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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 To link to this Article: DOI: 10.1080/01490450903485136 URL:

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Due to the inability of dead cells to precipitate these minerals. homeostasis. Hoehler. coralloids. HAB is supported in part by the Kentucky NSF EPSCoR Program (NSF0814194) and an NSF MIP/CAREER grant (NSF0643462). . but the toxicity of Ca2+ ions to bacteria. Banks. Within cave environments. we isolated 51 culturable bacteria from a coralloid speleothem and tested their ability to dissolve and precipitate CaCO3 . we used stable isotope probing with C13 O2 to determine whether atmospheric CO2 could be the source of these ions. In these environments. making a weak carbonic acid (H2 CO3 ) that dissolves limestone (calcium carbonate: CaCO3 ) rock. as well as a myriad of other potential forms (Hill and Forti 1997). 27:444–454. 2005a. Over geologic timescales. would be particularly beneficial to bacteria in Ca2+ -rich cave environments. It has been known since the 1970’s that CaCO3 deposition (calcification) is a general phenomenon among the Bacteria (Boquet et al. calcite. 2005b). John Roth and Dr. Moffett Field. Despite the broad taxonomic distribution of this phenotype. 2005b). Highland Heights. 1973). much of the work examining bacterially mediated CaCO3 precipitation has concentrated on cyanobacteria in Ca2+ -rich (∼40 mm) seawater. with infrastructure support by the NIH Kentucky INBRE program (NIH 5P20RR01648-05) and NSF Major Research Instrumentation award (MRI-0520921). Highland Heights. accepted 10 November 2009. Dave Bunnell for the image used in Figure 1. Given growth of these species on calcium acetate. particularly from the works of Dreybrodt and Kaufmann et al. being commonly white” (Beaumont 1676. Northern Kentucky University. Downloaded By: [Barton. The majority of these isolates could precipitate CaCO3 minerals. LLC ISSN: 0149-0451 print / 1521-0529 online DOI: 10. Highland Heights. Short et al. When this water drips into a cave. calcite and vaterite were produced in this process. Lubbers. (Buhmann and Dreybrodt 1984.1 Juan G. Badger and Price 1994. Such activity may also create the initial crystal nucleation sites that contribute to the formation of secondary CaCO3 deposits within caves. California Keywords To determine if microbial species play an active role in the development of calcium carbonate (CaCO3 ) deposits (speleothems) in cave environments. SC 204D Nunn Drive. Taylor. Kentucky 3 NASA Ames Research Center. EDB was supported by a SURF Fellowship from the ASM and a SURCA Award from NKU. Kubo for his assistance with the isotopic analyses and IRMS work. Michael D. photosynthesis alters the chemistry of the microenvironment by fixing CO2 (Aizawa and Miyachi 1986. this suggested that calcification requires metabolic activity. such as stalactites and stalagmites. suggesting that the need to remove Ca2+ ions from the cell may drive calcification. The physiological adaptation of removing toxic Ca2+ ions by calcification. Dreybrodt 1999. calcium speleothems caves. Address correspondence to Hazel A. we created a loss-of-function gene knock-out in the Ca2+ ion efflux protein ChaA.k. which lack 444 .1 Jason Gulley. Barton1 1 2 Department of Biological Sciences. This favors the formation of CO2− from HCO− and 3 3 leads to calcification.Geomicrobiology Journal. The resultant crystals were significantly enriched in this heavy isotope. Giarrizo.3 and Hazel INTRODUCTION When secondary cave deposits (speleothems. With no carbonate in the media used in the calcification studies. 2010 Copyright © Taylor & Francis Group. growth from the finest parts of clay.1 Heather A.1 Nicholas M. Bullen. a. Kentucky Department of Chemistry. suggesting that extracellular CO2 does indeed contribute to the mineral structure. albeit at a exceedingly slow rate (Hill and Forti 1997. Short et al. Department of Biological Sciences. Kaufmann 2003. Eric Kofoid for the Salmonella strains and Mr. Dr. cave formations) were described in 1676 by John Beaumont.a. with a high percentage of soil bacteria producing CaCO3 crystals when grown on media containing calcium acetate. scanning electron microscopy and X-ray diffractrometry demonstrated that aragonite. .2 Tori M. The authors would like to thank the landowners and cavers in the collection of the coralloid samples and strains. Northern Kentucky University. The description of cave formations as a vegetative growth seems quaint as we now know that such deposits form when surface water percolating through the soil acquires dissolved CO2 . Banfield and Nealson 1997). he classified them as a form of plant life with “. Northern Kentucky University. this deposition leads to the development of classic speleothems. KY 41099. the CO2 off-gases and the subsequent increase in the saturation index (SI) for Ca2+ and CO2− leads 3 to the precipitation of CaCO3 as calcite. leading to an increase in the local pH.1 Brad R. Hazel] At: 05:02 10 June 2010 Received 25 May 2009. The loss of this protein inhibited growth on media containing calcium. Dr. Barton. E-mail: bartonh@nku.1080/01490450903485136 Bacterial Calcium Carbonate Precipitation in Cave Environments: A Function of Calcium Homeostasis Eric D. The inorganic and physical chemistry that drives these phenomena are well understood. while useful in numerous environments. Hill and Forti 1997).

