to the plane surface seen in the silica glaze areas (sg).(B) Frequently small solitary coccus-like spheres (><) are seen. (C) Occasionally spheric assemblages are found that appear detached from the surrounding matrix of thev fossilsD divisiop ils tha

tae

4

ATtarfo

pv

arnish. Those assemblages (as) represent aggregates of coccus-like bacterial microivision structures of cocci (co), forming rod-like structures; two of the equatorialerpendicularly to the previous. (F) Filamentous structures of (assumed) microfoss

hese filaments were in the range of 35–45 nm, a dimension knownlso from filaments in environmental biofilms formed by bacteria,.g. for Desulfovibrio vulgaris (Clark et al., 2007).

. Discussion

Very recently newly discovered rock varnish material from theshikule Basin of Tibet had been studied by Krinsley et al. (2009).he Ashikule Basin, at an elevation in excess of 4000 m, is charac-erized by a warm arid desert climate. Based on the high-altitudend the high ultraviolet flux environment of the Tibetan locale thisock varnish had been considered as a suitable reference mineral

or a further understanding of (a)biogenic mineralization processesn early Mars (Krinsley et al., 2009; Xie et al., in press).

In the first part of our investigation, we performed EPMA map-ing to characterize the varnish bodies in the mineral layer of thearnish layer. They are rich in Fe, Mn, K, Ca and Al and clearly dis-

(co). (D) Higher magnification of spheroidal microfossils, coccoid bacteria (co). (E)n septa are oriented in the same direction, while the one in the terminal sphere is

t have changed the growth direction.

tinguished from the surrounding silica glaze by lower Si, O and Mglevels. This mapping allowed the identification of distinct islandswithin the varnish bodies that contained high levels of Fe, Mn, K,and Ca, which indicates that the composition of the varnish bodiesis not uniform. As analyzed in detail by HR-SEM these prominentislands within the varnish bodies are poor in Si and Al. In contrastto the minerals surrounding the varnish matrix and these varnishbodies, we found spherical assemblages of spheroidal microorgan-isms embedded in Fe/Mn/K/Ca-rich islands in the varnish minerals,which we interpret as aggregates of spheroidal microfossils. Wealso identified coccus-like microorganisms in the varnish matrixaround those varnish bodies, but found no bacterial-like forms in

the silica glaze islands. It is very common that microfossils becomeembedded into iron minerals, e.g. in deep sea nodules and crusts(Wang and Müller, 2009).

Krinsley et al. (2009) presented strong evidence that the surfaceof the varnish from Tibet is covered by three different morpho-

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X. Wang et al. / Micron 42 (2011) 401–411 409

Fig. 7. Indications for the formation of biofilm structures, produced by microorganisms in the varnish bodies. Outside the varnish bodies, but within the varnish matrix[ tructui in ano (ba)o ividua

tbuvpwbetscteecamc

varnish that surrounds the bodies] individual microorganisms with filamentous snterspersed in the matrix. (B) One solitary bacillus (ba) is depicted that is embeddedf such a region to accentuate the filaments (arrow) existing around one bacteriumne bacillus (b). (E and F) High resolution images from a biofilm net, displaying ind

ypes of Mn-depositing bacteria: (i) clusters of coccoidal forms, (ii)acterial hyphae, and (iii) budding bacteria. The varnish materialsed in this study (Krinsley et al., 2009) had been collected in Tibetery close to the region from where the varnish material in theresent study was obtained. In a comparative series of experimentse applied the HR-SEM technique and could identify the same

acterial forms like those that had been described recently. In anxtension of the report by Krinsley et al. (2009) we could resolve onhe surface of the varnish EPS filaments with smooth surfaces andurprisingly constant diameters of 90–150 nm. These structures areompatible with filaments found with soil biofilm-producing bac-eria, e.g. the filamentous exopolymer of Pseudomonas sp. (Mattisont al., 2002). These filaments were larger than the 30 nm thick flag-

lla identified in bacteria, e.g. in D. vulgaris (Clark et al., 2007), andlearly do not arise from one polar end of the microorganisms. Itlso appears unlikely that the filaments, observed in the varnishaterial, represent bacterial pili, even though pili have been impli-

ated in bacterial biofilm formation (O’Toole et al., 2000). Those

res are seen; SEM images. (A) The solitary bacilli/rod-like bacteria (ba) are foundarray of filaments forming a net (fne); pseudo-color image. (C) Higher magnification. (D). Imprints from suspected biofilm filaments (arrow) and perhaps fragments ofl filaments (arrows).

pilus appendages are much smaller (8 nm in diameter; Mu andBullitt, 2006) than the filaments seen in the varnish.

We applied the HR-SEM technique also to identify thosemicroorganisms entrapped inside the varnish mineral layers. Onlytwo bacteria-sized forms were identified within the varnish;spheric coccoid forms and bacillus-like microorganisms. The aggre-gates, consisting of clusters of cocci, had a size of 20–90 �m, andlacked any visible filaments. The interior of the clusters showedcocci that had undergone equatorial cell divisions. This finding is astrong indication that those cocci had been entrapped alive (Wanget al., 2009c). In addition, the finding that some of the bacteriawithin the mineral phase were in the state of cell division mightsuggest that the embedded and fossilized microorganisms had been

aerobic, chemolithotrophic bacteria, likely to be grouped with theiron or manganese-oxidizing bacteria (Ehrlich, 2002).

