Earth and Planetary Science Letters 285 (2009) 131–139

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Earth and Planetary Science Letters

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Presence of sulfate does not inhibit low-temperature dolomite precipitation

Mónica Sánchez-Román a,b,c,⁎, Judith A. McKenzie a, Angela de Luca Rebello Wagener d,Maria A. Rivadeneyra c, Crisógono Vasconcelos a

a ETH-Zürich, Geological Institute, 8092 Zürich, Switzerlandb NASA Astrobiology Institute and Savannah River Ecology Laboratory, University of Georgia, Drawer E, Aiken, SC 29808, USAc Dept. of Microbiology, Faculty of Pharmacy, University of Granada, 18071 Granada, Spaind Dept. of Chemistry, PUC, 22453-900 Rio de Janeiro, Brazil

⁎ Corresponding author. Present address: DepartmePharmacy, University of Granada,18071Granada, Spain. T958249486.

E-mail address: msanchezroman@ugr.es (M. Sánche

0012-821X/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.epsl.2009.06.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 April 2008Received in revised form 15 May 2009Accepted 3 June 2009Available online 9 July 2009

Editor: M.L. Delaney

Keywords:dolomitesulfate inhibition modelmoderately halophilic aerobic bacteriadumbbell and spheroid crystal morphology

The hypothesis that sulfate inhibits dolomite formation evolved from geochemical studies of porewatersfrom deep-sea sedimentary sequences and has been tested with hydrothermal experiments. We examinedthe sulfate inhibition factor using aerobic culture experiments with Virgibacillus marismortui and Halomonasmeridiana, two moderately halophilic aerobic bacteria, which metabolize independent of sulfate concentra-tion. The culture experiments were conducted at 25 and 35 °C using variable SO4

2− concentrations (0, 14, 28and 56 mM) and demonstrate that halophilic aerobic bacteria mediate direct precipitation of dolomite withor without SO4

2− in the culture media which simulate dolomite occurrences commonly found under theEarth's surface conditions. Hence, we report that the presence of sulfate does not inhibit dolomiteprecipitation. Further, we hypothesize that, if sedimentary dolomite is a direct precipitate, as in our low-temperature culture experiments, the kinetic factors involved are likely to be quite different from thosegoverning a dolomite replacement reaction, such as in hydrothermal experiments. Consequently, theoccurrence and, presumably, growth of dolomite in SO4

2−-rich aerobic cultures may shed new light on thelong-standing Dolomite Problem.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

The origin of dolomite remains a long-standing enigma insedimentary geology mainly because of its abundance in ancientrocks, whereas it is rarely found forming in modern carbonatesettings. Precipitation of dolomite inorganically in the laboratoryapparently requires special conditions, such as high temperature, pHand/or pressure (Lippman, 1973; Deelman, 1999). As such conditionsare not observed in the Earth's sedimentary environments, the originof dolomite has fueled, for more than 150 years, the conundrumcommonly referred to as the Dolomite Problem (Van Tuyl, 1916;McKenzie, 1991; Warren, 2000). Consequently, this has led to acontinuing search for chemical variables that might inhibit theprecipitation of dolomite.

Approximately 25 years ago, a new model for dolomite [CaMg(CO3)2] formation in sedimentary sequences appeared in theliterature (Baker and Kastner, 1981). Based on geochemical observa-tions in natural environments, such as organic carbon-rich hemi-pelagic sediments on continental margins, the model postulated thatsulfate ions inhibit dolomite precipitation. However, if the sulfate

nt of Microbiology, Faculty ofel.:+34 958 24 3874; fax:+34

z-Román).

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concentration were to be significantly reduced, for example, bybacterial sulfate reduction, dolomite would precipitate. The modelwas innovative because it provided a mechanistic approach leading tothe design of specific experiments conducted at elevated tempera-tures (200 °C), which indicated that the presence of small concentra-tions of sulfate inhibits the transformation of calcite to dolomite(Baker and Kastner, 1981). Thus, based on observations in naturalsystems and experimental results, it was inferred that the sulfateconcentration of solutions is the major kinetic factor limiting the low-temperature precipitation of dolomite (Kastner, 1984). Since itsformulation, the sulfate inhibition model has been broadly appliedin the study of modern and ancient sedimentary dolomite. Never-theless, the importance of themodel has often been debated because acontradiction exists in that most modern dolomite precipitates inenvironments where sulfate concentrations are approximately equalto that of seawater or greater, and argue against a dominant sulfatecontrol (Hardie, 1987). Dolomite formation is usually associated withsediments where sulfate reduction is active. The observation thatdissolved sulfate concentrations can remain high and dolomite canstill form argues against dominant control by dissolved sulfate (Morseet al., 2007). Also, it has been noted that SO4

2− may even be a catalystfor dolomite precipitation (Siegel, 1961; Brady et al., 1996; Vasconce-los andMcKenzie,1997). Disordered dolomite has been precipitated at25 °C from SO4

2−-rich solutions. Siegel (1961), for example, reportedusing 0.1 to 1 M MgSO4 solutions as a critical reactant to precipitate