(C) and (D) The same thin-section image as B. Such irregularities create the nucleation site for incorporation of additional crystal elements. from splashing of CaCO3 saturated water to the molecular movement of such water under capillary action (Hill and Forti 1997). Among these proposed methods. (A) Coralloids (cave popcorn) is a common speleothem found in caves. Hazel] At: 05:02 10 June 2010 photosynthetic activity and are found predominantly within limestone (CaCO3 ) rock. USA (cave conservation practices restrict naming the cave. Hill and Forti 1997). Among speleothems. FIG. for which no clear mechanism of formation has been described. coralloids are one of the most commonly observed formations (Figure 1A) (Hill and Forti 1997). Louis Formation. 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. 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. 2004. allowing subsequent CaCO3 deposition and speleothem development (Banfield and Nealson 1997. it is difficult to understand the evolutionary advantage of maintaining such a phenotype. MATERIALS AND METHODS Sample Site Samples were collected from an unnamed cave in Kentucky. we commonly identify such ‘surface irregularities’ as microbial colonies. no cause-and-effect for the presence of microbial species in speleothems has been elucidated (Barton and Northup 2007). Melim et al. Danielli and Edington 1983). Genevieve and St. 2006b). but location and access information can be obtained from the authors). Image A is ∼10 cm across. which enlarge up to the size of coralloids (Figure 1A). This ∼7 mile long cave is formed in Mississippian carbonate rocks of the St. 2008. These calcified colonies are often within close proximity to small CaCO3 mineral protrusions (∼1–2 mm). the need for surface irregularities on the base rock is consistent. Danielli and Edington 1983). Note the banding commonly observed in stromatolites. 2006a. 2004. 2001). Cacchio et al. 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. Various mechanisms for their formation have been described. or were somehow critical in the development of these speleothems. but using EDS analysis. (B) An example thin-section through cave popcorn under polarizing light. . Although bacterial fossils are found within speleothems (Melim et al. 1. Through our microbiology studies in caves. the observation of viable cells with these formations tends to be anecdotal (Baskar et al. when microorganisms become covered in an impermeable CaCO3 layer. If microbial species do play a role in the development of speleothems. Colors correspond the element being mapped.BACTERIAL PRECIPITATION OF CALCIUM CARBONATE 445 Downloaded By: [Barton. SEM examination of these colonies often reveals CaCO3 crystal deposition on the surface of the bacterial cells (Barton and Northup 2007). and whether such activity may lead to speleothem development. this prevents solute exchange with the environment and ultimately leads to death.

