Chemical Geology 617 (2023) 121266

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Chemical Geology
journal homepage: www.elsevier.com/locate/chemgeo

Calcification of cell membranes: From ions to minerals

Xiao Gao a, b, Jie Li a, Kaiming Hu a, Yanyang Zhao a, b, Yu Han c, Fang Liu d, e,
Maurice E. Tucker f, g, Zuozhen Han a, b, *
a
Shandong Provincial Key Laboratory of Depositional Mineralization and Sedimentary Minerals, College of Earth Science and Engineering, Shandong University of
Science and Technology, Qingdao 266590, China
b
Laboratory for Marine Mineral Resources, Center for Isotope Geochemistry and Geochronology, Qingdao National Laboratory for Marine Science and Technology,
Qingdao 266237, China
c
School of Geosciences, China University of Petroleum, Qingdao 266580, China
d
College of Chemical Engineering, China University of Petroleum, Qingdao 266580, China
e
State Key Laboratory of Petroleum Pollution Control, Beijing 102206, China
f
School of Earth Sciences, University of Bristol, Bristol BS8 1RJ, UK
g
Cabot Institute, University of Bristol, Cantock’s Close, Bristol BS8 1UJ, UK

A R T I C L E I N F O A B S T R A C T

Editor: Hailiang Dong The whole process of cell membrane calcification of Bacillus licheniformis DB1–9 was studied by molecular dy­
namics (MD) and microbially-induced carbonate precipitation (MICP) laboratory experiments. Typical metab­
Keywords: olitic products of Bacillus licheniformis DB1–9 were used as the calcium carbonate nucleation template to establish
Calcification the organic constituents and a water‑calcium carbonate two-phase system model for MD simulation, and to
Crystallization
characterize the early stages of calcium carbonate nucleation on the surfaces of extracellular polymeric sub­
Biological interface
stances (EPS). The surface minerals of calcified bacteria obtained by MICP were prepared by focused ion-beam
Molecular dynamics
Focused ion beam (FIB) and further analyzed using high-resolution transmission electron microscopy-selected area electron
diffraction (HRTEM-SAED). We propose that the evolution process of cell membrane calcification is: ions → ion-
pairs → multi-ion complexes (MIC) of large-size topological structures → pre-nucleated clusters (PNC) →
amorphous calcium carbonate (ACC) → carbonate minerals (monohydrocalcite, vaterite, aragonite). In addition,
ACC and the state before ACC were adsorbed on to the surface of the cell membrane after self-assembly in the
aqueous solution; the ACC then underwent maturation and crystallized into ordered carbonate minerals with a
crystal structure. Our study reveals the processes of microbial calcification, which has implications for the
calcification of microorganisms in modern aqueous environments and for the formation of microbialites
throughout the geological record.

1. Introduction Marine invertebrate exoskeletons including those of sponges, archae­

ocyathids and bryozoans are commonly found within some microbial
Carbonates are an important type of sedimentary rock and in many carbonates (Gandin and Debrenne, 2010; Lee and Riding, 2018; Riding
cases their formation has been directly or indirectly related to organ­ et al., 2019; Wood, 2000). In modern environments, the stromatolites in
isms. Bio‑carbonates are recorders of the biosphere and Earth surface the Bahamas, calcium carbonate deposits of hot springs at Yellowstone
processes reflecting biological evolution and long-term changes in and dolomite sediments in Qinghai Lake, China, are all examples of the
seawater chemistry (Riding and Aurélien, 2020). Microbialites are microbial involvement in the formation of carbonate deposits (Deng
sedimentary deposits formed through the activities of microorganisms et al., 2010; Reid et al., 1995). Consequently, the interactions between
and their biomineralisation (Riding, 2000; Riding and Aurélien, 2020; microorganisms and minerals are one of the important natural processes
Sondi et al., 2011). Microbial carbonates are not only widely distributed throughout Earth history and these are involved in environmental
in strata around the world from the early Precambrian onwards but they changes, element cycling, and the formation of mineral deposits (Dong
are also forming in modern lacustrine and marine environments today. et al., 2022).

* Corresponding author at: Shandong Provincial Key Laboratory of Depositional Mineralization and Sedimentary Minerals, College of Earth Science and Engi­
neering, Shandong University of Science and Technology, Qingdao 266590, China.
E-mail address: hanzuozhen@sdust.edu.cn (Z. Han).

https://doi.org/10.1016/j.chemgeo.2022.121266
Received 20 July 2022; Received in revised form 8 December 2022; Accepted 9 December 2022
Available online 14 December 2022
0009-2541/© 2022 Elsevier B.V. All rights reserved.
X. Gao et al. Chemical Geology 617 (2023) 121266

