This article was downloaded by: [Nanyang Technological University] On: 07 May 2012, At: 20:33 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.tandfonline.com/loi/ugmb20

Precipitation of Carbonates by Bacteria from a Saline Soil, in Natural and Artificial Soil Extracts
Jess Prraga , Mara Angustias Rivadeneyra , Juan Manuel Martn-Garca , Rafael Delgado & Gabriel Delgado
a a a a b c

Departamento de Edafologa y Qumica Agrcola, Facultad de Farmacia, Universidad de Granada, Spain

b c

Departamento de Microbiologa, Facultad de Farmacia, Universidad de Granada, Spain

Departamento de Geologa, Facultad de Ciencias Experimentales, Universidad de Jan, Spain Available online: 17 Aug 2010

To cite this article: Jess Prraga, Mara Angustias Rivadeneyra, Juan Manuel Martn-Garca, Rafael Delgado & Gabriel Delgado (2004): Precipitation of Carbonates by Bacteria from a Saline Soil, in Natural and Artificial Soil Extracts, Geomicrobiology Journal, 21:1, 55-66 To link to this article: http://dx.doi.org/10.1080/01490450490253464

PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, 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, 21:5566, 2004 Copyright C Taylor & Francis Inc. ISSN: 0149-0451 print / 1362-3087 online DOI: 10.1080/01490450490253464

Precipitation of Carbonates by Bacteria from a Saline Soil, in Natural and Articial Soil Extracts
Jesus P rraga,1 Mara Angustias Rivadeneyra,2 Juan Manuel Martn-Garca,3 a Rafael Delgado,1 and Gabriel Delgado1
1

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

Departamento de Edafologa y Qumica Agrcola, Facultad de Farmacia, Universidad de Granada, Spain 2 Departamento de Microbiologa, Facultad de Farmacia, Universidad de Granada, Spain 3 Departamento de Geologa, Facultad de Ciencias Experimentales, Universidad de Ja n, Spain e

The precipitation of carbonates by populations of bacteria from a saline soil cultivated in both solid and liquid culture media was studied. The soil was a Gley-Gypsic-Carbonated Solonchak from near an inland saltern in Granada, Spain. Two types of soil extract were prepared: 1:1 extract and saturation extract. The soil samples used for the bacteriological analysis were taken in situ from the A and C soil horizons. The culture media were made from (1) soil extract, (2) articial soil solutions, and (3) articial solutions of marine salts. The geochemical analysis of the solution was carried out using the program PHREEQC. Samples of puried bioliths were examined by X-ray diffraction and were observed with SEM. We found that the mineral precipitated from the natural and articial soil extracts was calcite. Calcite was also found in the secondary accumulations of this soil. However in the media derived from marine salts, mixtures of calcite, magnesian calcite, and aragonite were precipitated, depending on the Mg2+ concentration, the [Mg]/[Ca] molar ratio, and the type of medium. The morphology of the minerals precipitated shows bacterial involvement in the process of pedogenic bioprecipitation. The precipitation studied could be regarded as a process of induced biomineralization. Keywords biomineralization, biogenic carbonates, pedogenic carbonates, saline soil, soil bacteria, soil solution

INTRODUCTION Cases of bacterial and nonbacterial origins of carbonates in natural and experimental media have been studied in the literReceived 1 October 2002; accepted 3 April 2003. This study was supported by the Spanish Ministry of Science and Technology projects no. PB98-1361.0 and REN2003-09136. SEM images were carried out by the Servicio de Microscopa Electr nica de o la Facultad de Farmacia (University of Granada). We thank Robert Abrahams for translating the original manuscript into English. Address correspondence to Gabriel Delgado, Departamento de Edafologa y Qumica Agrcola, Facultad de Farmacia, Universidad de Granada, Campus Cartuja, 18071 Granada, Spain. E-mail: gdelgado@ ugr.es

ature and extensive reviews of published citations were compiled by Wright and Tucker (1991), Ehrlich (1995), Verrecchia et al. (1995), Castanier et al. (1999), Folk (1999), Kirkland et al. (1999), Riding and Awramik (2000), Warren et al. (2001) and Belnap and Lange (2001). The study of carbonate precipitation in soils is of great importance as it not only helps us to understand the genesis of soils but also to understand the pedogenic trapping of CO2 in the form of carbonates (Lal et al. 1999). In pedogenic environments the origin of the accumulations of secondary carbonates (pedogenic carbonates) has been traditionally ascribed to inorganic processes of precipitation (Gile et al. 1966; Rabenhorst et al. 1991). More recently some authors have referred to a biogenic precipitation (biomineralization processes); they include Edinger (1990), Monger (1990), and Monger et al. (1991), who reported this in Aridisols in California and New Mexico; Phillips et al. (1987) and Bruand and Duval (1999), who described calcied laments in calcretes of South Australia and a petrocalcic horizon in Beauce (France), respectively, and Amit and Harrison (1995), who described biogenic calcic horizons in Nizzana sand dunes (Israel). The biogenic precipitation in soil could involve any of several types of microorganisms among which invariably bacteria are found. Some workers studied carbonate precipitation by soil bacteria in articial culture media (Boquet et al. 1973; Rivadeneyra et al. 1997, 2000) and in soil columns (Monger 1990), but, until now, direct experimental evidence for microbial calcication in soil solution is lacking. Soil solution is dened as the aqueous liquid phase of the soil and its solutes (Soil Science Society of America 1978). According to Wolt (1994) one could consider soil solution as the hub about which other interacting compartments as biotic, liquid, solid, and gas phases of the soil revolve. As a result of these relationships it may seem that the biogenic precipitation of carbonates in soil must be related to the soil solution. The soil solution that represents the real status of this is that corresponding 55

