Microbial Communities Associated With Hydromagnesite and Needle-Fiber Deposits in a Karstic Cave (Altamira, Northern Spain)

J. C. CANAÄ VERAS Departamento Ciencias de la Tierra y del Medio Ambiente Universidad de Alicante Alicante, Spain and Museo Nacionales de Ciencias Naturales C.S.I.C. Madrid, Spain

M. HOYOS S. SANCHEZ-MORAL E. SANZ-RUBIO J. BEDOYA Museo Nacionales de Ciencias Naturales C.S.I.C. Madrid, Spain

V. SOLER Instituto de Productos Naturales y Agrobiologia de Canarias C.S.I.C. La Laguna, Spain

I. GROTH Hans-Knoll-InstitutÈ fuer Naturstoff-Forschung e.V. Jena, Germany Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 P. SCHUMANN Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Braunschweig, Germany

(continued)

Received 13 January 1998; accepted 23 September 1998. This work was supported by the European Commission, project ENV4-CT95-0104. Dr. J. A. Lasheras, Director ofAltamira Cave Research Centre and Museum, isacknowledged for his invaluablecollaborationthrough- out the whole period of investigation. Hydrogeochemical analyses were conducted with the assistance of M. A. Vallejo and M. I. Ruiz (MNCN-CSIC). R. Gonzalez (MNCN-CSIC) assisted with mineralogical determinations. The manuscript bene®ted greatly from critical comments provided by two anonymous reviewers. Address correspondencetoDr. Juan C. Canaveras,Ä Departamento Ciencias delaTierra y del MedioAmbiente, UniversidaddeAlicante, Campus San VincentedelRaspeig, E-03080,Alicante, Spain.E-mail: [email protected]

Geomicrobiology Journal, 16:9±25, 1999 Copyright C 1999 Taylor & Francis ° 0149-0451/99 $12.00 + .00 9 10 J. C. CanaverasÄ et al.

L. LAIZ I. GONZALEZ C. SAIZ-JIMENEZ Instituto de Recursos Naturales y Agrobiologia C.S.I.C. Sevilla, Spain

Microbial communities, where Streptomyces species predominate, were found in asso-

ciation with hydromagnesite, Mg5(CO3)4(OH)2 4H2O, and needle-®ber aragonite de- posits in an Altamira cave. The ability to precipitate¢ calcium carbonate in laboratory cultures suggests that these and other bacteria present in the cave may play a role in the formation of moonmilk deposits.

Keywords actinomycetes,Altamira cave, aragonite, biomineralization,, heterotrophic bacteria, hydromagnesite,moonmilk, , vaterite

Hydromagnesite, Mg5(CO3)4(OH)2 4H2O, a rare mineral in most caves, has been reported as a common constituent of moonmilk¢ deposits in caves; it is found as well in popcorn globulites, ªcauli¯owerº crusts, and exotic such as cave balloons, an extremely short-lived speleothem always associated with moonmilk (Fischbeck and MullerÈ 1971; Hill and Forti 1986; Ford and Williams 1989). On the other hand, aragonite and calcite are the most abundant cave minerals. Although needle-®ber habit is very common in carbonate soils and calcretes, it has also been reported in cave environments (Jones and Kahle 1993; Verrecchia and Verrecchia 1994). Microbial communities and evidences of microbial activity have been found in or on speleothems and underlying substrates in many karstic caves (Caumartin and Renault 1958; Williams 1959; Ohde and Takii 1978; Jones 1995). Processes of disintegration and forma- tion of speci®c types of speleothems such as cave saltpeter, mineral oxides accumulations, coloration,and moonmilk deposits have been related to microbial activity (White 1981; Hill and Forti 1986).Apparentlytwo main processes canbeinducedorcontrolledbymicroorgan- isms: destructive processes causing deterioration or surface disintegration of speleothems and other substrates, such as rock paintings; and constructive processes that fade or cover speleothems, rock paintings,and engravings. The aim of this article is to investigate the role of microorganisms in the formation of speci®c types of speleothems in an Altamira cave Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 and to consider their implications in the conservation of valuable prehistoric paintings.