132 M. Sánchez-Román et al. / Earth and Planetary Science Letters 285 (2009) 131–139

dolomite and claimed that SO42− was an essential component of the

reaction. Moreover, Rivadeneyra et al. (1993, 2000, 2006) conductedexperiments with halophilic bacteria using artificial seawater to finalconcentrations of 15% (120 mM SO4

2−) and 20% (160 mM SO42−) in

which dolomite precipitated at 32 °C. All of these observations andstudies provide evidence that SO4

2− may not inhibit dolomiteprecipitation at low temperatures (b50 °C). Siegel (1961) andRivadeneyra et al. (1993, 2000, 2006) did not conduct experimentswithout sulfate, thus, they could not study the influence of the lack ofsulfate on dolomite formation. It should be noted that Siegel (1961)and Rivadeneyra et al. (1993, 2000, 2006) were not unequivocally ableto evaluate the influence of sulfate on dolomite precipitation since nocomparative assays were conducted in the absence of sulfate.

However, anaerobic culture experiments have demonstrated thatsulfate-reducing bacteria can mediate dolomite precipitation at theEarth's surface conditions, arguably by increasing pH and removingthe inhibiting SO4

2− (Warthmann et al., 2000; van Lith et al., 2003;Wright and Wacey, 2005). Sulfate-reducing bacteria require andreduce high SO4

2− concentrations because they use the SO42− as a

terminal electron acceptor during anaerobic respiration. Hence,sulfate-reducing bacteria cannot be used to experimentally evaluatethe effects of sulfate on dolomite formation. For this reason, in thisstudy, we have used two moderately halophilic aerobic heterotrophicbacteria, Virgibacillus marismortui and Halomonas meridiana, becausethey do not require sulfate for their metabolism and because they areknown to form Ca–Mg carbonates, amongst them dolomite (Sánchez-Román et al., 2008, 2009). Since previous works only reportedprecipitation of dolomite in sulfate rich culture experiments (Rivade-neyra et al., 1993, 2000; Warthmann et al., 2000; van Lith et al., 2003;Wright and Wacey, 2005; Rivadeneyra et al., 2006) and did not testdolomite precipitation in cultures without sulfate: we have investi-gated the influence of sulfate concentrations on the bacteriallyinduced low-temperature precipitation of dolomite in aerobic semi-solid culture mediawith and without sulfate. Our culture experimentswere designed to simulate the specific Earth's surface conditions (likeshallow marine/lacustrine sediments rich in organic matter, biofilmsand/or mats, pore-space) where precipitation of dolomite occurs,with the goal of demonstrating that microbial dolomite precipitationis not inhibited by the presence of SO4

2−.

2. Material and methods

The experimental design took into consideration the fact thatsediments rich in organic matter and mats contain exopolymericsubstances (EPS), which are similar to a gel media where effectivediffusion coefficients of organic and inorganic substances are reducedin comparison to those in a free liquid media. This differencesignificantly affects ionic concentration and other physico-chemicalproperties relevant to solid phase formation at the microbial-mediuminterface during cell growth. Considering this phenomenon allexperiments were performed in a medium of agar gel.

2.1. Microorganisms

V. marismortui AJ009793 is a gram-positive, endospore-forming,rod shaped, chemoorganotrophic and strictly aerobic bacterium. Thegrowth temperature ranges from 15 to 50 °C (optimal at 37 °C) (Arahalet al., 1999). This strain was isolated from Brejo do Espinho (Rio deJaneiro, Brazil), a shallow hypersaline coastal lagoon in whichdolomite precipitates (Sánchez-Román, 2006; Sánchez-Román et al.,2009). It was first isolated from the Dead Sea and was originallydescribed as Bacillus marismortui (Arahal et al., 1999).

H. meridiana ACAM 246 (= UQM 3352) is a gram-negative,nonspore-forming, rod shaped, chemoorganotrophic and strictlyaerobic bacterium. Its maximum growth temperature is 45 °C (James

et al., 1990). H. meridiana was isolated in 1990 from Antarctic salinelakes (James et al., 1990).

Both of these microorganisms have been effective in experimentsto determine how the ionic composition of the environment affectsthe bacterial precipitation of minerals because both grow underwidely varying saline concentrations. Such halophilic organisms areabundant in hypersaline lakes and in other habitats characterized bysalt concentrations approaching halite saturation, and have a strongimpact on the ecosystems in which they thrive (Oren, 2002).

2.2. Culture experiments

The experiments were conducted using the D-1 medium (Sán-chez-Román, 2006; Sánchez-Román et al., 2008, 2009) designed withthe following composition, (%, wt/vol): 1% yeast extract; 0.5%proteose peptone; 0.1% glucose, and 3.5% NaCl, supplemented with84 mM Mg2+ and 11 mM Ca2+. The medium was modified by addingvarying amounts of Na2SO4 to obtain final SO4

2− concentrations of14 mM, 28 mM and 56 mM representing 0.5×seawater, seawater and2×seawater values. To obtain a gel medium, 20 g/l of Bacto-Agar wasadded. The pH was adjusted to 7.2 with 0.1 M KOH and the solutionwas sterilized at 121 °C for 20 min.