56

J. PARRAGA ET AL.

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

to eld moisture at a given moment, that is to say, variable in time (Rhoades 1990). Obtaining a representative sample of this solution is very complicated and historically standard soil extracts have been used to facilitate comparisons between different experiments (Wolt 1994). The most common soil extract has been the saturation extract (or saturated paste extract) and extracts of soil:water in the ratios of suspensions 1:1, 1:2 and 1:5, (w:w) (Richards 1982). Geochemical analysis using models such as PHREEQC (Parkhust and Appelo 1999) is a useful tool to predict the mineral species precipitated inorganically in aqueous media and is also extremely useful for comparing the processes of biomineralization with other inorganic processes. Previously, Rivadeneyra et al. (1993, 1997, 1999) have studied the carbonate precipitation by different groups of moderately halophilic bacteria (many of these isolated from saline soils) cultivated in articial culture media with different concentration of salts. Furthermore, P rraga et al. (1998) examined the possible a involvement of bacteria isolated from a saline soil in the authigenic formation of carbonates, using different types of soil solution as culture media. The intention with this work was to extend the study of biological carbonate precipitation in soils. The precipitation of carbonates by bacteria from a saline soil, using culture media prepared from natural and articial soil solution (soil extract) was investigated. Carbonate precipitation in culture media of soil extracts was compared with that in other articial culture media (media derived from solution of marine salt). The nature of the minerals precipitated in solid and liquid media was also compared. In this study it was found which culture media that would be the most suitable for future study of carbonate precipitation in soil by bacteria. MATERIAL AND METHODS Soil Samples (Horizons and Solutions) A and C horizons from a saline soil (Gypsic Aquisalid, Soil Survey Staff 1999 or Gley-Gypsic-Carbonated Solonchak, FAO 1998) close to an inland saltern (Malah , Granada, Spain), were a used. The characteristics of the soil (Table 1) were studied. Morphological features of soil horizons were studied in accordance with the Soil Survey Manual (Soil Survey Staff 1993). The procedures used in analysis are outlined by American Society of Agronomy and Soil Science Society of America (Page et al. 1982; Klute 1986): granulometric analysis was carried out by sieving and/or sedimentation (Robinson pipet); organic carbon content by the dichromate oxidation method; the nitrogen content by Kjeldhal method; CaCO3 equivalent by volumetry with Bernards calcimeter; pH in a 1:1 ne earth:water and ne earth:ClK 0.1 M suspensions (w/w); electrical conductivity (EC) in the saturation extract; water retention at 33 and 1500 kPa using a pressure membrane apparatus; the exchangeable bases (Ca2+ , Mg2+ , Na+ , K+ ) and cation exchange capacity (CEC) by

the ammonium acetate method (pH 7) and the sodium chloride method. The soil was chosen due to the presence of nodules and concretions of carbonates in A and C horizon and because its bacterial populations have been studied by Del Moral et al. (1987). They are comprised of moderately halophilic bacteria whose capacity for carbonate precipitation have been investigated previously in MH marine salts media (Ferrer et al. 1988; Rivadeneyra et al. 1991, 1994). The concretions and nodules present in the soil were studied by X-ray diffraction (XRD) using the technique and equipment described in the section Study of the crystals. Two types of soil extract were prepared; a 1:1 extract (1:1E) and a saturation extract (SE). The 1:1E was prepared by means of a soil/water suspension (1:1, w:w) and agitated for 24 h at 90 cycles/seg on a reciprocating shaker (Selecta, Rotatherm). The extracts were centrifuged at 2,000 rpm for 1 h using a Hermle ZK 510 refrigerated centrifuge with the temperature adjusted to 25 C. Saturation extract (SE) was prepared from saturated soil paste (Richards 1982) using a B chner lter funnel and lu tration ask connected to a vacuum pump with rotating blades in a oil bath (Telstar 2G-6, 6 m3 /h ow and 4 104 mbar). The displaced solutions were ltered through a cellulose acetate membrane lter (0.20 m pore size) and stored under refrigeration until all analyses were completed. All operations were carried out within 48 h of sampling. The determination of the ion concentration in soil extracts (Ca2+ , Mg2+ , Na+ , K+ , Cl , SO2 ) by chemically suppressed ion chromatography (Dionex 4 DX-120 IC) using conductivity detection and cation/anion exchange colums (CS12 and AS4A). Alkalinity was determined by titration with standard acid (HCl). Articial extracts (Art.1:1E and Art.SE) having the same ionic composition as the corresponding soil extracts, 1:1E and SE, were also prepared, their pH being adjusted to that of the natural soil extracts (7.27.4, Table 2). All chemicals were purchased from Sigma Chemical Co. (analytical grade). Geochemical Study The geochemical analysis of the solutions was carried out using the geochemical computer program PHREEQC Ver.2 (Parkhust and Appelo 1999). The activity of dissolved species was determined and the degree of saturation evaluated. The results of the PHREEQC are presented in terms of the saturation index (SI) for each predicted mineral, where SI = lg (IAP/Ksp ). IAP is the ion activity product of the dissolved mineral constituents in a solubility product (Ksp ) for the mineral. Thus, SI > 0 implies supersaturation with respect to the mineral, whereas SI < 0 means undersaturation. Micro-Organisms The experiments were performed with bacterial populations from the saline soil. The soil samples used for the

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

Table 1 Soil characteristicsa pH Bases and CEC (cmol (+) kg1 )

Horiz. White 23.0 0.39 0.04 22.33 8.1 7.6 16.5 0.99 0.10 20.73 7.7 7.3

Depth (cm) Structure

Color Munsell (moist) 13.8 47.0

Mottling Munsell (moist)

Sand Clay OC N CO2 EC SE H33 H1500 3 (%) (%) (%) (%) (%) H2 O ClK (dSm1 ) (%) (%) Na+ K+ Ca2+ Mg2+ CEC 24.6 27.8 10.9 13.4 1.7 198.0 12.7 38.8 2.3 116.1 3.4 8.1 10.1 10.4

Ah

Subangular 27.4 blocks Cgyk >20/27 5Y 5/27.5Y White and Massive and 11.8 5.5/0 10YR 6/6 laminar

020/27 2.5Y 4.5/2

General characteristics: Latitude: W 3 43 ; longitude: N 37 5 ; altitude: 700 m; orientation: W; slope: 35%; parent material: tertiary sediments of silty marl, gypsiferous and carbonated; vegetation: halophile (Suaeda sp., Salsola sp., Limonium sp., etc); annual precipitation: 508 mm; soil temperature regime: mesic; soil moisture regime: xeric. a Analytical values referred to ne earth fraction (<2 mm) at 105 C. Abbreviations: OC = organic carbon; N = total nitrogen; EC SE = electrical conductivity of the saturation extract; H33 = soil humidity (w/w) at 33 kPa; H1500 = soil humidity (w/w) at 1,500 kPa; CEC = cation exchange capacity.