Materials and Methods

Sampling Site The Altamira cave we studied, located near Santillana del Mar (northern Spain), was de- veloped on a small calcareous hill (at 158 m above sea level) composed by a succession of subhorizontal decimeter-thick beds of Cretaceous calcarenitic limestones (Valle et al. 1978). These limestones correspond to fossiliferous packstones-grainstones that are partly dolomitized (Hoyos 1983). Thin (2±10 cm thick) marly and clayey intercalations are found among the calcareous beds. The cave is placed in the vadose zone of a tabular polygenic system karst, in the so- called senile area of the karst, in which the processes of destruction are more important Microbial Communities in a Karstic Cave 11

than the processes of sedimentation and lithochemical reconstruction (Hoyos 1983). The hydrological dynamics in this cave are attributable exclusively to the rainwater that seeps directly into the cave through different strata of the ceiling. This Altamira cave possesses a remarkable collection of Paleolithic rock paintings and engravings along the halls and galleries; quite exceptional are those located in the Poly- chromes Hall, which is known as the Sistine Chapel of Quaternary art. The mineralogical, petrographical, and microbiological studies are based mainly on samples collected from moonmilk deposits and crusts situated on the walls and ceiling of the Polychromes Hall and the gallery to the Big Hall (Figure 1).

Petrological Analysis X-ray diffraction was used to determine the mineral composition in powdered samples. Petrographical conclusions were based on the examination of fresh-broken samples with a Philips scanning electron microscope (SEM) equipped with an energy-dispersive X-ray analytical system (EDS).

Water Analysis The water samples were collected in 200-mL bottles that had been prewashed in doubly distilled water. The temperature, electrical conductivity, and pH were measured when the 2 sample was taken; CO2, HCO3¡ , and CO3 ¡ contents were determined by using standard titration methods. These samples were stored at a constant temperature (12±13±C) until chemical analyses were performed in the laboratory with a Perkin-Elmer atomic absorption spectrometer. Chemical speciations and geochemical calculations were obtained by means of the computer program PHRQPITZ (version 0.2, 1990), which is a revision of the original version from Plummer et al. (1988).

Environmental Parameters An automatic in situ monitoring system for measuring microenvironmental parameters (e.g., temperature, relative humidity, carbon dioxide concentration, radon) installed in the Altamira cave provided climatic data of the cave atmosphere, which we used in the geo- chemical calculations. Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011

MicrobiologicalAnalysis For microbiological studies we selected two sampling points in the passage to the Big Hall (Figure 1): F-3/4, composed of 85% hydromagnesite and 15% calcite, and F-5, composed of 90% calcite, 5% hydromagnesite, and 5% aragonite. Samples were collectedin sterile tubes and were dividedinto two sets. One was used for direct inoculationina varietyofculturemedia: TSA; malt±yeast extract (MEY) (Laiz 1991); starch±casein (SC) (KusterÈ and Williams1964);glycerol±asparagin (GA) (Dietz and Thayer 1980); humic acid±agar (HA) (Hayakawa and Nonomura 1987); and water±agar (AA). Also, 1 g of sample was resuspended in sterile distilled water, shaken for 30 min, and inoculated in plates containing MEY for enumeration. The plates were incubated at 28±C for 48 h in both cases, although they were maintained for 8 weeks for slowly growing microorganisms. Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 12 z<

• WATER SAMPLES

.... MICROBIOLOGICAL SAMPLES X HYDROMAGNESITE

.&. ARAGONITE (CALCITE) NEEDLE-FIBERS

o 10 20 30m

FIGURE 1 Map of Altamira cave with location of samples collected. Microbial Communities in a Karstic Cave 13

To study the in¯uence of salt concentrations, we used a MEY medium with increasing concentrations of NaCl or MgSO 7H O. 4 ¢ 2

Identi®cation Individual colonies were randomly isolated and puri®ed by -plating onto TSA until pure-culture plates were obtained. Isolates were characterized by their morphological and physiological properties. Because most isolates were streptomycetes, the methods recom- mended in the International Streptomyces Project (Shirling and Gottlieb 1966) and forming the basis of the classi®cation of the genus Streptomyces in the eighth edition of Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons 1974) were applied. These practical approaches include, however, a limited number of intuitively chosen diagnostic features (Man®o et al. 1994).Therefore, identi®cation on the species level was possible only in some cases, and it was supported by comparison of the relevant isolates with the follow- ing type strains: S. roseoviridis (DSM 40175T), S. tuirus (DSM 40505T), S. xanthophaeus (DSM 40134T), S. ¯avogriseus (IMET 43576T), and S. ¯avotricini (IMET 42057T).