Microbially-induced carbonate precipitation (MICP) has attracted into the biogeochemical products: microbialites.
extensive attention in environmental and Earth sciences, engineering
and other fields due to its wide distribution and potential applications. 2. Methods
The capture of metal ions during MICP demonstrates its important po­
tential for environmental remediation (Li et al., 2014). In addition, 2.1. Models and simulations
minerals produced by MICP can cement surrounding materials and be
used in the field of construction for sand fixation (Sharma et al., 2021). A model system including aqueous ion speciation and concentrations
The MICP technique has been used to fill fissures to reduce the potential as well as the components of EPS was established to describe the calci­
of CO2 leakage, which is a new technology for industrial production fication of Bacillus licheniformis DB1–9. Based on metabolite analysis of
(Phillips et al., 2013). Furthermore, microbially-induced dolomite/ Bacillus licheniformis DB1–9, 17 types of amino acid (gly, ala, pro, glu,
proto-dolomite precipitation offers a new perspective to the “dolomite asp, val, ser, thr, leu, lys, arg, cys, phe, tyr, ile, met, and his) were
problem” (Gao et al., 2022; Kaczmarek and Thornton, 2017; Liu et al., selected as EPS components to represent the biological interface (Han
2020a; Liu et al., 2020b; Wang et al., 2022). et al., 2018). The model establishment process is as follows: the 17
Many researchers have undertaken laboratory experiments to un­ amino acids were first repeated six times and packed together using the
derstand the processes of MICP (Han et al., 2018; Zhao et al., 2021; amorphous cell tool to generate the initial configuration of the organic
Zhuang et al., 2018). However, microorganisms as participants involved interface (Supplementary Fig. S1). Secondly, the water - CO2− 3 - Ca
2+

in MICP are also affected by the ions in the aqueous solution, and the solution system was constructed with 2000 water molecules, 20 Ca2+
study of calcification of cell membranes has often neglected this aspect. ions and 20 CO2− 3 ions. Finally, the model for the EPS - water - CO3 -
2−
2+
Both chemical and biological factors are potentially involved in raising Ca solution system was established by the layers tool. All simulations
carbonate saturation and inducing biomineralisation (Riding and were performed with the Forcite package. The model system containing
Aurélien, 2020). It has been well established that bacteria can produce a 2000 water molecules, 20 carbonate ions, and 20 calcium ions with an
range of different organic components during their metabolic processes, initial size of 30 Å*50 Å*71.18 Å and a total of 9360 atoms (the con­
and the extracellular polymeric substances (EPS) on the bacterial sur­ centration in the model is slightly larger than the actual calcium car­
faces can sequester cations from solution and so lead to mineral pre­ bonate saturation, which is intended to increase to a nucleation event for
cipitation (Liu et al., 2020a; Pagliaccia et al., 2021; Qian Wang, 2022). easy observation), was then equilibrated by NPT (Liu et al., 2013) (N,
Thus, the classic chemical crystallization pathways may not be directly the molecular number is fixed; P, the pressure is constant; the T, tem­
applicable to carbonate precipitation at the biological interface. There­ perature is constant) to ensure the correctness of the density at 298 K, 1
fore, there is a case to rethink and explore further the nucleation and atm pressure. Moreover, the dynamics of NVT (Liu et al., 2013) (N,
crystallization of calcium carbonate at the biological interface. molecular number is fixed; V, volume is constant; T, temperature is
Compared with classical crystallization reactions in aqueous solutions, constant) were executed. The CompassII force field (CFF) was selected
various functional groups (e.g., carboxyl, amino and sulfuric acid.) on a for the calculation, and a Nose-Hoover Langevin (NHL) temperature
bacterial cell surface will affect the free energy of the nucleation inter­ controller was selected due to its resistance to Langevin friction and
face (Dobberschutz et al., 2018), resulting in different pathways for noise, thus providing a reliable equation calculation for a system in
cation absorption and mineral nucleation, leading to distinct crystalli­ balance or close to balance. The Berenson with decay constant 0.1 ps
zation behaviors at the biological interface. Moreover, the crystalliza­ was selected to realize pressure control by changing the coordinates of
tion process is also controlled by the salinity (Qiu et al., 2017), particles and the size of the unit cell which is applicable to the amor­
temperature (Zheng et al., 2021), pH (Guibaud et al., 2008), sedimen­ phous system to realize the rationality of the model. Finally, the system
tary environment (Fodor et al., 2020) and other factors (Dittrich and remained at 28 Å*47 Å*68.14 Å after equilibrium.
Obst, 2004; Zhang et al., 2015) of the aqueous solution. These various The total energy and temperature of the system tended to be stable
factors result in different crystallization modes and different mineral after equilibrating the system several times (Supplementary Fig. S2).
morphologies. The presence of calcium carbonate multi-ion complexes The nucleation process of Ca2+ ions and CO2− 3 ions at the EPS surfaces
(MIC) or pre-nucleation clusters (PNC) in aqueous solution has been was simulated with a total time of 1000 ps (ps) and step-size of 1
confirmed by cryotransmission electron microscopy and simulation femtosecond (fs). The electrostatic interaction was described by the
calculations (Chen et al., 2020; Pouget et al., 2007). However, the ex­ Ewald summation method. The atom-based summation method and
istence of PNC or MIC has been limited to aqueous solutions rather than Lennard-Jones 6–9 function was used to describe the van der Waals
at a biological interface. Enyedi et al. (2020) demonstrated that ACC can interactions in this study. In addition, a larger water - CO2− 3 - Ca
2+