57

58

J. PARRAGA ET AL.

Table 2 Composition of the soil extracts (soil solution) and solutions of marine salts at 2.5% and 7.5% as analyzed by ion chromatography Ion composition (m M) Solution/medium 1:1 extract (1:1E) Horiz. Ca
2+

Mg

2+

Na

Cl

SO2 4 12.33 24.29 21.64 66.72 24.35 73.93

HCO * 3 0.64 0.54 0.99 1.50 0.23 0.69

pH 7.3 7.4 7.2 7.4 7.2 7.2

Mg2+ /Ca2+ (molar) 0.10 0.50 0.10 0.50 1.65 4.75

Salinity (%) 0.7 2.3 1.0 6.2 2.5 7.5

A C Saturation extract (SE) A C Solution of 2.5% salts marine salts 7.5% salts

21.90 2.11 55.56 0.50 84.00 12.43 6.17 299.89 0.79 108.01 38.43 3.71 97.49 0.88 147.39 34.09 16.93 815.43 2.16 298.73 26.02 43.03 386.12 7.75 433.57 27.50 130.73 1172.47 23.53 1316.53

HCO by titration with standard acid. 3

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

bacteriological analysis were taken in situ from the A and C horizons with a sterile spatula and collected in a sterilized ask. Most of the halophilic strains appeared to be typical moderate halophiles. Del Moral et al. (1987) described six groups of moderately halophilic bacteria in this soil: Vibrio, DeleyaPseudomonas, Acinetobacter, Flavobacterium, Paracoccus, Planococcus and several nonclassied strains. Culture Media Table 3 shows the composition of all the culture media used in this study. Media based on the soil extracts described previously (1:1E and SE), both natural and articial, in addition to articial halophilic media (MH) with a solution of salts equivalent to a marine medium (Subow 1931) were used. Proteose-

pectone, yeast extract, and glucose, in different proportions were added. The articial seawater solution of Subow was a mixture of the following salts: NaCl; MgCl2 6H2 O; MgSO4 7H2 O; CaCl2 2H2 O; KCl; NaHCO3 ; and NaBr. Calcium acetate 4 g/L was added to the MH media. The ionic composition of these solutions at 2.5% and 7.5% is shown in Table 2. Salts employed were purchased from Sigma Chemical Co. (analytical grade). Solid media were obtained by addition of 20 g/L of agar (Difco). The pH was adjusted with 1 M KOH to 7.2 7.4. All the media were sterilised and autoclaved at 112 C for 30 min. Solid media were used for enumeration of the aerobic heterotrophic bacterial population and for studies of crystal formation. Liquid media were only used for the study of crystal formation.

Table 3 Composition of bacterial culture media Composition (g L1 ) Culture medium 1:1E/A-0 1:1E/A-1 1:1E/A-2 1:1E/A-3 Art.1:1E/A Art.SE/A 1:1E/C-0 1:1E/C-1 1:1E/C-2 1:1E/C-3 Art.1:1E/C Art.SE/C MH-2.5%a MH-7.5%a Proteose-peptone 0.5 2.5 5 5 5 0.5 2.5 5 5 5 5 5 Yeast extract 1 5 10 10 10 1 5 10 10 10 10 10 Glucose 0.1 0.5 1 1 1 0.1 0.5 1 1 1 1 1 Solvent (1000 mL) 1:1E hor. A 1:1E hor. A 1:1E hor. A 1:1E hor. A Articial solution like 1:1E hor. A Articial solution like SE hor. A 1:1E hor. C 1:1E hor. C 1:1E hor. C 1:1E hor. C Articial solution like 1:1E hor. C Articial solution like SE hor. C Solution at 2.5% of marine salts Solution at 7.5% of marine salts

Abbreviations: 1:1E = 1:1 (soil:water) extract; SE = saturation extract; Art. = articial; MH = halophilic media. a Suspensions of samples from A and C horizons were inoculated in MH.

CARBONATE BIOPRECIPITATION IN SOIL EXTRACTS

59

Table 4 Number of bacterial populations and percentages of crystal- and noncrystal-forming bacteria in solid media Horizon A Medium 1:1E/A-0 1:1E/A-1 1:1E/A-2 1:1E/A-3 Art.1:1E/A Art.SE/A MH-2.5% MH-7.5% 1:1E/C-0 1:1E/C-1 1:1E/C-2 1:1E/C-3 Art.1:1E/C Art.SE/C MH-2.5% MH-7.5% Bacteria/g of soil 123 105 115 105 114 105 110 105 120 105 116 105 110 105 108 105 90 104 105 104 107 104 102 104 110 104 95 104 108 104 112 104 Crystal-forming (%) 56 52 59 58 51 52 55 40 50 43 54 53 49 51 50 29 Noncrystal-forming (%) 44 48 41 42 49 48 45 60 50 57 46 47 51 49 50 71

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

Crystal Formation In solid media, plates containing the different culture media were surface inoculated with 0.1 mL of different dilutions of soil suspensions in sterile saline solution. The suspensions of samples from the A-horizon were inoculated in the media derived from A-horizon and in MH-2.5% and MH-7.5% media; the suspensions of samples from the C-horizon were inoculated in the culture media derived from the C-horizon and also in MH-2.5% and MH-7.5% media (Table 3). The inoculated agar plates (5 plates per dilution) were incubated aerobically at 25 C. Total heterotrophic bacterial counts were obtained for each soil sample on different culture media. The plates were daily examined for the presence of crystals using optical microscopy during 40 days of incubation. The percentage of crystal-forming and noncrystal-forming colonies was determined in all media (Table 4). The precipitation rate was determined with an optical microscope to denote the time required for the initiation of precipitation and the quantity of crystals formed. The precipitation rate will be greater when the time required for the initiation of precipitation decreases and/or the quantity of crystals formed increases. The minerals precipitated in the different media were recovered for later identication. They were removed from the agar medium by cutting out blocks and placing them in boiling water to dissolve the agar. The crystals in the resultant suspension were sedimented, resuspended and washed in distilled water to free the crystals of impurities. The washed crystals were dried at 37 C. In liquid media, a ask containing 100 mL of one of the different culture media (Table 3) was inoculated with 1 mL of a 102 dilution of soil from the A or C horizons (as in the case

of the solid culture media) and incubated aerobically at 25 C. Later, any crystals were collected by centrifugation, washed free of impurities in distilled water and air-dried. In all experiments, controls consisting of uninoculated cultures or cultures inoculated with a high concentration of dead bacteria, and thus lacking metabolic activity, were included.