The assemblages formed by compact aggregates of coccus-likeforms, deposited within the varnish material, were surrounded bybacterial aggregates of a different morphologies. The aggregates

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4 cron 4

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10 X. Wang et al. / Mi

ontained individual bacillus-like microorganisms (2.1 × 0.8 �m),hich were separated from each other by a distance of 20–50 �m.

uch bacteria have been described to exist in biofilms (Webb,007). The space between the microorganisms has been attributedo bacterial cell lysis during biofilm development (Mai-Prochnowt al., 2006). A characteristic feature of the bacillus-like microor-anisms is the arrangement/association with small chains. In theamples, analyzed here, the chains had diameters of 35–45 nm.hain-forming microorganisms have been frequently identified inacterial biofilms (Westall and Rince, 1994; Clark et al., 2007). Theiameter of 35–45 nm of the ropes of the microorganisms insidehe varnish material is smaller than the rope-like structures iden-ified around the bacteria that were detected on the surface of thearnish, which measure a thickness of around 100 nm. We attributehis difference in the size to the shrinking of those structures duringossilization from about 100 nm in the living state to about 40 nmn the fossilized form.

Taken together, the data presented in this study provide persua-ive results/observations that the formation of the varnish layers ismainly) driven biogenically, by microorganisms that form eitherolid aggregates (assemblages) of cocci or biofilm-producing soli-ary bacillus-like microorganisms. The close correlation/similarityetween the morphotypes of the living microorganisms that haveeen described to live on the surface of the same varnish mate-ial (Krinsley et al., 2009) that has been studied by us and theorphotypes of microorganisms (cocci and bacilli) within the min-

ral layer supports the view that biogenic processes contribute toarnish formation. Until now no age determinations have been per-ormed for the varnish samples from Tibet. It is also unknown how

uch time passed after the formation of the Sangsang volcanicock, approximately 50 million years ago (Lee et al., 2009), and theeriod of Pleistocene glaciations in that Tibetan Plateau (Derbyshiret al., 1991) until varnish formation started. However, the presentay climate (annual precipitation of approximately 300 mm; Dorn,998), the physical and chemical characteristics (high ultravio-

et irradiation, sulfate-rich, high elevation, low-pressure, and veryusty environment) display some similarities to the Martian sur-ace (Krinsley et al., 2009). In ongoing studies we are proceedingith an age determination of the Sangsang varnish rocks in order

o assess the age and the growth rate(s) of that Tibet varnish.

. Conclusion

In the present investigation we extend an earlier reportKrinsley et al., 2009) which described the existence of microorgan-sms on the surface of the varnish material, also collected from theshikule Basin in Tibet. We demonstrated microorganisms within

he varnish that have morphologies similar to those found on theurface of the varnish. Because we found bacterial cells within theineral layer that showed evidence of cell division, we propose

hat these bacteria, which we also found on the surface of the min-ral and must have at the time of fossilization, contributed to theineralization of the varnish layers. The implication of bacteria in

bio)mineral formation of deep sea manganese/polymetallic nod-les has recently been documented (Wang and Müller, 2009). Aroblem to be solved in future is to investigate if those bacteria

iving on and also within the rock layers precipitate the elementsn and Fe in an enzymatically driven manner (Ehrlich, 2002),

r by non-enzymatic chemical reactions. The latter process haseen proposed to occur by precipitation onto the S-layer proteins

f some bacteria (Wang et al., 2009d). A solution to this ques-ion will also help to assess further the potential implication ofacteria in the rock formation on Mars. Until now, the major-

ty of the micro-structural studies have been performed with thellan Hills (ALH) 84001 Martian meteorite, originating from the

2 (2011) 401–411

ancient Martian crust (about 4.5 Gyr) (Nyquist et al., 2001). Anal-yses with that meteorite provided some hints of an existence ofancient Martian life (McKay et al., 1996). Even though later inves-tigation by Barber and Scott (2002, 2003) and also by Brearley(2003) supported the view that thermal effects created the mag-netite “microfossils”, debate about and exobiological exploration ofMars will continue (Kerridge, 1997). Studies in our group are nowongoing, using that Martian meteorite, to explore with high reso-lution microscopical and spectroscopical techniques the structureand the sub-structures for potential biogenic traces, e.g. microfos-sils/biomarkers.

Acknowledgements

We thank Ms. M. Müller and Mr. G. Glasser (Research groups“Surface Chemistry” of Dr. M. Kappl and Dr. I. Lieberwirth; MaxPlanck Institute for Polymer Research; Mainz) for excellent helpin EDX and HR-SEM analyses. Furthermore we gratefully acknowl-edge the expert help of Dr. Dmitry V. Kuzmin (Max Planck Institutefor Chemistry; Mainz) during the EPMA analyses. This work wassupported by grants from the Bundesministerium für Bildung undForschung Germany [project: Center of Excellence BIOTEC marin],the Public Welfare Project of Ministry of Land and Resources of thePeople’s Republic of China (Grant No. 201011005-06) and the Inter-national Science and Technology Cooperation Program of China(Grant No. 2008DFA00980).

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