A mineralogical characterization of biogenic calcium ... · and their rates of change, result in...

A mineralogical characterization of biogenic calciumcarbonates precipitated by heterotrophic bacteria isolatedfrom cryophilic polar regionsJ . RONHOLM,1 D. SCHUMANN,2 , 3 , * H. M. SAPERS ,1 , * M. IZAWA,4 ,* D. APPLIN,4 B. BERG,4

P . MANN,4 H. VALI , 5 , 6 R . L . FLEMMING,2 E. A . CLOUTIS4 AND L. G. WHYTE1

1Department of Natural Resource Sciences, McGill University, Sainte-Anne-de-Bellevue, QC, Canada2Department of Earth and Sciences, Western University, London, ON, Canada3Fibics Incorporated, Ottawa, ON, Canada4Department of Geography, University of Winnipeg, Winnipeg, MB, Canada5Facility for Electron Microscopy Research, McGill University, Montreal, QC, Canada6Department of Earth and Planetary Sciences, McGill University, Montreal, QC, Canada

ABSTRACT

Precipitation of calcium carbonate (CaCO3(s)) can be driven by microbial activity. Here, a systematic

approach is used to identify the morphological and mineralogical characteristics of CaCO3(s) precipitated

during the heterotrophic growth of micro-organisms isolated from polar environments. Focus was placed

on establishing mineralogical features that are common in bioliths formed during heterotrophic activity,

while in parallel identifying features that are specific to bioliths precipitated by certain microbial phylotypes.

Twenty microbial isolates that precipitated macroscopic CaCO3(s) when grown on B4 media supplemented

with calcium acetate or calcium citrate were identified. A multimethod approach, including scanning elec-

tron microscopy, high-resolution transmission electron microscopy, and micro-X-ray diffraction (l-XRD),

was used to characterize CaCO3(s) precipitates. Scanning and transmission electron microscopy showed that

complete CaCO3(s) crystal encrustation of Arthrobacter sp. cells was common, while encrustation of Rhodo-

coccus sp. cells did not occur. Several euhedral and anhedral mineral formations including disphenoid-like

epitaxial plates, rhomboid-like aggregates with epitaxial rhombs, and spherulite aggregates were observed.

While phylotype could not be linked to specific mineral formations, isolates tended to precipitate either eu-

hedral or anhedral minerals, but not both. Three anhydrous CaCO3(s) polymorphs (calcite, aragonite, and

vaterite) were identified by l-XRD, and calcite and aragonite were also identified based on TEM lattice-

fringe d value measurements. The presence of certain polymorphs was not indicative of biogenic origin,

although several mineralogical features such as crystal-encrusted bacterial cells, or casts of bacterial cells

embedded in mesocrystals are an indication of biogenic origin. In addition, some features such as the for-

mation of vaterite and bacterial entombment appear to be linked to certain phylotypes. Identifying phylo-

types consistent with certain mineralogical features is the first step toward discovering a link between these

crystal features and the precise underlying molecular biology of the organism precipitating them.

Received 23 June 2014; accepted 13 August 2014

Corresponding author: J. Ronholm. Tel.: (514) 398-7823; fax: (514) 398-7990; e-mail: jennifer.

[email protected]

INTRODUCTION

The ability of heterotrophic bacteria to precipitate calcium

carbonate (CaCO3(s)) in certain environmental and in vitro

culture conditions has been well documented for some

time (Boquet et al., 1973). Although there are several

previously described mechanisms of prokaryotic CaCO3(s)

precipitation, most microbial heterotrophic CaCO3(s) pre-

cipitation is biologically induced and occurs as an indirect*Authors contributed equally to this study.

542 © 2014 John Wiley & Sons Ltd

Geobiology (2014), 12, 542–556 DOI: 10.1111/gbi.12102

result of metabolic activity. This is opposed to biologically

controlled precipitation where nucleation, crystal growth,

and final crystal location are directly moderated by bio-

logical processes (Dupraz et al., 2009). At least three

mechanisms are known to be partially responsible for bio-

logically induced mineralization. (i) Autotrophic or het-

erotrophic metabolic processes, such as photosynthesis

(Bundeleva et al., 2012), urea hydrolysis (Dhami, 2013),

sulfate reduction (Van Lith et al., 2003), or oxalate oxi-

dation (Braissant et al., 2004), alter the chemistry of the

cell-proximal environment and result in a microenviron-

ment with a higher pH causing a shift in the bicarbon-

ate–carbonate equilibrium to CO2�3 ðaqÞ production that

favors CaCO3(s) precipitation if Ca2+(aq) is present (Rodri-

guez-Navarro et al., 2003; Ben Chekroun et al., 2004;

Lian et al., 2006). Metabolically active bacteria may also

consume or fix aqueous components that inhibit mineral

precipitation, such as phosphate (Braissant et al., 2003;

Bundeleva et al., 2012) or sulfate (Wright & Wacey,

2005), and induce CaCO3(s) precipitation. Variability in

the physiochemical properties of the microenvironment,

and their rates of change, result in biogenic carbonates

with varying types of mineralogy (Hammes et al., 2003).

(ii) The cell surface hosts several molecules with a nega-

tive charge, such as proteins, lipid bilayers, and glycopro-

teins, that may absorb divalent cations (i.e., Ca2+),

providing sites for crystal nucleation. Beveridge (Beve-

ridge & Murray, 1976) first described the ability of

microbes to accumulate metals at the cell surface and pre-

cipitate minerals. Therefore, the specific molecular

makeup of individual cells, such as cell surface variation,

would affect the mineralogy of biogenic precipitates

(Contos et al., 2001). (iii) Extracellular polysaccharides

[exopolysaccharide (EPS) or capsular polysaccharide

(CPS)] can trap and concentrate cations (Braissant et al.,

2007, 2009). EPS may also contain nucleating proteins

that support biomineralization (Braissant et al., 2003).

The physiochemical properties of EPS, such as acidity and

functional group composition, will dictate if precipitation

occurs and will also influence the resultant mineralogy

(Kawaguchi & Decho, 2002; Braissant et al., 2003; Dup-

raz et al., 2009). Therefore, despite strong biological

influence on the precipitation of CaCO3(s), the formation

is passive and not directly genetically regulated by the

micro-organisms in these systems and CaCO3(s) precipi-

tates appear to have no specific biological function (Lian

et al., 2006).

Calcium carbonate is a ubiquitous mineral phase, occur-

ring naturally in marine, freshwater, and terrestrial settings,

as well as in extraterrestrial materials, such as the Phoenix

landing site (Smith et al., 2009). In order of increasing

stability at Earth’s surface, the CaCO3(s) polymorphs are

vaterite, aragonite, and calcite. Amorphous CaCO3(s),

which is less stable than vaterite, has also been identified

(Politi et al., 2008). Calcite is the dominant C-bearing

phase on Earth. Two hydrous phases occur naturally as

well: monohydrocalcite (CaCO3�H2O) and ikaite

(CaCO3�6H2O), although these form under very specific

conditions (Omelon et al., 2001).