Study of the Crystals The puried crystals were examined by X-ray diffraction (XRD) (powder diagram) in a Siemens D5005D diffractometer with CuK radiation with a graphite monochromator. Preparation of the samples involved pulverizing manually in an agate mortar before analysis. The mineral phases were identied in accordance with JCPDS and ASTM criteria (JCPDS and ASTM 1981). Fisher quartz was added to the samples as a standard to calibrate the diffractometer. Compositional semiquantitative analyses of mineral species were performed following the intensity factors methods (Klug and Alexander 1976). The diffraction peak corresponding to planes (104) (d104 0.3 nm) was used to determine the approximate magnesium content of calcite (Goldsmith et al. 1961). Samples of bioliths, puried as described previously, were observed with a Scanning Electron Microscope (SEM Hitachi S-510, operating at 25 kV) in order to study shape, size and internal structure. The bioliths were xed to adhesive paper, which was then xed to a sample-holder with colloidal silver. Metallization with gold in two orientations (2030 ; Bohor and Hughes 1971) was used in order to improve image quality.

60 RESULTS

J. PARRAGA ET AL.

Soil and Soil Solution The soil studied (Table 1) is scarcely evolved (prole A-C); physical properties, including color, mainly inherited from the parent rock (silty-marl); carbonated, gypsiferous; saline (electrical conductivity of 12 to 55 dS cm1 ) due to a uctuating ground layer of saline water with high chloride, sulphate, and calcium content (Del Moral et al. 1987), to which it owes its hydromorphic conditions. The A-horizon had large white masses or crystals, and very few small spherical white nodules; using X-ray diffraction, it was established that they were composed of 96% gypsum and 4% calcite. The C-horizon had mycelia and laminar deposits composed of 98% gypsum and 2% calcite, and nodules composed of 85% gypsum and 15% calcite. The calcite identied did not show detectable quantities of magnesium (d104 = 0.303 nm). These accumulations of secondary minerals are formed in the soil (pedogenic) or inherited from the sedimentary rock (marl). According to these characteristics it is a Gley-Gypsic-Carbonated Solonchak (FAO 1998). The soil extracts (Table 2) are in accordance with the mineralogical and chemical characteristics of the soil (Table 1) and with those of the ground waters, which are rich in chlorides and sulphates. Thus, the most abundant anion was Cl , which exceeded 10 g/L or 282.1 mM in SE of C-horizon, followed by SO2 (>6 g/L or >62.5 mM, in SE of C-horizon) and, nally, 4 HCO . The most abundant cation was Na+ , with quantities, in 3 the case of the SE, exceeding 18 g/L or 783 mM (C-horizon), followed by Ca2+ (0.51.5 g/L, or 12.537.4 mM, approx.), Mg2+ and K+ . Both in 1:1E and in SE, the concentration of almost all the ions analysed, increases from the A-horizon to the C-horizon. Thus, in the 1:1E the concentrations of Mg2+ were approximately 2.9 times greater in the C-horizon than in the A-horizon; the concentrations of Na+ , K+ , Cl , and SO2 were 4 respectively 5.4, 1.6, 1.3, and 2.0 times greater in the C-horizon than in the A-horizon. In the saturation extracts (SE) the concentrations of Mg2+ , Na+ , K+ , Cl , SO2 , and HCO were 4.6, 4 3 8.4, 2.5, 2.0, 3.1, and 1.5 times greater in the C-horizon than in the A-horizon. These differences are corroborated by different values of the electrical conductivity of the A and C horizons: 13.8 and 47.0 dSm1 respectively (Table 1). Another reason for these differences is that the C-horizon is more affected by the saline ground water and less washed by rainwater. However, one exception is that the concentration of Ca2+ was greater in the extracts from the A-horizon than in those from the C-horizon, 1.8 times greater in 1:1E and 1.1 times greater in SE. This may be because Ca2+ is the ion which is most retained by the organic matter (Duchaufour and Souchier 1983) and the content of organic carbon (OC) in the A-horizon is threefold that in the Chorizon (Table 1). The ionic concentrations registered in the 1:1E were lower than in the corresponding SE (Table 3), due to the lower quantity of water in the latter. Concentrations of ions in the A-horizon of the SE were approximately 1.8 times greater than those in the A-horizon of 1:1E, and were approximately 2.7 times greater in the C-horizon of SE than in the C-horizon of 1:1E.

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

Table 2 also shows the composition of the solutions of marine salts at 2.5% and 7.5%, which are ionic concentrations and salinities similar to those determined in the soil extracts of the saline soil studied although there are greater proportions in marine salts of Mg2+ , K+ , and Cl with respect to the soil extracts and consequently, the [Mg]/[Ca] ratio is also much higher (maximum of 4.75 in the solution of 7.5% salts compared to the minimum of 0.10 in the 1.1E). The mineral saturation indices (SI) of the solutions used as bacterial culture media (Table 5), calculated with the program PHREEQC, varied between 20.55 for lime (CaO) in the solution MH-2.5%, and +0.44 for gypsum (CaSO4 2H2 O) in the saturation extract (SE) of the C-horizon. The highest SI values for all the solutions used appeared in the SE of the A and C horizons. The mineral phases with SI values close to 0 (above and below the equilibrium point) were: anhydrite (CaSO4 ) (0.29 to +0.25), aragonite (CaCO3 ) (0.86 to +0.16), calcite (CaCO3 ) (0.73 to +0.30), dolomite (CaMg(CO3 )2 ) (1.13 to +0.39), gypsum (CaSO4 2H2 O) (0.08 to +0.44), and magnesite (MgCO3 ) (1.48 to 0.01). These values show that the solutions used to prepare the culture media are in thermodynamic elds close to the stability lines of these minerals, since values of SI = 0 denote stoichiometric saturation. This is a necessary condition for thermodynamic equilibrium and SI > 0 denotes that the solution phase is supersaturated with respect to the solid phase considered and inorganic precipitation may occur (Sparks 1999). Mineral Precipitation in Culture Media Formation of minerals was observed in all the culture media investigated, both solid and liquid. No precipitation was observed in the uninoculated controls, nor in those inoculated with a high concentration of dead bacteria and thus lacking metabolic activity. Table 4 shows the results of the enumeration of the aerobic heterotrophic bacteria population of the A and C horizons and the percentages of the carbonate-forming and non-carbonate forming bacteria carried out in the different solid culture media. The number of bacteria per gram of soil was around ten times higher in the A-horizon than in the C-horizon (108 105 to 123 105 bacteria/g of soil in the A-horizon and 90 104 to 112 104 bacteria/g of soil in the C-horizon). In almost all the media the percentage of carbonate-forming bacteria was slightly over 50% in the A-horizon (5159 %) and close to 50% in the C-horizon (4354%). The only exception was the MH 7.5% medium which showed 40% carbonate-forming bacteria in the A-horizon and 29% in the C-horizon. Generally, the precipitation rate was higher (greater number of crystallites evaluated in the microscope eld in shorter times) in the culture media inoculated with bacteria from the A-horizon than in the culture media inoculated with bacteria from the C-horizon. In the culture media with added organic compounds (1:1E/A-1, 1:1E/A-2, 1:1E/A-3, 1:1E/C-1, 1:1E/C-2, 1:1E/C-3) (Table 4), precipitation took place rapidly, crystal formation beginning 48 hours after inoculation (although only in those plates

