Bacterial Calcium Carbonate Precipitation in Cave Environments: A Function of Calcium Homeostasis

Abstract

To determine if microbial species play an active role in the development of calcium carbonate (CaCO ₃ ) deposits (speleothems) in cave environments, we isolated 51 culturable bacteria from a coralloid speleothem and tested their ability to dissolve and precipitate CaCO ₃ . The majority of these isolates could precipitate CaCO ₃ 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 activity. Given growth of these species on calcium acetate, but the toxicity of Ca ²⁺ ions to bacteria, we created a loss-of-function gene knock-out in the Ca ²⁺ ion efflux protein ChaA. The loss of this protein inhibited growth on media containing calcium, suggesting that the need to remove Ca ²⁺ ions from the cell may drive calcification. With no carbonate in the media used in the calcification studies, we used stable isotope probing with C ¹³ O ₂ to determine whether atmospheric CO ₂ could be the source of these ions. The resultant crystals were significantly enriched in this heavy isotope, suggesting that extracellular CO ₂ does indeed contribute to the mineral structure. The physiological adaptation of removing toxic Ca ²⁺ ions by calcification, while useful in numerous environments, would be particularly beneficial to bacteria in Ca ²⁺ -rich cave environments. Such activity may also create the initial crystal nucleation sites that contribute to the formation of secondary CaCO ₃ deposits within caves.

Keywords:

• calcite
• calcium caves
• coralloids
• homeostasis
• speleothems

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).