V. marismortui and H. meridiana were surface-inoculated onto gelmedia with SO4

2− concentrations of 0 mM,14 mM, 28 mM and 56mM.The cultures were incubated aerobically at either 25 or 35 °Cand experiments were carried out in triplicate. Parallel controlexperiments, without bacteria and with dead bacterial cells, wererun for all conditions. The cultures were examined periodically byoptical microscope for bacterial growth and for the appearance ofprecipitates.

pHmeasurementswere performed at the end of growth andmineralformation using indicator paper (Merck Spezial-Indikatorpapier)directly applied to the gel surface.

2.3. Mineral and medium analysis

The crystals formed in both V. marismortui and H. meridianacultures were isolated, purified and identified. Struvite (MgNH4-

PO4·6H2O) crystals were extracted from the solid agar by means of asmall spatula, washed with distilled water until perfectly clean andthen dried at 37 °C. The carbonate mineral precipitates at each SO4

2−

concentration were recovered from the medium by scraping thecolony from the agar surface. Subsequently, they were washed severaltimes with distilled water to eliminate the nutritive solution,remaining agar and cellular debris and dried at 37 °C. Microscopicobservations demonstrated that this treatment did not alter themorphology of the crystals.

The bulk precipitates cleaned of organic matter matrix wereexamined by X-ray diffraction analysis (Scintag). A LEO 1530 scanningelectron microscope (SEM), equipped with an energy dispersivedetector (EDS), was used for imaging and elemental analysis of singlecrystals.

The gel medium from sterile experiments and the one below thebacterial mass (after mineral precipitation) was cut into pieces of7 mm width and 1 cm length. The agar pieces were air dried at roomtemperature for twoweeks in order to remove thewater and the SO4

2−

content was measured as elemental S by electron microprobe JEOLJXA-8200 using 15 kV acceleration voltage, 5 nÅ beam current and 15–20 μm beam diameter. The bacteria used in the medium respireaerobically, thus, they do not use SO4

2− as final electron acceptor in therespiration process. They only reduce very small amounts of SO4

2− toform their cellular components and they do not release sulfide or anyother reduced form of S in the medium. Therefore sulfate wascalculated based on the conversion of S into SO4 using the molecularmasses. We also measured the loss of water on dried agar fromboth the sterile experiments and culture experiments after mineral

133M. Sánchez-Román et al. / Earth and Planetary Science Letters 285 (2009) 131–139

precipitation. The loss of water in both dried agar from sterileexperiments and from culture experiments (dried after mineralprecipitation) ranged from 91 to 92% depending on the mediacomposition.

The Ca and Mg contents of the dolomite crystal precipitates andthemediawere measured by Laser Ablation ICP-MS, LA-ICP-MS (ELAN6000 quadruple ICP-MSmass spectrometer equippedwith an Excimer193 nm ArF laser) using a pulse energy of 70–80 mJ and 10–14 μm pitsize. The 193 nm laser was calibrated using a NIST certified glassstandard reference material (SRM 610 glass standard from NIST,hereafter NIST 610).

We should note that: (1) we only used SEM/EDS for imaging andsemi-quantitative analyses of the mineral precipitates because theresolution and quality of the images by SEM is higher than by electronmicroprobe. (2) The S content in the media was analyzed withelectron microprobe and not with LA-ICP-MS because: the determina-tion of S by LA-ICP-MS is problematic due to molecular interferenceand some contamination in the ICP-MS. This interference arisesmainly because of the various combinations of the elements Ar, C, Cl,N, O, and S that are present in the relevant matrices and in the plasmagas.

2.4. Geochemical study

The activity of dissolved species and the degree of saturation in theinitial solutions assayed were determined using the geochemicalcomputer program PHREEQC, version 2 (Parkhust and Appelo, 1999).The results from PHREEQC are presented in terms of the saturationindex (SI) for each predicted mineral. SI is defined by SI=log (IAP/Ksp), where IAP is the ion activity product of the dissolvedconstituents and Ksp is the solubility product for the mineral. Thus,SI N0 implies supersaturation with respect to the mineral, whereas SIb0 means undersaturation. All calculations were performed applyingthe following starting values in the media (g/l): Mg2+=2, Ca2+=0.44, Na2+=14, Cl−=21.23, P=0.15 (PO4

3−=0.46), NH4+=1.73

and variable SO42− concentrations (1.34, 2.7 and 5.4 g/l). The values

of Na+, Cl−, P and NH4+ correspond to the addition of NaCl=35 g/l,

proteose peptone=5 g/l and yeast extract=10 g/l. Total nitrogen inthe culture media was determined by Kjeldhal's method, while totalphosphorus was determined colorimetrically in the nitrogen digests,

Fig. 1. X-ray diffraction diagrams of crystals formed in cultural experiments of Virgibacillus mconcentrations. Note that the major and secondary peaks for dolomite (D) are indicated, w

generating the phosphomolibdate complex (Page et al., 1982). Also,we should note that the initial CO2 value used in these geochemicalcalculations is 0.004 mg/l assuming that atmospheric CO2 was inequilibrium with saline medium.

