Download Influence of Bacillus subtilis Cell Walls and EDTA on Calcite

Environ. Sci. Technol. 2003, 37, 2376-2382
Influence of Bacillus subtilis Cell
Walls and EDTA on Calcite
Dissolution Rates and Crystal
Surface Features
A . K . F R I I S , †,§ T . A . D A V I S , †
M. M. FIGUEIRA,‡ J. PAQUETTE,† AND
A . M U C C I * ,†
Department of Earth and Planetary Sciences,
McGill University, 3450 University Street, Montreal, Quebec,
Canada H3A 2A7, and Biotechnology Research Institute, NRC,
6100 Royalmount Avenue, Montreal, Quebec,
Canada H4P 2R2
This study investigates the influence of EDTA and the
Gram-positive cell walls of Bacillus subtilis on the
dissolution rates and development of morphological
features on the calcite {101h4} surface. The calcite dissolution
rates are compared at equivalent saturation indicies (SI)
and relative to its dissolution behavior in distilled water (DW).
Results indicate that the presence of metabolically
inactive B. subtilis does not affect the dissolution rates
significantly. Apparent increases in dissolution rates in the
presence of the dead bacterial cells can be accounted
for by a decrease of the saturation state of the solution with
respect to calcite resulting from bonding of dissolved
Ca2+ by functional groups on the cell walls. In contrast,
the addition of EDTA to the experimental solutions results
in a distinct increase in dissolution rates relative to
those measured in DW and the bacterial cell suspensions.
These results are partly explained by the 6.5-8 orders
of magnitude greater stability of the Ca-EDTA complex
relative to the Ca-B. subtilis complexes as well as its free
diffusion to and direct attack of the calcite surface.
Atomic force microscopy images of the {101h4} surface of
calcite crystals exposed to our experimental solutions
reveal the development of dissolution pits with different
morphologies according to the nature and concentration of
the ligand. Highly anisotropic dissolution pits develop in
the early stages of the dissolution reaction at low B. subtilis
concentrations (0.004 mM functional group sites) and in
DW. In contrast, at high functional group concentrations (4.0
mM EDTA or equivalent B. subtilis functional group
sites), dissolution pits are more isotropic. These results
suggest that the mechanism of calcite dissolution is modified
by the presence of high concentrations of organic
ligands. Since all the pits that developed on the calcite
surfaces display some degree of anisotropy and dissolution
rates are strongly SI dependent, the rate-limiting step is
most likely a surface reaction for all systems investigated
in this study. Results of this study emphasize the
* Corresponding author telephone: (514)398-4892; fax: (514)3984680; e-mail: [email protected].
† McGill University.
‡ NRC.
§ Present address: Environment & Resources DTU, Technical
University of Denmark (DTU).
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 11, 2003
importance of solution chemistry and speciation in
determining calcite reaction rates and give a more accurate
and thermodynamically sound representation of dead
bacterial cell wall-mineral interactions. In studies of natural
aquatic systems, the presence of organic ligands is
most often ignored in speciation calculations. This study
clearly demonstrates that this oversight may lead to
an overestimation of the saturation state of the solutions
with respect to calcite and thermodynamic inconsistencies.
Introduction
The characterization of mineral-bacteria interactions contributes to our understanding of many low-temperature
geochemical processes, including dissolution, precipitation,
and adsorption reactions that govern the rates of chemical
weathering and regulate geochemical cycling. Numerous
laboratory and field studies have demonstrated that the
presence of certain bacteria influences the rate of mineral
dissolution (1-4), but the dissolution mechanisms in aqueous
solutions remain poorly characterized, even in abiotic
systems.
Bacteria are a common component in weathering environments. Metabolic activity, cell wall functional groups, and
exudate biopolymers may all influence the dissolution rates
of minerals (5). The bacterial cell wall contains several
different organic acid functional groups, the most important
of which are the carboxylic, phosphonic, and hydroxyl groups
(6). These cell walls carry no net charge at low pH (below
approximately 2) but become more negatively charged as
pH is increased (7). The sites can bind cations in solution,
but at the pH values most often encountered in natural
aquatic environments (i.e., pH 5-9), it is chiefly the carboxylic
and phosphonic sites that participate in metal sequestration
(6, 8-10).
