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Geomicrobiology Journal
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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
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
To link to this Article: DOI: 10.1080/01490450903485136
URL: http://dx.doi.org/10.1080/01490450903485136


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

logical Sciences, Northern Kentucky University, SC 204D Nunn Drive, cal pH. This favors the formation of CO2− 3 from HCO3 and
Highland Heights, KY 41099. E-mail: bartonh@nku.edu leads to calcification. Within cave environments, which lack


photosynthetic activity and are found predominantly within development (Banfield and Nealson 1997; Hill and Forti 1997).
limestone (CaCO3 ) rock, bacterial species demonstrate a higher Through our microbiology studies in caves, we commonly iden-
incidence of the ability to precipitate calcium carbonate and an tify such ‘surface irregularities’ as microbial colonies; SEM
overall increase in total CaCO3 precipitation levels (Cacchio examination of these colonies often reveals CaCO3 crystal de-
et al. 2004; Danielli and Edington 1983). position on the surface of the bacterial cells (Barton and Northup
Although bacterial fossils are found within speleothems 2007).
(Melim et al. 2008; Melim et al. 2001), the observation of vi- These calcified colonies are often within close proximity to
able cells with these formations tends to be anecdotal (Baskar small CaCO3 mineral protrusions (∼1–2 mm), which enlarge
et al. 2006a; Cacchio et al. 2004; Danielli and Edington 1983); up to the size of coralloids (Figure 1A). The close association
no cause-and-effect for the presence of microbial species in between calcified microbial colonies and coralloids suggested
speleothems has been elucidated (Barton and Northup 2007). that microbial cells were either being caught up in the coralloid
If microbial species do play a role in the development of formation processes (Baskar et al. 2006b), or were somehow
speleothems, it is difficult to understand the evolutionary ad- critical in the development of these speleothems. The goal of
vantage of maintaining such a phenotype; when microorganisms this study was therefore to test whether calcification by bacterial
become covered in an impermeable CaCO3 layer, this prevents species may be an important phenotype for survival in high
solute exchange with the environment and ultimately leads to calcium cave environments, and whether such activity may lead
death. to speleothem development.
Among speleothems, coralloids are one of the most com-
monly observed formations (Figure 1A) (Hill and Forti 1997). MATERIALS AND METHODS
Various mechanisms for their formation have been described,
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from splashing of CaCO3 saturated water to the molecular move- Sample Site
ment of such water under capillary action (Hill and Forti 1997). Samples were collected from an unnamed cave in Kentucky,
Among these proposed methods, the need for surface irregu- USA (cave conservation practices restrict naming the cave, but
larities on the base rock is consistent. Such irregularities create location and access information can be obtained from the au-
the nucleation site for incorporation of additional crystal ele- thors). This ∼7 mile long cave is formed in Mississippian car-
ments, allowing subsequent CaCO3 deposition and speleothem 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 and flow down onto the crystals, producing a ‘fizzing’ that in-
flood line. dicated the liberation of CO2 . Samples were then run on a CO2
vacuum extraction line to isolate the liberated gas and trapped
Bacterial Cultivation in glass ampoules for analysis against known standards.
In order to culture bacterial isolates, swabs were wetted in
filtered (0.2 µm) cave water and developing coralloids were Geology
swabbed. The swabs were then used to inoculate B-4 (Boquet To examine the physical structure of the coralloids, thin sec-
et al. 1973) media and B-4C media (this study) containing tions were prepared from three rock samples collected using
B-4 media with a 1.5% top agar with 2% calcium carbonate a hammer and chisel, dried at 100◦ C and impregnated with
(CaCO3 ) light powder (all chemicals, unless otherwise stated, Petropoxy (Burnham Petrographics, Rathdrum, ID) while hot.
were from Sigma Aldrich Chemicals, St. Louis, MO). Colonies The thin sections were prepared and photographed under plane
were picked and purified based on observation of CaCO3 pre- polarized light using a Nikon E400Pol polarizing microscope.
