Geomicrobiology Journal

ISSN: 0149-0451 (Print) 1521-0529 (Online) Journal homepage: https://www.tandfonline.com/loi/ugmb20

The Influence of Bacillus pasteurii on the

Nucleation and Growth of Calcium Carbonate

Andrew C. Mitchell & F. Grant Ferris

To cite this article: Andrew C. Mitchell & F. Grant Ferris (2006) The Influence of Bacillus�pasteurii
on the Nucleation and Growth of Calcium Carbonate, Geomicrobiology Journal, 23:3-4, 213-226,
DOI: 10.1080/01490450600724233

To link to this article: https://doi.org/10.1080/01490450600724233

Published online: 23 Feb 2007.

Submit your article to this journal

Article views: 537

Citing articles: 88 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=ugmb20
Geomicrobiology Journal, 23:213–226, 2006
Copyright

c Taylor & Francis Group, LLC
ISSN: 0149-0451 print / 1521-0529 online
DOI: 10.1080/01490450600724233
The Influence of Bacillus pasteurii on the Nucleation
and Growth of Calcium Carbonate
Andrew C. Mitchell and F. Grant Ferris
Department of Geology, University of Toronto, Toronto. Ontario, Canada
Microcosm experiments were performed to identify the influ-
ence of bacterial cell surfaces on the morphology, mineralogy, size
and solubility of CaCO3 precipitated in response to the enzymatic
hydrolysis of urea in an artificial groundwater (AGW) by the ure-
olytic bacteria, Bacillus pasteurii. In each microcosm, B. pasteurii
were contained within a cellulose dialysis membrane (10 K Dalton
MWCO), resulting in bacteria-inclusive and bacteria-free AGW
solution. Urea hydrolysis by B. pasteurii resulted in the production
of ammonium and an increase in pH in the whole AGW solution.
This initiated predominantly rhombohedral calcite precipitation
at the same critical saturation state (Scritical = 12) in the B. pas-
teurii-inclusive and bacteria-free zone of the AGW, indicating the
mineralogy and morphology of CaCO3 precipitation is not con-
trolled by B. pasteurii surfaces. However, the temporal evolution
of distinctly different lognormal crystal-size-distributions in the
B. pasteurii-inclusive and bacteria-free zone of the AGW resulted
from identical changes in bulk solution chemistry. Specifically, B.
pasteurii increased the size and size variance of crystals, and led
to a greater crystal growth rate throughout the experiments, rel-
ative to bacteria-free AGW. Calculated crystal solubility (ln K S0 )
was lower for crystals >4000 nm in diameter, reflecting smaller
molar surface areas. This suggests that the larger crystals gener-
ated in the presence of B. pasteurii have a lower affinity for re-
dissolution than those generated in the bacteria-free AGW, which
may act as a positive feedback to maintain larger crystal sizes in
the presence of B. pasteurii. During ureolysis, higher bacterial con-
centrations may therefore generate larger and less soluble carbon-
ate crystals. This has important implications for the adaptation of
bacterial ureolysis as a method for precipitating calcium carbon-
ate and co-precipitating metals and radionuclides in contaminated
aquifers.
Keywords Bacillus pasteurii, ureolysis, calcite precipitation, crystal-
size-distribution (CSD), groundwater, bio-remediation,
dialysis tubing, GALOPER
Received 6 October 2005; accepted 10 February 2006.
This work was supported by grant DE-FG07-99ER15025 from the
U.S. Department of Energy, Office of Energy Research Environmental
Management Science Program. Thanks to Lisa Magalhaes for under-
taking much of the crystal size measurement.
Address correspondence to Dr. Andrew C. Mitchell, Department of
Microbiology, Montana State University, 109 Lewis Hall, Bozeman,
Montana 59717-3520, USA. E-mail: mitchell@montana.edu
213
INTRODUCTION
Bacterially induced carbonate precipitation has long been of
interest, reflecting its role on carbon cycling at the geological
timescale (Vasconcelos et al. 1995), and more recently for in-
dustrial utilities such as mineral plugging (Ferris and Stehmeier
1992; Ferris et al. 1996; Bachmeier et al. 2002) and immobilising
calcium and contaminants in surface- and ground-water (Curti
1999; Hammes et al. 2003; Mitchell and Ferris 2005, 2006).
Bacterial carbonate precipitation results from both passive and
active nucleation (Schultze-Lam and Beveridge 1994; Schultze-
Lam et al. 1996; Stocks-Fischer et al. 1999; Warren et al. 2001).
Passive carbonate nucleation occurs from metabolically driven
changes in the chemistry of the bulk solution and in microenvi-
ronments around bacterial cells, which increases mineral satu-
ration and induces nucleation (Schultze-Lam et al. 1996). This
commonly reflects an increase in pH from the production of
−
CO2− 3 or HCO3 by bacterial ammonification (Stocks-Fischer
et al. 1999) or sulphate reduction in anoxic environments (Van
Lith et al. 2003), or from alkalisation by photosynthetic HCO−
3
consumption and OH− production by cyanobacteria (Ferris et al.
1997). Conversely, active carbonate nucleation occurs when bac-
terial cell surfaces are utilised as nucleation substrates, owing to
the chemically heterogeneous macromolecules that impart a net
electronegative charge, which favours the adsorption of cations
−
and the following attraction of CO2− 3 or HCO3 (Schultze-Lam
and Beveridge 1994; Bosak and Newman 2003; Van Lith et al.
2003). Carboxyl groups found at the surface of most gram-
positive and gram-negative bacteria exhibit the same spatial con-
figuration as the calcite lattice and share geometrical homologies
with carbonate ions, and are therefore critical to active calcite
formation (Braissant et al. 2003).
Although microorganisms are often associated with carbon-
ates in the natural environment, the extent to which they influ-
ence the size, morphology and mineralogy of carbonate minerals
is still poorly understood. Bacteriogenic macromolecules and or-
ganic acids (proteins, amino acids), and inorganic contaminants
have been shown to strongly influence the morphology and min-
eralogy of carbonates, by promoting distortions in the mineral
structure, which gives rise to a wide range of micro- and macro-
scale morphologies (Kitano and Hood 1965; Carter and Mitterer
1978; Falini 2000; Manoli and Dalas 2001; Meldrum and Hyde
214 A. C. MITCHELL AND F. G. FERRIS
2001; Sondi and Matijevic 2001; Braissant and Verrecchia 2002;
Braissant et al. 2003; Freij et al. 2004; Kamiya et al. 2004; Kralj
et al. 2004; Roque et al. 2004), as well as influencing the satu-
ration that can be attained and the rate at which carbonates are
precipitated, which controls the resulting carbonate polymorphs
produced (Meldrum and Hyde 2001; Bosak et al. 2004; Roque
et al. 2004; Bosak and Newman 2005). However, quantifying
the effect of bacteria on the size of precipitated carbonate crys-
tals has received little attention, despite the implications this has
for mineral solubility and stability (Mitchell and Ferris 2006;
Stumm and Morgan 1996), and determining the role of microor-
ganisms on carbonate precipitation in contemporary natural en-
vironments and the geologic record (Warren et al. 2001).
In this study we investigate CaCO3 precipitation in response
the enzymatic hydrolysis of urea by ureolytic bacteria (ureoly-
sis) (Stocks-Fischer et al. 1999; Fujita et al. 2000; Warren et al.
2001). This mechanism is being assessed as a potential method
for co-precipitating and immobilising divalent metals and ra-
dionuclides in situ in groundwater aquifers in the western USA
(Ferris et al. 2003; Fujita et al. 2004; Mitchell and Ferris 2005,
2006) which are contaminated as a legacy of Department of En-
ergy (DOE) operations (Knobel et al. 1992). Ureolysis results
in the production of ammonium (NH+ 4 ) and dissolved inorganic
carbon (DIC), and as increase in pH (Equation 1), which favors
calcite precipitation (Equation 2).
NH2 CONH2 + H+ + 2H2 O ⇔ 2NH+4 + HCO3
−
[1]
Ca2+ + 2HCO−
3 ⇔ CaCO3 (s) + CO2 + H2 O [2]
We consider the temporal evolution of crystal size-frequency
plots, or Crystal Size Distributions (CSDs), of calcium carbonate
precipitates induced by ureolysis from a bacteria-inclusive and
a bacteria-free artificial groundwater, mimicking the composi-
tion of the radionuclide contaminated Snake River Plain Aquifer
(SRPA) at the Idaho National Engineering and Environmen-
tal Laboratory (INEEL) (Mitchell and Ferris 2005). CSDs are
shaped by the fundamental kinetic, thermodynamic and trans-
port processes of crystal development (Eberl et al. 1998; McCoy
2001), and can therefore be used to elucidate the effect of bac-
terial surfaces and exudates on the nucleation and growth of
CaCO3 . CSDs first develop during nucleation, when crystals
equal to or greater than the critical nucleus size grow rapidly at
high levels of saturation. The CSD is then subsequently modified
by different rates at which crystals nucleate, or stop nucleating,
and the availability (surface-controlled growth) or unavailabil-
ity (supply-controlled growth) of solute for crystal development
(Eberl et al. 1998).
MATERIALS AND METHODS
Experimental Setup
Batch experiments were performed using an artificial ground-
water (AGW) mimicking the SRPA (Knobel et al. 1992), pre-
pared using ACS reagent grade chemicals and sterile ultrapure
water (SUPW). Specific inorganic constituents of the AGW
included KNO3 (0.0403 mM), MgSO4 (0.448 mM), CaCl2
(1.75 mM), NaNO3 (0.044 mM), NaHCO3 (1.1 mM) and
KHCO3 (0.0623 mM). Urea was added to the AGW at 16.5
mM to simulate the addition of urea to the aquifer. The AGW
was then acidified with analar grade HCl to a pH of 6.5 to
avoid premature calcium carbonate precipitation, and was ster-
ilised by filtration through 0.2 µm pore size cellulose nitrate
filters. Experiments were performed using the gram-positive,
urease positive endospore forming bacteria Bacillus pasteurii
ATCC 11859, and control experiments with the gram-positive
urease deficient Bacillus subtilis 168. It is instructive to note
that B. pasteurii has now been reclassified Sporosarcina pas-
teurii (Yoon et al. 2001). Cultures were grown overnight prior
to the experiment in Brain Heart Infusion, then centrifuged at
8500 × g for 10 minutes, washed four times with SUPW and
re-suspended in SUPW at an optical density of 0.14 at 600 nm
(OD600 ). The AGW was initially made at double strength to
be mixed in equal parts with SUPW or the bacterial suspen-
sions, resulting in a final strength bacteria-free AGW, and B. pas-
teurii-inclusive, or B. subtilis-inclusive AGWs with an OD600 of
0.07.
Individual microcosms were then immediately prepared:
100 ml of bacteria-free AGW was decanted into pre-autoclaved
acid washed 250 ml polypropylene cylinders containing acid
washed stir bars. 75 ml of either B. pasteurii-inclusive (test)
or B. subtilis-inclusive (control) AGW was then decanted into
a tube of inert Spectrum Spectra/Por 6 Regenerated Cellulose
Dialysis Membrane with a 29 mm diameter and a 10 K Dalton
molecular weight cut off (MWCO). This had been thoroughly
soaked for 1 day and rinsed 10 times in SUPW, and sealed
with autoclaved nylon clamps. This pore size will contain the
bacteria, and organic macromolecules, including high to low
molecular weight proteins and polysaccharides from bacterio-
genic exudates, but will allow inorganic ions to pass through
(Clapham et al. 1990). While amino acids <10 K MWCO from
the degradation of proteins may possibly pass through the dialy-
sis membrane, most organic molecules will be contained within
the B. pasteurii-inclusive AGW. Each dialysis tube containing
bacteria-inclusive AGW was immediately immersed into one of
the polypropylene cylinders containing bacteria-free AGW, cov-


