Biomediated precipitation of calcium carbonate metastable phases in hypogean environments: a short review

S. SANCHEZ-MORAL

Museo Nacional de Ciencias Naturales, CSIC, José Gutiérrez Abascal 2, 28006 Madrid, Spain

J.C. CAÑAVERAS

Departamento de Ciencias de la Tierra y del Medio Ambiente, Universidad de Alicante, Campus San Vicente del Raspeig, 03080 Alicante, Spain

L. LAIZ

C. SAIZ-JIMENEZ

Instituto de Recursos Naturales y Agrobiologia, CSIC, Apartado 1052, 41080 Sevilla, Spain

J.BEDOYA

L. LUQUE

Museo Nacional de Ciencias Naturales, CSIC, José Gutiérrez Abascal 2, 28006 Madrid, Spain

Abstract..

Natural precipitates of metastable polymorphs of CaCO₃, such as vaterite, are rarely found in nature, however they have been widely synthetised in laboratory under particular conditions (i.e. supersaturated solutions, relative high temperatures, etc.). By SEM and XRD we recognize vaterite spherulites from culturable microbial colonies isolated from hypogean environments. Spherical bodies (~10 m in diameter), probably composed of vaterite, occur in submilimetric microbial mats and biofilms on volcanic substrates (Saint Callixtus Catacombs, Rome, Italy) and karstic caves (Altamira, Candamo and Tito Bustillo caves, Spain, and Grotta dei Cervi, Italy) where cyanobacteria and actinomycetes are the major microbial components. These particles form beneath dense biofilms, where particular physicochemical conditions are developed by the microbial activity. Natural biofilms seems to generate microenvironments favoring the formation and preservation of metastable CaCO₃ polymorphs. This also shows a major role of microbes in processes of low-temperature alteration of different hypogean rock-substrates

Keywords: vaterite, monohydrocalcite, calcite, actinomycetes, karstic cave, catacombs

The research was supported by the European Commission, contract EVK4-CT2000-00028. This is part of the UNESCO/IUGS IGCP 448: “World Correlation on Karst Ecosystem”. We thank R. González for their excellent technical assistance. Many thanks are extended to Editor Ulrike Brehm and the two anonymous reviewers for their assistance and useful comments on the draft manuscript.

Address correspondence to Prof. J.C. Cañaveras, Dept. Ciencias de la Tierra y del Medio Ambiente, Universidad de Alicante, Ap. 99, 03080 Alicante, Spain. E-mail: jc.canaveras@ua.es

Microorganisms, particularly bacteria, inhabit all possible environments of the biosphere including subterranean ones, playing a role in geological processes such as mineral growth and dissolution, and influencing biogeochemical cycles (Krumbein, 1983; Ehrlich, 1998). In comparison with other environments, there are relatively few geomicrobiological studies dealing with hypogean environments (Northup et al., 1997; Northup and Lavoie, 2001). Microbes, are often harmful for cultural assets (e.g. Paleolithic paintings), because they are related to constructive (mineral precipitation) and destructive (substrate dissolution) processes affecting different substrates (host-rock, speleothems, paintings, etc.) (Monte and Ferrari, 1993; Saiz-Jimenez, 1995; Dornieden et al., 2000; Cañaveras et al., 2001). Therefore geomicrobiological studies are necessary to establish the role that microorganisms play in the microbial-mineral interactions that occur in hypogean environments, and to elaborate management tools for the conservation of show caves and tombs.

Laboratory experiments demonstrate the ability of bacteria isolated from caves to precipitate and dissolve carbonates (Danielli and Edington, 1983; Groth et al., 2001). However, the existence or recognition of the metastable phases in hypogean environments is rare and their organic or bioinduced origin is not always clear.

In the last years, our group has carried out multidisciplinary studies (geology, microbiology, hydrochemistry, microclimate) in different caves located in Spain, such as Altamira (Cañaveras et al., 1999; Sánchez-Moral et al., 1999), Tito Bustillo (Sánchez Moral et al., 1997; Cañaveras et al., 2001), Candamo (Hoyos et al., 1998), and Grotta dei Cervi, Italy (Groth et al., 2001). At present, we are carrying out similar studies in a different hypogean environment, the catacombs of Domitilla and Saint Callixtus in Rome, Italy. Microbial communities and evidences of microbial activity have been found in or on the different substrates sampled on Roman hypogea. This work documents the formation of metastable calcium carbonate mineral phases as an example of microbial-mineral interactions.

