geomicrobiology of carbonate–silicate microbialites from hawaiian basaltic sea caves

Ž .Chemical Geology 169 2000 339–355www.elsevier.comrlocaterchemgeo

Geomicrobiology of carbonate–silicate microbialites fromHawaiian basaltic sea caves

Richard J. Leveille), William S. Fyfe, Fred J. Longstaffe´ ´Department of Earth Sciences, The UniÕersity of Western Ontario, London, Ontario, Canada N6A 5B7

Received 6 May 1999; accepted 16 September 1999

Abstract

Modern microbial mats and microbialites are described from basaltic sea caves on the island of Kauai, HI. The mats growŽ .on the ceilings and walls in the photic zone of several open caves where fresh water seeps out of the rock. Scanning SEM

Ž .and transmission electron microscopy TEM showed that the active mats are dominated by filamentous and nonfilamentouscyanobacteria in the surface layers and heterotrophic bacteria in deeper layers. Energy dispersive X-ray analysis revealed

Ž .that copious amounts of extracellular polymeric substances EPS are rich in Mg, Si, O, and Ca, likely concentrated fromsolution. Petrographic microscopy and electron microprobe analysis of the mineralized microbialites showed textures

Ž .reminiscent of stromatolitic laminations, consisting mainly of alternating calcium carbonate calcite and aragonite andŽ .magnesium-rich silicate kerolite . Thin coatings rich in magnesite, hydromagnesite and monohydrocalcite surround the

microbialites on the rock surfaces and are likely inorganic in origin. Within the mats, minerals tend to form and concentratewithin, or around, dense matrices of EPS. Microenvironments with geochemical conditions favorable for mineral crystalliza-tion likely develop in the mats as a result of the mucilaginous extracellular material and the development of bacterialmicrocolonies. In addition, copious amounts of extracellular polymers bind ions from solution and provide nucleation sitesfor mineral crystallization and growth. This combination of biological and inorganic processes can explain the occurrence ofthe secondary minerals in these caves, as well as the stromatolitic textures of the microbialites. q 2000 Elsevier ScienceB.V. All rights reserved.

Keywords: Geomicrobiology; Stromatolites; Bacteria; Carbonates; Silicates

1. Introduction

Secondary minerals, or speleothems, are ubiqui-tous in caves, given the stable, undisturbed environ-

) Corresponding author. Tel.: q1-519-661-3187; fax: q1-519-661-3198.

Ž .E-mail address: [email protected] R.J. Leveille .´ ´

ment and continuous water supply that provide idealŽ .conditions for mineral formation White, 1976 .

These same conditions are ideal for microbial growthand numerous microorganisms, including hetero-trophic and phototrophic bacteria, have been de-

Žscribed from various caves see review by Cubbon,.1976 . Microbial mats and biofilms dominated by

bacteria or cyanobacteria have been described fromŽ .aerobic Abdelahad, 1989; Jones, 1995 and anaero-

Žbic limestone or dolostone caves Sarbu et al., 1996;

0009-2541r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0009-2541 00 00213-8

( )R.J. LeÕeille et al.rChemical Geology 169 2000 339–355´ ´340

Ž . Ž . Ž .Fig. 1. Location map showing: A the main Hawaiian Islands, B the island of Kauai and C the north shore of Kauai with the 3 mainŽ .caves studied here see text for descriptions .

.Mattison et al., 1998 ; in basaltic lava tubes, theyŽhave been called ‘‘lava-tube slime’’ Waters et al.,

. Ž .1990 and ‘‘cave slime’’ Greeley, 1971 .Although these microbial mats and biofilms are

commonly associated with various minerals, includ-Žing clays and carbonates e.g. Greeley, 1971; Waters

.et al., 1990 , few studies have focused on the role ofmicroorganisms, especially bacteria, in the formationof authigenic cave minerals. Because bacterial min-eralization is ubiquitous in near-surface environ-

Ž .ments e.g. Fortin et al., 1997 , it is likely to becommon in caves as well. Modern cyanobacteria-de-

posited stromatolitic speleothems have been de-scribed from the photic zone of limestone cavesŽ .Gomes, 1985; Cox et al., 1989 . Rousseau et al.Ž .1992 described organic biomarkers in flowstonesand suggested that bacterial activity may have beeninvolved in the formation of these deposits. Braith-

Ž .waite and Whitton 1987 suggested an indirectphysical control on gypsum and halite formation bythe cyanobacterial organic mucilage in stalactite-likestructures on the roof of an undercut cliff. In addi-tion, the formation of ‘‘Moonmilk’’, a soft, wet andpowdery deposit of magnesium and calcium carbon-

Ž . Ž .Fig. 2. Photographs of various microbial mats and microbialites. A Surface of thick, active microbial mat. B Cross-section view of thickŽ . Ž . Ž .mat in A showing vertical profile. Notice purple and green colors near the surface arrows and lighter color inside. C and D Thick,

mineralized microbialites of carbonate and kerolite. Dark area near center is wet; white mineralization surrounding microbialites isŽ . Ž .carbonate-rich i.e., magnesite, hydromagnesite, calcite, aragonite . E Polished slab of dry microbialite showing stromatolitic laminations.

