study of biomineral formation by bacteria from soil solution equilibria

ELSEVIER

REACTIVE &

FUNCTIONAL POLYMERS

Reactive & Functional Polymers 36 (1998) 265-271

Study of biomineral formation by bacteria from soil solution equilibria

J. P5rragaa, M.A. Rivadeneyrab, R. Delgado a, J. Ifiiguez a, M. Soriano a, G. Delgado a,* a Departamento Edafologia y Quimica Agricola, Facultad de Farmacia, Universidad de Granada, Campus Cartuja,

18071 Granada, Spain b Departamento Microbiologia, Facultad de Farmacia, Universidad de Granada, Campus Cartuja, 18071 Granada, Spain

Received 15 December 1996; accepted 12 October 1997

Abstract

The role of bacteria in mineral neoformation in a saline soil was studied. In this soil, authigenic precipitation of gypsum and calcite takes place. Bacteria isolated from the soil were cultivated ‘in vitro’ in solid and liquid media prepared from soil solutions (1 : 1 extract). Precipitation of calcite spherulites of between 20 and 50 pm diameter and of other bioliths was observed. From soil solutions (1 : 1 extract and saturation extract) activity coefficients and the states of reaction for 18 minerals were calculated using the SOLMINBQ 88 program. Calcite, dolomite, gypsum and aragonite were in equilibrium with the saturation extract while in the 1 : 1 extract no mineral phases in equilibrium were present. Therefore, bacteria must play an active role in calcite precipitation in this saline soil. 0 1998 Elsevier Science B.V. All rights reserved.

Keywords: Biominerahzation; Saline soil; Soil solution; Mineral equilibria; Halophilic bacteria; Calcite; Gypsum; SOLMINEZQ 88

1. Introduction

The formation of minerals by bacteria has been described by numerous authors. The precipitation of carbonates has been widely studied in laboratory cultures and carbonate formation by bacteria of different taxonomic groups and from different habitats has also been investigated [4,11,22,27].

It has been suggested that microbial activity could be related to the formation of marine cal- careous skeletons, carbonate sediments and carbonate rock [3,5,8,19]. Different mechanisms by which bacteria could control the precipitation of

*Corresponding author. Tel.: +34 (58) 243835, Fax: +34 (58) 243832.

minerals in natural habitats have been described; Edinger [7] in Orthids and Argids and Monger [23] in Argids. The possible role of bacteria in the precipitation of C03Ca and other minerals in saline soils has been one of the themes of in- vestigation by our research group: Ferrer et al. [ 10,111; Rivadeneyra et al. [27-3 11.

In natural inorganic environments, in living organisms, and in laboratory experiments, carbonate minerals are formed mainly from aqueous solutions [21]. Aqueous interstitial soil solution data have been used in many studies with considerable success to qualitatively pre- dict and explain the soil mineral weathering trends and thermodynamic relations of mineral- solution systems based on solubility-equilibrium principles [ 16,20,24]. Computer programs can be

1381-5148/98/$19.00 Q 1998 Elsevier Science B.V. All rights reserved. PZI S1381-5148(97)00158-2

266 J. Pdrraga et al. /Reactive & Functional Polymers 36 (1998) 265-271

used for geochemical modeling of water-rocks- soils-interactions [ 17,181.

The main objective of this work was to deter- mine whether bacteria play an important role in mineral precipitation in a saline soil. To achieve this objective, mineral stability in soil solution was compared to the precipitation of minerals in the same soil solution by laboratory cultures of bacteria isolated from these soils.

2. Material and methods

2.1. Soil and soil solution

Morphological and analytical features of the soil (Table 1) were studied in accordance with Soil Survey Staff [32], and the procedures used in analysis are outlined by A.S.A. and S.S.S.A. [1,2]. For the study of the soil solution, three samples representative of the three types of horizons present were used. These were: A, B and C which are the means weighted to the thicknesses of the seven horizons of which the soil is com- posed (e.g. sample A = mean of Ah1 and Ah2; B = 2Bw and C = mean of 2C, 3C1, 3C2 and 4c.

Two types of extract were considered as soil solution: the 1 : 1 extract and the saturation extract. The 1 : 1 water-soil suspension (1 : 1 extract) was dispersed for 1 h by a reciprocat-

Table 1 General and morphological characteristics of the haplic Solonchack

ing shaker. To obtain the extract the samples were centrifuged at 2000 rpm for 1 h using a HERMLE ZK 5 10 refrigerated centrifuge with the temperature adjusted to 25°C. Saturation extract was extracted from saturated soil paste [26] using a Buechner filter funnel and filtration flask connected to a vacuum source. The displaced solutions were filtered through a O-2 pm pore size cellulose acetate membrane filter and stored under refrigeration until all analyses were com- pleted. All these operations were carried out within 48 h of sampling.