Precipitation of Salts All isolates were tested for salt precipitation by using B-4 medium composed of (per liter) 2.5 g of calcium acetate, 4 g of yeast extract, 10 g of glucose, and 15 g of agar at pH 8 (Boquet et al. 1973). Precipitation was also tested in liquid medium for the isolates with higher precipitation capacity to collect crystals for further analysis. SEM with EDS, X- ray diffraction, and Fourier-transform infrared spectroscopy were routinely used for this purpose as described above.

Results

Hydromagnesite Deposits Hydromagnesite crystals have been found in the walls and ceilings of several halls and galleries in Altamira cave (Figure 1). They occur in moonmilk deposits, which mainly cover walls, ceilings, or other speleothems, such as popcorn globulites, and in thin crusts partly covering rock paintings in the ceiling of Polychromes Hall. Moonmilk deposits vary in thickness, ranging from 0.2 to 2 cm, whereas crusts rarely are thicker than 1±2 mm.

Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 When hydromagnesite is associated with popcorn globulites, these are usually composed of closely packed rhombohedral calcite crystals and radially arranged prismatic crystals of aragonite. Figure 2 shows the main occurrence patterns of hydromagnesite deposits. In the moonmilk deposits the hydromagnesite crystals are usually platy or blade-like in shape and are arranged as spherular or rosette-like clusters (resembling miniature ªdesert rosesº) (Figure 3A) or as ªhouse of cardsº aggregates. In the aggregates, two types of hydromagnesite crystal shapes are differentiated: small (2±10 mm wide) rectangular to trapezoidal crystals, and elongated subhexagonal crystals (5±80 mm wide). The individual crystals show smooth faces and generally are 0.1±1 mm thick. The spherular or rosette aggregates are usually composed of hexagonal platy crystals, 1±5 mm in diameter. In the ceiling of the Polychromes Hall two types of spatially related hydromagnesite (with or without other hydrous magnesium carbonates) crusts or coatings can be distin- guished: milky gelatinous coatings, formed by a loosely to tightly packed mosaic of spher- ical aggregates 1±3 mm in diameter (Figure 3B) associated with a dense mucous bio®lm, and transparent hard crusts composed by botryoidal or colloform aggregates, 100±500 mm 14 J. C. CanaverasÄ et al.

FIGURE 2 Scheme of the occurrence of hydromagnesite and aragonite deposits in Altamira cave. Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 in diameter (Figure 3C). The spherular aggregates or clusters are composed of stacked platelets, most of them rhombic in form and 0.2±1 mm thick; the botryoidal aggregates consist of closely packed interleaved sheet-like crystals in a radial arrangement. Small (1± 2 mm thick), disperse rhombohedric calcium carbonate crystals are recognized in the ®rst type of crust. Moonmilk deposits associated with these crusts are composed of spherular aggregates similar to those described before.

Needle-Fiber Crystals Accumulations of needle-®ber crystals, mainly composed of aragonite, usually occur as white nodular ®ne-grained (moonmilk) in association with popcorn globulites in the walls of several galleries and halls in the cave (Figure 1). Some of these walls are arti®cial, erected during conditioning works in the cave around 1940±50. The ®bers usually show a random microstructural arrangement, forming loosely to packed random meshes or bundles of crystals (Figure 3D). Microbial Communities in a Karstic Cave 15

FIGURE 3 (A) Rossette aggregates formed by bladed-like hydromagnesite crystals. (B) Mucous bio®lm embedding spherular clusters of hydromagnesite crystals. (C) Botryoidal aggregatescomposinghard hydromagnesitecrusts.(D) Needle-®beraragonitecrystal giving a random structure. (E) Detail of a paired monocrystalline rod. (F) Polycrystalline ®ber composed of stacked rhombohedra.