Ž .F Hand specimen of dry microbialite showing the knobby, irregular surface texture.

( )R.J. LeÕeille et al.rChemical Geology 169 2000 339–355´ ´ 341

ates, has often been attributed to bacterial activityŽ .Cubbon, 1976 .

Because of their high surface area to volumeratio, reliance on diffusion, production of chemically


active polymers and surface properties, bacteria actŽas highly reactive geochemical interfaces Beveridge,

.1989; Fein et al., 1997 . Bacteria and their extracel-lular polymers are especially effective at bindingions from solution and serving as nucleation surfaces

Žfor mineral formation Ferris et al., 1987; Beveridge,.1989; Fortin et al., 1997 . Their metabolic activities

can also lead to localized conditions that are favor-Žable for mineral precipitation Thompson and Ferris,

1990; Merz, 1992; Fortin et al., 1997; Barker and.Banfield, 1998 . Bacterial and cyanobacterial media-

tion of carbonate mineral formation has been exten-Žsively documented in freshwater environments Cox

et al., 1989; Thompson and Ferris, 1990; Merz,.1992 . Recently, the role of bacteria in the formation

Ž .of silicate minerals e.g. clays has also been demon-strated both experimentally and in natural systemsŽUrrutia and Beveridge, 1993, 1994; Fortin and Bev-eridge, 1997; Tazaki, 1997; Barker and Banfield,

.1998; Sanchez-Navas et al., 1998 .´In microbial mats, mineral deposition typically

leads to the formation of microbialites, which com-Žmonly show laminated stromatolitic structures Burne

.and Moore, 1987 . Modern mats and microbialitesare often considered as analogous for Archean andProterozoic stromatolites, which are some of the

Žoldest evidence for life on Earth Schopf and Klein,.1992 . By studying modern microbialite formation,

we can gain insights into similar ancient processes.Here we use petrographic microscopy, electron

microprobe analysis and electron microscopy to pro-vide the first detailed report of active microbial matsand associated mineralized microbialites on rock sur-faces inside Hawaiian basaltic caves. In addition toseveral carbonate phases, these deposits include, tothe best of our knowledge, the first known occur-rence of kerolite both in caves and associated withmicrobial mats.

2. Materials and methods

2.1. Sampling locations

Three large open caves are located at the base ofhigh cliffs just west of Haena on the north shore of

Ž .the island of Kauai, HI Fig. 1 . The caves all facenorth along Highway 560. These ancient sea caveswere cut into thin beds of the volcanic Napali For-mation when the sea level was higher than at pre-sent; the caves are the result of recessive erosionresulting from the constant assault of waves, whichexploited zones of weakness such as dykes and

Ž .fractures Macdonald et al., 1983; Liebman, 1992 .Maniniholo Dry Cave is the easternmost of the

three. The cave is approximately 20 m wide at itsopening with a maximum height of about 5 m. Themain cavern extends for about 50–60 m and narrowsto a much smaller tunnel, which continues furtherinto the cliff. Despite the name, the cave is relativelyhumid as water continually seeps out of the basalticrock; the walls and ceiling are often wet, especiallynear the entrance.

Waikapalae Wet Cave and Waikanaloa Wet Caveare located about 1 km west of the Dry Cave. Thesecaves are located further from the shore than the DryCave and are smaller, but they have higher ceilings.Both contain basal groundwater in standing pools,held back by deposits of impermeable alluviumŽ .Macdonald et al., 1960 .

Further westward along the Napali Coast, severalmore sea caves are actively forming by wave ero-sion. All of these caves are believed to be true sea

Ž .caves, as opposed to lava tubes Liebman, 1992 .Although we visually inspected these caves, no sam-ples were collected from them.

Our study thus far has been restricted to thephotic zone of these caves. There, we find secondarymineralization, or speleothems, on the interior wallsand ceilings, as well as on the exterior surfaces nearthe entrances. Many of these speleothems are wetand presumably actively forming from fresh watersseeping out of the basalt. Microbial mats are activelygrowing in many of the wet areas.

Samples for mineralogical analysis were collectedfrom the Dry Cave and the two wet caves. Detailedresults from powder X-ray diffraction and electronmicroprobe analysis will be reported elsewhereŽ .Leveille et al., 2000 . Microbial mat samples and´ ´microbiological samples were collected mainly fromthe Dry Cave, as the mats in the wet caves were notas easily accessible or as well developed as in theDry Cave.


