Biogeosciences, 16, 2147–2161, 2019
https://doi.org/10.5194/bg-16-2147-2019
© Author(s) 2019. This work is distributed under
the Creative Commons Attribution 4.0 License.
Kinetics of calcite precipitation by ureolytic bacteria under aerobic
and anaerobic conditions
Andrew C. Mitchell1,2, Erika J. Espinosa-Ortiz2, Stacy L. Parks2,3, Adrienne J. Phillips2,4, Alfred B. Cunningham2,4,
and Robin Gerlach2,3
1Department of Geography and Earth Sciences, Interdisciplinary Centre for Environmental Microbiology,
Aberystwyth University, SY23 3DB, UK
2Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA
3Department of Chemical and Biological Engineering, Montana State University, Bozeman, MT 59717, USA
4Department of Civil Engineering, Montana State University, Bozeman, MT 59717, USA
Correspondence: Andrew C. Mitchell (nem@aber.ac.uk) and Robin Gerlach (robin_g@coe.montana.edu)
Received: 8 November 2018 – Discussion started: 16 November 2018
Revised: 6 April 2019 – Accepted: 10 April 2019 – Published: 23 May 2019
Abstract. The kinetics of urea hydrolysis (ureolysis) and in-
duced calcium carbonate (CaCO3) precipitation for engineer-
ing use in the subsurface was investigated under aerobic con-
ditions using Sporosarcina pasteurii (ATCC strain 11859)
as well as Bacillus sphaericus strains 21776 and 21787. All
bacterial strains showed ureolytic activity inducing CaCO3
precipitation aerobically. Rate constants not normalized to
biomass demonstrated slightly higher-rate coefficients for
both ureolysis (kurea) and CaCO3 precipitation (kprecip) for B.
sphaericus 21776 (kurea = 0.10± 0.03 h−1, kprecip = 0.60±
0.34 h−1) compared to S. pasteurii (kurea = 0.07± 0.02 h−1,
kprecip = 0.25± 0.02 h−1), though these differences were
not statistically significantly different. B. sphaericus 21787
showed little ureolytic activity but was still capable of induc-
ing some CaCO3 precipitation. Cell growth appeared to be
inhibited during the period of CaCO3 precipitation. Trans-
mission electron microscopy (TEM) images suggest this is
due to the encasement of cells and was reflected in lower kurea
values observed in the presence of dissolved Ca. However,
biomass regrowth could be observed after CaCO3 precipi-
tation ceased, which suggests that ureolysis-induced CaCO3
precipitation is not necessarily lethal for the entire popula-
tion. The kinetics of ureolysis and CaCO3 precipitation with
S. pasteurii was further analyzed under anaerobic conditions.
Rate coefficients obtained in anaerobic environments were
comparable to those under aerobic conditions; however, no
cell growth was observed under anaerobic conditions with
NO−3 , SO
2−
4 or Fe
3+ as potential terminal electron accep-
tors. These data suggest that the initial rates of ureolysis and
ureolysis-induced CaCO3 precipitation are not significantly
affected by the absence of oxygen but that long-term ure-
olytic activity might require the addition of suitable electron
acceptors. Variations in the ureolytic capabilities and asso-
ciated rates of CaCO3 precipitation between strains must be
fully considered in subsurface engineering strategies that uti-
lize microbial amendments.
1 Introduction
Carbonate precipitation is a natural phenomenon that may
also be utilized in many subsurface engineering applica-
tions (Phillips et al., 2013a) including soil stabilization
(van Paassen et al., 2010), immobilization of radionuclides
(Mitchell and Ferris, 2005, 2006a, b; Tobler et al., 2012;
Warren et al., 2001), and mineral plugging for enhanced
oil recovery and carbon sequestration (Dupraz et al., 2009;
Ferris et al., 1996; Mitchell et al., 2010; Phillips et al.,
2013b). Mineral precipitation can be induced by bacteria as
a by-product of common microbial processes, such as urea
hydrolysis (ureolysis). In this process, bacteria hydrolyze
urea (CO(NH2)2), an important nitrogen compound found
in natural environments, to ammonia (NH3) and carbonic
acid (H2CO3) (Eqs. 1–3). The NH3 and H2CO3 equilibrate
in circumneutral aqueous environments to form bicarbonate
(HCO−3 ), two ammonium ions (NH
+
4 ) and one hydroxide ion
Published by Copernicus Publications on behalf of the European Geosciences Union.
2148 A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation
(OH−) (Eqs. 4–5), or at higher pH values, to one carbonate
ion (CO2−3 ) and two NH
+
4 (Eqs. 4–6). In the presence of dis-
solved calcium (Ca2+), this increase in carbonate alkalinity
shifts the saturation state of the system, allowing for solid
calcium carbonate (CaCO3) to form (Eq. 7). The overall re-
action from the hydrolysis of urea in the presence of Ca2+ is
summarized by Eq. (8).
CO(NH2)2+H2O→ NH2COOH+NH3 (1)
NH2COOH+H2O→ NH3+H2CO3 (2)
CO(NH2)2+ 2H2O→ 2NH3+H2CO3 (Eqs. 1 + 2 overall) (3)
2NH3+ 2H2O↔ 2NH+4 + 2OH− (4)
H2CO3↔ HCO−3 +H+ (5)
HCO−3 +H++ 2OH−↔ CO2−3 + 2H2O (6)
CO2−3 +Ca2+↔ CaCO3 (7)
CO(NH2)2+ 2H2O+Ca2+↔ 2NH+4 +CaCO3 (overall process) (8)
The use of ureolytic bacteria in biotechnological applications
is appealing for many reasons. Ureolytically active microor-
ganisms are common in a wide variety of soil and aquatic
environments; thus, indigenous microorganisms capable of
ureolysis can be either stimulated in situ or, alternatively,
they can be used to augment environments lacking ureolytic
microorganisms (Fujita et al., 2000; Warren et al., 2001).
Urea is a fairly inexpensive substrate and it is often con-
tained in wastewater (Hammes et al., 2003b), so this waste
product may be used to stimulate ureolysis in engineering
applications (Mitchell et al., 2010). Moreover, the controlled
increase in pH and alkalinity in the subsurface by ureolytic
bacteria is preferable to the injection of a basic solution (abi-
otic process), which could lead to instantaneous CaCO3 su-
persaturation and precipitation at the point of injection lim-
iting the radius of influence of the technology. The injection
of urea into the subsurface followed by microbially induced
ureolysis would allow for controlled, gradual ureolysis fur-
ther away from the injection point, promoting a wider spa-
tial distribution of CaCO3 in the subsurface and avoiding un-
controlled plugging at the point of injection (Cuthbert et al.,
2013; Ebigbo et al., 2012; Mitchell and Ferris, 2005; Schultz
et al., 2011; Tobler et al., 2012, 2014).
Among different ureolytic bacteria, Sporosarcina pas-
teurii (formerly known as Bacillus pasteurii) has been ex-
tensively used as the model urease-producing organism in
ureolysis-driven CaCO3 precipitation studies, due to its
high ureolytic activity and constitutive production of ure-
ase (Phillips et al., 2013a). The use of S. pasteurii for
CaCO3 precipitation is feasible under aerobic conditions
and the kinetics of ureolysis under different conditions have
been studied. Most studies have reported first-order ure-
olysis rates with respect to urea concentration, with the
rate constant (kurea) ranging between 0.002 and 0.090 h−1
under aerobic conditions in artificial groundwater without
nutrients added (Dupraz et al., 2009; Ferris et al., 2004;
Hammes et al., 2003a; Mitchell and Ferris, 2005; Tobler et
al., 2012) and 0.35 h−1 with the addition of nutrients (Lauch-
nor et al., 2015). Ureolysis rates have been suggested to
be temperature-dependent (Ferris et al., 2004), and it seems
to also be affected by cell concentration (inoculum size)
(Lauchnor et al., 2015; Tobler et al., 2011).
