Ecotoxicology and Environmental Safety 208 (2021) 111704

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Research paper

Clay-assisted protection of Enterobacter sp. from Pb (II) stress

Mu Su a, Feiyu Han a, Mengxiao Wang a, Jingxuan Ma a, Xuewei Wang a, Zhijun Wang a,
Shuijin Hu a, Zhen Li a, b, *
a
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
b
Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China

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

Edited by: Dr R Pereira Clay minerals can adsorb both microorganisms and heavy metals. In this study, typical soil bacterium, Entero­
bacter sp. was applied to investigate the potential protection of the bacterial cells from Pb2+ stress by clay
Keywords: minerals. The sorption by two representative types of montmorillonite (Mt) were contrasted, i.e., Mts/Mtw with
Clay mineral strong/weak CEC. There was no significant difference between the two clay minerals regarding their adsorption
Bacteria
of Pb2+ cations in water (i.e., ~55 mg L-1). However, the sorption of bacterial cells on the two clay minerals
Lead
showed evident contrasts, which resulted in the different capacity of Pb sorption. Mts with high CEC preferen­
Adsorption
Protection tially adsorbed abundant bacterial cells (rather than Pb2+) on its surface. The residual Pb2+ concentration in
solution actually raised by 7.5% after the addition of Enterobacter sp. In addition, both the Pb-contaminated cells
and “healthy” cells (with low Pb contamination) could be adsorbed onto Mt surface, whereas the latter domi­
nated the adsorbents on Mts. This was due to that the Mts with high CEC could provide more exchangeable
cations, building more cation bridging ligands between the microbial cells (whatever the types of cells) and clay
surface. Furthermore, the adsorbed “healthy” bacterial cells might escape from clay surface via “self-liberating”
mechanism, i.e., increasing electrostatic repulsion between the bacteria and clay during microbial decomposition
of the medium. This study hence elucidated the protection of microorganisms from Pb2+ stress by Mt.

1. Introduction
Clay minerals are common and indispensable components in
geologic and aqueous systems. They play a profound role in influencing
microbial metabolism, and physical and chemical environments (Lavie
and Stotzky, 1986; Li et al., 2019; McMahon et al., 2016; Sparks, 2003a).
Although variable in types and composition, most clay minerals have
large specific surface area and high cation exchange capacity (CEC) due
to their unique layered structure and isomorphous substitution (Borden
and Giese, 2001; Macht et al., 2011; Mermut and Lagaly, 2001).
Montmorillonite (Mt) is the most representative clay mineral with high
sorption capacity. It has been widely applied to environmental reme­
diation, medicine, and food production (Du et al., 2017; Wakai and
Almenar, 2015; Wang et al., 2019).
Microorganisms are ubiquitous in near-surface geologic systems,
linking the biosphere and pedosphere (Liu et al., 2011; Vogel et al.,
2014). In pedosphere, the growth and distribution of microorganisms
are significantly influenced by soil minerals, moisture, temperature, pH
and nutrients (Bashan et al., 1995; Elsas et al., 1991; Mander et al.,
2012). Microorganisms and clay minerals are often spatially associated
to form organic-inorganic complexes (Chenu et al., 2001; Huang, 2004).
In soil system, about 80–90% of microorganisms inhabit on mineral
surfaces (Hattori, 1973). Moreover, microorganisms are tightly involved
in the formation and transformation of clay minerals, which, in return,
affect their metabolism (Li et al., 2019; Vorhies and Gaines, 2009).
Microorganisms could adsorb metal cations (e.g., Pb, Cd, Cu, etc.) to
their cell membrane and wall (Fang et al., 2014; Guibaud et al., 2009;
Jiang et al., 2020; Qu et al., 2017; Sarı and Tuzen, 2009a, 2009b; Tian
et al., 2019). It reported that the maximum of adsorbed Pb and Cd on
montmorillonite were around 75 and 60 mmol Kg-1 respectively, while
the adsorption of Pb and Cd increased to 216 and 136 mmol Kg-1 in the
presence of bacteria-montmorillonite composites (Du et al., 2016).
Comparing with kaolinite and playgorskite, montmorillonite was able to
adsorb more Pb, which could reduce the Pb concentration from ~2000
to ~1000 mg Kg-1 (Li et al., 2017). Meanwhile, clay minerals can adsorb
microorganisms to their surface. Bacteria are more widely investigated
due to their high abundance, although fungi and actinomyces are also
associated with clay minerals. Metal cation sorption on clay minerals is

