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Bacterial Calcium Carbonate Precipitation in Cave Environments: A

Function of Calcium Homeostasis
Eric D. Banksa; Nicholas M. Taylora; Jason Gulleya; Brad R. Lubbersa; Juan G. Giarrizoa; Heather A.
Bullenb; Tori M. Hoehlerc; Hazel A. Bartona
a
Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky b
Department of Chemistry, Northern Kentucky University, Highland Heights, Kentucky c NASA Ames
Research Center, Moffett Field, California

Online publication date: 08 June 2010

To cite this Article Banks, Eric D. , Taylor, Nicholas M. , Gulley, Jason , Lubbers, Brad R. , Giarrizo, Juan G. , Bullen,
Heather A. , Hoehler, Tori M. and Barton, Hazel A.(2010) 'Bacterial Calcium Carbonate Precipitation in Cave
Environments: A Function of Calcium Homeostasis', Geomicrobiology Journal, 27: 5, 444 — 454
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 Geomicrobiology Journal, 27:444–454, 2010
Copyright © Taylor & Francis Group, LLC
ISSN: 0149-0451 print / 1521-0529 online
DOI: 10.1080/01490450903485136

Bacterial Calcium Carbonate Precipitation in Cave

Environments: A Function of Calcium Homeostasis
Eric D. Banks,1 Nicholas M. Taylor,1 Jason Gulley,1 Brad R. Lubbers,1
Juan G. Giarrizo,1 Heather A. Bullen,2 Tori M. Hoehler,3 and Hazel A. Barton1
1
Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky
2
Department of Chemistry, Northern Kentucky University, Highland Heights, Kentucky
3
NASA Ames Research Center, Moffett Field, California

Keywords calcite, calcium caves, coralloids, homeostasis,

To determine if microbial species play an active role in the de- speleothems
velopment of calcium carbonate (CaCO3 ) deposits (speleothems) in
cave environments, we isolated 51 culturable bacteria from a coral-
loid speleothem and tested their ability to dissolve and precipitate
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CaCO3 . The majority of these isolates could precipitate CaCO3 INTRODUCTION

minerals; scanning electron microscopy and X-ray diffractrometry
demonstrated that aragonite, calcite and vaterite were produced in
this process. Due to the inability of dead cells to precipitate these
minerals, this suggested that calcification requires metabolic ac-
tivity. Given growth of these species on calcium acetate, but the
toxicity of Ca2+ ions to bacteria, we created a loss-of-function gene
knock-out in the Ca2+ ion efflux protein ChaA. The loss of this pro-
tein inhibited growth on media containing calcium, suggesting that
the need to remove Ca2+ ions from the cell may drive calcification.
With no carbonate in the media used in the calcification studies,
we used stable isotope probing with C13 O2 to determine whether
atmospheric CO2 could be the source of these ions. The resultant
crystals were significantly enriched in this heavy isotope, suggest-
ing that extracellular CO2 does indeed contribute to the mineral
structure. The physiological adaptation of removing toxic Ca2+ ions
by calcification, while useful in numerous environments, would be
particularly beneficial to bacteria in Ca2+ -rich cave environments.
Such activity may also create the initial crystal nucleation sites that
contribute to the formation of secondary CaCO3 deposits within
caves.
Received 25 May 2009; accepted 10 November 2009.
The authors would like to thank the landowners and cavers in the
collection of the coralloid samples and strains, Dr. Dave Bunnell for
the image used in Figure 1, Dr. John Roth and Dr. Eric Kofoid for
the Salmonella strains and Mr. Michael D. Kubo for his assistance
with the isotopic analyses and IRMS work. EDB was supported by a
SURF Fellowship from the ASM and a SURCA Award from NKU.
HAB is supported in part by the Kentucky NSF EPSCoR Program
(NSF0814194) and an NSF MIP/CAREER grant (NSF0643462), with
infrastructure support by the NIH Kentucky INBRE program (NIH
5P20RR01648-05) and NSF Major Research Instrumentation award
(MRI-0520921).
Address correspondence to Hazel A. Barton, Department of Bio-
logical Sciences, Northern Kentucky University, SC 204D Nunn Drive,
Highland Heights, KY 41099. E-mail: bartonh@nku.edu
When secondary cave deposits (speleothems, a.k.a. cave for-
mations) were described in 1676 by John Beaumont, he classi-
fied them as a form of plant life with “. . . growth from the finest
parts of clay, being commonly white” (Beaumont 1676; Hill
and Forti 1997). The description of cave formations as a vege-
tative growth seems quaint as we now know that such deposits
form when surface water percolating through the soil acquires
dissolved CO2 , making a weak carbonic acid (H2 CO3 ) that dis-
solves limestone (calcium carbonate: CaCO3 ) rock. When this
water drips into a cave, the CO2 off-gases and the subsequent
increase in the saturation index (SI) for Ca2+ and CO2− 3 leads
to the precipitation of CaCO3 as calcite, albeit at a exceedingly
slow rate (Hill and Forti 1997; Short et al. 2005b). Over ge-
ologic timescales, this deposition leads to the development of
classic speleothems, such as stalactites and stalagmites, as well
as a myriad of other potential forms (Hill and Forti 1997). The
inorganic and physical chemistry that drives these phenomena
are well understood, particularly from the works of Dreybrodt
and Kaufmann et al. (Buhmann and Dreybrodt 1984; Dreybrodt
1999; Kaufmann 2003; Short et al. 2005a, 2005b).
It has been known since the 1970’s that CaCO3 deposition
(calcification) is a general phenomenon among the Bacteria
(Boquet et al. 1973), with a high percentage of soil bacteria
producing CaCO3 crystals when grown on media containing
calcium acetate. Despite the broad taxonomic distribution of
this phenotype, much of the work examining bacterially me-
diated CaCO3 precipitation has concentrated on cyanobacteria
in Ca2+ -rich (∼40 mm) seawater. In these environments, pho-
tosynthesis alters the chemistry of the microenvironment by
fixing CO2 (Aizawa and Miyachi 1986; Badger and Price 1994;
Banfield and Nealson 1997), leading to an increase in the lo-
−
cal pH. This favors the formation of CO2− 3 from HCO3 and
leads to calcification. Within cave environments, which lack