Although the ureolytic activity of S. pasteurii under anoxic
conditions has been observed (Martin et al., 2012; Mortensen
et al., 2011; Tobler et al., 2012), there is controversy regard-
ing the extent and duration of ureolytic activity that can be
achieved in the absence of oxygen. Mortensen et al. (2011)
and Tobler et al. (2012) observed extensive ureolytic activity
under anoxic conditions, suggesting that the anoxic environ-
ment does not inhibit urease activity. Conversely, Martin et
al. (2012) observed limited cell growth and poor ureolysis
under anoxic conditions and suggested that the ureolytic ac-
tivity observed was due to the urease already present in the
cells.
In this study, the ability of S. pasteurii to grow in the
absence of oxygen (with or without nitrate (NO−3 ), sulfate
(SO2−4 ) or ferric ion (Fe3+) as possible electron acceptors)
was investigated along with the kinetics of ureolysis and
CaCO3 precipitation. Moreover, this study investigates and
compares the ureolytic activity of S. pasteurii with differ-
ent strains of Bacillus sphaericus under aerobic conditions,
which have also been suggested to be capable of ureolysis-
induced CaCO3 precipitation (Dick et al., 2006; Hammes et
al., 2003a).
2 Materials and methods
2.1 Solutions
Kinetic experiments were carried out using the CaCO3 min-
eralizing medium (CMM) described by Ferris et al. (1996)
(see Table SI1.1 in the Supplement). Both Ca2+-inclusive
(CMM+) and Ca2+-exclusive (CMM−) versions of this
medium were used. Aerobic CMM− was prepared as fol-
lows. A double-strength solution of nutrient broth was pre-
pared and autoclaved. Nutrient broth was chosen in this
experiment to enable cell growth. A separate solution of
double-strength urea, ammonium chloride and sodium bicar-
bonate was prepared and stirred until completely dissolved.
These two solutions were combined and adjusted to a pH of
6.0 using concentrated HCl. CMM+ was prepared similarly,
but calcium chloride was added after the pH adjustment.
Media were filter-sterilized into sterile Pyrex bottles using
0.2 µm pore size filters (Nalgene, Rochester, NY). Anaerobic
CMM was produced in the same manner. However, all stock
solutions were made in an anaerobic chamber using water
that had been degassed by stirring overnight in the oxygen-
free atmosphere of the chamber (90 % N2, 5 % CO2, 5 %
H2). Solutions were filter-sterilized into serum bottles and
were then capped and sealed inside the chamber. Prior to the
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A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation 2149
experiments, the solutions were combined to reach the final
concentrations listed in Table SI1.1.
2.2 Bacterial strains and culturing conditions
Three strains of ureolytic bacteria were used: S. pasteurii
(ATCC 11859) and two isolates from a garden soil and land-
fill soil – B. sphaericus 21776 and B. sphaericus 21787
(Belgian Coordinated Collections of Microorganisms, Lab-
oratory of Microbiology, Ghent University) (Hammes et al.,
2003a). Bacillus subtilis strain 186 (ATCC 23 857), a non-
ureolytic organism, was used as a control species. Abiotic
(i.e., non-inoculated) controls were also set up and run in par-
allel. Pilot cultures were grown in flasks by adding 100 µL of
thawed stock to 100 mL of autoclaved brain heart infusion
(BHI) +2 % urea. S. pasteurii and B. sphaericus were grown
on an incubator shaker (New Brunswick Scientific, Edison,
NJ) at 30 ◦C and 150 rpm, while B. subtilis was grown on
an incubator shaker at 37 ◦C and 150 rpm. A total of 100 µL
of pilot cultures was transferred at 24 and 48 h to new flasks
containing 100 mL of BHI +2 % urea.
2.3 Aerobic experiments
Once the pilot cultures were ready for inoculation, 40 mL
of culture was added to a 50 mL centrifuge tube. This tube
was centrifuged at 4303× g using a Sorvall Instruments
(Asheville, NC) RC-5C centrifuge for 10 min at 4–6 ◦C. The
supernatant was poured off and the cell pellet was resus-
pended using about 40 mL of CMM− and again centrifuged
for 10 min. This process was repeated once more. After the
third run in the centrifuge, the supernatant was poured off
and enough CMM− was added to achieve a final optical
density reading at 600 nm (OD600) of 0.4 (CMM-blank cor-
rected, measured in a 96-well plate using a BioTek Syn-
ergy HT plate reader). A total of 1 mL of prepared S. pas-
teurii, B. sphaericus strain 21776 or strain 21787 culture
was added to 250 mL Pyrex bottles with 150 mL of media
(either CMM+ or CMM−; initial concentration of biomass
OD600 =∼ 0.015). After inoculation, the systems were stati-
cally incubated at 30 ◦C for kinetic experiments.
2.4 Anaerobic experiments
Pilot cultures for anaerobic experiments were limited to the
use of S. pasteurii and were grown in the same manner as
those used in aerobic experiments. However, cells were trans-
ferred into an anaerobic chamber and resuspended in anaer-
obically prepared CMM−, then transferred to a serum bot-
tle, sealed and crimped inside the anaerobic chamber. Opti-
cal density measurements for time zero were taken after the
final suspension, with the same initial density as the aero-
bic experiments (OD600 =∼ 0.015). The first set of experi-
ments investigated cell growth and ureolysis under oxygen-
free conditions with a range of potential terminal electron ac-
ceptors (TEAs). Experiments were run using a batch system,
consisting of 100 mL of CMM− media including 10 mM
NO−3 , SO
2−
4 or Fe
3+ as potential TEAs and inoculated with
1 mL of S. pasteurii in 150 mL serum bottles. Concentrated
stock solutions of each TEA were made in the anaerobic
chamber and filter-sterilized: (i) a 1 M solution of NaNO3;
(ii) a concentrated SO2−4 solution, made by combining 1 M
Na2SO4 and 1 M Na2S, where Na2S was added to quench
any residual oxygen and make SO2−4 reduction possible; and
(iii) a stock solution of Fe(III) citrate, using 50 mM Fe(III)
citrate as previously described (Gerlach et al., 2011). Ap-
propriate amounts of each stock solution were added to the
separate serum bottles containing CMM− and S. pasteurii.
The growth survey was also conducted in CMM− without
the addition of a TEA. After inoculation, the systems were
statically incubated at 30 ◦C. Abiotic control experiments,
without the inclusion of S. pasteurii, were also performed.
Aliquots were extracted from the systems in the anaerobic
chamber and monitored for pH and OD600 during the dura-
tion of the experiments. Comparative aerobic control exper-
iments were also performed with CMM− media including
10 mM NO−3 , SO
2−
4 or Fe
3+ and inoculated with 1 mL of S.
pasteurii in 150 mL serum bottles.