CARBONATE BIOPRECIPITATION IN SOIL EXTRACTS

61

Table 5 Values of the saturation index (SI) for some of the minerals investigated using the PHREEQC geochemical computer program, in the solutions used to prepare the culture media SI of solutions Mineral phase Anhydrite (CaSO4 ) Aragonite (CaCO3 ) Brucite (Mg(OH)2 ) Calcite (CaCO3 ) Dolomite (CaMg(CO3 )2 ) Epsomite (MgSO4 7H2 O) Gypsum (CaSO4 2H2 O) Halite (NaCl) Hydromagnesite (Mg5 (CO3 )4 (OH)2 4H2 O) Lime (CaO) Magnesite (MgCO3 ) Mirabilite (Na2 SO4 10H2 O) Natron (Na2 CO3 10H2 O) Nesquehonite (MgCO3 3H2 O) Periclase (MgO) Thenardite (Na2 SO4 ) 1:1E hor. A SE hor. A 1:1E hor. C SE hor. C Marine salts-2.5% Marine salts-7.5% 0.12 0.18 5.33 0.04 1.03 3.62 0.09 4.16 17.83 20.33 1.48 4.27 7.83 3.90 10.04 5.19 0.17 0.00 5.43 0.14 0.66 3.33 0.38 3.71 17.17 20.45 1.29 3.71 7.38 3.71 10.15 4.62 0.29 0.52 4.73 0.39 0.99 3.08 0.08 3.37 15.71 20.47 1.10 2.70 6.44 3.52 9.45 3.61 0.25 0.16 4.43 0.30 0.39 2.56 0.44 2.55 12.62 20.18 0.40 1.76 5.35 2.83 9.14 2.60 0.19 0.86 4.28 0.73 1.13 2.47 0.00 2.70 14.47 20.55 0.90 2.76 6.93 3.33 8.99 3.62 0.06 0.48 3.70 0.34 0.14 1.82 0.23 1.70 10.40 20.46 0.01 1.68 5.72 2.49 8.40 2.39

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

with abundant bacterial development), and the crystals were of large size principally in the culture media 1:1E/A-2, 1:1E/A-3, 1:1E/C-2 and 1:1E/C-3 (300 m, up to 500 m). In the culture media without added organic compounds (1:1E/A-0, 1:1E/C-0) precipitation took place more slowly (beginning after 7 days). The crystallites appeared sooner and more abundantly in the culture media of soil extracts (both natural and articial) than in the culture media made up with mixtures of marine salts (MH media). This was more pronounced in the MH at 7.5% salts inoculated with bacteria from C-horizon, where a signicant decrease from 40% to 29% of carbonate-forming bacteria was also noted.

Analysis of Crystals The results of the mineralogical analysis with XRD (Table 6) showed that the crystals precipitated in the cultures based on soil extracts, natural or articial, both solid and liquid, and with or without added organic material, were calcite without detectable quantities of magnesium (d104 0.303 nm). This nding is supported by morphological observation by SEM (Figures 1d and 1e). In the MH-7.5% media, mixtures with different proportions of magnesian calcites (d104 0.298 nm) and aragonites were precipitated; in MH-2.5% media, calcite (d104 0.303 nm) or magnesian calcite (d104 0.301 nm) with traces of aragonite was precipitated, depending on the type of medium (solid or liquid) and the bacterial populations with which they were in-

oculated (Table 6). From a mineralogical point of view it should be noted that calcite and metastable aragonite are polimorphic forms of CaCO3 , and that magnesian calcite is calcite containing quantities of magnesium substituing the calcium, generally <20 mol % of MgCO3 in biogenic species (Ca1n Mgn CO3 , n < 0.2) (Mackenzie et al. 1983; Lowenstam and Weiner 1989). A variety of shapes were observed by SEM (Figure 1), the most important being spheres, hemispheres, pseudopolyhedral forms, and, to a lesser extent, spindles, druses and dumbbells, which may be found isolated or in groups. The most frequent size was between 30 and 300 m (Figures 1c, 1d, 1f, 1g, and 1h). Some bioliths show a porous external surface (Figures 1c, 1d, 1e and 1f) while others are covered with a ne lm of secondary carbonate (Figures 1g and 1h). Their interior, which may be porous, is formed by grouping of roughly brousradiated crystals (Figures 1b and 1c). The interior may also show a more massive, brous-radiated appearance (Figures 1h). Among the morphologies and morphological features described there are also intermediate cases. The exact dependence of the morphologies on each type of medium is difcult to establish since a variety of morphologies is usually present. However, in the articial media of marine salts (MH) spherulitic forms appear to predominate (Figures 1g and 1h). These are larger, frequently grouped and covered with carbonate (Figure 1g). The different forms and their transitions were most easily observed in the liquid media derived from the soil solution extracts.