444
 BACTERIAL PRECIPITATION OF CALCIUM CARBONATE 445

photosynthetic activity and are found predominantly within
limestone (CaCO3 ) rock, bacterial species demonstrate a higher
incidence of the ability to precipitate calcium carbonate and an
overall increase in total CaCO3 precipitation levels (Cacchio
et al. 2004; Danielli and Edington 1983).
Although bacterial fossils are found within speleothems
(Melim et al. 2008; Melim et al. 2001), the observation of vi-
able cells with these formations tends to be anecdotal (Baskar
et al. 2006a; Cacchio et al. 2004; Danielli and Edington 1983);
no cause-and-effect for the presence of microbial species in
speleothems has been elucidated (Barton and Northup 2007).
If microbial species do play a role in the development of
speleothems, it is difficult to understand the evolutionary ad-
vantage of maintaining such a phenotype; when microorganisms
become covered in an impermeable CaCO3 layer, this prevents
solute exchange with the environment and ultimately leads to
death.
Among speleothems, coralloids are one of the most com-
monly observed formations (Figure 1A) (Hill and Forti 1997).
Various mechanisms for their formation have been described,
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development (Banfield and Nealson 1997; Hill and Forti 1997).
Through our microbiology studies in caves, we commonly iden-
tify such ‘surface irregularities’ as microbial colonies; SEM
examination of these colonies often reveals CaCO3 crystal de-
position on the surface of the bacterial cells (Barton and Northup
2007).
These calcified colonies are often within close proximity to
small CaCO3 mineral protrusions (∼1–2 mm), which enlarge
up to the size of coralloids (Figure 1A). The close association
between calcified microbial colonies and coralloids suggested
that microbial cells were either being caught up in the coralloid
formation processes (Baskar et al. 2006b), or were somehow
critical in the development of these speleothems. The goal of
this study was therefore to test whether calcification by bacterial
species may be an important phenotype for survival in high
calcium cave environments, and whether such activity may lead
to speleothem development.
MATERIALS AND METHODS

from splashing of CaCO3 saturated water to the molecular move-
ment of such water under capillary action (Hill and Forti 1997).
Among these proposed methods, the need for surface irregu-
larities on the base rock is consistent. Such irregularities create
the nucleation site for incorporation of additional crystal ele-
ments, allowing subsequent CaCO3 deposition and speleothem
Sample Site
Samples were collected from an unnamed cave in Kentucky,
USA (cave conservation practices restrict naming the cave, but
location and access information can be obtained from the au-
thors). This ∼7 mile long cave is formed in Mississippian car-
bonate rocks of the St. Genevieve and St. Louis Formation.