be preserved for a long time (at least half a year) due to the protective system was constructed to describe the assembly behavior of multi-ion
effect of EPS on the cell membrane. ACC research at the biological complexes (MIC), consisting of 2000 water molecules, 40 CO2− 3 ions
interface is extensive but provides little insight into any previous state. and 40 Ca2+ ions, with the same parameters and field as the organic
The mechanism of ion aggregation and nucleation at organic interfaces interface. The purpose of increasing the number of CO2− 3 and Ca
2+
ions is
and the nature of the mineral phases after nucleation remain unclear. To to increase the probability of ion-pair collision and speed up the reaction
the best of our knowledge, no such study has been conducted to char­ so as to reduce the computing time to realize the agglomeration of PNC.
acterize the entire bacterial calcification process from ions in aqueous In the statistical part of this work, the first 500 ps was used to equilibrate
solutions to mineral nucleation and crystallization at the molecular the structure again to ensure the stability and reliability of the system,
scale. Such information would help in our understanding of the mech­ and the last 500 ps was used for statistical calculation. The smoothed
anisms of bacterial calcification. field with width 3 Å and bins 12 Å were used to calculate the density
To understand the early stages of calcium carbonate nucleation, MD field of Ca2+ ions and CO2− 3 ions. The mobilities of Ca
2+
ions and CO2− 3
was used to simulate the binding of EPS with carbonate and calcium in ions perpendicular to the organic interface direction were described by
an aqueous solution. In particular, the modeling of aqueous calcium mean squared displacement (MSD).
carbonate in high concentrations in the absence of organic components
was conducted to explore the self-assembly of Ca-CO3 ion pairs in the 1 ∑N
MSD(t) = 〈|ri (t) − ri (0)2 |〉
aqueous solution. Experimental characterization, including wet chem­ N i=1
istry and electron microscopy, and MD simulation, successfully repli­
Where N is the number of target particles and ri (t) is the position of a
cated the whole bacterial calcification process. The aim of this study is to
particle at t time.
document the evolutionary path for the formation of calcified bacteria,
For the quantity of Ca2+ ions, the probability distribution
both in in modern and past environments, and thus to provide an insight

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X. Gao et al. Chemical Geology 617 (2023) 121266

Fig. 1. Snapshots of Ca2+-CO2− 3 on the surface of EPS. Initially, the Ca

2+
ions spontaneously coalesce with CO2−
3 ions in the solution, forming a topological structure
of first unit ion-pairs, which are then adsorbed on to the cell membranes surfaces. In addition, the ion-pairs are constantly being assembled, disaggregated and re-
assembled. Atoms are colored green, red, charcoal grey, blue and white for calcium, oxygen, carbon, nitrogen and hydrogen respectively. All the atoms in the
following figures are colored in the same way. Water molecules are hidden to facilitate observation of the kinetic processes. The ps stands for picosecond, which is a
unit of time. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