2.2. Analytical methods

Samples for transmission electron microscopyŽ .TEM were collected from the Dry Cave and pre-served on site. Hard, mineralized samples were ob-tained by scraping the surface with a sterile blade.Surfaces of soft, wet mats were sampled with an agar

‘‘sausage’’ made by placing warm agar in a plasticsyringe and allowing it to harden. For sampling, theend of the syringe was removed and the agar waspressed onto the mat surfaces. Mat pieces were alsoremoved with a sterile blade. All samples were fixedwith 10% glutaraldehyde diluted to 1–2% with either

Ž .cave water pH;8 or distilled water and kept cool.

Ž . Ž .Fig. 3. Backscattered electron microprobe image A and petrographic thin section image B of resin-impregnated microbialites showingstromatolitic laminations. Light-colored mineral is calcite or aragonite and dark mineral is kerolite.


Ž . Ž .Fig. 4. TEM micrographs of material from the surface of thick microbial mats. A Bacterial cells b are commonly enclosed in denseŽ . Ž .extracellular polymeric substances EPS; p , forming a microcolony. Mineral material m is concentrated around the edge of the

Ž . Ž .microcolony. B Microcolony where bacterial cells b with irregular shapes have been partially degraded or plasmolysed, whereas theŽ . Ž .abundant extracellular material p has resisted degradation. Ultrathin sections stained with uranyl acetate and lead citrate. C TEM-EDS

Ž . Ž . Ž .spectrum of dense extracellular material EPS surrounding cells similar to those seen in A and B . Major peaks are C, O, Mg and Si. Clis from the mounting medium and Ni is from the support grid. Unstained ultrathin section.

Samples were later processed in the laboratory byfirst placing in a buffer solution and centri-fuging for2–5 min. Pellets were then placed in warm agar andquickly cooled to harden. Once hard, the agar wassliced into small pieces and samples were furtherfixed with osmium tetroxide overnight. A step dehy-dration process of increasing acetone concentration

Žtreatments 20%, 50%, 70%, 90%, 10 min each;.100%, twice at 30 min each followed. Samples were

Žthen placed in acetone–plastic resin mixtures 3:1 for.90 min, 1:1 for 60 min, 1:3 for 60 min . Finally,

samples were placed in 100% resin overnight. Theywere then allowed to polymerize at 608C for 72 h.Ultrathin sections were prepared with a microtomeequipped with a diamond or glass knife and were

placed on nickel grids for TEM imaging. For X-rayanalysis, sections were placed on carbon-coatednickel grids with Formvar supporting films. Somesections were further stained with lead citrate anduranyl acetate to improve contrast of cellular mate-rial.

TEM was performed using a Phillips CM 10 TEMat an accelerating voltage of 80 kV. Energy disper-

Ž .sive X-ray spectroscopy EDS was carried out at100 kV on a Phillips EM400T equipped with a LinkLZ-5 X-ray spectrometer and analytical software.

Ž .For scanning electron microscopy SEM , smallfragments of the microbialites or dried mats werefixed onto aluminum stubs with two-way adherenttabs or conductive carbon paint, and allowed to dry


overnight. They were then gold-coated by sputteringfor approximately 2–3 min. All samples were exam-ined with a Hitachi S-4500 Field Emission SEMequipped with an EDAX energy dispersive X-rayspectrometer and analytical software. EDS analysiswas mainly qualitative because of the irregular sur-face topography of the samples. The SEM was oper-ated at 5 kV with a working distance of 15 mm foroptimum imaging and to minimize charging andsample damage. For X-ray analysis, an acceleratingvoltage of 15 kV was used to obtain sufficient X-raycounts.

Standard polished thin sections were preparedfrom resin-impregnated microbialite samples and ex-amined by petrographic microscope and electron mi-croprobe. Carbon-coated polished thin sections wereexamined with a Jeol JXA 8600 Superprobe electronmicroprobe in backscattered electron detection mode.Samples in thin section were also analyzed qualita-

Ž .tively by energy dispersive spectroscopy EDS andquantitatively by wavelength dispersive spectrometryŽ .WDS with four spectrometers and appropriate stan-dards.

3. Results

In all of the caves that we examined, microbialmats are growing within the photic zone on the wallsand ceilings where fresh water seeps out of the rock.

ŽThe mats are mostly located relatively close within.10–15m to the cave entrances. In the Dry Cave,

they tend to be concentrated slightly on the left sideŽ .facing west , possibly because more light reachesthere. However, because the cave entrances are wide,ample light reaches most areas in the main cavern.Further back in the Dry Cave, dry microbialites canbe found, though there were no water drips there andno obvious active microbial growth.