The second set of experiments investigated the detailed ki-
netics of ureolysis and CaCO3 precipitation with S. pasteurii
and CMM+ as described above, with NO−3 as the poten-
tial TEA, by monitoring pH, dissolved Ca2+ and NH+4 con-
centrations. Control experiments were also performed with
CMM+ without the addition of a TEA and CMM− with
NO−3 as a potential TEA. Here, a stock solution of 10 M
NaNO3 was made by mixing and filter-sterilizing in the
anaerobic chamber, and an appropriate amount was added to
the CMM+ or CMM− to reach a final concentration of 1 M
NO−3 .
2.5 Experimental sampling and analysis
At different time points, 3 mL of sample was aseptically ex-
tracted from the systems and measurements were made of
pH, biomass, NH+4 and Ca
2+ concentration. NH+4 concen-
trations were determined using the Nessler Assay (Mitchell
and Ferris, 2005). Urea concentrations were determined from
NH+4 concentrations, according to Eqs. (3) and (4). Ca
2+
concentrations were measured after appropriate dilution in
trace-metal grade 5 % HNO3 (Fisher Scientific) using an Ag-
ilent 7500ce inductively coupled plasma mass spectrome-
ter (ICP-MS). Bacterial biomass was determined using three
methods: plate counts, OD600 and protein assays. OD600 was
used as a growth indicator in experiments carried out in the
absence of Ca2+ (see Sect. 1.2 in the Supplement). Trans-
mission electron microscopy (TEM) images were taken us-
ing a LEO 912AB TEM and photographed with a Proscan
2048× 2048 charge-coupled device (CCD) camera from a
batch culture in CMM+ inoculated with S. pasteurii. At the
point of crystal formation (after approximately 2.5 h), a mix-
ture of CaCO3 crystals and cells were extracted from the sys-
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2150 A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation
tem. Separate samples of S. pasteurii grown in the absence
of Ca2+ were also collected and imaged. Further details are
given in the Supplement Sect. 1.3.
The PHREEQC (version 2) speciation–solubility geo-
chemical model (Parkhust and Appelo, 1999) was used to
calculate solution speciation and carbonate mineral satura-
tion. The simulation was performed using calcite as the only
precipitate as it was identified by X-ray diffraction (XRD)
as the calcium carbonate polymorph present in the systems.
The MINTEQ database was used for all calculations, and the
thermodynamic constants for urea (Stokes, 1967) were added
(more information is provided in SI1.4 in the Supplement).
2.6 Kinetics of ureolysis and CaCO3 precipitation
The rate coefficient for ureolysis was determined by integrat-
ing the following first-order differential equation assuming
constant biomass concentrations during the period of urea
hydrolysis (Ferris et al., 2004; Mitchell and Ferris, 2005):
d[Urea]
dt
=−kurea [Urea] [X] , (9)
to get:
[Urea]t = [Urea]oe−kureaXt , (10)
where kurea is the first-order rate coefficient for ureoly-
sis, t is the time and X is the concentration of biomass
(Sect. SI2.1 in the Supplement). First-order relationships
have been successfully used to describe microbial ureolysis
(Lauchnor et al., 2015; Connolly et al., 2015). Indeed, while
the Michaelis–Menten model has been used when evaluating
ureolysis, studies of ureolysis-based carbonate precipitation
have demonstrated that the first-order ureolysis rate model
fits well for urea concentrations of 330 mM or below (Lauch-
nor et al., 2015; Connolly et al., 2015), which is the concen-
tration range used in this study.
The change in urea concentration was determined accord-
ing to Eqs. (3) and (4) (Eq. 11).
1 [Urea]=−0.5 ·1[NH+4 ] (11)
Biomass-normalized ureolysis rates were calculated. Firstly,
it was assumed that biomass (X) is constant in Eqs. (10)
and (11), as performed in other studies of ureolysis kinetics
(Cuthbert et al., 2012; Ferris et al., 2004; Mitchell and Ferris,
2005, 2006; Schultz et al., 2011; Tobler et al., 2011). Sec-
ondly, the obtained first-order rate coefficients with respect
to urea concentration (kurea) were normalized to the biomass
concentration by dividing the ureolysis rate coefficient by
the biomass at the onset of precipitation (i.e., urea hydrol-
ysis rates were normalized to the initial biomass concen-
tration, determined as either OD600 or CFU mL−1; Supple-
ment Sect. 2.2), which was equivalent to the initial biomass
in each system (X =X0). This is an appropriate choice of
model, since the biomass analysis indicated that the cell den-
sity was constant for the duration of CaCO3 precipitation and
was equivalent to the initial biomass in the systems, as pre-
sented in the results section. The biomass-normalized ure-
olysis rates were compared to other parameters previously
published (Ferris et al., 2004; Fujita et al., 2000; Stocks-
Fischer et al., 1999; Tobler et al., 2011). The media used
in the different studies were similar to those used in this
study, all with 25 mM of Ca2+, 333 mM urea and includ-
ing nutrient-broth-based growth media, apart from (i) Fer-
ris et al. (2004), who used a dilute artificial groundwater
(nongrowth medium) with Ca2+ and urea concentrations of
1.75 and 6 mM, respectively, and (ii) Tobler et al. (2011),
who used different Ca2+ concentrations varying from 50 to
500 mM and urea concentrations between 250 and 500 mM.
The precipitation of CaCO3 from the system is dependent
on the saturation state of the system, as well as the growth
mechanism of CaCO3 (Ferris et al., 2004; Mitchell and Fer-
ris, 2005; Teng et al., 2000). The literature is ambiguous on
defining a set rate expression for CaCO3 precipitation, so a
non-affinity-based first-order rate law was applied to these
studies for both its simplicity and the fact that it seems to de-
scribe the data well, assuming that for every mole of Ca2+
removed from the solution 1 mol of CaCO3 formed (Teng et
al., 2000):
d
[
Ca2+
]
dt
=−kprecip
[
Ca2+
]
. (12)
Integration of the above equation yields
[Ca2+]t = [Ca2+]oe−kprecipt , (13)
where kprecip is the first-order rate coefficient for CaCO3 pre-
cipitation. Rate constants were found using a nonlinear re-
gression method utilizing the Solver function in Microsoft
Excel. Due to the significant lag phase, the data used for anal-
ysis excluded onset time before ureolysis and CaCO3 pre-
cipitation occurred, as previously documented (Tobler et al.,
2011).
3 Results and discussion
3.1 Aerobic experiments
3.1.1 Solution chemistry
Aerobic experiments with a CMM+medium inoculated with
S. pasteurii, B. sphaericus 21776 and B. sphaericus 21787
showed an increase in pH (Table 1) and NH+4 (displayed as
a stoichiometrically equivalent decrease in urea concentra-
tions; see Fig. 1) over time. These results support observa-
tions from previous studies confirming the ureolytic capabil-
ities of S. pasteurii (Ferris et al., 2004; Fujita et al., 2000;
Warren et al., 2001) and B. sphaericus species (Dick et al.,
2006; Hammes et al., 2003a). Differences in the rate of pH
change and the amount of urea hydrolyzed between the dif-
ferent bacterial species suggest differences in their ureolytic
Biogeosciences, 16, 2147–2161, 2019 www.biogeosciences.net/16/2147/2019/
A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation 2151
activity. After 30 h, 58 %–82 % and 72 %–80 % of the avail-
able urea was hydrolyzed by S. pasteurii and B. sphaericus
21776, respectively. B. sphaericus 21787 exhibited little uti-
lization of urea (12 %–15 % hydrolyzed) accompanied by a
smaller increase in pH values (∼ pH 8.7 by 24± 3 h) com-
pared to the other bacterial strains: ∼ pH 9.3 by 24± 3 h,
consistent with buffering by NH+4 ↔ NH3+H+, which has
a pKa value of 9.3 at 30 ◦C (Mitchell and Ferris, 2005). Con-
trol experiments, inoculated with the non-ureolytic organ-
ism B. subtilis and sterile controls, did not exhibit significant
changes in pH or urea concentrations (Table 1, Fig. 1). Geo-
chemical modeling suggested that no CaCO3 precipitation
should occur in the absence of ureolysis and that approxi-
mately 0.45 mM of urea would have had to be hydrolyzed to
achieve supersaturation and for CaCO3 precipitation to com-
mence (see Supplement Sect. SI1.4).