* Corresponding author at:College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China. 4399827.
E-mail address: lizhen@njau.edu.cn (Z. Li).

https://doi.org/10.1016/j.ecoenv.2020.111704
Received 6 May 2020; Received in revised form 14 October 2020; Accepted 19 November 2020
Available online 26 November 2020
0147-6513/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Su et al.
controlled by two mechanisms. The pH-independent adsorption is usu­
ally attributed to cation exchange within clay interlayers, resulting from
electrostatic interaction between the ions and the permanent charge
(Kraepiel et al., 1999; Mercier and Detellier, 1995; Sparks, 2003b). In
contrast, pH-dependent adsorption is resulted from surface complexa­
tion reactions (Kraepiel et al., 1999; Sparks, 2003b).
The sorption capacity of clay minerals is substantially influenced by
organic matters (Covelo et al., 2004; Dubbin, 2004; Schwab et al., 2005;
Town and Filella, 2002). It was proposed that there were inner-sphere
and outer-sphere adsorption of metals by clay minerals, and the latter
involve electrostatic coulombic interactions, which is more reversible
(Sparks, 2003b). Inner-sphere interactions include ligand exchange,
chelation and cation bridging. Hydrophobic interactions and other weak
interactions will result in outer-sphere interaction. In addition, the
inner-sphere complexation about Pb is favored at higher pH (> 6.3),
while outer-sphere complexation is favored at pH < 6.7 (Strawn and
Sparks, 1999; Warr et al., 2018). Sorption of metal cations by clay
minerals, either by inner- or outer-sorption, helps to reduce the
bioavailability of heavy metals (Dong, 2012).
The fate of heavy metals, such as Pb, in the soil-water environment
determines their potential mobility and toxicity in the ecosystem. Most
chemical forms of Pb are toxic, and hence can inhibit microbial growth
and activity (Li et al., 2016; Singh et al., 2006). Despite the considerable
Pb sorption by clay minerals (Li et al., 2017), desorption of Pb cations
from clay surface also happens (Singh et al., 2001; Rybicka et al., 1995).
In the complex system of clay minerals, heavy metals and microorgan­
isms, how do clay minerals influence growth of bacteria and Pb trans­
formation is still poorly understood. In particular, the influences caused
by variable sorption capacity of the clay minerals should be addressed.
In this study, we applied two types of montmorillonite, with strong
and weak adsorption capacity, to investigate bacterial activities under­
going Pb2+ stress. The concentrations of Pb and P were examined using
inductively coupled plasma optic emission spectrometry (ICP-OES). In
addition, attenuated total reflection infrared spectroscopy (ATR-IR), X-
ray diffraction (XRD), scanning electron microscopy (SEM) and trans­
mission electron microscope (TEM) were applied to observe the surface
morphology and compound changes of clay mineral.
2. Materials and methods
2.1. Sample collection
Two Mt samples (Mtw (SWy-1): with weak sorption capacity; Mts
(SAz-1): with strong sorption capacity) were provided by The Clay
Minerals Society (collection at Purdue University, Indiana, USA)
(Olphena and Fripiat, 2018). The CEC and specific surface area of Mtw
(Na-montmorillonite) are 76.4 meq 100 g-1 and 31.82 m2 g-1 respec­
tively, while the values are 120 meq 100 g-1 and 97.42 m2 g-1 for Mts
(Ca-montmorillonite) (Table S1). The variety of the exchangeable cat­
ions, i.e., Na and Ca, results in their different specific surface areas and
CEC (Olphena and Fripiat, 2018). In addition, it was proposed that
apatite is an excellent inorganic P source for bacterial growth in nature
(Su et al., 2019). Synthetic hydroxylapatite (Ap, Ca5(PO4)3OH >
99.999%) powder was purchased from Sigma-Aldrich® (St. Louis, MO,
USA). All the samples were stored in desiccator, and through 300 meshes
before the following experiments.
2.2. Bacterial strain and mediums
Enterobacter sp., with accession number of CGMCC (China General
Microbiological Culture Collection Center) No. 17428, was isolated from