Several studies have focused on CaCO3(s) precipitation

by photoautotrophic cyanobacteria that require light for

energy and fix their own carbon (Douglas & Beveridge,

1998; Dittrich & Sibler, 2010; Kranz et al., 2010; Marti-

nez et al., 2010; Bundeleva et al., 2012). Cyanobacteria-

induced CaCO3(s) precipitation can be either biologically

induced or biologically controlled. During biologically

controlled precipitation, cyanobacteria collect intracellular

amorphous carbonates (Couradeau et al., 2012). Biologi-

cally induced precipitation occurs in three stages: pH rise

coinciding with Ca2+ decrease, biomass production accom-

panied by carbonate precipitation, and finally bacterial lag

phase and mineral equilibrium (Bundeleva et al., 2012).

Certain phylotypes of cyanobacteria appear to actively

avoid whole cell carbonate encrustation by selectively con-

trolling the electrochemical potential at the cell surface to

reject Ca+ ions while attracting other nutrients (Martinez

et al., 2010; Bundeleva et al., 2011). In addition, some cy-

anobacteria actively shed precipitates from the cell surface

during cell division (Thompson & Ferris, 1990).

Calcium carbonate precipitation by sulfate-reducing bac-

teria (SRB) has also been extensively explored (Aloisi et al.,

2006; Braissant et al., 2007; Gallagher et al., 2012). Lithi-

fying microbial mats (microbial mats where calcium

carbonate precipitation occurs) can host SRB that are

involved in CaCO3(s) precipitation (Braissant et al., 2007;

Gallagher et al., 2012) and often form dolomite (a-CaMg

(CO3)2(s)) (Van Lith et al., 2003; Bontognali et al.,

2008). Through their metabolic processes, SRB create an

alkaline environment promoting CaCO3(s) precipitation

(Gallagher et al., 2012). Calcium carbonate nucleates on

the SRB’s cell surface, although the bulk of precipitation

nucleates on globules (60- to 200-nm-diameter spherical

objects that originate from the SRB’s cell surface and are

likely composed of EPS) where are released from the cell

into the culture medium (Aloisi et al., 2006). Active

release of globules is another mechanism to avoid cell

encrustation.

Comparatively, less work has focused on carbonate pre-

cipitation by chemoheterotrophic bacterial aerobes

(reviewed in Castanier et al., 1999) and much of this has

focused on Myxobacteria (reviewed in Gonzalez-Munoz

et al., 2010), ureolytic (Ferris et al., 2004; Mitchell &

Ferris, 2006; Achal & Pan, 2010; Okwadha & Li, 2010;

Burbank et al., 2012; Millo et al., 2012), and carbonic an-

hydrase-producing bacteria (Achal & Pan, 2010; W. Li

et al., 2011). Bacterial metabolic activities influence the

chemistry of the cell–media interface, and CaCO3(s) forma-

tion is strongly controlled by fluid chemistry. Therefore,

© 2014 John Wiley & Sons Ltd

Characterization of biogenic carbonate 543

different metabolic strategies will likely result in mineralog-

ically, structurally, or compositionally distinct calcium car-

bonate precipitates. Given the complexity and variety of

possible biomineralization processes linked to metabolism

and cell surface variation, which could be occurring during

heterotrophy, there are likely distinctive mineralogical dif-

ferences between CaCO3(s) precipitated by different

microbes and studies to date have not systemically focused

on characterizing these similarities and differences,

although others have called for this type of study (Dupraz

& Visscher, 2005).

In this study, we have focused on characterizing the

chemistry, morphology, and mineralogy of CaCO3(s) pre-

cipitated during growth of a variety of heterotrophic

microbes, isolated from similar (arctic and Antarctic) envi-

ronments, and demonstrated that this can result in the

production of all three CaCO3(s) anhydrous polymorphs

with a range of morphologies, some of which appear to

be linked to phylotype. This is the first systematic study

of variation in CaCO3(s) biolith morphology precipitated

by various heterotrophs isolated from polar environments.

This study complements previous work and widens the

known the range of environmental conditions where

CaCO3(s) biomineralization could occur in nature. In

addition, certain morphologies, such as the crystal

encrustation of cells, are linked to certain microbial phyl-

otypes, which indicates the presence of an underling

molecular explanation for at least some morphological

features.

MATERIALS AND METHODS

Bacterial growth, CaCO3(s) precipitation, and isolate

identification

All of the isolates used in this study are polar heterotro-

phic microbes isolated from active layer or permafrost soil

in the Canadian High Arctic (Steven et al., 2007; Ferrera-

Rodr�ıguez et al., 2012) or Antarctic permafrost and cryp-

toendoliths (J. Goordial & L. G. Whyte, unpublished

data). A culture collection (~300 isolates) was screened

for the ability to precipitate calcium carbonate crystals.

Cultures were removed from long-term storage (�80 °C)and streaked directly onto solid B4 media containing 4 g

of yeast extract, 10 g glucose, 15 g bacteriological agar,

and either 2.5 g calcium acetate or 2.5 g calcium citrate

per liter of media (Boquet et al., 1973). After 4 weeks of

growth in humidity-controlled environmental chambers at

22 °C, each isolate was inspected for CaCO3(s) precipita-

tion on both calcium acetate and calcium citrate-supple-

mented media. Several, but not all, isolates produced

macroscopic crystals on one or both media types

(Table 1). Isolates and crystals were selected for further

study if they precipitated enough material for to be ana-

lyzed by several techniques. Negative control B4 plates

were incubated under the same conditions without being

inoculated with bacterial cultures; crystals did not form on

these plates.

The 16S rDNA gene from isolates selected for further

study was amplified by colony PCR using the 27F and

758R primers (V1–V3 region). The PCR product was then

purified using the PureLink PCR Purification Kit (Life

Technologies, Burlingtion, ON, Canada) following the

manufacturer’s instructions and then sent to Plate-forme

d’Analyses Genomiques de l’Universite Laval for sequenc-

ing using the 27F primer. Sequences were compared to

the NCBI database using BLASTN. An Antarctic permafrost

yeast species, Rhodotorula mucilaginosa, also used in this

investigation, was identified as part of a whole-genome

sequencing project (J. Goordial, I. Raymond-Bouchard, J.

Ronholm, M. Huntemann, J. Han, A. Chen, N. Kyrpides,

V. Markowitz, K. Palaniappan, N. Ivanova, N. Mikhailova,

G. Ovchinnikova, A. Schaumberg, A. Pati, D. Stamatis, A.

Reddy, N. Shapiro, H. P. Nordberg, M. N. Cantor, S. X.

Hua, T. Woyke, C. Bakermans & L. G. Whyte, unpub-

lished data).

Mineral purification and storage

Precipitated CaCO3(s) was physically removed from agar

plates along with bacterial debris by scraping both off the

media with a sterile loop and suspending the mixture in

Table 1 Identification of calcium carbonate precipitating bacterial isolates

Environmental isolate ID

number and 16S rRNA

sequence closest match

Source

material

Optimal substrate for

crystal formation

Calcium

citrate

Calcium

acetate

Agromyces sp. ofe 5.5 Arctic Soil +

Arthrobacter sp. JG9 Antarctic

Endolith

+

Arthrobacter sp. oc 825 Arctic Soil +

Arthrobacter sp. ofe 4.2 Arctic Soil +




Arthrobacter sp. ofe 10.10 Arctic Soil



Arthrobacter sp. eur3 7.19 Arctic Soil +

Arthrobacter sp. CY7 Arctic Soil +

Ralstonia sp. ofe 1.3 Arctic Soil +

Rhodococcus sp. JG12 Antarctic

Endolith

+

Rhodococcus sp. MD.2 Arctic Soil +

Rhodococcus sp. ofe 9.29 Arctic Soil +

Rhodococcus sp. CY5 Arctic Soil +

Rhodococcus sp. CY6 Arctic Soil +

Rhodococcus sp. eur1 9.01.3 Arctic Soil +

Rhodotorula mucilaginosa

JG1 (yeast)

Antarctic

Permafrost

+


544 J. RONHOLM et al.

sterile Millipore water. Crystals were isolated through suc-

cessive rounds of sedimentation (P�arraga et al., 1998;

S�anchez-Rom�an et al., 2011) and then dried on the low

setting using a rotary evaporator. Purified precipitates were

stored at 4 °C until further examination.