Many types of crystalline morphologies of needle-®ber crystals have been found in the Altamira cave. They can be grouped in monocrystalline rods and polycrystalline chains. The rod surfaces are usually smooth, although some rods have slightly lumpy surfaces. These monocrystalline habits include single rods and paired rods that have a cross-section

Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 in the shape of an 8 (Figure 3E) or an X. Smooth single rods are 0.5±1 mm wide and 50 mm long.Polycrystalline®bers composed of chainsof ªen echelonº-stacked (Figure 3F), overlapping,staggeredrhombohedra.Polycrystallinechainsshow variabledimensions,with widths of 2±20 mm and lengths of 10±100 mm. Rhombohedron chains composed of stag- gered crystals usually show an irregular surface of spaced ridges and grooves, similar to those described by Jones and Ng (1988). Needle-®bers usually show epitaxial precipitates perpendicular to the growth axis or may be partly or completely encased with small (<2 mm long) calcium carbonate crystals. Single bodies or clusters of minute, calci®ed spherical-to-irregularbodiesplastered onto the ®bersurfaces are also recognized.All these ªconstructiveºfeatures modify the original crys- tal form and, in many cases, impede reliable recognition and classi®cation of needle-®bers.

Microbial Films Both hydromagnesite crystals and aragonite (calcite) needle-®bers are usually embedded in mucous or ®lamentous microbial ®lms. The former consist of thin (<5 mm) masses of 16 J. C. CanaverasÄ et al.

FIGURE 4 (A) Number of colony-forming units/g in sample F-3/4 vs increasing con- centrations of sodium chloride, after 72 h and after 1, 3, and 5 weeks. (B) Number of colony-forming units/g in sample F-3/4 vs increasing concentrationsof epsomite, after 72 h and after 1, 3, and 5 weeks.

organic materials (e.g., polysaccharides,proteins) that partly cover or embed the underlying substrate. These mucous bio®lms are relatively abundant in the gelatinous crusts composed

Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 of hydromagnesite (or other hydrous magnesium carbonates) in the ceiling of the Poly- chromes Hall (Figure 3B), although they also are present in the moonmilk deposits. The ®lamentous bio®lms generally consist of densely interwoven masses of irregular branching ®laments. The constituent®laments are 0.1±0.2 mm wide, can be up to 5 mm long, and usu- ally have smooth surfaces. The ®lamentousbio®lms are relatively abundantin the moonmilk deposits, especially those composed of calcium carbonate needle-®bers. Because bio®lms are complex and highly hydrated structures (Lawrence et al. 1991; Caldwell et al. 1992), one must address the problem of artifact production in this kind of SEM study. Prepar- ing samples for SEM may cause dehydration artifacts of two apparent bio®lm structures: mucoid or ®lamentous. This type of ®lamentous structure in bio®lms has generally been interpreted as an artifact (Costerton et al. 1995).

Hydrochemistry Table 1 shows the chemical composition of ®ve karstic waters belonging to dripping sites of several galleries of the Altamira cave (Figure 1). These dripping waters correspond Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011

TABLE 1 Chemical analyses (mg/L) of karstic waters

2 2+ 2+ + + + Sample Date T (±C) pH HCO3¡ SO4 ¡ Cl¡ Ca Mg Na K NH4 NO3¡ PAS(1) 2/5/96 14.5 6.97 797.0 12.0 56.0 260.7 20.3 10.5 9.1 3.6 0.4 PAS(2) 5/20/96 13.8 7.72 493.9 50.5 7.5 141.1 12.9 8.8 9.3 n.d. n.d. PAS(3) 5/21/96 13.5 7.45 475.6 51.3 7.5 132.8 12.4 8.6 9.0 n.d. n.d. EN(1) 2/5/96 13.5 7.88 242.0 58.0 47.0 112.0 14.0 22.2 8.1 0.0 85.4 EN(2) 5/20/96 13.7 7.67 320.1 54.5 5.5 86.2 16.1 15.5 7.9 n.d. n.d. PH(1) 2/5/96 13.5 7.79 291.0 48.0 29.4 106.3 7.9 12.7 5.2 0.0 16.1 PH(2) 5/20/96 13.6 7.85 326.2 55.1 5.0 94.9 9.0 13.3 3.7 n.d. n.d BH(1) 5/20/96 13.6 7.84 295.7 35.7 3.5 89.8 5.9 10.6 2.9 n.d. n.d. BH(2) 5/21/96 13.5 7.79 295.7 36.6 3.0 90.1 5.9 10.3 2.9 n.d. n.d. PC 5/21/96 13.6 7.91 305.4 24.1 5.0 32.0 38.0 9.1 6.1 0.0 22.2 See Figure 1 for location of samples. 17 18 J. C. CanaverasÄ et al.