3. Results

Our culture experiments conducted at 25 and 35 °C using V.marismortui and H. meridianawith SO4

2− concentrations ranging fromzero to 56 mM produced a mixture of minerals comprising dolomite[CaMg(CO3)2], hydromagnesite [Mg5(CO3)4(OH)2·4H2O] and struvite[NH4Mg(PO4)·6H2O)]. Struvite crystals were optically identified andonly the fraction composed of carbonate bioliths was analyzed by X-ray diffraction (Fig. 1). No precipitate formation was observed in thecontrol experiments. Table 1 contains the biochemical conditions ofthe culture media before and after mineral precipitation. Table 1shows the time in days required for growth of V. marismortui and H.meridiana, for the start of precipitation and for the production ofwidespread precipitation. In all of the culture experiments, the timerequired for the initiation and extensive precipitation decreased withincreasing temperature. A significant rise in pH occurred in thecultures with living bacteria, from the original pH of 7.2 in the D-1medium up to ~9. No change in pH was detected in the controlexperiments. BothMg2+ and Ca2+ concentrations were depleted withmineral precipitation, whereas the Mg/Ca molar ratio decreased by asmuch as a factor of 4 from an initial ratio of 7.5, indicating a greaterincorporation of Mg2+ in the precipitates. Also, we should note thatthe starting and final concentrations of SO4

2− remained almostconstant in all of the culture experiments, and the dissolved SO4

2−

(14, 28, 56 mM)was not consumed. Dolomite was found to be a majorconstituent in all experiments with live bacterial cells, with or withoutSO4

2−. We observed that as SO42− concentrations increased, the

proportion of hydromagnesite precipitated also increased. In allcultures, the quantity of crystals increased with increasing incubationtime and observations by optical microscope indicated that the rate ofcrystal growth was higher at 35°C than 25 °C.

When observed under the binocular microscope and SEM, mineralprecipitates formed: a) isolated or aggregated, colorless crystals ofdolomite with sub-spherical shapes (bioliths), b) isolated or aggre-gated, white crystals of hydromagnesite with spherical shapes and c)

arismortui (A) and Halomonas meridiana (B) at 25 and 35 °C with variable sulfate ionhereas only major peaks are indicated for hydromagnesite (HM).

Table 1Biochemical conditions of the culture media before and after mineral precipitation.

Culture T(°C)

aSO42−

(mM)

aCa2+

(mM)

aMg2+

(mM)

aMg2+/Ca2+ apH Time (days) bSO42−

(mM)

bCa2+

(mM)

bMg2+

(mM)

bMg2+/Ca2+ bpH

Growth Beginprecipitation

Extensiveprecipitation

V. marismortui 25 0 11.25 84 7.5 7.2 2 4 12 0 3.0 5.7 1.9 8.5V. marismortui 25 14 11.25 84 7.5 7.2 1 4 11 12 3.0 9.2 3.0 8.5V. marismortui 25 28 11.25 84 7.5 7.0 1 4 13 22 3.5 5.8 1.7 9.0V. marismortui 25 56 11.25 84 7.5 7.2 2 4 12 47 3.0 8.3 2.8 9.0V. marismortui 35 0 11.25 84 7.5 7.0 1 3 8 0 3.6 9.2 2.6 8.5V. marismortui 35 14 11.25 84 7.5 7.3 1 2 8 10 2.5 7.0 2.8 8.5V. marismortui 35 28 11.25 84 7.5 7.2 2 3 9 19 2.5 7.0 2.8 9.0V. marismortui 35 56 11.25 84 7.5 7.0 1 3 9 46 1.5 6.5 4.3 8.5H. meridiana 25 0 11.25 84 7.5 7.4 2 5 12 0 3.5 12.5 3.6 8.5H. meridiana 25 14 11.25 84 7.5 7.0 1 4 12 14 3.0 6.6 2.2 8.5H. meridiana 25 28 11.25 84 7.5 7.3 1 5 11 26 1.8 9.5 5.3 8.5H. meridiana 25 56 11.25 84 7.5 7.2 1 4 12 54 2.5 8.0 3.2 8.5H. meridiana 35 0 11.25 84 7.5 7.4 1 3 9 0 3.0 8.0 2.7 8.5H. meridiana 35 14 11.25 84 7.5 7.0 1 2 9 12 2.5 8.7 3.5 8.5H. meridiana 35 28 11.25 84 7.5 7.2 2 3 8 22 3.0 6.5 2.2 9.0H. meridiana 35 56 11.25 84 7.5 7.3 1 4 10 49 3.0 7.0 2.3 9.0

a Starting SO42−, Ca, Mg concentrations and pH in the medium.

b Final SO42−, Ca, Mg concentrations, Mg/Ca molar ratio and pH in the medium.

134 M. Sánchez-Román et al. / Earth and Planetary Science Letters 285 (2009) 131–139

isolated or aggregated, transparent to translucent crystals of struvitewith vitreous luster (Fig. 2).

An SEM investigation of the dolomite crystals shows that in all ofthe culture experiments, we obtained spheroidal, ovoidal and dumb-bell forms, up to 20 μm in diameter, appearing to be formed byaggregates of nano-crystalline particles (Figs. 3 and 4). EDS micro-analysis confirms the XRD results, showing the presence of dolomitein all samples. The stoichiometry of the dolomite crystals is welldefined by a constant intensity ratio of Mg and Ca peaks in EDS spectra(Fig. 3).