Light microscopy

To provide an initial characterization of bacterially induced

precipitates, and collect low-resolution overview images,

dried crystals were suspended in 10 lL Millipore water,

placed on a glass microscope slide, and examined under

multiple objectives using a phase-contrast BX41 micro-

scope (Olympus America Inc., Center Valley, PA, USA).

Scanning electron microscopy

Biogenic CaCO3(s) was transferred to carbon tape affixed to

titanium SEM imaging stubs. Each isolate was imaged

under 40 Pa on a Hitachi 3400-N (Schaumburg, IL, USA)

variable pressure scanning electron microscope at the Bio-

Tron advanced imaging facility, Western University,

Ontario, Canada, with an accelerating voltage of 10–15 kV

at a working distance of 5–10 mm. A subset of isolates (Ar-

throbacter sp. oc825, Arthrobacter sp. ofe 7.23, Arthrobact-

er sp. ofe 3.1, Ralstonia sp. ofe 1.3, and Arthrobacter sp.

JG9), representing a variety of precipitate morphologies,

were plasma-coated with 5 nm of amorphous osmium

using a Filgen OPC80T Osmium Plasma Coater with an

OsO4 source. Coating and subsequent high-resolution

imaging was carried out under high vacuum on a LEO

Zeiss (Jena, Germany) 1540XB FIB/SEM at the Western

University Nanofabrication Facility under an accelerating

voltage of 1 kV and a working distance of 3.8 mm.

Transmission electron microscopy

TEM was used to investigate the size, shape, and presence

of polymorphs in CaCO3(s) precipitates. Precipitates were

suspended in distilled water and transferred onto 300-mesh

copper TEM grids with carbon support film. The Philips

CM 200 TEM (Amsterdam, The Netherlands) equipped

with a Gatan Ultrascan 1000 2k 9 2k CCD camera system

Model 895 (Pleasanton, CA, USA) and an EDAX Genesis

energy dispersive X-ray spectroscopy (EDX) (Kennett

Square, CA, USA) system was used in bright-field mode at

an accelerating voltage of 200 kV. Lattice-fringe images

were taken at focus conditions. Measurements of d values

were taken on sets of several lattice fringes.

Micro-X-ray diffraction

Micro-X-ray diffraction (l-XRD) data were collected using

the Bruker D8 Discover diffractometer at the University of

Western Ontario. Operating conditions were 35 kV accel-

erating voltage and 45 mA beam current generating Co

Ka X-rays (k = 1.7 ADFS �A). G€obel mirror parallel beam

optics and a 300-lm snout were used. Scattered X-rays

were detected using a HiSTAR 2-dimensional detector

(Bruker, Billerica, MA, USA) [General Area Diffraction

Detection System (GADDS)]. All lXRD data were col-

lected in ‘coupled’ mode, that is, with source and detector

angles held constant at 20°, covering ~19°–60° 2h. Inte-

grated diffractograms were produced using Bruker GADDS

software (Bruker) and interpreted using the International

Center for Diffraction Data (ICDD) Powder Diffraction

File version 2 (PDF-2) and the Crystallography Open

Database (COD) with Bruker EVA 3.0 software. Samples

were held in 5-mm-diameter, 1.5-mm-deep wells drilled in

an aluminum block.

RESULTS

Isolate identification and crystal production

Sequence analysis of the 16s rRNA PCR amplicon was

used to identify isolates selected based on CaCO3(s) precip-

itation for additional study. BLASTN searches identified each

isolate to the genus level (Table 1). Most isolates that were

selected belonged to the Rhodococcus or Arthrobacter

genus, although, Agromyces, Ralstonia, and a yeast species

(R. mucilaginosa) were also selected.

Each microbial isolate was tested for the ability to pre-

cipitate CaCO3(s) on both calcium citrate- and calcium

acetate-amended B4 media. In most cases, isolates formed

CaCO3(s) precipitates on only one of the two amend-

ments. Three Arthrobacter sp. isolates ofe 3.1, ofe 1.1,

and ofe 8.9 (Table 1) precipitated CaCO3(s) on both sub-

strates; however, in these cases, the substrate that resulted

in the most visual CaCO3(s) precipitation was used for

precipitate production. R. mucilaginosa, Ralstonia, and

Agromyces only produced CaCO3(s) precipitate when cal-

cium acetate was used as the substrate. As groups, Rhodo-

coccus and Arthrobacter genuses did not clearly precipitate

more CaCO3(s) on either substrate. A clear link between

taxonomy and optimal CaCO3(s) precipitation substrates

was not shown.

Morphological characterization

Biogenic CaCO3(s) precipitates ranged in morphology from

mineral forms approximating euhedral crystals to spheroid

aggregates, and flaky plate-like irregular sheets (Fig. 1).

Cell-shaped voids (Fig. 2) and cells entombed in CaCO3(s)

are common (Fig. 3). Surface features included cell-cov-

ered, irregular organic matrices, voids, featureless, dentrit-

ic-like pits, and stepped growth faces. Precipitates fall into

two broad morphological classes: euhedral and anhedral



(Fig. 1). The former class consists of large (tens of

micrometers or larger) calcium carbonate crystals, while

the latter are composed of microcrystalline calcium carbon-

ate. Both are composed of crystalline material as supported

by XRD and TEM observations. SEM images show that

the euhedral CaCO3(s) precipitates are composed of

ordered aggregates of smaller euhedral subunits that appear

to share a common orientation (Fig. 4). Subunits are offset

giving rise to ‘pseudofaces’ in the mesocrystal such that,

for example, irregular quadrilateral subunits give rise to a

dodecahedron-like mesocrystal (Fig. 4). Mesocrystal mor-

phologies are transitional with endmember examples

depicted in Fig. 1. Mesocrystals may occur singly or aggre-

gated in complex, irregular forms (Fig. 1). Isolated meso-

crystals range in size from 20 to 200 lm. Precipitates

isolated from a particular microbial culture have a consis-

tent size, although the details of crystal aggregate mor-

phology vary, both within and between isolates (Fig. 1). A

variety of euhedral crystal forms are present among the cal-

cium carbonate precipitates, most of which appear to be

relative simple combinations or rhombohedral forms, con-

sistent with calcite (Fig. 1). Some crystal aggregates precip-

itated by Arthrobacter sp. ofe 3.1, Arthrobacter sp. ofe 8.9,

and Arthrobacter sp. eur3 7.19 showed a highly irregular

surface pattern that may have resulted from dissolution or

mesocrystal breaking (Fig. 5).

Spherulites and spherulite-like formations are dominant

among anhedral crystal forms. Spherulites occur both as

isolates or as aggregated into complex structures (Fig. 6A).