the soybean rhizosphere soil in Nanjing, China (Li et al., 2018). They
were activated in solid beef extract-peptone (BE) medium at 37 ◦ C for 3
d. Then, the Enterobacter sp. suspension was prepared based on their
growth curve for the subsequent analyses (Li et al., 2018). The BE me­
dium contains 3.0 g L-1 of beef extract, 10.0 g L-1 of tryptone, 5.0 g L-1 of
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Ecotoxicology and Environmental Safety 208 (2021) 111704
NaCl, with or without 20.0 g L-1 of agar, pH 7.2–7.6. In addition, 0.1 g
Ap or 0.03 g Mt was further added into liquid BE medium. All the me­
diums were sterilized by autoclaving at 121 ◦ C for 20 min.
2.3. Bacterial growth influenced by clay mineral and Pb
Bacterial suspension (1 mL) was inoculated in a 150 mL triangular
flask with 50 mL liquid BE medium, which contains 0.03 g Mtw and Mts
respectively. The treatments have 1000 mg L-1 Pb2+ by adding Pb(NO3)2
powder, denoted as Mtw+B+Pb and Mts+B+Pb (B = bacteria). To
investigate the influences of inorganic phosphate on bacterial growth,
another two treatments with 0.1 g Ap addition were set as the
Mtw+B+Ap+Pb and Mts+B+Ap+Pb. To explore the effect of clay min­
eral and Ap on Pb2+ transformation, treatments with no bacteria
included were also prepared, i.e., Mtw+Pb, Mts+Pb, Mtw+Ap+Pb, and
Mts+Ap+Pb. The control treatment with only Pb2+ addition in BE me­
dium was also performed. Additionally, a parallel experiment was car­
ried out in water (equivalent Pb2+ were added into water containing
Mtw or Mts) to test the sorption of Pb by the clays (without influences
from the medium), the two treatments were denoted as Mtw-wt and
Mts-wt respectively. All these treatments were shaken in an orbital
incubator (180 rpm at 37 oC) in dark for 72 h with three replicates.
P, Pb, and pH values in the medium were analyzed after centrifu­
gation at 8000 rpm for 2 min and filtration through 0.45 µm poly­
ethersulfone (PES) membrane. Precipitates in all the treatments were
rinsed twice in sterile water and centrifuged for ATR-IR, SEM and XRD
analysis after drying. In particular, for the treatments with Enterobacter
sp. involved, a small amount of homogeneous medium was diluted and
fixed in 2.5% (v/v) distilled glutaraldehyde. Then, they were rinsed
twice in sterile water and dropped onto a sheet of glass and air dried for
SEM analysis. Meanwhile, part of precipitates was dispersed by ultra­
sonic oscillation in alcohol and were loaded on copper grids for TEM
studies.
2.4. Instrumentation
All pH values were measured by SG98 InLab pH meter (Mettler
Toledo Int. Inc.) with an Expert Pro-ISM probe. The concentrations of
soluble Pb2+ and P in filtrate were determined using ICP-OES (Agilent
710). The filtrate was diluted 10 times and then determined. The cali­
bration of P was performed by 0, 0.5, 1, 2.5, 5, and 10 mg L-1 standards
while Pb2+ was 0, 5, 10, 25, 50 and 100 mg L-1 standards. Information
about the ATR-IR, XRD, SEM, and TEM can be referred to the Supple­
mentary Materials.
2.5. Statistical analysis
All the treatments were conducted in triplicates. The differences of
pH, P and Pb2+ concentrations among the four treatments with Mtw and
Mts addition respectively were evaluated by a one-way analysis of
variance (ANOVA) and multiple comparisons using SPSS 16.0. The re­
siduals from the models were examined for normality and homosce­
dasticity using graphical diagnostic plots (i.e. Q–Q plots and residuals
plots) (Quinn and Keough, 2002). Multiple comparisons with least sig­
nificant differences (LSD) test. The significance was set at the p < 0.05
level. Different letters indicated significant differences among the
treatments.
3. Results
3.1. pH values
After 72 h inoculation, pH value was 5.92 in the control treatment
(Fig. 1). In the presence of clay mineral, pH values maintained at
5.97–5.99 in Mtw+Pb and Mtw+Ap+Pb (Fig. 1A). However, in the
treatments with Enterobacter sp. inoculation (Mtw+B+Pb and
M. Su et al. Ecotoxicology and Environmental Safety 208 (2021) 111704