In all the experiments containing ureolytic bacteria, Ca2+
concentration decreased to ∼ 5 % of the initial Ca2+ con-
centration in the liquid medium after approximately 30 h
(Fig. 1). The decrease in Ca2+ concentrations suggests the
precipitation of CaCO3, which was identified by XRD as
the polymorph calcite. The onset of CaCO3 precipitation
occurred shortly after the start of the experiment (∼ 3–4 h)
in the S. pasteurii and B. sphaericus 21776 experiments,
whereas the onset of precipitation was delayed (∼ 9 h) for
B. sphaericus 21787. This supports differences in the rate
of urea hydrolysis and thus the time at which CaCO3 sat-
uration was exceeded (Eqs. 2, 4, 5 and 7). Differences in
ureolysis and CaCO3 precipitation rates can be attributed to
differences in the specific ureolytic activities of the organ-
isms or the number of ureolytically active cells (Anbu et al.,
2016; Hammes et al., 2003a). B. sphaericus 21776 and S.
pasteurii exhibit urease activities approximately twice that
of B. sphaericus 21787 when Ca2+ is present (Hammes et
al., 2003a), supporting our observations of limited ureolysis
and delayed CaCO3 precipitation by B. sphaericus 21787.
Nevertheless, the decrease in Ca2+ concentrations in the ab-
sence of significant urea hydrolysis for B. sphaericus 21787
suggests there was a sufficient carbonate ion concentration in
the CMM+ and from ureolysis to sustain CaCO3 precipita-
tion. Hammes et al. (2003a) observed B. sphaericus 21787
was also able to precipitate CaCO3 despite lower urease ac-
tivity in the presence of Ca2+, suggesting this strain may en-
hance precipitation via other mechanisms such as enhanced
nucleation on cell surfaces or via organic exudates (Mitchell
et al., 2006b).
3.1.2 Aerobic bacterial growth and ureolytic activity
Changes in biomass, measured as protein and colony-
forming units (CFUs), were observed during ureolysis in
both aerobic CMM+ and CMM− experiments (Fig. 2).
CFUs and protein concentrations exhibited similar trends for
S. pasteurii and B. sphaericus 21776, where in CMM− ex-
periments CFUs and protein increased over time asymptot-
Figure 1. Changes in urea (triangle) and dissolved calcium (cir-
cle) concentrations during ureolysis over time in calcium-inclusive
aerobic experiments for (a) S. pasteurii, (b) B. sphaericus 21776
and (c) B. sphaericus 21787. Urea concentrations for abiotic con-
trol experiments (x) are also shown. Data points are the averages
of triplicate experiments; vertical error bars represent the standard
deviations; horizontal error bars indicate standard deviation of the
sampling times; error bars are smaller than markers if not visible.
ically (Fig. 2). In CMM+ experiments, CFUs and protein
concentrations seemed to slightly decrease or remain quasi-
constant while CaCO3 precipitation occurred (< 10 h), fol-
lowed by an increase in CFUs and protein once Ca2+ had
been depleted (Figs. 1 and 2). Decrease in biomass growth
during CaCO3 precipitation has been suggested to occur due
to the encasement of bacteria within the CaCO3 precipi-
tates (Tobler et al., 2011). Encasement of S. pasteurii cells
in CaCO3 minerals has been reported (Cuthbert et al., 2012;
Ebigbo et al., 2012; Schultz et al., 2011; Stocks-Fischer et al.,
1999), and cell indentations in CaCO3 precipitates have been
observed (Mitchell and Ferris, 2005). The recovery (i.e., re-
growth) of biomass after CaCO3 precipitation suggests that
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2152 A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation
Table 1. Change in pH in aerobic calcium-inclusive (CMM+) and calcium-exclusive experiments (CMM−). Results are averages from
triplicate experiments unless stated otherwise. Data for hour 0 were taken immediately after inoculation.
Species 0 h 10(±1) h 24(±3) h
CMM+ S. pasteurii 6.66± 0.06 8.87± 0.08 9.33± 0.02a
B. sphaericus 21776 7.24± 0.30 8.80± 0.20 9.23± 0.09a
B. sphaericus 21787 6.87± 0.15 8.06± 0.12 8.70± 0.26a
B. subtilis 6.80b – 7.50b
Sterile control 7.08± 0.04 – 7.31± 0.05a
CMM− S. pasteurii 6.91± 1.01a 9.16± 0.12a 9.16b
B. sphaericus 21776 6.85± 0.91a 9.10b 9.30± 0.14a
B. sphaericus 21787 7.5b – 9.00b
B. subtilis 7.15a 7.40b –
Sterile control 6.3± 0.36 6.56± 0.35 6.60± 0.28a
a Data taken from only two experiments. bData taken from only one experiment. – No data available.
ureolysis-induced CaCO3 precipitation does not have to be
a lethal event for the population as a whole and that net cell
growth can resume after CaCO3 precipitation ceases (Fig. 2).
For both B. sphaericus strains, in contrast to S. pasteurii,
CFUs were higher in the CMM+ experiments, despite pro-
tein concentrations being lower in the CMM+ experiments.
This might suggest that cell mortality of B. sphaericus strains
is increased in the calcium-free experiments, which may re-
flect lower tolerance to the higher pH values generally ob-
served in the calcium-free experiments (Table 1, Fig. 2).
TEM images and electron energy-loss spectroscopy
(EELS) of material collected on 0.2 µm pore size filters from
the CMM+ S. pasteurii systems (Fig. 3a–c) confirm that
some cells are surrounded by a layer of calcium-containing
precipitates. Figure 3d shows S. pasteurii grown in CMM−
for comparison. The data suggest that cells are removed
from suspension and potentially inactivated by CaCO3 en-
casement, either in large crystals (Mitchell and Ferris, 2005,
2006a) or by a thin coating (Fig. 3a–c).