cipitation (P class) and/or dissolution (D class) and purified by The geochemistry of the thin sections was analyzed by EDS
passage by Tryptic Soy Agar (TSA; Becton, Dickinson, Co., mapping, as described in Spear et al. (2007).
Franklin Lakes, NJ). To compare the phenotype of non-cave de-
rived isolates, additional species were obtained from the ATCC Molecular Techniques
(Manassas, VA), including Microbacterium esteraromaticum Genomic DNA was extracted from each isolate in
(ATCC 51822), Stenotrophomonas maltophilia (ATCC 17666), order to identify precipitating bacterial strains using a
Streptomyces senoensis (ATCC 29794) and Raoultella planti- ZR Fungal/Bacterial DNA Kit (Zymo Research, Orange,
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cola (ATCC 33531) to supplement the lab strains Salmonella CA). The 16S ribosomal RNA gene sequence was PCR
entrica subsp. Typhimurium LT2 and Escherichia coli K12. amplified as previously described (Spear et al. 2007)
using the 8F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and
Mineral Analysis 1525R (5 -AAGGAGGTGATCCAGCC-3 ) primers. Sequenc-
In order to examine the mineral being precipitated, CaCO3 ing of purified PCR products using the primers 8F and
precipitating isolates were grown in 3 ml of liquid B-4 me- 1391R (5 -GACGGGCGGTGWGTRCA-3 ), with 515F (5 -
dia. CaCO3 crystals were collected by filtration on 5.0 µm fil- GTGCCAGCMGCCGCGGTAA-3 ) as an internal primer was
ters (Milipore, Billerica, MA), washed free of cells with filter- carried out commercially by Agencourt Bioscience (Beverly,
sterilized distilled water (pH 8.0), air dried for thirty minutes, MA). All sequences were deposited in the NCBI database under
sputter coated with Au/PD and analyzed using an FEI Quanta accession numbers FJ347997 – FJ348046. To identify isolates,
200 environmental SEM. To confirm mineral identity, CaCO3 gene sequences were compared to the Greengenes database
crystals were similarly collected for XRD analysis on a Rigaku (http://greengenes.lbl.gov) and confirmed by 16S rRNA phylo-
Ultima III powder XRD using Cu Kα-radiation (40 kV, 44 mA). genetic placement; DNA alignments were carried out using the
Data was collected over a 2θ range between 25–70◦ at scan rate ARB Software Package (http://mpi-bremen.de/molecol/arb).
of 0.1◦ /min. For distance calculations, the evolutionary model was GTR +
For isotopic probing to determine the source of the carbonate I + G (base (0.2402, 0.2366 0.3148), Nst/6, Rmat (0.7547,
in the precipitated minerals, 3 ml liquid B-4 media was inocu- 1.8102, 1.0282, 0.7316, 3.1058), Rates = gamma, Shape =
lated with the test strain and grown in a 2.4 liter glass chamber 0.6293, Pinvar = 0.2486), determined using MODELTEST
(Diagnostic Systems, Sparks, MD) fitted with an air tight gasket. (Posada 2003). Distance and parsimonious phylograms were
C13 O2 gas was then injected to generate an atmosphere that con- generated in PAUP* using a heuristic search (Wilgenbusch
tained 0.2% atmospheric CO2 and 0.2% C13 O2 . Cultures were and Swofford 2003). The highest scoring trees were tested by
maintained in this chamber for 2 weeks. Isotope ratio mass boostrap analysis using 1000 replicates. A maximum likeli-
spectrometry was carried out on a Nuclide 6-60 RMS dual-inlet hood phylogram was also generated using a Bayesian analysis
IRMS. To liberate the CO2 from the crystals, a degassed acid with the MrBayes program for 2 million generations (Ronquist
solution was prepared. Briefly, 100 µl concentrated (∼10 M) and Huelsenbeck 2003). In all cases, sequences from Aquifex
phosphoric acid was added to 5 ml ultraclean (MilliQ) water. pyrophilus and Thermoplasma acidophilium were used for
This acidified solution was degassed by repeated freeze/thaw outgroups.
events under vacuum, followed by 2 hours under vacuum.
The acidified solution was then kept frozen on a methanol/dry Construction of Deletion Mutants
ice slurry, the sample container was removed from vacuum, the To examine the role that microbial physiology had in precip-
sample crystals were placed on the side of the reaction ves- itation, deletion mutants of the genes chaA and nhaA were gen-
sel and the vessel was put back under vacuum to evacuate the erated using linear transformation in S. typhimurium TT22971
headspace for an additional hour. The vessel was then sealed, containing a lambda red background, based on the protocol of
taken off of vacuum, and the ice slurry was then allowed to melt Datsenko and Wanner (2000). To confirm that the insert had