Two main types of microbial mats can be differ-entiated in the Dry Cave. The first type is a thinŽ .about 1–2 cm mat with a hydrous, slime-like tex-ture. These mats range in color from dark brown tobright green to beige. Because of poor consolidation,intact samples of these mats could not be removedfrom the rock surface; only small portions could becollected. Although poorly mineralized, these matstend to contain mainly kerolite with some aragonite.

ŽThe second variety of microbial mat is a thick up.to ;15 cm , wet, soft, spongy mat with a bright

Ž .purple, brown or green exterior Fig. 2A–D . Themat surface is generally knobby with irregular pro-trusions. Some of these protrusions have a white tip,consisting of calcite, aragonite and hydromagnesite.The mats tend to form roughly circular patches up toapproximately 40 cm in diameter and some havedistinctive laminations with depth from the cavewall. For instance, approximately 1cm from the mat

Ž .surface lies a thin -0.5 cm green layer and belowŽ .that is a uniform beige-light green layer Fig. 2B .

The mats are somewhat coherent and resistant tobreakage; individual layers do not peel apart easily.The mats closely resemble the more mineralizedmicrobialites in texture. However, the dry micro-bialites have greater surface relief in the form ofsmall stromatolitic mounds, columns and protru-sions, which typically appear as concentrically lami-

Ž .nated structures in cross-section Fig. 2E, F .The bulk mineralogy of the mats, as determined

primarily by X-ray diffraction, is dominated by arag-onite, calcite and kerolite, but fine, white coatings ofhydromagnesite or monohydrocalcite are commonlypresent between the mounds and columns of a givenmat. In addition, thin coatings of magnesite, hydro-magnesite, gypsum, monohydrocalcite, calcite andaragonite also occur around the microbial mats on

Ž .the cave surfaces Fig. 2C, D . These thin depositslikely formed by inorganic processes.

Microscopic and electron microprobe examinationof thin sections of the dry, mineralized microbialites

Ž .revealed various laminated textures Fig. 3 . Lamina-tions are wavy, irregular and more or less continu-ous, with a thickness of a few micrometers to severaltens of micrometers. They are either subparallel toparallel or concentric, resembling stromatolites.These structures are typically columnar and cylindri-cal, range in size from less than 1 mm to 3 mm wideby 1–6 mm high, and are usually convex away fromthe growth surface. Electron microprobe analysisŽ .EDS and WDS confirmed that these structures

Žconsist of well-separated layers of CaCO calcite or3. Ž . Ž .aragonite and Mg Si O OH PnH O kerolite .3 4 10 2 2

The kerolite is usually fine-grained, massive andpoorly crystalline. The calcite and aragonite are alsofine-grained, though generally more crystalline andsometimes coarser than the kerolite.


Ž .Fig. 5. TEM micrographs of deep, mineralized portions of thick microbial mats. A Microcolony of bacterial cells showing well preservedŽ . Ž . Ž .EPS p around slightly desiccated cells. Wavy, poorly crystalline kerolite m surrounds the microcolony. B Another microcolony also

Ž . Ž .showing thick layers of EPS p around bacterial cells. Unidentified mineral material m is located around and within the microcolony.Ultrathin sections stained with uranyl acetate and lead citrate.


Although the older layers of the microbialitesclosest to the basalt surface are generally lightercolored, the younger surface layers commonly have adark brownish–reddish–yellowish color reflecting

Ž .higher clay kerolite , organic matter, or amorphouscontents. The presence of these phases is supportedby broad diffraction bands or high backgrounds inX-ray diffraction patterns.

Ž . Ž .Fig. 6. TEM micrographs of filamentous A and coccoid B cyanobacteria from the surfaces of thick microbial mats. Thylakoid-likeŽ . Ž .intracytoplasmic photosynthetic membranes th are well developed. The filamentous cyanobacterium in A is surrounded by a sheath and

Ž . Ž .relatively little EPS, while the coccoid cyanobacterium in B is surrounded by abundant EPS p . Ultrathin sections stained with uranylacetate and lead citrate.


Examination of the microbial mats by both TEMand SEM revealed a variety of microorganisms inassociation with different stages of mineralization.Nearly all of the microorganisms are surrounded bylarge amounts of extracellular polymeric substancesŽ .EPS , which has a fibrillar appearance and forms

Ž .thick ;1 mm layers of a mucilaginous matrix,occasionally in concentric layers around individual

Ž .cells or cell remnants Figs. 4 and 5 . A rare excep-tion to this observation is the presence of a filamen-tous, sheathed cyanobacterium that shows little or no

Ž .EPS Fig. 6A . In addition, several cells or cellremnants are commonly enclosed in a matrix of EPSforming a microcolony, which can be surrounded by

Ž .authigenic minerals Figs. 4 and 5 . The EPS isresistant to degradation as it is seen even in the moremineralized and dried samples and in cases where

Žcells are partially degraded or plasmolyzed Figs. 4B.and 5 .