FIG. 1. (A) Coralloids (cave popcorn) is a common speleothem found in caves, for which no clear mechanism of formation has been described. (B) An example
thin-section through cave popcorn under polarizing light. Note the banding commonly observed in stromatolites. (C) and (D) The same thin-section image as B,
but using EDS analysis. Colors correspond the element being mapped. Image A is ∼10 cm across.
 446 E. D. BANKS ET AL.
Samples were collected in a stream passage above the cave
flood line.
Bacterial Cultivation
In order to culture bacterial isolates, swabs were wetted in
filtered (0.2 µm) cave water and developing coralloids were
swabbed. The swabs were then used to inoculate B-4 (Boquet
et al. 1973) media and B-4C media (this study) containing
B-4 media with a 1.5% top agar with 2% calcium carbonate
(CaCO3 ) light powder (all chemicals, unless otherwise stated,
were from Sigma Aldrich Chemicals, St. Louis, MO). Colonies
were picked and purified based on observation of CaCO3 pre-
cipitation (P class) and/or dissolution (D class) and purified by
passage by Tryptic Soy Agar (TSA; Becton, Dickinson, Co.,
Franklin Lakes, NJ). To compare the phenotype of non-cave de-
rived isolates, additional species were obtained from the ATCC
(Manassas, VA), including Microbacterium esteraromaticum
(ATCC 51822), Stenotrophomonas maltophilia (ATCC 17666),
Streptomyces senoensis (ATCC 29794) and Raoultella planti-
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cola (ATCC 33531) to supplement the lab strains Salmonella
entrica subsp. Typhimurium LT2 and Escherichia coli K12.
Mineral Analysis
In order to examine the mineral being precipitated, CaCO3
precipitating isolates were grown in 3 ml of liquid B-4 me-
dia. CaCO3 crystals were collected by filtration on 5.0 µm fil-
ters (Milipore, Billerica, MA), washed free of cells with filter-
sterilized distilled water (pH 8.0), air dried for thirty minutes,
sputter coated with Au/PD and analyzed using an FEI Quanta
200 environmental SEM. To confirm mineral identity, CaCO3
crystals were similarly collected for XRD analysis on a Rigaku
Ultima III powder XRD using Cu Kα-radiation (40 kV, 44 mA).
Data was collected over a 2θ range between 25–70◦ at scan rate
of 0.1◦ /min.
For isotopic probing to determine the source of the carbonate
in the precipitated minerals, 3 ml liquid B-4 media was inocu-
lated with the test strain and grown in a 2.4 liter glass chamber
(Diagnostic Systems, Sparks, MD) fitted with an air tight gasket.
C13 O2 gas was then injected to generate an atmosphere that con-
tained 0.2% atmospheric CO2 and 0.2% C13 O2 . Cultures were
maintained in this chamber for 2 weeks. Isotope ratio mass
spectrometry was carried out on a Nuclide 6-60 RMS dual-inlet
IRMS. To liberate the CO2 from the crystals, a degassed acid
solution was prepared. Briefly, 100 µl concentrated (∼10 M)
phosphoric acid was added to 5 ml ultraclean (MilliQ) water.
This acidified solution was degassed by repeated freeze/thaw
events under vacuum, followed by 2 hours under vacuum.
The acidified solution was then kept frozen on a methanol/dry
ice slurry, the sample container was removed from vacuum, the
sample crystals were placed on the side of the reaction ves-
sel and the vessel was put back under vacuum to evacuate the
headspace for an additional hour. The vessel was then sealed,
taken off of vacuum, and the ice slurry was then allowed to melt
and flow down onto the crystals, producing a ‘fizzing’ that in-
dicated the liberation of CO2 . Samples were then run on a CO2
vacuum extraction line to isolate the liberated gas and trapped
in glass ampoules for analysis against known standards.
Geology
To examine the physical structure of the coralloids, thin sec-
tions were prepared from three rock samples collected using
a hammer and chisel, dried at 100◦ C and impregnated with
Petropoxy (Burnham Petrographics, Rathdrum, ID) while hot.
The thin sections were prepared and photographed under plane
polarized light using a Nikon E400Pol polarizing microscope.
The geochemistry of the thin sections was analyzed by EDS
mapping, as described in Spear et al. (2007).
Molecular Techniques
Genomic DNA was extracted from each isolate in
order to identify precipitating bacterial strains using a
ZR Fungal/Bacterial DNA Kit (Zymo Research, Orange,
CA). The 16S ribosomal RNA gene sequence was PCR
amplified as previously described (Spear et al. 2007)
using the 8F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and
1525R (5 -AAGGAGGTGATCCAGCC-3 ) primers. Sequenc-
ing of purified PCR products using the primers 8F and
1391R (5 -GACGGGCGGTGWGTRCA-3 ), with 515F (5 -
GTGCCAGCMGCCGCGGTAA-3 ) as an internal primer was
carried out commercially by Agencourt Bioscience (Beverly,
MA). All sequences were deposited in the NCBI database under
accession numbers FJ347997 – FJ348046. To identify isolates,
gene sequences were compared to the Greengenes database
(http://greengenes.lbl.gov) and confirmed by 16S rRNA phylo-
genetic placement; DNA alignments were carried out using the
ARB Software Package (http://mpi-bremen.de/molecol/arb).
For distance calculations, the evolutionary model was GTR +
I + G (base (0.2402, 0.2366 0.3148), Nst/6, Rmat (0.7547,
1.8102, 1.0282, 0.7316, 3.1058), Rates = gamma, Shape =
0.6293, Pinvar = 0.2486), determined using MODELTEST
(Posada 2003). Distance and parsimonious phylograms were
generated in PAUP* using a heuristic search (Wilgenbusch
and Swofford 2003). The highest scoring trees were tested by
boostrap analysis using 1000 replicates. A maximum likeli-
hood phylogram was also generated using a Bayesian analysis
with the MrBayes program for 2 million generations (Ronquist
and Huelsenbeck 2003). In all cases, sequences from Aquifex
pyrophilus and Thermoplasma acidophilium were used for
outgroups.
Construction of Deletion Mutants
To examine the role that microbial physiology had in precip-
itation, deletion mutants of the genes chaA and nhaA were gen-
erated using linear transformation in S. typhimurium TT22971
containing a lambda red background, based on the protocol of
Datsenko and Wanner (2000). To confirm that the insert had
 BACTERIAL PRECIPITATION OF CALCIUM CARBONATE 447