4 liter glass chamber (Diagnostic Systems. BANKS ET AL. 44 mA). In all cases. NJ). Construction of Deletion Mutants To examine the role that microbial physiology had in precipitation. determined using MODELTEST (Posada 2003). thin sections were prepared from three rock samples collected using a hammer and chisel. ID) while hot. washed free of cells with filtersterilized distilled water (pH 8. unless otherwise stated.0282. All sequences were deposited in the NCBI database under accession numbers FJ347997 – FJ348046. Shape = 0. producing a ‘fizzing’ that indicated the liberation of CO2 . MA).lbl. the evolutionary model was GTR + I + G (base (0. Louis. MA).. Rates = gamma.446 E. The thin sections were prepared and photographed under plane polarized light using a Nikon E400Pol polarizing microscope. 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. gene sequences were compared to the Greengenes database (http://greengenes. 0. MD) fitted with an air tight gasket. 3 ml liquid B-4 media was inoculated with the test strain and grown in a 2. The vessel was then sealed. deletion mutants of the genes chaA and nhaA were generated using linear transformation in S.2366 0. Distance and parsimonious phylograms were generated in PAUP* using a heuristic search (Wilgenbusch and Swofford 2003). Typhimurium LT2 and Escherichia coli K12. Isotope ratio mass spectrometry was carried out on a Nuclide 6-60 RMS dual-inlet IRMS. a degassed acid solution was prepared.6293.8102.0 µm filters (Milipore. The acidified solution was then kept frozen on a methanol/dry ice slurry.0). based on the protocol of Datsenko and Wanner (2000).1◦ /min.7316. The geochemistry of the thin sections was analyzed by EDS mapping. typhimurium TT22971 containing a lambda red background. This acidified solution was degassed by repeated freeze/thaw events under vacuum. Franklin Lakes. A maximum likelihood phylogram was also generated using a Bayesian analysis with the MrBayes program for 2 million generations (Ronquist and Huelsenbeck 2003). Mineral Analysis In order to examine the mineral being precipitated. were from Sigma Aldrich Chemicals. 1973) media and B-4C media (this study) containing B-4 media with a 1. Co. To liberate the CO2 from the crystals. and the ice slurry was then allowed to melt and flow down onto the crystals. CaCO3 crystals were similarly collected for XRD analysis on a Rigaku Ultima III powder XRD using Cu Kα-radiation (40 kV. Bacterial Cultivation In order to culture bacterial isolates. air dried for thirty minutes. Sequencing of purified PCR products using the primers 8F and 1391R (5 -GACGGGCGGTGWGTRCA-3 ). dried at 100◦ C and impregnated with Petropoxy (Burnham Petrographics. The highest scoring trees were tested by boostrap analysis using 1000 replicates. Billerica. followed by 2 hours under vacuum. DNA alignments were carried out using the ARB Software Package (http://mpi-bremen. C13 O2 gas was then injected to generate an atmosphere that contained 0. the sample container was removed from vacuum. Becton. Data was collected over a 2θ range between 25–70◦ at scan rate of 0. swabs were wetted in filtered (0. To confirm mineral identity. Stenotrophomonas maltophilia (ATCC 17666). 1. CaCO3 precipitating isolates were grown in 3 ml of liquid B-4 media. Briefly. 3. Streptomyces senoensis (ATCC 29794) and Raoultella planticola (ATCC 33531) to supplement the lab strains Salmonella entrica subsp. Rathdrum. 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.2402. Colonies were picked and purified based on observation of CaCO3 precipitation (P class) and/or dissolution (D class) and purified by passage by Tryptic Soy Agar (TSA. 1.2 µm) cave water and developing coralloids were swabbed.3148). CaCO3 crystals were collected by filtration on 5. the sample crystals were placed on the side of the reaction vessel and the vessel was put back under vacuum to evacuate the headspace for an additional hour. 100 µl concentrated (∼10 M) phosphoric acid was added to 5 ml ultraclean (MilliQ) water.1058).5% top agar with 2% calcium carbonate (CaCO3 ) light powder (all chemicals.2% atmospheric CO2 and 0. additional species were obtained from the ATCC (Manassas. Hazel] At: 05:02 10 June 2010 . VA). (2007). Samples were collected in a stream passage above the cave flood line. Orange. D. For distance calculations. The swabs were then used to inoculate B-4 (Boquet et al. To identify isolates. MO).2486). Dickinson. 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. with 515F (5 GTGCCAGCMGCCGCGGTAA-3 ) as an internal primer was carried out commercially by Agencourt Bioscience (Beverly. taken off of vacuum. Geology To examine the physical structure of the coralloids. To confirm that the insert had Downloaded By: [Barton. St. Sparks. sequences from Aquifex pyrophilus and Thermoplasma acidophilium were used for outgroups. CA).2% C13 O2 . For isotopic probing to determine the source of the carbonate in the precipitated minerals. Rmat (0. Pinvar = 0. 0. as described in Spear et and confirmed by 16S rRNA phylogenetic placement. Cultures were maintained in this chamber for 2 weeks. including Microbacterium esteraromaticum (ATCC 51822). sputter coated with Au/PD and analyzed using an FEI Quanta 200 environmental SEM. Nst/6. To compare the phenotype of non-cave derived

