PLEASE SCROLL DOWN FOR ARTICLE This article was downloaded by: [Barton, Hazel] On: 10 June 2010 Access details: Access Details: [subscription number 922886987] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37- 41 Mortimer Street, London W1T 3JH, UK Geomicrobiology Journal Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713722957 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: http://dx.doi.org/10.1080/01490450903485136 Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

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 1Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky 2Department of Chemistry, Northern Kentucky University, Highland Heights, Kentucky 3NASA Ames Research Center, Moffett Field, California To determine if microbial species play an active role in the de- 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 CaCO3. The majority of these isolates could precipitate CaCO3 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 C13O2 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 Keywords calcite, calcium caves, coralloids, homeostasis, speleothems INTRODUCTION 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 (H2CO3) 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 CO3��� 2 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 CO3��� 2 from HCO3 ��� and leads to calcification. Within cave environments, which lack 444