Chemical analyses performed on solution sub- samples included pH and electrical conductivity (EC) (immediately after extraction). Concentra- tions of Na, K, Ca and Mg were measured by atomic absorption spectrometry, of SOd2- and Cl- by high pressure liquid chromatography and of HC03- by titration with standard acid.

Mineralogical analyses of significant macro- scopic salt accumulations (such as nodules, concretions, powdery pockets) were carried out by X-ray diffraction (Philips PW 1730, Cu KU). The mineral phases were identified in accordance with JCPDS and ASTM criteria [ 14,151.

Interstitial soil solution data were computed using the version renamed SOLMINEQ 88 from the original program SOLMNEQ [ 17,181. The principal computations carried out by SOLMINEQ 88 are aqueous speciation and sat-

Horizon Thickness Color

(cm) (Munsell) Mottling (Munsell)

Structure Nodules concretions deposits

Ah1 Ah2

2Bw

2Cg

3Clg 3c2g

4Cg

O-7 2.5Y 3.512 - bl sa, me/gr, mo 7-20127 2.5Y 4.512 - bl sa, mo gr,

20127-35 5Y 512 and 7.5YR 5.510 white, 2.5Y 4.514 and bl sa, ma/de me, 7.5YR 416

35-47 5Y 512 and 7.5YR 5.510 1OYR 616, white, massive and laminar 1OYR 5/l and 7,5YR 4/6

47-57 5Y 512 and 7.5YR 5.5/O white and 1OYR 616 massive and laminar 57-80 5Y 512 and 7.5YR 5.5/O 1OYR 616 and white massive and lam&r

>80 5Y 512 and 7.5YR 5.5/O white and black massive

masses or crystals, large, white very few nodules, small, spherical and white mycelium of carbonates and gypsum

laminar deposits and frequent nodules

laminar deposits and abundant nodules frequent laminar deposits and diffuse nodules frequent and diffuse nodules

Latitude: W 3’43’; longitude: N 37”5’; altitude: 700 m; orientation: W; slope: 35%; parent material: Tertiary sediments of variable texture, carbonated (>20% de COs2-); vegetation: halophile (Suaeda sp., SaZsoZa sp., Limonium sp., etc).

J. Pdrraga et al. /Reactive & Functional Polymers 36 (1998) 265-271 261

uration states of minerals. The present version has a revised thermodynamic data base with more inorganic (260) and organic (80) aqueous species and minerals (220), and computes pH, mineral solubilities at subsurface and pressures. SOLMINEQ 88 computes the activity coefficients of the aqueous species using Pitzer equations [ 13,251 and the equilibrium constants are computed from an integrated form of the Van? Hoff equations.

2.2.5. Analysis of crystals The purified crystals were examined by X-ray

diffraction (powder diagram) as described previously for soil mineralogy. Preparation of the samples involved pulverizing purified crystals in an agate mortar before analysis. For the morphological study, the crystals were examined in a Hitachi S-5 10 Scanning Electronic Microscope (SEM), operated at 25 kV. The samples were coated with gold.

2.2. Crystal formation by bacteria 3. Results and discussion

2.2.1. Microorganisms The experiments were performed with bacte-

rial strains isolated from the soil studied.

2.2.2. Culture media Liquid media are the same soil solutions (only

1: 1 extract). Solid media are the soil solutions (1 : 1 extract) with added 1.6% Bacto-Agar (Difco). The medium was sterilized at 112°C for 30 mm.

2.2.3. Crystal formation in liquid media

The soil sampled was saline (haplic Solonchak, [9]) and with a profile type A, Bw, Cg (Table 1) with lithological discontinuities due to both col- luvial and geological material. The origin of the salinity of the soil (CE between 12 and 55 dS m-l, Table 2) is a fluctuating water table of salt water which also creates hydromorphic conditions in the C horizons with mottled colour (alternating grays and o&es) and segregation of iron minerals. The semi-arid climate, which is characteristic of the zone, does not favour the elimination of the ex- cess of soluble salts in the soil.

100 ml of medium (soil solution) in 250~ml Erlenmeyer flasks were inoculated and incubated at 25°C and periodically examined for the presence of bioliths. After 30 days of incubation the culture medium was decanted and crystals were resuspended and washed in distilled water to free the crystals of impurities. Washed crystals were air-dried at 37°C.

2.2.4. Crystalformation in solid media The bacterial strains were surface inoculated

into solid media. The plates were examined periodically up to 30 days after inoculation by optical microscopy for the presence of crystals. Crys- tals formed were removed from the medium by cutting out agar blocks and placing them in a boiling water bath until the agar dissolved. The supernatants were decanted and the sediments resuspended and washed in distilled water until crystals were free of impurities. Finally the washed crystals were air-dried at 37°C.