There was also what appeared to be an accumulation of silica.BACTERIAL PRECIPITATION OF CALCIUM CARBONATE 447 TABLE 1 Primers used in this study Primer Name yadF-forward yadF-reverse yadF-FPE yadF-RPI yarF-RPE chaA-forward chaA-Reverse chaA-FPE chaA-RPI chaA-RPE Sequence 5 -ATTGGTGGGGAATGTATTTCAGACTTAGGGCGGAAATACCACCA AACACCCCCCAAAACC-3 5 -CCCAACGCACTTATTTGGTTACAGGTCGTTAACTTCCATCACACAACCACACCACACCAC-3 5 -TTGGCTTTTCGATTCCGGACG-3 5 -ATCTGAACTGTCTCTCCGTGG-3 5 -ATTTCCCTGCTCCATTTGGC-3 5 -ATGACACATGCCTGTGAGGCGGTGAAAACCCGCCATAAGCACCAAACACCCCCCAAAACC-3 5 -TGCGGTTTTTAGCTTGTGTAGGCCGGATAAGATACTATCCACACAACCACACCACACCAC-3 5 -CCCAGTCGAGGAGGATTT-3 5 -GACCAATAATGCCTGACCG-3 5 -AATGCCGCGACCCATTTG-3 Downloaded By: [Barton. which often allows the separation of mixed cultures. it was noted that virtually all of the crystals included pits and depressions that matched the structure of the precipitating bacteria. the mineral surface (Figure 3F). energy-dispersive spectroscopy (EDS) mapping of the thin-sections was carried out (example in Figures 1C and 1D). 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 . A number of these depressions also included structures that were indicative of cell growth and division. To ensure that the crystals produced by these isolates on B-4 media were indeed CaCO3 mineral polymorphs. this dark material was coated with layers of what appeared to be CaCO3 (by gross examination) in a stromatolitic-like growth. aluminum and iron signatures of clays observed in the St. which are generally not soluble. which is only transiently found in nature (Figure 4). laminated layer between the base of each coralloid and the host rock (Figure 1B). nodular coralloids (an example is shown in Figure 1B–D). Using this approach we identified 19 additional (10 P-class and 9 D-class) isolates. quite distinct from the mineral structure of the host rock (as shown in Figure 1B). polarizing-light microscopy revealed a dark. Genevieve Formation host rock (Figures 1B–D) (McGrain and Dever 1967). Although examining these crystals using SEM. RESULTS Analysis of Coralloid Thin Sections Eight thin sections were prepared from rock samples that contained both observed microbial colonies and 56 small. generating 51 unique bacterial cultures isolated from these developing coralloids. respectively (Figure 2). lacking the trace silica. One strain that contained either a deletion of chaA or nhaA was then transduced by P22 into a wild-type S. they were examined using SEM microscopy and X-ray diffraction (XRD). To examine the chemistry of this deposition. including vaterite. typhimurium TR10K background and verified by PCR to generate the respective knock out mutant. These data suggest that the structure and chemistry of these coralloids is different than the host rock and in-line with a depositional origin. which matched the thin-section appearance through colonies that had been visually identified on the surface of these samples. bacteria were isolated from coralloids in close proximity to the rock samples. The chemistry of this material matches the chemistry of clay minerals in the Ste. vaterite and aragonite (Lippmann 1973). such as the septa observed in Figure 3E. except under extremely low pH. Genevieve Formation. and emerging from. aluminum and ironrich material between all examined coralloids and the host rock (Figure 1C and 1D). and those able to dissolve calcite were D-class isolates. The organic nature of these cells was confirmed by EDS. Within these thin sections. isolates were identified based on unique colony morphology and phenotype. many crystals also contained obvious cells closely associated with. XRD analysis confirmed the presence of these multiple mineral forms. species able to precipitate CaCO3 were considered P-class. These colonies were then picked and streaked for pure culture on TSA media. The SEM results (Figure 3) demonstrated the presence of mineral polymorphs. PCR was carried out using a combination of a forward external primer (FPE) and either a reverse internal (RPI) or external (RPE) primer (Table 1). both in terms of size and morphology (Figures 3D and 3E). Hazel] At: 05:02 10 June 2010 knocked out the gene being studied. Distribution of the Calcification Phenotype Among Bacteria Isolated From Speleothems To assess whether microbial activity played a role in the CaCO3 deposition seen in coralloid development. In all cases. including calcite. Even though these crystals were washed free of bacterial cells. The results demonstrate that the coralloids were calciumand oxygen-rich in chemistry (indicative of CaCO3 ). Following growth at room temperature for 2–4 weeks. This initial screen on B-4 and B-4C media identified 15 P-class and 17 D-class colonies. which indicated the presence .