Quasi-constant biomass concentrations during CaCO3
precipitation (Fig. 2) suggest that cell growth might not
have to be considered in kinetic descriptions of bacteri-
ally induced CaCO3 precipitation. However, it is unclear
whether (i) CaCO3-encased cells are ureolytically active or
(ii) CaCO3 precipitates surrounding the cells effectively act
as a barrier to urea reaching the cell or to NH3, OH− or NH+4
formed by the hydrolysis of urea from diffusing through the
CaCO3 to the bulk solution. Therefore, a theoretical analysis
of urea diffusion in CaCO3 was performed. The diffusion of
oxygen in CaCO3 at high temperatures has been documented
(Farver, 1994), but, to the best of our knowledge, information
on urea diffusion in CaCO3 at 30 ◦C has not been reported. A
number of assumptions were made for the estimates in this
study: (1) since urea has a lower diffusion coefficient than
oxygen in aqueous solutions at 25 ◦C (Stewart, 2003), it was
assumed that this will hold true at other temperatures and
through other substances, like CaCO3; (2) since the diffusion
coefficient of oxygen through CaCO3 at 400 ◦C and 100 MPa
is 2.66× 10−22 m2 s−1 (Farver, 1994), and diffusion coef-
ficients generally increase with increasing temperature and
pressure, it can be assumed that the diffusion coefficient of
urea in CaCO3 at atmospheric pressure and 30 ◦C is smaller
than 2.66×10−22 m2 s−1; (3) assuming that the geometry of
the CaCO3 is a uniformly thick slab with a thickness of ap-
proximately 200 nm, as determined from the TEM images
(Fig. 3b), the time it will take to reach 5 % of the bulk urea
concentration can be calculated using the relation presented
by Carslaw and Jaeger (1959):
t5 = 0.1L
2
De
, (14)
where L is the slab thickness, De is the (estimated) effective
diffusion coefficient in CaCO3 and t5 is the amount of time
it will take to reach 5 % of the bulk concentration. Using the
above assumptions, it would take at least 175 d for 5 % of
the urea to diffuse through the CaCO3 surrounding the cells.
Because CaCO3 precipitation takes place over the course of
approximately 1 d, it can safely be assumed that even if the
encased cells are still alive, urea is not able to diffuse through
the CaCO3 fast enough for them to hydrolyze significant
amounts and contribute to the increase in solution alkalinity.
Therefore, it is argued that, at least in the systems described
here, cell growth does not have to be considered in kinetic ex-
pressions describing ureolysis during CaCO3 precipitation.
Instead, the biomass at the onset of precipitation, which is
equivalent to the initial biomass in the system (Fig. 2), can
be used to normalize the observed ureolysis rates to biomass
concentration.
While a physical association between cells and CaCO3
precipitates is evident, precipitation is likely to occur from
a combination of (i) homogeneous nucleation in the bulk so-
lution in alkaline microenvironments around bacterial cells
(Schultze-Lam et al., 1996; Stocks-Fischer et al., 1999), and
(ii) heterogeneous nucleation on nascent crystals, bottle walls
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A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation 2153
Figure 2. Change in protein concentrations and CFU mL−1 over time for calcium-inclusive (solid markers) and calcium-exclusive (open
markers). Aerobic media: (a) S. pasteurii, (b) B. sphaericus 21776 and (c) B. sphaericus 21787. Data points are the average of triplicate
experiments; vertical error bars represent the standard deviation of triplicate experiments; horizontal error bars indicate standard deviation of
the sampling times.
and the bacterial cell surfaces (Rodriguez-Navarro et al.,
2012).
3.1.3 Kinetics of ureolysis and CaCO3 precipitation
Kinetic analyses were performed on the individual CMM+
and CMM− experimental data for all bacterial strains
(Fig. SI2.1 in the Supplement). A summary of the param-
eters estimated is shown in Table 2 (for detailed results
on individual experiments see Table SI2.1 in the Supple-
ment). kurea values for the bacterial species varied accord-
ing to the presence (CMM+) or absence (CMM−) of Ca2+
in the medium. S. pasteurii and B. sphaericus 21776 exhib-
ited statistically insignificant differences in kurea values (t test
p value= 0.27) from each other in both CMM− and CMM+
systems with kurea values being between 1.6 and 2.5 times
higher in the absence of Ca2+ (S. pasteurii: kurea,CMM+ =
0.07±0.02 h−1, kurea,CMM− = 0.19±0.10 h−1; B. sphaericus
21776: kurea,CMM+ = 0.10± 0.03 h−1, kurea,CMM− = 0.16±
0.05 h−1). This is likely due to the encasement of cells by
CaCO3 and their inactivation in CMM+ experiments. Some
data points were excluded for S. pasteurii CMM− kurea cal-
culations because of an estimated increase in urea concen-
tration (based on a decrease in NH+4 concentration) likely
due to significant volatilization of NH+4 ↔ NH3+H+ that
can occur at pH > 9 (at 34 h; open marker, Fig. SI2.1b). B.
sphaericus 21787 exhibited low kurea values in both CMM+
(kurea = 0.02 h−1) and CMM− (kurea = 0.05 h−1). From trip-
licate experiments, only one experiment showed values that
could be used for kinetic analysis (Fig. S2.1), and some out-
lying data points were not used for the kinetic calculations;
hence, no standard deviations can be provided (Table 2).
Thus, rate coefficients obtained for B. sphaericus 21787 are
not statistically valid but were estimated for the purpose of
comparison to the other studied bacterial strains. B. sphaeri-
cus 21787 has been shown to have urease activity about half
that of S. pasteurii and B. sphaericus 21776 in the presence
of Ca2+, which could explain the low kurea values. How-
ever, in the absence of Ca2+, urease activity for B. sphaericus
21787 is about twice that of B. sphaericus 21776 (Hammes
et al., 2003a) that does not support our experimental results.
This suggests while B. sphaericus 21787 has high potential,
they generate comparable rates of urea hydrolysis to the other
strains under the experimental conditions used in this study
B. sphaericus 21787 exhibits limited ureolytic capabilities.
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2154 A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation
Table 2. Summary of kinetic parameters for urea hydrolysis (kurea) and calcite precipitation (kprecip) in aerobic and anaerobic experiments in
calcium-inclusive (CMM+) and calcium-exclusive (CMM−) experiments inoculated with S. pasteurii, B. sphaericus 21776 and B. sphaeri-
cus 21787. Anaerobic experiments were incubated with or without nitrate as the terminal electron acceptor (TEA). Results are averages from
triplicate experiments unless stated otherwise.
Microorganism Initial biomass kurea Lag time kurea normalized to kprecip Lag time
(OD600) (h−1) (h) OD600 (OD−1600 h−1) CFU (mL CFU−1 h−1) (h−1) (h)
Aerobic
CMM+ S. pasteurii 0.014± 0.001 0.074± 0.021 5.0± 1.0 5.251± 1.273 3.22× 10−8± 6.54× 10−9 0.253± 0.021 3.3± 0.6
B. sphaericus 21776 0.014± 0.001 0.107± 0.038 4.0± 0.0 8.020± 3.786 5.30× 10−8± 3.23× 10−8 0.604± 0.344 3.3± 0.6
B. sphaericus 21787 0.015± (n/a)a 0.023± (n/a)a 3± (n/a)a 1.526± (n/a)a 8.52× 10−9± (n/a)a 0.219± (n/a)a 8± (n/a)a
CMM− S. pasteurii 0.017± 0.000b 0.192± 0.104b 4.0± 0.0b 11.227± 5.988b 5.88× 10−8± 2.02× 10−8b – –
B. sphaericus 21776 0.015± 0.001b 0.168± 0.050b 3.5± 0.71b 10.818± 2.797b 6.20× 10−8± 1.33× 10−8b – –
B. sphaericus 21787 0.015± (n/a)a 0.067± (n/a)a 6.0± (n/a)a 3.196± (n/a)a 1.75× 10−8± (n/a)a – –
Anaerobic
CMM+ S. pasteurii/NO−3 0.014± 0.002b 0.048± 0.018b 6.5± 0.7b 3.617± (1.092)b 2.34× 10−8± 4.67× 10−9
b
0.360± 0.223 6.5± 0.6
S. pasteurii/no TEA 0.014± 0.002 0.082± (n/a)a 10.0± (n/a)a 6.616± (n/a)a 4.68× 10−8± (n/a)a 0.191± 0.050 3.3± 0.6
CMM− S. pasteurii/NO−3 0.014± 0.002b 0.071± 0.017b 8.5± 2.1b 5.278± 0.875b 3.45× 10−8± 2.05× 10−9
b
– –
a One experiment used in analysis. b Two experiments used for kinetic analysis. n/a=No data available.