Materials and methods

Site

The four caves studied (Altamira, Tito Bustillo, Candamo and Grotta dei Cervi) are world-famous for having a remarkable collection of paleolithic and neolithic paintings and engravings. From a geologic point of view, all these caves are located in the unsaturated water zone of karstic systems where processes of dissolution/destruction of carbonate substrates (mainly limestone) are more important than precipitation/lithochemical reconstruction processes. The tunnels that constitute both Domitilla and Saint Callixtus catacombs were dug through the relatively soft volcanic sediments of Roma. These deposits are tuffs and the so-called pozzuolana (scoriae in an ash matrix) of alkaline and potash composition. Building materials are coating part of the hypogean surface, mostly with a decorative purpose. These materials are usually a mixture of rock fragments in a carbonated matrix forming a mortar and a thin layer of slaked lime or plaster over which some paints were made. These cover some cubicles, and crypts.

Mineralogy (XRD), chemical composition (A.A.S., ICP, EDS), porosity (Hg porosimetry) and petrographic features (SEM and transmitted light microscopy) of hypogean substrata have been characterized. Special attention was given to biofilm/substrate interfaces. Particular care was taken during sample preparation for SEM. Most of the samples were fixed in situ with glutaraldehyde and later, in the laboratory, were gradually dehydrated and dried to prevent the formation of artifacts.

Hydrochemical characterization of hypogean waters (dripping, condensation, etc.) were carried out. Temperature, electrical conductivity and pH of water samples were measured in situ, and CO₂, HCO₃^- and CO₃^2- contents were analyzed using standard titration methods. Further, chemical analyses were performed in the laboratory using a Perkin-Elmer atomic absorption spectrometer or by an ionic capillary electrophoresis technique with a QUANTA 4000 capillary electrophoresis instrument. Chemical speciations and geochemical calculations were obtained by means of the computer program PHRQPITZ (version 0.2, 1990), which is a modification of the original version from Plummer et al. (1988).

Microenvironmental data (temperature, CO₂ concentration, relative humidity, ²²²Rn concentration) were obtained from automatic in situ monitoring systems installed in the studied sites (Sánchez-Moral et al., 1999). The climatic data of the cave atmosphere were used in geochemical calculations. The results obtained over annual cycles in different caves showed that the microclimate conditions were stable and similar in relative air humidity, with some differences in temperature and carbon dioxide concentrations. The relative humidity values are always close to saturation point (above 95%). The mean air temperature in Grotta dei Cervi (about 18°C) is higher than that measured in Spanish caves (12.5-14.5°C), and the mean annual air CO₂ concentration is smaller than in the case of the Spanish caves. In order to study the microenvironment of the catacombs, a microclimate monitoring system, measuring the temperature (16-17°C), relative humidity (>95%) and CO₂ concentrations (1500-3500 ppm) was operating during 2001 for one day each in Domitilla (5 March) and Saint Callixtus (6 March). Since March 2002 a microclimate monitoring system, measuring lighting time, air/rock temperature, air relative humidity, CO₂ and ²²²Rn concentrations was installed in Saint Callixtus. One sensor was installed specifically in the Oceanus cubiculum for detecting temperature differences between the air and rock surface. The automatic recording system is programmed to store record every 20 minutes. First results recorded show that the temperature of the rock surface during the studied period (March-May) is always slightly higher than the air temperature ranging from 0.05 to 0.2°C. The combined effect of an increase in CO₂ concentration, water vapour emission and temperature variations induced by visitors can directly affect the intensity and even the development of bio-colonization. Biogeochemical cycles must be influenced by the continuous availability of water originated by means of induced condensation processes.

Sampling and sample location

Sampling have been carried out in Altamira, Candamo, Tito Bustillo and Grotta dei Cervi caves since 1997 and in Roman Catacombs (Domitilla and Saint Callixtus) since 2000 to investigate the microbial population and their interaction with hypogean substrate. Active stalactites, wall concretions, microbial mats and dripping waters from different areas were collected aseptically in sterile tubes and kept at 4°C for two days until microbiological analyses were carried out.