Samples from thin wet mats and the outer sur-faces of thick mats contain a large number and widevariety of microorganisms. They are usually domi-nated by phototrophic microbes, which are deeplyembedded in copious amounts of EPS and poorly

Ž .crystalline minerals in many cases Figs. 6B and 7 .Cyanobacteria can be identified based on either theirfilamentous morphology or on the presence of thyl-

Ž .akoid-like intercytoplasmic membranes ICMs usedfor photosynthesis. These ICMs are typically ar-ranged concentrically in the filamentous andpseudo-concentrically or irregularly in the nonfila-

Ž .mentous, coccoid variety Fig. 6 . The excellentpreservation of these ICMs suggests the cyanobacte-ria are actively photosynthesizing in the caves. SmallŽ .-1 mm in diameter coccoid bacteria are alsocommon, typically in microcolonies of several cells

Ž .surrounded by extracellular material Figs. 4 and 5 .We were unable to identify conclusively the natureof these bacteria. Smaller heterotrophic bacteria alsooccur in association with larger cyanobacteria insome samples. Small amounts of diatom frustules are

Ž .sometimes present Fig. 7 , but they are not believedto be an active component in the mats. The diatomsare few in numbers and little cytoplasmic materialhas been preserved in them compared to some of thebacteria. In many samples, mineral material is con-centrated in and around the extracellular polymers of

Ž .individual cells and microcolonies Fig. 4 . How-

Ž .Fig. 7. A SEM micrograph of surface material of thick matshowing filamentous cyanobacteria deeply embedded in amor-phous kerolite-like material. A diatom frustule is present in the

Ž .top-right corner of image. B SEM-EDS spectrum of keroliteŽ . Ž . Ž .arrows in A , consisting mainly of C–O–Mg–Si arrows . CSEM-EDS spectrum of similar material, but also including Ca, S,and increased amounts of C. The additional elements likely repre-sent carbonate, organic matter and minor amounts of gypsum. Aupeaks are from gold coating.

ever, the amount of mineralized material is relativelysmall in these layers compared to deeper inside thethick mats.

In the inner layers of the thick mats, there is aŽdecrease in the overall abundance of cells especially

.the cyanobacteria relative to the outer surface lay-ers, but an increase in mineralized material. Some ofthe cells are similar to those in the surface layers,whereas others are unique to the mat interiors. Com-monly, cells or poorly preserved cell remains are

Žcompletely encrusted with mineral material e.g. Fig..5 . Fine-grained, flaky kerolite is common through-

out these layers. In addition, crystalline materialŽ .possibly carbonate is locally concentrated around

Ž .cells and microcolonies Figs. 5B and 8A, B . Fine,


linear features in the EPS may represent protocrys-Žtals, which have nucleated on the polymers Fig.

.8A . None of the mineralization is directly associatedwith cell surfaces. Instead, mineralization is concen-trated in and around the thick layers of EPS.

Desiccated and mineralized microbialites containfew preserved cells, and instead are dominated bymineralized material, which is typically laminated on

Ž .a micrometer scale Fig. 8C . Concentric structuresapproximately 1–2 mm in diameter most likely cor-respond to the previous locations of cells, whichhave been completely encrusted without preservationof the organic cellular material.

EDS analysis with both SEM and TEM revealedmost of the mat samples to be rich in Mg, Si, O, Ca

Ž .and C Figs. 4C and 7B, C . This composition isconsistent with our electron microprobe results, themineralogy of the samples and the chemistry ofwater in the caves. These elements are present in theEPS layers around cells in the mat surface layers andthroughout the lower, more mineralized layers. Insome cases, the only elements detected are Mg, Siand O, which is indicative of kerolite or a precursor

Ž .material Figs. 4C and 7B . This composition isespecially common in the thick mat interiors. In

Žother cases, more Ca, O and C are detected Fig..7C , likely because of higher CaCO or organic3

matter content. Analysis of cells and cell surfacesrevealed only minor concentrations of these ele-ments, which suggests that the EPS layers are moreeffective than the cells at concentrating ions fromsolution. Small amounts of S are also present, possi-bly from gypsum or organic matter; minor quantitiesof gypsum were detected by X-ray diffraction. Thepresence of purple sulfur bacteria could also explainthe S peaks.