perpendicular to the organic interface direction was statistically calcu­ the mineral surface were removed (Yan et al., 2020). In-situ analysis of
lated in a time unit of every 200 ps. The statistical results can be used to minerals on the surface of bacteria was performed. First, calcified bac­
understand the evolution of Ca2+ ions along the Z-axis with time, and teria were identified under a focused ion beam scanning electron mi­
then provide insight into the precipitation process of Ca2+ ions on the croscope (Crossbeam 540, ZEISS, Oberkochen, Germany). Secondly, the
surface of the cell membrane. calcified bacteria were sprayed with Pt and sliced with an ion mill by a
HXe plasma beam (Helios G4 PFIB HXe, FEI, Hillsboro, OR, USA). The
lattice parameters and diffraction spots were observed by HRTEM-SAED
2.2. Experiments and characterizations
(JEM-F200) to determine the mineral phase in -situ.
The bacterium Bacillus licheniformis DB1–9 was selected for this work
3. Results
based on our previous experiments showing that it can induce carbonate
mineral formation (Han et al., 2018). The MICP components include the
3.1. Molecular dynamics simulation
nutritional materials required for bacterial growth, beef extract, 5.0 g/L;
sodium chloride, 5.0 g/L; tryptone,10.0 g/L, and precipitation compo­
Carbonate and calcium ions were randomly placed in the box to
nents, calcium chloride (0.01 M), Na2CO3 (0.006 M) and NaHCO3
correspond to their actual state in an aqueous solution. At the beginning
(0.006 M) (Han et al., 2018). Firstly, the nutrient components and the
of the operation, the first unit ion-pair (n (Ca-CO3), n = 1) is formed after
calcium chloride solution were sterilized in an autoclave, and sodium
the combination of carbonate and calcium. Over time, the first unit ion-
carbonate and sodium bicarbonate were sterilized using a filtration
pair assembles to form MIC (multi-ion complexes) with a larger size,
membrane. Secondly, 0.9 mL Na2CO3 (1 M) and 0.9 mL NaHCO3 (1 M)
eventually attaching itself to the cell membranes (Fig. 1). It is not
were added to the mixed solution of precipitate and nutrient compo­
difficult to see that the topological structure of MIC is dynamic with the
nents (150 mL), and the solution was maintained to a pH of 7.0 with HCl.
nature of continuous assembly or separation (the complete kinetics
Finally, 1.5 mL of isolated and purified Bacillus licheniformis DB1–9 seed
process can be seen in the Supplementay video 1).
liquid was added to the mixed solution and cultured at 25 ◦ C for 15 days
The different locations of Ca2+ ions on the EPS surfaces captured in
in a constant temperature incubator (Zhao et al., 2021). All culture ex­
this dynamic process were described as concentration profiles. The
periments were carried out in conical bottles and three groups of parallel
concentration profile shows that Ca2+ ions have a higher concentration
samples were set up.
in the aqueous solution at the initial state (Supplementary Fig. S3a). The
The minerals were harvested by centrifugation from the murky
Ca2+ ions were gradually adsorbed onto the surface of the EPS with this
liquid at the bottom of the bottles and washed with distilled water 3
dynamic process (Supplementary Fig. S3a-g). The Ca2+ ions maintain a
times and anhydrous ethanol 3 times, and then air-dried. The mineral
high concentration on the EPS (Supplementary Fig. S3g) after dynamics
phases were determined by powder X-ray diffraction (XRD) using CuKα
terminated (blue represents areas of calcium enrichment). The supple­
radiation with diffraction patterns of 2-theta = 20 to 60◦ at a rate of 8◦
mentary Fig. S3i and j are detailed maps corresponding to the concen­
s− 1 and a step-size of 0.02◦ (XRD, D/Max-RC, Rigaku, Tokyo, Japan).
tration profile capturing the dynamic process. Next, dynamical statistics
The organic components of the minerals were detected by FTIR using the
were performed on the particles (Ca2+ ions and CO2− 3 ions) to elaborate
KBr method with the scanning range of 500–4000 cm− 1 (FTIR, Nicolet
on the nucleation process from solution to the EPS interface. The density
380, Thermo Fisher Scientific Inc., Waltham, MA, USA). To circumvent
distribution results show that large amounts of carbonate and calcium
the organic contamination by culture medium components, the
accumulate on the surface of the EPS after the operation ceases, but
collected mineral pellets were cleaned with ~5% sodium hypochlorite
relatively little is formed in the solution (Fig. 2a). Based on the MSD of
for 5 min before FTIR testing to ensure that the organic components on

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X. Gao et al. Chemical Geology 617 (2023) 121266

Collecting snapshots of ion-pairs during the kinetic process described

above, it was found that CO2− 3 and Ca
2+
ions are paired in chains by
electrostatic interaction (Fig. 3a and Supplementary Fig. S4). The
dihedral angle, length, and the number of ion-pairs are regulated by the
aqueous solution and the coordination of CO2− 3 and Ca
2+
ions are 1–1,
1–2, and 2–3 (the number before the dash indicates the number of CO2− 3
ions and the number after the dash means the number of Ca2+ ions). In
fact, the angles and coordination numbers of the anions in the solution
are complex and varied, and they are not limited to those listed in
Fig. 3a. Therefore, it may be more accurate to describe the diversity of
the space of structural variability by referring to MIC as topologies.
Moreover, the ion-pairs with different lengths or dihedral angles will
evolve and aggregate over time. To prove that ion-pairs can be assem­
bled into MIC or even to the size of PNC, a larger water - CO2− 3 - Ca
2+

system was constructed to verify our views. As can be seen from the
dynamic snapshots captured, the ion-pairs of CO2− 3 - Ca
2+
gradually
increase to PNC with a large size by combining with other ion-pairs over
time (Fig. 3b and Supplementary video 2 for a completely dynamic
process). Previous studies have shown that the size of PNC and ACC is
about 0.5–2 nm and 30–70 nm, respectively (Dey et al., 2010). It is
worth noting that the size of PNC (~40 Å*20 Å) in Fig. 3b may be a
precursor of ACC. In particular, dehydration has been going on since the
formation of the first unit of the CO2− 3 - Ca
2+
ion-pair. It is only with
constant dehydration that new CO3 and Ca2+ ions assemble into large
2−

clusters and this process is spontaneous thermodynamically as sup­

ported by existing data (Demichelis et al., 2011; Politi et al., 2006;
Raiteri et al., 2015).
Therefore, it is reasonable to believe that the formation of CaCO3 on
the surface of the cell membrane begins with the ion-pairs of CO2− 3 - Ca
2+

in solution as the first unit, and they continue to assemble to the size of
PNC. ACC will be formed when the PNC grow to a certain critical state.
In addition, ACC, as a precursor of mineral nucleation, will develop into
different mineral phases, depending on developing ambient conditions.
In principle, thermodynamically unstable minerals with high entropy
should be formed first, such as vaterite (Chaturvedi et al., 2018; Lyu
et al., 2021).