Isolation and identification

Samples were added to a saline solution (0.9% NaCl), serially diluted and spread on different culture media. The media were selected taking into account that actinomycetes were the most abundant microorganisms in these caves (Groth et al. 1999; Laiz et al. 2000; Groth et al. 2001) and that their colonies, distributed all over the caves, were partially visible to the naked eye. Tryptone soy agar (TSA), malt-yeast extract-agar, starch-casein-agar, glycerol-asparagin-agar and water-agar were used as culture media. The composition of the media is described elsewhere (Laiz et al. 1999; Groth et al. 2001). The plates were incubated at 28°C for at least 48 h, and up to 8 weeks to record fast and slowly growing bacteria respectively.

Characterization of the isolates

The isolates were classified by morphological and chemotaxonomic methods. Carbon utilisation (95 carbon sources) was tested using the Biolog-identification system, but only in a few cases was identification at the species level possible on the basis of the Biolog database.

Total cellular fatty acid methyl esters (FAME) were analyzed using the MIDI system in accordance with the protocols for cultures grown on solid medium and instrument specifications recommended by Microbial Identification System, Inc. Delaware, USA. Comparison with the Sherlock Standard Aerobe database allowed automatic identification of bacteria. This method was used for identification of bacteria other than actinomycetes. The identification of streptomycete isolates was performed using morphological and physiological methods as recommended by Shirling and Gottlieb (1966). Methods for chemotaxonomic analyses are described elsewhere (Groth et al. 1999).

Precipitation of salts

The isolates were tested for salt precipitation using B-4 medium composed of 2.5 g calcium acetate, 4 g yeast extract, 15 g agar in 1 L distilled water, pH 8 (Boquet et al., 1973). Precipitation was also tested in liquid medium for the isolates with higher precipitation capacity in order to collect the crystals for further analysis. SEM and EDX, X-ray diffraction and FT-IR were routinely used for this propose.

Crystal analysis

Three Acinetobacter sp. strains were isolated from Altamira cave. One isolate grew very fast and produced a large amount of crystals surrounding the colonies and visible after 24 h. After one week, the crystals were distributed all over the plate. This isolate was only able to use acetate and pyruvate but not citrate or glucose; the other two strains were able to used citrate.

In solid (agar) media structural and morphological analyses were performed on crystals collected from the colonies surface of a 20-day culture of the most active Acinetobacter isolate. In a similar way, crystals were obtained in the longest incubated plates from a Rhodococcus isolate. Calcium carbonate polymorphs were identified by FT-IR spectroscopy (Falini et al. 1996), SEM and X-ray diffraction. The mineral composition of the precipitates was characterized by XRD and EDS.

Results and discussion

Grouped or isolated vaterite hemispherical aggregates precipitated in laboratory cultures of Acinetobacter spp. when B-4 medium with calcium and/or magnesium acetate was used. Vaterite spherulites ranged from 5 to 20 mm in diameter and showed a radial internal structure (Figure 1A). Some of the spherulites were hollow (Figure 1B). The production of crystals only appeared when the Ca acetate/Mg acetate ratio was higher than 1, indicating the inhibiting role of magnesium in the precipitation of vaterite. When this ratio was lower than 1, no CaCO₃ crystals are produced. The inhibitory effect of Mg ions on carbonate precipitation by bacteria was also observed by Rivadeneyra et al. (1985). In Grotta dei Cervi samples, hemispheroidal vaterite was also obtained from Rhodococcus isolates (Groth et al., 2001). Similar hemispherical morphologies in CaCO₃ precipitates were obtained by Rivadeneyra et al. (1998) from soil bacteria. They suggested that the spherulites may be the end stage of a progressive calcification process. XRD analysis indicated that Acinetobacter spp. isolated from Altamira cave and Rhodococcus sp. from Grotta dei Cervi produced vaterite and calcite. XRD analysis of powdered samples carried out several days after collection revealed a decrease in the proportion of vaterite with respect to calcite in moist samples, indicating the partial transformation of unstable vaterite into stable calcite.

In solid culture media of Grotta dei Cervi isolates (Rhodococcus sp.), spherulitic crystals were also produced, but XRD analysis revealed monohydrocalcite as the main mineral composition. The spherulites range 30 to 70 mm in diameter and also showed a radial internal structure (Figure 1C).