SEM images of calcite crystals generated by GGC cultivars. all 51 isolates demonstrated a calcification phenotype on B-4 media.448 E. The calcium carbonate precipitates demonstrating different mineral polymporphs can be seen within each colony. (A) Original colony morphologies of isolates streaked on B-4 media. 3. 2. including cell-shaped pits (D) and septa (E). D. (B) aragonite (D15) and (C) vaterite (P11). Although these crystals were washed free of bacterial cells prior to SEM. 2004). 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). including (A) calcite (P9B). along with CaCO3 precipitation in the center of isolated colonies. FIG. .and Gammaproteobacteria. Barns and Nierzwicki-Bauer 1997. Hazel] At: 05:02 10 June 2010 of phosphorous. Beta. BANKS ET AL. Numerous calcite polymorphs are produced by microbial activity. nitrogen and sulfur peaks when compared to the calcium carbonate mineral (data not shown). Downloaded By: [Barton. Colonies range in size from 2–4 mm. the Firmicutes and Actinobacteria. While this distribution does not represent the total bacterial population in this environment. (B) Calcite dissolution by D17D when grown on B-4C media. The close associate between microbial growth and the precipitation of these minerals can also be seen. the divisional representation is in line with previous cultivation and non-cultivation based studies in cave and subsurface environments (Amann et al. Once calcification by the bacterial isolates was confirmed. indicated by arrow). 1995. irrespective of whether FIG. PCR amplification of the 16S rRNA gene sequence was used to determine their identity. Together these data demonstrate a close association between the bacterial cells and the formed crystals. Precipitation and dissolution phenotypes of cave isolates. representing members of the Alpha-. The zone of clearing of the CaCO3 around each colony can be seen. The results (Figure 5) demonstrate a broad distribution of isolates. With the exception of GGC-D3. Barton et al.

This phenotype was observed for all dissolving phenotypes.12‰. To assess whether Ca2+ ions were providing the selection pressure for maintenance of the calcification phenotype. were in line with speleothem studies. To differentiate the contribution of bacterial structure to crystal formation. however. B4 media does not contain CO2− ions. In order to assess the role that these mutations had on calcium carbonate precipitation. It is known that microbial species from cave environments demonstrate an enhanced . Yet. Killed cells were unable to precipitate CaCO3 crystals in liquid B-4 media. The results (Figure 5) demonstrate that the phenotype for these species does indeed match those for the clade to which they belong.1% glucose/0. The results for the control sample (D7A).3‰ ± 0. While this mutant did grow weakly on the glucose/yeast extract media amended with CaCO3 . Stenotrophomonas maltophilia and Streptomyces senoensis. 2007). removing its function within the cell. In support of a genetic basis. typhimurium LT2. Hazel] At: 05:02 10 June 2010 FIG. with the exception of the Bacilli (Figure 5). Cave isolates D7A.BACTERIAL PRECIPITATION OF CALCIUM CARBONATE 449 Downloaded By: [Barton. suggesting that cells must be metabolically active for calcification and that cell structure alone is not sufficient to promote this process. while D9A was too enriched in C13 to be accurately measured. the CaCO3 precipitation phenotype for many isolates on B-4 was lost when they were cultivated on TSA media. D11 was measured at δ 13 Cvpdb + 403. D11 and D9A were grown in liquid B-4 media under normal atmospheric air (control) or an atmosphere amended with 0. 