This study focuses on the influence of the cell wall
functional groups of a common, Gram-positive soil bacterium
(Bacillus subtilis) on calcite dissolution rates, solute speciation, and micro-topographic dissolution features that
develop on the {101h 4} surface of calcite crystals in aqueous
solutions. An independent set of parallel experiments was
also carried out in EDTA solutions at equimolar functional
group concentrations to assess if bacterial cell wall functional
groups could be viewed merely as dissolved organic acids
(i.e., as studied by Orme et al.; 11). The objective was to
determine whether dissolution rates are affected by a shift
in the saturation state of the solution or a specific interaction
with the bacterial cell walls.
The influence of EDTA and B. subtilis on the calcite
dissolution mechanism was assessed on the basis of the
morphology of surface features (i.e., dissolution pits) that
developed on the calcite {101h 4} crystal face upon reaction
with our experimental solutions. These features were imaged
by atomic force microscopy (AFM).
Methods
Bacterial Growth, Preparation and Viability. A culture of
Bacillus subtilis (168) was kindly provided by T. J. Beveridge
(University of Guelph, Canada). The bacteria were incubated
for 24 h on trypticase soy agar and then transferred to 0.3
L of trypticase soy broth to grow for another 48 h while being
stirred on an orbital shaker (180 rpm) at 32 °C. Both the
broth and the agar contained 0.5 w/v % of yeast extract. The
B. subtilis cells were separated from their growth media by
centrifugation before they were rinsed with a 0.1 N HNO3
solution and then with distilled, deionized water (DDW).
10.1021/es026171g CCC: $25.00
 2003 American Chemical Society
Published on Web 05/03/2003
They were then rinsed in a 0.001 M EDTA solution and finally
5 times in DDW. This protocol, described by Fein et al. (9),
serves to strip the cell walls of calcium ions and other
substances acquired from the growth medium. The total
bacterial concentration is reported as wet weight per liter of
bacterial suspension after centrifugation at 6000 rpm (RCF
) 2560g; 12) for 30 min. This measurement was used to
estimate the number of functional groups on the cell walls
in units of functional group sites per wet weight of bacterial
suspension (g), as reported by Fein et al. (9).
The number of viable cells was determined by counting
the number of cfu (colony-forming units/volume in agar
plates). Approximately 1% of the cells were microbiologically
viable before rinsing (estimated from 10 individual determinations). After multiple rinses, less than 1% of the initially
viable cells remained viable, thus, less than 0.01% of the
bacterial cells were microbiologically viable at the start of
the experiments.
Calcite Dissolution. Calcite crystal fragments of similar
size (approximately 10 mm2) were cleaved parallel to the
{101h 4} face from a large, optical-grade Iceland spar crystal
using a razor blade. They were mounted on a glass slide with
acetone-soluble Crystalbond 509 (obtained from SPI Metallography Supplies) to ensure that only the {101h 4} face was
exposed to the solution. Tests carried out in the presence
and absence of the adhesive revealed that it did not affect
the pH of the solution or the surface-normalized rate of calcite
dissolution in DDW. The surface area of each cleavage
rhombohedron was estimated by measurement of the crystal
dimensions with an optical microscope and a ruler with an
error of (0.1 mm.
Calcite dissolution, free-drift experiments were carried
out in 0.5 L Erlenmeyer flasks in which three calcite crystals
were immersed in 0.100 L of the experimental solutions. The
flasks were stirred at 120 rpm on an orbital shaker at room
temperature (22-25 °C). They were closed to the atmosphere
(covered by Parafilm) to avoid microbial contamination from
the ambient air and evaporation of the solution. The
dissolution experiments were carried out from 5 min to 14
days, with most lasting 5 days. Experiments were conducted
in either distilled water, EDTA in distilled water, or rinsed B.
subtilis suspensions in distilled water. EDTA (ethylenediaminetetraacetic acid, disodium salt dihydrate, >99%, purchased from Aldrich) was used over the concentration range
of 0.004-2.39 mM. The range in wet weight of centrifuged
bacteria used in the experiments was 0.10-58.2 g/L. This
corresponds to approximately 4.1 × 1011-2.3 × 1014 cells/L
and concentrations that are comparable to the abundance
of microbial life in natural aquifer systems (i.e., 7 × 109-7
× 1011 cells/L; 13). The low and intermediate bacterial
concentrations used in this study fall within the range of
microbial abundances encountered in natural weathering
environments (i.e., 3 × 109-3 × 1013 cells/L; 14).