to sampling campaigns carried out in February and May 1996 and are considered to be representativeofthe hydrochemistryof Altamira cave. The chemicalanalysesof percolation water samples at different points of the cave indicate that the karstic waters are mainly of

the Ca-HCO3¡ type with a relatively high concentration of total dissolved species; in the Polychromes Hall (PC), however, the waters are of the Ca-Mg-HCO3¡ type. In the Passage to the Big Hall (PAS) and in the Entrance (EN), the magnesium concentration is slightly higher than in the Well Hall (PH) and the Big Hall (BH). The relatively high contents of Cl and Na in the waters are related to the proximity of the Altamira cave to the Cantabrian Sea and re¯ect the input of sea salts, an aerosol effect. In addition to all of the aforementioned analytical determinations, we also analyzed + chemical pollutants in four samples (Table 1). The NO3¡ and K contents of these samples indicate that the in®ltrating waters pass through fertilizer-enriched pastures (Moore 1991) + before reaching the cavity. The high NH4 concentration (3.6 mg/L) in the PAS(1) sample is consistent with the presence of high amounts of dissolved organic matter in these karstic + waters (R. van Grieken, personal communication). The presence of NH4 in both surface and subsurface waters, together with PO4, Fe, and Mn, is considered a chemical signal of + recent contamination, because the NH4 has not yet oxidized to nitrites or nitrates.

Microbial Communities Figures 4 and 5 show the total number of colony-forming units (CFU) in samples F-3/4 and F-5. The number of colonies in F-5 was 30 times higher than on F-3/4. In addition, the colonies of F-3/4 grew more slowly and showed a great difference in number in subsequent weeks. The microbial communities in these samples were moderately halophilic,and the num- ber increasedin the presenceofa lowpercentageofNaCl (1%); when the NaCl was increased to 5%, however, growth was practicallyabsent. The isolatesseems to adapt better to increas- ing amounts of Mg2+ than of Na+ , as denoted the growth seen in epsomite concentrations up to 15%. A total of 30 different colonies were isolated from F 3-4, and 32 from F 5, from which 97% and 84%, respectively,were gram-positive.Of these,73%and 63%, respectively, were identi®ed as actinomycetes on the basis of their clear morphological characteristics.

Bacteria Identi®cation Chemotaxonomy is generally considered a valuable tool for delineating genera within the order Actinomycetales (Embley and Stackebrandt 1994; Stackebrandt et al. 1995). The

Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 actinomycetes are classi®ed according to the presence or absence of the isomers of di- aminopimelic acid (DAP) in the peptidoglycan. Thus, all isolates identi®ed by the occur- rence of LL-DAP in combinationwith the menaquinonesof the type MK-9(H4), MK-9(H6), and MK-9(H8) were members of the genus Streptomyces (Table 2). Streptomyces species are the dominant bacteria in all Altamira cave walls and ceilings (Groth et al. 1997; Laiz et al. 1997),in contrastto those isolated from drippingwaters, where gram-negative bacteria dominate (Laiz et al. 1997). In addition,in sample F-3/4, an isolate with white aerial mycelium and the menaquino- nes MK-9(H4), MK-7(H4), MK-10(H4), and MK-8(H4) was assigned to the genus Amyco- latopsis. An isolate from sample F-5 grew very slowly and formed a black soluble pigment on oatmeal agar, developing a white aerial mycelium that changed to gray with age and was completely fragmented into irregular ovoid spores with a smooth surface. In addition, the hyphae had a characteristic zig-zag morphology. Menaquiones MK-9(H4), MK-8(H4), MK-7(H4), and MK-10(H4) were found in ratios of79:9:6:4,respectively.These data placed the isolate into the genus Saccharothrix. Microbial Communities in a Karstic Cave 19

FIGURE 5 (A) Number of colony-forming units/g in sample F 5 vs increasing concen- trations of sodium chloride, after 72 h and after 1, 3, and 5 weeks. (B) Number of colony- forming units/g in sample F 3-4 vs increasing concentrations of epsomite, after 72 h and after 1, 3, and 5 weeks.