3.2. MICP experiments

The XRD results show that the minerals induced by Bacillus lichen­
iformis DB1–9 are monohydrocalcite, vaterite and aragonite (Fig. 4a). In
addition, abundant functional groups were detected by FTIR, including
O-C=O, C– – N, C– – O, C–H and O–H (Fig. 4b), which confirms the
presence of organic components in the formation of surface minerals.
SEM observation of the precipitated minerals obtained by MICP after
cleaning and drying shows that a large number of tubular calcified
bacteria were distributed within the matrix of the mineral precipitates
(Fig. 5). The cell membranes also calcify to different degrees. Previous
studies have shown that vaterite and aragonite with porous spherical
Fig. 2. Statistics of Ca2+ and CO2− 3 ions. (a) The density of Ca
2+
and CO2−
3 ions
shapes can be formed in the presence of organic constituents (Islam
near the EPS is higher than that in the solution, and the density curve of Ca2+ et al., 2013; Song et al., 2021). Elemental composition was identified as
and CO2−3 ions near the EPS is almost the same, indicating that binding of Ca
2+ predominantly Ca, O, C, P and S through EDS analysis. In particular, the
to CO2−
3 ions into clusters is occurring. The simulation was run for 1000 ps, with presence of P and S likely originated from the bacterial cell membrane.
the first 500 ps for structural balance and the last 500 ps for MSD statistics. In Supplementary Table 1 shows the weight percentage and atomic per­
the statistical time of 500 ps, it can be seen that the MSD of Ca2+ and CO2−
3 ions centage of elements at the five sites in Fig. 5d.
is almost synergistic. Moreover, the MSD of Ca2+ and CO2− 3 ions increases at the Furthermore, HRTEM-SAED was utilized to characterize the mineral
end of the operation as shown in (b). The number of Ca2+ ions collected in 200
phases on the bacteria surfaces in-situ. A cross-section of calcified bac­
ps as a time unit, and Ca2+ ions gradually adsorbed from the solution to the
teria was obtained by cutting the calcified bacteria with a focused ion
surface of EPS as time went by are seen in (c). The ps = picosecond, PNC = pre-
beam at the position of the white line (Fig. 6a). An area of the surface of
nucleated clusters and MSD = mean square displacement.
the cell membrane was selected for SAED analysis and marked as a
dashed red line (Fig. 6a). Moreover, the outer surface of the cell mem­
carbonate and calcium between 500 and 1000 ps (Fig. 2b), the MSD
brane in the marked area was selected to measure the interplanar
curves of carbonate and calcium ions are almost the same, which con­
spacing (Fig. 6b). The SAED results show that the calcified bacteria are
firms the formation of the carbonate and calcium ion-pairs in the solu­
composed of major mineral phases: the (211), (300), (003), (113) and
tion. Fig. 2c shows the occurrence state of Ca2+ ions over time. As time
(223) crystal faces of monohydrocalcite (Fig. 6b1). In view of the small
goes by, the number of Ca2+ ions adsorbed on the EPS surfaces increases.
nucleation size of the minerals nucleated on the surface of the cell

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X. Gao et al. Chemical Geology 617 (2023) 121266

Fig. 3. Coordination information of Ca2+ and CO2− 3 ions. (a) Topological structure of ion-pair units. The ion-pair model captures the dynamic evolution of Ca
2+
and
CO2−
3 ions on the EPS, and the ball-stick model shows the structure. The topological structure shows that Ca 2+
-CO 2−
3 forms in chains before PNC, and that the number
and angle of coordination of the Ca2+-CO2−3 ions are different. In addition, even in the case of the same coordination number, such as 2–1 (the number before the dash
indicates the number of CO2−3 , and the number after the dash indicates the number of Ca
2+
ions), the coordination angles of CaCO3 and Ca2+ ions are different. (b)
The aggregation process of ion-pairs (b). Snapshots taken from the Ca2+-CO2− 3 -water system show that the ions form ion-pairs by electrostatic interaction and then
assemble into PNC of a larger size. A PNC with sizes of 40 Å*20 Å was captured in this simulation system, which may be critical to ACC (amorphous cal­
cium carbonate).

MHC (113) respectively (Supplementary Fig. S5). The elemental map­

ping indicates that Ca, O and C are distributed along the surface of the
cell membrane, and P is present in a circular distribution (Fig. 6c).

4. Discussion

It is well understood how calcium carbonate is formed at the organic

interface through MD simulation. Based on the MD simulation results,
we interpret that the Ca2+ − CO2−
3 pair is the first nucleation unit, which
then assembles into MIC and this evolves into PNC at critical points.
With the calcified bacteria obtained by the MICP experiments, the
minerals are identified as monohydrocalcite, vaterite and aragonite by
HRTEM-SAED.