R
ered with Parafilm , and placed on a magnetic stirring plate at a
controlled room temperature of ∼20◦ C. This resulted in control
and test microcosms with equivalent starting AGW chemistry,
with bacteria-inclusive AGW inside the dialysis membrane, and
bacteria-free AGW outside the membrane (Figure 1). Reverse
experiments were also undertaken in duplicate as above, but
with the bacteria-free AGW inside the dialysis tubing, and the
bacteria-inclusive AGW outside the dialysis tubing, in order to
assess any experimental artefacts.
Solution Chemistry and Reaction Kinetics
At time 0 and on days 1, 2, 4 and 7, the solution and crystals
that may have formed were decanted from the bacteria-inclusive
 INFLUENCE OF B. PASTEURII ON CaCO3 PRECIPITATION
FIG. 1. Schematic diagram of experimental setup.
and bacteria-free AGW of the control and test experiments, into
separate polypropylene sterile containers. 20 ml of solution was
extracted from each of the containers with a sterile syringe, and
filtered through a 0.2 µm pore-size polycarbonate filter. From
this solution, pH was measured on a Ross Sure-Flow electrode
calibrated with NIST traceable buffers, and dissolved NH+ 4 was
measured by the Nessler method using a HACH DR-2000 spec-
trophotometer (precision ±6%). Ca2+ was determined using a
Perkin-Elmer 4000 flame atomic adsorption spectrophotometer
(precision ± 4%). From the solution data, the calcite saturation
state was calculated from the product of the activities of dis-
solved Ca2+ and CO32− divided by the equilibrium calcite solu-
bility product. The critical saturation state at which precipitation
began and the absolute reaction rate of calcite precipitation was
determined as previously described (Ferris et al. 2003; Mitchell
and Ferris 2005) using affinity-based rate laws applied to the ex-
perimental data using unconstrained non-linear estimation and
a quasi-Newton optimisation routine for parameter estimation
in STATISTICA v 6.0.
Crystal Collection and Analysis
Decanted bacteria-inclusive and bacteria-free AGW solutions
were immediately fixed using glutaralderhyde to a final concen-
tration of 2%, and were vacuum filtered through 0.2 µm pore-
215
size polycarbonate filters. No other buffers or preparations were
used to avoid any possible acid dissolution of the precipitated
crystals. Filtered precipitates were mounted on carbon slips, gold
coated on a Cressington 108 sputter coater, and imaged by scan-
ning electron microscopy (SEM) on a JEOL 840 in secondary
electron mode for improved contrast between the crystals and the
background filter paper. For days 1 and 7, a random area of the
coated precipitates was imaged at ×50, and from this image <20
randomly chosen areas were imaged at ∼×1.5 k. All crystals in
the field of view were manually measured at their maximum
diameter using ImageJ v.1.33. This generated between 157 and
646 individual crystal diameter measurements for each system
on each day of the experiments. Mineralogical analysis of the
precipitates was performed by powder X-ray diffraction (XRD)
using a Phillips XRD system. Crystals were mounted on silicon
gel on glass slides, and were imaged between 20 and 60 2θ.
Modelling Nucleation and Crystal Growth
Raw crystal diameter data was entered into the USGS
CRYSTAL COUNTER software (Eberl et al. 2000) and a CSD
with a 500 nm bin size was generated for days 1 and 7 of the
experiment. The observed CSDs from the bacteria-inclusive
or bacteria-free AGW were then matched with simulated
CSDs, modelled using distinct crystal nucleation and growth
216 A. C. MITCHELL AND F. G. FERRIS
mechanisms using the USGS GALOPER (Growth According
to the Law of Proportionate Effect) software (Eberl et al. 2000).
GALOPER assumes crystal growth rates are proportional to
crystal size, rather than the same linear growth rate once crystals
have been nucleated (Kirkpatrick 1981; Marsh 1988). Size pro-
portionate growth is the only mechanism by which CSDs can
be created and maintained under the conditions of many natural
systems (Eberl et al. 2002). This Law of Proportionate Effect
(LPE) is defined as:
XJ+1 = XJ + εJ XJ ,
where XJ is the linear crystal dimension after J calculation cy-
cles, and εJ is a random number that generally varies between
0 and 1 which differs for each crystal and each growth cycle.
Equation 3 is iterated many times for each crystal with each
iteration termed a growth cycle (Kile et al. 2000). Nucleation
mechanisms that can be simulated using GALOPER include a
constant-rate of nucleation, or a decaying-rate of nucleation, fol-
lowed by surface-controlled growth, supply-controlled growth,
constant-rate and random growth, Ostwald ripening, random
ripening, and crystal coalescence (Eberl et al. 2000; see Mitchell
and Ferris 2006 [Supporting Information] for details).
Mass, Surface Area and Solubility
The solubility of individual CaCO3 precipitates was esti-
mated as a function of crystal diameter. Smaller crystals tend to
have a larger free surface energy than larger crystals that makes
them thermodynamically less stable and more soluble (Schindler
FIG. 2. Ammonium NH+ 4 concentrations, (b) ureolysis rate, (c) pH, and (d) Ca
over the duration of the ureolysis experiments.
1967; Morse and Mackenzie 1990; Stumm and Morgan 1996).
The solubility of individual crystals was estimated as the solu-
bility constant of a given molar surface area (Ks0(A) )(Mitchell
and Feris 2006; Schindler 1967; Stumm and Morgan 1996), ac-
cording to;
2γ A
ln Ks0(A) = ln Ks0(A=0) + [4]
3RT
where ln Ks0(A=0) is the published solubility constant at equi-
librium (for an infinite molar surface area, A, =0), γ is the
[3] solid-liquid interfacial free energy for calcite (0.094 joules m−2 )
(Stumm and Morgan 1996), R is the gas constant and T
is the temperature in Kelvin. The molar surface area (moles
m−2 ) for individual crystals was calculated according to A =
Moles/Surface Area (m−2 ). The mass and surface area of indi-
vidual crystals was estimated assuming spherical precipitates.
RESULTS
Aqueous Chemistry in the Bacteria-Inclusive
and Bacteria-Free Artificial Groundwater
In experiments containing B. pasteurii-inclusive AGW,
NH+ 4 concentrations increased identically in both the bacteria-
inclusive and bacteria-free AGW over the duration of the exper-
iments, until nearly all the urea had been hydrolysed by day 7
(Figure 2a). The rate of ureolysis decreased gradually over the
duration of the experiments to negligible rates by day 7 in the
B. pasteurii-inclusive AGW (Figure 2b; Table 1). Ammonifica-
tion caused a rapid increase in pH from ∼6.5 at time 0 to 9.1 by
2+ concentration in the B. pasteurii-inclusive and bacteria-free artificial groundwater
 INFLUENCE OF B. PASTEURII ON CaCO3 PRECIPITATION 217