Primers used in this study
Primer Name Sequence

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 CaCO3 deposition seen in coralloid development, bacteria were
transduced by P22 into a wild-type S. typhimurium TR10K back- isolated from coralloids in close proximity to the rock samples.
ground and verified by PCR to generate the respective knock This was done by streaking swab samples onto either B-4 media
out mutant. or B-4C media, which allow us to identify bacterial species that
demonstrate the ability to either precipitate or dissolve CaCO3 ,
RESULTS respectively (Figure 2). Following growth at room temperature
for 2–4 weeks, isolates were identified based on unique colony
Analysis of Coralloid Thin Sections morphology and phenotype; species able to precipitate CaCO3
Eight thin sections were prepared from rock samples that con- were considered P-class, and those able to dissolve calcite were
tained both observed microbial colonies and 56 small, nodular D-class isolates. This initial screen on B-4 and B-4C media
coralloids (an example is shown in Figure 1B–D). Within these identified 15 P-class and 17 D-class colonies. These colonies
thin sections, polarizing-light microscopy revealed a dark, lam- were then picked and streaked for pure culture on TSA media,
inated layer between the base of each coralloid and the host which often allows the separation of mixed cultures. Using this
rock (Figure 1B), which matched the thin-section appearance approach we identified 19 additional (10 P-class and 9 D-class)
through colonies that had been visually identified on the surface isolates, generating 51 unique bacterial cultures isolated from
of these samples. In all cases, this dark material was coated with these developing coralloids.
layers of what appeared to be CaCO3 (by gross examination) To ensure that the crystals produced by these isolates on
in a stromatolitic-like growth, quite distinct from the mineral B-4 media were indeed CaCO3 mineral polymorphs, they were
structure of the host rock (as shown in Figure 1B). To examine examined using SEM microscopy and X-ray diffraction (XRD).
the chemistry of this deposition, energy-dispersive spectroscopy The SEM results (Figure 3) demonstrated the presence of
(EDS) mapping of the thin-sections was carried out (example in mineral polymorphs, including calcite, vaterite and aragonite
Figures 1C and 1D). (Lippmann 1973). XRD analysis confirmed the presence of
The results demonstrate that the coralloids were calcium- these multiple mineral forms, including vaterite, which is only
and oxygen-rich in chemistry (indicative of CaCO3 ), lacking transiently found in nature (Figure 4). Although examining these
the trace silica, aluminum and iron signatures of clays ob- crystals using SEM, it was noted that virtually all of the crys-
served in the St. Genevieve Formation host rock (Figures 1B–D) tals included pits and depressions that matched the structure of
(McGrain and Dever 1967). These data suggest that the struc- the precipitating bacteria, both in terms of size and morphol-
ture and chemistry of these coralloids is different than the host ogy (Figures 3D and 3E). A number of these depressions also
rock and in-line with a depositional origin. There was also what included structures that were indicative of cell growth and di-
appeared to be an accumulation of silica, aluminum and iron- vision, such as the septa observed in Figure 3E. Even though
rich material between all examined coralloids and the host rock these crystals were washed free of bacterial cells, many crystals
(Figure 1C and 1D). The chemistry of this material matches also contained obvious cells closely associated with, and emerg-
the chemistry of clay minerals in the Ste. Genevieve Formation, ing from, the mineral surface (Figure 3F). The organic nature of
which are generally not soluble, except under extremely low pH. 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 Alpha-, Beta- and Gammaproteobacteria, the Firmicutes and
the calcium carbonate mineral (data not shown). Together these Actinobacteria. While this distribution does not represent the to-
data demonstrate a close association between the bacterial cells tal bacterial population in this environment, the divisional repre-
and the formed crystals. sentation is in line with previous cultivation and non-cultivation
Once calcification by the bacterial isolates was confirmed, based studies in cave and subsurface environments (Amann et
PCR amplification of the 16S rRNA gene sequence was used al. 1995; Barns and Nierzwicki-Bauer 1997; Barton et al. 2004).
to determine their identity. The results (Figure 5) demonstrate With the exception of GGC-D3, all 51 isolates demonstrated a
a broad distribution of isolates, representing members of the 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).