The initial pH of the distilled water and EDTA solutions
was adjusted to match the initial pH (i.e., approximately 4.5)
of the bacterial suspensions by adding a maximum of 40 µL
of 0.993 N HCl to the experimental solutions. The pH was
measured with a combination glass electrode (Orion 910600)
connected to a pH/ISE meter (Orion 710A) and calibrated
with three NIST (National Institute of Standards and Technology) traceable buffers (i.e., 4.01, 7.00, and 10.0) at 25 °C.
The dissolution experiments were interrupted after 1, 2,
6, 12, 24, 48, 72, 96, and 120 h by removing the calcite crystals
from the solutions. At the end of each experiment, the pH
and temperature were measured. Approximately 20 mL of
the solution were recovered from the Erlenmeyer flask,
transferred to polyethylene plastic containers, acidified with
0.167 mL of 12 M HNO3, and sealed with snap-on lids.
Solutions isolated from the crystals and containing the dead
bacterial cells were acidified and left to react for a minimum
of 1 h in order to liberate calcium attached to the cell walls
(15). Subsequently, the bacterial suspensions were centrifuged at 3000 rpm (RCF ) 1280g; 12) for 15 min, and the
supernatant was decanted and stored for later analyses. The
procedure was repeated a second time to ensure that all the
calcium had been stripped from the cell walls and the
supernatants were combined. The total calcium concentration in the resulting solutions was determined by flame
atomic absorption spectrophotometry (AAS) with a PerkinElmer 3100 spectrophotometer (50 ppb detection limit,
reproducible to >95%).
Estimates of the Calcite Dissolution Rates. The dissolution rates were estimated from the change in the total calcium
concentration in the solution ([Ca2+]) divided by the time (t)
and normalized to the total surface area of the three crystals
(A):
rate )
∆[Ca2+]
tA
(1)
The uncertainty on the rate measurements is estimated at
(5% based on the cumulative errors of the calcium analyses
((3%) and crystal size measurements ((3%). Buhmann and
Dreybrodt (16) demonstrated that the presence of a variety
of ionic compounds (e.g., Na+, Cl-, Mg2+, and SO42-) at a
concentration up to 1 mM does not significantly affect the
calcite dissolution rate constants; thus, the ionic strength
should not significantly influence the kinetics of dissolution
(i.e., mechanism) over the range (i.e., I < 0.007 m) of our
experimental solutions.
Saturation State of the Experimental Solutions. PHREEQC (17) was used in this study to estimate the saturation
index (SI) or saturation state (Ω) of our experimental solutions
with respect to calcite. PHREEQC uses ion-association and
Debye-Hückel expressions to account for the non-ideality
of aqueous solutions (i.e., estimate ion activity coefficients)
as a function of ionic strength. This aqueous speciation model
is entirely adequate at the low ionic strengths of our
experimental solutions (i.e., I < 0.007 m), but it does break
down at higher ionic strengths (i.e., I > 0.5 m). The SI is
defined as:
(
SI ) log
)
{Ca2+}{CO32-}
) log Ω
K °sp
(2)
where { } represents the activities of the species and Ksp° is
the thermodynamic calcite solubility constant at 25 °C and
1 atm total pressure, 10-8.48 (18). According to the above
definition, the SI is zero when the solution is saturated with
respect to calcite, SI < 0 when undersaturated, and SI > 0
when supersaturated. The error on SI is estimated as the
cumulative uncertainties on the calcium analysis ((3%), the
activity coefficient estimates obtained by PHREEQC ((4%),
and the carbonate ion concentration ((3%). The cumulative
uncertainty on the SI is estimated at (0.025 log unit. In the
presence of organic ligands, the uncertainty will be larger
due to errors in the ligand concentrations and their calcium
binding constants.