TABLE 1
Average ureolysis rate, calcite saturation state (S), calcite
precipitation rate, and rate constant for calcite precipitation
(k p ) in response to bacterial ureolysis in the B.
pasteurii-inclusive and bacteria–free artificial groundwater
B. pasteurii- bacteria-
Mixture inclusive AGW free AGW
Ureolysis rate (mmol L−1 day−1 ) 3.2 —
S (calcite) 3.8 3.7
Calcite precipitation rate (mmol 0.055 0.054
L−1 day−1 )
kp 3.12 3.11

day 1 in both the B. pasteurii-inclusive and bacteria-free AGW,

consistent with buffering by NH+ +
4 = NH3 + H , which has a pKa
value of ∼9.3 at the temperature of the experiments (Stumm and
Morgan 1996; Figure 2c). Increases in NH+ 4 concentration and
pH were associated with a decrease in the concentration of Ca2+
in both the B. pasteurii-inclusive and bacteria-free AGW (Figure
2d). Identical changes in the concentration of NH+ 4 , Ca
2+
and H+
in both the B. pasteurii-inclusive and bacteria-free AGW dur-
ing the experiment demonstrates there was rapid ionic diffusion
across the dialysis membrane.
The decrease in the concentration of Ca2+ correlated with the
precipitation of visible crystals both inside and outside the dial-
ysis membrane. Crystals were visually apparent slightly earlier
in the bacteria-free AGW. XRD analysis of crystals from the
B. pasteurii-inclusive and bacteria-free AGW exhibited strong
calcite peaks. While peaks which had an approximate match
with aragonite were apparent, they were very small and very
poorly defined, indicating ureolysis in waters with the composi-
tion of the SRPA lead predominately to the formation of calcite.
Experiments containing B. subtilis-inclusive AGW exhibited no
NH+ 4 production, and while pH drifted upwards to ∼8, probably
reflecting CO2 degassing from the AGW, Ca2+ concentrations
remained constant at ∼1.5 mM, and no crystals were formed
(data not shown). CaCO3 formation is therefore exclusively as-
FIG. 3. Calcite saturation state (S) as a function of time, (b) Calcite precipi-
sociated ureolysis driven by B. pasteurii, on which the following
tation rate as a function of time and (c) Calcite precipitation rate as a function
sections will focus. of S.
The calculated saturation state for calcite (S) increased
equally in both the B. pasteurii-inclusive and bacteria-free AGW culated from solution chemistry using affinity based rate laws
from time 0 to day 2, indicating the rate of CO2− 3 production by
(Ferris et al. 2003; Mitchell and Ferris 2005), increased from day
ureolysis was greater than the rate of removal by CaCO3 precip- 1 to 2, and then decreased over the remainder of the experiments
itation. S then decreased over the remainder of the experiments in the B. pasteurii-inclusive and bacteria-free AGW (Figure 3b).
in both the B. pasteurii-inclusive and bacteria-free AGW, in- Rates were equivalent in the B. pasteurii-inclusive and bacteria-
dicating the rate of CO2−3 production was now smaller than the
free AGW (Table 1) and were fundamentally controlled by, and
rate of removal (Figure 3a). The critical saturation state at which increased with S (Figure 3c).
precipitation was initiated (Ferris et al. 2003; Mitchell and Ferris
2005) was equal in the B. pasteurii-inclusive and bacteria-free Calcium Carbonate Morphology
AGW (Scritical = 12 ± 5%), occurring between time 0 and day 1, Examples of calcite crystals precipitated in the B. pasteurii-
consistent with the observed decline in dissolved Ca2+ from the inclusive and bacteria-free AGW indicate crystal morphology
beginning of the experiments. Rates of calcite precipitation, cal- is predominantly rhombohedral (Figure 4a to d). However,
218 A. C. MITCHELL AND F. G. FERRIS

FIG. 4. Example SEM images of calcite precipitates generated in response to bacterial ureolysis. Calcite crystals generated by day 1 in the (a) B. pasteurii-
inclusive AGW, and (b) and in the bacteria-free AGW, and by day 7 in the (c) B. pasteurii-inclusive AGW, and (d) and in the bacteria-free AGW. (e) and (f) show
the stepped surface topography of crystals in both the B. pasteurii-inclusive and bacteria-free AGW that causes somewhat rounded crystal edges. (c) and (e) also
show surface indentations of B. pasteurii encased within the growing crystals from the B. pasteurii-inclusive AGW. (g) and (h) show examples of calcite crystals
generated by bacterial ureolysis from reverse experiments from the B. pasteurii-inclusive and bacteria-free AGW.
 INFLUENCE OF B. PASTEURII ON CaCO3 PRECIPITATION 219