4.1. Ion aggregation

Our results show that even in the presence of an organic interface,

the first unit ion-pairs of Ca2+ − CO2−3 were formed in the solution. Large
clusters are created by the continuous assembly of ion-pairs to each
other through electrostatic interaction and some negatively charged
clusters in the form of electrostatic force are adsorbed to the EPS surface
(Fig. 1 and Supplementary Fig. S4). These clusters are continuously
assembled and separated, as seen in the video of dynamics (Supple­
mentary video 1). This conclusion is also supported by the fact that Ca2+
ions are not densely clustered on the EPS surface during 800–1000 ps in
Fig. 2c. In general, the ion pairs are still attached to the EPS surfaces
(Fig. 2a). To observe the assembly behavior of ion-pairs with high
definition, a water - CO2− 3 - Ca
2+
system with more CO2−3 and Ca
2+
ions
was simulated. Through this process the large-scale assembly behavior
of ion-pairs was demonstrated: the MIC evolved into PNC, followed by
the formation of ACC once the critical point was reached (Fig. 3b).
Under the effects of solvation free energy and ionic strength (Demichelis
et al., 2011; Shang et al., 2020), PNC will exist in a stable form for a
certain time before ACC. It should be noted that the PNC is composed of
Fig. 4. The minerals composed of monohydrocalcite, vaterite and aragonite fluffy MIC with lots of water. According to the existing data, the size of
(a). The organic components detected by FTIR from minerals include carboxyl, MIC is <0.5 nm, the size of PNC is between 0.5 and 2 nm, and the size of
hydroxyl, carbonyl and nitrogenous groups (b). ACC is between 30 and 70 nm (Chen et al., 2020; Dey et al., 2010). Only
after dehydration can these hydrated substances be gradually ordered to
membrane, the crystal plane spacing was determined by averaging at generate more Ca-CO3 coordination. The size (~40 Å*20 Å) of PNC
least three bright and dark stripes by FFT (Supplementary Fig. S5). The shown in Fig. 3b may have reached the range observed by SEM. The
mineral species and crystal faces from the inner circle to the outer circle difference between the critical point state of PNC and ACC is the content
identified by the HRTEM are MHC (301), MHC (103), MHC (203) and of water in the cluster. The coordination of Ca2+ ions with water was
replaced by CO2− 3 when the water molecules were desorbed from the

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X. Gao et al. Chemical Geology 617 (2023) 121266

Fig. 5. Nucleation of minerals on the surface of cell membranes. A large number of tubular calcified cell membranes are stacked together. The calcium carbonate
nanoparticles are packed closely to each other and precipitated on the membrane surface (a, b). Calcified bacteria are cemented with precipitated mineral particles
(c). The calcified cell bacteria are clumped together in a tubular shape and the cross-sections are circular (d). The calcified layer on the surface of the cell membrane is
smooth and has good crystallinity; this is different from the calcium carbonate nanoparticles (d compared with b). (e) Calcified bacterial shells are cemented with
minerals. (f) Numberous calcified bacterial shells.

hydrated Ca2+ ions. A higher coordination number of CO2− 3 with Ca

2+
is surfaces (Fodor et al., 2020).
a characteristic feature of ACC and its crystals (Czapla and Freza, 2018; The protonation process of organic functional groups cannot be
Mergelsberg et al., 2020; Pondelak et al., 2020). Only after significant realized due to the limitation of MD, but it is undeniable that functional
dehydration, will the structure, size and degree of order of PNC be groups can adsorb Ca2+ ions independently to form organic‑calcium
further increased to form ACC; this is, called the nucleation event. In complexes (Lyu et al., 2021). In addition, even in the absence of pro­
addition, the assembly and separation of ion-pairs are mediated by tonation, hydroxyl and other functional groups will absorb Ca2+ ions
solvation, which means that a large amount of water surrounds them. through the Van der Waals effect (Petrash et al., 2017; Zhang et al.,
However, the adsorption effect of carbonate can destroy the water layer 2020; Zhao et al., 2021). In Fig. 2b, at first glance, the MSD curves of
to achieve spontaneous assembly with other ion-pairs. The binding en­ carbonate and calcium are consistent, but the MSD of calcium will be
ergy of CO2−
3 and Ca
2+
is − 20.3 kJ/mol (Demichelis et al., 2011), and it larger in the later stages of the system’s operation. We believe that some
is very easy to observe the rapid generation of precipitates after the organic functional groups will be adsorbed on to calcium after the
sodium carbonate solution is added to the calcium chloride solution in cluster adsorption to the organic interface, resulting in a stronger
the experiments. Combined with the evolution of the occurrence state of binding between the organic components and Ca2+ ions. In general,
Ca2+ ions over time and the density distribution, it can be shown that Ca- clusters formed in solution adsorb on to the surface of the cell membrane
CO3 was formed in the solution first and then adsorbed on to the surface to form precipitates, which is the main process of cell membrane calci­
of the EPS. The results of CaCO3 nucleation at the biological interface are fication. However, Ca2+ ions may also interact with organic components
consistent with the mechanism of CaCO3 precipitation in the aqueous as free radicals to form organic-Ca2+ complexes; this is an additional
system without organic constituents (Demichelis et al., 2011). The way of calcium precipitation. Based on the above MD results, it is sug­
mechanism of interface adsorption has also been explained by molecular gested that the nucleation process of CaCO3 at the organic interface is as
dynamics of CaMg(CO3)2 mineral precipitation induced onto clay follows: ions, ion-pairs, MIC, PNC, ACC with the gradual decrease of