Crystal Precipitation Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 Fifty percent of the isolates from sample F 3-4 and 34% of those from F 5 produced crystals in medium B-4 (Figure 6). These percentages are higherthan those observed inisolatesfrom the Polychromes Hall and its gallery (Laiz et al. 1997). Some of the isolates characterized by crystal precipitationare Streptomyces rishiriensis, Amycolatopsissp., Acinetobactersp., and Chryseomonas sp.

Discussion Hydromagnesite is recognized as the ®nal-phase mineral in a Mg-rich mineral sequence in many caves (Hill and Forti 1986). Its formation is mainly related to Mg-enriched solu-

tions (Mg/Ca ratio > 16 according to Fischbeck and MullerÈ 1971) and low PCO2 values

(at high PCO2 values, nesquehonite is the stable mineral phase). These conditions are con- sistent with highly alkaline waters and high evaporation conditions, as is the case of some ªhydromagnesiteº lakes (Walter et al. 1973; Braithwaite and Zedef 1996). Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 20

TABLE 2 Actinomycetes with LL-diaminopimelic acid in the cell wall

Utilization of carbon sourcesa Aerial Substrate Soluble Melanin Identi®cation mycelium mycelium pigment production Ara Glu Fru Ino Man Raf Rha Sac Xyl Sample F-3/4 Streptomyces sp. Absent Yellowish + + + ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ Streptomyces sp. Gray Dark brown + + + + ¡ ¡ ¡ ¡ ¡ ¡ ¡ /¡ Streptomyces sp. Reddish gray Yellowish + + + + + + + + ¡ ¡ /¡ /¡ /¡ ¡ /¡ /¡ Streptomyces sp. Pale reddish gray Yellowish brown + + + + ¡ ¡ ¡ ¡ ¡ ¡ ¡ S. ¯avotricini Reddish gray Brown + (brown) + + + ¡ ¡ ¡ ¡ ¡ ¡ ¡ Sample F-5 S. rishiriensis Gray Gray, red + (red) + + + + + + + + + ¡ S. xanthophaeus Gray Yellow + + ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ S. xanthoplaeus Pale gray Yellow + + ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ S. ¯avotricini Gray Yellowish + + + ¡ ¡ /¡ ¡ ¡ ¡ ¡ ¡ ¡ Streptomyces sp. Dark grey Gray brown + + + + + + ¡ ¡ ¡ ¡ ¡ S. cinereoruber Gray Gray, olive + + + + + + + ¡ /¡ ¡ ¡ ¡ /¡ aAra: arabinose, Glu: glucose, Fru: fructose, Ino: inositol, Man: mannose, Raf: raf®nose, Rha: rhamnose, Sac: saccharose, Xyl: xylose. Microbial Communities in a Karstic Cave 21

FIGURE 6 Isolates of samples F-3/4 and F-5 and ability to precipitate calcium carbonate.

Although hydromagnesite is a common constituent in moonmilk Altamira deposits, and an organic origin has never been ruled out, most authors consider hydromagnesite moonmilks to be an example of inorganic precipitation from karstic waters. To check this possible inorganic origin, taking into account the pH, temperature, and chemical composi-

tion of the sampled waters (Table 1), we calculated the saturation states and PCO2 values of the karstic waters at the Altamira cave (Table 3). The geochemical calculation was carried out with the PHRQPITZ program (Plummer et al. 1988). The data obtained indicate that all analyzed samples are oversaturated with respect to calcite, aragonite, and dolomite but are strongly undersaturated with respect to hydromagnesite and others magnesium carbonates such as nesquehoniteand . The conditions of high alkalinity,which are necessary for the precipitation of hydromagnesite,seem to be reached only by means of a strong decrease

in the PCO2 of the karstic waters. In the Altamira cave, the water supply is exclusively from the direct in®ltration of rainwater; thus, before entering the cave, this water can dissolve the CO2 produced in the upper soil horizons by biological degradation of organic matter. When this water reaches