FIG. 5. Crystal size distributions of calcite crystals generated from bacterial ureolysis from B. pasteurii-inclusive and bacteria-free artificial groundwater, on (a)
day 1 and (b) day 7 of the experiment, generated using the USGS CRYSTAL COUNTER software (Eberl et al. 2000). Log-normal fits of the raw CSDs from the B.
pasteurii-inclusive and bacteria-free artificial groundwater, generated in the CRYSTAL COUNTER software, are shown in (c) and (d) respectively. The strength
of the lognormal CSD fit is expressed as the residual squared of the crystal size frequency between the raw and fitted CSD (n = 100, significant always at 99%
confidence level).

morphology is not clean, and often appears poorly ordered, with
a stepped surface topography which causes somewhat rounded
crystal edges (Figure 4e and f). By day 7, crystals from the
bacteria-free AGW appear more poorly ordered, with rougher
crystal faces (Figure 4d) than those precipitated in the presence
of B. pasteurii (Figure 4c). Bacteria are also evidently encased
within the growing crystals from the bacteria-inclusive AGW,
as the crystals become larger than the individual B. pasteurii
(Figure 4c and e). Reverse experiments, undertaken with the
bacteria-free AGW inside the dialysis tubing, and the B. pas-
teurii-inclusive AGW outside the dialysis tubing, exhibited the
same morphologies associated with the presence and absence of
bacteria (Figure 4g and h), demonstrating crystal morphologies
were influenced by B. pasteurii, and were not an artefact of the
experimental design and the influence of the dialysis membrane.
SEM images confirmed the complete absence of bacteria in the
bacteria-free AGW.
Crystal Size and Mass
CSDs from the B. pasteurii-inclusive and bacteria-free AGW
on days 1 and 7 of the experiment are shown in Figure 5. Crys-
tal diameters exhibit an apparent lognormal distribution in the
presence and absence of B. pasteurii (Figure 5a and b). This was
confirmed by lognormal CSD curves generated by CRYSTAL
COUNTER software (Eberl et al. 2000), which provided a excel-
lent fit of the raw crystal diameter data (Figure 5c and d). These
data demonstrate that distinctly different CaCO3 CSDs evolve
in the B. pasteurii-inclusive and bacteria-free zone of the AGW
from the identical changes in solution chemistry described.
Specifically, by day 1, crystals from the B. pasteurii-inclusive
220 A. C. MITCHELL AND F. G. FERRIS

TABLE 2 by only 900, 1900 and 3600 nm respectively in the bacteria-

Summary crystal diameter data (nm) for calcite precipitates free AGW (Table 2). The median crystal diameter at the end
induced by bacterial ureolysis from the B. pasteurii-inclusive of the experiments was 46% larger in the B. pasteurii-inclusive
and bacteria-free AGW than the bacteria-free AGW. Estimates of crystal mass, assuming
spherical precipitates, suggest this increase in crystal diameter
B. pasteurii- Bacteria-free accounts for increase in median crystal mass of 2.9 × 10−7 mg
inclusive AGW AGW in the presence of B. pasteurii, and only 8.5 × 10−8 mg in the
Day 1 Day 7 Day 1 Day 7 absence of bacteria (Figure 6b). While sizes increased, the vari-
ance of the CSDs remained near constant from day 1 to 7 in
No. particles measured 225 555 646 157 both the B. pasteurii-inclusive (0.72 to 0.70) and bacteria-free
Mean 4200 7400 2400 5000 AGW (0.23 to 0.29). The reverse experiments exhibited approx-
Natural Log mean size (a) 7.8 8.6 7.7 8.2 imately the same CSD relationship in the presence and absence
Median 2200 6000 2200 4100 of bacteria to those described above, demonstrating the CSDs
Minimum 300 335 380 600 are not an artefact of the experimental setup, and the effect of
Maximum 74000 28000 11000 120000 the dialysis membrane (Figure 7).
10th Percentile 900 2000 1200 1900 While crystals occur most frequently in the <20,000 nm di-
90th Percentile 8400 15000 3700 7300 ameter range (Figure 5), 17 crystals larger than 20,000 nm were
99th Percentile 36000 20000 7200 11000 present. While these only accounted for 1% of the total number
Size variance (b2 ) 0.72 0.70 0.23 0.29 of crystals measured, individual crystals larger than 20,000 nm
r2 of CSD fit∗ 0.87 0.87 0.97 0.85 in diameter account for between 1 and 32% of the total crystal
∗
mass precipitated on days 1 or 7 in the B. pasteurii-inclusive
CSD (Figure 5c and d) generated in USGS CRYSTAL COUNTER
or bacteria-free AGW. There does not appear to be any clear
software (Eberl et al. 2000). The strength of the lognormal CSD fit is
expressed as the residual squared of the crystal size frequency between relationship with the size and frequency of these anomalously
the raw and fitted CSD (n = 100, significant always at 99% confidence large crystals with either the duration of crystal growth or the
level). presence or absence of bacteria (data not shown).
It is instantly apparent that the larger diameter of the crystals
AGW exhibit a lower mode and minimum crystal diameter, developed in the B. pasteurii-inclusive AGW may reflect the
an equal 10th percentile and median crystal diameter, but a physical mass of bacterial cells encased at the surface and in-
higher 90th percentile and maximum crystal diameter than in ternally within the growing precipitate (Figures 4c and e), since
the bacteria-free AGW. These differences are reflected by the far bacteria often appear to retain their original volume within grow-
higher CSD variance in the B. pasteurii-inclusive AGW (0.72) ing biominerals (Stocks-Fischer et al. 1999; Konhauser et al.
than the bacteria-free AGW (0.23) (Figure 5; Table 2). 2001; Dittrich et al. 2003). In order to assess this affect, the
Crystal growth is apparent from day 1 to 7, with greater crystal dimensions of 20 individual B. pasteurii surface indentations
growth in the B. pasteurii-inclusive than the bacteria-free AGW were measured, and an average volume estimated, according to
(Figures 5 and 6a). Specifically, the 10th percentile, median and the volume of a cylinder (π r2 h), where h is the length of the
90th percentile crystal diameter increases by 1100, 3800 and bacteria, and r is the radius, assumed to be half the width of
7600 nm respectively in the B. pasteurii-inclusive AGW, and the bacteria. The number of bacteria encased in the surface of

FIG. 6. (a) Crystal size and (b) estimated spherical mass as a function of calcite precipitation rate in the B. pasteurii-inclusive and bacteria-free AGW.
 INFLUENCE OF B. PASTEURII ON CaCO3 PRECIPITATION 221

FIG. 7. Comparison of calcite crystal size distributions generated by day 1 in the (a) normal and (b) reverse experiments.