Fig. 1. pH values (A, B), concentrations of P (C, D), and Pb2+ (E, F) in the eight treatments after 72 h incubation (N = 3). The dotted line indicated the pH values in
the control treatment (with only Pb2+ addition in medium). The initial pH values in all the treatments were maintained 5.80–6.00. Concentrations of P and Pb2+ in
the control treatment were 98.63 mg L-1 and 930.32 mg L-1 respectively. Different letters above the bars indicate a significant difference at 0.05 probability level.
Error bars represent ± SE.

Mtw+B+Ap+Pb), pH values were significantly increased to 8.24 and
7.55, respectively (Fig. 1A). This was caused by ammonia production via
breaking down organic nitrogen by the bacterium in the medium (pH
increased from neutral to >8) (Chen et al., 2019; Jiang et al., 2020). Mts
has the same trend as Mtw, i.e., pH values were 5.95 and 5.97 in the
treatments of Mts+Pb and Mts+Ap+Pb, while they significantly
increased to 8.33 in Mts+B+Pb (p < 0.05) and 8.11 in Mts+B+Ap+Pb (p
< 0.05) (Fig. 1B). In particular, the pH value in Mts+B+Pb was higher
than that in Mts+B+Ap+Pb (p < 0.05).
3.2. Pb2+ removal by bacterium and clay mineral
The P concentration was 98.63 mg L-1 in the control treatment. For
the treatments with addition of Mtw, there were no evident difference in
P concentrations, i.e., from 91.51 to 86.68 mg L-1 (Fig. 1C). However,
the P content decreased after Enterobacter sp. addition, which was
mainly due to the consumption of P by the bacterial cells. As well as Mtw,
P concentrations were 95.42 and 95.66 mg L-1 in the Mts+Pb and
Mts+Ap+Pb treatments. In the presence of Enterobacter sp., they
decreased to 92.98 and 91.27 mg L-1 in Mts+B+Pb and Mts+B+Ap+Pb
respectively (Fig. 1D).
After 72 h incubation, the residual Pb2+ in treatments of Mtw-wt and
Mts-wt were 942.94 and 946.97 mg L-1 (initial Pb2+ concentration =
1000 mg L-1), respectively. In the control treatment, the concentration
of residual Pb2+ was 930.32 mg L-1. The Pb2+ contents in Mtw+Pb and
Mtw+Ap+Pb were 853.12 and 858.88 mg L-1 respectively (Fig. 1E),
indicating no significant difference. In the treatments of Mtw+B+Pb and
Mtw+B+Ap+Pb, the values declined to 808.83 and 838.21 mg L-1.
There was no evident microbial enhancement of Pb2+ removal
compared with the values from Mtw+Pb and Mtw+Ap+Pb.
The concentrations of Pb2+ in Mts+Pb and Mts+Ap+Pb were 872.48
and 888.91 mg L-1 (Fig. 1F). However, the residual Pb2+ content was