Vaterite, a kinetically favored, metastable calcium carbonate polymorph, is rarely found in nature but it has been identified in hypersaline lakes (Giralt et al., 2001), fluvio-lacustrine sediments (Ito et al., 1999; Lu et al., 2000); drilling muds (Friedman and Schultz, 1994), mortars (Signorelli et al., 1996; Gleize et al., 2000); metamorphic aureoles (McConnell, 1960), urinary calculi (Prien and Fondel, 1947; Diaz-Espineira et al., 1995), and tissues of some organisms (Lowestam and Abbot, 1975; Falini et al., 1996; Lenaz and Miletic, 2000). Monohydrocalcite (CaCO₃·H₂O) is also a scarce Ca-carbonate mineral. It has been identified in carbonate-silicate microbialites (Léveillé et al, 2001), carbonate hardgrounds (Last, 1992), polymetallic mineralizations (Ridkosil et al., 1991), lake sediments (Bird et al., 1991) and salt efflorescences (Jones et al., 1999 and references therein). Vaterite and monohydrocalcite are also rare in hypogean environments, only having been reported in a few karstic caves as constituents of moonmilk, crusts or coralloids (Hill and Forti, 1997 and references herein).

Vaterite has been synthesized in the laboratory from oversaturated aqueous solutions (e.g.: Plummer and Busenberg, 1982; Easton and Claugher, 1986; Ogino et al., 1987), in organic media (e.g.: Mann et al., 1988; Pach et al., 1990; Falini 2000), in microbial cultures (Rivadeneyra et al., 1985; Groth et al, 2001), by electrocrystallization techniques (Gabrielli et al., 1999; Euvrard et al., 2000) or using gas-liquid-solid reactive crystallizers (Chen et al., 1997). Most vaterite crystals reported in laboratory experiment shows a globular or spherical morphology, ranging from a few microns to more than one hundred microns in diameter. Exceptions to this spherical morphology were mainly the result of experiments looking for habit modification or specific morphological changes (e.g. Sims et al., 1995; Dupont et al. 1997). In nature, vaterite commonly shows spherulitic and radial-fibrous arrangement (Friedman and Schultz, 1994; Giralt et. al 2001). Laboratory synthesis of monohydrocalcite crystals from bacterial isolates have also been reported (Rivadeneyra et al., 1998).

Vaterite is a metastable CaCO₃ polymorph. Due to its instability, vaterite irreversibly transform into more stable polymorphs (Bischoff, 1968; Ogino et al., 1987; Kralj et al., 1997). Factors influencing the nucleation and crystal growth of vaterite are complex. Experimental reports have shown that less stable carbonate polymorphs appear due to addition of impurities such as some inorganic ions (Tsuno et al. 2000) and organic substances (Kitano and Hood, 1965; Falini et al., 1996; Pach et al., 1990; Mann et al., 1988) or due to kinetic factors (Ogino et al., 1987; Gabrielli et al., 1999; Elfil and Roques, 2001). However, experimental data have also indicated some factors favoring the preferential precipitation of a stable CaCO₃ polymorph; these factors are, for example, organic substances (e.g. fulvic acid) and inorganic suspended impurities (e.g. montmorillonites) (Vdocic and Kralj, 2000; Kralj and Vdocic, 2000).

Laboratory experiments have revealed that vaterite preservation is favored by the presence of organic substances, such as amino acids or mucopolysaccharides (Manoli and Dalas, 2000). Vaterite preservation is also favored in phosphorus-enriched medium, because dissolved phosphorus inhibit the precipitation of calcite and/or aragonite; the stability of vaterite in the presence of phosphate ions was ascribed to the retardation of both the dissolution of vaterite and the crystallization of calcite (Katsifaras and Spanos, 1999).

SEM study of more than one hundred samples from caves and catacombs studied since 1997, has revealed the existence of microbial assemblages, directly or indirectly able to mediate a variety of constructive and/or destructive processes resulting in the formation of distinctive fabrics (Cañaveras et al., 2001). Constructive fabrics include calcified microbes and bioinduced crystalline precipitates.

Spheroidal bodies, similar to those synthesized in laboratory cultures, have been recognized at all the studied sites. In laboratory experiments, crystal nucleation occurs on a substrate where a complete sphere could not form resulting in a hemisphere. These spherical bodies, ~10 mm in diameter, were observed in association with biofilms and bacterial colonies (Figure 1E). Their uniformity in size and their presence in different substrates (geological and ecological) seem to indicate a microbial origin.