calcite crystals from day 7 of the B. pasteurii-inclusive AGW
was then measured as a function of surface area for 50 crystals.
The total bacteria volume was then estimated as a percentage
of crystal volume, assuming the observed bacterial volumes at
the surface occurred at different crystal thicknesses throughout
the entire crystal, from 0.5 to 10 µm (i.e., different bacterial
densities internally within the entire precipitate). The maximum
bacteria volume estimated as a percentage of crystal volume for
the different crystal thicknesses was then subtracted from the ob-
served crystal volumes, and the effect on crystal size calculated
(Table 3).
These calculations demonstrate that even if the highest mea-
sured bacterial density is assumed to occur internally every
0.5 µm through the entire crystal (the equivalent of the max-
imum measured density of bacteria occuring the thickness of
one cell throughout the entire crystal), only 12% of total crystal
volume comprises of bacteria, which would only account for
4.4% of the crystal diameter. The ∼46% larger median crystal
diameter exhibited by day 7 in the B. pasteurii-inclusive, than the
bacteria-free AGW, demonstrates that the observed differences
in CSD, generated from equal changes in solution chemistry and
precipitation rates, cannot simply reflect the mass of the encased
B. pasteurii.
Modelling Crystal Nucleation and Growth
Crystal evolution comprises of a nucleation and growth stage
(Lasaga 1998), as can be modelled on GALOPER (see Mitchell
and Ferris 2006 for details). Combinations of all nucleation
mechanisms and all subsequent crystal growth mechanisms
were modelled. The shape, variance and mean crystal diame-
ter of the observed lognormal CSDs from day 1 of both the
B. pasteurii-inclusive and bacteria-free AGW could only be

TABLE 3
Estimates of the effect of encased B. pasteurii at the surface and internally within calcite precipitates, on the diameter
of resulting crystals
Mean Median Min Max
Area of calcite (µm2 ) 95 93 66 117
Number B. pasteurii 13 13 8 18
Total B. pasteurii volume (µm3 )∗ 4.6 4.5 2.8 6.2
B. pasteurii as % of crystal volume∗∗ Per 0.5 µm thick Per 1 µm thick Per 5 µm thick Per 10 µm thick
Mean 9.6 4.8 0.96 0.48
Median 9.7 4.8 0.97 0.48
Min 7.7 3.9 0.77 0.39
Max 12 6 1.2 0.6
Estimated % difference in crystal diameter between Per 0.5 µm thick Per 1 µm thick Per 5 µm thick Per 10 µm thick
bacteria-inclusive and bacteria-free crystals,
assuming maximum bacterial density for different
crystal thickness
4.4 2.1 0.4 0.2
∗
Average individual B. pasteurii volume estimated as 0.35 µm3 .
∗∗
Assuming the observed bacterial volumes for different crystal thicknesses, from 0.5 to 10 µm (i.e., different bacterial densities throughout
the entire crystal).
222 A. C. MITCHELL AND F. G. FERRIS

FIG. 8. Results of crystal nucleation and growth evolution modelling using USGS GALOPER software (Eberl et al. 2000). (A) B. pasteurii-inclusive AGW:
(i) Initial nucleation and surface-controlled growth with a critical nucleus size of 3 nm and a 0.63 probability of nucleation. (ii) Growth from nucleation to observed
day 1 CSD by supply-controlled growth. (iii) Growth from day 1 to day 7 by supply-controlled growth then Ostwald ripening, with a 0.2 probability of crystal
coalescence. (B) Bacteria-free AGW: (i) Initial nucleation and surface-controlled growth with a critical nucleus size of 3 nm and a 0.8 probability of nucleation.
(ii) Growth from nucleation to observed day 1 CSD by supply-controlled growth. (iii) Growth from day 1 to day 7 by continued supply-controlled growth. Quality
of simulation expressed as the residual squared of the crystal size frequency between the raw and simulated CSD (n = 100, significant always at 99% confidence
level).