62

J. PARRAGA ET AL.

Table 6 Mineralogy (XRD) of the bioliths precipitated Mineral (%) Culture medium All culture media derived from soil solution (natural or articial) MH-2.5% MH-2.5% MH-2.5% MH-2.5% MH-7.5% MH-7.5% MH-7.5% MH-7.5% Bacterial Consistency population Solid Solid Liquid Liquid Solid Solid Liquid Liquid Solid Solid Liquid Liquid A-horizon C-horizon A-horizon C-horizon A-horizon C-horizon A-horizon C-horizon A-horizon C-horizon A-horizon C-horizon Calcite (CaCO3 ) 100 100 100 100 100 100 100 Mgcalcite (Ca1n Mgn CO3 ) 100 100 55 60 10 Aragonite (CaCO3 ) d104 (nm) tr tr 45 40 90 0.303 0.303 0.303 0.303 0.303 0.303 0.303 0.301 0.298 0.298 0.298 0.298 Formula of the Mgcalcite Ca0.910 Mg0.090 CO3 Ca0.805 Mg0.195 CO3 Ca0.805 Mg0.195 CO3 Ca0.805 Mg0.195 CO3 Ca0.805 Mg0.195 CO3

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

Abbreviations: Mgcalcite = magnesian calcite; tr = trace. 1:1E/A-0; 1:1E/A-1; 1:1E/A-2; 1:1E/A-3; Art.1:1E/A; Art.SE/A; 1:1E/C-0; 1:1E/C-1; 1:1E/C-2; 1:1E/C-3; Art.1:1E/C; Art.SE/C.

DISCUSSION The most signicant nding of this work is that the bacterial populations of a soil, when cultivated in the laboratory in media derived from the soil solution, induce precipitation of carbonates; in this case, of calcite. Previous studies also show the active role of bacteria in carbonate precipitation in the soil (Monger et al. 1991; P rraga et al. 1998). However, the use of the soil a solution, which is the natural aqueous medium where the soil bacteria interact with other solid and gaseous components of the soil, as a culture medium, is an important advance in the study of biomineralization in soils. Numerous studies have reported that supercial soil horizons (epipedons), which are richer in organic material, contain up to 103 times more bacteria than the underlying horizons. According to Barns and Nierzwicki-Bauer (1997) the number of microorganims in soil habitats typically ranges from 106 to 109 bacteria per gram. The results in the current study would tend to conrm these ndings, since, in A-horizon the bacterial counts were roughly ten times higher than those in C-horizon (Table 4). It was also observed that in the bacterial populations of both horizons, a greater number of bacteria were capable of precipitating carbonates in non-modied extracts (1:1E/A-0, 1:1E/C-0) from the soil (56% A-horizon and 50% C-horizon). However, the fact that the precipitation rate reached was slightly higher in the media from A-horizon than in those from C-horizon, and that the number of bacteria was higher in A-horizon, suggests that this horizon is more suitable for carbonate precipitation than the C-horizon. It was also observed that better precipitation rates were obtained in the extract media (both natural and articial) from A and C horizons, compared to the media MH made up

with mixtures of marine salts, mainly in the medium with 7.5% salts. This suggests that in the A and C horizons of the soil studied precipitation of carbonates through bacterial action could take place in soil in situ. Furthermore, secondary accumulations of carbonates (calcite) are frequent in this soil. Evidence for the biomineralizing role of the bacteria is that in the uninoculated culture media or in those inoculated with dead bacteria, no mineral precipitation was detected. According to the results of the geochemical analysis (PHREEQC) (Table 5) anhydrite, aragonite, calcite, dolomite, and gypsum could be precipitated inorganically in the solutions studied, since their SI is greater than 0. However, on cultivating the halophilic bacteria isolated from the soil used in the media under study, calcite was biomineralized in the media derived from the soil solution (1:1E and SE) and calcite, magnesian calcite and aragonite in the MH media of marine salts (Table 6). The differences in mineralogical composition between the soil extract media and the MH media can be attributed to the concentration of magnesium and particularly to the [Mg]/[Ca] ratio (Gonz lez 1989; Rivadeneyra et al. 1985, 1991). It has been a shown that an excess of magnesium has a dominant inhibiting effect on calcite precipitation (Cailleau et al. 1979). This would affect nucleation and growth of calcite, delaying its crystallization and favoring kinetically the formation of the metastable polymorph aragonite. However, the inuence of magnesium concentration and the [Mg]/[Ca] ratio must be a threshold, above which their inuence is clear, since important differences in mineralogical composition were only observed in the marine salts medium at 7.5% with magnesium concentrations of 130.73 mM and [Mg]/[Ca] ratio of 4.75 (Table 2). It also seems that, above

CARBONATE BIOPRECIPITATION IN SOIL EXTRACTS

63

this threshold of the [Mg]/[Ca] ratio and of the concentration of magnesium, the physical state of the culture medium (solid or liquid) affects the polymorph of carbonate precipitated, liquid media favouring aragonite precipitation. These trends are supported by the previous studies of Ferrer et al. (1988) and Rivadeneyra et al. (1998, 2000). The shapes and morphological features described using SEM (Figure 1) are typical of bacterial carbonate precipitation, and are related to a process of biomineral formation through accretion of calcied bacterial bodies, under highly supersaturated condi-

tions, nally leading to the spherical shape; these ndings have been reported previously by Rivadeneyra et al. (1994, 1998). The possibility that the biolith undergoes posterior recrystallization with greater or lesser contribution of the bacteria should not be ruled out. In studies on petrocalcic horizons Bruand and Duval (1999) and Schmittner and Giresse (1999) observed the same morphologies as those regarded as having a biogenic origin in the present study. All the results conrm that bacterial activity has an important role in the formation of minerals studied in vitro, and that this