4. the media could not be cleared of this mineral (Figure 6) when compared to WT. we have long questioned the mechanisms that allow bacterial species to survive under the very high levels of calcium encountered in these environments (Barton et al.10‰). five bacterial species isolated from non-cave environments were tested for their ability to precipitate and/or dissolve CaCO3 . wild type (WT). If Ca2+ ions are transported out of the cell by chaA during growth on calcium-rich substrates. DISCUSSION In studying cave microorganisms. The chaA mutant also grew poorly on B-4 media when compared to wild type. 1994). Escherichia coli. The pattern of both CaCO3 precipitation.5% yeast extract media amended with 4g/l CaCO3 . 2004. except GGC-D10A.2% C13 O2 . The nhaA control did not demonstrate a significant phenotype under any of the conditions used. Microbacterium esteraromaticum. In comparison. To determine whether the relationship between members of a clade and phenotype is sufficiently robust to be predictive of phenotype. likely due to the large amount of acid production necessary to observe this phenotype. they were initially identified based on a phenotype of precipitating or dissolving calcium carbonate (Figure 5). While the chaA and nhaA mutants grew as well as WT on TSA media. GGC-D10B1 and GGC-P11. the gene for the Ca2+ /2H+ antiporter chaA (Ivey et al. the source of CO2− ions for 3 subsequent CaCO3 precipitation remained unclear. typhimurium. 1993) was knocked out in S. these included S. in which the atmosphere was not supplemented with C13 O2 . which generated a very large zone of clearing. the crystals of D11 and D9A grown in the presence of a C13 O2 were highly enriched for the heavier isotope. 2001. many of the D-class isolates also precipitated CaCO3 crystals on top of their colonies during dissolution (Figure 5). chaA and nhaA strains were streaked on B4-media and a 0. the loss of cell viability using this method was confirmed by plating. with a slight enrichment for the lighter isotope (δ 13 Cvpdb = −12. it was noted that during the course of these experiments. The corresponding peaks are labeled. Representative XRD analysis of calcium carbonate crystals (from isolate P11) shows presence of vaterite. despite the fact that they were not isolated from caves. the precipitation capabilities of a random group of isolates killed with paraformaldehyde was tested. Dissolution activity was not so broadly represented. The Physiology of Calcium Carbonate Precipitation The close association between clade and phenotype shown in Figure 5 suggests that either a structural or genotypic relationship is responsible for the CaCO3 phenotypes observed. 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. dissolution and a combined dissolving/precipitating phenotype appeared to be similar for all isolates within a particular clade. We therefore tested whether at3 mospheric CO2 played a role in crystal formation using stable isotope probing.53‰ ± 0. 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. Klebsiella planticola. their growth was inhibited on media containing calcium (Figure 6). calcification on B-4 media could be restored by prior passage of the culture on media containing either calcium acetate or CaCO3 powder.

D. 5. The ∗ symbol indicates that crystals were also precipitated during dissolution. Phylogenetic placement of GGC cultivars with their corresponding calcite precipitation and dissolution phenotypes: = strong phenotype. while indicates a very high level of CaCO3 dissolution around the colony. . = intermediate phenotype.450 E.1 substitutions per site. Downloaded By: [Barton. BANKS ET AL. Hazel] At: 05:02 10 June 2010 FIG. 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. = absent. The scale bar represents 0.