In the Saint Callixtus catacombs, a good example of these kind of spherical bodies association has been observed within a 100-150 mm thick subaerial biofilm, (Figure 1E). The biofilm structure consists of a 5-10 mm thick surface layer of dense filamentous mucus and a 100-140 mm thick needle mat layer. This mat is composed of aragonite needle-fiber crystals (up to 5 mm long, 0.75 mm wide) in a loosely packed random arrangement (Figure 1E). The spherical bodies are located in the lower part of the needle mat, in contact with the rock substratum, and are commonly associated with beaded filaments. The spheroids show a 0.5-2 mm thick coating composed of radially-arranged submicron-sized crystals of CaCO₃ that probably precipitated around spherical bacterial colonies (Figure 1F). Unfortunately, the abundance of these CaCO₃ spheroids is insufficient for discriminating their mineralogy using available methodology. Needle-fiber mats are probably related to biomineralization processes by actinomycetes (Cañaveras et al., 1999) or fungi (Callot et al., 1985; Verrecchia and Verrecchia, 94).

Karstic waters of the studied caves are mainly of Ca²⁺-HCO₃^- and Mg-Ca-HCO₃^- types. They have relatively high concentrations of total dissolved species, and the presence of high amounts of dissolved organic matter, NO₃^- and NH₄⁺ in these karstic waters is a consequence of infiltrational waters passing through fertiliser-enriched pastures before reaching the cavity (Sánchez-Moral et al., 1997; Hoyos et al., 1998; Sánchez-Moral et al., 1999; Cañaveras et al., 1999). Geochemical estimates indicate that hypogean waters (dripping water, condensation, etc.) are able to precipitate calcite or aragonite. However, for the precipitation of metastable Ca-carbonates phases, such as monohydrocalcite or vaterite, higher supersaturation is needed. As previously mentioned, organic substances, which can be present in biofilm structures, favor the precipitation of unstable carbonate phases (Falini et al., 1996; Pach et al., 1990; Mann et al., 1988).

Hydrochemical and microclimatic data confirm that Pco₂ values of waters in and around the microbial mats and spheroidal carbonate particles are lower than air Pco₂. Thus, precipitation of carbonate species must be related to some process controlling the CO₂ degassing of thewater. This phenomenon can only occur in a semi-closed carbonate system (biofilm) where the CO₂ removed from the water cannot be replenished from the cave atmosphere even though CO₂ solution in water from air is very fast. Recent studies have demonstrated that heterotrophic bacteria are capable of chemolithoautotrophic assimilation of CO₂, and that the carbon is incorporated into all major cell biopolymers (Buzolyova and Somov, 1999). Cañaveras et al. (1999) demonstrated the biomediated precipitation of hydromagnesite in crust and moonmilk deposits in Altamira cave. As a result, microbial activity is able to control the CO₂ content of intergranular water in biofilms, leading to supersaturation in both calcium and magnesium carbonate minerals. An example of the bacterial capability for sequestering CO₂ in solution is the enzyme carbonic anhydrase. Carbonic anhydrase, an enzyme that catalyzes the reversible hydration of CO_2, is widely distributed among phylogenetically and physiologically diverse prokaryotes. Carbonic anhydrase could be responsible for the consumption of CO₂ by heterotrophic bacteria in cave systems. This would indicate a far greater role for this enzyme in nature than previously recognized (Tripp et al. 2001). The dependence of bacteria on the presence of CO₂ for growth or for overcoming long lag times is known from long time, and CO₂ and HCO₃^- are required for growth (Smith and Ferry, 2000).

Conclusions

Several facts encourage us to consider as microbial induced precipitates some carbonate particles found in hypogean environments in association with heterotrophic bacteria. They probably consist of metastable calcium carbonate composition, such as vaterite. (i) Some of the naturally occurring precipitates on the studied hypogean substrates are essentially identical to those produced in the laboratory. The similarity between experimental and natural spherical particles (~ 10 m size spheroids) can be considered as a demonstration of an active process of bacterial carbonate precipitation. (ii) Samples collected from these sites were used to isolate microorganisms able to produce crystal precipitates in laboratory experiments. Although the vaterite composition of natural spheroids have not been analytically tested, hydrochemical, microclimatic, petrological and microbiological data seem to indicate that small quantities of CO₂ must be consumed by heterotrophic microorganisms in order to explain the physico-chemical modifications of intergranular fluids that lead to create supersaturation conditions suitable for neoformation of metastable minerals (with the highest solubility). This kind of spherical particle could be a typical representation of the precipitation of metastable carbonate mineral phases in hypogean environments.