6
X. Gao et al. Chemical Geology 617 (2023) 121266

Fig. 6. In-situ analysis of calcified cell membranes by HRTEM-SAED. The Bacillus licheniformis DB1–9 was sprayed with Pt and sectioned along the white line by FIB
(a). The area inside the red box in Fig. 6a is used for crystal plane measurement and spot diffraction, respectively (b and b1). SAED analysis of the minerals on the
surface of the cell membrane reveals the presence of monohydrocalcite (b1). MHC represents monohydrocalcite. Mapping shows that the main components of the
surface are Ca, O, C and P (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

entropy and continuous removal of water. Moreover, the previous state membrane after 15 days of culture (Fig. 4b and d). After 15 days of
of the MIC mainly takes place in the aqueous solution. The simulation cultivation, there was still monohydrocalcite, vaterite and aragonite on
results have revealed the presence of CaCO3 in an early stage of this the cell membrane based on XRD result; this can be explained by the
process at the organic interface. organic components having a delaying effect on the phase transition of
this spherical porous mineral. The EPS metabolized by a strain of bac­
teria isolated from a cave was able to store ACC for >26 weeks (Enyedi
4.2. The formation of carbonate minerals et al., 2020). In other words, the conversion of mineral phases is slow at
the organic interface under the action of organic components. EPS can
ACC, as a precursor (Politi et al., 2006; Shang et al., 2020), will be accumulate a lot of ACC by providing a local alkaline environment (Liu
transformed into ordered minerals with a crystal structure in a relatively et al., 2020a; Zhang et al., 2020); the conversion of the ACC will be
short time. However, the time for ACC to transform into minerals is controlled by the EPS, and the phase conversion will be slow under the
delayed in the presence of organic components, which are considered to action of organic components, so the phases of monohydrocalcite,
be a result of the protective effect of EPS on ACC (Aloisi et al., 2006). The vaterite and aragonite can co-exist. In particular, monohydrocalcite as
study of ACC has shown it can to be stored for at least half a year in the the most predominant product (~90%) induced by Bacillus licheniformis
presence of organic components and it can even keep the original DB1–9, the EPS and lipid bilayer on the cell surface may have contrib­
diffraction parameters after several wetting and drying sessions (Enyedi uted to the presence of hydrated minerals after 15 days of cultured. The
et al., 2020; Garvie et al., 2022). The growth and phase transition of mechanism of action of foreign ions (Mg2+ ions) on monohydrocalcite
crystals are controlled by thermodynamics. Vaterite, aragonite, and lies in the hydration layer of Mg2+ ions, which delays the coordination of
calcite are almost always converted by the dehydration of unstable ACC carbonate with calcium ions, allowing the calcium ions to carry water
(Czapla and Freza, 2018; Mergelsberg et al., 2020; Obst et al., 2009; molecules (Nishiyama et al., 2013; Zeyen et al., 2021). Similarly, EPS
Simonič et al., 2021). Monohydrocalcite, as an incomplete product of and the lipid layer’s preservation of aqueous substances (minerals) is
dehydration, is also common as an intermediate mineral in MICP pro­ consistent with the action mechanism of magnesium ions. The adsorp­
cesses (Zhao et al., 2021). Meanwhile, due to this instability, ACC will be tion effect formed by EPS, lipid layer and the mineral surface delays the
affected by the interface to improve its stability or maintain itself for a diffusion of water molecules in water bearing minerals to the solution
long time after aggregation at the biological interface (Enyedi et al., system (Enyedi et al., 2020). Moreover, in-situ identification (SAED) of
2020; Lyu et al., 2021; Mergelsberg et al., 2020). After accumulating a the surface minerals of calcified bacteria shows that the mono­
large amount of ACC on the surface of the cell membrane, a thermo­ hydrocalcite (211), (300), (003) and (113) and (223) spots are present;
dynamically sub-stable vaterite will crystallize, firstly under the control this supports this conclusion again. On the other hand, ACC will grad­
of the composition of the cell membrane surface and the properties of ually convert to calcite in a solution free of foreign impurities (Bots
the solution. ACC is less expensive to crystallize as nanoparticles than it et al., 2012).
is to grow indefinitely (Bruno, 2019). Then, the vaterite changes to Through experiments, we have determined the mineral phase of
thermodynamically stable minerals such as aragonite, mono­ CaCO3 at the organic interface after nucleation. We suggest that ACC is
hydrocalcite or calcite. In addition, the spherical vaterite and aragonite the precursor mineral to the formation of monohydrocalcite, vaterite
can form under the action of organic components (Islam et al., 2013; Lyu and aragonite based on our results and combined with the previous
et al., 2021). According to the SEM observations, carbonate minerals studies on the differential thermodynamic stability of CaCO3 (Lyu et al.,
with varying degrees of crystallization coated the surface of cell

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X. Gao et al. Chemical Geology 617 (2023) 121266

Fig. 7. Schematic diagram of cell membrane calcification.