The chief constraints imposed on the model include the
assumption that the system is closed to the atmosphere as
well as the assignment of acid dissociation and metal
complexation constants for the functional groups present
on the bacterial cell walls. The system was considered closed
to the atmosphere because Parafilm, which was used to cover
the reaction flasks, was deformed during the experiment as
the headspace gas expanded and contracted with changes
in temperature. This assumption was verified by testing the
internal consistency of the carbonate system (i.e., measured
VOL. 37, NO. 11, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Deprotonation and Metal Stability Constants for EDTA and Functional Groups on B. subtilis Cell Walls Used in the
PHREEQC Model
Log K
Reference
EDTAH3- S EDTA4- + H+
EDTAH22- S EDTA4- + 2H+
EDTAH3- S EDTA4- + 3H+
EDTAH4 S EDTA4- + 4H+
B-COOH0 S B-OO- + H+
B-POH0 S B-PO- + H+
B-OH S B-O- + H+
Deprotonation Constants
-11.25
-18.08
-20.36
-22.56
-4.82 ( 0.14
-6.9 ( 0.5
-9.4 ( 0.6
Daniele et al. in ref 31
Daniele et al. in ref 31
NIST in ref 31
NIST in ref 31
8
8
8
EDTA4- + Na+ S EDTANa3EDTA4- + H+ + Na+ S EDTAHNa2EDTA4- + Ca2+ S EDTACa2EDTA4- + H+ + Ca2+ S EDTAHCaB-COO- + Ca2+ S B-COOCa+
B-POO- + Ca2+ S B-POOCa+
Metal Stability Constants
2.7
11.44
10.7
16.0
2.8
4.2
Daniele et al. in ref 31
Daniele et al. in ref 31
31
Morel et al. in ref 31
15
21
Reaction
and calculated pH from alkalinity and pCO2) using PHREEQC.
The stability constants used in the speciation model are listed
in Table 1.
Complexation reactions and constants with EDTA and
cell wall functional groups were added to the PHREEQC
database. The deprotonation constants for the cell wall
functional groups of B. subtilis are similar to those reported
in other studies (19, 20). The complexation constant for the
calcium-phosphonyl complex of B. subtilis was estimated by
the correlation technique described by Langmuir (21). This
was necessary because the complexation constant of calcium
to B. subtilis or other related bacterial species has not been
measured experimentally and estimates are not available in
the literature. The stability constants for various metals
complexed with phosphoric acid (22) were correlated to the
stability constants of metal-phosphonyl groups of the same
metals bound to the cell wall of B. subtilis (9) and a value for
the B-POOCa+ complex (log K ) 4.2, Table 1) was interpolated.
Atomic Force Microscope (AFM). Reacted crystals were
taken out of the Erlenmeyer flasks and briefly rinsed with a
minimal amount of DW in order to remove solution salts,
bacterial cells, exudates, etc. This avoided adherence of, for
example, cells to the cantilever tip of the AFM and/or
precipitation of salts that would have created morphological
artifacts on the surface of the reacted crystals. The crystals
were dried by holding a tissue at one of their edges in order
to absorb most of the residual water from the surface. The
crystals were air-dried at room temperature and stored in
closed Petri dishes.
AFM imaging of the reacted surfaces was carried out within
5 days of sampling using a Digital Instrument Dimension
3000 scanning probe microscope. The probe is a combined
assembly of a single-crystal silicon tip (model TESP) attached
to the end of a single beam cantilever mounted on a
piezoelectric scanner. The probe was operated in tapping
mode (4), and both height and phase images were captured.
The scan parameters (i.e., scan angle, scale, and speed) as
well as the original position of the sample (i.e., samples were
physically rotated 90°) were varied before image capture in
order to test for image artifacts (23). The digital images were
processed (3rd order of flattening) using the image treatment
software supplied by Digital Instruments.