3
M. Su et al.
937.90 mg L-1 after addition of Enterobacter sp. in the Mts+B+Pb treat­
ment, which was significantly higher than that in the treatment of
Mts+Pb by 7.5% (p < 0.05). This was different from the result with Mtw
involved. In the Mts+B+Ap+Pb treatment, even with phosphate addi­
tion, the residual Pb2+ was 886.68 mg L-1, which had no difference with
that in Mtw+Ap+Pb. These results suggested that the strong sorption of
Mts did not benefit the Pb sorption (onto Mt) after bacterial incubation.
3.3. ATR-IR analysis and XRD
ATR-IR spectra showed multiple peaks of phosphates. The peaks at
600, 960 and ~1089 cm-1 were assigned to the P-O (PO4) vibration of
phosphates (Chen et al., 2017; Jastrzebski et al., 2011; Rehman and
Bonfield, 1997). In addition, the peak at 626 cm-1 was assigned to the
vibrations of OH- groups in hydroxylapatite (Jastrzebski et al., 2011).
Four peaks located at 600, 628, 960, and 1089 cm-1 in Mts+Ap+Pb were
more intense than that in Mtw+Ap+Pb. This indicated that more un­
dissolved Ap was left in the treatment with addition of Mts, due to their
sorption of Ap.
The peaks at 884, 916, and 1117 cm-1 were assigned to the Si-O vi­
bration of Mtw (Fig. 2A). These peaks in Mtw+Ap+Pb and
Mtw+B+Ap+Pb treatments were weaker than those in other treatments.
Specially, the peak located at 1117 cm-1 disappeared in Mtw+B+Ap+Pb.
This might be due to the coverage of Mtw surface by the adsorbed sub­
stances. In addition, the peak at 1636 cm-1 was assigned to the vibra­
tions of O-H (H2O) groups in Mtw (Zhang et al., 2018), and it was also
decreased in Mtw+B+Ap+Pb. Comparing with Mtw, all the peaks at 835,
916 and 1104 cm-1 (Si-O vibrations) were decreased/disappeared in the
four treatments with the addition of Mts (Fig. 2B).
XRD patterns showed predominant components among various
treatments. The three strongest peaks located at 31.77o, 32.20o and
32.90o in the treatments with the addition of Ap (Fig. 3). For the four
treatments with the addition of Mtw, quartz (with the dominant peak at
26.24o) and Mt (19.94o) can be identified (Fig. 3A). It was shown that
pyromorphite (29.90o and 30.18o) only appeared in treatments of
Mtw+B+Pb, Mts+B+Pb and Mts+B+Ap+Pb (Fig. 3B). Therefore, the
inoculation of Enterobacter sp. promoted the formation of pyromorphite
from mobile Pb2+ (Li et al., 2018).
3.4. SEM and TEM analysis
The EDS semi-quantitative analysis showed that the average
Fig. 2. ATR-IR spectra (500–2000 cm-1) of the original Mt samples and the precipitation collected from the eight treatments: A for Mtw and B for Mts.
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Ecotoxicology and Environmental Safety 208 (2021) 111704
adsorption of Pb cations by the two types of clay mineral had no sig­
nificant differences (Fig. 4A and B), which was consistent with our ICP
results. In addition, in the treatments of Mtw+B+Pb and Mts+B+Pb,
some rod-like particles with abundant Pb and P were observed on the
surface of Mt (Fig. 4C and D). These are ascribed to pyromorphite
mineral, also confirmed by the above XRD results (Li et al., 2016).
Fig. 5 A showed the morphology of Enterobacter sp. (collected from
the medium after incubation) in the treatment of Mtw+B+Pb. EDS
mapping indicated that the distribution of Pb matched the location of
bacterial cells. In addition, in the treatment of Mtw+Ap+B+Pb, many
cells showed evident damage (Fig. 5B). However, a relatively large
number of “healthy” bacterial cells (no evident Pb signal under EDS
mapping) were discovered in both Mts+B+Pb, and Mts+B+Ap+Pb
treatments (Fig. 5C, D). Therefore, the decreased sorption ability of Pb to
bacterial cells contributed to the enriched Pb in the medium of
Mts+B+Pb, which is confirmed by ICP results.
On Mt surface, some bacterial cells covered with Pb were also
adsorbed on Mtw in the treatment of Mtw+B+Pb (Fig. 6A and B). These
cells were near-spherical, which adsorbed on the depression and surface
of Mtw (Fig. S1A, B). There also existed some damaged cells, which
might be attributed to the demise of bacterial cells under high Pb2+
stress (Fig. S1A, B). In addition, the cells that covered with Pb and
“healthy” bacterial cells were both appeared on the surfaces of Mtw in
Mtw+B+Ap+Pb treatment (Fig. S1C, D). Additionally, many Pb-
contaminated bacterial cells were identified on the surface of Ap
(Figs. 6C and S1E, F). In the Mts+B+Pb treatment, it showed a relatively
less Pb-contaminated bacterial cells on Mts, whereas more “healthy”
bacterial cells on Mts surface (Fig. 6D). Similarly, in the Mts+B+Ap+Pb
treatment, more “healthy” bacterial cells rather than Pb-contaminated
bacterial cells were adsorbed on Ap (Fig. 6E and F) and Mts (Fig. 6G
and H). In contrast to Mtw, relatively more “healthy” bacterial cells (not
plump and smooth) were adsorbed on Mts surface in both treatments of
Mts+Ap+Pb and Mts+B+Ap+Pb (Fig. S2).
In the treatment of Mtw+Ap+Pb, we observed small particles con­
taining Pb on Mtw surface via TEM-EDS (Fig. 7A). In the presence of
Enterobacter sp., there were no difference in Pb distribution on the Mtw
surface (Fig. 7B, C). In contrast, the counts of Pb nano-particles (NPs)
that adsorbed on Mts surface in the treatment of Mts+Ap+B+Pb (Fig. 7E,
F) were significantly less than that in the Mts+Ap+Pb treatment (see
Fig. 7D). This might be attributed to the preferential adsorption of
bacterial cells (rather than Pb2+ cations) on the Mts surface.
M. Su et al. Ecotoxicology and Environmental Safety 208 (2021) 111704