2021). The experimental results provide evidence for mineral crystalli­ molecules and the formation of more Ca2+ and CO2− 3 coordination. The
zation and the mineral phases at the organic interface. calcified bacteria were obtained by the MICP experiments, and then the
Above all, combining the results of the molecular simulations and the sections of the calcified bacteria were prepared by FIB technology. The
experiments, it is concluded that the process of cell membrane calcifi­ microbially-induced production of a variety of carbonate minerals such
cation proceeds from ion to ion-pairs, MIC, PNC, ACC and then the as monohydrocalcite, vaterite and aragonite is caused by the particu­
carbonate minerals (monohydrocalcite, vaterite and aragonite) (Fig. 7). larity of the biological interface. Moreover, the crystal faces of MHC
Along this pathway, as the water is being lost, the entropy is going down (211), (300), (003), (113) and (223) were identified by SAED in-situ.
and the mineral stability is going up. Combined with the results of molecular dynamics and MICP experi­
ments, the process of cell membrane calcification is: ions → ion-pairs →
4.3. Geological significance multi-ion complexes → pre-nucleated clusters → amorphous calcium
carbonate → carbonate minerals (monohydrocalcite, vaterite and
Few examples of ancient bio-minerals composed of these unstable aragonite).
minerals, like vaterite and monohydrocalcite have been discovered, Supplementary data to this article can be found online at https://doi.
since they have changed into stable minerals (generally calcite) and org/10.1016/j.chemgeo.2022.121266.
possibly others (e.g. dolomite) through diagenetic processes, mostly
soon after their formation (Dupraz et al., 2009; Walton et al., 2021). In CRediT authorship contribution statement
addition to the magnesium ion possessing a high hydration energy,
biological activities may also affect monohydrocalcite in nature. As an Xiao Gao: Formal analysis, Investigation, Writing - original draft. Jie
example the calcimicrobe Girvanella, common in the Cambrian of Li: Formal analysis. Kaiming Hu: Formal analysis. Yanyang Zhao:
western Shandong, and elsewhere, is composed of the relatively stable Resources, Visualization. Yu Han: Formal analysis. Fang Liu: Concep­
calcite (Supplementary Figure S6). Calcified microorganisms preserve tualization, Software. Maurice E. Tucker: Writing – review & editing.
records of the sedimentary environment, and may provide useful in­ Zuozhen Han: Methodology, Formal analysis, Resources, Writing - re­
formation on the temperature and composition of seawater at the time view & editing.
(through stable isotope and REE analyses). As can be seen from the
studies reported here, there are advantages of undertaking laboratory Declaration of Competing Interest
experiments to describe accurately the biogeochemical processes
involved rather than postulating how minerals were and are being The authors declare that they have no known competing financial
precipitated without data. Microbial processes and the formation of interests or personal relationships that could have appeared to influence
microbialites and bio-minerals are one of the most important aspects of the work reported in this paper.
the biogeochemical cycle; it is useful to have a more-in-depth under­
standing of the bacterial calcification and biomineralisation in terms of Data availability
biogeochemical cycles in these times of environmental and climate
changes. Data will be made available on request.

5. Conclusions Acknowledgements

In this study, the whole process of cell membrane calcification of We would particularly like to thank editor, Prof. Hailiang Dong for
Bacillus licheniformis DB1–9 was examined by molecular dynamics (MD) expeditious editorial handing and three anonymous reviewers for
and laboratory experiments on microbially-induced carbonate precipi­ constructive and helpful comments which greatly improved the clarity
tation (MICP). The aggregation process of CaCO3 on the surface of EPS and presentation of the manuscript. This work was supported by the
produced by the bacteria was deduced by using molecular dynamics, National Natural Science Foundation of China [41972108, 42102134]
and a calcium carbonate - aqueous solution model with no biological and the Natural Science Foundation of Shandong Province
interface was established to prove that MIC could be assembled into [ZR2021QD094].
larger PNC. From the molecular dynamics results, Ca2+ − CO2−3 exists in
the form of ion-pairs in aqueous solution and continuously evolves into
MIC, PNC, and ACC. The state after PNC may occur on the surface of the
EPS, and this process is accompanied by the dissociation of water

8
X. Gao et al. Chemical Geology 617 (2023) 121266

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