Void spaces observed on the surfaces of many spherulitic

aggregates are consistent with bacterial casts due to the

correspondence between the size and shape of the void

spaces, and the types of bacteria present (Fig. 6B). Surface

features of spherulites range from irregular organic matri-

ces (Fig. 6C–E) to what appear to be euhedral crystallo-

graphically controlled units (Fig. 6D,F). Secondary

electron SEM images showed that several bacterial isolates

(Arthrobacter sp. ofe 7.23, Arthrobacter sp. ofe 10.25, Ar-

throbacter sp. ofe 3.1, Arthrobacter sp. ofe 8.9, and Ar-

throbacter sp. CY7) became encrusted within a network of

very fine crystals (Figs 3 and 7A,B). Bright-field TEM

imaging revealed that these networks are composed of ara-

gonite nanocrystals (Fig. 7C,D). Several families of lattice

planes of aragonite (e.g., {200}, {130}, {112}) could be

identified in lattice-fringe images (Fig. 7D). The TEM sep-

arates of these encrusted bacterial isolates also contained

crystal aggregates that seem to have grown independently

from the cellular surfaces (Fig. 7E). Lattice-fringe images

show that these aggregates are also composed of aragonite

nanocrystals (Fig. 7F).

Several of the bacterial isolates formed up to 5 lm larger

mesocrystals (e.g., Arthrobacter sp. JG9) (Fig. 8A). These

mesocrystals were too thick to obtain lattice-fringe images

from their interior parts (Fig. 8A). However, the outer

areas of the crystals were thin enough for the acquisition

of lattice-fringe images (Fig. 8B–D). The outer rim of

these mesocrystals is composed of nanocrystals of aragonite

(Fig. 8C,D). High-resolution TEM imaging revealed the

{112}, {012}, {022}, and {121} families of lattice planes

of aragonite (Fig. 8C,D). The TEM investigations con-

ducted on these samples were not able to determine

whether the central parts of these mesocrystals were also

composed of aragonite nanocrystals.

Energy dispersive X-ray spectroscopy point analyses of

this aragonite nanocrystal network reveal that the crystals

were primarily composed of Ca, C, and O with trace

amounts of Si, P, and Fe (Fig. 8E,F). Trace amounts of Fe

could have contributed to the red coloration of these

nanocrystals (Fig. 8G).

Identification of CaCO3(s) polymorphs

Each anhydrous CaCO3(s) polymorph was detected at least

once using lXRD (Table 2). Calcite was detected in every

precipitate characterized in this study (Fig. 9A), although

Arthrobacter sp. ofe 7.23 had trace amounts of aragonite

(Fig. 9B) and Arthrobacter sp. JG9 had trace amounts of

vaterite (Fig. 9C). Characteristic calcite peaks were missing

from the l-XRD patterns (Fig. 9), because of the coarse-

grained nature of the crystals. X-rays diffracted by coarse

crystals do not uniformly fill the Debye rings corresponding

to each lattice plane; therefore, diffracted rays corresponding

to the some lattice planes do not fall on the detector. Simi-

larly, the relative intensities of the detected X-ray maxima

for coarse-grained samples are not always identical to the

reference patterns that are collected from fine-grained, iso-

tropically oriented crystals (or simulated from single-crystal

patterns to match such a powder). Because the chemical

composition of the samples is well constrained, the diffracto-

grams can be interpreted in terms of which CaCO3 poly-

Fig. 1 Observed CaCO3(s) mineral morphologies. SEM revealed a variety of crystal aggregate morphologies for microbially precipitated CaCO3(s). Several of

these were euhedral to subhedral crystal forms, and SEM images were used to generate categories to which precipitates were assigned based on the mor-

phology of the euhedral/subhedral crystal aggregates. Wireframe drawings representing the ideal crystal forms for each category are shown for reference.

Some precipitates were anhedral or composed of submicroscopic crystals, and occurred in a variety of aggregation states. SEM images were also used to gen-

erate categories for these precipitates. Microbial isolates were able to precipitate more than one type of morphology. Representative SEM images for each

category are shown, and the isolates that precipitated minerals with similar morphologies are listed to the right of the image. Isolates denoted in bold indicate

precipitated mineral in the shown SEM image.





morph(s) are present despite these imperfections of the data

by simple peak position matching.

DISCUSSION

Approximately 300 polar microbes isolated from polar soil

and endolithic communities were screened, and twenty iso-

lates that precipitated the largest volumes of macroscopic

CaCO3(s) were selected. These twenty isolates were used to

produce biogenic CaCO3(s) for the systematic study of

mineralogical similarities and differences of CaCO3(s) pre-

cipitated by the growth of environmental heterotrophs.

Arthrobacter and Rhodococcus genera were identified at a

disproportionately high rate when compared to their

occurrence our strain library, but these genera are known

to precipitate calcium carbonates at a high rate (Rusznyak

et al., 2012), explaining why they were selected during this

screen. Ralstonia and Agromyces, also identified in this

screen, are also known to precipitate calcium carbonates

(Groth et al., 2001; Braissant et al., 2003). Other genera

such as Bacillus (Hammes et al., 2003; Dhami, 2013),Brachybacterium (Groth et al., 2001), and Planococcus (N.

C. S. Mykytczuk, J. R. Lawrence, C. R. Omelon, G.

Southam & L. G. Whyte, unpublished data), known to

precipitate CaCO3(s), were also in our culture library, but

were not identified in the screen. Several factors could have

accounted for this: species/strain-specific differences

(Hammes et al., 2003), the requirement of urea (Dhami,

2013), or failure of organisms to precipitate CaCO3(s)

under the narrow growth conditions and time scale used in

the screen. Prior to this study, R. mucilaginosa has not

been shown to precipitate CaCO3(s), although other fungal

species have and there is a growing appreciation for the

role that fungi play in the biogeochemical carbon cycle

(Burford et al., 2006; Gadd, 2013).

Heterotrophic bacterial growth passively promotes bio-

logically induced CaCO3(s) precipitation through several

mechanisms, and specific physiological attributes of individ-

A B

C

D

E

Fig. 2 Pitting of CaCO3 crystals by bacterial

cells. Several SEM images of crystals, (A)

Arthrobacter sp. oc825 and (B) Rhodococcus

sp. CY5 for example, showed a pitting

pattern. Pits are consistent with the size and

shape of bacterial cells. High-resolution SEM

imaging of Arthrobacter sp. oc825 samples

showed bacterial cells were sometimes present

within crystal pits (C and D). Although

bacterial cell casts are clearly preserved in the

crystal structure, it is unclear whether the

bacterial cell is dissolving the crystal at the

cell–crystal interface and forming a pit, or

whether the crystal is growing around the cell.

(E) A high-resolution image of Arthrobacter

sp. ofe 7.23 simultaneously shows bacterial

cell-sized pits, crystal-encrusted cells, and

broken cell encrustations, which have each

been indicated by arrows.



ual microbes affect mineral formation (Hammes et al.,

2003). However, microbial growth can create different

microenvironments within the culture media that likely

result in different mineral morphologies in the same cul-

ture. Differences in the visual appearance of the CaCO3

crystals are described in terms of morphology knowing

that crystal habit, crystal form, and crystal texture may all

contribute to these variations. Several isolates in this study

A

D E F

B C

Fig. 3 Mesocrystal formation by aggregation of nanocrystal-encrusted bacterial cells. In several samples that included bacterial cells encrusted with

nanocrystals, (A) Arthrobacter sp. ofe 4.2, (B) Arthrobacter sp. ofe 7.23, (C) Arthrobacter sp. ofe 1.1, and (D) Arthrobacter sp. CY7, encrusted cells

agglomerated to form large mesocrystals. Mesocrystals are shown for under lower magnification for (E) Arthrobacter sp. ofe 7.23 and (F) Arthrobacter sp.