generated by a decaying rate of nucleation and surface-
controlled growth (where the growth rate is limited by the
available surface area), followed by supply-controlled growth
(where the growth rate is limited by the availability of reactants)
(Figure 8A(i) and (ii) and B(i) and (ii)). Thereafter, the evolution
of the day 7 bacteria-free CSD was achieved by further supply-
controlled growth (Figure 8B(iii)), whereas the B. pasteurii-
inclusive CSD was achieved by further supply-controlled growth
followed by Ostwald ripening (Figure 8A(iii)). The modelled
CSDs provided an excellent match with the measured CSDs.
Therefore, modelling suggested the larger CSD variance
generated by day 1 in the B. pasteurii-inclusive AGW
than the bacteria-free AGW was formed in the nucle-
ation stage. This was controlled by a lower probability of
nucleation in the B. pasteurii-inclusive AGW (0.63) than
the bacteria-free AGW (0.8) after the critical saturation
had been reached. This generated an initial CSD in the
<100 nm diameter range, where the B. pasteurii-inclusive
CSD was wider (variance = 0.37) and exhibited a greater
mean crystal diameter (38 nm) than the bacteria-free CSD
(variance = 0.19; mean crystal diameter = 14 nm) (Figure 8A(i)
and B(i)). Modelling therefore suggests crystal nucleation is
more probable away from the bacteria.
Surface Area and Solubility
The estimated surface area of individual calcite precipi-
tates increased asymptotically with increasing crystal diameter
(Figure 9a). However, large crystals >20,000 nm that represent
only 1% of the total number of crystals measured, still account
for between 1 and 28% of the total crystal surface area precipi-
tated on days 1 or 7 in the B. pasteurii-inclusive or bacteria-free
AGW. Nevertheless, the Molar Surface Area (A) exhibits an ex-
ponential decay with increasing crystal diameter, reflecting a
decreasing surface area—mass ratio (Figure 9b). This impacts
greatly on the theoretical solubility of the precipitated crystals, ln
Ks0(A) (Equation 4) (Stumm and Morgan 1996), which exhibits
and exponential increase for crystals smaller than ∼4000 nm,
due to the increase in A in both the B. pasteurii-inclusive and
 INFLUENCE OF B. PASTEURII ON CaCO3 PRECIPITATION
FIG. 9. Estimated spherical crystal surface area, (b) molar surface area, and
(c) solubility (ln Ks0) as a function of crystal diameter, from the bacteria-free
and B. pasteurii-inclusive AGW on days 1 and 7.
bacteria-free AGW (Figure 9c). Since 66% of the crystals mea-
sured are in the <4000 nm diameter range (Figure 5), the solu-
bility of the majority of CaCO3 crystals formed in response to
bacterial ureolysis are likely to be affected by surface area.
DISCUSSION
Crystalline properties in the presence of B. pasteurii that are
distinct from crystals formed in the bacteria-free AGW must
result from the effect bacterial surfaces and organic macro-
molecules. B. pasteurii surfaces are comprised of several lay-
223
ers of peptidoglycan polymers, with intermixed proteinaceous
and teichoic acids, and the outer surface of the plasma mem-
brane, typical of such gram positive bacteria (Schultze-Lam et al.
1996; Madigan and Martinko 2005). Consequently nucleation
and growth in the presence of B. pasteurii will likely be affected
by this milieu of high to low molecular weight proteins, polysac-
charides and organic acids >10 K Dalton MWCO. Equally the
aqueous microenvironments around bacterial cells, which are
different from the measured changes in bulk solution chemistry,
may also effect nucleation and growth relative to the bacteria-
free AGW (Schultze-Lam et al. 1996), as discused below.
The consistency of calcite formation, the stable CaCO3 poly-
morph, in both the B. pasteurii-inclusive and bacteria-free AGW
suggests CaCO3 mineralogy is not physically controlled by bac-
terial surfaces or the influence of bacteriogenic organic macro-
molecule exudates in our experiments. Indeed, the incorpora-
tion of organic macromolecules within the mineral structure,
including proteins and organic acids, often promotes the for-
mation and stabilisation of the metastable CaCO3 polymorphs,
vaterite, aragonite, as well as impure calcite (Falini 2000; Raz
et al. 2000; Manoli and Dalas 2001; Braissant and Verrecchia
2002; Braissant et al. 2003; Sanchez-Moral et al. 2003; Kralj
et al. 2004; Roque et al. 2004). Calcite formation in our ex-
periments is more likely to reflect the slow rates of CaCO3
precipitation induced by bacterial ureolysis under the condi-
tions of our experiments (Mitchell and Ferris 2005). Calcite
is usually favoured at lower CaCO3 precipitation rates, which
can be slowed by the complexation of Ca2+ or carbonate ions
with organic and inorganic species which causes a reduction
of ionic activity, mineral supersaturation and precipitation rates
(Kitano and Hood 1965; Meldrum and Hyde 2001; Braissant
and Verrecchia 2002; Braissant et al. 2003; Bosak et al. 2004;
Bosak and Newman 2005). This contrasts to the formation of
metastable CaCO3 polymorphs, vaterite and aragonite, often fa-
vored at high levels of supersaturation and high rates of pre-
cipitation (Plummer and Busenberg 1982; Ogino et al. 1987).
Indeed, previous investigation into CaCO3 precipitation by bac-
terial ureolysis has demonstrated the precipitation of vaterite as
well as calcite, which probably reflected high CaCO3 saturation
states and precipitation rates induced by the concentrated so-
lutions of CaCl2 , NaHCO3 (25 mM) and urea (330 mM) used
(Warren et al. 2001).
The predominantly rhombohedral morphology in the experi-
ments is consistent with the standard polyhedral form of calcite
(Deer et al. 1992) often precipitated without the presence of
bacteria or organic macromolecules (Meldrum and Hyde 2001;
Braissant et al. 2003; Roque et al. 2004). This suggests the in-
fluence of B. pasteurii surfaces and exudates on crystal mor-
phology is somewhat limited. The stepped and rounded edges
of crystals from both the B. pasteurii and bacteria-free AGW
probably reflect the effect of inorganic ions within the precipi-
tating solid, which have been shown to distort CaCO3 mineral
structures (Tracy et al. 1998; Raz et al. 2000; Kontrec et al. 2004;
Kralj et al. 2004). Interestingly, this morphology is very similar
224 A. C. MITCHELL AND F. G. FERRIS
to mature calcite crystals precipitated from solutions contain-
ing urea and CaCl2 in the presence of purified urease enzyme
(Sondi and Matijevic 2001). This suggests NH+ 4 or urea may be
controlling the dominant morphology of the crystals, since ex-
uded urease should be contained within the dialysis membrane
and only organic macromolecules <10 K Dalton MWCO will be
present in both the B. pasteurii and bacteria-free AGW. However
the more ordered nature and smoothed surfaces of crystals from
the B. pasteurii-inclusive AGW suggests the presence of bac-
teria and organic macromolecules is somewhat stabilising the
rhombohedral form of the crystals. Warren et al. (2001) demon-
strated a wider range of crystal morphologies than in our current
study, including pseudopolyhedral dumbbell, globular, as well
as rhombohedral morphologies. Some of this variation is likely
to reflect the formation of vaterite, which often exhibits a more
spherical or globular morphology (Braissant et al. 2003; Roque
et al. 2004). However, the high concentration of urea and NH+ 4 bacteria, reflecting the smaller diffusion distances of carbonate
generated (<660 mM) in the experiments of Warren et al. (2001)
is probably responsible for much of the deviation of morphology
away from standard rhombohedral calcite (Sondi and Matijevic
2001).
The larger crystal size and variance of CSDs that evolved
from identical changes in bulk solution chemistry and precipi-
tation rates in the presence of B. pasteurii indicates the direct
influence of bacterial surfaces and bacteriogenic organic macro-
molecules, since estimates suggest this size difference cannot
simply reflect the mass of encased bacteria. Warren et al. (2001)
also noted the formation of larger CaCO3 crystals with a greater
size variance in the presence of B. pasteurii relative to bacteria-
free solutions, although this was not quantified.
Bacterial surfaces are typically thought to lower the activa-
tion energy barrier that normally retards spontaneous nucleation,
through the uptake and retention of metal ions, thereby reduc-
ing the critical saturation state at which precipitation can begin
(Schultze-Lam et al. 1996; Ferris et al. 1997). However, the ear-
lier observation of crystals in the bacteria-free AGW suggest this
is not the case. It seems more plausible that CSD differences may
be propagated in the nucleation stage. Modelling suggests this
results from a reduction in the probability of nucleation in the
B. pasteurii-inclusive AGW relative to the bacteria-free AGW