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-
FIG. 4. Representative XRD analysis of calcium carbonate crystals (from cose/0.5% yeast extract media amended with 4g/l CaCO3 . While
isolate P11) shows presence of vaterite. The corresponding peaks are labeled. the chaA and nhaA mutants grew as well as WT on TSA
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media, their growth was inhibited on media containing calcium

they were initially identified based on a phenotype of precipi- (Figure 6). The chaA mutant also grew poorly on B-4 media
tating or dissolving calcium carbonate (Figure 5). when compared to wild type. While this mutant did grow weakly
Dissolution activity was not so broadly represented, likely on the glucose/yeast extract media amended with CaCO3 , the
due to the large amount of acid production necessary to ob- media could not be cleared of this mineral (Figure 6) when
serve this phenotype; however, many of the D-class isolates compared to WT. The nhaA control did not demonstrate a
also precipitated CaCO3 crystals on top of their colonies dur- significant phenotype under any of the conditions used.
ing dissolution (Figure 5). This phenotype was observed for all If Ca2+ ions are transported out of the cell by chaA during
dissolving phenotypes, except GGC-D10A, GGC-D10B1 and growth on calcium-rich substrates, the source of CO2− 3 ions for
GGC-P11, which generated a very large zone of clearing. subsequent CaCO3 precipitation remained unclear; B4 media
The pattern of both CaCO3 precipitation, dissolution and does not contain CO2− 3 ions. We therefore tested whether at-
a combined dissolving/precipitating phenotype appeared to be mospheric CO2 played a role in crystal formation using stable
similar for all isolates within a particular clade, with the ex- isotope probing. Cave isolates D7A, D11 and D9A were grown
ception of the Bacilli (Figure 5). To determine whether the in liquid B-4 media under normal atmospheric air (control) or
relationship between members of a clade and phenotype is suf- an atmosphere amended with 0.2% C13 O2 . These cultures were
ficiently robust to be predictive of phenotype, five bacterial incubated for 2 weeks at room temperature and the crystals that
species isolated from non-cave environments were tested for formed were harvested and tested for the presence of heavier
their ability to precipitate and/or dissolve CaCO3 ; these included carbon isotopes using IRMS.
S. typhimurium, Escherichia coli, Klebsiella planticola, Mi- The results for the control sample (D7A), in which the at-
crobacterium esteraromaticum, Stenotrophomonas maltophilia mosphere was not supplemented with C13 O2 , were in line with
and Streptomyces senoensis. The results (Figure 5) demonstrate speleothem studies, with a slight enrichment for the lighter iso-
that the phenotype for these species does indeed match those for tope (δ 13 Cvpdb = −12.53‰ ± 0.10‰). In comparison, the crys-
the clade to which they belong, despite the fact that they were tals of D11 and D9A grown in the presence of a C13 O2 were
not isolated from caves. highly enriched for the heavier isotope. D11 was measured at
δ 13 Cvpdb + 403.3‰ ± 0.12‰, while D9A was too enriched in
The Physiology of Calcium Carbonate Precipitation C13 to be accurately measured.
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. DISCUSSION
To differentiate the contribution of bacterial structure to crys- In studying cave microorganisms, we have long questioned
tal formation, the precipitation capabilities of a random group the mechanisms that allow bacterial species to survive under
of isolates killed with paraformaldehyde was tested; the loss the very high levels of calcium encountered in these environ-
of cell viability using this method was confirmed by plating. ments (Barton et al. 2001, 2004, 2007). It is known that micro-
Killed cells were unable to precipitate CaCO3 crystals in liquid 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.