TABLE 1
Primers used in this study
Primer Name Sequence
yadF-forward 5 -ATTGGTGGGGAATGTATTTCAGACTTAGGGCGGAAATACCACCA AACACCCCCCAAAACC-3
yadF-reverse 5 -CCCAACGCACTTATTTGGTTACAGGTCGTTAACTTCCATCACACAACCACACCACACCAC-3
yadF-FPE 5 -TTGGCTTTTCGATTCCGGACG-3
yadF-RPI 5 -ATCTGAACTGTCTCTCCGTGG-3
yarF-RPE 5 -ATTTCCCTGCTCCATTTGGC-3
chaA-forward 5 -ATGACACATGCCTGTGAGGCGGTGAAAACCCGCCATAAGCACCAAACACCCCCCAAAACC-3
chaA-Reverse 5 -TGCGGTTTTTAGCTTGTGTAGGCCGGATAAGATACTATCCACACAACCACACCACACCAC-3
chaA-FPE 5 -CCCAGTCGAGGAGGATTT-3
chaA-RPI 5 -GACCAATAATGCCTGACCG-3
chaA-RPE 5 -AATGCCGCGACCCATTTG-3

knocked out the gene being studied, PCR was carried out using Distribution of the Calcification Phenotype Among
a combination of a forward external primer (FPE) and either a Bacteria Isolated From Speleothems
reverse internal (RPI) or external (RPE) primer (Table 1). One To assess whether microbial activity played a role in the
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strain that contained either a deletion of chaA or nhaA was then
transduced by P22 into a wild-type S. typhimurium TR10K back-
ground and verified by PCR to generate the respective knock
out mutant.
RESULTS
Analysis of Coralloid Thin Sections
Eight thin sections were prepared from rock samples that con-
tained both observed microbial colonies and 56 small, nodular
coralloids (an example is shown in Figure 1B–D). Within these
thin sections, polarizing-light microscopy revealed a dark, lam-
inated layer between the base of each coralloid and the host
rock (Figure 1B), which matched the thin-section appearance
through colonies that had been visually identified on the surface
of these samples. In all cases, this dark material was coated with
layers of what appeared to be CaCO3 (by gross examination)
in a stromatolitic-like growth, quite distinct from the mineral
structure of the host rock (as shown in Figure 1B). To examine
the chemistry of this deposition, energy-dispersive spectroscopy
(EDS) mapping of the thin-sections was carried out (example in
Figures 1C and 1D).
The results demonstrate that the coralloids were calcium-
and oxygen-rich in chemistry (indicative of CaCO3 ), lacking
the trace silica, aluminum and iron signatures of clays ob-
served in the St. Genevieve Formation host rock (Figures 1B–D)
(McGrain and Dever 1967). These data suggest that the struc-
ture and chemistry of these coralloids is different than the host
rock and in-line with a depositional origin. There was also what
appeared to be an accumulation of silica, aluminum and iron-
rich material between all examined coralloids and the host rock
(Figure 1C and 1D). The chemistry of this material matches
the chemistry of clay minerals in the Ste. Genevieve Formation,
which are generally not soluble, except under extremely low pH.
CaCO3 deposition seen in coralloid development, bacteria were
isolated from coralloids in close proximity to the rock samples.
This was done by streaking swab samples onto either B-4 media
or B-4C media, which allow us to identify bacterial species that
demonstrate the ability to either precipitate or dissolve CaCO3 ,
respectively (Figure 2). Following growth at room temperature
for 2–4 weeks, isolates were identified based on unique colony
morphology and phenotype; species able to precipitate CaCO3
were considered P-class, and those able to dissolve calcite were
D-class isolates. This initial screen on B-4 and B-4C media
identified 15 P-class and 17 D-class colonies. These colonies
were then picked and streaked for pure culture on TSA media,
which often allows the separation of mixed cultures. Using this
approach we identified 19 additional (10 P-class and 9 D-class)
isolates, generating 51 unique bacterial cultures isolated from
these developing coralloids.
To ensure that the crystals produced by these isolates on
B-4 media were indeed CaCO3 mineral polymorphs, they were
examined using SEM microscopy and X-ray diffraction (XRD).
The SEM results (Figure 3) demonstrated the presence of
mineral polymorphs, including calcite, vaterite and aragonite
(Lippmann 1973). XRD analysis confirmed the presence of
these multiple mineral forms, including vaterite, which is only
transiently found in nature (Figure 4). Although examining these
crystals using SEM, it was noted that virtually all of the crys-
tals included pits and depressions that matched the structure of
the precipitating bacteria, both in terms of size and morphol-
ogy (Figures 3D and 3E). A number of these depressions also
included structures that were indicative of cell growth and di-
vision, such as the septa observed in Figure 3E. Even though
these crystals were washed free of bacterial cells, many crystals
also contained obvious cells closely associated with, and emerg-
ing from, the mineral surface (Figure 3F). The organic nature of
these cells was confirmed by EDS, which indicated the presence
 448 E. D. BANKS ET AL.