once a critical saturation has been attained. This generates a
lower frequency of smaller crystals but a higher frequency of
larger crystals than in the bacteria-free AGW, with subsequent
size-dependent supply-controlled growth propagating this size
difference throughout the growth process. B. pasteurii may re-
duce the probability of crystal nucleation once Scritical has been
attained though complexing of Ca2+ , HCO− 2−
3 or CO3 with or-
ganic macromolecules, which could reduce the relative satura-
tion and inhibit spontaneous homogenous nucleation, although
the exact controls cannot be ascertained from this study. Other
controlled calcite nucleation and growth experiments, where
reactants were added continuously to solutions supersaturated
with calcite (S = 22 to 41) for 170 min, also demonstrate
that lognormal CSDs are generated during the nucleation stage
and are preserved by subsequent size dependent growth (Kile
et al., 2000).
The modelling requirement of surface-controlled growth dur-
ing the nucleation processes suggests the initial rate of growth
was limited by the available surface area and not the supply of
Ca2+ , HCO− 2−
3 or CO3 , which is plausible given the low surface
area of calcite at the beginning of these unseeded experiments.
Conversely, subsequent supply-controlled growth, even though
Ca2+ and carbonate ions were at significant concentrations in
the bulk solution suggests the transport of reactants to the crys-
tal surface was limited. This is most likely to reflect advection
limited transport, since solution velocity is considerable relative
to the mean crystal size for small crystals (<100 µm in diam-
eter) due to convection and Brownian motion (Kile and Eberl
2003). Equally, crystal growth proximal to bacteria is likely to
be subject to smaller reactant supply limitations than away from
ions away from bacterial cells, and may further account for the
larger crystals generated in the presence of bacteria.
Modelling also suggests larger crystals may be further devel-
oped in the presence of B. pasteurii by Ostwald ripening between
days 1 and 7, where larger crystals grow at the expense of smaller,
more unstable and soluble crystals, forming a more positively
skewed CSD (Lasaga 1998; Kile et al. 2000). Crystal solubility
calculations support these inferences and indicate that by the end
of the experiments the proportion of crystals that are apparently
affected by surface area controlled solubility (<4000 nm diam-
eter) in the B. pasteurii inclusive AGW are only 35% compared
to 48% in the bacteria-free AGW. Indeed, the lower solubility
of larger crystals suggests that mature crystals developed in the
presence of B. pasteurii have a lower affinity for re-dissolution
than those generated in the bacteria-free AGW, which may act
as a positive feedback to maintain larger crystal sizes in the B.
pasteurii-inclusive AGW. Therefore, the larger crystal size and
crystal size variance developed in the presence of B. pasteurii
appears to be a consequence of nucleation inhibition, lower re-
actant supply limitations for crystals proximal to bacteria, and
subsequent size related solubility.
The validity of the modelling results is based upon the as-
sumption that crystal growth obeys the LPE, and is proportional
to crystal size (Eberl et al. 2000). This assumption has been
proven to be valid for most natural crystal growth mechanisms,
since log-normal CSDs, which are the most common observed
in nature, can only be realistically generated and maintained by
proportional growth mechanisms (Kile et al. 2000; Eberl et al.
2002). This contrasts to the common assumption in petrology
studies that once crystals are nucleated, all crystals will grow
at the same linear rate, independent of crystal size (Kirkpatrick
1981; Marsh 1988). This is highly unlikely, since a log-normal
CSD would require an initially slow nucleation rate, to generate
the right-hand side of the CSD, then an increase in nucleation
rate to generate the modal crystal diameter, then a decrease in nu-
cleation rate to generate the left-hand tail of the CSD (Eberl et al.
2002). In reality, nucleation generally occurs over a short period
 INFLUENCE OF B. PASTEURII ON CaCO3 PRECIPITATION
at high levels of saturation, driving a decrease in saturation as nu-
clei appear and grow, making continued nucleation less likely
(Lasaga 1998; Eberl et al. 2002). This suggests the decaying
rate of nucleation required to generate the observed CSDs in our
study (Figure 8) at such a supersaturated state (Figure 3a) is valid.
CONCLUSIONS
The novel use of dialysis membranes allowed investigation
of the influence of B. pasteurii on the mineralogy, morphology
and crystal size distributions of CaCO3 precipitated in response
to ureolysis. Ureolysis induced CaCO3 precipitation in both the
B. pasteurii-inclusive and bacteria-free region of the AGW, in
response to identical changes in bulk solution chemistry and
precipitation rates. Rhombohedral calcite, the standard poly-
hedral prismatic form of calcite, was precipitated in both the
presence and absence of B. pasteurii. It is reasonable to assume
this reflects the slow precipitation rates exhibited over the dura-
tion of the experiments, which are inhibited by organic macro-
molecules and inorganic species in the AGW. While CaCO3
mineralogy and morphology was not strongly influenced by the
B. pasteurii surfaces or organic exudates, the evolution of dis-
tinctly different lognormal crystal-size-distributions in the B.
pasteurii-inclusive and bacteria-free zone of the AGW resulted
from identical changes in bulk solution chemistry. Specifically,
B. pasteurii increased the size and size variance of crystals,
and led to a greater crystal growth rate throughout the experi-
ments, relative to bacteria-free AGW. Estimates of the mass of
B. pasteurii encased within the mature calcite crystals suggest
B. pasteurii can only account for <4.4% of the crystal diameter,
and was not sufficient to account for the ∼46% larger median
crystal diameter exhibited by day 7 in the B. pasteurii-inclusive,
than the bacteria-free AGW.
The larger crystals which developed in the presence of B.
pasteurii are likely to be propagated in the nucleation stage, by
a reduction in the probability of initial nucleation, as suggested
by crystal growth modelling on GALOPER. This may be in-
duced by complexing of Ca2+ , HCO− 2−
3 or CO3 with organic
macromolecules. Modelling suggests supply-controlled growth
despite the availability of Ca2+ and carbonate ions, implying
crystal growth is limited by the rate of solute advection to the
crystal surface. Supply limitations are likely to be lower for crys-
tal growth proximal to bacteria, reflecting the smaller diffusion
distances of carbonate ions away from bacterial cells, and may
further account for the larger crystals generated in the presence
of bacteria. Calculated crystal solubility (ln K S0 ) was lower for
larger crystals, reflecting smaller molar surface areas, suggest-
ing that crystals generated in the presence of B. pasteurii have
a lower affinity for re-dissolution than those generated in the
bacteria-free AGW, which may act as a positive feedback to
maintain larger crystal sizes in the presence of B. pasteurii.
The results imply that during ureolysis, greater bacterial con-
centrations will generate larger carbonate crystals. The lower
solubility of these larger crystals suggests that co-precipitated
metals and radionuclides will be more effectively immobilised
225
in bioremediation strategies using ureolysis induced carbonate
precipitation, if bacterial concentrations in the natural system are
higher. Further experiments are required to test these assertions.
REFERENCES
Bachmeier KL, Williams AE, Warmington JR, Bang SS. 2002. Urease activity
in microbiologically-induced calcite precipitation. J Biotechnol 93:171–181.
Bosak T, Newman DK. 2003. Microbial nucleation of calcium carbonate in the
Precambrian. Geology 31:577–580.
Bosak T, Newman DK. 2005. Microbial kinetic controls on calcite morphology
in supersaturated solutions. J Sediment Res 75:190–199.
Bosak T, Souza-Egipsy V, Newman DK. 2004. A laboratory model of abiotic
peloid formation. Geobiology 2:189–198.
Braissant O, Cailleau G, Dupraz C, Verrecchia AP. 2003. Bacterially induced
mineralization of calcium carbonate in terrestrial environments: the role of
exopolysaccharides and amino acids. J Sediment Res 73:485–490.
Braissant O, Verrecchia EP. 2002. Microbial biscuits of vaterite in Lake Issyk-
Kul (Republic of Kyrgyzstan)—Discussion. J Sediment Res 72:944–946.
Carter PW, Mitterer RM. 1978. Amino-acid composition of organic-matter as-
sociated with carbonate and non-carbonate sediments. Geochim Cosmochim
Acta. Vol. 104. 42:1231–1238.
Clapham L, McLean RJC, Nickel JC, Downey J, Costerton JW. 1990. The