Various types of accumulations were identified, according to their size, internal structure and other morphological characteristics (Table 1, last column). Some of them were authigenic according to their morphological characteristics. The mineral phases detected (DBX) were predomi- nantly calcite and gypsum. Significant amounts of carbonates, particularly of Ca were present in the soil (Table 2). The pH was slightly basic and the cation exchange complex in the soil was saturated with high percentages of Na and Ca (Ta- ble 2). The fact that the sum of extractable bases was much higher than the value of the CEC must be taken into account. There must therefore have been a large quantity of cations in the soil solution which were extracted together with the exchange cations. The presence in the soil of large quantities of neutral soluble salts (sulphates and chlorides of alkaline and alkaline-earth cations) (Table 3) and of calcium carbonate impedes the strong alkalin- ization of the soil (pH 8.2).

268 J. Pdrraga et al. /Reactive & Functional Polymers 36 (1998) 265-271

Table 2 Analytical characteristics of the haplic Solonchack

Horizon Thickness Sand clay CO N CO:- pH 1: 1 CE dS m-l Bases and exchange capacity

(%) (%) (a) (%) (%) H20 ClK (S. extract) cmol (+) kg-’

Na+ K+ Cazf Mg2+ CEC

Ah1 O-7 21.3 19.5 1.60 0.15 23.18 7.5 7.2 12.7 11.7 2.6 164.1 3.3 16.2 Ah2 7-20127 30.5 15.0 0.68 0.08 19.50 7.8 7.4 14.3 14.3 1.2 215.0 3.5 7.1 2Bw 20127-35 10.5 26.5 0.44 0.05 24.15 7.8 7.5 42.0 32.1 2.4 251.3 7.2 12.5 2Cg 35-47 8.7 28.5 0.42 0.05 25.17 8.0 7.6 55.6 40.0 2.7 130.8 10.1 17.1 3Clg 47-57 18.7 16.5 0.28 0.03 16.26 8.2 7.7 41.1 33.8 1.9 176.4 7.4 19.4 3c2g 57-80 10.5 19.7 0.39 0.03 22.13 8.2 1.7 51.9 46.3 2.4 180.8 8.1 10.2 4Cg >80 11.8 27.0 0.40 0.03 24.12 8.2 1.7 39.6 34.2 2.1 138.0 7.6 13.3

Values referred to fine earth fraction (~2 mm) (105°C). CO = organic carbon; N = total nitrogen; CE = electrical conductivity; and CEC = cation exchange capacity.

Table 3 Ion activities in saturation extract and 1: 1 extract, expressed as negative logarithms

Sample Solution ion activities in saturation extract Solution ion activities in 1 : 1 extract

pCa’+ pMg’+ pNa+ pK+ pCl- pSOd2- pHCO3- pH+ pCazf pMg2+ pNa+ pK+ pCl- pSOd2- pHCO3- pH+

A 1.97 2.95 1.17 3.23 0.99 2.56 3.22 7.15 2.15 3.14 1.40 3.45 1.21 2.67 3.37 7.30 B 2.19 2.49 0.62 2.99 0.61 2.30 2.99 7.15 2.55 2.89 1.07 3.43 1.05 2.48 3.41 7.36 C 2.12 2.34 0.24 2.86 0.76 2.42 3.13 7.39 2.46 2.71 0.65 3.26 1.15 2.55 3.48 7.36

The results of the soil solution analysis (Ta- ble 3) were processed using the SOLMINEQ 88 program (pressure: 1 atm, temperature: 25°C).

In general, all single-ion activities exhibited high values given the saline characteristics of the soil, being lower in the 1 : 1 extract than in the saturation extract. Na and Cl are the ions which showed the highest activities as a consequence of the ionic composition of the water table affecting the soil. With the exception of the Ca ion the single-ion activities were lower in the superficial horizons than in those corresponding to the subsurface. This could be due to the greater influence of the water table on the latter. The differences in single-ion activity reached almost 0.93 units in the case of the Na ion in the saturation extract. The behaviour of the Ca ion could be explained by the presence in all the profile of gypsums and carbonates. On the other hand, the Ca ion showed a great affinity for the organic compounds to form complexes and these compounds are most abundant in horizon A.

The high single-ion activity of Na in the

soil solutions resulted in a large quantity of extractable Na (Table 2) which increased pro- gressively with depth. In contrast, K and Mg ions, less abundant in the soil solutions, were the least represented in the exchange complex. The extractable Ca, present in significant quantities (Table 2), probably proceeds from the carbonates and the gypsum.

Another feature of the standard output of the SOLMINEQ 88 program is a table showing the states of reactions for 26 minerals considered: log (AP) (ion activity product), log K(T) (equilibrium constant at the specified temperature), log(AP/K T) , and another column with A G (free energy variation, kcal/mole).