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
FIG. 6. Phenotypes of knockout mutants grown on B-4 media or glucose/yeast Chafetz 1991), therefore suggests that bacterial metabolism
media containing CaCO3 . Each of the mutants (chaA and nhaA) is compared plays a dominant role in calficifaction. In attempting to deduce
to wild-type (WT). The dark zones seen on the CaCO3 media represents clearing a metabolic role for calcification where entombment would be
of this mineral from the media, allowing the dark background to be observed. ultimately detrimental to survival, it is important to examine the
context of the environment in which the cell is found. Under
capability to precipitate calcium carbonate minerals, both from the nutrient limitation of cave environments, any reduced com-
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the total number of species displaying this phenotype to the pounds excreted by he cell are likely to be reassimilated as an
extent they can precipitate this mineral (Danielli and Edington energy source, as suggested by the presence of high-affinity ac-
1983). The survival of these species under such conditions is etate transporters encoded by a large number of bacterial species
even more remarkable given the predominance of heterotrophic (Wolfe 2005). Indeed, in a past study, we saw the assimilation
microbial species in caves, the metabolic activity of which may of a number of metabolic products during the growth of E. coli
lead to the formation of organic acids. If such acids are produced, in the presence of calcite crystals (Bullen et al. 2008).
these can dissolve the host rock (CaCO3 ) as shown in Figure 2. However, species that uptake these potential carbon and en-
If microbial activity in caves is dissolving the host rock, ergy sources in calcium-rich environments must also deal with
this would generate the calcium salt of that acid. In support of toxic Ca2+ ions (Anderson et al. 1992). The ChaA antiporter,
this hypothesis, we have previously observed the in situ pro- which is conserved across many bacterial phyla (Ivey et al.
duction of both calcium acetate and calcium pyruvate in cave 1993), can secrete these ions into the extracellular environment,
environments (Bullen et al. 2008). This observation is signifi- but must do so against an ever-increasing concentration gradient
cant, given that calcium acetate has been the staple of assessing (Anderson et al. 1992).
the bacterial calcification phenotype since the 1970s (Boquet An alternative approach for these cells could be to remove ex-
et al. 1973). In this study, we suggest that such dissolution ac- cess Ca2+ ions as they are excreted by the cell, via calcification.
tivity is further evidenced by the accumulation of insoluble clay The high distribution of this phenotype among these isolates
residues underneath apparent microbial colonies on the host rock in accordance with the results of previous speleothem cultiva-
(Figure 1C–D). While such clay deposits are also formed during tion studies (Danielli and Edington 1983). Indeed, the majority
the process of speleogenesis, they were not detected elsewhere of the CaCO3 dissolving isolates also re-precipitated CaCO3
on the samples and only directly below the apparent microbial on the surface of the colony during growth (Figures 2 and 5).
colonies. Similar clay accumulation under speleothem deposits The only exceptions to this were GGC-D10A, GGC-D10B1 and
has been observed in other caves (Barton and Northup 2007; GGC-P11; however, these species demonstrated a remarkable
Blyth and Frisia 2008; Borsato et al. 2000). amount of CaCO3 dissolution and presumably a very high level
Past investigators have argued that passive structural com- of acid production by these strains is not compatible with CaCO3
ponents of the bacterial cell play the most important role in co-precipitation. This could also explain the widespread CaCO3
calcification (Barabesi et al. 2007; Wolfe 2005). Nonetheless, precipitation phenotype among bacteria as Ca2+ ions are found
many of these studies were carried out in Bacilli species, which in a wide variety of habitats.
contain numerous mineral precipitating polymeric surface struc- The work of Barabesi et al. (2007) has recently identified
tures (Ercole et al. 2007; Sleytr and Beveridge 1999) and have a potential role for fatty acid metabolism in CaCO3 precipi-
the least consistent precipitation phenotype (Figure 5). Such tation by B. subtilis. A mutation in fadD, which regulates the
studies have argued against a metabolic role in calcification, as β-oxidation of fatty acids to acetyl-CoA, prevented calcifica-
killed cells also promote CaCO3 precipitation (Martinez et al. tion on B-4 media. While the exact role of this gene in CaCO3
2008; Yates and Robbins 1998); however these studies killed precipitation remains to be elucidated, these investigators car-
cells by autoclaving or using metabolic poisons, such as potas- ried out experiments on B-4 media containing calcium acetate.
452 E. D. BANKS ET AL.