CY7. Only members of the Arthrobacter genus were observed to become entirely encrusted by nanocrystals, although cell encrustation was not observed for

all Arthrobacter isolates.

Fig. 4 Crystal subunits. Crystal subunits are offset in several of the biologi-

cally precipitated minerals characterized here. The subunit offsets cause

‘pseudofaces’ to be present mesocrystal. In this example, Rhodococcus sp.

(JG12) forms small rhombohedral subunits which aggregate into a roughly

scalenohedral aggregate.

Fig. 5 Patterning of biological precipitates. Similar irregular patterns were

observed on three of the biological precipitates formed in this study: Ar-

throbacter sp. ofe 3.1, Arthrobacter sp. ofe 8.9, and Arthrobacter sp. eur3

7.19. A high-resolution image of one such pattern on Arthrobacter sp. ofe

3.1 is shown here. It is ambiguous as to whether these patterns were

formed by biologically induced dissolution, chemically induced dissolution

during one of the washing steps, or a break pattern where large mesocrys-

tals broke during processing.



were responsible for precipitating multiple mineral mor-

phologies, although individual isolates tended to produce

only euhedral or anhedral minerals (Fig. 1). Arthrobacter

sp. JG9 was one of three isolates that contradicted this

rule (Arthrobacter sp. cy7, Rhodococcus sp. JG12), and

formed tetragonal disphenoid mesocrystals, irregular mes-

ocrystals, individual spherulites, and globular mineral mor-

phologies. Myxococcus xanthus has also been shown to

produce euhedral and anhedral precipitates, including

spherulites and globular mineral morphologies. Previous

studies have shown that M. xanthus precipitates a combi-

nation of calcite rhombohedra-containing intact imprints

of bacterial cells and aggregates of vaterite spherulites

(Gonzalez-Munoz et al., 2010) similar to our observa-

tions of the chemistry and morphology observed for

Arthrobacter sp. JG9. This may indicate that these two

physiologically different isolates, Arthrobacter sp. JG9

(Gram-positive) and M. xanthus (Gram-negative), share a

common CaCO3(s) precipitation mechanism or combina-

tion of mechanisms.

The use of B4 media in the precipitation experiments

introduced certain biases into mineral formation, as avail-

ability of nutrients may influence which CaCO3(s) mineral

is precipitated (Rusznyak et al., 2012). Previous work has

reported that the formation of vaterite is not favored dur-

ing microbial CaCO3(s) during nutrient-limited in situ

growth, although organisms from the same environment

may precipitate vaterite during nutrient-rich in vitro

growth (Rusznyak et al., 2012). Other studies have

avoided this bias using a less nutrient-rich media consisting

of soil extract and agar (P�arraga et al., 1998; Braissant

et al., 2004). However, each of the isolates in this investi-

gation was grown in identical culture conditions, and vate-

rite was only identified in the precipitates generated by one

isolate. This strongly indicates that phylotype-specific attri-

butes contribute to polymorph selection.

There were discrepancies between l-XRD and lattice-

fringe d value measurements in determining which CaCO3

(s) polymorph was formed by some cultures. In these cases,

l-XRD indicated that calcite was the most common min-

A B

C

D F

E Fig. 6 Surface features of spherulite and

spherulite-like formations. Arthrobacter sp.

JG9 formed both irregular and spherulite-like

CaCO3(s) precipitates. Spherulite-like

formations tended to aggregate into complex

structures (A). High-resolution SEM imaging

of these spherulite-like aggregates revealed

void spaces at the surface, consistent with the

size and shape of Arthrobacter cells (B). An

organic matrix, probably exopolysaccharide

(EPS), was visible on spherulite-like features,

which may have contributed to the

stabilization of the trace vaterite detected in

these samples (C). This organic material is also

visible on the surface of the spherulite-like

feature, precipitated by Ralstonia sp. ofe 1.3,

as well (D). Ralstonia consistently formed

individual spherulite-like precipitates with

organized crystals extending from spherulite

periphery. Bacterial cells are also present –

despite several rounds of washing (E). High-

resolution imaging of the surface of the

spherulite feature shows a texture that is

consistent with a crystallographically

controlled unit (F).



eral phase, while the TEM lattice fringes were consistent

with aragonite (Table 2). This apparent discrepancy is due

to the volume of material probed by the two different

measurements. The l-XRD has a nominal spot size of

300 lm, and diffracted rays are detected from at least

several micrometers below the ‘surface’ of each sample,

corresponding to an excitation volume of order 10�12 m3,

whereas TEM lattice-fringe measurement is produced by

electron diffraction within an excitation volume corre-

sponding to the cross section through a thin (several ~10 s

of angstroms) flake of mineral with an area of a few ang-

stroms at most (an excitation volume of ~10�18 m3). The

nanoscopic crystals imaged by TEM may be more likely to

be aragonite as a sequential transformation process, which

is governed by the Ostwald-Lussac law of stages, drives

carbonate polymorphism selection through a succession of

polymorphs, where polymorphs with the highest solubility

product (SP) (vaterite: �7.60 logKsp, aragonite:

�8.22 logKsp) are formed first and followed by polymor-

phs (calcite: �8.42 logKsp) in order of decreasing solubility

(Mann, 2001). The diffraction patterns of biogenic calcite

indicate that it does not differ in its bulk long-range lattice

properties from abiotic calcite, although this does not

exclude the possibility of short range or microscopic/nano-

scopic differences. Therefore, based on these experiments,

long-range crystallographic order effects alone cannot

unambiguously identify these particular CaCO3 precipitates

as biogenic.

Vaterite is more soluble than either calcite or aragonite

and commonly undergoes rapid polymorphic transitions to

calcite (or less commonly aragonite) outside of its stability

field, was detected in CaCO3(s) precipitated by Athrobacter

sp. JG9, and is known to be precipitated by the microbial

growth activity of M. xanthus (Rodriguez-Navarro et al.,

A B

C D

E F

Fig. 7 Cell encrustation by calcium carbonate

nanocrystals. Cells entirely encrusted in

calcium carbonate nanocrystals were

observed. SEM images of the hemi-spherulite

mesocrystals formed by nanocrystal-encrusted

Arthrobacter sp. ofe 10.25 cells show that

cell-sized voids are present on the flat side,

while crystal-encrusted cells continue to be

added to the spherulite-like side (A). SEM also

shows crystal-encrusted cells and cell voids are

present on the imaged mesocrystal (B). TEM

image shows Arthrobacter sp. ofe 10.25 cells

entirely encrusted by a network of

nanocrystals (C). Lattice-fringe images of the

area marked with an arrow in (C) show the

network of aragonite nanocrystals (D). Crystal

aggregate from the same TEM separate that

occurred separately from the encrusted cellular

surfaces (E). Lattice-fringe images of the area

marked with a black arrow in (E) reveal that

this crystal aggregate is also composed of

aragonite nanocrystals (F).