References

Bird MI, Chivas AR, Radnell CJ, Burton HR. 1991. Sedimentological and stable-isotope evolution of lakes in the Vestfold Hills, Antarctica. Palaeogeogr Palaeoclim Palaeoecol 84: 109-130.

Bischoff JL. 1968. Catalysis, inhibition and the calcite-aragonite problem. II The vaterite-aragonite transformation. Amer J Sci 266: 80-90.

Boquet E, Bordonat A, Ramos Cormenzana A. 1973. Production of calcite crystals by soil bacteria is a general phenomenon. Nature 246:527-528.

Buzolyova, L.S., Somov, G.P. 1999. Autotrophic assimilation of CO₂ and C₁-compounds by pathogenic bacteria. Biochemistry (Moscow) 64:1146-1149.

Callot G, Guyon A, Mousain D. 1985. Inter-relations entre aiguilles de calcite et hyphes mycéliens. Agronomie 5: 209-216.

Cañaveras JC, Hoyos M, Sanchez-Moral S, Sanz-Rubio E, Bedoya J, Soler V, Groth I, Schumann P, Laiz L, Gonzalez I, Saiz-Jiménez C. 1999. Microbial communities associated with hydromagnesite and needle-fiber aragonite deposits in a karstic cave (Altamira, Northern Spain). Geomicrobiol J 16:9-25.

Cañaveras JC, Sánchez-Moral S, Soler V, Saiz-Jiménez C. 2001. Microorganisms and microbially induced fabrics in cave walls. Geomicrobiol J 18: 223-240.

Chen P-Ch, Tai CY, Lee KC. 1997. Morphology and growth rate of calcium carbonate crystals in a gas-liquid-solid reactive crystallizer. Chem Engin Sci 52: 4171-4177.

Danielli HM, Edington MA. 1983. Bacterial calcification in limestone caves. Geomicrobiol J 3:1-16.

Diaz-Espineira M, Escolar E, Bellanato J, Medina JA. 1995. Crystalline composition of equine urinary sabulous deposits. Scan Microsc 9: 1071-1079.

Dornieden Th, Gorbushina AA, Krumbein WE. 2000. Biodecay of cultural heritage as a spece/time-related ecological situation- an evaluation of a series of studies. Int Biodeter Biodegrad 46: 261-270

Dupont L, Portemer F, Figlarz M. 1997. Syntehesis and study of a well crystallized CaCO₃ vaterite showing a new habitus. J Mater Chem 7: 797-800.

Easton AJ, Clauger D. 1986. Variations in a growth form of synthetic vaterite. Mineral Mag 50: 332-336.

Ehrlich HL. 1998. Geomicrobiology: its significance for geology. Earth-Sci Rev 45: 45-60.

Elfil H, Roques H. 2001. Role of hydrate phases of calcium carbonate on the scaling phenomenon. Desalination 137: 177-186.

Euvrard M, Filiatre C, Crausaz E. 2000. A cell to study in situ electrocrystallization of calcium carbonate. J Cryst Growth 216: 466-474.

Falini G. 2000. Crystallization of calcium carbonates in biologically inspired collagenous matrices. Int J Inorg Mat 2: 455-461

Falini G, Albeck S, Weiner S, Addadi L. 1996. Control of aragonite or calcite polymorphism by mollusk shell macromolecules. Science 271:67-69.

Friedman GM, Schultz DJ. 1994. Precipitation of vaterite (CaCO₃) during oil field drilling. Mineral Mag 58:401-408.

Gabrielli C, Maurin G, Poindessous G, Rosset R. 1999. Nucleation and growth of calcium carbonate by an electrochemical scaling process. J Cryst Growth 200: 236-250.

Giralt S, Juliá R, Klerkx J. 2001. Microbial biscuits of vaterite in Lake Issyk-Kul (Republic of Kyrgyzstan). J Sed Res 71: 430-435.

Gleize P, Silva DA, Nappi S. 2000. Ancient rendering mortars from a Brazilian place. Its characteristics and microstructure. Cement Concrete Res 30: 1609-1614.

Groth I, Vettermann R, Schuetze B, Schumann P, Saiz-Jimenez, C. 1999. Actinomycetes in karstic caves of Northern Spain (Altamira and Tito Bustillo). J Microbiol Meth 36:115-122.

Groth I, Schumann P, Laiz L, Sanchez-Moral S, Cañaveras JC, Saiz-Jimenez, C. 2001. Geomicrobiological study of the Grotta dei Cervi, Porto Badisco, Italy. Geomicrobiol. J. 18:241-258.

