Cave bacteria-induced amorphous calcium carbonate formation

Abstract

Amorphous calcium carbonate (Acc) is a precursor of crystalline calcium carbonates that plays a key role in biomineralization and polymorph evolution. Here, we show that several bacterial strains isolated from a Hungarian cave produce Acc and their extracellular polymeric substance (epS) shields ACC from crystallization. The findings demonstrate that bacteria-produced ACC forms in water-rich environment at room temperature and is stable for at least half year, which is in contrast to laboratoryproduced ACC that needs to be stored in a desiccator and kept below 10 °C for avoiding crystallization. the Acc-shielding epS consists of lipids, proteins, carbohydrates and nucleic acids. in particular, we identified large amount of long-chain fatty acid components. We suggest that ACC could be enclosed in a micella-like formula within the EPS that inhibits water infiltration. As the bacterial cells lyse, the covering protective layer disintegrates, water penetrates and the unprotected Acc grains crystallize to calcite. our study indicates that bacteria are capable of producing Acc, and we estimate its quantity in comparison to calcite presumably varies up to 20% depending on the age of the colony. Since diverse bacterial communities colonize the surface of cave sediments in temperate zone, we presume that Acc is common in these caves and its occurrence is directly linked to bacterial activity and influences the geochemical signals recorded in speleothems. Amorphous calcium carbonate (ACC) is known as a precursor phase of crystalline CaCO 3 that plays a key role during calcium carbonate precipitation and biomineralization 1. It is the least stable CaCO 3 modification that rapidly transforms to crystalline calcium carbonate polymorphs. Laboratory-synthesized ACC crystallization can be delayed by keeping the physisorbed H 2 O below the critical level and storing the material in a desiccator and keeping it below 10 °C 2. Additives such as Mg 2+ , phosphate, and organic macromolecules can retard its crystallization 3-5. According to Purgstaller et al. 6 , the metastability of Mg-ACC is associated with the formation kinetics (pH and the Mg/Ca ratio) of the less soluble crystalline phase, i.e., the physico-chemical conditions of the environment. Biogenic activity can also modify the physico-chemical conditions, and thus can enhance the preservation of ACC. It has been reported from tissues of various eukaryotic organisms and several organic molecules have been associated with its occurrence. According to Aizenberg et al. 7 , the skeletal parts of calcareous sponge Clathrina and the spicules of ascidian Pyura pachydermatina contain ACC and its formation is associated with polysaccharides and proteins enriched in glutamic acid (and/or glutamine), serine and glycine. ACC was also described from the intraskeletal organic matrix of numerous scleractinian corals 8 , the spicules of the embryos of Strongylocentrotus purpuratus sea urchins 9 and the shell of Biomphalaria glabrata snail embryos 10. Amines, glycosylated proteins and phosp...

Bacterial and abiogenic carbonates formed in caves–no vital effect on clumped isotope compositions

“…FTIR and micro-XRD analyses show that the 1–2 months old carbonate samples collected from the glass plates of the control and UV-illuminated samples are dominated by calcite. These analyses did not detect ACC, contradicting the observation that this amorphous phase occurs in relatively fresh (1–7 days) cave carbonate precipitates [ 15 ] and on microbial colonies [ 18 ].…”

“…Cultivation and cultivation-independent analyses demonstrate that the surface of dripstones is inhabited by a diverse bacterial community [ 18 , 35 ], but their role in the precipitation of cave carbonates remains uncertain. Although a small percentage of bacteria can be cultivated from environmental samples [ 36 ], germ count estimation methods are usually applied to detect microbial contamination on surfaces.…”

“…The flowstone occurrence (drill core BNT-2) is approximately 5 km away from the Baradla Cave and has been extensively monitored [ 20 ] and geochemically analyzed [ 17 ]. Additionally, the carbonate produced by cultivated bacteria ( Rhodococcus degradans strain BaTD-248 [ 18 ]) was treated with 5% NaClO solution to remove the agar medium used for cultivation. The treatment was conducted at room temperature in nitrogen atmosphere (to avoid artificial carbonate precipitation from air CO 2 ) for one day, after which the sample was washed 10 times in distilled water and dried overnight at 40°C.…”

“…At a preliminary stage of this pilot study, the flowstones yielded close-to-“equilibrium” clumped isotope compositions, while the stalagmites’ Δ 47 data were strongly shifted to lower values. Additionally, it was discovered that bacterial carbonate precipitation produced amorphous calcium carbonate in the Baradla Cave [ 15 , 18 ], which was preserved by a covering of an extracellular polymeric substance (lipids, proteins, carbohydrates, and nucleic acids, [ 18 ]). This observation raised the question of whether microbial carbonate production exerted a vital effect on clumped isotope compositions, or whether the low stalagmite Δ 47 values were attributed solely to abiogenic isotope fractionations.…”

“…They attributed the shift in Δ 47 values (up to –0.27‰) to ureolytic DIC (dissolved inorganic carbon) production in the bacterial cells. This mechanism can be expected in the bacteria thriving in the Baradla Cave, where heterotrophic bacterial strains that precipitate carbonate through ureolysis were identified [ 18 ]. The amount of microbial carbonate is estimated to be 10–50% from textural observations and Fourier Transform Infrared Spectroscopy (FTIR) [ 15 , 19 ].…”