2007; Gonzalez-Munoz et al., 2010) and Xanthobacter

autotrophicus (Braissant et al., 2003). Biogenic vaterite pre-

cipitation can be driven by the presence of EPS with a high

xanthan content and is further induced by the presence of

acidic amino acids, namely aspartic and glutamic acid

(Braissant et al., 2003). Stabilization of biogenic vaterite is

likely due to adsorption of macromolecules such as in EPS

(Mann, 2001), which occurs at the bacterial cell surface and

in the extracellular space. EPS was observed as a coating

on the surface crystals formed by Athrobacter sp. JG9

(Fig. 6C), and this might explain for why vaterite formed

in this sample was stable enough to be detected by l-XRD.

The calcite crystal structure readily accommodates Mg2+

into its lattice and can have magnesium concentrations of

up to 30 mol %. In contrast, very little Mg2+ enters the

aragonite lattice (Mann, 2001). A trace amount of magne-

sium was observed in CaCO3(s) precipitates formed by

Arthrobacter sp. ofe 3.1, and trace silicon, phosphorus, and

iron were more common (Fig. 8F). These minor and trace

elements are most likely associated with adsorbed,

included, or otherwise associated materials (e.g., nanophase

inclusions, adsorbed materials). The incorporation of dif-

ferent trace element signatures in biogenic and abiogenic

CaCO3 polymorphs may provide additional constraints on

the conditions of precipitation of those phases, and it is a

worthy subject for future investigation.

Biogenic minerals are often characterized by having elab-

orate architecture where crystal nucleation and growth has

A B

C

E

G

F

D

Fig. 8 Representative (Arthrobacter sp. JG9)

sample demonstrating TEM, lattice-fringe

d-spacing measurements, and EDX.

Arthrobacter sp. JG9 formed large

mesocrystals (A). The outer rim of the

mesocrystals was composed of nanocrystals

(B). Lattice-fringe measurements indicated

that this outer rim area is composed of a

network of aragonite nanocrystals (C and D).

The lattice-fringe images were acquired from

the areas marked with (C) and (D) in image

(B). Black arrow indicates the area within the

network of nanocrystals from which an EDX

point analysis was acquired (E). EDX spectra

of the area marked with the black arrow in (E)

and (F). The presence of iron likely results in

the red coloration observed for this crystal

during light microscopy (G).



been affected by association with organic material, while

abiogenic minerals tend to feature classical shapes with

angular faces that reflect the atomic order of the underly-

ing crystal face (Kellermeier et al., 2012a). However, dur-

ing abiogenic CaCO3(s) precipitation, the presence of silica

has the effect of creating CaCO3(s) precipitates that resem-

ble traditionally biogenic crystal morphologies, which have

been deemed ‘silica biomorphs’ (Kellermeier et al.,

2012a). Silica biomorphs with complex morphology that

resembles each of the mesocrystal morphologies observed

in this study (Fig. 1) have been observed, including nano-

spherulites (Prieto et al., 1981; Bella & Garcia-Ruiz, 1986;

Kellermeier et al., 2010, 2012a,c). However, silica bio-

morph spherulites (generally < 1 lm) (Kellermeier et al.,

2012a,c) are much smaller than the spherulites (~ 20 lm)

and nanocrystal-entombed bacterial cells observed in the

current study. In addition, during the formation of silica

biomorphs, silica tends to coat the agglomerating CaCO3

(s) particles in a silica shell (Kellermeier et al., 2012b), such

that prolonged exposure to an electron beam (Kellermeier

et al., 2012b) or leaching in a dilute acid solution (Keller-

meier et al., 2010) will dissolve the CaCO3(s) core and

leave a ‘ghost’ silica shell. The existence of silica biomorphs

draws attention to the fact that structural features indica-

tive of biogenic origin would only be a preliminary step in

identifying a true biomineral, and additional experimenta-

tion would be required to unequivocally conclude the min-

eral was biogenic in origin.

Bacterial cell encrustation by nanocrystals was common

for Arthrobacter isolates in this investigation (Figs 2, 3 and

7). Precipitation of CaCO3(s) by cyanobacteria can result in

filamentous microfossils being preserved as stromatolites

and thrombolites (Arp, 2001). However, there appears to

be some taxonomic control over cyanobacteria encrusta-

tion: Synechoccus sp. actively sheds mineral precipitates dur-

ing cell division (Thompson & Ferris, 1990), Dichothrix

sp. produces encrusted filaments (Planavsky et al., 2009),

and calcified cyanobacteria filaments have been observed in

situ directly adjacent to well-laminated stromatolites (Plan-

Table 2 Identification of CaCO3(s) polymorphs by lXRD and direct lattice-

fringe measurements

Isolate

CaCO3(s) polymorphs identified in

biological precipitates

lXRD

Direct lattice-fringe

measurements

Agromyces sp. ofe 5.5 Calcite

Arthrobacter sp. JG9 Calcite,

Vaterite

Aragonite

Arthrobacter sp. oc825 Calcite

Arthrobacter sp. ofe 4.2 Calcite

Arthrobacter sp. ofe 10.25 Calcite Aragonite

Arthrobacter sp. ofe 7.23 Calcite,

Aragonite

Aragonite





Arthrobacter sp. eur3 7.19 Calcite Aragonite

Arthrobacter sp. CY7 Calcite

Ralstonia sp. ofe 1.3 Calcite Aragonite

Rhodococcus sp. JG12 Calcite

Rhodococcus sp. MD.2 Calcite

Rhodococcus sp. ofe 9.29 Calcite Calcite

Rhodococcus sp. CY5 Calcite

Rhodococcus sp. CY6 Calcite

Rhodococcus sp. eur1 9.01.3 Calcite

Rhodotorula mucilaginosa

JG1 (yeast)

Calcite Aragonite

A

B

C

Fig. 9 Three representative samples lXRD patterns. Three integrated back-

ground-subtracted diffractograms are displayed on the left figure panel. On

the right panel, the corresponding lXRD 2-dimensional diffraction patterns

and photomicrographs showing the approximate footprint of the lXRD

beam shown as a black circle are shown. Arthrobacter sp. ofe 10.10 con-

sisted of fine-grained calcite (A). Precipitates formed by Arthrobacter sp.

ofe 7.23 contained a mixture of calcite and aragonite (B). Arthrobacter sp.

JG9 formed precipitate that is dominated by calcite, but trace levels of

vaterite also appear (C). Vaterite is a relatively rare polymorph of CaCO3

and occurs under restricted physicochemical conditions.



avsky et al., 2009). SRB also release globules from their

cellular surface into the culture medium (Aloisi et al.,

2006) as a mechanism to avoid cell encrustation. Environ-

mental parameters may also play a role in inducing or

repressing cell encrustation (Arp, 2001). As encrustation

was not observed for Rhodococcus sp. isolates, and the cells

were grown in the same conditions as the Arthrobacter sp.

cells, it is possible that Rhodococcus has an avoidance mech-

anism absent in Arthrobacter sp., which would be an inter-

esting area for future investigation. Extensive cell

encrustation of Arthrobacter has not been previously

documented.