Ito T, Matsubara S, Miyawaki R 1999. Vaterite after ikaite in carbonate sediment. Ganko 94: 176-182.

Hill CA, Forti P. 1997. Cave minerals of the world. Huntsville: National Speleological Society.

Hoyos M, Soler V, Cañaveras JC, Sanchez-Moral, S, Sanz-Rubio E. 1998. Microclimatic characterization of a karstic cave: human impact on microenvironmental parameters of a prehistoric rock art cave (Candamo Cave, northern Spain). Environ Geol 33:231-242.

Jones MS, Wakefield RD, Forsyth G. 1999. The occurrence of rare minerals on decayed medieval Scottish building sotone colonised by biological growths. Mat Constr 49 (256): 3-14.

Katsifaras A, Spanos N. 1999. Effect of inorganic phosphate ions on the spontaneous precipitation of vaterite and on the transformation of vaterite to calcite. J Cryst Growth, 204:183-190.

Kitano Y, Hood DW. 1965. The influence of organic material on the polymorphic crystallization of calcium carbonate. Geochim Cosmochim Acta 29: 29-41.

Kralj D, Brecevic L, Kontrec J. 1997. Vaterite growth and dissolution in aqueous solution III. Kinetics of transformation. J Cryst Growth 177: 248-257.

Kralj D, Vdovic N. 2000. The influene of some naturally occurring minerals on the precipitation of calcium carbonate polymorphs. Wat. Res. 34:179-184.

Krumbein WE. 1983. Microbial Geochemistry. Blackwell Sci Publ. Oxford. 315 pp.

Last WM. 1992. Petrology of modern carbonate hardgrounds fron East Basin Lake, a saline maar lake, Southern Australia. Sedim Geol 81: 215-229.

Laiz L, Groth I, Gonzalez I, Saiz-Jimenez C. 1999. Microbiological study of the dripping waters in Altamira cave (Santillana del Mar, Spain). J Microbiol Meth 36:129-138.

Laiz L, Groth I, Schumann P, Zezza F, Felske A, Hermosin B, Saiz-Jimenez C. 2000. Microbiology of the stalactites from Grotta dei Cervi, Porto Badisco, Italy. Int Microbiol 3:25-30.

Lenaz D, Miletic M. 2000. Vaterite otoliths in freshwater fishes of the Lower Friuli Plain (NE Italy). Neues Jb Miner Monat 11: 522-528.

Levéillé RJ, Fyfe WS, Longstaffe FJ. 2000. Geomicrobiology of carbonate-silicate microbilites from Hawaiian basaltic sea caves. Chem Geol 169: 339-355.

Lowestam HA, Abbot DP. 1975. Vaterite: a mineralization product of the hard tissues of a marine organism (Ascidiacea). Science 188: 363-365.

Lu G, Zheng C, Donahoe RJ, Berry Lyons W. 2000. Controllong processes in a CaCO3 precipitating stream in Huanglong Natural Scenic District,Sichuan, China. J Hydrol 230: 34-54.

Mann S, Heywood BR, Rajam S, Birchall JD. 1988. Controlled crystallization of CaCO₃ under stearic acid monolayers. Nature 334:692-695.

Manoli F, Dalas E. 2000. Spontaneous precipitation of calcium carbonate in the presence of chondroitin sulfate. J Cryst Growth 217: 416-421.

McConnell JDC. 1960. Vaterite from Ballycraigy,Larne, Northern Ireland Mineral Mag 32: 535-544.

Monte M, Ferrari R. 1993. Biodeterioration in subterranean environments. Aerobiologia 9:141-148.

Northup DE, Lavoie KH. 2001. Geomicrobiology of caves: a review. Geomicrobiol J 18: 199-222.

Northup DE, Reysenbach A, Pace N. 1997. Microorganisms and speleothems. In Hill C, Forti P (eds): Cave Minerals of the World. NSS, Huntsville: 261-266.

Ogino T, Suzuki T, Sawada R. 1987. The formation and transformation mechanism of calcium carbonate in water. Geochim Cosmochim Acta 51: 2757-2767.

Pach L, Hrabe Z, Komarneni S, Roy R. 1990. Controlled crystallization of vaterite from viscous solutions of organic colloids. J Mater Res 5: 2928-2932.