Results and Discussion
Calcite Dissolution Rates. The calcite dissolution rates in
distilled water, EDTA, and B. subtilis decrease exponentially
with time irrespective of the functional group concentration
(Figure 1). The measured dissolution rate in distilled water
is not sensitive to the initial pH over the range studied (i.e.,
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FIGURE 1. Calcite dissolution rates in distilled water and in the
presence of organic ligands at equimolar functional group concentrations.
4.14-5.75) because the buffer capacity of this solution is
negligible but increases drastically following the dissolution
of calcite. The pH increased rapidly during the first 24 h of
reaction until it reached a plateau value, between 4.6 and
6.5, which differed according to the nature and concentration
of the ligand. Over the range investigated (i.e., 0-2.39 mM
EDTA and 0-58.2 g B. subtilis/L), calcite dissolution rates
increased linearly with the ligand concentration (not shown).
Rates determined in the presence of EDTA or dead
bacterial cells are compared at equimolar acid functional
group concentrations. For comparison purposes, only the
carboxylic and phosphonic sites of the bacteria cell walls are
FIGURE 3. Calcite dissolution rates as a function of SI at equivalent
functional group concentrations and in distilled water. Note that
the rate scale is logarithmic.
FIGURE 2. Evolution of SI during calcite dissolution in distilled
water and in the presence of organic ligands at equimolar functional
group concentrations.
taken into consideration, since hydroxyl sites are not
abundant and do not contribute significantly to the sequestration of calcium ions. Rates are compared at low, intermediate, and high functional group concentrations (i.e., 1.6
× 10-5, ∼4.0 × 10-3, and 9.5 × 10-3 M, respectively),
corresponding to the experimental EDTA or bacterial cell
concentrations.
Results of experiments performed at the low functional
group concentration (not shown; 0.004 mM EDTA or 0.10 g
B. subtilis/L) show no significant difference between the
calcite dissolution rates measured in the presence or absence
of organic ligands (EDTA or B. subtilis). At intermediate (1.00
mM EDTA ) 4.0 mM sites; two concentrations of bacteria:
22.5 and 26.3 g/L corresponding to 3.7 and 4.3 mM of sites,
respectively) and high (2.39 mM EDTA or 58.2 g B. subtilis/L)
but equivalent functional site concentrations, the dissolution
rates are higher in the presence of EDTA than in the bacterial
cell suspensions (Figure 1A,B). The faster calcite dissolution
rate in the presence of EDTA can be explained by the much
greater stability of the calcium-EDTA complex. Its complexation constant is 6.5-8 orders of magnitude greater than
the calcium complexes that form with functional groups on
the bacterial cell walls (Table 1).
Chemical Evolution of the Experimental Solutions. The
SI was chosen as the master variable to compare the
dissolution rates in the different experimental solutions
because it provides an unbiased characterization of the degree
of disequilibrium of the system. Thereby, a comparison of
rates normalized to SI allows one to distinguish if factors
other than the calcium and carbonate ion activity product
or the saturation state of the solution influence the dissolution
rates. Under the free-drift conditions of our experiments,
values of SI evolve toward saturation (i.e., SI ) 0) as dissolution
proceeds (Figure 2), but it is possible to compare instantaneous rates at equivalent SI values.
The calculated SI values reveal that all solutions remain
undersaturated with respect to calcite (Figure 2) throughout
the duration of the experiments. In all cases, the logarithm
of the dissolution rate is inversely proportional to SI (Figure
3), thus, the calcite dissolution rate accelerates at higher
degrees of undersaturation and is most likely dominated by
surface reactions (24). The calcite dissolution rate is barely
influenced by the presence of either EDTA or dead B. subtilis
cells at the low functional group concentration (∼1.6 × 10-5
M sites, SI-normalized, not shown). At the intermediate and
high functional group concentrations (Figure 3A,B), there is
a distinct difference between dissolution rates measured in
the presence of EDTA and the bacterial cell suspensions at
comparable SI values. In contrast, the rates measured in the
presence of B. subtilis cells are, within the uncertainty of our
rate and SI estimates, undistinguishable from those obtained
in distilled water.