This study provides the first systematic study of the vari-

ety of mineralogies and morphologies of CaCO3(s) that can

be precipitated by different microbial phylotypes isolated

from a unique environment. While all bioliths produced in

this work tended toward having elaborate mesocrystal

architecture, phylotype-specific precipitate morphologies

were also observed. Future work should be directed toward

elucidating the underlying molecular etiology behind these

complex mineralogical features.

ACKNOWLEDGMENTS

The University of Winnipge’s HOSERLab was established

with funding from the Canada Foundation for Innovation,

the Manitoba Research Innovations Fund, and the Cana-

dian Space Agency, whose support is gratefully acknowl-

edged. This study was supported by research grants from

NSERC, the Canadian Space Agency, and the University

of Winnipeg. JR, HS, DS, and MRMI gratefully acknowl-

edge funding from the NSERC CREATE Canadian Astro-

biology Training Program. MRMI also acknowledges the

Mineralogical Association of Canada.

REFERENCES

Achal V, Pan X (2010) Characterization of urease and carbonic

anhydrase producing bacteria and their role in calciteprecipitation. Current Microbiology 62, 894–902.

Aloisi G, Gloter A, Kr€uger M, Wallmann K, Guyot F, Zuddas P

(2006) Nucleation of calcium carbonate on bacterial

nanoglobules. Geology 34, 1017.Arp G (2001) Photosynthesis-induced biofilm calcification and

calcium concentrations in phanerozoic oceans. Science 292,1701–1704.

Bella SD, Garcia-Ruiz JM (1986) Textures in induced

morphology crystal aggregates of CaCO3: sheaf of wheat

morphologies. Journal of Crystal Growth 79, 236–240.Ben Chekroun K, Rodriguez-Navarro C, Gonzalez-Munoz MT,Arias JM, Cultrone G, Rodriguez-Gallego M (2004) Precipitation

and growth morphology of calcium carbonate induced by

Myxococcus xanthus: implications for recognition of bacterial

carbonates. Journal of Sedimentary Research 74, 868–876.Beveridge TJ, Murray RG (1976) Uptake and retention of metals

by cell walls of Bacillus subtilis. Journal of Bacteriology 127,1502–1518.

Bontognali TRR, Vasconcelos C, Warthmann RJ, Dupraz C,

Bernasconi SM, McKenzie JA (2008) Microbes produce

nanobacteria-like structures, avoiding cell entombment. Geology36, 663.

Boquet E, Boronat A, Ramos-Cormenzana A (1973) Production

of calcite (calcium carbonate) crystals by soil bacteria is a general

phenomenon. Nature 246, 527–529.Braissant O, Cailleau G, Dupraz C, Verrecchia EP (2003)Bacterially induced mineralization of calcium carbonate in

terrestrial environments: the role of exopolysaccharides

and amino acids. Journal of Sedimentary Research 73,485–490.

Braissant O, Cailleau G, Aragno M, Verrecchia EP (2004)

Biologically induced mineralization in the tree Milicia excelsa(Moraceae): its causes and consequences to the environment.Geobiology 2, 59–66.

Braissant O, Decho AW, Dupraz C, Glunk C, Przekop KM,

Visscher PT (2007) Exopolymeric substances of sulfate-reducing

bacteria: interactions with calcium at alkaline pH andimplication for formation of carbonate minerals. Geobiology 5,401–411.

Braissant O, Decho AW, Przekop KM, Gallagher KL, Glunk C,

Dupraz C, Visscher PT (2009) Characteristics and turnover ofexopolymeric substances in a hypersaline microbial mat. FEMSMicrobiology Ecology 67, 293–307.

Bundeleva IA, Shirokova LS, B�en�ezeth P, Pokrovsky OS,Kompantseva EI, Balor S (2011) Zeta potential of anoxygenic

phototrophic bacteria and Ca adsorption at the cell surface:

possible implications for cell protection from CaCO3

precipitation in alkaline solutions. Journal of Colloid andInterface Science 360, 100–109.

Bundeleva IA, Shirokova LS, B�en�ezeth P, Pokrovsky OS,

Kompantseva EI, Balor S (2012) Calcium carbonate

precipitation by anoxygenic phototrophic bacteria. ChemicalGeology 291, 116–131.

Burbank MB, Weaver TJ, Williams BC, Crawford RL (2012)

Urease activity of ureolytic bacteria isolated from six soils inwhich calcite was precipitated by indigenous bacteria.

Geomicrobiology Journal 29, 389–395.Burford EP, Hillier S, Gadd GM (2006) Biomineralization of

fungal hyphae with calcite (CaCO3) and calcium oxalate mono-and dihydrate in carboniferous limestone microcosms.

Geomicrobiology Journal 23, 599–611.Castanier S, Le M�etayer-Levrel G, Perthuisot JP (1999)

Ca-carbonates precipitation and limestone genesis – themicrobiogeologist point of view. Sedimentary Geology 126,9–23.

Contos JM, James B, Heywood A (2001) Morphoanalysis ofbacterially precipitated subaqueous calcium carbonate from

Weebubbie Cave, Australia. Geomicrobiology Journal 18, 331–343.

Couradeau E, Benzerara K, Gerard E, Moreira D, Bernard S,Brown GE, Lopez-Garcia P (2012) An early-branching

microbialite cyanobacterium forms intracellular carbonates.

Science 336, 459–462.Dhami NK (2013) Biomineralization of calcium carbonatepolymorphs by the bacterial strains isolated from calcareous

sites. Journal of Microbiology and Biotechnology 23, 707–714.Dittrich M, Sibler S (2010) Calcium Carbonate Precipitation byCyanobacterial Polysaccharides, Geological Society, London,Special Publications 336, 51–63.

Douglas S, Beveridge TJ (1998) Mineral formation by bacteria in

natural microbial communities. FEMS Microbiology Ecology 26,79–88.



Dupraz C, Visscher PT (2005) Microbial lithification in marine

stromatolites and hypersaline mats. Trends in Microbiology 13,429–438.

Dupraz C, Reid RP, Braissant O, Decho AW, Norman RS,Visscher PT (2009) Earth-science reviews. Earth Science Reviews96, 141–162.

Ferrera-Rodr�ıguez O, Greer CW, Juck D, Consaul LL, Mart�ınez-

Romero E, Whyte LG (2012) Hydrocarbon-degrading potentialof microbial communities from Arctic plants. Journal of AppliedMicrobiology 114, 71–83.

Ferris FG, Phoenix V, Fujita Y, Smith RW (2004) Kinetics ofcalcite precipitation induced by ureolytic bacteria at 10 to 20°Cin artificial groundwater. Geochimica et Cosmochimica Acta 68,1701–1710.

Gadd GM (2013) Geomycology: fungi as agents ofbiogeochemical change. Biology & Environment: Proceedings ofthe Royal Irish Academy 113B, 1–15.

Gallagher KL, Kading TJ, Braissant O, Dupraz C, Visscher PT

(2012) Inside the alkalinity engine: the role of electron donorsin the organomineralization potential of sulfate-reducing

bacteria. Geobiology 10, 518–530.Gonzalez-Munoz MT, Rodriguez-Navarro C, Martinez-Ruiz F,

Arias JM, Merroun ML, Rodriguez-Gallego M (2010) Bacterialbiomineralization: new insights from Myxococcus-inducedmineral precipitation. Geological Society, London, SpecialPublications 336, 31–50.

Groth P, Schumann L, Laiz S, San I (2001) Geomicrobiological

study of the Grotta dei Cervi, Porto Badisco, Italy.