1992). they were not detected elsewhere on the samples and only directly below the apparent microbial colonies. Nonetheless. therefore suggests that bacterial metabolism plays a dominant role in calficifaction. Yates and Robbins 1998). 2008. However. this would generate the calcium salt of that acid. The work of Barabesi et al. The ChaA antiporter. which contain numerous mineral precipitating polymeric surface structures (Ercole et al. 1993). 1992). (2007) has recently identified a potential role for fatty acid metabolism in CaCO3 precipitation by B. Lichstein and Soule 1943) or the ability of fermentation reactions to maintain cellular viability. many of these studies were carried out in Bacilli species. the majority of the CaCO3 dissolving isolates also re-precipitated CaCO3 on the surface of the colony during growth (Figures 2 and 5). Indeed. such as potas- sium cyanide and sodium azide. 1973). capability to precipitate calcium carbonate minerals. the metabolic activity of which may lead to the formation of organic acids. Past investigators have argued that passive structural components of the bacterial cell play the most important role in calcification (Barabesi et al. these can dissolve the host rock (CaCO3 ) as shown in Figure 2. In this study treatment with paraformaldehyde. The high distribution of this phenotype among these isolates in accordance with the results of previous speleothem cultivation studies (Danielli and Edington 1983). subtilis. can secrete these ions into the extracellular environment. Such studies have argued against a metabolic role in calcification. Similar clay accumulation under speleothem deposits has been observed in other caves (Barton and Northup 2007. This work. in a past study.BACTERIAL PRECIPITATION OF CALCIUM CARBONATE 451 FIG. however these studies killed cells by autoclaving or using metabolic poisons. An alternative approach for these cells could be to remove excess Ca2+ ions as they are excreted by the cell. any reduced compounds excreted by he cell are likely to be reassimilated as an energy source. but must do so against an ever-increasing concentration gradient (Anderson et al. In support of this hypothesis. Sleytr and Beveridge 1999) and have the least consistent precipitation phenotype (Figure 5). which regulates the β-oxidation of fatty acids to acetyl-CoA. Borsato et al. This could also explain the widespread CaCO3 precipitation phenotype among bacteria as Ca2+ ions are found in a wide variety of habitats. If such acids are produced. coli in the presence of calcite crystals (Bullen et al. In this study. The only exceptions to this were GGC-D10A. The dark zones seen on the CaCO3 media represents clearing of this mineral from the media. Blyth and Frisia 2008. While the exact role of this gene in CaCO3 precipitation remains to be elucidated. Indeed. Phenotypes of knockout mutants grown on B-4 media or glucose/yeast media containing CaCO3 . 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. Downloaded By: [Barton. While such clay deposits are also formed during the process of speleogenesis. 2007. we saw the assimilation of a number of metabolic products during the growth of E. The ability of metabolic poisons to kill bacterial cells can be limited by cyanide/azide resistant cytochrome oxidases (Cunningham and Williams 1995. in support of other investigators (Buczynski and Chafetz 1991). we suggest that such dissolution activity is further evidenced by the accumulation of insoluble clay residues underneath apparent microbial colonies on the host rock (Figure 1C–D). Wolfe 2005). as killed cells also promote CaCO3 precipitation (Martinez et al. If microbial activity in caves is dissolving the host rock. 2008). GGC-D10B1 and GGC-P11. these investigators carried out experiments on B-4 media containing calcium acetate. which maintains cellular structure by cross-linking proteins. via calcification. 2007. species that uptake these potential carbon and energy sources in calcium-rich environments must also deal with toxic Ca2+ ions (Anderson et al. prevented calcification on B-4 media. A mutation in fadD. we have previously observed the in situ production of both calcium acetate and calcium pyruvate in cave environments (Bullen et al. The survival of these species under such conditions is even more remarkable given the predominance of heterotrophic microbial species in caves. Under the nutrient limitation of cave environments. we found that a large number of our isolates remained viable following similar cyanide/azide treatments (data not shown). appeared to consistently kill the cells. allowing the dark background to be observed. however. It is hard to determine what consequence autoclaving would have on the chemistry of CaCO3 precipitation as it leads to cell lysis and gross structural changes. 2008). This approach may explain why our results were more consistent in ruling out a role for metabolic activity. Hazel] At: 05:02 10 June 2010 . 2000). Each of the mutants ( chaA and nhaA) is compared to wild-type (WT). In attempting to deduce a metabolic role for calcification where entombment would be ultimately detrimental to survival. which is conserved across many bacterial phyla (Ivey et al. Indeed. given that calcium acetate has been the staple of assessing the bacterial calcification phenotype since the 1970s (Boquet et al. it is important to examine the context of the environment in which the cell is found. This observation is significant. 6. both from the total number of species displaying this phenotype to the extent they can precipitate this mineral (Danielli and Edington 1983). as suggested by the presence of high-affinity acetate transporters encoded by a large number of bacterial species (Wolfe 2005).