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

Figure 1. Scanning electron micrographs illustrating the morphology of bioliths formed by soil bacteria. (A) Articial medium 1:1 E/A (A horizon). Liquid. Field of bioliths where spherulitic and pseudopolyhedral forms predominate. (B) Medium 1:1 E/C-0 (C horizon). Liquid. Dumbbell shapes. The bioliths are formed by large numbers of bers with varying degrees of recrystallization. (C) Medium 1:1 E/A-0 (A horizon). Liquid. Drusiform biolith composed of several aggregation units of crystals. Fibrous radiated internal structure. (D) Medium 1:1 E/C-0 (C horizon). Liquid. Spherulite with trigonal crystals (calcite) on surface. (E) Medium 1:1 E/A-0 (A horizon). Liquid. Detail of surface of biolith in Figure lC. Small crystals with trigonal symmetry (calcite). (F) Articial medium SE/C (C horizon). Solid. Spherical and hemispherical bioliths with reticulated surface. (G) Medium MH-7.5% (A horizon). Solid. Group of intergrowing spherulites with individual diameters between 50 and 60 m, forming a ring-shaped structure. Surface covered with a ne carbonate lm. Supercial ssures are experimental artefacts due to heating of the sample during metallization and observation. A more compact group of smaller forms can be seen at the bottom left. (H) Medium MH-2.5% (A horizon). Liquid. Field of bioliths. Size varies from approximately 50 to 500 m. Hemispherical forms predominate with a tendency to form bunches that are intergrowing groups. Surface covered with a ne lm of secondary carbonate. (Continued)

64

J. PARRAGA ET AL.

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

Figure 1. (Continued) is consequently a biomineralization process. Having shown that the conditions of the culture medium composition also have an effect on the result of the process, this must be bacterially induced biomineralization, according to the denition of Lowenstam and Weiner (1989). The formation of mainly calcite (Table 6) in the majority of the media provides further evidence for the possible effect of bacteria on the in situ precipitation of carbonates in soil since the accumulation of secondary carbonates of the soil studied is also calcite. Another interesting point is that both, 1:1E (natural or articial extract) and the saturation extract SE (articial) are equally suitable for the study of biomineralization in this soil since all gave very similar results. These articial media are necessary, especially in the case of SE, since the quantity extracted is so small that its use as a culture medium is unviable. CONCLUSIONS In this paper it was shown that when the bacteria from the A and C horizons of a saline soil were cultivated in media based on natural soil extracts, calcite without an appreciable magnesium content was precipitated. This also occurs in articial media with ionic composition similar to the natural media. This was true both in liquid and in solid media and takes place independently of the quantities of organic material added. It should be emphasized that the accumulation of secondary carbonates of the soil studied is also calcite. This result provides further evidence in support of the previous hypothesis that bacteria could play a part in carbonate precipitation in their natural habitats, in this case, the soils. This process can be dened as induced biomineralization, according to Lowenstam and Weiner (1989), since the inuence of the ionic medium on the process can be observed. For the study of biomineral precipitation by bacteria in soils, it was demonstrated that it is possible to use the soil solution (1:1 extract), thus allowing the investigation to be carried out in an environment most similar to natural conditions. REFERENCES
Amit R, Harrison JBJ. 1995. Biogenic calcic horizon development under extremely arid conditions Nizzana Sand Dunes, Israel. Adv Geoecol 28:65 88. Barns SM, Nierzwicki-Bauer SA. 1997. Microbial diversity in ocean, surface and subsurface environments. In Baneld JF, Nealson KH, editors. Geomicrobiology: interactions between microbes and minerals. Rev Mineral 35:3579.

CARBONATE BIOPRECIPITATION IN SOIL EXTRACTS Belnap J, Lange OL (editors). 2001. Biological soil crust: structure, function, and management. Ecological Studies 150. Berlin: Springer. 503 p. Bohor BF, Hughes RE. 1971. Scanning electron microscopy of clay and clay minerals. Clays Clay Min 19:4954. Boquet E, Boronat A, Ramos-Cormenzana A. 1973. Production of calcite (calcium carbonate) crystals by soil bacteria is a general phenomenon. Nature 246:527529. Bruand A, Duval O. 1999. Calcied fungal laments in the petrocalcic horizon of Eutrochrepts in Beauce, France. Soil Sci Soc Am J 63:164169. Cailleau P, Jacquin C, Dragone D, Girou A, Roques H, Humbert L. 1979. Inuence des iones etrangers et the la matiere organique sur la cristallisation des carbonates de calcium. Etude pr liminare de la cimentation des roches e carbonnatees. Revue de lInstitut Fran ais du Petrole 1:83112. c Castanier S, Le M tayer-Levrel G, Perthuisot JP. 1999. Ca-carbonates precipie tation and limestone genesisthe microbiogeologist point of view. Sed Geol 126:923. Del Moral A, Quesada E, Ramos-Cormenzana A. 1987. Distribution and types of bacteria isolated from an inland saltern. Ann. Inst. Pasteur/ Microbiol. 138:5966. Duchaufour P, Souchier B. 1983. P dologie. 1. P dogen` se et classication. e e e Paris: Masson. 459 p. Edinger SB. 1990. Polygenesis and calcite micromorphology of Aridisols on a granitic pediment, East Mojave Desert, California. Ann Arbor, MI: University Microlm International. 165 p. Ehrlich HL. 1995. Geomicrobiology. New York: Marcel Dekker. 719 p. FAO. 1998. World reference base for soil resource. World Soil Resources Reports 84. Rome: FAO. 91 p. Ferrer MR, Quevedo-Sarmiento J, B jar V, Delgado R, Ramos-Cormenzana A, e Rivadeneyra MA. 1988. Calcium carbonate formation by Deleya halophila: effect on salt concentration and incubation temperature. Geomicrobiol J 6:49 57. Folk RL. 1999. Nannobacteria and the precipitation of carbonate in unnusual environments. Sed Geol 126:4755. Gile LH, Peterson F, Grossman RB. 1966. Morphological and genetic sequence of carbonate accumulation in desert soils. Soil Sci 101:347360. Goldsmith JR, Graf DL, Heard HC. 1961. Lattice constants of the calciummagnesium carbonates. Am Miner 46:453457. Gonz lez LA. 1989. Inorganic calcium carbonate precipitation: controls on mina eralogy, morphology and trace elemental composition. Ann Arbor, MI: University Microlm International. 180 p. JCPDS (Joint Committee on Powder Diffraction Standars) and ASTM (American Society for Testing and Materials). 1981. Selected Powder Diffraction Data for Minerals, Supplement. JCPDS, Park Lane, Swarthmore, PA. Kirkland B, Lynch F, Rahnis M, Folk R, Molineux I, McLean R. 1999. Alternative origins for nannobacteria-like objects in calcite. Geology 27:327 350. Klug HP, Alexander LEC. 1976. X-Ray Diffraction Procedures for Polycrystalline and Amorphous Material. New York: John Wiley and Sons. 716 p. Klute A (editor). 1986. Methods of soil analysis. Part 1Physical and mineralogical methods. Madison, WI: American Society of Agronomy and Soil Science Society of America. 1188 p. Lal R, Kimble JM, Eswaran H, Stewart BA (editors). 1999. Global climate change and pedogenic carbonates. Boca Raton, FL: Lewis. 305 p. Lowenstam HA, Weiner S. 1989. On Biomineralization. Oxford: Oxford University Press. 324 p. Mackenzie FT, Bischoff WD, Bishop FC, Loijens M, Schoonmaker J, Wollast R. 1983. Magnesian calcites: low-temperature occurrence, solubility and solid-solution behavior. In: Reeder RJ, editor. Carbonates: mineralogy and chemistry. Rev Mineral 11:97144. Monger HC. 1990. Soil mineral transformations in a Southern New Mexico Aridisols. Ann Arbor, MI: University Microlm International. 151 p. Monger HC, Daugherty LA, Lindemann WC, Liddell CM. 1991. Microbial precipitation of pedogenic calcite. Geology 19:9971000.