Although all three systems display a strong dependency
on SI, the dissolution in the presence of EDTA clearly proceeds
at an accelerated rate, likely because the reaction mechanism
is modified. Fredd and Fogler (25) interpreted the accelerated
dissolution of calcite in the presence of various organic
chelators, including EDTA, as a direct “attack” on the crystal
surface. This attack may only proceed as long as the functional
groups of the EDTA are not completely saturated with calcium
ions. Our speciation calculations reveal that, within the first
hour, enough calcite was dissolved to saturate the EDTA sites
at the low ligand concentration (i.e., 0.004 mM EDTA).
Accordingly, at the low functional group concentration (not
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FIGURE 5. (A) Idealized {101h4} calcite rhombohedron with the c
axis oriented vertically. Nonequivalent edges and corners of type
P (polar) and E (equatorial) of the {101h4} face are shown. (B)
Schematic growth hillock or dissolution pit on an idealized single
{101h4} face, with step A′ moving toward edges of type P (After ref
32).
FIGURE 4. Calcite dissolution rates as a function of the “true” and
“apparent” SI, illustrating the influence of the organic ligands on
the solution speciation. The lines correspond to the linear leastsquares fit to the rates measured in the presence and absence of
bacterial cells in distilled water presented in Figure 3.
shown), there is no significant difference between the calcite
dissolution rates measured in the three systems under
investigation. In contrast, in the intermediate and high EDTA
concentration solutions, the ligand remained unsaturated
throughout the dissolution experiments, with its carboxylic
sites free to attack the calcite surface and enhance the
dissolution rates relative to those measured in distilled water
(Figure 3A,B).
The solution speciation model described previously, used
to estimate the saturation state of the experimental solutions
with respect to calcite, accounts for the complexation of
dissolved calcium with the organic ligands. In characterizing
the speciation of natural solutions, however, the presence of
organic ligands is generally ignored because their nature and
concentration are not readily determined. Thus, the saturation state of the solutions estimated from a purely inorganic
speciation model may be inaccurate and lead to observational
inconsistencies (e.g., dissolution of calcite in an apparently
supersaturated solution). To illustrate this, results of our
dissolution rate experiments in distilled water and in the
presence of dead B. subtilis cells at intermediate and high
functional group concentrations are reproduced in Figure 4
as a function of the “true” (i.e., including organic complexation) and “apparent” (excluding organic complexation) SI.
They clearly show that the complexation of dissolved Ca2+
by B. subtilis cell wall functional groups decreases the
saturation state of the solutions and that the calcite dissolution rates measured in the presence of these ligands are
nearly identical to those measured in distilled water (Figure
3) at equivalent, “true” SI. Ignorance of the complexation by
the cell wall functional groups would have led us to conclude
that their presence accelerates the calcite dissolution rate.
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FIGURE 6. Nomenclature for section analysis. (A) Asymmetric
dissolution pit on an idealized {101h4} face. (B) Cross-section of
idealized asymmetric dissolution pit (x/y ) degree of anisotropy).
These results will likely be modified by the metabolic activity
and the presence of exudates in systems with live bacteria.
Atomic Force Microscopy. In most of the earlier AFM
studies, dissolution features that developed on the surface
of calcite were imaged after short reaction times (i.e., seconds
to minutes; e.g., refs 26-28). This section focuses on the
morphology of the calcite {101h 4} surface and more specifically on the anisotropy of the dissolution pits generated
during the reaction following dissolution for between 5 min
and 1 h in distilled water, EDTA solutions, and aqueous
suspensions of dead B. subtilis cells.
As dissolution takes place, pits typically develop as
discrete, straight-edge features on the mostly flat {101h 4}
cleavage surface of calcite (29). Figure 5 depicts the plan
view of a rhombohedral dissolution pit (Figure 5B) with edges
parallel to those of the morphological (or cleavage) plane of
calcite (Figure 5A) (30). As dissolution proceeds further, the
pits (Figure 5B) grow wider and deeper but usually at different
rates along edges that are parallel and opposite to each other,
and the resulting morphology can be described by its degree
of anisotropy. The pit morphology (i.e., aspect ratios) that
develops early on is preserved as the pit grows (27).