FIG. 2. Precipitation and dissolution phenotypes of cave isolates. (A) Original colony morphologies of isolates streaked on B-4 media. The calcium carbonate
precipitates demonstrating different mineral polymporphs can be seen within each colony. Colonies range in size from 2–4 mm. (B) Calcite dissolution by D17D
when grown on B-4C media. The zone of clearing of the CaCO3 around each colony can be seen, along with CaCO3 precipitation in the center of isolated colonies.
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of phosphorous, nitrogen and sulfur peaks when compared to
the calcium carbonate mineral (data not shown). Together these
data demonstrate a close association between the bacterial cells
and the formed crystals.
Once calcification by the bacterial isolates was confirmed,
PCR amplification of the 16S rRNA gene sequence was used
to determine their identity. The results (Figure 5) demonstrate
a broad distribution of isolates, representing members of the
Alpha-, Beta- and Gammaproteobacteria, the Firmicutes and
Actinobacteria. While this distribution does not represent the to-
tal bacterial population in this environment, the divisional repre-
sentation is in line with previous cultivation and non-cultivation
based studies in cave and subsurface environments (Amann et
al. 1995; Barns and Nierzwicki-Bauer 1997; Barton et al. 2004).
With the exception of GGC-D3, all 51 isolates demonstrated a
calcification phenotype on B-4 media, irrespective of whether