TABLE 3 Saturation indices of main phases and PCO2 values of the analyzed waters Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 Saturation Index PCO2 Sample (atm) Aragonite Calcite Vaterite Hydromagnesite Huntite

1.16 PAS(1) 10¡ + 0.32 + 0.52 0.11 15.41 3.64 2.12 ¡ ¡ ¡ PAS(2) 10¡ + 0.62 + 0.82 + 0.20 12.54 2.16 1.87 ¡ ¡ PAS(3) 10¡ + 0.32 + 0.51 0.11 14.31 3.36 2.59 ¡ ¡ ¡ EN(1) 10¡ + 0.38 + 0.57 0.04 12.59 2.70 2.26 ¡ ¡ ¡ EN(2) 10¡ + 0.20 + 0.39 0.22 12.97 2.92 2.42 ¡ ¡ ¡ PH(1) 10¡ + 0.36 + 0.56 0.06 13.96 3.44 2.43 ¡ ¡ ¡ PH(2) 10¡ + 0.43 + 0.62 0 13.13 2.89 2.46 ¡ ¡ BH(1) 10¡ + 0.36 + 0.56 0.06 14.20 3.61 2.41 ¡ ¡ ¡ BH(2) 10¡ + 0.32 + 0.51 0.11 14.51 3.81 2.51 ¡ ¡ ¡ PC 10¡ + 0.01 + 0.21 0.41 9.64 1.26 ¡ ¡ ¡ 22 J. C. CanaverasÄ et al.

the cavity, partial outgassing is produced by the imbalance between air CO2 and water CO2 contents. This ªinorganicº outgassing mechanism is effective until the equilibrium is 3.0 1.55 attained.PCO2 valuesinthe cave atmosphererange between 10¡ and10¡ atm; however,

the PCO2 value necessary to reach the hydromagnesite saturation in the PC sample, the most 4.96 Mg-rich water, was calculated as 10¡ atm. Thus ªinorganicº outgassing does not seem a feasible mechanism for causing hydromagnesite precipitation. Nonetheless, before a water 4.96 sample can reach PCO2 = 10¡ atm, the precipitation of calcium carbonate (aragonite and/or calcite) takes place. On the other hand, the relative humidity values in the cave atmosphere ranges from 95% to 100% and for the majority of the year is at saturation point; therefore, evaporationprocesses also can beruled out as being responsiblefor the outgassing and carbonate precipitation.All these facts seem to indicate that microbial activity may play a role, directly or indirectly, in the precipitation of hydromagnesite deposits. Hydromagnesite crystals are usually partly embedded in bio®lms, and several calcite/ vaterite-precipitating bacteria have been isolated from either dripping waters or hydro- samples. In addition, Mann et al. (1988) reported that the use of stearic acid monolayers in controlled crystallization of calcium carbonate from supersaturated solu- tions gives rise to formation of oriented vaterite. Fatty acids are cellular components of bacteria, and free (extractable) lipids typically account for 5±10% of the dry cell mass. Nevertheless, the activity of microorganisms must be re¯ected as a modi®cation of water