Plummer LN, Busenberg E. 1982. The solubilities of calcite, aragonite and vaterite in CO₂-H₂O solutions between 0 and 90ºC, and an evaluation of the aqueous model form the system CaCO₃-CO₂-H₂O. Geochim Cosmochim Acta 46: 1011-1040.

Plummer LN, Parkhurst DL, Fleming GW, Dunkle SA. 1988. PHRQPITZ, a computer program incorporating Pitzer's equations for calculation of geochemical reactions in brines. US Geological Survey Water Resources Investigation Report 88-4153. Washington: Goverment Printing Office.

Prien EL, Frondel C. 1947. Studies in urolithiasis; I. The composition of urinary calculi. J Urology 57: 949-994.

Ridkosil T, Sejkora J, Ondrus P. 1991. Monohydrocalcite from polymetallic vein of the Vranice Deposit, near Pribram, Czechoslovakia. Neues Jb Miner Monat 7: 289-295.

Rivadeneyra MA, Ramos-Cormenzana A, García-Cervigón A.1985. Etude de l'influence du rapport Mg/Ca sur la formation de carbonate par des bacteries telluriques. Can J Microbiol 31: 229-231.

Rivadeneyra MA, Delgado G, Delgado R, Ramos-Cormenzana A. 1998. Biomineralization of carbonates by Halomonas eurihalina in solid media with different salinities: crystal formation sequence. Res Microbiol 149: 277-287.

Saiz-Jimenez C (Ed.) 1995. The deterioration of monuments. Special Issue, Sci Total Environ 167, 400 pp.

Sánchez-Moral S, Cañaveras JC, Sanz-Rubio E, Hoyos M, Soler V. 1997. Hydrogeochemical characterization of Tito Bustillo Cave (northern Spain) Proceedings of the 12^th International Congress of Speleology, Neuchâtel, Suisse. Vol 2: 103-108.

Sánchez-Moral S, Soler V, Cañaveras JC, Sanz-Rubio E, Van Grieken R, Gysels K. 1999. Inorganic deterioration affecting the Altamira Cave, N Spain: quantitative approach to wall-corrosion (solutional etching) processes induced by visitors. Sci Total Environ 243/244: 67-84.

Shirling EB, Gottlieb D. 1966. Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313-340.

Signorelli S, Peroni C, Camaiti M, Fratini F. 1996. The presence of vaterite in bonding mortars of marble inlays from Florence Cathedral. Mineral Mag 60: 663-665.

Sims SD, Didymus JM, Mann S. 1995. Habit modification in synthetic crystals of aragonite and vaterite. J Chem Soc Chem Commun: 1031-1032.

Smith KS, Ferry JG. 2000. Prokaryotic carbonic anhydrases. FEMS Microbiol Rev. 24: 335-366.

Tripp BC, Smith K, Ferry JG. 2001. Carbonic anhydrase: new insights for an ancient enzyme. J Biol Chem 276: 48615-48618.

Tsuno H, Kagi H, Akagi T. 2000. Effects of trace lanthanum ion on the stability of vaterite and transformation from vaterite to calcite in an aquatic system. Goldschmidt 2000 Journal of Conference Abstracts 5(2): 1022-1023.

Vdovic N, Kralj D. 2000. Electrokinetic properties of spontaneously precipitated calcium carbonate polymorphs: the influence of organic substances. Colloids Surf 161: 499-505.

Verrecchia EP, Verrecchia KE, 1994. Needle-fiber calcite: a critical review and a proposed classification. J Sedim Res A64: 650-664

Figure caption.

Figure 1. Scanning electron micrographs showing Ca-carbonate precipitates. A) Vaterite hemispherulite showing fibro-radial internal structure (Acinetobacter sp., Altamira Cave, grown in liquid medium, scale bar: 5 mm). B) Hollow vaterite hemispherulite (Acinetobacter sp., Altamira Cave, grown in liquid medium, scale bar: 5 mm). C) Monohydrocalcite spherulites in agar media (Rhodococcus sp., Grotta dei Cervi, scale bar: 50 mm). D) Spheroidal particle in Altamira cave (scale bar: 5 mm). E) Spheroidal particles associated with needle-fiber aragonite crystals and beaded filaments. Biofilm from Saint Callixtus Catacombs (scale bar: 5 mm). F) Detail of spheroidal particle fragments. Saint Callixtus Catacombs (scale bar: 5 mm).

Figure 1