Figure 3. Transmission electron microscopy images of S. pasteurii
cells in calcium-inclusive (a–c) and calcium-exclusive (d) media
with cell walls (CWs) and calcium-containing precipitate (CCP) la-
beled. Panels (a) and (d) show end cross sections though S. pasteurii
cells, with CWs clearly exhibited. A calcium-containing precipitate
coverage that encapsulates the cells is exhibited as darker regions
beyond the cell wall in (a)–(c). The labeled CCP cross section is
measured in (b) and is approximately 280 nm thick.
A lag time of 3–5 h before the onset of ureolysis was de-
tectable for all bacterial strains, which was slightly longer
for CMM+ experiments (CMM+= 5 and 4 h; CMM−= 4 h
and 3.5 for S. pasteurii and B. sphaericus 21776, respec-
tively). Connolly et al. (2013) observed longer lag times to
the onset of ureolysis of ∼ 15 h for S. pasteurii, ∼ 6 h for
Pseudomonas aeruginosa MJK1 and ∼ 4 h for Escherichia
coli MJK2 when cultivated with 0.16 mM urea in similarly
composed CMM−, likely reflecting the lower urea concen-
trations used that would limit reactant supply to urease and
increase the time before urea hydrolysis was detectable.
In the present study, kurea values normalized to the initial
biomass concentration were higher for B. sphaericus 21776
than S. pasteurii (Table 2) but differences were not statisti-
cally significant, suggesting similar cell specific urease ac-
tivity between the strains. The standard deviations of kurea,
initial biomass and lag time were small between replicate
experiments with S. pasteurii and B. sphaericus 21776, and
R2 values for the fit to Eq. (10) were greater than 0.9 (Ta-
ble SI2.1).
The kinetic parameters obtained in this study were com-
pared to other parameters previously published (Tobler et
al., 2011; Ferris et al., 2004; Fujita et al., 2000; Stocks-
Fischer et al., 1999). kurea values of S. pasteurii obtained
in this study as well as in previous publications were stan-
dardized to the initial cell concentrations (Table 3). Values
of kurea were higher in the present study for both S. pasteurii
and B. sphaericus 21776 (kurea = 0.07 and 0.11 h−1, respec-
tively) than those from S. pasteurii in other studies (kurea =
0.005 to 0.095 h−1). This was also apparent once normal-
ized to biomass (this study, B. sphaericus 21776 kurea =
8.02 OD−1600 h−1 and S. pasteurii 5.251 OD
−1
600 h
−1, compared
to kurea = 0.11 to 2.80 OD−1600 h−1 in previous studies). The
generally higher kurea values in this study appear to reflect
the higher temperature (30 ◦C) used compared to the previ-
ous studies, which ranged from 20 to 25 ◦C. Higher temper-
atures generally increase reaction rates where chemical re-
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A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation 2155
actions are advanced through a transient activated complex
(Stumm and Morgan, 1996). In urease, the transitional state
involves coordination of urea and water at the active catalytic
site of the enzyme (Jabri et al., 1995). Formation of such
an activated complex tends to impart a greater temperature
dependency on the absolute reaction rate than would be en-
countered if the reactions were mediated solely by collisions
arising from molecular diffusion (Ferris et al., 2004; Mitchell
and Ferris, 2005).
The biomass concentration-normalized kurea values from
Fujita et al. (2000) are much lower. This could be due to
the highest biomass concentrations (OD600 = 0.072) used in
these studies; very high biomass concentrations could shift
the primary kinetic dependency from being catalysts (i.e.,
enzyme-limited) to substrate-limited. However, this appears
to be opposite to what Tobler et al. (2011) reported indi-
cating kurea increased with increasing inoculum density. The
rate constants obtained with the three organisms used in the
present study are similar to the range of values measured in
deeper vadose zone mineral subsoils which were between
0.00375 to 0.07 h−1 (Swensen and Bakken, 1998), suggest-
ing natural levels of ureolytic bacterial activity were reason-
ably approximated in the aerobic experiments.
On average, B. sphaericus 21776 had the highest kprecip
(0.60±0.34 h−1), although considering its high standard de-
viation, kprecip for S. pasteurii is not significantly different
(kprecip = 0.25± 0.02 h−1; t test p value 0.21). R2 values of
the fit to Eq. (12) were relatively high (0.84–0.93) for B.
sphaericus 21776 and S. pasteurii (Table 2). The kprecip for B.
sphaericus 21787 was lower than the other strains (0.21 h−1);
although, as noted, rate constants for this strain are not sta-
tistically significant. The lag time for CaCO3 precipitation
was 3.3 h for B. sphaericus 21776 and S. pasteurii, which
reflects the similar kurea values, and thus the similar time
it took to reach CaCO3 saturation and induce precipitation,
whereas the longer lag time for B. sphaericus 21776 reflects
the significantly lower kurea value. Tobler et al. (2011) ob-
served similar lag times until CaCO3 precipitation (2–3 h)
in aerobic experiments (artificial groundwater with no nutri-
ents added, 250–500 mM urea and 50–500 mM Ca) with S.
pasteurii, as observed here for B. sphaericus 21776 and S.
pasteurii. First-order rate constants for CaCO3 precipitation
observed here for B. sphaericus 21776 and S. pasteurii were
also higher (0.21 h−1 < kprecip < 0.60 h−1) compared to other
studies (0.01 h−1 < kprecip < 0.11 h−1) (Table 3). This is likely
associated with the greater kurea values observed in this study
than in previous studies. Temperature is unlikely to account
for this variation given the modest decrease in calcite solubil-
ity (∼ 27 %) that occurs between 20 and 30 ◦C (Miller, 1952;
Stumm and Morgan, 1996). Overall, kurea values are lower
than kprecip values in this and previous studies, with the ex-
ception of Ferris et al. (2004), indicating urea hydrolysis is
the rate limiting step during ureolysis-induced CaCO3 pre-
cipitation and that CaCO3 precipitation rates are rapid and
controlled by the rate of urea hydrolysis, once the critical su-
persaturation is exceeded (Mitchell and Ferris, 2005).
3.2 Anaerobic experiments
3.2.1 Anaerobic ureolysis and bacterial growth
Given the potential for anaerobic conditions in subsurface
environments, screening experiments were performed to as-
sess the capability of S. pasteurii to grow and/or increase pH
in CMM− in the presence of NO−3 , SO2−4 and Fe3+ as po-
tential TEAs. There are contradicting reports in the literature
regarding S. pasteurii’s ability to grow and hydrolyze urea in
the absence of oxygen; some studies suggest that the anoxic
environment does not hinder urease activity (Mortensen et
al., 2011; Tobler et al., 2011), whereas other studies report
limited microbial growth and poor ureolysis (Martin et al.,
2012).