FIG. 3. SEM images of calcite crystals generated by GGC cultivars. Numerous calcite polymorphs are produced by microbial activity, including (A) calcite
(P9B), (B) aragonite (D15) and (C) vaterite (P11). The close associate between microbial growth and the precipitation of these minerals can also be seen, including
cell-shaped pits (D) and septa (E); indicated by arrow). Although these crystals were washed free of bacterial cells prior to SEM, the close association between the
bacteria and the crystals can be seen by the emergence from and continued adherence of cells to the crystals (F).
 BACTERIAL PRECIPITATION OF CALCIUM CARBONATE
FIG. 4. Representative XRD analysis of calcium carbonate crystals (from
isolate P11) shows presence of vaterite. The corresponding peaks are labeled.
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they were initially identified based on a phenotype of precipi-
tating or dissolving calcium carbonate (Figure 5).
Dissolution activity was not so broadly represented, likely
due to the large amount of acid production necessary to ob-
serve this phenotype; however, many of the D-class isolates
also precipitated CaCO3 crystals on top of their colonies dur-
ing dissolution (Figure 5). This phenotype was observed for all
dissolving phenotypes, except GGC-D10A, GGC-D10B1 and
GGC-P11, which generated a very large zone of clearing.
The pattern of both CaCO3 precipitation, dissolution and
a combined dissolving/precipitating phenotype appeared to be
similar for all isolates within a particular clade, with the ex-
ception of the Bacilli (Figure 5). To determine whether the
relationship between members of a clade and phenotype is suf-
ficiently robust to be predictive of phenotype, five bacterial
species isolated from non-cave environments were tested for
their ability to precipitate and/or dissolve CaCO3 ; these included
S. typhimurium, Escherichia coli, Klebsiella planticola, Mi-
crobacterium esteraromaticum, Stenotrophomonas maltophilia
and Streptomyces senoensis. The results (Figure 5) demonstrate
that the phenotype for these species does indeed match those for
the clade to which they belong, despite the fact that they were
not isolated from caves.
The Physiology of Calcium Carbonate Precipitation
The close association between clade and phenotype shown
in Figure 5 suggests that either a structural or genotypic re-
lationship is responsible for the CaCO3 phenotypes observed.
To differentiate the contribution of bacterial structure to crys-
tal formation, the precipitation capabilities of a random group
of isolates killed with paraformaldehyde was tested; the loss
of cell viability using this method was confirmed by plating.
Killed cells were unable to precipitate CaCO3 crystals in liquid
449
B-4 media, suggesting that cells must be metabolically active
for calcification and that cell structure alone is not sufficient
to promote this process. In support of a genetic basis, it was
noted that during the course of these experiments, the CaCO3
precipitation phenotype for many isolates on B-4 was lost when
they were cultivated on TSA media. Yet, calcification on B-4
media could be restored by prior passage of the culture on media
containing either calcium acetate or CaCO3 powder.
To assess whether Ca2+ ions were providing the selection
pressure for maintenance of the calcification phenotype, the
gene for the Ca2+ /2H+ antiporter chaA (Ivey et al. 1993) was
knocked out in S. typhimurium LT2, removing its function within
the cell. The gene nhaA (which encodes a Na+ /H+ antiporter)
was also knocked out as a control against the effects of a gene
knockout on the general precipitation phenotype (Ohyama et al.
1994). In order to assess the role that these mutations had on
calcium carbonate precipitation, wild type (WT), chaA and
nhaA strains were streaked on B4-media and a 0.1% glu-
cose/0.5% yeast extract media amended with 4g/l CaCO3 . While
the chaA and nhaA mutants grew as well as WT on TSA
media, their growth was inhibited on media containing calcium
(Figure 6). The chaA mutant also grew poorly on B-4 media
when compared to wild type. While this mutant did grow weakly
on the glucose/yeast extract media amended with CaCO3 , the
media could not be cleared of this mineral (Figure 6) when
compared to WT. The nhaA control did not demonstrate a
significant phenotype under any of the conditions used.
If Ca2+ ions are transported out of the cell by chaA during
growth on calcium-rich substrates, the source of CO2− 3 ions for
subsequent CaCO3 precipitation remained unclear; B4 media
does not contain CO2− 3 ions. We therefore tested whether at-
mospheric CO2 played a role in crystal formation using stable
isotope probing. Cave isolates D7A, D11 and D9A were grown
in liquid B-4 media under normal atmospheric air (control) or
an atmosphere amended with 0.2% C13 O2 . These cultures were
incubated for 2 weeks at room temperature and the crystals that
formed were harvested and tested for the presence of heavier
carbon isotopes using IRMS.
The results for the control sample (D7A), in which the at-
mosphere was not supplemented with C13 O2 , were in line with
speleothem studies, with a slight enrichment for the lighter iso-
tope (δ 13 Cvpdb = −12.53‰ ± 0.10‰). In comparison, the crys-
tals of D11 and D9A grown in the presence of a C13 O2 were
highly enriched for the heavier isotope. D11 was measured at
δ 13 Cvpdb + 403.3‰ ± 0.12‰, while D9A was too enriched in
C13 to be accurately measured.
DISCUSSION
In studying cave microorganisms, we have long questioned
the mechanisms that allow bacterial species to survive under
the very high levels of calcium encountered in these environ-
ments (Barton et al. 2001, 2004, 2007). It is known that micro-
bial species from cave environments demonstrate an enhanced
 450 E. D. BANKS ET AL.
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FIG. 5. Phylogenetic placement of GGC cultivars with their corresponding calcite precipitation and dissolution phenotypes:
= strong phenotype; =
intermediate phenotype;  = absent. The ∗ symbol indicates that crystals were also precipitated during dissolution, while indicates a very high level of CaCO3