Fig. 3. X-ray powder diffraction patterns of the precipitates collected from the eight treatments: A for Mtw and B for Mts.

Fig. 4. SEM imaging on the surface of Mtw and Mts. A-B: Imaging on the surface of Mtw (A) and Mts (B) after adding Pb2+ in water solution (the parallel treatments).
The table at the bottom of A & B showed the EDS results of Pb contents in the corresponding circular regions in A and B. C-D: Distribution of Pb and P on the surface
of Mt in Mtw+B+Pb (C) and Mts+B+Pb (D). The squares show the selected areas (circular) analyzed by EDS.

4. Discussion primary Pb precipitates on bacterial cell walls were likely transformed

4.1. Hazardous effects of Pb on bacterial cells
Microorganisms are present in a wide range of terrestrial and aquatic
environment. They are inevitably subjected to heavy metal stress. Under
high Pb2+ stress, the adsorbed Pb cations could penetrate into cells
(Guibaud et al., 2009; Jiang et al., 2020). These were harmful to mi­
crobial normal growth and metabolism. The reactive sites on bacterial
surfaces can promote mineral nucleation (Dong, 2012). Then, the
into hard "shells". The dead bacterial cells would still own the
Pb-containing shells (Figs. 5B and S1A and B). Although harmful to
bacteria, the shell allows physical-chemical techniques to identify these
contaminated cells (considering the Pb2+ with high atomic weight and
the identifiable morphology).
4.2. Adsorption of Pb2+ by clay mineral
Montmorillonite bears constant negative charges due to the

5
M. Su et al. Ecotoxicology and Environmental Safety 208 (2021) 111704

Fig. 5. SEM and EDS mapping images of Enterobacter sp. in Mtw+B+Pb (A), Mtw+B+Ap+Pb (B) and Mts+B+Pb (C), Mts+B+Ap+Pb (D) treatments after 72 h
incubation. The EDS mapping (based on Pb signal) was shown in the middle of the figure. The samples were collected from the medium after incubation.

Fig. 6. SEM and EDS mapping images of

Enterobacter sp. in Mtw+B+Pb (A, B),
Mtw+B+Ap+Pb (C) and Mts+B+Pb (D),
Mts+B+Ap+Pb (E, F, G, H) treatments after
72 h incubation.
A-B: Enterobacter sp. on the surface of Mtw. C, E-
F: Enterobacter sp. on the surface of Ap. D, G-H:
Enterobacter sp. on the surface of Mts. The bac­
terial cells in the circle were the “healthy”
Enterobacter sp. with no evident Pb signal under
EDS mapping. The samples were collected from
the precipitates after incubation.