65

Page AL, Miller RH, Keeney DR (editors). 1982. Methods of soil analysis. Part 2chemical and microbiological properties. Madison, WI: American Society of Agronomy and Soil Science Society of America. 1159 p. Parkhust DL, Appelo CAJ. 1999. Users guide to PHREEQC (Version 2). A computer program for speciation, batch-reaction, one-dimensional transport and inverse geochemical calculations. Water Resources Investigations. Report 99-4959. Denver, Colorado: U.S. Geological Survey. 312 p. P rraga J, Rivadeneyra MA, Delgado R, I iguez J, Soriano M, Delgado G. 1998. a n Study of biomineral formation by bacteria from soil solution equilibria. React Funct Polym 36:265271. Phillips SE, Milnes AR, Foster RC. 1987. Calcied laments: An example of biological inuences in the formation of calcrete in South Australia. Aust J Soil Res 25:405428. Rabenhorst MC, West LT, Wilding LP. 1991. Genesis of calcic and petrocalcic horizons in soils over carbonate rocks. In: Nettleton WD, editor. Occurrence, Characteristics and Genesis of carbonate, gypsum and silica accumulations in soils. Madison, WI: Soil Sci Soc Am, Spec. Publ. 26. pp. 6174. Rhoades JD. 1990. Monitoring soil salinity: A review of methods. In: Elprince A, editor. Chemistry of soil solutions, Malabar, FL: Krieger Publ. pp. 359374. Richards LA (editor). 1982. Suelos salinos y s dicos: diagn stico y rehabilo o itaci n. M xico: Limusa. 172 p. o e Riding RE, Awramik SM (editors). 2000. Microbial sediments. Berlin: Springer. 331 p. Rivadeneyra MA, Ramos-Cormenzana A, Garca-Cervig n A. 1985. Etude de o linuence du rapport Mg/Ca sur la formation de carbonate par des bacteria telluriques. Can J Microbiol 31:229231. Rivadeneyra MA, Delgado R, Quesada E, Ramos-Cormenzana A. 1991. Precipitation of calcium carbonate by Deleya halophila in media containing NaCl as sole salt. Curr Microbiol 22:185190. Rivadeneyra MA, Delgado R, Delgado G, Ferrer M, Del Moral A, RamosCormenzana A. 1993. Carbonate precipitation by Bacillus sp. isolated from saline soils. Geomicrobiol J 11:175184. Rivadeneyra MA, Delgado R, Del Moral A, Ferrer M, Ramos-Cormenzana A. 1994. Precipitation of calcium carbonate by Vibrio sp. From an inland saltern. FEMS Microbiol Ecol 13:197204. Rivadeneyra MA, Delgado G, Ramos-Cormenzana A, Delgado R. 1997. Precipitation of carbonates by Deleya halophila in liquid media: Pedological implications in saline soils. Arid Soil Res Rehabil 11:3549. Rivadeneyra MA, Delgado G, Ramos-Cormenzana A, Delgado R. 1998. Biomineralization of carbonates by Halomonas eurihalina in solid and liquid media with different salinities: crystal formation sequence. Res Microbiol 149:277 286. Rivadeneyra MA, Delgado G, Soriano M, Ramos-Cormenzana A, Delgado R. 1999. Biomineralization of carbonates by Marinococcus albus and Marinococcus halophilus isolated from the Salar de Atacama (Chile). Curr Microbiol 39:5357. Rivadeneyra MA, Delgado G, Soriano M, Ramos-Cormenzana A, Delgado R. 2000. Precipitation of carbonates by Nesteronkonia halobia in liquid media. Chemosphere 41:617624. Schmittner KE, Giresse P. 1999. Micro-environmental controls on biomineralization: supercial processes of apatite and calcite precipitation in quaternary soils, Roussillon, France. Sedimentology 46:463476. Soil Science Society of America. 1978. Glossary of soil science terms. Madison, WI: Soil Science Society of America. 36 p. Soil Survey Staff. 1993. Soil survey manual. Washington, DC: US Department of Agriculture. 437 p. Soil Survey Staff. 1999. Keys to soil taxonomy. 8th ed. Soil Conservation Service, USDA. Blacksburg, Virginia: Pocahontas Press. 545 p. Sparks DL. 1999. Soil Physical Chemistry. 2th edition. Boca Raton, FL: CRC Press. 409 p. Subow NN. 1931. Oceanographical tables. USSR Oceanographic Institute, Hidrometereological Commission, Moscow.

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012

66

J. PARRAGA ET AL. itation and for solid-phase capture of inorganic contaminants. Geomicrobiol J 18:93115. Wolt JD. 1994. Soil solution chemistry: applications to environmental science and agriculture. New York: Wiley. 345 p. Wright VP, Tucker ME (editors). 1991. Calcretes. Reprint series v. 2 of the International Association of Sedimentologists. Oxford: Blackwell. 352 p.

Verrecchia EP, Freytet P, Verrecchia KE, Dumont JL. 1995. Spherulites in calcrete laminar crust: Biogenic CaCO3 precipitation as a major contributor to crust formation. J Sediment Res A65:690 700. Warren LA, Maurice PT, Parmar N, Ferris FG. 2001. Microbially mediated calcium carbonate precipitation: implications for interpreting calcite precip-

Downloaded by [Nanyang Technological University] at 20:33 07 May 2012