4. Discussion

4.1. Microbial mats and mineralization

The metabolic activities of microorganisms cansignificantly alter the geochemical composition of

their surroundings by exchanging ions with a solu-tion, or by producing various inorganic and organicproducts that cause changes in pH or redox condi-

Žtions Simkiss and Wilbur, 1989; Chafetz and.Buczinski, 1992; Fortin et al., 1997 . Thus, microbial

activity may create localized supersaturation condi-tions, which in turn may lead to crystallization ofminerals. In microbial mats, this behaviour is espe-cially common as decreased diffusion in the water-rich mucilaginous matrix allows microenvironments

Žto be maintained more easily Chafetz and Buczin-.ski, 1992; Arp et al., 1998 . In addition, the develop-

ment of microcolonies within a mat can lead tofurther isolated and unique geochemical mi-

Ž .crodomains Ransom et al., 1999 .Photosynthetic microbes, for example, can greatly

affect the local carbonate dynamics as the uptake andfixation of dissolved inorganic carbon from solutiontends to drive carbonate precipitation by increasingpH when HCOy is the dominant inorganic carbon3

Ž .species Thompson and Ferris, 1990; Merz, 1992 .Cyanobacterial photosynthesis has been shown to beresponsible for CaCO production in both laboratory3

studies and in natural environments. For example,Ž .Thompson and Ferris 1990 demonstrated that the

cyanobacterium Synecchococcus was directly re-sponsible for alkalinization of its microenvironmentby using HCOy as a primary source of inorganic3

carbon. Other bacterial processes that may inducecarbonate precipitation include oxidation and degra-dation of organic carbon and nitrogen compounds,with release of NH and CO , and reduction of3 2

Ž .sulfate to sulfide Chafetz and Buczinski, 1992 .Bacterial cell surfaces and EPS are also highly

effective at binding dilute ionic species from solu-Ž .tions Ferris et al., 1987; Beveridge, 1989 . Usually,

binding of metal cations takes place on the cell wallor on polysaccharide-rich extracellular polymerswhere exposed carboxyl and phosphoryl functional

Žgroups provide negatively charged sites Beveridge,.1981, 1989 . Once complexed, bound metals may

serve as nucleation sites for further metal complexa-Žtion and mineral crystallization Ferris et al., 1987;

Ž . Ž .Fig. 8. TEM micrographs of progressively mineralized bacteria from microbialites. A Single bacterium showing dense EPS p withŽ . Ž . Ž . Ž .unidentified minerals m . The fine, linear features arrows within the EPS may be proto-crystals, possibly of Mg-silicate kerolite . B

Ž . Ž . Ž .Single bacterium with unidentified minerals m completely surrounding the EPS layer p. C Completely mineralized microbialiteŽ .showing concentric remnants arrows of bacterial cells.


.Beveridge, 1989; Fortin et al., 1997 . Positivelycharged compounds, such as amine groups, along

Žwith metal ion-bridging, can also lead to anion e.g..silicate, carbonate binding at near neutral pH condi-

Ž .tions Urrutia and Beveridge, 1993, 1994 .This binding ability is not limited to active or live

bacteria. Experimental evidence suggests that deadcells can be even more effective at binding metals

Žthan live ones Ferris et al., 1988; Chafetz and.Buczinski, 1992; Urrutia and Beveridge, 1993 . For

Ž .example, Chafetz and Buczinski 1992 demon-strated that mineralization of microbial mats in mod-ern tidal flats occurs preferentially on deadcyanobacterial filaments rather than on livecyanobacteria, and only in the presence of associatedbacteria. They suggested that bacteria may help tobreak down cyanobacterial remains in deeper, moremineralized parts of the mats and alter the microen-vironment such that crystallization is favored. Be-cause most microbial mats in modern tidal flats lackmineralization at the surface where cyanobacteria aredominant, and because mineralization tends to in-crease with depth in the mat, Chafetz and BuczinskiŽ .1992 concluded that bacteria, and not cyanobacte-ria, are responsible for much of the lithification ofmicrobial mats into microbialites.

4.2. Microbialite formation in Kauai CaÕes

Although the term ‘‘microbialite’’ was originallyŽ .used by Burne and Moore 1987 to designate ben-

Ž .thic microbial deposits, Cox et al. 1989 suggestedthe extension of its meaning to nonbenthic microbialdeposits such as cyanobacterially depositedspeleothems. We suggest that this term is also bestsuited to describe the microbial deposits in the KauaiCaves, despite a few differences relative to conven-tionally defined microbialites. For example, micro-bialites generally contain some detrital materialŽ .Burne and Moore, 1987 , whereas the minerals inthe deposits that we studied are believed to bemainly authigenic. No carbonates or kerolite occurabove the caves, and the waters from which thesedeposits are forming carry little inorganic particulatematter. Similarly, no common soil minerals werefound in the cave deposits. Also, the kerolite ismassive and poorly crystalline, characteristics thatare typical of an authigenic origin.