Table 2 shows the mineral phases with SI values positive or veryclose to 0 (above or below the equilibrium point), suggesting thepossibility of inorganic precipitation in the media assayed. These datawere obtained by applying the geochemical computer programPHREEQC to the ionic composition of the various culture media(Table 2). According to the results, the media without SO4

2− areundersaturated with respect to aragonite, calcite, dolomite andhydromagnesite, and saturated with respect to hydroxyapatite andstruvite. Thus, these media should form struvite and hydroxyapatiteand at 35 °C are in fact supersaturated with respect to struvite. At25 °C, media with 14, 28, and 56 mM of SO4

2− are undersaturated withrespect to aragonite, calcite, dolomite, hydromagnesite, hydroxyapa-tite, struvite, anhydrite and gypsum. In contrast, the same media at35 °C are undersaturated with respect to aragonite, calcite, dolomite,hydromagnesite, hydroxyapatite, anhydrite and gypsum but saturatedwith respect to struvite. A decrease of SO4

2− concentration slightly

Fig. 2. Photomicrograph showing crystals formed in Virgibacillus marismortui cultureexperiment at 25 °C and 28 mM SO4

2−. The mineral assemblage of dolomite (D, smallspheroids), hydromagnesite (HM, larger spheroids) and struvite (S) is typical for all ofthe experiments reported herein.

favours the formation of struvite, whereas an increase favours theformation of anhydrite and gypsum. An increase in temperature alsofavours the formation of struvite.

4. Discussion

4.1. Sulfate inhibitor

Because V. marismortui and H. meridiana induce dolomite pre-cipitation with or without the presence of SO4

2−, we conclude thatdissolved SO4

2− apparently does not inhibit dolomite formation, evenat double the concentration of seawater (56 mM). The present studysupports the results of Siegel (1961) and Rivadeneyra et al. (1993,2000), which indicated that dolomite can precipitate in SO4

2−-richsolutions and is in accordance with the observation that most naturaldolomite-forming environments are rich in SO4

2− (see Hardie, 1987).Furthermore, the SO4

2− concentration in the experiments afterdolomite precipitation is nearly the same as the starting SO4

2−

concentration in the media (see Table 1). The small differencesobserved may be due to the bacterial assimilation by reduction ofsmall amounts of SO4

2− to produce their cellular components. Ourresults contrast with those of anaerobic experiments (Warthmannet al., 2000; van Lith et al., 2003; Wright and Wacey, 2005) whichreport that sulfate-reducing bacteria remove the kinetic SO4

2−

inhibitor, and thereby promote dolomite formation.In fact, sulfate-reducing bacteria may induce dolomite precipita-

tion in a similar way as halophilic aerobic bacteria. In our cultureexperiments, dolomite forms independently of the SO4

2− concentra-tion present in the medium, and although the decrease in SO4

2−

concentration is due to microbial metabolism, this does notnecessarily allow or favour dolomite precipitation. This view is inaccordance with the suggestions of Morse et al. (2007) that reactionproducts of sulfate reduction, such as increased alkalinity, may play amore important role in increasing the formation rate of dolomite thansulfate does in inhibiting it. In fact, it has been observed in moderndolomite precipitating environments that the re-oxidation of sulfideions by phototrophic sulfur bacteria actually maintains high sulfateconcentrations (Moreira et al., 2004). Moreover, van Lith et al. (2002)investigated the surface water annual cycle (April 1996 to June 1997)from Lagoa Vermelha and Brejo do Espinho (two hypersaline lagoonswhere dolomite occurs) and reported that in both lagoons sulfateconcentrations were always higher than seawater sulfate concentra-tion (28 mM). All this implies that the sulfate inhibition model is notapplicable to many modern dolomite-forming environments and we

Fig. 3. SEM photomicrographs of dolomite precipitated in Virgibacillus marismortui culture experiments using variable SO42− and at two temperatures [0 mM, 25 °C (A); 28 mM, 35 °C

(B); 56 mM, 25 °C (C)], and of dolomite precipitated in Halomonas meridiana culture experiments using variable SO42− and two temperatures [0 mM, 25 ° C (D); 28 mM, 35 °C (E);

56 mM, 25 °C (F)]. (A) Dolomite dumbbell showing surface projection of the crystals forming the dumbbell. Rod shaped bacterium (arrow) is closely related to the dolomitedumbbell. (B) Dolomite dumbbell embedded in an extracellular matter (eom). (C) Dolomite dumbbell showing surface projection of the crystals forming the dumbbell. (D,E)Dolomite with spheroidal morphology. (F) Dolomite with ovoidal and spheroidal morphology. EDS scans indicate the relatively equal proportions of Mg and Ca in the selectedsamples (hatched arrows).

135M. Sánchez-Román et al. / Earth and Planetary Science Letters 285 (2009) 131–139

therefore conclude that the importance of sulfate inhibition must bereevaluated.

However, and in contrast to the experiments of Baker and Kastner(1981), it is clear from the data presented here that variations in SO4

2−

concentration do not have a discernible influence on the precipitationof dolomite. There are several potentially important differencesbetween the experimental conditions of this study and those adoptedby Baker and Kastner (1981), including differences in temperature,solution composition, solution properties and the addition of themicrobial factor. Whereas our results are more relevant for primarydolomite precipitation under the Earth's surface conditions, the workof Baker and Kastner (1981) might be applied more directly to the

interpretation of ancient examples where massive dolomitization bythe replacement of calcite probably occurred at temperatures from150° to 250 °C.