chemistry, creating conditions of high alkalinity, low PCO2 , and a high Mg/Ca ratio, which are suitable for hydromagnesite precipitation. Growth of heterotrophicbacteria that use organic carbon from dissolved organic matter provokes an increase in alkalinity in their microenvironment and favors hydromagnesite stability. However, hydromagnesite precipitation also requires a previous precipitation of calcium carbonate phases to achieve a high Mg/Ca ratio in the solution. In vitro studies revealed the capacity for calcium carbonate (vaterite, aragonite, and calcite) precipitationof a large number ofbacteria isolated from environmentalsamples. Various bacterial metabolic activities, such as carbon dioxide ®xation, nitrate reduction, sulfate reduction, or oxidative deamination of amino acids, may induce calcium carbonate precipitation (Simkiss and Wilbur 1991), and all of these are possible in the Altamira cave environment. Geochemical calculations based on the hydrochemistry and microclimatic character- istic of the Altamira cave show that inorganic precipitation of calcium carbonate mineral phases is possible (Table 3). Polycrystalline chains have been attributed to inorganic pro- cesses under evaporation conditions,and the mineralogy (aragonite) and the acicular habits 2+ + 2 are controlled by inhibition action of adsorbed ions (Mg , Na , SO4 ¡ ), clays, or organic matter (James 1972; Pouget and Rambaud 1980; VergesÁ et al. 1982; Ducloux et al. 1984; Downloaded by [Fac Psicologia/Biblioteca] at 03:46 21 July 2011 Verrecchia and Verrecchia 1994). In our case, all of these ªinhibition factorsº favoring aci- cular habits are available in the karstic waters and substrates; moreover, the microclimatic conditions and hydrochemistry of karstic waters are in agreement with the precipitation of aragonite ®bers as a part of the mineral sequence of the cave. Thus, an inorganic origin for polycrystalline chains cannot be rejected. Nevertheless, the common association between aragonite needle-®ber and microbial activity (heterotrophic bacteria and bio®lms) leads us to consider an organic vs inorganic origin for all or part of the types of needle-®bers identi®ed. This type of crystal is extensively reported in the literature (Jones and Kahle 1993; Verrecchia and Verrecchia 1994 and references therein) and is commonly associated with plants, fungi, and bacteria. For this reason, needle-®ber crystals have been frequently interpreted as being biogenic in origin. Inorganic precipitationof needle-®bers or acicular crystals has been achieved in labora- tory experiments, revealing that different factors, such as ¯uid saturation rates and temper- ature, control the process (Strickland-Constable 1968). However, these experiments do not Microbial Communities in a Karstic Cave 23

necessarily indicate that biological precipitation may be ruled out. Bacterially induced cal- cium carbonate crystals have been precipitated in culture studies (Krumbein 1968; Boquet et al. 1973; Monger et al. 1991, Laiz et al. in press). The af®nity between Ca2+ and organic substances and the capability of microorganisms to foster or catalyze the precipitation of calcium carbonate are well known (Thompson and Ferris 1990; Merz 1992; Folk 1993). These studies indicate that microbial activity would be able to directly or indirectly modify the physicochemicalcharacteristics of ¯uids and create conditionssuitable for precipitation of needle-®ber . On the other hand, an inorganicorigin would ªexplain neither the structural organization nor the variety of [these] habitsº (Verrecchia and Verrecchia 1994). Monocrystalline rods are attributed to bacterial calci®cation or to precipitation in the interior of fungal mycelium bundles in different conditions (Boquet et al. 1973; Callot et al. 1985, Phillips and Self 1987; Verrecchia and Verrecchia 1994). The ®nding of mycelia in cave samples has usually been attributed to fungi. However, microscopic observation of mycelia cannot always be attributed to fungi, as myceliar morphology is also typical of actinomycetes, a term that now encompasses a wide range of bacteria. In fact, sporoactino- mycetes are bacteria with branching hyphae and specialized spore-bearing structures, and nocardioform actinomycetes present a fragmenting substrate mycelium and limited aerial mycelium that can bear chains of arthrospores. These bacteria are widely distributed in caves and represent the most abundant microorganisms, as reported here. A preliminary differentiation between actinomycete and fungal mycelium can be achieved by noting the morphology and diameter of hyphae, which in Streptomyces (usually the most abundant genus in caves) is <1±2 mm in diameter, and in fungi is between 3±5 and 6±10 mm in diameter. The ®nding of hyphae of 0.1±0.2 mm (Figure 3E), the identi®cations provided in Table 2 and elsewhere (Groth et al. in press; Laiz et al. in press), and the ability of actinomycetes to precipitate calcium carbonates indicate that the role of actinomycetes in the colonization of speleothems and cave rock surfaces and in the processes of mineral precipitation should emerge from the neglect they have faced until now.

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