Several lines of evidence indicate that the forma-tion of the microbialite minerals was influenced bythe presence of microorganisms and microbial matsin the caves. In the microbial mats, various elementsŽ .Mg, Si, O, Ca and C are concentrated aroundbacteria and cyanobacteria in dense layers of EPS.Similarly, in the more mineralized mats and micro-bialites, microbial cells are intimately associated withmineralized material; even in completely mineralized

Ž .samples, ‘‘remnant cells’’ Fig. 8C are observed.Also, calcite, aragonite, and kerolite are most abun-dant in, though not limited to the microbial mats andmicrobialites. Kerolite was not detected in thin, pre-sumably inorganically deposited, carbonate-rich coat-ings that surround many of the mats and micro-bialites. Finally, the wavy and irregular laminatedtextures of the deposits resemble small stromatolites,which are generally indicative of microbial activityŽBurne and Moore, 1987; Chafetz and Buczinski,

.1992 .Because few cells are well preserved in the micro-

bialites and minerals do not occur directly at cellsurfaces in the partially mineralized mats, we believethe abundant extracellular material is most crucial inmediating the mineralization in the microbialites. Intheir study of microbially formed minerals, Ransom

Ž .et al. 1999 often observed little direct contactbetween cell surfaces and minerals formed around

Žthem. Instead, the minerals especially layered sili-.cates tended to be tangentially oriented at the outer

limits of the EPS layers surrounding the cells. BarkerŽ .and Banfield 1998 also observed tangentially ori-

ented clay minerals within the EPS of bacteria cells.These authors concluded that the minerals nucleatedand grew within the extracellular matrices. We foundsimilar small, linear features in the EPS surrounding

Ž .many cells e.g. Fig. 8A . These features may beproto-crystals that originate at sites on the fibrillarpolymers where ions have been concentrated. Wewere unable to determine conclusively the composi-tion of these proto-crystals because of their smallsize.

The poor crystallinity, massive nature, and smallgrain size of the kerolite in the mats and micro-bialites suggests that it may have formed from aMg–Si–O gel or other precursor. The EPS in themicrobial mats would have facilitated developmentof the gel and concentration of ions from solution; as


it dehydrated, kerolite could begin to crystallize.Extracellular bacterial formation of silicates can be-gin as fine-grained, hydrous and poorly ordered pre-cipitates, which may progressively evolve to true

Žcrystalline phases Urrutia and Beveridge, 1993;.Fortin et al., 1997; Tazaki, 1997 . Sanchez-Navas et´

Ž .al. 1998 studied Fe-rich smectites intimately inter-grown with Fe–Si–Al amorphous phases withinancient phosphate stromatolites. Based on this asso-ciation, they concluded that the smectite formedauthigenically from the precursor Fe–Si–Al amor-phous gel-like material, which could have originatedfrom bacterial extracellular polymeric mucilage

Ž 3y 2q 2y .where ions e.g. PO , Ca , CO , Fe, Al, Si4 3

were bound and concentrated from solution. Similarprocesses may be responsible for kerolite formationin our samples. Degradation and dehydration of apolysaccharide-rich matrix within the mats couldproduce a precursor gel, which would ultimatelydevelop into poorly crystalline kerolite, possibly withextracellular polymers helping nucleation. Labora-tory studies in which kerolite was synthesized from

Ž .Mg–Si-rich gels Decarreau et al., 1989 and field-Ž .based studies Brindley et al., 1977 support this

hypothesis.The small grain size of the calcite and aragonite is

also consistent with rapid precipitation from locallyŽsupersaturated solutions. Calcium carbonate arago-

.nite and calcite is slightly undersaturated in allKauai meteoric waters that we analyzed, including

Ž .cave waters unpublished data . However, conditionswithin the microbial mats may be more favourablefor carbonate formation. For example, the binding ofCa2q, CO2y or HCOy by bacterial EPS could lead3 3

to localized areas within the mats where calcite oraragonite precipitate. Direct precipitation of calciteor aragonite near the mat surface could also beinduced by an increase in pH levels caused bycyanobacterial photosynthesis. Other phototrophicbacteria may also be present in these mats, possiblyin lower, anaerobic layers. For instance, the purplecolour of some of the mats and the presence of sulfurŽ .Fig. 7C suggest that purple sulfur bacteria arepresent. These bacteria are common in marine micro-

Ž .bial mats Burne and Moore, 1987 , but we were notable to positively identify any such organisms basedsolely on electron microscopy. Similarly, anaerobicdecay of organic matter by heterotrophic bacteria

could affect the pH in lower layers of mats byproducing NH and by reduction of sulfate to sulfide3Ž .Chafetz and Buzinski, 1992 . An increase in pHalso increases the potential for metal cation bindingbecause metals tend to bind to deprotonated func-

Ž .tional groups Fein et al., 1997 . The isolation ofbacterial microcolonies within the mats by the EPSthat surrounds them could amplify all these effects.