Additionally, the original hypothesis of Baker and Kastner (1981)evolved, in part, from geochemical observations in natural environ-ments, such as organic carbon-rich hemipelagic sediments oncontinental margins. Furthermore, based on carbon isotopic studies,it has been proposed that diagenetic dolomite forms in themethanogenesis zone of deep-sea sediments after the porewatersulfate has been removed by bacterial sulfate reduction (e.g. Kelts andMcKenzie, 1982; Coleman, 1985). It has been postulated that methaneoxidation in anoxic sediments is mediated by a consortium of

Fig. 4. SEM photomicrographs of dolomite spheroids showing variable surface textures.(A,B) Spheroids with rough, knobby surfaces and high porosity formed in H. meridianacultures at 35 °C. (C) Interior of fibrous radial spherulite formed in V. marismortuicultures at 35 °C.

136 M. Sánchez-Román et al. / Earth and Planetary Science Letters 285 (2009) 131–139

methane-oxidizing archaea and sulfate-reducing bacteria (Boetiuset al., 2000; Orphan et al., 2001; Birgel et al., 2008). As a consequenceof this process, the anaerobic oxidation of methane, the alkalinity willincrease leading to the precipitation of different carbonate mineralsincluding dolomite, calcite and aragonite (Ritgel et al., 1987; Orphanet al., 2001; Peckmann and Thiel, 2004; Campbell, 2006). Although,this process could decrease the sulfate concentration of the micro-environments around the consortium of methane-oxidizing archaeaand sulfate-reducing bacteria, sulfate concentration would not bedepleted enough to stop this association. Meister et al. (2007)

demonstrate that the dolomite precipitates predominantly in themicrobially active sulfate–methane transition zone. In other words,the process of anaerobic methane oxidation requires the presence ofsulfate ions and, likewise, promotes dolomite precipitation byincreasing alkalinity. All these observations suggest that reactionsthat produce an increase of alkalinity may play a more important rolein the precipitation of dolomite than sulfate does in inhibiting it(Morse et al., 2007), in a manner similar to what we have observed inour culture experiments.

The coeval precipitation of dolomite and hydromagnesite in ourculture experiments and the fact that the amount of hydromagnesiteprecipitated increases with increasing SO4

2− concentration in theculture are undoubtedly significant. These results link the bacterialprecipitation of dolomite and hydromagnesite, which commonlyoccur together in natural Mg–Na–SO4 brines and lakes (Renaut, 1990;Warren, 1990; Last and Ginn, 2005) and in Holocene stromatolites(Coshell et al., 1998).

4.2. Role of cell surface, bacterial metabolism and conditions forprecipitation

Whereas purely inorganic chemical precipitation of dolomite isdifficult to achieve in natural or sterile laboratory environments, thepresence of bacteria can induce precipitation of minerals by (1)modifying the conditions of the medium and/or concentrate ions inthe bacterial cell envelope and (2) acting as nucleation sites. Bacteriacan act as nuclei for mineral precipitation by adsorbing cations aroundthe cellular surface membrane, cell wall or EPS layer (Morita, 1980;Ferris et al., 1991; Castanier et al., 1999; Braissant et al., 2003).Rivadeneyra et al. (2004) proposed that different bacteria couldcreate, in their cellular envelopes, microenvironments with varyingproportions of Ca2+ and Mg2+ ions, leading to the precipitation ofdifferent minerals. Commonly, various bacteria mediate precipitationof different minerals under identical culture conditions.

XRD analyses of the minerals precipitated and geochemical studiesof the initial conditions of the culture media (Fig. 1 and Table 2)indicate that media without SO4

2− at 25 and 35 °C should providegeochemical conditions ideal for the formation of hydroxyapatite andstruvite, whereas media with varied concentrations of SO4

2− (14, 28,56 mM) at 35 °C should precipitate struvite. In fact, only dolomite,hydromagnesite and/or struvite precipitated in the media investi-gated. These results show that bacteria exert some control in theprocess of precipitation, i.e., bacterial metabolism acts as source ofchemical conditions for precipitation to occur and cells act asnucleation sites. Although all of the media were initially under-saturated in carbonate minerals, the bacteria investigated promotedthe precipitation of dolomite and hydromagnesite.

Thermodynamic limitations mean that solid phases are formedonly if supersaturation exists. It is evident that the metabolic activityof the bacteria imposes such conditions in the medium adjacent to thecell walls, the areas in which the solids formed in our experiments.The importance of bacterial metabolic activity to the precipitation ofcalcium and magnesium minerals is clear. Bacterial metabolic activitychanges the pH, ionic strength and ionic composition of the mediumand influences the cell surface charge (Ahimou et al., 2002), thusmodifying the Ca2+ versus Mg2+ ratio at the cell surface. Thedegradation of organic matter in the medium by halophilic aerobicbacteria produces the CO3

2−, NH4+ and PO4

3− ions necessary for theobserved precipitation of carbonate and phosphate minerals. Noprecipitates are observed in abiotic media, firstly, because carbonateions are not generated (equilibrium with atmospheric CO2 of theinitial solution, before gelification, leads to carbonate concentration ofthe order of 10−8 M) and, secondly, because all the Ca2+ and Mg2+

ions are strongly bound to ligand groups in the amino acids thatcontain oxygen and nitrogen atoms as electron donors.