In this study, pH increased (pH > 9.0) in all experiments
with and without potential TEAs added (Fig. 4), suggesting
ureolysis by S. pasteurii took place in the absence of oxygen.
The pH of the abiotic controls did not exceed pH 7, except for
the medium containing SO2−4 as the added TEA, which had
an initial pH of 7.7 and remained constant throughout the ex-
periments (Fig. 4c). Growth in the CMM− anaerobic exper-
iments quantified by OD600 absorbance was lower than that
observed in aerobic experiments regardless of the presence
or absence of TEAs (Fig. 4). Although some growth might
have occurred during the initial period of the experiments (in-
creased absorbance by ∼ 20 h), the lack of sustained growth
over time suggests the inability of S. pasteurii to grow un-
der anaerobic conditions. These findings are in agreement
with those by Martin et al. (2012), who observed limited cell
growth in the absence of oxygen. In the present study, once
oxygen was allowed to diffuse into the systems (at 120 h), op-
tical density in all inoculated systems increased, indicating
that even though no significant growth was observed in the
absence of oxygen, the bacteria were still viable after 120 h
of oxygen depletion and can be resuscitated.
Sustained growth of S. pasteurii in the absence of oxygen
does not appear to be feasible which might limit the poten-
tial use of S. pasteurii for inducing CaCO3 precipitation in
the subsurface to only short-term purposes. However, the po-
tential regrowth of microbes, even after prolonged periods of
exposure to oxygen-free conditions, suggests that S. pasteurii
could be resuscitated and restimulated through the injection
of oxygenated fluids, which could enable bacterial growth
and thus ureolytic activity over longer periods of time.
3.2.2 Kinetics of anaerobic ureolysis and CaCO3
precipitation
After the screening experiments with different TEAs, studies
were performed to determine the kinetics of ureolysis and
CaCO3 precipitation in the absence of oxygen. Since there
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2156 A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation
Table
3.Sum
m
ary
ofkinetic
coefficients
and
initialgrow
th
conditions
foraerobic
calcium
-inclusive
experim
ents
perform
ed
in
this
study
and
previous
studies.
A
erobic
T
his
study
T
his
study
T
his
study
Stocks-Fischer
Fujita
et
Ferris
et
Tobleretal.(2011)
conditions
etal.(1999)
al.(2000)
al.(2003)
B
.sphaericus a
B
.sphaericus
S.pasteurii
S.pasteurii
S.pasteurii
S.pasteurii
S.pasteurii
21787
21776
A
T
C
C
11859
A
T
C
C
6453
A
T
C
C
11859
A
T
C
C
11859
A
T
C
C
11859
Tem
perature
30
30
30
25
20
20
20
20
20
20
20
20
( ◦C
)
Initial
6.7
6.7
6.7
8.0
6.5
6.5
6.5
6.5
6.5
6.5
6.5
6.5
pH[Ca 2+
]
25.2
25.2
25.2
25.2
25
1.75
50
200
500
50
200
500
(m
M
)
[U
rea]
333
333
333
333
333
6
250
250
500
250
250
500
(m
M
)
[cells]
0.015
0.014
0.014
0.010
0.072
0.070
0.03
0.03
0.03
0.07
0.07
0.07
(O
D
600 ) b
kurea
0.023
0.107
0.074
0.028
0.008
0.038
0.007
0.005
0.005
0.074
0.095
0.044
(h −
1)
kurea
1.526
8.020
5.251
2.800
0.111
0.543
0.250
0.180
0.180
1.065
1.363
0.630
(O
D −
1
600
h −
1)
kurea
8
.52×
10 −
9
5
.30×
10 −
8
3
.22×
10 −
8
3
.00×
10 −
8
3
.73×
10 −
10
1
.81×
10 −
9
9
.86×
10 −
10
7
.13×
10 −
10
7
.13×
10 −
10
3
.56×
10 −
9
4
.56×
10 −
9
2
.11×
10 −
9
(m
L
C
FU −
1
h −
1)
kprecip
0.219
0.604
0.253
0.116
0.112
0.014
0.007
0.011
–
–
0.065
–
(h −
1)
a
R
ate
coefficients
obtained
forthis
strain
are
notconclusive
and
should
be
taken
only
as
a
guide
forcom
parison
purposes
in
this
study. b
O
D
600
values
w
ere
converted
to
a
1
cm
path
length
equivalentw
here
necessary.
–
N
o
reported
values.
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A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation 2157
Figure 4. Changes in pH and OD600 over time in anaerobic (square) and aerobic (circle) calcium-exclusive medium for S. pasteurii with
(a) NO−3 , (b) Fe3+ and (c) SO
2−
4 as terminal electron acceptors (TEAs); experiments without added TEAs are shown in (d). Abiotic controls
(triangle) are also shown. Open markers indicate values for time points after the bottles were opened to the environment allowing oxygen to
enter the system.
were similarly low levels of growth in the initial anaero-
bic screening experiments with different TEAs and no TEA
(Fig. 4), kinetic experiments in the absence of oxygen were
conducted with no TEA, as well as with NO−3 as a poten-
tial TEA (Fig. 5). Urea was hydrolyzed under all experimen-
tal conditions and CaCO3 precipitation was observed in the
presence of Ca2+ (Fig. 5). However, ureolysis and CaCO3
precipitation did not occur in all replicates for each experi-
ment, which accounts for the high standard deviations.
Rate coefficients were estimated as previously described
for aerobic experiments (Fig. SI2.2) and a summary of the
results is presented in Table 2 (for detailed results for the
individual experiments see Table SI2.2). Data points which
preceded the onset and completion of ureolysis and calcite
precipitation were excluded (Fig. SI2.2) kurea seems to be
lowest in CMM+ with NO−3 (kurea = 0.04± 0.01 h−1), fol-
lowed by CMM− with NO−3 (kurea = 0.07± 0.01 h−1) and
CMM+ without TEA (kurea = 0.08 h−1). The same relative
rates were apparent when kurea values were normalized to
biomass (Table 2). The presence of NO−3 appears to have
slightly decreased ureolysis rates. While the reasons for this
possible decrease in the ureolytic activity due to the presence
of NO−3 are unclear, it could be perceivable that the ureolytic
activity is downregulated in the presence of the alternative ni-
trogen source NO−3 . Longer lag times to the onset of ureoly-
sis (ranging from 6.5 to 10 h) were observed in the anaerobic
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2158 A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation
Figure 5.Changes in urea (triangle markers) and dissolved calcium (circle markers) concentrations over time in anaerobic experiments with S.
pasteurii in calcium-inclusive medium with NO−3 (black solid markers), calcium-exclusive medium with NO
−
3 (open markers) and calcium-
inclusive medium without added terminal electron acceptor (grey solid markers). Data points are the averages of triplicate experiments;
vertical error bars represent the standard deviations of measurements; horizontal error bars indicate standard deviation of the sampling times;
error bars are smaller than markers if not visible.
experiments relative to the aerobic ones. Moreover, compar-
ing the experiments containing NO−3 , the presence of Ca
2+
in the medium results in a lower kurea value (Table 2). This
could be due to the encasement of cells by CaCO3.