6
M. Su et al.
Fig. 7. Distribution of Pb on the surface of Mt in Mtw+Ap+Pb (A) and Mtw+B+Ap+Pb (B, C), Mts+Ap+Pb (D) and Mts+B+Ap+Pb (E, F). The table at the bottom
showed the EDS results of Pb contents in the corresponding circular regions.
substitution for Si4+ or Al3+ by lower valence cations (such as Fe2+,
Mg2+) at the tetrahedra/octahedra sites. Free Pb2+ were hence adsorbed
on clay mineral surface for charge balance. In addition, metal cations
can also co-precipitate with clay minerals, and even incorporated into
their structure (Brandt et al., 2007; Dong, 2012; Sparks, 2003b). How­
ever, the Pb2+ adsorption capacity of the two clay minerals were
approximately 55 mg L-1, which was relatively low (considering the
initial Pb2+ concentration of 1000 mg L-1). The previous research
noticed that Pb2+ could also form complexes with organic molecules
(Schwab et al., 2005; Town and Filella, 2002), reducing the Pb adsorbed
by clays. Moreover, low-molecular-weight organic substance and mac­
romolecules, such as enzymes, proteins and DNA, could be adsorbed
onto clay minerals (Jiang et al., 2007). These organic matters could
further reduce Pb sorption as they occupy the available sites on clay
surface. Therefore, there was no significant difference in the adsorption
properties for Pb between the two clay minerals in BE medium (see
Fig. 1 E and F).
In natural environments, phosphorus (P) is the major limiting
nutrient for microbial growth, as well as tightly related to Pb2+ removal
via the formation of stable pyromorphite (Ma et al., 1993; Shen et al.,
2018; Tian et al., 2020; Zeng et al., 2017). In particular, Pb2+ was able to
react with Ap to form pyromorphite on Mt surface (Li et al., 2017).
Although the Ap addition resulted in the formation of a small amount of
pyromorphite, it showed weak effect on altering P and Pb2+ concen­
trations due to its low solubility under alkaline environment (Fulmer
et al., 2002).
4.3. Adsorption of bacterial cells onto Mt
Microorganisms were able to adhere on the surface of clay minerals
via a variety of mechanisms, including electrostatic interactions, extra­
cellular polymers, protein-binding receptors and hydrophobic in­
teractions (Jiang et al., 2007; Yan et al., 2019). The adsorption was
influenced by the pH and cation concentration (Li et al., 2019; Qu et al.,
2017). Clay mineral could reduce bacterial activity via inhibiting their
mobility, while contributing to their growth in long-term (Su et al.,
2019). In addition, clay minerals could also adsorb small molecular
metabolites secreted by microorganisms (Zhang et al., 2018). Therefore,
microorganisms could in turn alter physiochemical properties of clay
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Ecotoxicology and Environmental Safety 208 (2021) 111704
minerals (Yan et al., 2019). They can even accelerate the dissolution of
clay minerals via redox reactions and release of metabolite (Dong,
2012).
Comparing with Mtw, Mts, with higher CEC, had greater potential to
release more cations (such as Mg2+ and Ca2+) into solution (Table S1).
The elevation of these cations in solution could enhance the adsorption
of “healthy” bacterial cells to Mts via the formation of bridging
structures.
4.4. Competition between Pb and bacterial cells
In a system with mobile Pb2+ cations and bacterial cells, there
existed competition between the adsorption of bacterial cells and Pb2+
by clay minerals. Based on the evidences from 4.2 and 4.3, the differ­
ences of sorption ability of the clay minerals did not cause their different
adsorption to Pb2+. However, it resulted in the evident contrast of the
sorption of microbial cells. These cells hence successfully “beat” Pb2+
cations on the clay surface. Moreover, the adsorption of Pb onto mineral
followed pseudo-second order kinetics very well (Sarı and Tuzen, 2009a,
2009b). The bacterial cells built biofilms once adsorbed on mineral
surface, which further shield the reactive sites on clay mineral surface
for Pb2+ (Omarova et al., 2018; Poorni and Natarajan, 2013). In addi­