Table 2Saturation index values (SI) for different minerals in all media assayed.

Mineral phase SO42− concentrations in the media

Media at 25 °C Media at 35 °C

0 mM 14 mM 28 mM 56 mM 0 mM 14 mM 28 mM 56 mM

Aragonite, CaCO3 −6.23 −7.41 −8.54 −9.21 −6.71 −7.81 −8.89 −9.45Calcite, CaCO3 −6.09 −7.27 −8.39 −9.06 −6.57 −7.68 −8.75 −9.31Dolomite, CaMg(CO3)2 −11.10 −13.46 −15.72 −17.07 −11.98 −14.18 −16.34 −17.49Halite, NaCl −2.38 −2.39 −2.39 −2.39 −2.41 −2.41 −2.42 −2.42Hydromagnesite, Mg5(CO3)4(OH)2•4H2O −34.37 −44.82 −50.49 −53.88 −35.90 −46.64 −52.06 −54.96Hydroxyapatite, Ca5(PO4)3OH 7.15 −10.50 −14.47 −16.81 7.84 −11.71 −15.48 −17.44Struvite, MgNH4PO4•6H2O 0.83 −4.17 −5.31 −5.99 21.79 16.25 15.16 14.58Anhydrite, CaSO4 – −1.53 −1.08 −0.73 – −1.58 −1.10 −0.74Gypsum, CaSO4•2H2O – −1.31 −0.88 −0.53 – −1.43 −0.94 −0.59

Results of the geochemical computer program PHREEQC. SI is defined by SI=log (IAP/Ksp), where IAP is the ion activity product of the dissolved mineral constituents in a solubilityproduct (Ksp) for the mineral. Thus, SI N0 implies supersaturation with respect to the mineral, whereas SI b0 means undersaturation.Note: All calculations were performed applying the following starting values in the media (g/l): Mg2+=2, Ca2+=0.44, Na2+=14, Cl−=21.23, P=0.15 (PO4

3−=0.46), NH4+=1.73

and variable SO42− concentrations (1.34, 2.7 and 5.4 g/l). For more details, see Section 2.4.

137M. Sánchez-Román et al. / Earth and Planetary Science Letters 285 (2009) 131–139

When the gel is formed and water molecules are bound to thecolloidal particles (stiff agar gel may contain up to 100 molecules ofwater per molecule of agar), these ions, immobilized in the amino acidarray, can only be liberated by significantly changing the physical andchemical conditions. Such changes occur in the presence of bacteria,which, by metabolizing the organic substances (particularly, thoserich in ligands for calcium and magnesium ions), liberate NH3, H2O,HPO3

2−, Ca2+, Mg2+ and CO2. The presence of “free” water is essentialfor the hydration of CO2 and protonation of ammonia that lead to themeasured alkaline pH. Under these conditions, of high pH andavailability of inorganic carbon, carbonate ions can be formed at thebacteria–medium interface. Due to obstructing effects (Borhade andPatil, 2000; Rajurkar and Gokarn, 2005), the agar medium slows thediffusion of ions away from the interface, which, as the bacteriagrowth proceeds, becomes progressively enriched in the ions ofinterest (Ca2+, Mg2+ and CO3

2−) until supersaturation is reached. Inaddition, the activity at the bacterial surfaces tends to lower theactivation energy required for nucleation of dolomite. Similarmechanisms have been described for bacterial precipitation in naturalhabitats (Ehrlich, 2002) and in laboratory aerobic culture experiments(Rivadeneyra et al., 1993, 1998, 2004; Sánchez-Román et al., 2007,2008, 2009).

In summary, the precipitation of dolomite is a complex phenom-enon, whereby bacterial metabolic activity, the physico-chemicalproperties of the cellular envelope and the characteristics of themediaall play important roles in the process.

4.3. Dolomite morphology and internal structure

Although three different variables were incorporated in our study(SO4

2− concentration, temperature and bacteria), it is significant thatno differences in morphology or crystal shape were observed. In all ofthe culture experiments, we obtained dolomite with spheroidal anddumbbell morphologies, up to 20 μm in diameter (Fig. 3). At highmagnification, it is possible to observe different surface structures onthe dolomite microbioliths (Fig. 4) that may indicate differences in themechanisms of precipitation, growth and/or recrystallization of theVirgibacillus and/or Halomonas bioliths. Most appear to be formedby aggregates of nano-crystalline particles (Fig. 3). Some (Fig. 4C)have laminated and/or radial internal structures, making them appearvery similar to some ooids, peloids and/or spherulites formed innatural carbonate environments. Fig. 4A–B shows bioliths with rough,knobby surfaces with some open spaces and high porosity. Theseopenings are of similar size and shape to the bacteria (~1 μm long and500 nmwide). Surface roughness is also observed in the bioliths witha radial structure (Fig. 4C). EDS analysis of the bioliths and dumbbellsconfirms the XRD results showing the presence of dolomite in all

samples. The stoichiometry of dolomite crystals is well defined by aconstant intensity ratio of Mg and Ca peaks in EDS spectra (Fig. 3).