The onset of CaCO3 precipitation occurred after approx-
imately 6 h, which was twice the lag time observed for pre-
cipitation under aerobic conditions (Table 2); this was ex-
pected, as slower ureolysis was observed in the anaerobic
experiments (Table 2). Rate constants (kprecip) for anaero-
bic CaCO3 precipitation were not statistically different for
CMM+ with NO−3 (0.36 h−1± 0.22) and CMM+ without
TEA (0.19 h−1± 0.05). Tobler et al. (2011) reported a similar
kurea value (0.09 h−1) for experiments with S. pasteurii under
anoxic conditions in natural groundwater containing com-
parable concentrations of urea and Ca2+ (250 and 50 mM,
respectively); kprecip was not reported due to a poor fit of
the data with first-order kinetics. Experiments containing
only indigenous bacteria exhibited far lower rates of ureol-
ysis (kurea = 0.0016 h−1) and CaCO3 precipitation (kprecip =
0.009 h−1) than observed in the present study. Differences in
ureolysis and CaCO3 precipitation rates between this study
and Tobler et al.’s (2011) study are likely due to the low initial
biomass of the indigenous ureolytic population in the natural
groundwater.
The comparison of kinetics from aerobic and anaerobic
experiments in the present study demonstrates that rates are
on the same order of magnitude (Table 2). In CMM+, urea
hydrolysis rates for S. pasteurii under aerobic and anaer-
obic conditions (with or without TEA) were not signifi-
cantly different (Pvalue = 0.274), even when normalized to
initial biomass concentrations (OD600 or CFU mL−1). pH
increases in these screening experiments suggest anaerobic
ureolysis occurred at the same rate as under aerobic condi-
tions (Fig. 4). Similarly, kprecip in aerobic and anaerobic ex-
periments are comparable (0.19 and 0.25 h−1, respectively).
This suggests that oxygen-free environments do not signif-
icantly impact the rate of ureolysis or CaCO3 precipitation
initially but that anaerobic growth cannot be conclusively
demonstrated under the conditions of this present study. This
supports observations by Tobler et al. (2011), who reported
similar rates of ureolysis under both oxic and anoxic con-
ditions when amending natural groundwater with S. pas-
teurii (kurea0.10 h−1 in oxic conditions, kurea0.097 h−1 in
anoxic conditions with 50 mM Ca2+ and 250 mM urea; Ta-
ble 3). Martin et al. (2012) also observed ureolytic activity
by S. pasteurii under anoxic conditions but to a lesser ex-
tent compared to the extensive activity reported by Tobler et
al. (2011); however, rate constants were not reported. The
current study therefore suggests ureolytic activity observed
under anoxic conditions corresponds to the urease already
present in the cells, as suggested by Martin et al. (2012). S.
pasteurii could therefore be used for CaCO3-induced pre-
cipitation in the subsurface in the short term, and bacterial
growth could be stimulated through multiple injections of
bacterial cells or oxygenated medium to re-enable ureolytic
activity and thus CaCO3 precipitation. This is supported by
our calcite precipitation rate constants under anaerobic con-
ditions, the first to be reported for a ureolytic strain, which
are comparable to aerobic rate constants, suggesting anaero-
Biogeosciences, 16, 2147–2161, 2019 www.biogeosciences.net/16/2147/2019/
A. C. Mitchell et al.: Aerobic and anaerobic microbial ureolysis and calcite precipitation 2159
bic conditions will not significantly inhibit CaCO3-induced
precipitation in the subsurface.
4 Conclusions
All three ureolytic strains studied, S. pasteurii and B. sphaer-
icus strains 21776 and 21787, were capable of inducing
CaCO3 precipitation under aerobic conditions. Data obtained
in this study suggest that rates of ureolysis and ureolysis-
induced CaCO3 precipitation are affected by differences in
the ureolytic species. This information should be consid-
ered in subsurface engineering strategies utilizing microbial
amendment or stimulation. Specifically, rates of ureolysis
and CaCO3 precipitation were highest and comparable for
S. pasteurii and B. sphaericus 21776. Although B. sphaer-
icus 21787 showed poor ureolysis, some CaCO3 precipita-
tion was observed, suggesting this strain may enhance pre-
cipitation via other mechanisms such as enhanced nucleation
on cell surfaces or via organic exudates. When rate coeffi-
cients were normalized to cell numbers, B. sphaericus ex-
hibited 21776 higher rates of ureolysis per cell compared to
S. pasteurii, but the differences were not statistically signifi-
cant, suggesting that both of these strains are good candidate
species for subsurface augmentation if maximizing rates of
ureolysis and precipitation are desirable.
S. pasteurii was capable of ureolysis in anaerobic envi-
ronments with and without the addition of potential electron
acceptors; however, sustained growth of S. pasteurii over
time in the absence of oxygen did not appear to be possi-
ble. Comparison of kinetics from aerobic and anaerobic ex-
periments demonstrates that rates are on the same order of
magnitude, suggesting that oxygen-free environments do not
significantly impact the initial rate of ureolysis or CaCO3
precipitation. Apparent rate coefficients for ureolysis were
reduced in CMM+ relative to CMM−. The limited increase
in cell biomass during the period of CaCO3 precipitation
and TEM images reveals that this may be due to the en-
casement and inactivation of cells. However, populations can
recover and regrow once CaCO3 precipitation has ceased.
Therefore, ureolysis-induced CaCO3 precipitation is likely
to be efficient in aerobic and anaerobic subsurface systems.
However, our data and other recent studies under flow con-
ditions demonstrate that if only one injection of microbes
is to occur but longer-term ureolysis is desired in subsur-
face applications, resuscitation and regrowth of microbes,
e.g., through the injection of growth media, will be neces-
sary since CaCO3 precipitation greatly inhibits cell growth
(Cuthbert et al., 2012; Phillips et al., 2013b).
Data availability. Raw experimental data is deposited at Dryad
repository https://datadryad.org/ (last access: 1 May 2019) at
https://doi.org/10.5061/dryad.5r01k2k (Mitchell et al., 2019). Data
files: Data Appendix_compiled/.
Supplement. The supplement related to this article is available
online at: https://doi.org/10.5194/bg-16-2147-2019-supplement.
Author contributions. ACM, EJEO, SLP and RG wrote the
manuscript. RG, ACM, SLP, AJP and ABC designed experiments.
SLP performed experiments.
Competing interests. The authors declare that they have no conflict
of interest.
Acknowledgements. Funding was provided from the US Depart-
ment of Energy (DOE) Zero Emissions Research Technology
Center (ZERT), award no. DE-FC26-04NT42262, DOE EPSCoR
award no. DE-FG02-08ER46527, DOE Office of Science, Sub-
surface Biogeochemical Research (SBR) Program, contract no.
DE-FG-02-09ER64758, DOE NETL contract no. DE-FE0004478
and DE-FE0009599, European Union Marie-Curie Reintegration
Grant, award no. 277005 (CO2TRAP), National Science Founda-
tion, award no. DMS-0934696, and a Sêr Cymru National Research
Network for Low Carbon, Energy and the Environment Grant from
the Welsh Government and Higher Education Funding Council for
Wales. Support for the Environmental and Biofilm Mass Spectrom-
etry Facility through DURIP, contract no. W911NF0510255 and
the MSU Thermal Biology Institute from the NASA Exobiology
Program Project NAG5-8807 is acknowledged. TEM images were
kindly taken by Susan Brumfield at Montana State University in the
Plant Science and Plant Pathology Laboratory.
Review statement. This paper was edited by Denise Akob and re-
viewed by two anonymous referees.
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