Table 4 shows the changes in standard Gibbs energy of the 18 most frequent minerals in soil. Of these 18, only those with negative AG values are viable, which indicates that the solution is supersaturated with respect to these mineral phases. The saturation extract reflects the reality of the soil solution better than the 1 : 1 extract [33]. In the former it was observed that the minerals


Table 4 Changes in standard Gibbs energy (k&mole)

Phase

Anhydrite Aragonite Brucite Calcite Dolomite Gypsum Halite Huntite Hydromagnesite Magnesite MgCl2 Mirabilite Natron Nesquehonite Periclase Sylvite Thenardite Trona

Saturation extract 1 : 1 extract

horizon A horizon B horizon C horizon A horizon B horizon C

0.309 0.238 0.343 0.693 0.992 0.971 0.042 0.023 -0.182 0.285 0.814 0.796 7.047 6.436 5.594 6.890 6.390 6.164

-0.149 -0.168 -0.373 0.094 0.624 0.605 -0.466 -1.415 -1.949 0.035 0.207 0.049 -0.081 -0.141 -0.027 0.301 0.602 0.585

5.125 3.848 3.547 5.676 5.068 4.631 7.903 5.096 3.901 8.922 8.378 7.940

12.422 8.419 6.404 13.293 11.382 10.514 1.770 0.841 0.511 2.029 1.671 1.531

36.949 35.280 35.513 37.805 37.034 37.081 5.242 3.433 2.622 5.978 4.853 3.810

10.765 9.007 7.886 11.358 10.464 9.424 5.463 4.551 4.233 5.718 5.363 5.229

13.865 13.249 12.403 13.710 13.208 12.981 6.998 6.153 6.193 7.597 7.365 7.264 6.305 4.440 3.585 7.053 5.918 4.855

17.069 14.207 12.729 18.183 16.900 15.359

which can be formed in all these soil horizons are calcite, dolomite and gypsum, and, in horizon C, aragonite too. In the accumulations of salts in the soil, described in Table 1 the presence of two of these phases; calcite and gypsum is shown. Dolomite and aragonite are not at all common as authigenic phases in soils [6,20] and for this reason do not appear in the accumulations. However, the medium is supersaturated in these phases. This is not contradictory since AG indicates that the reactions can proceed but does not mean that they will proceed in the specified direction. It is possible for a given solution to be supersaturated in terms of AG but without precipitation of any mineral. It must also be taken into account that the saturated paste does not cor- respond exactly to the normal situation of the soil with respect to its water content which is gener- ally lower. Consequently, the supersaturation of mineral species shown in Table 4 would be more accentuated in ‘in situ’ soil [ 161.

In the 1: 1 extract it can be observed that all the values of AG are positive, so the possibility of precipitation of these minerals in these conditions can be discounted.

The result of the bacterial culture in solid and

liquid media, obtained from 1 : 1 extracts, was the formation of bioliths after 7 days incubation. Study of the mineralogy (DRX) of the bioliths precipitated by the bacteria in these cultures re- vealed that in all cases the mineral precipitated was always calcite with no traces of other minerals. The SEM morphology of these bioliths is in the form of spherulites (Fig. 1A) of between 20 and 50 urn in diameter and spindle-shaped (Fig. 1B). The spindle-shaped forms eventually generated spherulites through association and growth (Fig. 1C). The internal structure of the bioliths was fibroradial. Calcified bacterial bodies adhering externally to the bioliths were identified (Fig. 1D) thus confirming their biological origin.

The precipitation of calcite when the soil bacteria were cultivated in 1 : 1 extracts in vitro con- firms their active role in the process, since, as has been shown (Table 4), inorganic mineral formation cannot be expected at these concentrations.

Amongst the bacterial mechanisms which may induce biomineralization are the following: the rise in partial pressure of CO:! and of pH as a result of bacterial metabolism and the selective accumulation of Ca in the cell walls [ 121.

The active role of bacteria in mineral forma-

270 J. Pa’rraga et al. /Reactive & Functional Polymers 36 (1998) 265-271

Fig. 1. Scanning electron micrographs illustrating the morphology of calcite biolitbs formed by bacteria. (A) Photo 9012: spherical biolith. (B) Photo 9038: spindle-shaped biolith. (C) Photo 9008: biolith association to form a spherulite. (D) Photo 9003: calcified bacterial bodies on biolith surface.

tion in laboratory culture media has been reported previously [ 10,11,28-3 l] and furthermore the re- lationship between mineral type and bacterial species has been proved. The results obtained in this study tend to confirm this.

The results included here suggest that bacteria play an active part in the authigenic formation of calcite in the soil studied and that they induce the precipitation of this mineral, even in conditions where supersaturation has not been reached.

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study of biomineral formation by bacteria from soil solution equilibria

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