Geomicrobiology Journal 18, 241–258.Hammes F, Boon N, de Villiers J, Verstraete W, Siciliano SD(2003) Strain-specific ureolytic microbial calcium carbonate

precipitation. Applied and Environment Microbiology 69, 4901–4909.

Kawaguchi T, Decho AW (2002) A laboratory investigation ofcyanobacterial extracellular polymeric secretions (EPS) in

influencing CaCO 3 polymorphism. Journal of Crystal Growth240, 230–235.

Kellermeier M, Melero-Garc�ıa E, Glaab F, Klein R, Drechsler M,

Rachel R, Garc�ıa Ruiz JM, Kunz W (2010) Stabilization of

amorphous calcium carbonate in inorganic silica-rich

environments. Journal of the American Chemical Society 132,17859–17866.

Kellermeier M, C€olfen H, Garc�ıa Ruiz JM (2012a) Silica

biomorphs: complex biomimetic hybrid materials from “Sand

and Chalk”. European Journal of Inorganic Chemistry 2012,5123–5144.

Kellermeier M, Gebauer D, Melero-Garc�ıa E, Drechsler M,

Talmon Y, Kienle L, C€olfen H, Garc�ıa Ruiz JM, Kunz W(2012b) Colloidal stabilization of calcium carbonate

prenucleation clusters with silica. Advanced Functional Materials22, 4301–4311.

Kellermeier M, Melero-Garcia E, Kunz W (2012c) The ability ofsilica to induce biomimetic crystallization of calcium

carbonate. In Advances in Chemical Physics, Volume 151:Kinetics and Thermodynamics of Multistep Nucleation and Self-Assembly in Nanoscale Materials, 1st edn (eds Gregoire N,Dominique M). John Wiley & Sons, Inc., Hoboken, NJ,

USA, pp. 277–307.Kranz S, Wolf-Gladrow D, Nehrke G (2010) Calcium carbonate

precipitation induced by the growth of the marinecyanobacteria Trichodesmium. Limnology and Oceanography 55,2563–2569.

Li W, Liu LP, Zhou PP, Cao L, Yu LJ (2011) Calciteprecipitation induced by bacteria and bacterially produced

carbonic anhydrase. Current Science (Bangalore) 100,502–508.

Lian B, Hu Q, Chen J, Ji J, Teng HH (2006) Carbonate

biomineralization induced by soil bacterium Bacillusmegaterium. Geochimica et Cosmochimica Acta 70,14–14.

Mann S (2001) Biomineralization: Principles and Concepts inBioinorganic Materials Chemistry, Oxford University Press,Oxford.

Martinez RE, Gard�es E, Pokrovsky OS, Schott J, Oelkers EH

(2010) Do photosynthetic bacteria have a protective mechanismagainst carbonate precipitation at their surfaces? Geochimica etCosmochimica Acta 74, 1329–1337.

Millo C, Ader M, Dupraz S, Guyot F, Thaler C, Foy E, M�enez B

(2012) Carbon isotope fractionation during calcium carbonateprecipitation induced by urease-catalysed hydrolysis of urea.

Chemical Geology 330–331, 39–50.Mitchell AC, Ferris FG (2006) The influence of Bacilluspasteuriion the nucleation and growth of calcium carbonate.Geomicrobiology Journal 23, 213–226.

Okwadha G, Li J (2010) Optimum conditions for microbial

carbonate precipitation. Chemosphere 81, 1143–1148.Omelon CR, Pollard WH, Marion GM (2001) Seasonal formationof ikaite (caco3 & #xB7; 6h2o) in saline spring discharge at

expedition fiord, Canadian high arctic: assessing conditional

constraints for natural crystal growth. Geochimica etCosmochimica Acta 65, 1429–1437.

P�arraga J, Rivadeneyra MA, Delgado R (1998) Study of

biomineral formation by bacteria from soil solution equilibria.

Reactive and Functional Polymers 36, 265–271.Planavsky N, Reid RP, Lyons TW, Myshrall KL, Visscher PT

(2009) Formation and diagenesis of modern marine calcified

cyanobacteria. Geobiology 7, 566–576.Politi Y, Metzler RA, Abrecht M, Gilbert B, Wilt FH, Sagi I,Addadi L, Weiner S, Pupa G (2008) Transformation mechanism

of amorphous calcium carbonate into calcite in the sea urchin

larval spicule. PNAS 105(45), 17362–17366.Prieto M, Garcia-Ruiz JM, Amor�os JL (1981) Growth of calcite

crystals with non-singular faces. Journal of Crystal Growth 52,864–867.

Rodriguez-Navarro C, Rodriguez-Gallego M, Ben Chekroun K,Gonzalez-Munoz MT (2003) Conservation of ornamental

stone by Myxococcus xanthus-induced carbonate

biomineralization. Applied and Environment Microbiology 69,2182–2193.

Rodriguez-Navarro C, Jimenez-Lopez C, Rodriguez-Navarro A,

Gonzalez-Munoz MT, Rodriguez-Gallego M (2007) Bacterially

mediated mineralization of vaterite. Geochimica et CosmochimicaActa 71, 17–17.

Rusznyak A, Akob DM, Nietzsche S, Eusterhues K,

Totsche KU, Neu TR, Frosch T, Popp J, Keiner R,

Geletneky J, Katzschmann L, Schulze ED, Kusel K(2012) Calcite biomineralization by Bacterial isolates from

the recently discovered pristine karstic Herrenberg

Cave. Applied and Environment Microbiology 78,1157–1167.

S�anchez-Rom�an M, Romanek CS, Fern�andez-Remolar DC,

S�anchez-Navas A, McKenzie JA, Pibernat RA, Vasconcelos C

(2011) Aerobic biomineralization of Mg-rich carbonates:

implications for natural environments. Chemical Geology 281,143–150.

Smith PH, Tamppari LK, Arvidson RE, Bass D, Blaney D,

Boynton WV, Carswell A, Catling DC, Clark BC, Duck T,Dejong E, Fisher D, Goetz W, Gunnlaugsson HP, Hecht MH,



Hipkin V, Hoffman J, Hviid SF, Keller HU, Kounaves SP,

Lange CF, Lemmon MT, Madsen MB, Markiewicz WJ,

Marshall J, McKay CP, Mellon MT, Ming DW, Morris RV, Pike

WT, Renno N, Staufer U, Stoker C, Taylor P, Whiteway JA,Zent AP (2009) H2O at the Phoenix landing site. Science 325,58–61.

Steven B, Briggs G, McKay CP, Pollard WH, Greer CW, Whyte

LG (2007) Characterization of the microbial diversity in apermafrost sample from the Canadian high Arctic using culture-

dependent and culture-independent methods. FEMSMicrobiology Ecology 59, 513–523.

Thompson JB, Ferris FG (1990) Cyanobacterial precipitation of

gypsum, calcite, and magnesite from natural alkaline lake water.

Geology 18, 995.Van Lith Y, Warthmann R, Vasconcelos C, McKenzie JA (2003)Microbial fossilization in carbonate sediments: a result of the

bacterial surface involvement in dolomite precipitation.

Sedimentology 50, 237–245.Wright DT, Wacey D (2005) Precipitation of dolomite usingsulphate-reducing bacteria from the Coorong Region, South

Australia: significance and implications. Sedimentology 52,987–1008.



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