dissolution around the colony. The phenotypes from a number of previously characterized bacterial strains are also included (in green). The phylogram represents
a consensus tree generated from distance and maximum likelihood tree-building algorithms. The scale bar represents 0.1 substitutions per site.
 BACTERIAL PRECIPITATION OF CALCIUM CARBONATE
FIG. 6. Phenotypes of knockout mutants grown on B-4 media or glucose/yeast
media containing CaCO3 . Each of the mutants (chaA and nhaA) is compared
to wild-type (WT). The dark zones seen on the CaCO3 media represents clearing
of this mineral from the media, allowing the dark background to be observed.
capability to precipitate calcium carbonate minerals, both from
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the total number of species displaying this phenotype to the
extent they can precipitate this mineral (Danielli and Edington
1983). The survival of these species under such conditions is
even more remarkable given the predominance of heterotrophic
microbial species in caves, the metabolic activity of which may
lead to the formation of organic acids. If such acids are produced,
these can dissolve the host rock (CaCO3 ) as shown in Figure 2.
If microbial activity in caves is dissolving the host rock,
this would generate the calcium salt of that acid. In support of
this hypothesis, we have previously observed the in situ pro-
duction of both calcium acetate and calcium pyruvate in cave
environments (Bullen et al. 2008). This observation is signifi-
cant, given that calcium acetate has been the staple of assessing
the bacterial calcification phenotype since the 1970s (Boquet
et al. 1973). In this study, we suggest that such dissolution ac-
tivity is further evidenced by the accumulation of insoluble clay
residues underneath apparent microbial colonies on the host rock
(Figure 1C–D). While such clay deposits are also formed during
the process of speleogenesis, they were not detected elsewhere
on the samples and only directly below the apparent microbial
colonies. Similar clay accumulation under speleothem deposits
has been observed in other caves (Barton and Northup 2007;
Blyth and Frisia 2008; Borsato et al. 2000).
Past investigators have argued that passive structural com-
ponents of the bacterial cell play the most important role in
calcification (Barabesi et al. 2007; Wolfe 2005). Nonetheless,
many of these studies were carried out in Bacilli species, which
contain numerous mineral precipitating polymeric surface struc-
tures (Ercole et al. 2007; Sleytr and Beveridge 1999) and have
the least consistent precipitation phenotype (Figure 5). Such
studies have argued against a metabolic role in calcification, as
killed cells also promote CaCO3 precipitation (Martinez et al.
2008; Yates and Robbins 1998); however these studies killed
cells by autoclaving or using metabolic poisons, such as potas-
451
sium cyanide and sodium azide. It is hard to determine what con-
sequence autoclaving would have on the chemistry of CaCO3
precipitation as it leads to cell lysis and gross structural changes.
The ability of metabolic poisons to kill bacterial cells can be lim-
ited by cyanide/azide resistant cytochrome oxidases (Cunning-
ham and Williams 1995; Lichstein and Soule 1943) or the ability
of fermentation reactions to maintain cellular viability. Indeed,
we found that a large number of our isolates remained viable
following similar cyanide/azide treatments (data not shown). In
this study treatment with paraformaldehyde, which maintains
cellular structure by cross-linking proteins, appeared to consis-
tently kill the cells. This approach may explain why our results
were more consistent in ruling out a role for metabolic activity.
This work, in support of other investigators (Buczynski and
Chafetz 1991), therefore suggests that bacterial metabolism
plays a dominant role in calficifaction. In attempting to deduce
a metabolic role for calcification where entombment would be
ultimately detrimental to survival, it is important to examine the
context of the environment in which the cell is found. Under
the nutrient limitation of cave environments, any reduced com-
pounds excreted by he cell are likely to be reassimilated as an
energy source, as suggested by the presence of high-affinity ac-
etate transporters encoded by a large number of bacterial species
(Wolfe 2005). Indeed, in a past study, we saw the assimilation
of a number of metabolic products during the growth of E. coli
in the presence of calcite crystals (Bullen et al. 2008).
However, species that uptake these potential carbon and en-
ergy sources in calcium-rich environments must also deal with
toxic Ca2+ ions (Anderson et al. 1992). The ChaA antiporter,
which is conserved across many bacterial phyla (Ivey et al.
1993), can secrete these ions into the extracellular environment,
but must do so against an ever-increasing concentration gradient
(Anderson et al. 1992).
An alternative approach for these cells could be to remove ex-
cess Ca2+ ions as they are excreted by the cell, via calcification.
The high distribution of this phenotype among these isolates
in accordance with the results of previous speleothem cultiva-
tion studies (Danielli and Edington 1983). Indeed, the majority
of the CaCO3 dissolving isolates also re-precipitated CaCO3
on the surface of the colony during growth (Figures 2 and 5).
The only exceptions to this were GGC-D10A, GGC-D10B1 and
GGC-P11; however, these species demonstrated a remarkable
amount of CaCO3 dissolution and presumably a very high level
of acid production by these strains is not compatible with CaCO3
co-precipitation. This could also explain the widespread CaCO3
precipitation phenotype among bacteria as Ca2+ ions are found
in a wide variety of habitats.
The work of Barabesi et al. (2007) has recently identified
a potential role for fatty acid metabolism in CaCO3 precipi-
tation by B. subtilis. A mutation in fadD, which regulates the
β-oxidation of fatty acids to acetyl-CoA, prevented calcifica-
tion on B-4 media. While the exact role of this gene in CaCO3
precipitation remains to be elucidated, these investigators car-