while the growth of coralloids . 2003). past investigators have argued that calcification generates protons for nutrient acquisition. In the case of coralloids. Although the extrusion of Ca2+ ions would certainly contribute to bacterial precipitation of CaCO3 . bacterial metabolic activity in these environments appears to lead to the precipitation of various CaCO3 mineral forms on the microbial colony. to allow conservation of CO2 for gluconeogenesis. When the concentrations 3 of both Ca2+ and CO2− ions exceed the solubility product for 3 these ions (Ksp = 4. acetate is also used for fatty acid synthesis. Our stable isotope probing analyses suggest that atmospheric CO2 is contributing. 2001). 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. in part. Whatever the pathway. however. The glyoxylate cycle (also known as the glyoxylate bypass or glyoxylate shunt) used in the consumption of acetate for central metabolism. Our results therefore suggest that. This would also correlate well with the extreme nutrient limitation of cave environments. it is possible that they would be consumed as part of cellular metabolism (contributing to the proton motive force). the source of CO2+ ions is more difficult to discern.5 × 10−9 for calcite). This activity leads to a sharp increase in pH. affecting the overall CO2 budget for the cell. Together. 2003). Such activity would generate the distinct lines of coralloid formation often seen in caves with such airflow patterns. The role of microbial species in the development of secondary. 2003). The production of acetyl-CoA from fatty acid catabolism by FadD also requires the cell to use the glyoxylate bypass for growth (Figure 7). In addition to energy production. however. personal communication. Merlin et al.452 E. while oxaloacetate is consumed for gluconeogenesis. The putative roles of yadA and chaA are also shown. The chaA gene knock-out generated in this study and the observed phenotype on calcium containing media provides the first evidence for a link between calcium metabolism in bacteria and calcification. limiting the availability of CO2 for cellular processes such as fatty acid synthesis (Figure 7). Given that our results suggest that atmospheric CO2 is at least partially responsible for the CO2− ions found in the 3 calcium carbonate crystals. with the phenotype becoming more pronounced under nutrient limitation (McConnaughey and Whelan 1997). carbonate deposits in caves has remained controversial for quite some time (Barton et al. calcification occurs. 3 This may occur through the equilibrium of CO2 between the atmosphere and as bicarbonate ions in the medium surrounding the cell (Formula 1). Such activity would draw Equation 1 to the right and favor the formation of carbonate ions. Calcification experiments have also been carried out in ureolytic bacterial species. it is unclear how altering the cellular CO2 budget may affect this process. these bacterially deposited crystals can then form the nucleation site for subsequent mineral accumulation through abiotic processes (Banfield and Nealson 1997).33 ↔ HCO− + H+ ↔ CO2− + 2H+ H2 O + CO2 3 3 [1] Downloaded By: [Barton. D. The fate of the protons would influence subsequent carbonate ion formation. promoting calcite precipitation. acetate must enter the TCA cycle via the glyoxylic acid bypass. Using calcium acetate as a carbon and energy source for growth presents a problem for bacterial cells due to the assimilation of acetate. pKa = 6. which would aid in the transport of Ca2+ ions into the extracellular medium. BANKS ET AL. 2007). Alternatively. Indeed. especially given that 3 the carbonic anhydrase gene (yadF) appears to be an essential gene for a large number of bacterial species under normal atmospheric conditions (Kusian et al. with mostly anecdotal evidence of microbial association within speleothems (Baskar et al. these protons could be utilized by the ChaA Ca2+ /2H+ antiporter. In the formation of coralloids. 2002. which breakdown urea with the generation of ammonia (Hammes et al. CO2− ions to the precipitated minerals. such secondary processes may include the moisture-driven flow of CaCO3 saturated water from areas of dissolution to precipitation in caves containing convention airflow currents (M. 7.35 pKa = 10. 2006a) and the lack of a cause-and-effect rationale (Barton and Northup 2007). Hazel] At: 05:02 10 June 2010 FIG. the loss of the protons would generate more alkali conditions and favor the formation of CO2− ions. Queen.

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