In spite of possible differences in detail, we consider that thecarbonate precipitation processes induced by the studiedmicroorgan-isms can be described as biologically induced biomineralization(Lowenstam and Weiner, 1989; Chafetz and Buczynski, 1992). Thespheroidal growth morphology is similar to that of natural dolomitesfound in many modern settings, including saline lakes (Von der Borchand Jones, 1976; De Deckker and Last, 1988; Wright, 1999), the tidalflats of Florida (Carballo et al., 1987) and beneath the Abu Dhabisabkha (Bontognali et al., 2005). Spheroidal dolomite has also beenfound in ancient rocks (Guanatilaka et al., 1987; Amiri-Garroussi,1988; Guanatilaka, 1989; Nielsen et al., 1997; Mastandrea et al., 2006).These spherulitic structures have been interpreted as being ofmicrobial origin (Nielsen et al., 1997; Lee and Golubic, 1999;Mastandrea et al., 2006). Biogenic dolomite with a spheroidalstructure has also been reported for uroliths produced in the urinarybladder of a male Dalmatian, Canis familiaris (Mansfield, 1980).According to Buczynski and Chafetz (1991), spheroidal and dumbbellhabits imply bacterially mediated precipitation and can be used toidentify such precipitates in the rock record. However, we do notrecommend the use of spheroidal and dumbbell structures asmorphological biosignatures in ancient rocks. It has been demon-strated that similar structures, as well as a large variety of unusualmorphologies, may form in response to abiotic processes or in thepresence of organic molecules, whose origin may not be linked to abiological process (Tracy et al., 1998; Golden et al., 2001; Fernandez-Diaz et al., 2005).

4.4. Involvement of bacteria in dolomite formation in naturalenvironments

Recent dolomite formation in natural environments commonlyoccurs in organic carbon-rich sediments (e.g., Baker and Burns, 1985;Compton, 1988; Mazzullo, 2000). The gel medium used in ourexperiments contains Ca2+, Mg2+ and abundant organic matter,having both yeast extract and peptone (sources for CO2 and NH4

+) thatreplicate organic carbon-rich sediments. Thus, diagenetic dolomiteprecipitation in natural environments can be explained as a by-product of the bacterial degradation of organic matter producing CO2

and ammonia, which supply the high pH conditions necessary forprecipitation (Table 1). Bacterial activity is also responsible for localsupersaturation in Ca2+and Mg2+ ions in the microenvironment at oraround cell surfaces, overcoming low-temperature kinetic barriers toprecipitation (e.g., Sánchez-Román et al., 2008, 2009). This mechan-ism, increasing alkalinity, explains the formation of dolomite under(hyper)saline conditions and could explain the formation of dolomite

138 M. Sánchez-Román et al. / Earth and Planetary Science Letters 285 (2009) 131–139

in natural environments. In dolomitic sediments containing abundantprotein-rich organic matter and SO4

2−, the pH is approximately 10[e.g., Pellet Lake in the Coorong region (Rosen et al., 1988)].

The formation of dolomite in natural environments under aerobicconditions, with or without SO4

2−, can be explained by the degradationof organic matter by halophilic aerobic bacteria and, especially, by theproduction of ammonia which is vital to dolomite formation. This issupported by the fact that we isolated V. marismortui from a dolomiticsediment rich in organic matter and SO4

2−, with a pH approaching 9(Sánchez-Román, 2006; Sánchez-Román et al., 2009).

5. Summary and conclusions

We have tested the validity of the sulfate inhibition model usinglow-temperature aerobic cultures experiments. Our results demon-strate that it is possible to form dolomite in the presence of SO4

2−

under the Earth's surface conditions, as has been observed to occur inmany modern environments. We propose that dolomite can form innatural environments with or without significant SO4

2− concentra-tions, under conditions similar to those used in our culture experi-ments. Bacterial activity will produce local supersaturation in themicro-environment around cell surfaces, overcoming any low-temperature kinetic barriers to dolomite precipitation. Further, thepresence of dolomite and hydromagnesite in our cultures leads us toconsider that the co-occurrence of these twominerals inmany naturalSO4-rich habitats may reflect aerobic halophilic bacterial mediation.

Our results suggest that the sulfate inhibition model proposed byBaker and Kastner (1981) may only apply to inorganic dolomiteformation at higher temperatures. Although their model has beenwidely applied to dolomite formation, the results of our aerobicculture experiments imply that the importance of SO4

2− inhibition indolomite precipitation must be reevaluated.

Acknowledgements

The Swiss Science National Foundation (SNF) is gratefullyacknowledged for financial support through Grant No. 20-067620and 20-105149.We acknowledge the assistance of Thomas Pettke, EricReusser, Luca Caricchi and Anne Greet Bittermannwith Laser AblationICP-MS, Electron Microprobe and SEM analyses. Peggy Delaney, DavidT Wright, Max Coleman and three anonymous reviewers providedcomments that greatly improved the earlier versions of thismanuscript.

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