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− +

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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
FIG. 7. The glyoxylate cycle (also known as the glyoxylate bypass or gly- would also correlate well with the extreme nutrient limitation
oxylate shunt) used in the consumption of acetate for central metabolism. In of cave environments. Alternatively, these protons could be uti-
addition to energy production, acetate is also used for fatty acid synthesis, while lized by the ChaA Ca2+ /2H+ antiporter, which would aid in the
oxaloacetate is consumed for gluconeogenesis. The putative roles of yadA and
chaA are also shown.
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
Using calcium acetate as a carbon and energy source for growth of both Ca2+ and CO2− 3 ions exceed the solubility product for
presents a problem for bacterial cells due to the assimilation these ions (Ksp = 4.5 × 10−9 for calcite), calcification occurs.
of acetate; to allow conservation of CO2 for gluconeogenesis, The role of microbial species in the development of sec-
acetate must enter the TCA cycle via the glyoxylic acid bypass, ondary, carbonate deposits in caves has remained controversial
limiting the availability of CO2 for cellular processes such as for quite some time (Barton et al. 2001), with mostly anecdotal
fatty acid synthesis (Figure 7). evidence of microbial association within speleothems (Baskar
The production of acetyl-CoA from fatty acid catabolism et al. 2006a) and the lack of a cause-and-effect rationale (Barton
by FadD also requires the cell to use the glyoxylate bypass and Northup 2007). Whatever the pathway, bacterial metabolic
for growth (Figure 7), affecting the overall CO2 budget for activity in these environments appears to lead to the precipita-
the cell. Given that our results suggest that atmospheric CO2 tion of various CaCO3 mineral forms on the microbial colony.
is at least partially responsible for the CO2−
3 ions found in the In the case of coralloids, these bacterially deposited crystals can
calcium carbonate crystals, it is unclear how altering the cellular then form the nucleation site for subsequent mineral accumula-
CO2 budget may affect this process. Calcification experiments tion through abiotic processes (Banfield and Nealson 1997). In
have also been carried out in ureolytic bacterial species, which the formation of coralloids, such secondary processes may in-
breakdown urea with the generation of ammonia (Hammes et al. clude the moisture-driven flow of CaCO3 saturated water from
2003). This activity leads to a sharp increase in pH, promoting areas of dissolution to precipitation in caves containing con-
calcite precipitation; however, this process is primarily geared vention airflow currents (M. Queen, personal communication,
toward the solid-phase capture of organic contaminants and 2007). Such activity would generate the distinct lines of coral-
the high levels of urea used in these experiments (approaching loid formation often seen in caves with such airflow patterns.
600 mm) are not normally found in nature (Hammes et al. 2003). Our results therefore suggest that, while the growth of coralloids

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