Consequently, the steepness of the pit walls is directly related
to its degree of anisotropy.
A 3D image of the dissolution pit is generated by the AFM,
from which a cross-sectional reconstruction along the
symmetry axis is obtained (Figure 6). From this cross-section,
the length, (x + y), is determined by the image treatment
software (Digital Instruments). The degree of anisotropy is
calculated as x, the horizontal length of the shortest flank,
divided by y, the horizontal length of the longest flank (Figure
6). A value of x/y ) 1 represents a perfectly isotropic pit,
whereas values deviating from 1 represent an increasing
anisotropy is expected given the fact that parallel steps on
opposite sides of the pit are not related by the face symmetry
of the calcite rhombohedron. At the low B. subtilis cell
concentration (0.004 mM functional group sites), the average
degree of anisotropy of the pits was 0.44 ( 0.12 (n ) 5; image
not shown), but the surfaces were not imaged at the low
EDTA concentration. At higher functional group concentrations (i.e., 4.0 mM sites as EDTA, Figure 7B, or 4.3 mM sites
on B. subtilis cell walls, Figure 7C), the average values of pit
anisotropy were found to be 0.81 ( 0.19 (n ) 4) and 0.94 (
0.05 (n ) 5), respectively. These results suggest that, above
a critical organic ligand concentration (i.e., >0.004 mM
functional group sites), the dissolution mechanism changes.
Despite the enhanced dissolution rates in the presence of
EDTA compared to those measured in suspensions of B.
subtilis cells, the anisotropy of dissolution pits revealed by
AFM images is very similar. Isotropic dissolution pits probably
did not form in the low concentration B. subtilis solution
because all the cell wall binding sites were rapidly saturated
with calcium.
The use of AFM to quantitatively characterize the morphology of calcite dissolution pits that develop in the presence
of natural (i.e., dead bacteria/cell walls) and synthetic organic
ligands (i.e., EDTA) provides insights into the mechanisms
that govern these reactions. This study shows that increasing
concentrations of both bacterial cells and EDTA result in the
development of different micro-topographic features on
calcite mineral surfaces, including an increased isotropy of
the dissolution pits.
Acknowledgments
Financial support for this research was provided by the
Natural Sciences and Engineering Research Council of
Canada (NSERC) through individual grants to A.M. Additional
funds were provided by GEOTOP-UQAM-McGill, Knud
Højgårds Fond, Ingeniørforeningen Danmark, Rudolph Als
Fondet, Frants Allings Legat, and UniDanmark Fondet. The
authors wish to thank Terry Beveridge for providing a
laboratory stock culture of Bacillus subtilis 168. A.K.F. would
also like to thank Prof. H. Vali for stimulating discussions on
the interactions of biomolecules with the calcite surface;
Associate Professor Rasmus Jakobsen for guidance on the
use of PHREEQC; Glenn Poirier for operation of the AFM
and interpretation of images; Constance Guignard, Glenna
Keating, and Sandra Lalli for their technical assistance in the
laboratory and analytical/intrumental instructions. Finally,
we would like to acknowledge the three anonymous reviewers
who provided critical and constructive comments on a
previous version of this manuscript.
Literature Cited
FIGURE 7. Representative pits developed on calcite crystals upon
dissolution in some of our experimental solutions: (A) distilled water,
(B) 1 mM EDTA (i.e., 4.0 × 10-3 M functional groups), and (C) 26.3
g B. subtilis/L (i.e., 4.3 × 10-3 M functional groups).
degree of anisotropy.
A pit that typically develops following dissolution in
distilled water displays an anisotropic morphology (Figure
7A). The average degree of anisotropy (i.e., x/y) of pits that
developed in distilled water was 0.44 ( 0.20 (n ) number of
distinct measurements ) 5). As indicated earlier, this
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Received for review September 19, 2002. Revised manuscript
received February 26, 2003. Accepted March 13, 2003.
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