influence of bacteria on struvite crystal habit and its importance in urinary
stone formation. J Crysl Growth 475–484.
Curti E. 1999. Coprecipitation of radionuclides with calcite: estimation of parti-
tion coefficients based on a review of laboratory investigations and geochem-
ical data. App Geochem 14:433–445.
Deer WA, Howie RA, Zussman J. 1992. An Introduction to Rock Forming
Minerals. Englewood Cliffs, NJ.:Prentice Hall, 712 p.
Dittrich M, Muller B, Mavrocordatos D, Wehrli B. 2003. Induced calcite pre-
cipitation by cyanobacterium Synechococcus. Acta Hydrochim Hydrobiol
31:162–169.
Eberl DD, Drits VA, Srodon J. 1998. Deducing growth mechanisms for minerals
from the shapes of crystal size distributions. Amer J Sci 298:499–533.
Eberl DD, Drits VA, Srodon J. 2000. User Guide to Galoper—A program for
simulating the shapes of crystal size distributions—and associated programs:
U.S. Geological Survey. Open File Report: OF 00-505. Denver, Colorado.
Eberl DD, Kile DE, Drits VA. 2002. On geological interpretations of crystal
size distributions: constant vs. proportionate growth. Amer Mineral 87:1235–
1241.
Falini G. 2000. Crystallization of calcium carbonates in biologically inspired
collagenous matrices. Inter J Inorg Mater 2:455–461.
Ferris FG, Phoenix V, Fujita Y, Smith RW. 2003. Kinetics of calcite precipitation
induced by ureolytic bacteria at 10 to 20 degrees C in artificial groundwater.
Geochim Cosmochim Acta 68:1701–1710.
Ferris FG, Stehmeier LG. 1992. Bacteriogenic mineral plugging. Patent number:
5,143,155. U.S. Patent Office. Washington DC.
Ferris FG, Stehmeier LG, Kantzas A, Mourits FM. 1996. Bacteriogenic mineral
plugging. J Can Petrol Technol 35:56–61.
Ferris FG, Thompson JB, Beveridge TJ. 1997. Modern freshwater microbialites
from Kelly lake, British Columbia, Canada. Palaios 12:213–219.
Freij SJ, Putnis A, Astilleros JM. 2004. Nanoscale observations of the effect of
cobalt on calcite growth and dissolution. J Crysl Growth 267:288–300.
Fujita Y, Ferris FG, Lawson RD, Colwell FS, Smith RW. 2000. Calcium carbon-
ate precipitation by ureolytic subsurface bacteria. Geomicrobio J 17:305–318.
Fujita Y, Redden GD, Ingram JC, Cortez MM, Ferris FG, Smith RW. 2004. Stron-
tium incorporation into calcite generated by bacterial ureolysis. Geochim
Cosmochim Acta 68:3261–3270.
Hammes F, Seka A, Van Hege K, Van de Wiele T, Vanderdeelen J, Siciliano
SD, Verstraete W. 2003. Calcium removal from industrial wastewater by bio-
catalytic CaCO3 precipitation. J Chem Technol Biotechnol 78:670–677.
Kamiya N, Kagi H, Tsunomori F, Tsuno H, Notsu K. 2004. Effect of trace
lanthanum ion on dissolution and crystal growth of calcium carbonate. J Crysl
Growth 267:635–645.
226 A. C. MITCHELL AND F. G. FERRIS
Kile DE, Eberl DD. 2003. On the origin of size-dependent and size-independent
crystal growth: influence of advection and diffusion. Amer Mineral 88:1514–
1521.
Kile DE, Eberl DD, Hoch AR, Reddy MM. 2000. An assessment of calcite crystal
growth mechanisms based on crystal size distributions. Geochim Cosmochim
Acta 64:2937–2950.
Kirkpatrick RJ. 1981. Kinetics of crystallisation of igneous rocks. In: Lasaga
AC, Kirkpatrick RJ, editors. Kinetics of Geochemical Processes. Washington,
DC: Mineralogical Society of America.
Kitano Y, Hood DW. 1965. Influence of Organic Material on Polymorphic Crys-
tallization of Calcium Carbonate. Geochimica Et Cosmochimica Acta 29:29–
41.
Knobel LL, Bartholomay RC, Cecil LD, Tucker BJ, Wegner SJ. 1992. Chemical
Constituents in the Dissolved and Suspended Fractions of Ground Water from
Selected Sites, Idaho National Engineering Laboratory and Vicinity, Idaho,
1989. U.S. Geological Survey Open File Report: OF 92-51. Denver, Colorado.
Konhauser KO, Phoenix VR, Bottreal SH, Adams DG, Head IM. 2001.
Microbial-silica interactions in Icelandic hot spring sinter: possible ana-
logues for some Precambrian siliceous stromatolites. Sedimentology 48:415–
433.
Kontrec J, Kralj D, Brecevic L, Falini G, Fermani S, Noethig-Laslo V,
Mirosavljevic K. 2004. Incorporation of inorganic anions in calcite. Euro-
pean Journal of Inorganic Chemistry 23:4579–4585.
Kralj D, Kontrec J, Brecevic L, Falini G, Nothig-Laslo V. 2004. Effect of inor-
ganic anions on the morphology and structure of magnesium calcite. Chem
Eur J 10:1647–1656.
Lasaga AC. 1998. Reaction Kinetics in Geoscience. Princeton, US: Princeton
University Press. 811 p.
Madigan M, Martinko J. 2005. Brock Biology of Microorganisms, 11th ed.
Englewood Cliffs, NJ: Prentice Hall. 1088 p.
Manoli F, Dalas E. 2001. Calcium carbonate crystallization in the presence of
glutamic acid. J Crys Growth 222:293–297.
Marsh BD. 1988. Crystal size distribution (CSD) in rocks and the kinetics and
dynamics of crystallisation I. Theory. Cont Mineral Petrol 99:277–291.
McCoy BJ. 2001. A new population balance model for crystal size-distributions:
reversible, size-dependent growth and dissolution. J Coll Interf Sci 240:139–
149.
Meldrum FC, Hyde ST. 2001. Morphological influence of magnesium and or-
ganic additives on the precipitation of calcite. J Crys Growth 231:544–558.
Mitchell AC, Ferris FG. 2005. The co-precipitation of Sr into calcite precip-
itates induced by bacterial ureolysis in artificial groundwater—temperature
and kinetic dependence. Geochim Cosmochim Acta 69:4199–4210.
Mitchell AC, Ferris FG. 2006. Effect of strontium contaminants on the size and
solubility of calcite crystals precipitated by the bacterial hydrolysis of urea.
Environ Sci Tecnolol 40:1008–1014.
Morse JW, Mackenzie FT. 1990. Geochemistry of Sedimentary Carbonates: In:
Developments in Sedimentology 48. Elsevier, Amsterdam, 707 p.
Ogino T, Suzuki T, Sawanda R. 1987. The formation and transformation mech-
anism of calcium carbonate in water. Geochim Cosmochim Acta 51:2757–
2767.
Plummer L, Busenberg E. 1982. The solubilities of calcite, aragonite and va-
terite in CO2 -H2 O solutions between 0 and 90◦ C, and an evaluation of the
aqueous model for the system CaCO3 -CO2 -H2 O. Geochim Cosmochim Acta
46:1011–1040.
Raz S, Weiner S, Addadi L. 2000. Formation of high-magnesian calcites via
an amorphous precursor phase: possible biological implications. Adv Mater
12:38–42.
Roque J, Molera J, Vendrell-Saz M, Salvado N. 2004. Crystal size distribu-
tions of induced calcium carbonate crystals in polyaspartic acid and Mytilus
edulis acidic organic proteins aqueous solutions. J Crysl Growth 262:543–
553.
Sanchez-Moral S, Canaveras JC, Laiz L, Saiz-Jimenez C, Bedoya J, Luque L.
2003. Biomediated precipitation of calcium carbonate metastable phases in
hypogean environments: a short review. Geomicrobiol J 20:491–500.
Schindler PW. 1967. Heterogeneous Equilibria Involving Oxides, Hydroxides,
Carbonates and Hydroxide Carbonates. In: Equilibrium Concepts in Natu-
ral Water Systems, Adv. Chem. Ser., No. 67. Washington, DC: American
Chemical Society. p 196.
Schultze-Lam S, Beveridge TJ. 1994. Physicochemical characteristics of the
mineral-forming S-layer from the Cyanobacterium Synechococcus strain
Gl24. Can J Microbiol 40:216–223.
Schultze-Lam S, Fortin D, Davis BS, Beveridge TJ. 1996. Mineralization of
bacterial surfaces. Chem Geol 132:171–181.
Sondi I, Matijevic E. 2001. Homogeneous precipitation of calcium carbonates
by enzyme catalyzed reaction. J Coll Interf Sci 238:208–214.
Stocks-Fischer S, Galinat JK, Bang SS. 1999. Microbiological precipitation of
CaCO3 . Soil Biol Biochem 31:1563–1571.
Stumm W, Morgan JJ. 1996. Aquatic Chemistry, 3rd ed. New York: John Wiley
& Sons. 1040 p.
Tracy SL, Francois CJP, Jennings HM. 1998. The growth of calcite spherulites
from solution I. Experimental design techniques. J Crysl Growth 193:374–
381.
Van Lith Y, Warthmann R, Vasconcelos C, McKenzie JA. 2003. Microbial fos-
silization in carbonate sediments: a result of the bacterial surface involvement
in dolomite precipitation. Sedimentology 50:237–245.
Vasconcelos C, McKenzie JA, Bernasconi S, Grujic D, Tien AJ. 1995. Microbial
mediation as a possible mechanism for natural dolomite formation at low
temperatures. Nature 377:220–222.
Warren LA, Maurice PA, Parmar N, Ferris FG. 2001. Microbially mediated
calcium carbonate precipitation: implications for interpreting calcite precip-
itation and for solid-phase capture of inorganic contaminants. Geomicrobiol
J 18:93–115.
Yoon JH, Lee KC, Weiss N, Kho YH, Kang KH, Park YH. 2001. Sporosarcina
aquimarina sp nov., a bacterium isolated from seawater in Korea, and
transfer of Bacillus globisporus (Larkin and Stokes 1976), Bacillus psy-
chrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1889) to the
genus Sporosarcina as Sporosarcina globispora comb. nov., Sporosarcina
psychrophila comb. nov and Sporosarcina pasteurii comb. nov., and emended
description of the genus Sporosarcina. Inter J System Evolution Microbiol
51:1079–1086.