Thus, several processes appear to be acting to-gether to induce the mineralization observed in thesemats. As the mats develop, ions are bound fromsolution by the polymers in the extracellular mate-rial, and organic matter begins to decay and producean ion-rich, gel-like phase. The role of the EPS isespecially important when one considers that evenunder conditions of near saturation or supersatura-tion, mineral precipitates do not always spontaneousform from solution unless nucleation centres are

Žprovided for their crystallization and growth Stumm.and Morgan, 1981; Simkiss and Wilbur, 1989 . Mi-

croenvironments begin to develop as microcoloniesform and as the mats become stratified. This situa-tion can lead to localized zones with conditions morefavourable for mineral formation. Kerolite may beginas an amorphous phase in the gel-like, EPS-richmatrix. Proto-crystals may nucleate on extracellularpolymers where ions are concentrated and grow pro-gressively as the gel dehydrates and degrades. Thekerolite likely forms continuously throughout themat, whereas carbonate precipitation may dominatein zones or periods of greater phototrophic activityor, simultaneously, as a result of the decay of or-ganic matter by heterotrophic bacteria within themat. Carbonates may also nucleate on extracellularpolymers where ions are concentrated. Thus, forma-tion of carbonate may be favoured at times, whereaskerolite formation proceeds at other times. Such acyclic pattern of mineral formation explains the lam-

Ž .inated i.e. stromatolitic structures characteristic ofthe mineralized microbialites. The increase in miner-alization with depth in these mats is consistent withprevious findings on microbial mat mineralizationŽChafetz and Buczinski, 1992; Schultze-Lam et al.,

.1995 . As the mats become extensively mineralized,they may divert the flow of water to other areas ofthe cave surface and progressively desiccate. Evapo-ration of water at the mat surfaces and around themats could partially explain the formation of smaller


amounts of magnesite, hydromagnesite, monohydro-calcite and gypsum; these minerals are commonlyformed in evaporative settings.

4.3. PreserÕation of microbes in microbial deposits

In the most mineralized samples of Kauai micro-bialites, few cells are well preserved. The cellularorganic matter is likely degraded fairly rapidly, pos-sibly by anaerobic bacteria in deeper parts of themats. This process can explain the tendency for thedeeper, more mineralized parts of the mats to be oflighter colour than the surface, organic-rich layers.

Ž .Cox et al. 1989 also found few preserved cells incyanobacterially deposited speleothems. Conversely,the extracellular polymers appear to be more resis-tant to degradation as they are commonly well pre-

Ž .served even around highly degraded cells Fig. 4B .Metal binding by microbial surfaces has been in-ferred to be a significant benefit for preservation oforganic cellular material and EPS by inhibiting cellu-lar autolytic enzymes, which normally degrade the

Žorganic matter of dead cells Ferris et al., 1988;.Ransom et al., 1999 . Because metal binding seems

to be mainly related to the EPS in our samples andnot the cell surfaces, we would therefore not expectmuch cell preservation. Also, metal binding has oc-curred in an environment without any silicification,which is considered to be an important prerequisitefor long-term preservation of cells as microfossilsŽ .Ferris et al., 1988; Schultze-Lam et al., 1995 .Presumably, kerolite is the stable silicate phase inthis system; no silica was detected in any of oursamples other than in a few diatom frustules. Onlythe extracellular polymers appear somewhat intact inmineralized samples, most likely because of thismetal binding. Therefore, metal binding alone with-out silicification does not lead to abundant and easilyrecognizable preservation or fossilization of micro-bial cells.

5. Conclusions

Unique deposits of calcium and magnesium car-bonates mixed with kerolite are forming from freshwaters on the surfaces of basaltic sea caves onKauai, HI. Microbial mats, dominated by bacteria,

cyanobacteria and extracellular polymeric substancesare intimately associated with these cave deposits.Hand specimen, geochemical and microscope evi-dence suggests that the microbes and their extracellu-lar material have influenced the formation of theseminerals by acting as nucleation sites and by alteringthe local aqueous geochemistry within the matsthrough their metabolic processes or as a result oftheir physical properties. The microbial mats likelydevelop and maintain geochemical conditionsfavourable for mineral precipitation. Bacterial extra-

Žcellular material binds ions e.g. Mg, Ca, silicate,.carbonate from solution and serves as nucleation

sites for mineral formation. In addition, inorganicŽ .processes e.g. evaporation complement these bio-

logical ones. This combination of processes actingtogether can explain the stromatolite structures andthe variety of minerals formed in these caves. Suchmicrobially influenced mineral deposition may be afeature common to the photic zone of many caves.

Acknowledgements

Ž . Ž .We thank R. Smith UWO and B. Harris Guelphfor help with the TEM work, A. Pratt for help withthe SEM, and Y. Thibault for assistance with opera-tion of the electron microprobe. We are grateful to S.Koval and T. Beveridge for their helpful comments.We are also grateful for critical reviews of thismanuscript by H. Chafetz and an anonymous re-viewer. This research was funded by the NaturalSciences and Engineering Research Council of

Ž .Canada NSERC with grants to WSF and FJL, and apostgraduate scholarship to RJL.

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