ried out experiments on B-4 media containing calcium acetate.
 452 E. D. BANKS ET AL.
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FIG. 7. The glyoxylate cycle (also known as the glyoxylate bypass or gly-
oxylate shunt) used in the consumption of acetate for central metabolism. In
addition to energy production, acetate is also used for fatty acid synthesis, while
oxaloacetate is consumed for gluconeogenesis. The putative roles of yadA and
chaA are also shown.
Using calcium acetate as a carbon and energy source for growth
presents a problem for bacterial cells due to the assimilation
of acetate; to allow conservation of CO2 for gluconeogenesis,
acetate must enter the TCA cycle via the glyoxylic acid bypass,
limiting the availability of CO2 for cellular processes such as
fatty acid synthesis (Figure 7).
The production of acetyl-CoA from fatty acid catabolism
by FadD also requires the cell to use the glyoxylate bypass
for growth (Figure 7), affecting the overall CO2 budget for
the cell. Given that our results suggest that atmospheric CO2
is at least partially responsible for the CO2−
3 ions found in the
calcium carbonate crystals, it is unclear how altering the cellular
CO2 budget may affect this process. Calcification experiments
have also been carried out in ureolytic bacterial species, which
breakdown urea with the generation of ammonia (Hammes et al.
2003). This activity leads to a sharp increase in pH, promoting
calcite precipitation; however, this process is primarily geared
toward the solid-phase capture of organic contaminants and
the high levels of urea used in these experiments (approaching
600 mm) are not normally found in nature (Hammes et al. 2003).
The chaA gene knock-out generated in this study and the ob-
served phenotype on calcium containing media provides the first
evidence for a link between calcium metabolism in bacteria and
calcification. Although the extrusion of Ca2+ ions would cer-
tainly contribute to bacterial precipitation of CaCO3 , the source
of CO2+3 ions is more difficult to discern, especially given that
the carbonic anhydrase gene (yadF) appears to be an essential
gene for a large number of bacterial species under normal atmo-
spheric conditions (Kusian et al. 2002; Merlin et al. 2003). Our
stable isotope probing analyses suggest that atmospheric CO2
is contributing, in part, CO2−
3 ions to the precipitated minerals.
This may occur through the equilibrium of CO2 between the
atmosphere and as bicarbonate ions in the medium surrounding
the cell (Formula 1).
pKa = 6.35 pKa = 10.33
H2 O + CO2 ↔ HCO−
3 + H+
↔ 3 + 2H
CO2− +
[1]
The fate of the protons would influence subsequent carbonate
ion formation; however, it is possible that they would be con-
sumed as part of cellular metabolism (contributing to the proton
motive force). Such activity would draw Equation 1 to the right
and favor the formation of carbonate ions. Indeed, past investiga-
tors have argued that calcification generates protons for nutrient
acquisition, with the phenotype becoming more pronounced un-
der nutrient limitation (McConnaughey and Whelan 1997). This
would also correlate well with the extreme nutrient limitation
of cave environments. Alternatively, these protons could be uti-
lized by the ChaA Ca2+ /2H+ antiporter, which would aid in the
transport of Ca2+ ions into the extracellular medium. Together,
the loss of the protons would generate more alkali conditions
and favor the formation of CO2− 3 ions. When the concentrations
of both Ca2+ and CO2− 3 ions exceed the solubility product for
these ions (Ksp = 4.5 × 10−9 for calcite), calcification occurs.
The role of microbial species in the development of sec-
ondary, carbonate deposits in caves has remained controversial
for quite some time (Barton et al. 2001), with mostly anecdotal
evidence of microbial association within speleothems (Baskar
et al. 2006a) and the lack of a cause-and-effect rationale (Barton
and Northup 2007). Whatever the pathway, bacterial metabolic
activity in these environments appears to lead to the precipita-
tion of various CaCO3 mineral forms on the microbial colony.
In the case of coralloids, these bacterially deposited crystals can
then form the nucleation site for subsequent mineral accumula-
tion through abiotic processes (Banfield and Nealson 1997). In
the formation of coralloids, such secondary processes may in-
clude the moisture-driven flow of CaCO3 saturated water from
areas of dissolution to precipitation in caves containing con-
vention airflow currents (M. Queen, personal communication,
2007). Such activity would generate the distinct lines of coral-
loid formation often seen in caves with such airflow patterns.
Our results therefore suggest that, while the growth of coralloids
 BACTERIAL PRECIPITATION OF CALCIUM CARBONATE
may be a physiochemical phenomenon, the precise location of
these deposits may be influenced by the need of extant microbial
species to maintain calcium homeostasis.
CONCLUSIONS
The bacterial precipitation of calcium carbonate minerals has
been dismissed by some investigators as a passive process, given
the lack of material to link calcium metabolism with microbial
ecosystem energetics. Here, by examining the calcification phe-
notypes of numerous bacterial species and using a knock-out
in the chaA calcium antiporter protein, we suggest that calcium
toxicity provides both the physiological basis and selection pres-
sure for the calcification phenotype. We propose that Ca2+ ions
are detoxified by calcification, which allows the cell to survive
an immediate metabolic insult. While this approach might ul-
timately prove fatal, from cellular entombment and death, the
wide distribution of this phenotype suggests that the short-term
benefits may outweigh the potential cost. If the pathway we have
proposed is correct, then this could have a profound impact on
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our understanding of both microbially derived calcium precip-
itation in a variety of habitats and the potential for long-term,
geologic sequestration of atmospheric CO2 .
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