tion, the adsorption of Pb2+ depends greatly on mineral/bacteria ratio
and their different affinities towards Pb2+. The preferential adsorption
of bacterial cells by clay minerals decreased the adsorption of Pb2+ by
bacterial cells in solution, which caused the relatively high Pb2+ in
solution.
4.5. Critical role of CEC
CEC was proposed as a critical factor in interactions between clays
and microorganisms (Zhang et al., 2019). Other impurities were not
detected in the Mt samples according to the information from The Clay
Minerals Society, except for quartz and scant feldspar, which are both
inactive minerals. Then, the CEC should be ascribed to the clay minerals.
The clay mineral with high CEC had more exchangeable cations on their
surface, building more cation bridging ligands. Then, bacterial cells,
whatever healthy or Pb-contaminated, could be more easily adsorbed
onto clay mineral surface (Du et al., 2017; Warr et al., 2018).
M. Su et al.
Extracellular polymeric substances (with abundant negative charges e.
g., hydroxyl and carbonyl) of the microorganism could further enhance
such bridging effect (Yan et al., 2019). For the clay minerals with low
CEC (e.g., Mtw), the mobile Pb2+ worked as bridging cations, which
caused abundant Pb-contaminated cells on its surface. Therefore, clay
minerals with high CEC could have higher ability to protect bacterial
cells from Pb stress.
There was also a “self-liberating” mechanism after sorption of the
microbial cells by clay minerals. The bacterial cells could produce
ammonia via breaking down organic nitrogen and increased the pH of
solution (Chen et al., 2019; Letoffe et al., 2014). Subsequently, increased
pH promoted the electrostatic repulsion between bacterial cells and clay
minerals in the solution (Qu et al., 2017), as well as easily displaced
Ca-cation bridge (Sparks, 2003a). Then, the adsorbed bacterial cells
might be re-released from clay surface, due to the increased electrostatic
repulsion with mineral surface.
5. Conclusions
A complicated connection existed among clay minerals, bacteria and
heavy metals. Bacterial cells could adsorb Pb on their surface when
exposed to high concentration of Pb2+, which was not positive to bac­
terial growth and metabolism. There was no significant difference
regarding the adsorption of Pb2+ cations by the two clay minerals.
Remarkably, the residual Pb2+ concentration in solution with the Mts
(with high CEC) addition actually raised by 7.5% after inoculating
Enterobacter sp. In addition, the clay mineral with high CEC preferen­
tially adsorb abundant “healthy” cells. It is due to that the high CEC of
clay minerals can provide more exchangeable cations, building more
cation bridging ligands between the microbial cells and clay surface. The
adsorption of “healthy” cells, in addition to the “self-liberating”
pathway, reduced the exposure of bacteria to Pb stress. The in-field work
based on this preliminary study should be encouraged in future.
CRediT authorship contribution statement
Mu Su: Conceptualization, Methodology, Investigation, Writing -
original draft. Feiyu Han: Writing - original draft, Investigation, Formal
analysis. Mengxiao Wang: Investigation, Validation. Jingxuan Ma:
Investigation, Validation. Xuewei Wang: Investigation, Validation.
Zhijun Wang: Investigation, Data Curation. Shuijin Hu: Writing. Zhen
Li: Conceptualization, Writing - review & editing.
Declaration of Competing Interest
All authors declare no competing financial interest.
Acknowledgements
We thank Dr. Juan Li and Dr. Jiani Chen at Nanjing University State
Key Laboratory for the technical assistance with data collection and
analyses. This work was partially supported by National Key Research
and Development Program of China (No. 2017YFC0503902), Funda­
mental Research Funds for the Central Universities (No. KYZ201712),
and Program for Student Innovation through Research and Training (No.
1813C15 & 201910307090P).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2020.111704.
8
Ecotoxicology and Environmental Safety 208 (2021) 111704
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